Smart Hydrogels Based Platforms for Investigation of Biochemical Reactions

Smart hydrogels based platforms for investigation of biochemical reactions

DISSERTATION

zur Erlangung des akademischen Grades

Doctor rerum naturalium

(Dr. rer. nat.)

vorgelegt

der Fakultät Mathematik und Naturwissenschaften

der Technischen Universität Dresden

von

Nidhi C. Dubey

geboren am 18.10.1985 in Nashik, India

Die Dissertation wurde in der Zeit von Februar 2011 bis März 2015

am Leibniz-Institut für Polymerforschung Dresden e.V. angefertigt

Gutachter: Prof. em. Dr. rer. nat. Manfred Stamm

Prof. Dr. rer. nat. habil. Karl-Heinz van Pée

Eingereicht am: 13 May 2015

Tag der Verteidigung: 20 August 2015

TABLE OF CONTENT

1 Introduction ...... 1 1.1 Motivation ...... 1 1.2 Goals ...... 6 1.3 Outline ...... 7

2 Fundamentals and Methods ...... 9 2.1 Smart polymer hydrogel ...... 9 2.1.1 Introduction ...... 9 2.1.2 Types of smart polymers ...... 10 2.1.3 Core shell microgel and synthesis ...... 17 2.2 Biochemical reaction ...... 25 2.2.1 Acetyl CoA synthetase ...... 25 2.2.2 Pyruvate kinase and lactate dehydrogenase ...... 28 2.3 Enzyme immobilization ...... 31 2.3.1 Introduction ...... 31 2.3.2 Factors affecting enzyme stability ...... 35 2.3.3 Michaelis Menten kinetics ...... 36 2.4 Characterization techniques ...... 39 2.4.1 Dynamic light scattering ...... 39 2.4.2 Zeta potential ...... 41 2.4.3 Scanning electron microscopy ...... 43 2.4.4 UV visible absorbance spectroscopy ...... 46 2.4.5 Fluorescence spectroscopy ...... 48 2.4.6 Circular dichroism ...... 49 2.4.7 Enzyme assays ...... 51

3 Results and discussions ...... 53 3.1 Thermo-responsive microgels synthesis and characterization ...... 53 3.1.1 Introduction ...... 53 3.1.2 PNIPAm-AEMA core shell microgel preparation ...... 53 3.1.3 PNIPAM-PEI core shell microgel preparation ...... 56 3.1.4 Conclusion ...... 59 3.1.5 Experimental section ...... 59 3.2 PNIPAm-AEMA core-shell microgels support for Acs ...... 63 3.2.1 Introduction ...... 63 3.2.2 Results and discussion ...... 63

3.2.3 Conclusion ...... 71 3.2.4 Experimental section ...... 72 3.3 Adsorptions of Acetyl CoA synthetase on PNIPAm-PEI 1 microgels ...... 77 3.3.1 Introduction ...... 77 3.3.2 Results and discussion ...... 78 3.3.3 Conclusion ...... 87 3.3.4 Experimental section ...... 88 3.4 Sequential enzymes reactions on thermo-responsive microgels ...... 91 3.4.1 Introduction ...... 91 3.4.2 Results and discussion ...... 92 3.4.3 Conclusion ...... 102 3.4.4 Experimental section ...... 103

4 Summary ...... i

5 Future outlook ...... v

6 Appendix ...... ix 6.1 Abbreviations ...... xi 6.2 List of figures ...... xiii 6.3 List of tables ...... xix

7 Bibliography ...... xxi

Acknowledgements ...... xliii

1. Introduction

1 Introduction

1.1 Motivation

Principally the life on earth is driven by the cascades of biochemical reactions taking place inside the micro-compartments of the cell in presence of the biocatalyst called ‘enzymes’.1,2 Many of the metabolic reactions are catalyzed via one or more membrane-associated multi-enzyme complexes and not actually by free-floating ‘soluble’ enzymes.3,4 By sitting close to each other enzymes carry out reaction in tandem by micro-channeling the molecules, thus overcoming the diffusion barrier due to viscous interior of the cell.3,5,6 Under physiologically mild conditions the enzymes interplay different class of reactions (amidation, acetylation, esterification, oxidation, hydrogenation, etc.) to fulfill the metabolism.1,7,8 The high specificity, efficiency and green nature of the enzyme catalyzed reaction are the beneficial characteristics for the development of clean processes in synthesis of the chiral building blocks and biologically active compounds.9–12

Pharmaceutical 7 6 21 Biotech R&D Diagnostic 9 Biocatalyst Food and Beverage 12 15 fabric and Houshold Biofuel 6 3 Animal feed 21

Figure 1.1. The chart shows the World enzyme market in 2010 for different sectors (The Freedonia Group Inc.).13,14

1 1.1 Motivation

The World enzyme market is growing and is expected to rise to $8 billion in 2015 with annual 6.8 % increase in the demand.14 Figure 1.1 depicts the sectors that employee enzymes for their purposes, and clearly indicates pharmaceutical industries as one of the most prominent user of the enzymes. Straathof and co- worker analyzed 134 different industrial transformations data and presented an overview of the use of different enzyme classes and whole cells for the conversions (Figure 1.2).15 Among the six classes of enzyme (oxido-reductase, transferase, hydrolase, lyase, isomerase, ligase), most in vitro explored reactions were by hydrolases that perform reactions with relatively simple chemistry.16,17

Transferases Oxido- reductase

Hydrolases Oxidizing cells

Reducing cells Isomerases Lyases

Figure 1.2: The chart represents the contribution of different classes of enzymes in industrial bio-transformations.15

On the other hand enzymes belonging to the other classes have potential to carry complex reactions of commercial importance.17 But the obligatory requirement of the cofactor and high cost of cofactors limits their wide scale use.17,18 Generally cofactors generating (enzymatic) systems are simultaneously used with the process to provide continuous supply of the molecule.17–19One such pharmaceutically important process is Polyketide-based drugs synthesis. Polyketides are secondary metabolites synthesized by microorganism and plants as the defense molecules. The drugs like tetracycline and erythromycin 2 1. Introduction

(antibacterial), doxorubicin and mithramycin (anticancer), EGCG and resveratrol (antioxidants), and Zocor (drug lowering cholesterol levels) comprise this class. The polyketide-based drugs and products constitute one third of the drug market of which cephalosporin alone marked $11.9 billion sale in 2009.20,21 Therefore, pharmaceutical companies attach an enormous value to the polyketide compound libraries collected from bacteria, fungi, and plants, and this has led to a rapid increase in commercial bio-combinatorial research activity.22,23Acetyl Co- enzyme A (ACoA) is a biomolecule central to the metabolic process, as it is connecting link between glycolysis and tri-citric acid cycle and in the later pathway it is oxidized to produce energy.16 It plays vital role as a precursor molecule in various primary (fatty acid) and secondary metabolite (polyketide) syntheses.16,24,25 The high cost and increasing demand of ACoA for the synthesis of these drugs makes the process highly expensive. Acetyl CoA synthetase is most commonly used enzyme for regeneration of AcoA.17,26 Similarly, the synthesis of polyketide is highly co-factor (ATP, NADH etc) dependent for reduction and group transfer reactions. ATP is a high-energy biological compound and is stepwise cleaved into adenosine di phosphate (ADP) and adenosine mono phosphate (AMP) to obtain chemical energy in the cell.16 Numerous enzymatic reactions in both anabolic and catabolic metabolism require ATP as the cofactor, specifically in the formation of P—O bonds.27 Enzymes belonging to the class ligase require ATP for accomplishing the reaction.28 The in-vitro ATP-dependent enzymatic reaction are accomplished by either their direct addition in stoichiometric amounts or by the inclusion of a cofactor regeneration system.27 The regeneration of ATP from ADP is performed using different kinase-substrate combinations (pyruvate kinase-phosphoenol pyruvate (Pk-PEP), hexokinase-

29–31 glucose 6-phophate (Hk-G-6P), acetate kinase-acetyl phosphate (Ak-AcPO4). In spite of high cost of PEP, the high donor capacity and solubility in aqueous solution has made it most suitable substrate for ATP generation.27 The Pk-PEP reaction is further probed by another enzyme lactate dehydrogenase (Ldh) by monitoring NADH oxidation at 340 nm. This minimizes the effect of product inhibition by pyruvate to Pk and provides continuous monitoring of the reaction. The Pk-Ldh coupled enzymes are well known assay method to detect ADP,

3 1.1 Motivation phosphoenol pyruvate (PEP).32 Therefore the enzymes (e.g. acetate kinase) reaction-producing ADP can be followed using these coupled enzymes reaction. Nevertheless the benefits of the enzymes as the superior catalysts, the short life span of the enzymes in the solution may allow only one time use of the enzymatic regeneration system. The enzymes are tailored to catalyze the reaction under biological conditions, but for the industrial biotransformation the conditions may significantly differ from the natural state.33 The preservation of catalytic complex molecular structures of enzyme and the economics of the process are key issues in their wide scale usage.33,34 There are many ways to increase robustness of biocatalysts through genetic manipulation, direct evolution, and immobilization.33 Compared to the labor-intensive molecular biological techniques, immobilization of the enzymes is alternative and feasible approach. The immobilization of enzymes on artificial carrier may also provide better understanding of enzymes and their interaction in the confined spaces.35 In today’s world, the designing and synthesis of materials that simulate the supra-molecular architecture of the naturally occurring assemblies is possible.36 The living cells harnesses polymers not only for their structural framework but also as the vital functional components. In this respect, for the in-vitro use of enzymes, the selection of a suitable carrier system that mimics natural conditions and provide high stability is desirable for sustainable biocatalysis.3,9 The process- dependent design of carrier and choice of appropriate conjugation methods, has gained great impetus in the field of immobilization techniques. The anchoring of the biomolecules [DNA, RNA, proteins (enzymes, antibodies, etc.), substrate and co-factors] is an imperative arena of research for various applications like biosensing, bio-separation, biocatalysis, tissue engineering, etc.37–41 Mannens et al. reported Acs immobilization on glass beads for synthesis of C11 labelled acetyl CoA and further column reactor was fabricated to obtain complete conversion of 1µmol of acetate to AcoA.42,43 Reports are also available where Acs was immobilized on different support materials (e.g. Nafion membrane, cellulose fibres, etc) to study immobilization and biochemical reactions.44,45 Sundaram and others have reported the immobilization of Pk-Ldh enzymes on the membrane reactor and studied their detection ability to probe ADP, PEP and pyruvate.46,47 The glass beads were also utilized as a carrier for Pk-Ldh and their ATP

4 1. Introduction regeneration ability were studied.48 Recently complex carrier with agarose core and silica-polyethyleneimine shell was used to study this sequential enzyme reaction.2 The polymer-based particles of Nano and submicron size have become attractive material for their role in the life sciences. Smart colloidal particles are particularly interesting due their stimuli responsive properties and are generally known as microgels.49,50 Microgels are gel particles with size <1000 nm, composed of network of polymers that have high water holding capacity.51 With the advances in synthetic protocols of the microgels and commercial availability of many of the monomers, it is feasible to tune the properties of the particles as per the process requirement.52 The microgels can be designed in multi-structural level with core-shell or yolk-shell morphologies.53 The core shell microgel with functional shell allows high loading of ligands onto the microgel particles due to increased availability of functional group on the outer surface.54 Small size of the particles tends to respond fast to the external signal (temperature, pH, redox signal, magnetic, chemical, or biomolecules) by changing the conformation of the polymers.55,56 Also the advantage of large surface to volume ratio of these particles provides large interfaces with surrounding medium, which is an important criterion for biocatalysis.37,57 Obtaining highly stable, active and reusable biocatalyst are the major requirements for the industrial use of the enzyme.58,59 Various immobilization methods (EDC/NHS chemistry, site specific, adsorption, encapsulation, etc.) have been used to immobilize different enzyme on PNIPAm based smart polymers. 59–64

5 1.2 Goals

1.2 Goals

Owing to the beneficial characteristics of smart microgel, like colloidal dimensions, high water holding capacity, tunable properties and high interface with surrounding medium the objective of this thesis is to explore thermo- responsive microgels platform to study biochemical reactions as represented in Figure 1. 3.

(a)$ (b)$ CH3COOH#+# PEP#+#ADP$ CoA#+ATP# NADH$

Acetyl)CoA) Pyruvate)kinase. synthetase)(Acs)) Lactate)dehydrogenase)) (Pk.Ldh))

NAD+$ CH3CO*SCoA$ ATP$ (Acetyl#CoA)#

Core)#Shell#cationic#PNIPAm#Microgel#

Figure 1.3: Sketch representing core shell microgel-enzyme(s) bio-conjugate for (a) synthesis of acetyl CoA using S acetyl CoA synthetase (Acs); and (b) Sequential reaction of pyruvate kinase (Pk) and lactate dehydrogenase (Ldh).

The main aims of the work are 1) Design and synthesis of thermo-responsive microgels for biocatalyst immobilization. 2) Immobilization of acetyl CoA synthetase on different microgels and their application in synthesis of the acetyl CoA. 3) Immobilization of multi (dual)-enzymes to study sequential reactions for application in cofactor dependent process.

6 1. Introduction

1.3 Outline

The work is summarized in the thesis into five main chapters, which are outlined as follows; Chapter 1: The Introduction consists of the motivation, and goal of the work. Chapter 2: The Fundamentals and theory describes the theoretical background and experimental methods Chapter 3: The Result and discussion part consists of 4 main sections; Section 3.1: This section consists of synthesis of the thermo-responsive microgels using different approaches. The poly-(N- isopropylacrylamide-aminoethylmethacrylate) (PNIPAm-AEMA) microgels was synthesized using two-step free radical polymerization and poly (N-isopropylacrylamide-ethyleneimine) PNIPAm-PEI microgels was synthesized via one pot grafting-from co-polymerization of PEI. Section 3.2: The section describes the study of covalent immobilization of Acs on cationic PNIPAm-AEMA microgels. The enzyme was covalently immobilized on microgels via 1-ethyl-3-(3- NN- dimethylaminopropyl) carbodiimide (EDC) chemistry at standardized condition. The reaction and stability parameters were evaluated comparing with the soluble enzyme. Section 3.3: Adsorption based immobilization of Acs on PNIPAm-PEI microgels was studied. After thorough investigation of microgel-enzyme bioconjugate, the bioconjugate was immobilized on already prepared microgel support on polyethylene track etched membrane. The prepared membrane was used in a dead end filtration device to monitor the bioconversion efficiency and operational stability of the bioconjugate. Section3.4: Sequential reaction of Pk-Ldh enzyme system on PNIPAm-PEI microgel is described with respect to the single and dual enzyme(s) microgel bioconjugates. The enzymes were covalently immobilized

7 1.3 Outline

via glutaraldehyde coupling and different approaches were used to prepare dual enzyme bioconjugates. The optimized conjugates were checked for their kinetic, reaction and stability parameters. After thorough characterization of the bioconjugates, the dual enzymes systems were probed for their role in cofactor dependent processes. Chapter 4:The main output of the work is concluded in the Summary. Chapter 5: The Future outlook is presented.

8 2. Fundamentals and Methods

2 Fundamentals and Methods

2.1 Smart polymer hydrogel

2.1.1 Introduction

Hydrogels are gels composed of cross-linked polymeric networks, which swell in presence of water.65–67 They can absorb and retain water up to 99% (w/w) of their dry weights without dissolution.67,68 They are characteristically prepared from hydrophilic organic monomer/polymer that is cross-linked into a 3D network by either covalent or non-covalent interactions.65,66

Bio/Nano reactor

Valve, Tissue Gate engineering

Smart polymer hydrogel

Bio/ Drug chemical delivery separation

Sensor and actuator

Figure 2.1.1: Schematic representation showing different applications of the smart polymer hydrogel.66,69

This cross-linking provides the dimensional stability, where as high content of solvent gives rise to the fluid-like transport properties.66,70 Hydrogels composed of smart polymers undergo a volume-phase transitions upon exposure to the

9 2.1 Smart polymer hydrogel respective stimulus due to molecular interactions with surrounding medium resulting in changes in the network such as swelling, collapse or solution-to-gel transitions.67 In the era of nanotechnology, working in smaller dimensions imparts size dependent properties to the gel particles. The term ‘microgel’ was given by Baker in 1949 to describe cross-linked poly-butadiene latex particles.51 The word ‘micro’ describes the size whereas gel refers to the ability of the particles to swell in the solvents. In the lack of universal definition of these particles the microgel can be described as colloidal dispersion of gel particles, with size range of 10 – 1000 nm.51,71 Gels falling in the lower size range are also termed as nanogels.37 Colloidal gel particles comparing to bulk gel show faster response to changes in environment.72 These properties are due to fact that time constant of the swelling and de-swelling process is directly proportional to square of the geometric dimension of gel particles.37,73,74 The properties like large surface area, low viscosity and mechanical flexibility of microgels, which are comparable to human tissue, make this stimuli-responsive microgel an ideal candidate for various applications as depicted in figure 2.1.1. 57,69,75,76

2.1.2 Types of smart polymers

The “smart or intelligent” behavior of the hydrogels come from the type of the polymers or monomers used in the synthesis of these gels.66 Smart polymers are classified based on the type of stimulus they respond to like temperature, pH, light, ionic strength, biomolecules, etc. These properties are imparted to the polymers due to specific chemical groups/molecules/biomolecules present or attached on the polymer.77 In the present work, the PNIPAm-based microgels were studied consisting of the shell prepared out of cationic co- monomer/polymer. These polymers basically fall into categories of either temperature or pH sensitive polymers and therefore these two groups of polymers are further discussed in details.

10 2. Fundamentals and Methods

Poly(N-alkyl(alkyl)acrylamide) Poly(N, N’-dialkylaminoethyl(alkyl)acrylate)

PNIPAM: R=R1=H; R2= -CH(CH3)2 PDMAEMA: R=R1=R2= CH3 PDMA: R=H; R1=R2=CH3 PDEAEMA: R= CH3, R1=R2= -CH2CH3 PtBuMA: R=R1=H; R2= -C(CH3)3

Poly(ethylene oxide) Poly(propylene oxide) Poly(N-vinylcaprolactame)

Poly(vinyl ether)

R= alkyl chain or alkyloxy chain

Figure 2.1.2: Figure showing chemical structures of the different thermo-responsive polymers.78

2.1.2.1 Thermo-sensitive polymers Thermo-responsive polymers are the most widely studied smart polymers; few of the representative polymers structures are depicted in figure 2.1.2. 71,78For a given polymer, the cloud point is marked by the temperature at, which the polymer changes its conformation. This transition temperature can be either a lower critical solution temperature (LCST) or an upper critical solution temperature (UCST) as shown in figure 2.1.3.78,79 The LCST is the temperature above which a polymer phase separates from solvent while the reverse solubility transition is called UCST, i.e. the polymer is insoluble at low temperatures and completely miscible upon heating.78,80

11 2.1 Smart polymer hydrogel

a.# homogeneous)mixture) b.# two)phase)region)

UCST) LCST) temperature) temperature)

two)phase)region) homogeneous)mixture)

0) composi6on) 1) 0) composi6on) 1)

Figure 2.1.3: Phase diagrams of the thermo-responsive polymer with the characteristic (a) UCST and (b) LCST where the transition temperature is indicated by red arrow.79,81

Hydrogel prepared from thermo-responsive polymers undergo change in volume to the thermal response near the cloud point of the polymer. This is marked by swollen and collapsed structure, and is know as volume phase transition temperature (VPTT) (figure 2.1.4).66,76 Generally the hydrophilic-hydrophobic balance of polymer structure is responsible for the characteristic structural change, therefore by varying this balance the transition temperature can be finely manipulated.82,83

T>VPTT

Collapsed Swollen Hydrophobic interaction Hydrophilic interaction

Figure 2.1.4: Schematic representation of volume phase transition temperature (VPTT exhibited by PNIPAM microgels due to hydrophobic-hydrophilic balance at different temperature conditions.37,84

12 2. Fundamentals and Methods

2.1.2.1.1 PNIPAm The vast studies on the Poly (N-isopropylacrylamide) (PNIPAM) have made it a synonym for thermo-responsive polymers. It possess LCST at around 32 oC, which is close to physiological temperature and this value can be easily manipulated near to the body temperature by incorporating co-monomers or hydrophilic groups.85 Though this property makes PNIPAM a prominent candidate for biomedical applications, the inherent limitation with the polymer like non-degradability, phase transition hysteresis, and a significant end group influence on thermal behavior divert great deal of research in developing PNIPAm based materials for desired application.86 Based on the hydrophobic- hydrophilic groups in the NIPAm, the structure can be viewed in two distinct parts, which has prominent effect on the polymer transition temperature. The hydrophilic part consists of amide group, in aqueous system this group are bonded to water molecule via hydrogen bonding.87 On the other hand the hydrophobic part includes isopropyl group and is surrounded by structured water. This effect of structured water around the nonpolar moiety is known as hydrophobic effect.88,89 When temperature is raised above the LCST, water turns to be a bad solvent for the polymer dissolution, because at this condition polymer-polymer interactions are stronger than the polymer-solvent interactions and consequently the polymer phase is withdrawn from the aqueous phase.90,91 The dominance of hydrophobic bonds and breaking of hydrogen bonds with the water molecules, entropically favors release of the water molecule ultimately causing coil to globule structural transition.83,91 This transition can be probed using UV-VIS spectroscopy (transmittance), differential scanning calorimetry, dynamic light scattering, viscometry, small-angle neutron scattering (SANS) and fluorescence spectroscopy technique.91–94

2.1.2.1.2 Theory for degree of microgel swelling The swelling behavior of polymer is determined by de-swelling ratio (α) (equation 2.1.1) that is the ratio of volumes of microgels in collapsed state and swollen state.79 For spherical microgels radius can be used for determining the de-swelling ratio.

13 2.1 Smart polymer hydrogel

V R3 α = collapsed = collapsed 3 (2.1.1) Vswollen Rswollen

Theoretical explanation for the swelling behavior can be described by the

95 classical Flory-Rehner theory. The free energy of the charged gel Fgel is collective contribution of three components96

Fgel = Fmix + Felasticity + Fion (2.1.2)

where, Fmix is the free energy of mixing of the gel and the surrounding solvent,

Felasticity is the free energy of polymer network elasticity and Fion is the free energy of charged gels.79 The change in volume causes a change in free energy, which can be described by the internal osmotic pressure of a gel particle. 96

$ ∂F ' Πgel = −& ) = Πmix + Πelasticity + Πion (2.1.3) % ∂V (T

For neutral gels, the osmotic pressure is given by two factors ‘elastic’ pressure

(∏elasticity) of the cross linked polymer chains counteracting the osmotic swelling; and osmotic pressure ‘mixing’ (∏mix) of the network chains and solvent molecules, which is the polymer solvent interaction. The ∏mix contribution can be explained by Flory Huggins theory or by scaling concept as follows,

N AκBT 2 Πmix = − φ + ln(1− φ) + χφ (2.1.4) ϑ ( )

where, NA is Avogadro’s constant, κB is the Boltzmann constant, T is the absolute temperature, ϑ the molar volume of the solvent, φ is the volume fraction of polymer, χ is the Flory- Huggins interaction parameter and is dependent on polymer concentration.

14 2. Fundamentals and Methods

Further, using the theory of rubber elasticity, the osmotic pressure associated with elastic deformation of polymer network ∏elasticity can be identified with the shear modulus G, which scales with the volume ϕ1/3. The shear modulus is direct consequence of crosslinking.

1/3 Πelasticity = −ARTνφ = −G (2.1.5)

Where, R is gas constant, ν is the concentration of elastic chains and A is pre- factor depends on the functionality of junction. In the polyelectrolyte gels there is the electrostatic potential set by the network charge due to inhomogeneous distribution of mobile charges (ions) in and outside the (fixed) gel charge and is termed as Donnan potential ∆φ. The osmotic pressure for polyelectrolyte gel is given by

N gel sol Πion = RT ∑(cj − cj ) (2.1.6) j=1

Where, N is the number of mobile ions in the system while c gel and c sol represent the concentrations of the ions in the gel and the equilibrium solution,

96 respectively. In equilibrium condition net osmotic pressure of microgel (∏gel) is zero. The sign on osmotic pressure in non-equilibrium state indicates swelling (positive) or collapse (negative) of the microgels. All the three component of osmotic pressure for polyelectrolyte microgels collectively contributes to the size of microgels at given condition. 97

2.1.2.2 pH responsive polymers The polymers whose physical properties (solubility, volume, configuration and conformation) are sensitive to the external pH are termed as pH responsive polymers.98 These polymers contain functional groups that ionise by accepting or releasing protons with the change in pH figure 2.1.5.99,100

15 2.1 Smart polymer hydrogel

Acidic%Solu*on% Basic%Solu*on% (a)$

+% 2% 2% H +% OH 2% OH H+% H 2% 2% H+% H+% 2% +% 2% +% H 2% 2% H OH2% 2% OH2% Collapsed% Swollen% Anionic%polyelectrolyte%microgel%

(b)$ +% H +% 2% +% +% H OH2% OH +% 2% OH2% +% +% OH 2% OH2% H+% +% H+% OH Swollen% Collapsed% Ca*onic%polyelectrolyte%microgel%

Figure 2.1.5: pH responsive swelling and de-swelling of (a) anionic and (b) cationic microgels.100

The pH responsive polymers are characterized into anionic and cationic polyelectrolytes depending on the respective pendant group present in the polymer structure, which are generally weak acids or bases. The acidic pendant group includes carboxylic, phosphoric and sulfonic acids while basic groups comprise of amines.99,101 The pH-sensitive polymers synthesized with either acidic or basic components demonstrate reversible swelling-deswelling properties depending on the pH of the environment. Microgels prepared from these polymers reversibly swell and collapse depending on the pH of the medium. Cationic polyelectrolytes, such as poly (aminoethyl methacrylate) (pAEMA), poly(ethyleneimine) (PEI), chitosan have higher solubility at a lower pH due to their ionization. On the other hand, anionic polyelectrolytes like poly (acrylic acid) (PAA) are more soluble in water at a high pH.99,100 The main driving force leading to swelling of gels is the electrostatic repulsion between the charges present on the polymer chain.101 Therefore the extent of swelling is influenced by those conditions that alter the electrostatic repulsion such as pH. Among the different applications of the pH responsive microgel glucose sensing

16 2. Fundamentals and Methods and drug delivery are two most widely explored and interesting applications.102,103

2.1.2.2.1 PEI polymer Poly (ethyleneimine) (PEI) is a polymer of ethylamine and is highly soluble in the water.104 PEI is available in the form of linear and branched structures; linear polymer is composed only of secondary amino groups.105 Whereas, branched PEI (figure 2.1.6) contains primary, secondary, and tertiary amino groups in 1:2:1 proportion and the branching point exist at tertiary amines.104,106

Figure 2.1.6: The chemical structure of the branched poly (ethyleneimine) (PEI).

Branched PEI is synthesized from aziridine by cationic polymerization. All initial polymer species are formed through proton transfer from monomer.107 This cationic polyelectrolyte is very promising material for designing artificial enzyme and as a non-viral vector for gene delivery.106 The cationic property of PEI is dependent on the degree of protonation; nearly 80 % nitrogen atoms are available for protonation, which increases to 90 % in presence of poly-anion. This affinity of PEI towards poly-anion is useful in flocculation of anionic colloidal particles from the solution.107 The PEI has also been established as a good stabilizing agent for biomolecules like enzymes and therefore has been utilized to coat immobilizing supports.108,109

2.1.3 Core shell microgel and synthesis

Recently, there has been a great deal of interest in design of core-shell (CS) particles that are able to respond to the changes in surrounding condition.53,78,110 Hughes and Brown were the first to investigate the physical properties of core

17 2.1 Smart polymer hydrogel shell particles in the year 1961.111

Core!shell!hydrogel!

! !!Core!material! ! ! ! Non!Hydrogel! Hydrogel! ! !

Polymer!par5cle! Inorganic!par5cles! e.g.!Polystyrene! e.g.!Gold,!Silica!

Figure 2.1.7: The classification of the core shell hydrogels depending on the material used for core preparation while the shell is of hydrogel like material.66,112

Core-shell particles are multilayer composite particles consisting of atleast two different components, one in principle forms the core and another forms the shell of the particles.112 The superior properties of individual components of core and shell are blended in single morphological unit thus transmitting new or improved functionality to the CS hydrogel.66,112 For example a microgel with hydrophobic core covered with hydrophilic shell has good colloidal stability and functionality due to hydrophilic shell while the hydrophobic core provides mechanical properties (hard/soft) to the particle.113 There are various categories to classify the core shell particles, depending on the type of material (inorganic/organic)114 used for core and shell preparation, state (hydrogel/non hydrogels)66,112 and size (nano/micro)112, etc. The work on core-shell hydrogels reported till date can be generally classified based on the core material and is described in figure 2.1.766. Based on the material used for core preparation the CS microgels can be divided into two main classes first where core is prepared from non-hydrogel material and the shell is made from hydrogel while the second category includes both the core and shell made of the hydrogel-like material.66,112

18 2. Fundamentals and Methods

2.1.3.1 Synthesis of core shell particles The choices of material for core shell microgels are mainly dependent on the end application. The parameters like functional group, size, shape, cross-linking density has impact on the composite properties of CS microgels, and need to be controlled during the synthesis of the particles.49,55 There are various approaches to obtain core shell particles, which are summarized in figure 2.1.8.115 Two-step seed based radical polymerization method (I) is most widely used to generate core shell microgel.66,116 The conventional radical polymerization (suspension, emulsion, dispersion, and precipitation) methods can be differentiated based on the initial state of polymerization mixture, overall kinetics, molecular structure and size.117,118

Radical'ini5ator' Monomer'2' (I)' Monomer'1' Emulsion/dispersion/'precipita5on'

Reac5ve'surfactant' (II)' Monomer'' Emulsion/dispersion/'precipita5on'

Monomer'1'' (III)' Monomer'2'' Heterogeneous'coagula5on'

Core'crosslinking' Micelle' (IV)' forma5on'

Amphiphilic'block' Shell'crosslinking' copolymer'' Self'assembly'

(V)' Gra@ing'to/'gra@ing'from' ATRP/'RAFT'

Figure 2.1.8: Scheme representing the different approaches to synthesize core shell particles.113

The fundamental difference between these heterogeneous polymerization systems is enlisted in Table 2.1.1. The monomer is referred to as monomer or 19 2.1 Smart polymer hydrogel dispersed phase and liquid phase consisting of the monomer phase is described as polymerization or continuous or outer phase.

Table 2.1.1: The characteristics difference between various types of free radical polymerization.115

Precipitation Dispersion Emulsion Suspension polymerization polymerization polymerization polymerization

Reaction Water Organic Water/oil Water/oil medium Solvent

Particle size 0.1-10 0.5-10 0.05-0.3 >10 (μM)

Loci of Continuous phase Polymer and Monomer Monomer polymerization continuous swollen latex droplet on phase polymer

Initiator Water soluble Water soluble Water soluble Oil soluble

Monomer Water soluble Water soluble Water insoluble Water insoluble

Stabilizing - Oligomers and Surfactant Non micelle agent polymers Micelle forming forming

Kinetics Rate of Similar to Rate of Rate of polymerization precipitation polymerization polymerization proportional to and emulsion proportional to increase with polymer surfactant and decrease in concentration initiator degree of concentration polymerization

Core shell particle synthesized via heterogeneous polymerization is similar to seed polymerization except the shell synthesis must be strictly carried under following condition; core particles must not swell in monomer, the shell polymer must form on the core surface and both core and shell must not diffuse into each other.119 Radical polymerization can also yield core shell particles in one step approach by introducing reactive surfactant (II), which can copolymerize with monomers resulting thin-shelled latex particles.112,115 In another approach Heterogeneous coagulation of oppositely charged polymer particles followed by heat treatment above Tg of shell particles (III) produces core shell particles, similarly layer by layer deposition of polyelectrolyte on charged core surface

20 2. Fundamentals and Methods generate C-S particles with multilayered shell.113,120 Furthermore block copolymers (IV) with units having different miscibility in solvent are allowed to form micelles by self-assembly and further cross-linked to obtain the core shell morphology.112,115 Well defined structures can be obtained by attaching preformed polymers onto the core surface or first activating the core surface and subsequently employing controlled polymerization to obtain shell could achieve this achieved by graft polymerization (V). Free-living polymerization (atom- transfer radical polymerization (ATRP), and reversible addition-fragmentation chain-transfer (RAFT) methods are commonly used to obtain brush like composite particles. In this work two different core-shell microgels are reported, where, AEMA-PNIPAm is synthesized by two-stage precipitation polymerization whereas PEI-PNIPAm is obtained by graft copolymerization of PEI and PNIPAm.

The PNIPAm-AEMA microgels are synthesized by typical precipitation polymerization in two steps. Initially core particles were synthesized using monomer, cross-linker, surfactant and initiator (Figure 2.1.9, step 1).54

I II III IV

Step1:'Core'synthesis' Step2:'Shell'synthesis'

Figure 2.1.9: Two-step reaction of PNIPAm-AEMA core shell microgel synthesis.

Next the core solution was used as seed for shell synthesis (Figure 2.1.9, step 2). The high temperature for polymerization, which is above LCST of PNIPAm is essential to obtain collapsed precursor particles and also for activation of initiator; where sulfate radicals formation takes place. Particles are formed by homogenous nucleation, i.e. the entire polymerization ingredients are in same (continuous) phase. The general mechanism behind the microgel synthesis includes first the NIPAM monomer is initiated to polymerize (I), the growing polymer collapse due to temperature effect (II) and further continues to grow till

21 2.1 Smart polymer hydrogel stable colloidal particles are formed in the solution (III). The mechanism for formation of core shell is similar to core synthesis (IV). AT temperature above VPTT, the core is collapsed and due to increased hydrophobic effect, it captures growing polymers ending up into shell structure (V). The charge, size and stability of microgel are very much dependent on the selection and use of co- monomer, initiator and surfactant. 91,116

PNIPAm-PEI core shell microgel are synthesized by one-step graft co- polymerization of PNIPAm from PEI at temperature (70 oC) above the LCST of PNIPAm (figure 2.1.10). Tertiary butyl hydro peroxide (tBuOOH) initiates the reaction by forming tBuOO- and amino radicals (I). The grafting from polymerization of NIPAm is initiated from the PEI surface with simultaneous cross-linking with N,N bis methyl acrylamide (MBA) (II).121

NH2&

NH2& I III NH2& II tBuOOH& NH2& NH2&

Polyethyleneimine+ Core+shell+microgel+ (PEI)+

Figure 2.1.10: Schematic for PNIPAm-PEI microgel synthesis in one step graft co polymerization method.

The amphiphilic PEI-g-PNIPAM polymer generated act as polymeric surfactants by forming micelle-like micro domains (III). These micro-domains further promote the emulsion polymerization of the NIPAM leading to core shell microgel.121,122

2.1.3.2 Core shell microgel in biocatalysis Ever since the Hoffmann’s group in 90’s used the PNIPAm polymer to immobilize different enzymes, this polymer has been explored in different forms (polymer chain, cross-linked microgels or coating as brush structure) for biocatalyst immobilization.123,124 Specifically for microgels that are assumed as the close resemblance to the cellular environment, a number of enzymes have been 22 2. Fundamentals and Methods attached to study the efficiency of microgels as the carrier system as enlisted in Table 2.1.2. The initial reports on the enzyme immobilization on the microgels appear in 1990’s, the PNIPAm hydrogel beads immobilized with β-D-glucosidase were used to study the thermal cycling effects on the enzymatic conversions. Yasui M. et al. developed microsphere with hairy structures using carboxylated PNIPAm, and demonstrated temperature dependent activity of trypsin covalently immobilized on these microspheres.125–127 Nearly in every reports on microgel- enzyme bioconjugates temperature dependent activity control and enhanced catalytic properties with respect to the free enzyme were reported.125Magnetic nanoparticles have also been introduced into the microgel for their easy recovery from the solution.64 Shamim et al. had studied the adsorption/desorption of lysozyme on PNIPAm coated nano-magnetic particles.128 Interestingly in one of the approaches enzyme was employed to regulate the microgels collapse to improve the membrane permeability.129 Recently solvent exchange method was used to immobilize enzymes (HRP and lipase) into the PNIPAm microgels and improved activity were observed in non- aqueous solvents.130,131 Though core- shell microgels are not very new concept but the amount of the literatures available on the enzyme immobilization are limited. The detailed work on adsorption of the proteins and enzymes (lysozyme, β-D-glucosidase, RNase) on spherical polymer brush particles (core shell microgels) from the Ballauff group have established microgels as the ‘nanoreactor’ for bioconversion reaction.61,132,133

23 2.1 Smart polymer hydrogel

Table 2.1.2: The use of different PNIPAm based microgels for enzyme immobilization.

Microgel Polymerization Enzyme Immobilization Reference method PNIPAm/PEI Graft co- Horseradish Adsorption on Jing Xu et al. polymerization, peroxidase, gold nanoparticles 2007134 tBuOOH,* MBA** Urease PS-PNIPAm Photo-emulsion β-D-glucosidase Adsorption Welsch et al. polymerization, 200961 MBA** PS-P (NIPAm- Emulsion and seed Lysozyme Adsorption Welsch et al. AAc) polymerization, 2012132

P(S-NIPAm- Surfactant-free α-chymotrypsin Covalent bonding Chen et al. NAS) emulsion through ester 135 polymerization groups PNIPAm / PEG- Self-assembly and Trypsin Inside the core by Harada et al. PAA redox self assembly 2007136 polymerization.

TEMED* and MBA.** Caboxylated Soap free emulsion Trypsin Covalent binding Yasui et al. PNIPAm-latex polymerization EDC-NHS 1997125 particles GMA- St-DVB PNIPAM- Precipitation Alcohol Microgel- Wise et al. PNIPMAM polymerization dehydrogenase Stabilized Smart 2013137 Emulsions

PNIPAm Inverse emulsion Hemoglobin, Suction Wu et al. polymerization Horseradish 2013138 peroxidase

PNIPAm-PVIm water/oil inverse Laccase, Encapsulation Schachschal emulsion, APS and peroxidase et al. 201163 TEMED,* MBA, ** PNIPAm Surfactant free Lipase B Solvent exchange Gawlitza et emulsion al. 2012130 polymerization

Note: * initiator, **cross linker, p N,Isopropyl acrylamide (PNIPAm), polyethyleneimine (PEI), poly vinyl imidazole (pPVIm), N-acryloxysuccinimide (NAS) ammonium peroxodisulfate (APS), N′,N′,N′,N′-tetramethylethylenediamine (TEMED), poly(ethylene glycol)-block-poly(a,b- aspartic acid) (PEG-PAA), poly styrene (PS), Glycidyl methacrylate (GMA), styrene (St), Divinylbenzene (DVB)

24 2. Fundamentals and Methods

2.2 Biochemical reaction

Bio-chemical reactions are reactions carried out in biological system in presence of biocatalyst/enzyme.1,16 These reactions play vital role for the organism from transferring genetic information, to energy production and also providing immunity.1,2,139 The enzymes also act similar to the chemical catalyst by reducing the activation energy of reaction and thus making the complex reactions in the cell possible at mild conditions of temperature, pH and atmospheric pressure.1,140 Many of these reactions have been capitalized for the applications in food, processing, pharmacy, biofuel production etc.141,142 In the present work two-biochemical reactions were studied, one work was focused on the biosynthesis of precursor acetyl CoA using acetyl CoA synthetase enzyme; and another part was focused on bi-enzymatic reactions carried by pyruvate kinase and lactate dehydrogenase.

2.2.1 Acetyl CoA synthetase

2.2.1.1 Introduction Acetyl Coenzyme A synthetase (Acs) also known as acetate: CoA ligase (AMP); belongs to the ATP- dependent AMP binding enzyme family with denoted EC 6.2.1.1.143 The enzyme has wide occurrence from bacteria to humans.144 It catalyses the two-step acetyl CoA synthetic reaction as depicted in figure 2.2.1.

Figure 2.2.1: Two step reaction for acetyl CoA synthesis catalyzed by acetyl CoA synthetase (Acs); Step I: Acetate is activated to Acetyl-AMP by ATP hydrolysis at lysine residue of enzyme active site with release of pyrophosphate (PPi). Step II: Marks the synthesis of Acetyl CoA from CoA and Acetyl AMP.

25 2.2 Biochemical reaction

The reaction proceeds through formation of an enzyme bound acetyl adenylate intermediate (step1) which subsequently reacts with CoA to produce AMP and acetyl CoA (step2).143,145 Acs possesses high level of substrate specificity and is an important supplier of acetyl CoA for various biochemical reactions in the cell. 146 Commercially available acetyl CoA synthetase is obtained from the yeast (Saccharomyces cerevisiae). Two isoforms of Acs exist in eukaryotes, one in the cytosol and other one in the mitochondria. In yeast, these isoform are expressed on their (aerobic/ anaerobic) growth conditions.147 Acs1 is aerobic isoform present in mitochondria, which supports growth on non-fermentable carbon sources like glycerol, etc., while Acs 2 is an anaerobic isozyme required for growth on fermentable sugars such as glucose.148 The Acs is a trimer, with each monomer unit of 78,000 molecular weight.149 The crystal structure of Acs in a binary complex with AMP revealed the enzyme structure during first half of the reaction. The study established that the conserved Lys 675 residue in the small domain plays important role in orientation of substrates and is in close proximity to AMP. 145 The enzyme structure during the second step of reaction is not clearly understood, but the crystal structure of enzyme in presence of CoA and AMP inhibitor shows that the lysine residue is not a part of the active site.143 The small domain undergoes a large conformational reorganization, which leads to the formation of the CoA binding pocket to further facilitate enzyme conformation for the second step of the reaction. 144,145

Figure 2.2.2: The structure of yeast ACS (ribbon diagram), the trimer consists of large domains (yellow, cyan, and green,) and small domains (grey) respectively.145

26 2. Fundamentals and Methods

2.2.1.2 Biochemistry of acetyl CoA The Acetyl CoA plays an essential role in the central metabolic reaction as depicted in figure 2.2.3. The molecule is centre to metabolism, as it is metabolized through citric acid or Kerb cycle for energy production.16 It serves as initial units for fatty acid, lipid, and cholesterol synthesis.24,150,151 Acetylation/deactylation process is one of the important mechanisms to regulate various biochemical reactions, for such reactions acetyl group is provided by the cofactor, e.g. DNA packaging on Histone proteins.16 It also takes part in synthesis of numerous secondary metabolite like terpenes, biofuel and polyketides.25,152 The diverse class of polyketides are obtained by incorporation of different precursor and monomer units generated from acetyl CoA in various secondary metabolic pathways. 25

Protein( Glucose( Fats( Cholesterol(

Glycolysis(

Amino(acid( Pyruvate(( Fatty(acid(

Oxida-on(

ACETYL'COA''

Biofuel( ((1BHexanol,(( 1B(Butanol)(

Kreb(cycle( Precursors:(Malonyl( Mavalonate(pathway( CoA,((Acetoacetyl(CoA(( intermediate(

Polyketide( CO2(+(H2O(+(energy((ATP)( terpenes(

Figure 2.2.3: The flowchart shows anabolic and catabolic pathways of acetyl CoA metabolism.152,153

27 2.2 Biochemical reaction

2.2.2 Pyruvate kinase and lactate dehydrogenase

2.2.2.1 Introduction Pyruvate kinase (Pk)(EC 2.7.1.40) and Lactate dehydrogenase (Ldh) (EC 1.1.1.27, L-lactate; NAD+ oxido-reductase) are two coupled enzymes, used for ATPase assay and regeneration of different cofactors (ATP, NAD+).32,154 These enzymes have been utilized in fabrication of various biosensors for detection of lactate, pyruvate etc. that find important applications in the fields of medicine, critical care, food science, and biotechnology.155,156 Pk catalyzes the last reaction in glycolysis, in which phosphoenol-pyruvate is converted to pyruvate and ATP, in presence of ADP and proton.157 In anaerobic respiration, the Ldh converts the final product of glycolysis (pyruvate) to lactic acid.16 Figure 2.2.4 shows the sequential reactions catalyzed by Pk and Ldh where in two cofactors (ATP and NAD+) are regenerated after completion of the reactions.

Figure 2.2.4: Represent sequential reaction catalyzed by pyruvate kinase and lactate dehydrogenase.158

2.2.2.1.1 Lactate dehydrogenase Ldh is ubiquitous enzyme found in animals, plants and prokaryotes. Due to its abundant nature, it is medically important indicator for cell/tissue damage, tumor and many other diseases.159 The enzyme activity in forward or reverse order can be measured simply by spectrophotometer at 340 nm wavelength.160 Ldh is well-known multimeric enzymes, and is a tetramer i.e. it consist of four subunits to form a functional enzyme. The Ldh exist in basically two forms; H- type and M-type depending on the source of extraction i.e. heart and muscle

28 2. Fundamentals and Methods respectively.161 The subunits in the tetramer in all vertebrates are found to be similar in molecular weight (35,000) but differ in amino acid composition, structure and kinetic properties.160 Extensive homology between the subunit is apparent from in vitro ready hybridization of subunits from different species to form functional tetramer. The two types of polypeptide readily associate to form five different isozymes of enzyme; two homomer H4, and M4 and hybrids are

162 referred to as H3M, H2M2 and HM3. The co-enzyme (NADH) and substrate (pyruvate) form adduct, which at higher concentration of pyruvate leads to substrate inhibition.163 The ternary structure of enzyme (Ldh)-pyruvate-NADH is shown in figure.

Figure 2.2.5: The structure of Ldh (a) tetrameric structure, and (b) binding of the substrates pyruvate and NADH in the active site.164,165

2.2.2.1.2 Pyruvate kinase Pk is multimeric enzyme (figure 2.2.6) constituted of identical subunits and has molecular weight of 2,37,000.166 Each subunit is formed of four domains namely; N-terminal, A, B and C domain. Further in between the subunits two distinct interface is formed, which are Y and Z respectively.167 PK employ inter-domain movement for catalyzing the reaction, domain A and B forms the cleft. The mobility of domain B decides the open and closed conformation or say the

29 2.2 Biochemical reaction inactive and active state of the enzyme.157,167The functions of C and N terminal domains are not well understood.167 For catalysis Pk requires monovalent cation (K+, NH4+) and divalent metal ion (Mg+2) for catalysis which balances the high density of negative charge by the anionic substrates.168 Pk reaction proceeds in two chemical steps. The first step commences with formation of ATP and enolate form of pyruvate from phosphoenol-pyruvate (PEP). In the second step the enolate form accepts proton to produce the keto-form of the pyruvate.157

Figure 2.2.6: The crystal structure of Pk tetramer containing domains and interfaces.167

30 2. Fundamentals and Methods

2.3 Enzyme immobilization

2.3.1 Introduction

The high rate of enzymatic reaction, enantio-selectivity and specificity makes them important candidate for green and sustainable technology.34,169 Enzyme immobilization is important approach to obtain stable and reusable biocatalyst.170,171 The general immobilization approaches with and without carrier system are depicted in figure 2.3.1.10

E E" E" E E E E E" E E E" E" E E E" E E E E" E E"

(a)$Encapsulation/$ (b)$Bound$to$carrier$ (c)$Carrier7free:$crosslinked$ entrapment$

Figure 2.3.1: Scheme representing different types of immobilization techniques

The carrier- dependent approaches include encapsulation/ entrapment or attachment of enzyme in/on the surface of the support. The first method is enormously utilized and immobilization of enzyme is achieved by preloading of enzyme during the formation of the support; as in case of formation of sol-gel,172 or using amphiphilic block copolymers,173 or liposomes.174 However this technique also experiences inherent limitations of enzyme leakage and higher diffusion barrier for substrate and product.37,59 The direct attachment of enzymes on preformed carrier is another possibility, which can be performed by physisorption (via hydrophobic or ionic interaction) or by covalent coupling. The adsorption of enzyme generally does not hamper the enzyme activity and is considered one of the simplest method.10,170 But desorption of enzyme is always a concern, which to some extent is possible to overcome by using dense charged network e.g. polymeric bed which allow strong interaction with enzymes and limits the enzyme loss.59,171 Covalent immobilization surpasses the enzyme leakage challenge. Upon covalent immobilization, though there is initial loss of 31 2.3 Enzyme immobilization the enzyme activity but the rigidification of enzyme structure provides high overall stability and reusability of enzymes.59,175 Carrier-less system is also developed where enzymes are cross-linked to each other using reagent like gluteraldehyde.10 In this work, the enzymes were immobilized on microgels via covalent binding or through adsorption.

2.3.1.1 Covalent immobilization Covalent immobilization is most widely used method to immobilize enzymes on support through different chemistry. The advantage of covalent immobilization is that the enzymes are bound firmly to the support and thus leakage and contamination of enzymes with the product in the reaction media is minimized.176 This method is generally adapted where multipoint attachment improves thermal stability and provides limited flexibility for accomplishment of the reaction. But the drawback of this method also includes the chemical modification of enzymes that causes loss in the activity.10,176 The primary amines, carboxylic, sulfhydryl groups etc. of enzymes are targeted to attach them to carrier via amide bond formation, amidation, Schiff’s base formation, and disulfide bond formation.177 Many cross-linkers are available that are used to link enzyme to the carrier such as glutaraldehyde, carbodiimides (EDC), maleimides.

2.3.1.1.1 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC)

Enzyme Microgel Enzyme-microgel conjugate

EDC$ amine-reactive O- acylisourea intermediate

Figure 2.3.2: Schematic representation of EDC coupling of enzyme and carrier (cationic microgel).178

32 2. Fundamentals and Methods

EDC is water-soluble, zero-length cross-linker. That is, it readily forms amide bond between carboxylic group (-COOH) and primary amines (-NH2) without itself being part of the final conjugates (figure 2.3.2). As proteins are abundant in possessing these groups, this cross-linker forms a well-known choice for protein immobilization. EDC crosslinking is most efficient in acidic conditions (pH 4.7-6) in buffer without the reactive groups. At higher pH slightly more amount of cross-linker is added to achieve the conjugation.178,179

2.3.1.1.2 Glutaraldehyde (GA) Glutaraldehyde was well used as the fixative in tissue sample preparation and also to obtain stable crystals for X-ray diffraction studies.180 The GA as protein cross-linker is widely used for designing biocatalyst by activation of carrier or in cross-linked enzyme aggregate (CLEA) preparation.181 GA is a homo bi- functional cross-linker that reacts with mainly primary amine but may react with other groups (thiols, phenols and imidazoles). The reaction between glutaraldehyde and protein occurs through different mechanism because of the various GA species existing in the solution. Under acidic and neutral conditions the GA exists in main five forms free GA (I), linear mono (II), dehydrates (III), a cyclic hemiacetal (IV), oligomer (V). The aldehyde groups from GA can react with proteins by formation of Schiff bases as described in figure 2.3.3.180,181

Figure 2.3.3: Schematic representation of glutaraldehyde coupling of enzyme and carrier (cationic microgel).

2.3.1.2 Adsorption This method is inexpensive way of enzyme immobilization. The enzyme leakage in aqueous solution is a concern, therefore this method is well suited for the bioconjugates in non-aqueous medium.182 Adsorption of protein on the support

33 2.3 Enzyme immobilization is contribution of different interactions such as van der Wall’s forces (hydrophobic), hydrogen bonding (hydrophilic) etc. The advantages of the method includes no pre treatment of the enzymes is required before immobilization and allows straightforward manipulation of the enzyme’s properties.176 Adsorption on ionic exchangers allows multi-point interaction and prevents sub-unit dissociation.183 In this respect particles possessing ionic polymer coating or shell of PEI can provide maximum interactive surface to fix the enzyme on the support. 184

2.3.1.2.1 Adsorption isotherm The non-competitive adsorption of protein is conveniently modeled using Langmuir isotherm. Irving Langmuir proposed the model for describing adsorption of gas molecules on surface that was assumed to be in equilibrium with the molecules in gas phase at a given pressure.185 This model has been previously applied to understand enzyme immobilization on different microgel supports. 60,61 According to the model the solid is considered to have limited adsorption capacity Γmax, and following assumption are taken into account (1) all sites are considered identical, (2) each site must contain only one molecule and (3) all sites are energetically and satirically independent of the adsorbed quantity.186 At the equilibrium binding the Langmuir equation is given by θ i.e. fraction of adsorption sites as following;187

K C θ = d 2.3.1 1+ KdC

Further θ is ratio of amount of adsorbent adsorbed to the maximum binding site available.

Γ θ = 2.3.2 Γmax

Therefore the Langmuir isotherm equation 2.3.1 becomes. 188

34 2. Fundamentals and Methods

Γ C Γ = max K C d + 2.3.3

To determine the maximum binding sites and dissociation constant this equation was linearized by multiplying both sides by (Kd+C) and further dividing by Γ, this results in equation (2.3.4) with which the experimental data were fit.

C ⎛ 1 ⎞⎛ K d ⎞ = ⎜ ⎟⎜C + ⎟ Γ ⎝ Γmax ⎠⎝ Γmax ⎠ (2.3.4)

where Γ is amount of Acs adsorbed on microgel (μg/mg), Γmax is the maximum binding capacity (μg/mg), c is the Acs concentration in solution (µg/mL), and Kd is the dissociation constant (mL/µg).

2.3.2 Factors affecting enzyme stability

The activity and stability of enzyme are key points for in-vitro use of the biocatalyst. Protein as a macromolecule, its role is essentially dependent on its structure. The catalytic properties of enzymes completely rely on their specific architecture adapted for the purpose. The polypeptide chains folds in sequential ordered structure via intra and inter molecular interactions (hydrogen bonds, electrostatic interactions, hydrophobic interactions) and bonds (disulfide bonds, and metal binding) forming a protein scaffold. The appropriate geometry of amino acid in the scaffold formation coincides with the stability and activity of the enzymes.189 Enzyme outside its natural environment experiences number of inactivation steps during isolation, purification, storage and application. The phenomenon leading to enzyme inactivation includes multimeric enzymes subunits dissociation, denaturation, aggregation, coagulation and chemical decomposition.190,191 The unfolding of enzyme structure to disordered polypeptide causing the loss of functional and structural native conformation is known as denaturation.191 The enzyme inactivation is given by following scheme 2.3.5, where N is native, U is unfolded and I is a inactivated enzyme. The N to U

35 2.3 Enzyme immobilization transition is reversible and corresponds to thermodynamic stability, this is followed by irreversible N to I transition which relates to kinetic stability.

NDUDI 2.3.5

The thermodynamic stability is the resistance of the folded protein conformation to denature and long-term or kinetic stability is the resistance to irreversible inactivation. 191,192 Immobilization is most widely used approaches to improve enzyme stabilization. The bound enzyme resembles the state of enzymatic protein within the cellular milieu and therefore could be established as a good model to mimic the in vivo condition.193 Nevertheless in vitro study, the enzyme in solution are considered as native enzyme and many characteristics of bound enzymes differs from native enzymes due to the after effect of immobilization. Irrespective of the immobilization method, several factors are responsible for the normal functioning of enzymes that can be generalized as accessibility of the substrate to the active site (enzyme orientation and diffusion limitation), conformational integrity of enzymes and carrier effect.189 Hence evaluation and comprehension of probable structural and functional alterations of enzymes in presence of carrier are important in choice and optimization of immobilization method. 189

2.3.3 Michaelis Menten kinetics

Leonor Michaelis and Maud L. Menten in 1913 provided the mathematical framework for enzyme kinetics based on the work of French chemist Victor Henri.194 The enzyme-catalyzed reaction is considered as a two steps reaction as shown in scheme 2.3.6 and in step 1, enzyme (E) and enzyme- substrate (ES) complex exist in pre-equilibrium state. 195

k E + S ES cat E + P K s 2.3.6

36 2. Fundamentals and Methods

S and P stand for substrate and product respectively. The rate constant Ks is the ratio of k-1 to k1, which are first order rate constant for the formation and dissociation of ES complex and kcat describes the latter part of the reaction. The steady state kinetics given by George Briggs and John Haldane, in 1925, the ES complex formation was assumed to reach a steady state with the product formation.196 From this assumption, the Michaelis-Menten equation is derived as equation 2.3.7.

υmax [S] υ = 2.3.7 Km + [S]

Where, υ initial rate of catalyzed reaction, υmax is the maximum initial rate at infinite substrate concentration and Km is Michaelis constant. The equation explains the dependency of initial rate of reaction on substrate concentration as shown in figure 2.3.4. The rate of reaction increases proportionally with the increase in substrate, ultimately reaching to maximum velocity where all the active sites in enzymes are saturated with substrate and further increase in [S] concentration does not affect the velocity and reaction follows zero order kinetics. 195

0.5# 100# (a)( (b)( vmax# 0.4# 80# ( 1 0.3# 60#

(mM/min)( 0.2# 40# Km#

ν v 1# max# 0.1# 1/n(((mM/min)- 20# vmax# Km# 0.0# 0# 0.0# 0.2# 0.4# 0.6# 0.8# 1.0# 0# 50# 100# 150# [S]((mM)( 1/[S]((mM)-1(

Figure 2.3.4: (a) Michaelis-Menten plot; (b) Lineweaver Burk plot

Generally the first hand data are subjected to non-linear regression to calculate kinetic parameters. Another way is to linearize the MM equation, Lineweaver Burk method is the most widely used one and is the reciprocal of MM equation

37 2.3 Enzyme immobilization given by equation 2.3.8. The double reciprocal plot is 1/ν vs 1/[S], and the slope of the line gives Km/υmax and intercept is 1/υmax.

1 K 1 1 = m . + υ υm [S] υmax 2.3.8

Km is referred to as Michaelis constant, and is defined as substrate concentration that provides a reaction velocity that is half of the maximal velocity obtained

195 under saturating substrate conditions. Km is equivalent to Ks at condition k2

38 2. Fundamentals and Methods

2.4 Characterization techniques

2.4.1 Dynamic light scattering

Dynamic Light Scattering (DLS) or Photon Correlation Spectroscopy (PCS) is most widely used technique for measuring the size of colloidal particles in a liquid. The process is quasi-elastic when photons are scattered by particles in motion therefore this technique was initially known as Quasi-Elastic Light Scattering (QELS).198 QELS measurements yield information on the dynamics of scatterer, which gave rise to the term dynamic light scattering (DLS).199

2.4.1.1 Theory of dynamic light scattering

Two theoretical framework Rayleigh scattering and Mie scattering are used to interpret light scattering phenomenon by small and large particles. According to Rayleigh theory (for small particles, less than one tenth of the wavelength of light) intensity of the scattered light for particles is uniform in all directions.

(a)$Rayleigh$Sca.ering$$ (b)$Mie$Sca.ering,$ (c)Mie$Sca.ering,$ small$par7cles$$ large$par7cles$

Figure 2.4.1: The Rayleigh and Mei theory of scattering by spherical particles.200

Whereas, the Mie theory provide exact description of how spherical particles of all size scatter light. With the particles size approaching near to wavelength of incident light, the isotropic scattering becomes forwardly distorted (Figure 2.4.1.) and scattering becomes complex function having maxima and minima with respect to the angle. 201

The particles in constant Brownian motion scatter light with fluctuations in the intensity with respect to the time. The analysis of the time dependent

39 2.4 Characterization techniques intensity fluctuation yields the diffusion coefficient of the particles. Further using the Stokes-Einstein equation (equation 2.4.1) the size of the particles with known viscosity can be determined,201,202

κT d(H) = 2.4.1 3πηD

Here, d(H) is hydrodynamic diameter of the particles, D is translational diffusion coefficient, κ is Boltzmann constant, T absolute temperature, η medium viscosity. The intensity fluctuation over time is co-related and compared by correlator of the DLS instruments. It determines the degree of similarity between the two signals by quantifying the degree of correlation (correlation function (G)) in the signal as an exponential function of the correlation time delay (τ);

G (τ) = A%$1+ Bexp(−2Γτ)'& 2.4.2

Where A is the baseline of the correlation function, B is intercept of the correlation function, and decay rate (Γ) is given by following equation;

Γ = Dq2 2.4.3

Where D = translational diffusion coefficient, and wave vector (q) is given as follows;

4πn θ q = sin 2.4.4 λ0 2

Where, n is the refractive index of dispersant, θ is the scattering angle, and λo is the wavelength of the laser. The equation for the poly-dispersed samples is the

202,203 sum of all the exponential decays g1(τ) as described by following equation;

G( ) A"1 Bg 2 $ 2.4.5 τ = # + 1 (τ) %

40 2. Fundamentals and Methods

2.4.1.2 Instrumentation:

A typical set up for dynamic light scattering system comprises of six main components shown in figure 2.4.2. The sample is illuminated using a laser (He- Ne red light, wavelength 633 nm), the attenuator regulates the amount of incident on the sample in the cell and also scattered light reacting the detector. The detector is positioned at particular angle (173o or 90o) to measure the scattered light. The signals are further processed in correlator that compares the scattering intensity with time and derives the rate of intensity fluctuations. Finally data is analysed by the computer to furnish size information of the particles. 202

Figure 2.4.2: Optical configurations of the PCS instrument for DLS measurements.

In this work dynamic light scattering (DLS) measurements were performed using Zetasizer Nano ZS, (He-Ne-laser 4 mW, 632,8 nm), collecting the scattering information at 173° (back scattering), NIBS®-Technology, Malvern Instruments. Before the size measurements, the samples were thermally equilibrated for 10 min, and data were acquired by averaging 30 measurements, with a 10 s integrating time for each measurement. The volume phase transition temperature was determined with respect to temperature (24−40 °C).

2.4.2 Zeta potential

In the DLS instrument setup, electrophoretic light scattering (ELS) technique

41 2.4 Characterization techniques is utilized to measure zeta potential of particles in solution (Malvern technical note).204 The zeta potential of the particles gives information about the colloidal stability of the particles in solution. In the year 1940, scientist B.V. Derjaguin, and L. Landau; and E. J. W. Verway and J. Overbeek developed independently the theory for the stability of colloidal system, which is famously known as DLVO theory.205 The theory suggest that the stability of particles is contribution of combined effect of van der Waals of attraction (VA) and electrical double layer repulsive forces (VR) existing between the particles as they approach each other. The repulsive forces (electrostatic repulsion) prevent particles from aggregation by maintaining the energy barrier between them, but when the particles colloid with one another with sufficient energy, attractive forces predominates and leads to aggregation.

Poten(al"(mV)" +" ψ "Surface"poten(al" +" +" !" !" +" ϕ" +" +" +" !" +"+" +"+" +" !" +" +" +" +" +" +" !" !" +" !" ψS""Stern"poten(al" +" +" !" +" !" +" +" +" +" +" +" +" +" !" !" +" +" "ζ!""–"zeta"poten(al"+" !" !" +" +" +" +" +" !" +" +" !" +"+"+" +" +" +" +" +" +" +" +" !" +" +" !" !" !" Distance"from" Stern"layer" Slipping"plane" par(cle"surface"

Figure 2.4.3: Structure of the electrochemical double layer on a negative particle surface.79

The charged particles dispersed in a solvent are solvated and electric double- layer (EDL) is formed on the particle surface. The EDL phenomenon is described by Stern model developed in 1924 by combining earlier models of Helmholtz and Gouy-Chapman. Figure 2.4.3, schematically shows the distribution of ions at the interface of a negatively charged particle surface. The EDL consists of two regions, the dormant layer of counter ions neutralizing the charge on the particles known as the Stern layer while the outer layer consist of the slipping plane or diffuse layer where ions diffuse under the influence of electric and thermal motion. The electric potential decreases linearly from surface potential 42 2. Fundamentals and Methods

(ψ0) to Stern potential (ψδ). Further it decays exponentially to zero in the diffuse layer. The EDL potential at this shear plane is defined as the zeta potential (ζ). 206 The principle of zeta potential measurement is based on the mobility of the charged particles under constant electric field. Hence the zeta potential is

207 related to electrophoretic mobility (UE) by Henry equation.

2εζf (κa) U = 2.4.6. E 3η

where ζ is the zeta potential, ε is the dielectric constant, η is the viscosity and f(κa) is the Henry function which considers the thickness of the double layer (κ Debye length) and 'a' refers to the particle diameter. Depending on the Henry function two models are used for determining the zeta potential from electrophoretic mobility. Smoluchowski's approximation is commonly used for determining zeta potential in aqueous media containing moderate electrolyte concentration. f(κa) in this case is 1.5 and model works good for particles larger than 0.2 microns. For small particles dispersed in non-aqueous media the f (κa) becomes 1 through simple calculation known as Huckel approximation.207 The electrophoretic mobility is measured in the Zetasizer Nano series by combination of laser Doppler velocity and phase analysis light scattering (PALS) (Malvern technical note).

2.4.3 Scanning electron microscopy

Scanning electron microscopy (SEM) was developed in early 1990's and was first commercialized in 1965 by Cambridge Scientific instruments.208 It is powerful imaging technique that uses electron beam to form image. The microscope allows excellent visualization of surface topography by providing high magnification (100-100,000 X) and at highest magnification; resolution of 1nm can be achieved on conducting surfaces.209

2.4.3.1 Instrumentation

The typical setup of SEM (figure 2.4.4.) consists of a source of electron (electron gun), electron beam manipulation system (condenser and apertures), beam

43 2.4 Characterization techniques specimen interaction (specimen chamber), detection of electrons and X-ray (detection system), signal processing (image generation), and display and recording. The vacuum system is vital for electron generation and its control in the microscope. Air molecules in column can oxidize the filament, and can interfere with electron beam and block it before reaching the specimen. Therefore air molecule must be removed to obtain better functioning of the microscope. Generally SEM operates at 10-4-10-6 Torr pressure. The electron gun generates electrons via thermal or field emission. Thermal emission uses heat to emit electrons from the filament by supply of current, while in the field emission the electrons are drawn in strong electric field. The electron gun essential consists of 3 components; filament or cathode (tungstun / Lanthanum hexaboride (LaB6)/ Cerum hexaboridethermionically emit electrons, a grid cap or Wehnelt cylinder (controls flow of electrons) and anode plate.210

Electron)Gun)

Anode)

Magne1c)Lens) TV) Scanner)

Scanning)Coils)

Secondary) Backsca9ered) Electron) Electron)Detector) Detector)

Specimen) Stage)

Figure 2.4.4: Schematic representation of the SEM instrumentation.211

The electron manipulation system consists of copper coils and deflector plates beam, which generate magnetic fields. These fields are the lenses of the electron microscope also known as electromagnetic lenses. The condenser lenses are involved in demagnification; the objective lens focuses beam on the specimen.

44 2. Fundamentals and Methods

The beam in the final condenser lens is moved by two sets of magnetic coils along x and y axes so that it scans in a raster fashion over a rectangular area of the sample surface.

Electron beam

Auger electrons (~1nm) Secondary electrons (~100nm)

Inelastically backscattered electrons (~1 µm)

Characteristic X-rays (~10 µm)

Continuum X-rays (~10 µm) Fluorescent X-rays (~10 µm)

Figure 2.4.5: Interaction volume is the depth range probed by various types of scattered electrons and X-rays. 212

Detection of signal begins when the primary electron beam interacts with the sample, and scattering event takes place within a teardrop-shaped volume of the specimen known as the interaction volume (100 nm- 5 μm). The beam interacts with the specimen and generates different type of signals like backscattered electrons, secondary electrons, X rays, Auger electrons, cathado-luminescence. When beam interacts with electric field of specimen electrons, energy is transferred and in this inelastic events secondary electron (< 50 eV), and X rays are produced. In elastic event the beam interacts with the nucleus of the specimen atom causing change in the direction of the beam known as backscattered electrons (50 eV). The size of the interaction volume depends on the atomic density, topography of specimen, and acceleration potential of the primary electrons. Electronic amplifiers of various types are used to enhance the signals, which are displayed as variations in brightness on a cathode ray tube. The raster scanning of the CRT display is synchronized with that of the beam on the specimen in the microscope, and the resulting image is therefore a distribution map of the intensity of the signal being emitted from the scanned area of the specimen. The image is digitally captured and displayed on a computer monitor.213 45 2.4 Characterization techniques

In this work scanning electron microscopy was carried out using Ultra 55 (Carl Zeiss NTS, Oberkochen, Germany) operating at 3 kV in the secondary electron (SE) mode. The microgel samples were dip coated on cleaned silicon wafer and dried under vacuum for overnight. All the samples were coated with 5 nm of platinum layer before SEM imaging using low vacuum spatter coater.

2.4.4 UV visible absorbance spectroscopy

2.4.4.1 Theory Light absorbance is energy transfer phenomenon from light to matter (atom, electrons) mostly in the form of thermal energy. Electromagnetic waves in ultraviolet (200-400 nm) and visible (400-800 nm) range are able to induce electronic transitions in molecules.214 During this course electrons get excited and attain its high energy levels from their respective ground state. The absorbance in UV-visible range causes the electron transition mostly between n→ π∗ and π → π∗. Molecules or parts of molecules that absorb light strongly in the UV-vis region are called “chromophore”.215 The conjugated systems, where the molecule consists of alternating single and double bonds, posses de-localized electrons due to overlap of the p orbitals in the double bonds. The delocalization reduces the energy gap between the bonding orbitals and π anti-bonding orbitals and therefore light of lower energy, and longer wavelength will be absorbed. Further increase in delocalization results in highly colored compound and eventually absorbs light in visible rage.215 Similarly in case of transition metal complexes, the ligands and metal ion complex solution exist in beautiful color. This is due to the splitting of the d orbitals, i.e. some of the d orbitals gain energy and some lose energy.216 The deciding factor for the particular wavelength of light to be absorbed relies on the amount of energy required to attain the electron transition. Light of lower wavelength has high energy and vice versa. The colored compounds absorb light with opposite color are depicted in figure 2.4.7 and hence from this range of wavelength of light for absorbance can be predicted.217

46 2. Fundamentals and Methods

E Visible Light λ

Color of the sample absorbing respective wavelength of light

Figure 2.4.7: White light spectra in visible range (E is energy and λ is the wavelength) and color of the samples absorbing respective wavelength.217

The Lambert and Beer’s law relates the absorbance of a solution to the concentration of the absorbing species by following equation;

I A = log 0 = εcl 2.4.8. 10 I

Where A is the absorbance, I0 is the intensity of the incident beam of light, I is intensity of the transmitted light after passing through the sample, ε is the extinction coefficient, c is the concentration of the absorbing species in the solution, l is the path length of the light through the sample.214

2.4.4.2 Instrumentation The instrumentation of microtiter plate for UV visible measurement consists of light source, excitation monochromator, and absorbance optics and measurement module. Condenser type optics focus light through the monochromator where specific wavelength of light is focused through the fiber optics into the microtiter plate well. The absorbance MTP module located beneath the plate carrier measures the transmitted through the sample. In this work, monochromator based microtiter plate reader Tecan infinit pro M200 (Tecan, Austria) was used to perform UV-visible absorbance and fluorescence emission studies. Absorbance of sample was done in 96 well flat bottom plates (Carl Roth, Germany), and 96 well UV transparent plates (Sigma, Germany) and fluorescence was done in 96 well round bottom black plates (Carl Roth, Germany).

47 2.4 Characterization techniques

Condensor& Excita5on& Monochromator&& Flash& Filter& Fiber& lamp& Wheel& Fiber& Ref;&Diode& switch&

Abs;&Diode&

Figure 2.4.8: Schematic representation of instrumentation of UV-visible setup in microtiter plated reader (Reference: User Manual of Tecan infinit pro M200 (Tecan, Austria)).

2.4.5 Fluorescence spectroscopy

This is a complementary technique to absorption spectroscopy that analyses fluorescence from the sample. The phenomenon of fluorescence exhibited by a compound that absorbs light at one wavelength and re-emit the light at higher wavelength, therefore the compound shows characteristic two spectra: an excitation and emission spectrum. In the course, the molecules are excited to higher energy levels by absorbing photons and through random collision the molecule loses vibrational energy till it reaches the ground state by emitting the photon. This electronic transition is given by Jablonski diagram.218 Aromatic amino acids (tryptophan, tyrosine and phenylalanine) figure 2.4.9 have fluorescence properties and proteins consist of different numbers of these amino acids in its structure. Owing to the high sensitivity of tryptophan to its local environment provide valuable structural information in the form of intrinsic protein fluorescence. The conformational transitions, subunit association, substrate binding or denaturation affects the emission spectra.

48 2. Fundamentals and Methods

- COO- COO COO- + C H +H N C H + H3N 3 H3N C H CH CH2 2 CH2 C CH NH OH

Tryptophan Tyrosine Phenylalanine

Figure 2.4.9: Structures of aromatic amino acids (Tryptophan, tyrosine and phenylalanine).

These interactions can affect the local environment surrounding the indole ring. Tryptophan fluorescence can be selectively excited at 295-305 nm. 219 In this work temperature dependent fluorescence emission spectra were studied to determine the thermal stability of immobilized enzyme compared to the free enzyme.

2.4.5.1 Instrumentation In this work, monochromator based microtiter plate reader Tecan infinit pro M200 (Tecan, Austria) was used to perform UV-visible absorbance and fluorescence emission studies. Absorbance of sample was done in 96 well flat bottom plates (Carl Roth, Germany), and 96 well UV transparent plates (Sigma, Germany) and fluorescence was done in 96 well round bottom black plates (Carl Roth, Germany).

2.4.6 Circular dichroism

2.4.6.1 Theory CD spectroscopy is powerful technique to probe the protein structure in the solution.220CD spectroscopy is a variant of adsorption spectroscopy, where it measures the difference in absorbance between left (AL) and right (AR) circularly polarized components given by equation 2.4.9.221

49 2.4 Characterization techniques

ΔA = AL − AR 2.4.9 The result in CD is given in terms of the ellipticity (θ) (unit is in degrees), ellipticity is related to ΔA by simple numerical term,

θ = 32.98 ΔA 2.4.10 Where θ =tan-1 (b/a), b and a are minor and major axes of resulting ellipse. The chromophores include peptide bond (absorbance below 240 nm), aromatic amino acid (260 to 320 nm) and disulphide bonds (260 nm). The transition from n→ π* is determined near 220nm and more intense transition from π→π* is marked at 190 nm. The secondary structures of protein have typical spectra for each type (alpha helix, beta sheet and random coil) as shown in figure 2.4.10.

(A)' * * α!helix' (B)' !! π→π ! n→!π !

β!'sheet' amide&bond& 190&nm& 220&nm& $ random'coil'

mdeg α.helix& 190&nm&(+)& 222&nm&(.)&

β.&sheet& 218&nm&(.)& 196&nm&(+)&

random&coil& 212&nm&(+)& 195&nm&(.)& 180$ 190$ 200$ 210$ 220$ 230$ 240$ 250$ Wavelength$(nm)$

Figure 2.4.10: CD spectroscopy (A) CD spectra of different protein secondary structures; (B) table depicting wavelength maxima for respective electron transitions.222

2.4.6.2 Instrumentations The plane-polarized rays are produced by passing light produced from light source through series of prisms, mirrors, and slits. The ordinary rays are focused by lens and passed through filter to the modular. The circularly polarized light are then passed through the shutter to the sample compartment and subsequently detected by photo-multiplier.221

50 2. Fundamentals and Methods

Figure 2.4.11: Schematic representation of instrumentation of CD spectroscopy.223,224

In this work, UV-CD spectra of native and immobilized enzyme were measured on a Jasco 810 (Jasco, Germany) using a quartz cuvette with 1 mm path length. Spectra were corrected by subtracting the buffer baseline. CD spectroscopy was applied to examine the secondary structure of the proteins. The spectra (190 to 250 nm) were analyzed using the tool “CD-PRO-Analysis” of Jasco Software Spectra Manager Version 2 (Version 2.06.00 [Build 6]) from JASCO GmbH, Groß- Umstadt, Germany.

2.4.7 Enzyme assays

2.4.7.1 Pyrophosphate assay

Pyrophosphate (PPi) assay or molybdate assay is a chemical assay to detect inorganic phosphate in presence of thiol agent.225 This assay is sensitive to PPi in presence of ten-fold excess of inorganic orthophosphate (Pi). PPi reacts with the molybdate reagent to produce phosphomolybdate and PPi-molybdate complexes. The complex is reacted with thiol reagents (2-mercaptoethanol) to develop the color. The PPi and Pi complexes are distinguished by use of two reducing agents Eikonogen and 2-mercaptoethanol and then measuring the absorbance at 580 nm.225,226 The reaction is done at room temperature and the color develops in 10 min and is stable up to 1 hr. The extinction coefficients of chromophore is 2.70 × 104 for PPi.225 This method was applied to measure the activity of Acs.226

2.4.7.2 Coupled enzyme assay The coupled enzyme assay is one way to detect the enzymatic reaction whose product is difficult to detect (e.g. optically inactive in UV-visible range for absorbance spectroscopy). Principally the product of main reaction is substrate

51 2.4 Characterization techniques for the coupled enzyme/s, which further convert it stoichometrically into detectable signal/product. The generalized reaction can be stated as follows;

v1 v2 A B C 2.4.11

A is the substrate for the main reaction producing product B, the rate of this reaction is v1, The v2 is the velocity for the coupling reaction, coupled enzyme utilizes B as its substrate to give product C which finally measured. For optimum measurement of B using coupled assay, v1 must be rate limiting i.e is v2 must be

195 very higher than v1 and B must attain its steady state concentration. In this work we used malate dehydrogenase and citrate synthase coupled enzyme assay reaction to monitor acetyl CoA formation.

52 3. Results and discussions.

3 Results and discussions.

3.1 Thermo-responsive microgels synthesis and

characterization

3.1.1 Introduction

The methods and approaches to synthesize thermo-responsive microgels are numerous. Each method has its own advantages and limitations; hence the selection of a particular method basically relies on achieving desired quality of microgels for its end application.49 Surfactant free emulsion polymerization (SFEP) and precipitation polymerization are the most commonly used methods that yields microgels with very narrow distributed size.66 The cationic microgels can be synthesized by using cationic initiator, or using cationic monomers /polymers added during the synthesis of microgels or by post-synthesis modification.227,228 The basis for using core-shell morphology is to obtain high amino group density on the surface of microgel particles, which enable better enzyme load.91 In this chapter, the cationic core-shell PNIPAm microgels synthesis using either cationic co-monomer (AEMA) or polymer (PEI) is described.

3.1.2 PNIPAm-AEMA core shell microgel preparation

The PNIPAm-AEMA core shell microgel was synthesized using free radical based precipitation polymerization in two stages (figure 3.1.1).54

53 3.1 Thermo-responsive microgels synthesis and characterization

O O HN 70 oC, 4 h, Argon HN + HN SDS, APS O

PNIPAm Core

Step I: Core Synthesis Step I: Core N-isopropyl acrylamide N,N′-Methylene (NIPAm) bis(acrylamide) (MBA)

NH2.HCl

70 oC, 4 h, Argon + O Core Solution, PNIPAm, O MBA, SDS, APS

CH2

H3C Step II: Synthesis Shell 2-Aminoethyl methacrylate PNIPAm-AEMA core shell microgel (AEMA)

Figure 3.1.1: Schematic representation of the two steps synthesis of PNIPAm-AEMA core-shell microgel; Step 1: PNIPAm core synthesis and Step 2 cationic shell synthesis.

3.1.2.1 Characterization of PNIPAm-AEMA microgel The microgels was characterized with respect to the size, thermo-responsive behavior, cationic nature and morphology. Dynamic light scattering method was used to probe the size and thermal sensitive characteristics of the PNIPAm- AEMA microgels in the solution. Figure 3.1.2 shows the hydrodynamic size of microgel with respect to the temperature. The size of core and core-shell microgels in water at 24 °C were found to be 150 nm and 320 nm, respectively.

54 3. Results and discussions.

PNIPAM"-core"microgel" 400" PNIPAm-"AEMA"core-shell"microgel"

300"

200"

100" Hydrodynamic+size+(nm)+

0" 22" 27" 32" 37" 42" Temperature+(°C)+

Figure 3.1.2: Temperature dependent dynamic light scattering measurement of PNIPAm-AEMA microgels in water (0.01 mg/mL)

While at 40 °C, the size changed to 60 and 180 nm for core and core-shell microgel, respectively. The microgels exhibit volume phase transition from a swollen state to a collapsed state at around 32 °C and this transition temperature was not significantly affected by the presence of functional co-monomer in the structure.54,78 The morphology of core-shell microgel was characterized with the help of SEM and the image is depicted in figure 3.1.3. It is evident from the SEM image that the microgels synthesized were nearly uniform in size and shape.

200nm

Figure 3.1.3: SEM image of PNIPAm-AEMA microgels dried under vacuum and sputter coated with 3 nm platinum.

55 3.1 Thermo-responsive microgels synthesis and characterization

The cationic nature of PNIPAm-AEMA microgel was confirmed by measuring the zeta potential. The zeta potential (Figure 3.1.4) value of diluted microgel sample in deionized water at pH 7 was found to be 8.60 mV, which clearly indicates the cationic chracteristic of microgel particles. This behaviour of the PNIPAm-AEMA microgels was originated due to the presence of free –NH2 groups in shell. The presence of free –NH2 group of AEMA co-monomer in the shell was also confirmed by Ninhydrin assay.229 The free amino group concentration was obtained as 71 µM/mg of dry microgel with respect to glycine as the standard. Thus, the positive value of zeta potential and free amine content in microgel, confirms that the microgels were sufficiently positive charged.227,228

15#

10#

zeta%potential%(mV)% 0#

!5# 6.5# 7.5# 8.5# 9.5# 10.5# pH%

Figure 3.1.4: Zeta potential measurement with respect to pH of microgel in water (0.01 mg/mL)

3.1.3 PNIPAM-PEI core shell microgel preparation

The microgels were prepared by graft co-polymerization of the NIPAM and MBA from the PEI in presence of initiator t-BuOOH in aqueous media as depicted in figure 3.1.5.121

56 3. Results and discussions.

70)oC/)2)h/)Ar)

tBuOOH)

Polyethyleneimine) (PEI))

N!Isopropyl) N"N’!Methylene) PNIPAm!PEI)) Acrylamide) Bisacrylamide) core)shell)microgel) (NIPAm)) (MBA))

Figure 3.1.5: Schematic representation of PNIPAm-PEI core shell microgel synthesis.

This synthetic protocol results in colloidal microgels with PNIPAm core and PEI shell.45, 47 Also the method gives high rate of monomer conversion and due to use of high molar amount of cross-linker, the microgel contains dense core.47, 52 The two different microgels PNIPAm-PEI 1 and 2 were prepared by varying temperatures and initiator concentrations.

3.1.3.1 Characterization of PNIPAm-PEI microgel The two PNIPAm-PEI microgels were analyzed for their size, charge properties and morphology characteristics. The dynamic light scattering measurement for size of the microgels with respect to temperature is depicted in figure 3.1.6. The PNIPAm-PEI 1 has hydrodynamic size of 320 nm in water at 24 °C, which upon increase in temperature is reduced to 234 nm at 40 °C. Similarly for PNIPAm-PEI 2 the corresponding sizes at 24 and 40 °C were 736 and 500 nm respectively. These results demonstrate the thermo-responsive property of the microgels. PNIPAm-PEI microgels exhibit the volume phase transition temperature (VPTT) near to the LCST of the PNIPAm, confirming that the transition temperature was not affected by the copolymer.71

57 3.1 Thermo-responsive microgels synthesis and characterization

340# 800# (a)# (b)# 320# 750# 700# 300# 650# 280# 600# 260# 550# Hydrodynamic#size#(d.nm)# Hydrodynamic#size#(d.#nm)# 240# 500#

220# 450# 18# 22# 26# 30# 34# 38# 42# 22# 27# 32# 37# 42# Temperature#(oC)# Temperature#(°C)#

Figure 3.1.6: Dynamic light scattering measurement of microgels prepared at different temperatures and initiator concentrations: (a) PNIPAm-PEI 1 and (b) PNIPAm-PEI 2 microgels (0.01mg/mL in water) sizes with respect to the temperature.

The zeta potential measurements of the microgels were positive (table 3.1.1) in water and the isoelectric point of microgel was found to be around pH 10. These results indicated that the cationic microgels were synthesized and the positive charge was contributed from the PEI polymer to the microgel structure.

Table 3.1.1: Zeta potential values of PNIPAm-PEI microgels.

Microgel Zeta potential (mV) Size in dry state* PNIPAm-PEI 1 11.5 250 nm PNIPAm-PEI 2 11.1 500 nm

Note: * from SEM image (given on page number 59)

The morphological characteristics of dried microgels were imaged with the help of SEM technique figure 3.1.7 (a and b). It is evident from the microscopic images that the microgels were highly mono-dispersed. The size of microgels PNIPAm- PEI 1 and PNIPAm-PEI 2 in dried state were found to be 250 nm and 500 nm respectively.

58 3. Results and discussions.

(a) (b)

Figure 3.1.7: SEM images of PNIPAm-PEI microgels (a) PNIPAm-PEI 1 and (b) PNIPAm- PEI 2 dried under vacuum and sputter coated with 3 nm platinum.

3.1.4 Conclusion

The cationic core shell microgels PNIPAm-AEMA and PNIPAm-PEI were synthesized using two different approaches. Both methods yielded microgels with highly uniform size distribution and possessed thermo-responsive behavior. The microgel particles exhibit the phase transition at around 32 °C which is near to the LCST of PNIPAm. The demonstration of high density of cationic groups on the microgels surface was confirmed by zeta potential measurements. The PNIPAm-AEMA possessed sufficient amino groups due to AEMA that can be used for covalent binding of enzymes whereas the branched PEI structure in PNIPAm- PEI could be utilized for enzyme immobilization via ionic exchange and covalent binding.

3.1.5 Experimental section

3.1.5.1 Materials N-isopropylacrylamide (97 %) (NIPAm) was recrystallized from n-hexane and dried in vacuum before use. N, N’-methylenebis (acrylamide) (≥ 99.5 %) (MBA), sodium dodecyl sulfate (SDS), ammonium persulfate (≥ 98.0 %) (APS), 2-

59 3.1 Thermo-responsive microgels synthesis and characterization aminoethyl methacrylate hydrochloride (90 %) (AEMA), Branched poly (ethyleneimine) (PEI) with an average molecular weight of 25,000 (50 wt.% solution in water), and tert-butyl hydroperoxide (70% solution in water, t- BuOOH). Water used throughout the investigation was purified to a resistance of 18 MΩ (Millipore), and filtered through a 0.45-μm nylon filter to remove particulate matter.

3.1.5.2 Method

3.1.5.2.1 Synthesis of PNIPAm-AEMA core shell microgels The microgels were synthesized by two-step free radical precipitation polymerization method.91 The total monomer concentrations were 64 mM and 37 mM for core particles and shell preparation respectively. Except for the initiator all ingredient were first dissolved in water and filtered through Whatman filter paper and heated to 70 °C under stirring condition and gentle stream of argon for 1 h. First core was synthesized by rapid addition of 1ml of APS solution (2 mM) into monomer solution (700 mg NIPAm and 21 mg MBA) containing SDS (2 mM). The reaction was continued for 4 h at 70 °C under continuous stirring condition. After the completion of reaction, core solution was filtered through 0.45-µm pore size filter. For synthesis of shell, 20 mL solution of core microgel was diluted to 75 mL using water. SDS (0.7 mM) was added to core solution and the solution was further heated to 70 °C, under stirring in Ar atmosphere. 25 mL of monomer solution (400 mg NIPAm, 12 mg MBA, and 10 mg AEMA) for shell synthesis was added to the heated core microgel solution and the reaction was initiated with the addition 1 ml APS solution (1.5 mM). The reaction mixture was continuously stirred for 4 h at 70 °C. Thus obtained core- shell microgel solution was allowed to cool at room temperature and filtered with suitable membrane. The un-reacted monomers and small molecular weight polymers were removed from the microgel solution by continuous dialysis for a week using 10 KDa MWCO dialysis tube against daily change of water. The microgel was characterized by Dynamic light scattering (DLS), Zeta potential, SEM and ninhydrin assay.

60 3. Results and discussions.

3.1.5.2.2 Ninhydrin assay

Free amino group content on microgel particles was determined using ninhydrin test in a 96-well plate format. The ninhydrin reagent was prepared in ethanol at concentration 50 mg/mL. Equal volumes of microgel solution and ninhydrin reagent were mixed in a 96-well plate and incubated at 50 °C for 20 min. The absorbance was checked at 590 nm using plate reader. The concentration of amino group was determined from standard plot of glycine. 229

3.1.5.2.3 Synthesis of PNIPAM-PEI Core-Shell Microgels

The cationic thermo-responsive PNIPAm-PEI microgel was synthesized via graft co-polymerization of NIPAm and MBA from PEI.121 Polymerization was carried out in three-necked round bottomed flask equipped with a reflux condenser, a thermometer and argon inlet under continuous magnetic stirring (300 rpm). A mixture of NIPAm monomer (800 mg) and MBA (80 mg) in water were added to the flask and the solution was treated with gentle stream of argon for 30 min. Subsequently, PEI (0.4 g) was dissolved in water and solution was neutralized to pH 7 using 1 M HCl solution. The PEI solution was then mixed with the monomer solution (total volume 50 mL) and the solution was heated to 70 °C under gentle stream of argon. After the desired temperature of the solution was reached, diluted tBuOOH solution in water (0.5 mL, 10 mM) was added drop wise to the mixture to initiate polymerization reaction and the reaction was allowed to proceed for 2 h, at 70 °C with constant stirring under argon atmosphere. For synthesis of PNIPAm-PEI 2 all conditions were similar except temperature for synthesis 63 oC and initiator concentration (11 µL in 1mL). After the completion of the reaction, the dispersion of microgels was carefully purified by repeated centrifugation at 13,000 rpm for 30 min and further purified by dialyzing against water using dialysis tubing (Cellulose membrane; 14,000 Da molecular weight cut-off) for one week at room temperature. Lyophilized microgel was dispersed to concentration of 1 wt %. The microgel suspensions were shaken for 24 h to get evenly dispersed microgel solution.

61 3.1 Thermo-responsive microgels synthesis and characterization

62 3. Results and discussions.

3.2 PNIPAm-AEMA core-shell microgel support for Acs

3.2.1 Introduction

The beneficial accounts of covalent immobilization such as multipoint attachment and prevention of enzyme leakage is being explored in this section for the immobilization of acetyl CoA synthetase (Acs).10 The methods for immobilization of Acs on respective microgels (PNIPAm-AEMA and PNIPAm-PEI) were selected based on the results of conjugation standardization (Appendix figure A1). The cationic thermo-responsive core shell PNIPAm-AEMA microgels (preparation details section 3.1.2) was used to immobilize Acs using 1-ethyl-3- (3-NN-dimethylaminopropyl) carbodiimide (EDC) coupling (figure 3.2.1). The carboxyl groups of Acs were activated to immobilize on amino functionalized PNIPAm-AEMA microgels via amide bond formation. The enzyme immobilization conditions on the microgel were standardized and different reaction and stability parameters were evaluated comparing with the soluble enzyme.

O" O" RN" C" NR" OH" +"H2N" N" H"

Figure 3.2.1: Schematic representation of bio-conjugation of Acs on PNIPAm-AEMA microgels using EDC coupling.

3.2.2 Results and discussion

3.2.2.1 Standardization of the immobilization parameters. The first step of Acs reaction was sensitive to amino-group activating crosslinker due to activation of vital lysine group at the active site.145 Therefore zero length cross linker EDC was used to covalently bind enzymes to the microgels.230 The carboxylic acid of enzyme was activated by EDC , for this pH and time for

63 3.2 PNIPAm-AEMA core-shell microgel support for Acs conjugation was optimized to prevent self crosslinking of the enzymes. The immobilization conditions were standardized with respect to EDC concentration, microgel and salt concentration. The effect of EDC concentrations on Acs immobilization to microgel were studied from 0.25 to 5 mg/mL concentration. As shown in Figure 3.2.2 a, the enzyme activity increased with increase in EDC concentration up to 1 mg/ml, above this concentration there was a sharp decline in the enzyme activity. Since EDC is a cross linker, at higher concentration, it cross link enzyme molecules to one another at faster rate leading to decrease in enzyme activity.

0.10 0.10 0.12 (a) (b) (c) 0.10 0.08 0.08 0.08

0.05 0.05 0.06

0.04 0.03 0.03

Specific activity (U/mL/mg) (U/mL/mg) activity Specific 0.02 Specific activity (U/mL/mg) (U/mL/mg) activity Specific Specific activity (U/mL/mg) (U/mL/mg) activity Specific

0.00 0.00 0.00 0.25 0.5 1 2 5 1 2 5 10 0 1 10 100 1000 EDC concentration (mg/ml) Microgel concentration (mg/ml) NaCl concentration (mM)

Figure 3.2.2: Standardization of immobilization conditions with respect to; (a) effect of EDC, (b) microgel, and (c) salt on activity of Acs.

To determine the working concentration of the microgels for enzyme immobilization different concentrations of microgel from 1 to 10 mg/mL were incubated with 1 mg/ml of enzyme in presence of EDC. The highest concentration of microgel for this study was maintained to be 10 mg/mL to avoid very viscous solution above this concentration. From figure 3.2.2 b, it can be seen that the enzyme activity increases with increase in microgel concentration. Therefore for further use, working concentration of 10 mg/mL microgel was found suitable, which is 1:10 proportion of enzyme to microgel for conjugation protocol. To obtain the bioconjugate after immobilization process, the microgels were precipitated from reaction mixture at room temperature with the help of salt. 64 Hence it was important to study the effect of salt concentration on enzyme 64 3. Results and discussions. activity. The conjugate was subjected to different concentration of NaCl solution and activity was determined. At given centrifugal condition, 1 M NaCl solution was optimum to obtain complete bioconjugate. Also figure 3.2.2 (c), shows that no significant reduction in enzyme activity at 1 M NaCl concentration was observed as compared to 10-1000 folds less concentration of NaCl. After standardization of immobilization conditions, the conjugation was carried out at optimized conditions. From the Bradford method, the amount of Acs immobilized onto microgel was determined to be 0.02 mg per mg of dry microgel particles. From this protein data ~24 Acs molecules were calculated to be bound on the surface of each microgel particle using equation (3.2.1 to 3.2.3).229 The activity of immobilized Acs was found to be 0.23 mU/mg of the carrier. The attachment of enzyme to microgel using carbodiimide chemistry resulted in more active enzyme with respect to the total bound enzyme. The decrease in enzyme activity after immobilization is effect of several factors such as change in enzyme conformation or modification of important residues due to covalent immobilization and addition to this immobilization matrix also imparts additional factors like steric hindrance, partition effects and mass transfer constraints.231

3.2.2.2 Effect of temperature and pH on Acs Activity. Temperature and pH are two important parameters that influence the activity of the enzymes. It is important to study these factors to determine any changes in the conformation of enzyme on binding to the support.188 The temperature dependence on the activity of free and immobilized Acs was studied in the temperature range of 25 °C to 55 °C. The results presented in figure 3.2.3 shows that the immobilized enzyme was active at a broad temperature range and exhibited optimum temperature of reaction at 37 °C that was the similar to free enzyme.232 These results can be attributed to the rigidification of protein conformation due to covalent immobilization of enzyme making it less susceptible to the temperature-induced conformational changes.175

65 3.2 PNIPAm-AEMA core-shell microgel support for Acs

0.4 120" Immobilized enzyme 100" 0.3 Free enzyme 80"

0.2 60"

40"

0.1 Relative"activity"(%)" Specific activity (U/mL/mg) (U/mL/mg) activity Specific 20" Immobilized"enzyme" Free"enzyme" 0.0 0" 20 30 40 50 60 20" 30" 40" 50" 60" Temperature (oC) Temperature"(°C)"

Figure 3.2.3: Effect of different temperatures on the activities of free and immobilized Acs was studied in borate buffer pH 8. (Left figure: the specific activity, and Right figure: the normalized specific activities of the respective enzymes)

pH dependent activity of enzyme was studied from pH range 5 to 9 and the properties of immobilized enzyme were compared with those of free enzyme. It can be seen from figure 3.2.4 that free enzyme has optimal pH at 8.0, which is close to previous report on the same enzyme,149 while for immobilized Acs the pH was shifted towards more alkalinity.

0.6 Immobilized enzyme Immobilized"enzyme" 100" Free enzyme Free"enzyme" 80" 0.4 60"

40" 0.2 Relative"activity"(%)"

Specific activity (U/mL/mg) (U/mL/mg) activity Specific 20"

0.0 0" 4 6 8 10 4" 5" 6" 7" 8" 9" 10" pH pH"

Figure 3.2.4: Effect of different pH on the activities of free and immobilized Acs was studied in borate buffer at 37 oC. (Left figure: the specific activity, and Right figure: the normalized specific activities of the respective enzymes). 66 3. Results and discussions.

The electrostatic interactions of enzyme with the matrix leads to unequal partitioning of H+ and OH- between the microenvironment of the immobilized enzyme and the bulk phase. This is attributed to Donnan partition effect where charged support often leads to displacements in the pH dependent activity profile of immobilized enzymes.175

Michaelis-Menten kinetics was studied to determine the apparent Km and Vmax using Lineweaver-Burk plot. From table 3.2.1, apparent Km values of acetate for immobilized enzyme and free enzyme were found to be close to each other.

Table 3.2.1. Kinetic parameters of immobilized and free enzyme for acetate substrate at 37 °C.

Vmax/Km Enzyme Km (mM) Vmax(U/mL) (min-1)

Immobilized enzyme 0.18 ± 0.02 5.6 × 10-3± 0.1 × 10-3 0.03 ± 0.003

Free enzyme 0.17 ± 0.03 8.5 × 10-3± 0.4 × 10-3 0.05 ± 0.008

The Michaelis constant (Km) is a direct measure of the enzyme substrate affinity.

The marginal increase of Km of the immobilized enzyme indicates that the Acs- substrate binding continues to be efficient after immobilization. While the maximum velocity of immobilized enzyme was reduced compared to the free enzymes. As Vmax is dependent on active enzyme concentration, the decrease may be due to inactivation of enzyme particles or due to limited diffusion of substrate to the immobilized enzyme.233,234

3.2.2.3 Fluorescence Study Tryptophan (Trp) fluorescence study provides information about the structures of protein adsorbed to optically transparent substrates.60 The changes observed in fluorescence intensity and λmax can be attributed to the change in Trp environment, hinting to altered enzyme conformation.235

67 3.2 PNIPAm-AEMA core-shell microgel support for Acs

75000 60000 (a) 25oC25 oC (b) 25oC25 oC 30! oC 30! oC 60000 30oC 50000 30oC 32oC32! oC 32oC32! oC 37oC37 oC 40000 37oC37 oC ! o ! o 45000 42oC42 C 42oC42 C ! 30000 ! 30000 20000 Intensity units units Intensity 15000 units Intensity 10000

0 0 300 350 400 300 350 400 Wavelength (nm) Wavelength (nm)

Figure 3.2.5: Fluorescence emission spectroscopy (Ex280/Em 310-390)) at different temperatures of free (a) and immobilized (b) Acs in 0.01 M phosphate buffer (protein concentration = 0.05 mg/mL)

From figure 3.2.5 the fluorescence spectra of both free and immobilized Acs shows λmax at 330 nm, which indicates that there is no significant change in the immobilized enzyme native conformation. Further the temperature dependent intensity profile was studied from temperature range (25 °C to 42 °C) to determine conformational stability of enzyme after immobilization. The temperature dependent fluorescence spectra of free enzyme (Figure 3.2.5 a) indicate a linear decrease in the intensity with increasing temperature. About 30% decrease in intensity of free enzyme was recorded with the change of temperature from 25 to 42 °C. Whereas in case of immobilized enzyme (Figure 3.2.5b), no prominent change in intensity was observed below VPTT of the microgel. Moreover with the increase in temperature beyond 32 °C, only 12% reduction in intensity was seen at 42 °C. The comparatively low reduction in intensity of immobilized enzyme shows improved thermal stability of enzyme after immobilization on microgel because of structural rigidity.175,236

3.2.2.4 Stability of immobilized Acs. The attachment of enzyme to a suitable polymer matrix provides longer shelf life and thermal stability to the biocatalyst. Thermal stability of enzyme after immobilization was checked in temperature range 32-60 °C. The thermal

68 3. Results and discussions. stability of conjugate enzyme was found comparatively higher than the free enzyme figure 3.2.6.

0.5# 110#

100# 0.4#

90# 0.3# 80# 0.2# 70# Relative#activity#(%)# 0.1# 60#

SpeciHic#activity#(U/mL/mg)# Immobilized#enzyme# Immobilized#enzyme# Free#enzyme# Free#enzyme# 0.0# 50# 25# 30# 35# 40# 45# 50# 55# 60# 65# 30# 35# 40# 45# 50# 55# 60# 65# Temperature#(°C)# Temperature#(°C)#

Figure 3.2.6: Thermal stability studies of free and immobilized Acs (Left figure: the specific activity, and Right figure: the normalized specific activities of the respective enzymes). The thermal stability of prepared conjugate and free enzymes was assessed by determining the activity under standard conditions after subjecting to various temperature ranges from 32-60 °C for 10 min followed by cooling on ice

The enhancement of rigidity of enzyme structure upon attachment to support resists change in the native conformation of enzyme. Protection of enzyme conformation from environmental changes due to restricted mobility of the covalently immobilized enzyme on support imparts enhanced thermal stability.175 The similar behaviour of PNIPAm-enzyme bioconjugate with lysozyme, horse radish peroxidase (HRP) has also been reported which shows higher thermal stability upon immobilization.126,132,237

69 3.2 PNIPAm-AEMA core-shell microgel support for Acs

0.5 120 Immobilized enzyme Free enzyme 0.4 100

0.3 80

0.2 60 Relative activity (%) activity Relative

Specific activity (U/mL/mg) (U/mL/mg) activity Specific 0.1 40 Immobilized*enzyme*Conjugate Free enzyme 0.0 20 1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 Days Days Figure 3.2.7: Storage stability studies of free and immobilized Acs in borate buffer (0.1 M, pH 8) at 4 °C; (Left figure: the specific activity, and Right figure: the normalized specific activities of the respective enzymes)

In solution the native enzyme tends to acquire thermodynamically stable conformation which leads to distortion of structure and inactivation of enzyme. At a temperature below the LCST of polymer, it probably prevents the unfolding of enzyme by forming a hydrated surface layer, like a protective colloid.64,175 Figure 3.2.7. shows the data of storage stability for free and immobilized enzymes. The immobilized Acs maintains more than 90% of its initial activity after stored for 9 days at 4 °C, while free Acs demonstrates only 63% of its initial activity under the similar conditions. This indicates that the storage stability of enzyme was improved remarkably after the immobilization on PNIPAm-AEMA core-shell microgel. Further this result is comparable to the report on Acs immobilized on CNBr activated glass beads where they obtain relative activity between 80-90% on 9th day of storage.42

70 3. Results and discussions.

7" 140"

6" 120"

5" 100"

4" 80"

3" 60"

Activity"(mU/mL)" 2" 40" Relative"activity"(%)" 1" 20"

0" 0" 1" 2" 3" 4" 5" Number"of"cycles"

Figure 3.2.8: Operation stability of immobilized Acs was checked for 5 cycles at standard reaction conditions (FE=12mU/mL).

Reusability of bioconjugate depends on extent of stabilization which is directly related to the extent of covalent bond formation and electrostatic interactions. Immobilization of enzyme allows switching of conjugate from soluble to insoluble form in the solution making easy recovery from the reaction mixture.62,238The operation stability of this immobilized Acs was studied to estimate the number of times immobilized enzyme can be reused. From figure 3.2.8, it can be seen the immobilized Acs preserved 50% of its initial activity after 4 consecutive operations.

3.2.3 Conclusion

The novelty of work lies in investigation of core shell thermal responsive PNIPAm-AEMA microgel as a support for acetyl CoA synthetase to obtain reusable biocatalyst. The cationic microgel synthesized was nearly uniform in size and contained sufficient amino group for enzyme attachment. After immobilization, Acs possessed optimum activity at wide range of temperature and pH. Its thermal, storage and operational stability were significantly improved after immobilization, indicating that PNIPAm-AEMA core shell microgel is the favourable carrier for enzyme immobilization. Moreover, the immobilized enzyme could be reused for 4 consecutive operation cycles with more than 50% initial activity that opens new perspective for efficient synthesis of many drugs.

71 3.2 PNIPAm-AEMA core-shell microgel support for Acs

3.2.4 Experimental section

3.2.4.1 Materials S-Acetyl-coenzyme A synthetase (E C 6.2.1.1, >3 units/mg, from baker's yeast) (Acs), 1-ethyl-3-(3-NN-dimethylaminopropyl) carbodiimide (EDC) and all other chemicals were obtained from Sigma-Aldrich (Germany). All the chemicals were used as received or else stated. 30 and 10 KDa molecular weight cut off (MWCO) centrifugal filter tubes were purchased from Millipore.

3.2.4.2 Methods

3.2.4.2.1 Immobilization of enzyme on microgel. Acs was covalently immobilized to PNIPAm-AEMA core shell microgel using carbodiimide chemistry in borate buffer (0.1 M) at pH 8 (by using higher EDC concentration than generally used for lower pH). 64,230 Inorganic phosphate present in commercial enzyme preparation (interferes with Molybdate assay) was removed by filtration using 30 kDa centrifuge filter tubes (Millipore). The immobilization conditions were standardized before conjugation with respect to EDC, microgel, and salt concentration. A total 6 ml of mixture solution containing microgel solution (10 mg/ml), enzyme solution (1 mg/ml), and EDC (1 mg/ml) were incubated at 10 oC for 4 h. After the completion of incubation period, equal volume of tris-Cl buffer (0.1 M, pH 8) was added to the reaction mixture to quench extra EDC. Thus bioconjugate formed was precipitated by adding salt solution (1 M NaCl), and obtained by centrifugation at 12,000 rpm for 5 min at 25 oC. Finally the bioconjugate was desalinated by repeated centrifugation with borate buffer using 10 KDa MWCO centrifuge filter tubes. The amount of enzyme loaded to microgel was determined by subtracting the residual protein content in the supernatant from the initial protein content. The enzyme and bioconjugate were stored in borate buffer (0.1 M) containing glutathione (1 mM) and magnesium chloride (1 mM) (pH 8).

The number of molecules per unit volume (Nm and Ne) for microgel and enzyme were calculated using following formula 229,239

72 3. Results and discussions.

V N = 3.2.1 vi

where V is the total volume of particles per unit volume of dispersion (mL) and νi is the mean volume of a particle. The value of V was found as;

W V = 3.2.2 ρ

W is the mass of wet microgel or enzyme, ρ is the density, since the microgel particles were highly swollen in water ρ for microgel was assumed to be 1.00 g

-1 -1 mL and average protein density for enzyme 1.35 g mL . The value of νi was determined as

! 4 $ v = # &πR3 i " 3 % h 3.2.3

where Rh is the hydrodynamic radius. Using these data the number of enzyme molecules on single microgel particles was calculated from Ne /Nm.

3.2.4.2.2 Determination of the Acs activity.

The activity of free acetyl CoA synthetase and their bioconjugates with PNIPAm- AEMA microgel was studied in borate buffer (0.1 M, pH 8), in presence of potassium acetate (20 mM), MgCl2 (4 mM), glutathione (4 mM), ATP (1 mM), and co-enzyme A (500 µM). The reaction was terminated by addition of acidic molybdate reagent. The pyrophosphate-molybdate complex formed was further reduced by addition of mercaptoethanol and Eikonogen reagent. The obtained coloured product was quantified by measuring the absorption at 590 nm and pyrophosphate concentration was determined (ε= 2.7 × 104 M-1 cm-1). 225 One unit of enzyme activity was defined as the amount of enzyme required to form 1µmol pyrophosphate per min at pH 8 and 37oC.226 Relative activities are

73 3.2 PNIPAm-AEMA core-shell microgel support for Acs normalized specific activities of enzymes with respect to the highest activity of respective enzymes and the experiment. Each experiment was done in triplicate and error bars in figures show standard deviations calculated with three data points. The variation in the activities of enzymes for different experiments were due to variable activity of enzyme during storage at -20 oC and different batches.

3.2.4.2.3 Determination of the protein concentration.

Bradford micro assay with sensitivity of (1–10 μg/mL) was used to determine protein concentration using bovine serum albumin as the standard protein. 240

3.2.4.2.4 Effect of pH.

Optimum pH condition for both immobilized and free enzymes was evaluated in the range of 5 to 9. The enzymes were assayed in 0.1 M 2-(N- morpholino)ethanesulfonic acid (MES) buffer (pH 5-6.5) and 0.1 M borate buffer (pH 7-9) at 37 oC for 15 min for respective pH.

3.2.4.2.5 Effect of temperature.

The effect of temperature on enzymes reactivity was studied at different temperature ranging from 25 to 55 oC in 0.1 M borate buffer (pH 8) for 15 min. The enzymes activity was normalized with respect to maximum activity for given experimental condition and the final data were represented as relative activity.

3.2.4.2.6 Effect of substrate concentration.

The kinetic constants for acetate as a substrate were obtained by varying its concentration from 0.5 to 10 mM at constant cofactor concentration (500 μM ATP and 100 μM Co enzyme A).241 Assuming Michaelis-Menten kinetics for Acs, the Km and Vmax values were estimated with the help of Lineweaver-Burk plot.

3.2.4.2.7 Determination of thermal stability of Acs.

The thermal stability of prepared conjugate and free enzymes was assessed by determining the activity under standard conditions after subjecting to various temperature ranges from 32-60 °C for 10 min followed by cooling on ice.231

74 3. Results and discussions.

3.2.4.2.8 Determination of storage stability of Acs.

The storage stability was monitored by measuring the activity of enzyme stored at 4 oC in 0.1 M borate buffer. For control experiments, assay for free enzyme was run in parallel.

3.2.4.2.9 Determination of operational stability of Acs.

Reusability of immobilized enzyme was also evaluated by consecutive operation cycles of enzymatic reaction for 15 min, conjugate were obtained as mentioned in immobilized enzyme preparation and further washing with 0.1M Borate buffer. The procedure was repeated using fresh aliquot of substrate.

75 3.2 PNIPAm-AEMA core-shell microgel support for Acs

76 3. Results and discussions.

3.3 Adsorptions of Acetyl CoA synthetase on PNIPAm- PEI 1 microgels

3.3.1 Introduction

In this Chapter, we report Acs immobilization on thermo responsive PNIPAm-PEI 1 core shell microgels via ionic adsorption and explore the potential of bioconjugate for acetyl CoA synthesis in membrane reactor platform (figure 3.3.1). Detail quantitative study on enzyme activity and structural characteristic of enzyme after immobilization is presented. Further an ultrathin membrane is fabricated to assess the acetyl CoA formation and operational stability in flow through condition.

(a) pH 8 in 0.1 M Tris buffer +

(b)

I. Filtration of II. Filtration of Microgel Cd(OH)2 nanostrand PET membrane III. Microgel crosslinking and removal of nanostrand

IV. Filtration of bioconjugate

Figure 3.3.1: Schematic representation of preparation of (a) Acs-PNIPAm PEI 1 conjugate, and (b) microgel-PET (polyethylene terephthalate) membrane for bioconversion study.

77 3.3 Adsorptions of Acetyl CoA synthetase on PNIPAm-PEI 1 microgels

3.3.2 Results and discussion

3.3.2.1 Adsorption of acetyl CoA synthetase on PNIPAm-PEI 1 microgel Preliminary experiments were performed to determine optimal conditions like pH, temperature and microgel concentration for enzyme coupling. The adsorptions studies were then performed at standard conditions of temperature (<10 °C), and pH (8.0) using microgel solution (10 mg/ml). The adsorption isotherm studies were carried out by investigating the Acs binding to microgel as a function of enzyme concentrations. Figure 3.3.2 (a) suggest the adsorption behavior of Acs on microgel follow Langmuir-type model. The Kd (dissociation constant) and Гmax (maximum binding capacity) was calculated to be 0.019 mL/µg and 286 µg/mg of microgel respectively. The maximum amount of enzyme adsorbed at the standardized conditions on the microgels was 278 μg/mg, this value was found to be close to the maximum binding capacity of microgels. These results indicate that high load of enzymes on the microgel particles were achieved. The linear graph (figure 3.3.2 b) of the enzyme adsorption showed excellent fit with relatively high R2 values (0.99) further confirms that the model predicts well with the adsorption behavior.

300 6 (a) (b) 5

250 4

3 г / C 200 2 y = 0.0035x + 0.1856 1 R² = 0.99929 (µg enzyme / mgmicrogel) mgmicrogel) / enzyme (µg г 150 0 0 300 600 900 1200 1500 0 500 1000 1500 2000 C (µg/ml) C (µg/ml)

Figure 3.3.2: Adsorption isotherm study of Acs on PNIPAm-PEI 1 microgels. (a) the plot of adsorbed amount of Acs on microgel vs Acs in solution. (b) Linearized Langmuir plot. 78 3. Results and discussions.

The respective concentration of enzyme at which maximum enzyme load was obtained was used to prepare the bio-conjugate and was subsequently subjected for further studies. PNIPAm-PEI microgel can be viewed as a support covered with flexible ionic polymer (PEI) bed.242 PEI shell provides multiple sites for the attachment of enzymes that interact with various charged sites on the enzyme surface and hence stabilizes enzyme and its subunits from denaturation and dissociation respectively.109,183Since the adsorption was carried out at pH 8, which is close to the isoelectric point of Acs (pH 7.5), and the overall charge on the microgel is positive due to protonated amino group of PEI shell.122,149 The adsorption of Acs on microgel occurs due to ionic exchange between these anionic and cationic groups of respective molecules.183 This assumption was confirmed by zeta potential results, high positive zeta potential value of microgel (ζ = 8.87 mV) was significantly reduced after immobilization of Acs (ζ = 0.0841 mV). Furthermore, the temperature for adsorption was below the cloud point or LCST of PNIPAm, this finally states that the strong binding of enzyme on microgel particles occurs in swollen state of the microgel.61,109

3.3.2.1.1 Circular dichroism study.

The conformational stability of the enzymes after immobilization was studied using CD spectroscopy. The experiment with enzyme loaded on microgel was not successful due to high interference from microgel particles.243 Therefore the enzyme that were desorbed from microgel in 0.01 M potassium phosphate solutions (pH 4) at 37 °C were subjected for this study. Figure 3.3.3 shows CD spectra of free enzymes and enzymes desorbed from PNIPAm-PEI 1 under same conditions.

79 3.3 Adsorptions of Acetyl CoA synthetase on PNIPAm-PEI 1 microgels

15 Desorbed Acs 10 Acs

CD/mdeg -5

-10

-15 180 200 220 240 Wavelength (nm)

Figure 3.3.3: CD spectra of free enzymes and enzymes desorbed from PNIPAm-PEI 1 at pH 4 (protein concentration (0.05mg/mL)

The spectra obtained were then analyzed using Jasco Software Spectra Manager Version 2 (Jasco GMBH, Groß-Umstadt, Germany) and the respective secondary structure contributions are given in table 3.3.1. The free enzyme possessed 48.8 % of α- helical structure, which reduced to 35 % for immobilized Acs. This results shows that nearly 72 % of native structure of Acs was retained after immobilization.

Table 3.3.1. The contributions of secondary structure elements of free and immobilized Acs.

α-helical β-sheet Turn Unordered

(%) (%) (%) (%)

Free enzyme 48.8 6.4 20.8 24

Immobilized 35 12.3 19.5 34.6 enzyme

Further correlating these results with immobilization output of the bioconjugate, it was found that the conjugate possessed nearly 60% of the initial activity of enzyme added to prepare the bioconjugate, which almost corresponds to the amount of enzyme immobilized on the microgel. Therefore from these results it can be concluded that though there were structural changes (indicated by CD

80 3. Results and discussions. spectroscopy) in enzyme after immobilization, it had no adverse effect on the activity of enzyme.

3.3.2.1.2 Acs reaction kinetics.

The reaction kinetics of free and immobilized enzymes were studied at various concentrations of acetate (5 mM to 0.3 mM) as a substrate while keeping ATP and CoA concentration constant (1 mM and 0.5 mM) respectively. The dependence of the initial rate on the substrate concentration was measured and the experimental data were analyzed by Lineweaver-Burk plots. The two important kinetic parameters Michaelis constant Km and maximum velocity Vmax were calculated from linear regression curves and are represented in Table 3.3.2.

The Km values of immobilized enzyme were higher compared to the free enzyme, which indicates a decrease in substrate-enzyme binding affinity. The Km value for free enzyme obtained was close to the earlier report on Acs from yeast.244

Table 3.3.2. The kinetic parameters of immobilized and free enzymes for acetate substrate at 37 °C.

Vmax/Km Enzyme Km (mM) Vmax(U/mL) (min-1)

Immobilized enzyme 0.43 ± 0.01 0.044 ± 0.002 0.1 ± 0.008

Free enzyme 0.18 ± 0.02 0.016 ± 0.001 0.08 ± 0.008

The increase in Km after immobilization on charged particles is expected, as the carrier plays important role in availability of the substrate to the enzyme. By virtue of these effects high amount of substrate in required to attain the maximum rate of reaction and hence contributing to higher value of Km compared to that of free enzyme. On the other hand, positive effect was observed on rate of reaction (Vmax) which shows significantly increase, marked by ~2.5 times higher value compared to the free enzyme. This indicates that the enzyme-substrate complex is moreover directed towards product formation. From these values

Vmax/ Km for free and immobilized enzymes were calculated to determine the rate

81 3.3 Adsorptions of Acetyl CoA synthetase on PNIPAm-PEI 1 microgels at which enzymes capture substrate into productive complex.197 Here also the value for immobilized enzyme was relatively better than the free enzyme, which further states PEI-PNIPAm microgel as a superior carrier for Acs immobilization.

3.3.2.1.3 Effect of temperature and pH on Acs activity.

The temperature and pH have profound effect on the activity of enzymes, study on the effect of these parameters on the activity of enzymes points out to the changes in conformation of the enzyme on binding to the support.188 In the current work, the enzymes were subjected to temperature dependent study in the range of 25 °C to 65 °C and reaction was carried out for 15 min. It is clear from figure 3.3.4 that the activity of both the immobilized and free Acs exhibits temperature dependence. Free enzyme had maximum activity at 37 °C which was similar to the literature value and the activity decreased upon further increase in the temperature.149

2.5 Free enzyme 100" 2.0 Immobilized enzyme 80" 1.5 60"

1.0 40" Immobilized" enzyme" 0.5 Relative"activity"(%)"" 20" Free"enzyme" Specific activity (U/mL/mg) (U/mL/mg) activity Specific 0.0 0" 20 30 40 50 60 70 20" 30" 40" 50" 60" 70" o o Temperture C Temperature" C"

Figure 3.3.4: Effects of different temperature on the activities of free and immobilized Acs was studied in tris buffer pH 8. (Left figure: the specific activity, and Right figure: the normalized specific activities of the respective enzymes).

The results also shows that immobilized enzymes showed higher activity at temperatures below the LCST of PNIPAm (32 °C) and the activity gradually decreased with increase in the temperature. The loss in enzyme activity signifies decrease in the active enzyme concentration on the microgel. With the increase

82 3. Results and discussions. in temperature the microgel core structure collapses which lead to removal of enzymes and also entrapment of remaining enzymes within the polymer network in microgel blocking the substrate accessibility.125 Furthermore, the similar thermal profile at higher temperature of immobilized and free enzymes indicate that the native enzyme structure is maintained after adsorption to microgel. Activity of enzyme as a function of pH was studied from pH range 6 to 9 and the properties of immobilized enzyme were compared with those of free enzyme. It can be observed from figure 3.3.5 that free enzyme has optimal pH in between 8.0 to 8.5 that was close to the earlier report on the Acs extracted from yeast, while immobilized Acs showed higher activity at pH 9.

2.5" Immobilized"enzyme" 100" Free"enzyme" 2.0" 80" 1.5" 60"

1.0" 40"

0.5" 20" Relative"activity"(%)"" Immobilized"enzyme" SpeciDic"activity"(U/mL/mg)" Free"enzyme" 0.0" 0" 5.5" 7.5" 9.5" 5.5" 6.5" 7.5" 8.5" 9.5" pH" pH"

Figure 3.3.5: Effect of different pH on the activities of free and immobilized Acs was studied in tris buffer at 37 oC. (Left figure: the specific activity, and Right figure: the normalized specific activities of the respective enzymes).

The shift in optimum pH is attributed to charged support, which often leads to displacements in the pH activity profile of immobilized enzymes. As pH is govern by H+ and OH- ions in the solution, the PEI shell of microgel leads to unequal partitioning of these ions between the microenvironment of the immobilized enzyme and the bulk phase.175,234 Due to the relatively better activity profile of Acs adsorbed on PNIPAm-PEI 1 microgel compared to the covalent binding on

83 3.3 Adsorptions of Acetyl CoA synthetase on PNIPAm-PEI 1 microgels

PNIPAm-AEMA, Acs-PNIPAm-PEI 1 bio-conjugates were used for membrane based reactor studies.

3.3.2.2 Fabrication, bioconversion, and operational stability of membrane bioreactor The enzyme membrane reactor serves as a good platform for biocatalysis and bio-separation. Membrane directs the mass transport across itself thus keeping the enzymes inside the reactor and also achieving some level of product separation. Moreover the continuous removal of product will maintain the equilibrium of a reaction towards the product side and thereby improving the productivity of the whole process, which is an exceptional advantage of membrane reactor.58 Keeping the above facts in mind, enzymatic membrane reactor was constructed by covalently anchoring the immobilized enzyme- microgel bioconjugate on microgel-PET support. The membrane characterization using SEM and the flux data are described in Figure 3.3.6.

160 Microgel-enzyme layer (c)

120

80 Flux (L/m2/h) Flux (L/m2/h)

(a) 200 nm (b) 200 nm 40 Track etched support etched Track 0 1 2 3 4 5 Pressure (bar)

Figure 3.3.6: (a & b) cross-section SEM image of ultrathin bioconjugate-membrane on PET track etched support. (c) Flux versus pressure relation of bioconjugate-membrane PET membrane.

The cross-section SEM images of the membrane system in Figure 3.3.6 (a & b) indicate that the pores of the PET support were intact and a thin film of microgel- bioconjugate was successfully formed over this support. The thickness of this film was about 600 nm. Under high magnification, the microgel layer looks like interpenetrating porous network. After successful membrane fabrication, a 84 3. Results and discussions. membrane bioreactor was prepared using dead end filtration device and employed to study the biocatalytic synthesis of acetyl CoA. The permeability of prepared membrane was assessed by recording the PBS buffer flux at varying transmembrane pressure from 1 to 4 bar at room temperature. The flux versus pressure relationship is depicted in figure 3.3.6(c), which showed linear behavior with pressure. Since the membrane showed sufficient flux at moderate transmembrane pressure, operation of membrane reactor was checked at 2 bar under constant stirring condition. The membrane performance was studied in stirred condition and respective activity was compared to free enzyme (FE) and conjugate (CB) in batch mode. Using equation 3.3.3, the percent yield was calculated for FE, CB and CM. The ‘actual yield’ is the acetyl CoA formed (determined by correlating with coupled assay) to the ‘theoretical yield’ i.e. the expected acetyl CoA formed from 0.05 mM CoA, in presence of 0.05 mM ATP and 5 mM acetate as co-substrate.

Actual yield Yield (%) = ×100 Theoretical yield (3.3.3)

The plots for acetyl CoA formed with respect to time for FE and conjugate CB in batch mode and conjugate on the membrane is depicted in Figure 3.3.7. In the batch conditions, the acetyl CoA formation was similar for both FE and CB and the conversion reaction was linear till 40 min of reaction thereafter saturation limit was attained as depicted by a plateau. Whereas the membrane immobilized conjugate (CM) maintained the linear trend of product formation till the end of the reaction. The comparatively low amount of product formed in case of CM with respect to FE and CB can be attributed to the diffusion limitation and unavailability of enzyme towards the membrane surface.58,123,171

85 3.3 Adsorptions of Acetyl CoA synthetase on PNIPAm-PEI 1 microgels

30 Yield (%) Yield 20 Free enzyme 10 Conjugate (batch) Conjugate (membrane) 0 0 20 40 60 80 100 time (min)

Figure 3.3.7: Acetyl CoA formation is given as percent yield with respect to time by free Acs (¾) and conjugate (●) in batch condition and conjugate on the membrane (¿)

The initial rate of reaction was found to be 1.42 µM/min, 1.69 µM/min and 0.68 µM/min for FE, CB and CM respectively from nearly 2 U/mL activity of the enzymes. Our result can be positively comparable with the previously reported acetyl CoA formation using immobilized Acs, where Mannens et al. had reported complete 1µM acetate conversion in 1-2 min time using 6.12 U reactor column reactor.42 At the end of the reaction, the calculated percent yield was FE (61.5 ± 0.37 %), CB (64.76 ± 3.41 %) and CM (62 ± 0.1 %) respectively. Additionally the storage stability of membrane reactor was checked, for which the membrane was stored in refrigerator (~4 °C) and on 4th day of storage the enzyme possessed >50% of initial activity. To study the efficacy of immobilized enzyme as a reusable biocatalyst for acetyl CoA synthesis, the membrane was subjected for operation stability studies. The catalyst reusability was determined by measuring stability of enzyme on the membrane reactor as a function of number of reuses. Consecutive reaction cycles were operated with alternate washing steps. The residual activity after each cycle is depicted in figure 3.3.8. The membrane showed consistent performance and maintained >70% initial activity of bioconjugate till the last cycle.

86 3. Results and discussions.

120 0.09

80 0.06

Activity (U/mL) (U/mL) Activity 0.03 Relative activity (%) activity Relative

0.00 0 1 2 3 4 5 6 7 Number of cycles

Figure 3.3.8: Operational stability of Acs on PNIPAm-PEI microgel membrane. Relative activities are normalized activities of enzymes with respect to the highest activity of respective enzymes and experiment.

The better and consistent performance of enzyme reactor can be attributed to the stabilizing effect of PEI shell of microgel on enzyme and also the membrane, which maintain the total enzyme concentration on the surface.

3.3.3 Conclusion

In this study, PNIPAm-PEI microgel was investigated as a support for Acs immobilization for acetyl CoA synthesis. High adsorption of enzyme was obtained due to strong ionic interaction between the PEI shell and enzyme. The adsorption of enzyme to microgel improved the catalytic efficiency over free enzyme. Enzyme parameters and conformation studies demonstrated the maintenance of structural integrity of enzyme after immobilization. The bioconjugate was used to fabricate thin biocatalytic membrane and the conversion was monitored in flow through operation condition. The membrane reactor gave consistent performance maintaining more than 70% of initial activity after multiple usage and high rate bioconversion of acetate with respect to time. These results suggest that the prepared membrane serve as a good platform for acetyl CoA synthesis and can be explored for other precursor biomolecule production.

87 3.3 Adsorptions of Acetyl CoA synthetase on PNIPAm-PEI 1 microgels

3.3.4 Experimental section

3.3.4.1 Materials. S-acetyl-coenzyme A synthetase (Acs) (E C 6.2.1.1, >3units/mg, from baker's yeast), Malic acid dehydrogenase (Mdh) from porcine heart (≥600 units/mg protein), Citrate synthase (Cs) from porcine heart (≥100 units/mg protein), and all other chemicals were obtained from Sigma-Aldrich (Germany). BCA Protein Assay Kit (Thermo scientific (Pierce protein)), acetyl co-enzyme A (Roche, Germany), β-nicotinamide adenine dinucleotide, NADH disodium salt (Carl Roth, Germany), and 30 kDa MWCO centrifugal filter tubes (Millipore) were purchased from respective suppliers. The polyethylene terephthalate (PET) track etched micro porous membranes (diameter: 47 mm, pore size: 0.3-0.4 µm, pore density: 1.5×108 pores/cm2) were obtained from it4ip, Belgium.

3.3.4.2 Method

3.3.4.2.1 Adsorption experiment.

The immobilization of Acs on PNIPAm-PEI 1 microgel was achieved via adsorption in 0.1 M tris-Cl buffer at pH 8 for 6 h at 4 °C. The conjugate was obtained by centrifugation at 12,000 rpm for 15 min at 25 °C and subsequently washed with same buffer to remove non-adsorbed enzymes. Using BCA (for protein concentrations 20-2000 μg/mL) assay kits the amounts of adsorbed enzyme loaded to microgel were determined by blanking out the microgel absorbance from the enzyme-microgel bioconjugate. The enzyme and conjugate were stored in 0.1 M tris buffer (pH 8) containing 1 mM glutathione (an antioxidant) and 1 mM magnesium chloride (divalent cation)245 for further use.

3.3.4.2.2 Enzyme activity assay.

The activity of free and immobilized Acs was determined spectrophotometrically by following acetyl CoA synthesis at 340 nm wavelength by coupling with citrate synthase (Cs) and malate dehydrogenase (Mdh) assay in 96 well format. The assay medium composition for enzyme activity consisted of 20 mM potassium acetate, 4 mM MgCl2, 4 mM glutathione, 1 mM ATP, 500 µM coenzyme A, 1 mM NAD+ 4 mM malate, 16.6 U/mL Mdh and 5 U/mL Cs in 100 mM

88 3. Results and discussions. tris-Cl buffer at pH 8. The interference from microgel solution was blanked out for all the measurements. The acetyl CoA synthesized was correlated to

-1 -1 concentration of NADH formed in coupled assay using ε340 = 0.6220 M cm . One unit of enzyme activity was defined as the amount of enzyme which is required to form 1 µmol of NADH per min at pH 8 and 37 °C.246 The temperature dependent enzyme activities of immobilized and free enzymes were studied from 25 to 65 °C in 100 mM tris-Cl buffer at pH 8. The effect of pH on enzyme activities were studied for the pH range 6 to 9 at 37 °C in the respective buffers 0.1 M phosphate buffers (pH 6, 6.5, 7 and 7.5) and 0.1 M tris-Cl buffer (pH 8, 8.5 and 9). Kinetic parameters of enzymes were determined by varying acetate concentration (0.3 mM to 5 mM) while keeping concentrations of ATP and CoA constant at 1 mM and 0.5 mM, respectively. The values for Km and Vmax, were estimated with the help of Lineweaver-Burk plot. The activity of enzymes was studied in triplicate, and the standard deviation was used as the error.

3.3.4.2.3 Enzyme immobilized membrane fabrication.

The enzyme immobilized ultrathin membrane surface was prepared on PET track etched membrane support using enzyme-immobilized microgels.247 The permeation behavior of the prepared microgel-based membrane was evaluated by measuring the pure water flux under pressure range from 1 to 4 bar. The catalytic activity of the membrane was evaluated by determining the acetyl CoA formation at different time point for enzyme immobilized on membrane and free enzyme at 25 °C. For this purpose the reactants were supplied to dead end filtration device and permeate was collected at definite time interval. The reaction buffer composition consisted (total volume 4 mL) of 5 mM potassium acetate, 4 mM MgCl2, 4 mM glutathione, 50 µM ATP, 50 µM co-enzyme A, in 100mM tris-Cl buffer at pH 8 and further acetyl CoA formed was assayed with 0.5 mM NAD+ 2.5 mM malate, 16.6 U/mL Mdh and 5 U/mL Cs in tris buffer pH 8. Furthermore the operational stability or the reusability of the membrane was checked for consecutive reaction cycles with 1 mL reaction buffer under similar assay condition. After each cycle the membrane was washed 2- 3 times with total 5 mL of 0.1 M tris-Cl buffer containing 10% glycerol, 1mM glutathione and 1mM

MgCl2 to remove any entrapped reactant or product.

89 3.3 Adsorptions of Acetyl CoA synthetase on PNIPAm-PEI 1 microgels

90 3. Results and discussions.

3.4 Sequential enzymes reactions on thermo-responsive microgels

3.4.1 Introduction

Immobilization has been thought as a good tool for metabolon (metabolic enzyme clusters) formation.4 In cells the proteins are fixed in an environment where diffusion is a limiting factor for many processes, thus bio-conjugation of the enzyme in a way can imitate such conditions. In this section, we study the sequential enzyme reactions (figure 3.4.1) on PNIPAm-PEI 2 microgels carried by two enzymes pyruvate kinase (Pk) and L-lactic dehydrogenase (Ldh) as model systems. These coupled enzymes are commonly used as an assay and cofactors (ATP, NAD+) regeneration system.32,154 These enzymes have been utilized in fabrication of biosensor for detection of lactate, pyruvate etc. that find wide range of application in the fields of medicine, critical care, food science, and biotechnology.155,156

Pyruvate)kinase) PEP## +# ADP# Pyruvate# +# ATP# (1)#

Lactate)dehydrogenase) +# Pyruvate# +# NADH# Lactate# +# NAD (2)#

Figure 3.4.1: The sequential enzymatic reactions catalyzed by pyruvate kinase and lactate dehydrogenase, where phosphoenol pyruvate (PEP) is converted finally to lactate with generation of ATP in the first reaction and oxidation of NADH in the later step of reaction.

The PNIPAm-PEI 2 microgels were activated with glutaraldehyde (GA) and enzymes were immobilized under optimized condition to obtain single and dual enzyme/s-microgel bioconjugates as shown in the figure 3.4.2. The effects of sequential and simultaneous enzyme additions on preparation of dual enzyme bioconjugates were studied. The single enzyme and dual enzyme systems were characterized for their activity in ratio-metric approach. The optimized conjugates were subjected for various temperature, pH dependent reaction 91 3.4 Sequential enzymes reactions on thermo-responsive microgels parameters and stability studies. Finally the conjugate were checked for their application in ADP assay and ATP generation.

Figure 3.4.2: Schematic representation of preparation of Pk and Ldh bioconjugates; (i) single enzyme-microgel conjugates (Pi and Li) and dual enzymes systems PiLi (enzymes on different microgel particles), (ii) PL (simultaneous addition) and (iii) P3Lo and L3Po (sequential addition) for enzymes immobilized on same microgel particles.

3.4.2 Results and discussion

3.4.2.1 Optimization of bio-conjugation on PNIPAm-PEI 2 microgel

3.4.2.1.1 Dual enzyme-microgel bioconjugate (enzymes on the same

microgel particles PL, P3Lo and L3Po) The immobilization conditions were initially standardized with respect to pH, glutaraldehyde and enzyme concentration for preparation of single enzyme conjugates. At standardized conditions (pH 7.4, 0.5 % glutaraldehyde and 0.4 mg/mL enzyme concentration) the single and dual enzyme conjugates were prepared. For dual enzyme conjugates co-immobilized on same particles, different initial ratios of Pk and Ldh were added to the microgel solution to

92 3. Results and discussions. determine the binding behaviour of the two enzymes on the microgel. Two coupling approaches were investigated based on the addition of enzymes, simultaneous (microgel, Pk and Ldh added at one time) and sequential (where both enzymes were added at different intervals). The optimum conjugates were screened based on the highest sequential reaction (Pk activity). Figure 3.4.3 (a) shows the Pk (sequential activity) and Ldh activities for respective co- immobilized conjugates. It is worth noticeable that the Ldh activity in all cases is 10 folds higher than the sequential (Pk) activity, which is an essential criterion for effective assaying of the Pk reaction. Also the actual Pk activity was reconfirmed by addition of ten fold excess of free Ldh, the corresponding activity was close to the obtained by co-immobilized Ldh. Therefore, such particles can have promising outcome for applications related to Pk where the product ADP and PEP can be monitored instantaneously.47,155 The dual enzyme bioconjugate obtained by simultaneous addition of 1:1 (v/v) Pk: Ldh ratio showed highest Pk activity (0.8 U/mL), thus this bioconjugate was subjected for further studies and was denoted as PL.

8 Pk (Sequential reaction) (b) Pk (Sequential reaction) Sequential reaction (c) 8(a) (a) Ldh Ldh 1.0 6 6 0.8

4 4 0.5

2 2 Enzyme activity (U/ml) (U/ml) activity Enzyme Enzyme activity(U/mL) activity(U/mL) Enzyme

Enzyme activity (U/mL) (U/mL) activity Enzyme 0.3

0 0 0.0 1:4 2:3 1:1 3:2 4:1 P3Lo L3Po PL 1:1 1:2 1:4 2:1 4:1 Pk:Ldh ratio Dual conjugates Ratio of Pi:Li

Figure 3.4.3: Screening of dual enzymes system: enzyme activities of Pk and Ldh in dual enzyme microgel particles prepared by different initial enzyme ratios (a) added simultaneously and (b) sequentially and (c) Pk (sequential reaction) activities of respective ratios of single enzyme conjugates (Pk and Ldh) added during the assay.

For the preparation of sequentially added bioconjugate, the initial amounts of Pk and Ldh were similar to the enzyme concentration used for the preparation of PL

93 3.4 Sequential enzymes reactions on thermo-responsive microgels bioconjugate. The conjugates were named depending on the sequence of enzymes added, for example, P3Lo was prepared by initially allowing Pk to immobilize on microgel for 3 hours and thereafter Ldh was immobilized on the complex for overnight. The activity results for both the conjugates were compared to the activity of PL, figure 3.4.3 (b). The P3Lo possessed similar Ldh activity as in the case of PL while the Pk activity was reduced to half. Beside the layer of Pk bound on microgels first, the maintenance of similar ldh activity indicates that the binding sites for Ldh was still intact to achieve its maximum immobilization. On the other hand for L3Po, a significant decrease in both Ldh and Pk activity was observed. In both the sequential bioconjugates (P3Lo and

L3Po) the decreased in activities of enzymes added first could be because of the low substrate availability due to coverage of the second enzyme.248 Overall the conjugate prepared sequentially possessed lower activities compared to the PL. The comparative better activity of PL could be due to the effects of proper orientation and better substrate channelling by co-localization of the enzymes.249,250 Therefore due to better outcome for PL, only this conjugate was subjected for next level studies.

3.4.2.1.2 Dual enzymes system with single-enzyme bioconjugates (enzyme on separate microgel particles PiLi) The single enzyme bioconjugates (Li and Pi) were added in different proportions and the optimum ratio was decided based on the highest sequential activity. From figure 3.4.3(c) it can be observed that the equal ratios of both the conjugates give sufficient activity of Pk, which slightly increased with the increase in Li amount. The maximum Pk activity was obtained by using Pi:Li combination in 1:4 proportion and hence this ratio (denoted as PiLi) was used for further evaluation. It is important to mention that from figures 3.1.4 (a and c) the corresponding activities of dual-enzymes bioconjugates PL (0.8 U/mL) and PiLi (0.74 U/mL) was found to be close to each other. But comparative specific activity (i.e. unit activity per mg of enzymes) of PL was 1.6 times higher than PiLi. The possible reason for higher activity in PL can be due to the co-immobilization of both the enzyme on same particle, this causes sufficiently higher local concentration of the substrate (the product of the first enzyme) to the second 94 3. Results and discussions. enzyme for the reaction.171 These results prove that the enzymes immobilized on the same particles provide better accessibility to the substrates and reduce diffusion barrier of substrate to the enzymes.

3.4.2.1.3 Immobilization of enzymes on microgel At the optimized immobilization conditions, single (Li and Pi) and dual PL enzyme-microgel-conjugates were prepared and immobilization results obtained are summarized in Table 3.4.1. The corresponding immobilization yield values obtained for Li, Pi and PL bioconjugates were 28 % of Pk, 20 % of Ldh and 13 % (Pk) and 14 % (Ldh) for PL of the initial enzyme activities respectively. Although the immobilization yield was low but it was nearly similar for all the conjugates. These activity results were further correlated to determine the approximate amount of enzyme immobilized on the microgel using standard plot (Figure A2 in appendix) of enzyme concentration to their activity and results are given as enzyme load.251

Table 3.4.1: Immobilization yield (from equation 3.4.1) and enzyme load for single and dual enzyme microgel bioconjugate.

Immobilization yield Enzyme load* Conjugates (µg/mg)

Pi 0.28 ± 0.04 88

Li 0.20 ± 0.09 16

0.13 ± 0.03 41 (Pk) PL 0.14 ± 0.03 12 (Ldh) Note: *enzyme load is µg of enzyme/mg of microgel and was calculated from activity standard plot of the enzymes.

95 3.4 Sequential enzymes reactions on thermo-responsive microgels

3.4.2.2 Enzyme reaction parameter study

3.4.2.2.1 Enzyme kinetic studies:

The kinetic parameters Michaelis constant (Km), and maximum velocity (Vmax) for free enzyme and conjugates were calculated using Lineweaver-Burk plot and data are given in the Table 3.4.2. For the Pk kinetics, the concentrations of substrate adenosin diphosphate (ADP) (0.05-0.6 mM) were varied keeping PEP concentration (1 mM) constant. Table 3.4.2 (A) represent the kinetic parameters for the free enzyme (Pk) and the conjugates (Pi, PL, and PiLi).

Table 3.4.2: Kinetic parameters of free enzyme and conjugates: (A) kinetic parameters for ADP at constant PEP concentration for free and immobilized Pk. (B) kinetic parameters for pyruvate at constant NADH concentration for free and immobilized Ldh.

Km Vmax Vmax/ Km (A) (mM) (mmol/g/min) (g-1 L min-1)

Pk 0.263 ± 0.023 45 ± 2 169

Pi 0.478 ± 0.058 63 ± 8 132

PL 0.448 ± 0.091 73 ± 9 162

PiLi 0.427 ± 0.031 29 ± 3 69

Km Vmax Vmax/ Km (B) (mM) (mmol/g/min) (g-1 L min-1)

Ldh 0.331 ± 0.021 220 ± 7 665

Li 0.38 ± 0.091 565 ± 77 1486

PL 0.26 ± 0.029 270 ± 18 1037

96 3. Results and discussions.

The apparent Km(adp) for all the bioconjugates was found to be two times higher than the soluble enzyme. The increase in apparent Km is an expression of loss of affinity of the enzyme for its substrate because of the structural changes leading to restricted enzyme flexibility and also lower accessibility of substrate to the active site.252 Hence more ADP is required to achieve the maximum activity. The

Vmax results showed increased activity for Pi and PL but a 34 % decrease for PiLi compared to the free enzymes. Further the Vmax/ Km for conjugates with respect to the free Pk showed slight decrease in the case Pi and PL, while more than two- folds reduction for PiLi. These activity results of Pi and PL suggests that most of the enzyme-substrate complex formed during the reaction was directed towards product formation. Additionally the better channelling of substrate to second enzymes could also be the reason in case of PL.171 While the decrease in rate of reaction for PiLi may be due to the effects of microgel causing mass transfer

171,242 constraints, steric hindrance and microenvironment effects. On the other hand for Ldh all the kinetic parameters showed favourable effects on the enzymes after the immobilization. The apparent Km(pyruvate) for native Ldh and Li were similar and was even lower for PL. These results suggested improved affinity of Ldh towards the substrate after immobilization. The maximum velocity and catalytic efficiency was also significantly improved for conjugates Li and PL. Similar improved kinetic parameters results were previously reported for Ldh immobilized on magnetic particles and glass beads.253–255 Thus, the positive results can be attributed to proper orientation of enzymes and stabilization of subunits via glutaraldehyde coupling. 171,181

3.4.2.2.2 Fluorescent emission spectra study Tryptophan (Trp) fluorescence study provides information about the structures of protein immobilized to optically transparent substrates.60 The alteration in maximum wavelength of emission and intensity is associated with changes in Trp environment, hinting to altered enzyme conformation.235 From figure 3.4.4 (a and c) the maximum fluorescence emission spectra for Ldh and Pk was found to be 334 and 315 nm respectively. The λmax remained same in case of conjugates Li and Pi. The maximum intensity for dual enzyme systems PL and PiLi were near to

97 3.4 Sequential enzymes reactions on thermo-responsive microgels

334 nm, which indicated dominance of Ldh (trp) structures in the dual enzyme bioconjugates.

12000 6000 12000 25 (a) (c) (e) 28 30 9000 4500 9000 32 35 6000 3000 6000 37 40 3000 1500 3000 42

(au) (au) 0 0 0 295 315 335 355 375 395 295 315 335 355 375 395 295 315 335 355 375 395

Intensity 9000 7500 12000 (b) (d) (f) 6000 9000 6000 4500 6000 3000 3000 3000 1500

0 0 0 295 315 335 355 375 395 295 315 335 355 375 395 295 315 335 355 375 395 Wavelength (nm)

Figure 3.4.4: Temperature dependent fluorescent spectra of (a) free Ldh; (b) immobilized Ldh; (c) free Pk, (d) immobilized Pk, (e) PL and (f) PiLi

Further the temperature dependent intensity profile was studied from temperature range (25 °C to 42 °C) to determine conformational stability in enzyme after immobilization. The temperature dependent fluorescence spectra of free enzyme (Ldh and Pk, figure 3.4.4 a and c) indicate a linear decrease in the intensity with increasing temperature. The decrease in the fluorescent intensity with the increase in temperature, was higher for free enzymes (Ldh and Pk) compared to the bioconjugates (Li, Pi, PL and PiLi; figure 3.4.4 b, d, e and f). The comparatively low reduction in the intensity of immobilized enzyme shows improved thermal stability of enzyme after immobilization on microgel because of the structural rigidity.175

3.4.2.2.3 Effect of Temperature and pH on Ldh and Pk activity. The diversion from optimum pH and temperature for enzyme activity provide insight into the structural changes in the enzyme. The pH dependent activities of

98 3. Results and discussions. the enzymes were studied from pH range 6 to 9 for free enzymes (Pk and Ldh) and the conjugates (Li, Pi, PL and PiLi).

1.0 100 60 (a) (b) Free pk (c) PL ) Immobilized pk (Pi) 50 PiLi

ug 80 / 0.8 /mg) mL /mg)

mL 40 mL (U/ 60 (U/

0.5 (U/ 30 activity 40 activity

activity

20 0.3 Specific 20 Free Ldh Specific 10 Specific Immobilized Ldh (Li) 0.0 0 0 5.5 6.5 7.5 8.5 9.5 5.5 6.5 7.5 8.5 9.5 5.5 6.5 7.5 8.5 9.5 pH pH pH

Figure 3.4.5: Effect of pH on the activities of (a) free and immobilized Ldh; (b) free and immobilized Pk, (a) PL and PiLi at 37 °C in buffers with different pH.

From figure 3.4.5. (a and b) it can be seen that the free enzymes Ldh and Pk had optimum pH at 8 and 7.5 respectively which is close to previous report on same enzymes.256 After immobilization, the optimum pH for all the conjugates were shifted towards lower pH (figure 3.4.5. (a,b and c). These shifts can be attributed to the presence of charged carrier which creats a new micro-environment around the enzymes affecting the assesibility to the substrate. The new microenvironment leads to Donnan partitioning effect and hence causing the shift in the optimum pH.175 Likewise, the temperature dependence on the activity of free and immobilized enzymes were probed in the temperature range of 30 °C to 42 °C. The results are presented in figure 3.4.6, which shows that the immobilized enzymes were active at broad range of the temperatures beyond the optimum temperatures (25-37 °C) of soluble enzyme. The activity of Ldh was stable till 40 °C after which it significantly declined, whereas in case of the Li the activity trend was increasing with the increase in the temperature. In case of Pk the activity profile was similar for both the free and immobilized Pk. The corresponding activities of PL and PiLi also followed the increasing temperature trend. The covalent immobilization using glutaraldehyde reduces enzyme susceptiblility to thermal changes by rigidification of enzyme structure and preventing subunits disscociation.171,181

99 3.4 Sequential enzymes reactions on thermo-responsive microgels

1 70 60 (a) (b) (c) 60 0.75 50 40 40 0.5 30 20 20 0.25 Specific activity (U/mL/ug) (U/mL/ug) activity Specific Specific activity (U/mL/mg) (U/mL/mg) activity Specific

Free Ldh (U/mL/mg) activity Specific 10 Free Pk PL Immobilized Ldh (Li) Immobilized Pk (Pi) PiLi 0 0 00 28 30 32 34 36 38 40 42 44 28 30 32 34 36 38 40 42 44 22 26 30 34 38 42 Temperature (oC) Temperature (oC) Temperature (oC)

Figure 3.4.6: Effect of temperature on the activities of (a) free and immobilized Ldh; (b) free and immobilized Pk, (c) PL and PiLi at different temperature in phosphate buffer pH 7.4.

3.4.2.2.4 Storage and reusability studies In solution the native enzyme tends to acquire thermodynamically stable conformation which leads to distortion of structure and inactivation of enzyme. Figure 3.4.7 (a and b) shows the data of storage stability for Pk and Ldh immobilized enzymes monitored for 2 weeks. For both the enzymes systems the stability profile was improved with respect to the soluble enzymes.

Figure 3.4.7: Storage stability of free and immobilized enzymes at 6 oC in PBS buffer pH 7.4 (a) Pk activity, (b) Ldh activity.

In the case of Pk activity, the conjugates (Pi and PL) showed similar stability trend as in case of soluble enzymes till the end of day 14. While the activity in

100 3. Results and discussions. case of Ldh, the activity of immobilized enzymes (Ldh and PL) was 68 % of initial activity till the 14th day of storage while the free enzyme lost the activity significantly.

Figure 3.4.8: Reusability of Pi and PL for Pk activity and Li and PL for Ldh activity (100% activity for were 1.1 (Pi), 0.7 (PL_Pk) and 5.5 U/mL (Li and PL_Ldh) respectively).

The operational stability of bio-conjugates were checked with respect to the number reuse of the particles with subsequent washing step (figure 3.4.8). 70 and 55 % Pk activity was maintained for the Pi and PL respectively after second cycle of the reaction, whereas the Ldh activity was consistently maintained after 5th cycle for Li and PL.

3.4.2.3 Application of the dual enzyme bioconjugates The Pk-Ldh coupled enzymes are commonly used to assay ADP forming enzyme (ATPase) reaction.32,47 The suitability of this enzyme system also needs higher activity of Ldh to avoid limiting Pk activity. During the preparation of the dual enzyme conjugates (enzymes on same microgel particles), the activity of Ldh was always found to be higher compared to Pk activity, therefore the dual enzymes system PL and PiLi were subjected for ADP detection. The reaction of acetate kinase (Ak) was run and the conjugates were added to determine the initial velocity of Ak. Ak catalyses acetyl phosphate and ADP formation by consuming

101 3.4 Sequential enzymes reactions on thermo-responsive microgels acetate and ATP. The ADP was probed using dual enzymes conjugates PL and PiLi and free enzymes (Pk) as standard. The figure 3.4.9 (a) shows the initial velocity of Ak, the reaction proceeded linearly with the time as reported by Pk.

10 50 Pk (a) PL (b) PL PiLi 40 P_L

/L) /L) 8 PiLi

p_L /L) µmol µmol

( 30 5 y = 0.4624x R² = 0.99597 20

concentration 3 y = 0.4202x R² = 0.95882 10 ATP concentration ( concentration ATP ADP ADP y = 0.3044x R² = 0.95565 0 0 0 5 10 15 20 25 0 1 2 3 4 Time (min) Enzyme units (U/mL)

Figure 3.4.9: Application of Dual enzyme bioconjugates for (a) determining initial velocity of Ak activity. (b) Bioconjugate concentration dependent ATP formation

The initial velocity of Ak determined by Pk, PL and PiLi were 0.46, 0.42 and 0.3 µmol/L/min respectively. This indicated that dual enzymes bioconjugate PL performed equally well as free enzyme compared to the single enzyme bioconjugates (PiLi) dual system. Secondly the efficiency of conjugates for ATP formation for further application in co-factor regeneration was studied by varying the amount of bioconjugates in the reaction figure 3.4.9 (b). The amount of NADH oxidized was correlated to ATP produced, for both the dual enzyme systems the linear trend of ATP formation was observed with the increased bioconjugate concentration (PL and PiLi). But the higher amount of ATP formed in case of PL shows efficacy of this dual enzyme system for further co-factor synthesis.

3.4.3 Conclusion

Sequential reaction of Pk and Ldh enzymes were studied on PNIPAm-PEI microgels. The glutaraldehyde activated microgel showed selective binding of Ldh over Pk. The single and dual enzyme bioconjugates were studied, and the dual enzyme particles were optimized with respect to the ratios and approach of 102 3. Results and discussions. binding of the enzymes. The bioconjugate (PL) prepared by simultaneous addition of Pk and Ldh in 1:1 ratio was selected to study dual enzyme microgel system. In this respect, different combinations of single enzyme bioconjugates (Pi and Li) were also optimized with respect to their ratios. The activity results showed positive effect on all the bioconjugates except for PiLi. The reaction parameters studies showed shift in the optimum pH and temperature for all the enzymes after immobilization. Further more all the bioconjugates showed improved storage and operational stabilities over time compared to the free enzymes. Finally the dual enzyme bioconjugates were efficiently used for application in co-factor dependent processes where PL showed better functioning compared to PiLi.

3.4.4 Experimental section

3.4.4.1 Materials L-lactic dehydrogenase (Ldh) from rabbit muscle Type XI, (600-1,200 units/mg protein). pyruvate kinase (Pk) from rabbit muscle Type III, (350-600 units/mg protein), acetate kinase from Escherichia coli (≥150 units/mg protein), adenosine di-phosphate (ADP), phosphoenol pyruvate (PEP) and ADP standard assay kit were procured from Sigma. BCA Protein Assay Kit (Thermo scientific (Pierce protein)), β-Nicotinamide adenine dinucleotide, disodium salt (NADH) was obtained from Carl Roth.

3.4.4.2 Methods

3.4.4.2.1 Enzyme activity assay The activity of the Ldh was determined spectrophotometrically at 340 nm (NADH,ε= 6.23 × 103 M-1 cm-1), the assay medium composition consisted of 1mM pyruvate, 0.4 mM NADH in 100mM PBS buffer pH 7.4. While the activity of Pk was measured by coupling to Ldh assay, using assay medium containing 1 mM ADP, 1 mM PEP, 0.4 mM NADH in 100mM PBS buffer pH 7.4. One unit of enzyme activity was defined as amount of enzyme, which convert 1 µmol of NADH per min at pH 7.4 and 37 oC. The activity of enzyme was studied in triplicate, and the standard deviation was used as the error.

103 3.4 Sequential enzymes reactions on thermo-responsive microgels

3.4.4.2.2 Enzyme immobilization Ldh and Pk were covalently immobilized to PNIPAm-PEI core shell microgel using glutaraldehyde coupling in PBS buffer (0.1 M, pH 7.4). The enzymes were initially desalted by filtration using 30 kDa centrifuge filter tubes (Millipore) and reconstituted in PBS buffer (0.1 M, pH 7.4). The immobilization conditions were standardized before conjugation with respect to pH, enzyme concentration, and glutaraldehyde concentration. For every 1 ml of conjugate mixture, 500 µl microgel solution (20 mg/ml), 50 µl of enzyme solution (~10 mg/ml), and 50 µl of glutaraldehyde (10%) were used. The microgel was first activated with glutaraldehyde solution for 10 min at room temperature, and subsequently washed with PBS buffer by centrifugation at 15,000 rpm for 10 min to remove excess of glutaraldehyde. The enzyme was added to activate microgel solution and was allowed to conjugate for 3 h at 6 oC. After the completion of incubation period, bioconjugate formed was obtained by centrifugation at 15,000 rpm for 10 min at 4 oC. Using BCA (for protein concentrations 20-2000 μg/mL) assay kit protein was estimated from bovine serum albumin (BSA) as standard. The enzyme and bioconjugate were stored in PBS buffer. The immobilization yield was calculated from equation 3.4.1.140

U Immobilization yield = immobilized (2) Uinitial 3.4.1

Where,U immobilized and U initial are the activities of immobilized enzyme and initially added free enzyme.

3.4.4.2.3 Effect of Temperature, pH, and substrate concentration The effect of various reaction parameters such as temperature, pH, and substrate concentration on immobilized and free enzymes reactivity was studied. Optimum pH condition for both immobilized and free enzymes was evaluated in the range of 6 to 9. The enzymes were assayed in 0.1 M phosphate buffer (pH 6, 6.5, 7, 7.5 and 8) and 0.1 M tris buffer (pH 8.5 and 9) at 37 oC for respective pH. The effect of temperature on enzymes reactivity was studied at different temperature ranging from 25 to 42 oC. The enzyme activity was normalized with

104 3. Results and discussions. respect to maximum activity for given experimental condition and the final data were represented as relative activity. The kinetic constants for Pk was studied for the substrate ADP by varying concentrations from 0.05 to 1 mM and keeping PEP concentration at 500 μM respectively; and for Ldh, pyruvate concentration (0.05 to 1 mM) was varied at constant NADH concentration (1mM). Assuming

Michaelis-Menten kinetics for both the enzymes, the values for Km and Vmax, were estimated with the help of Lineweaver-Burk plot.

3.4.4.2.4 Fluorescence study The structural changes in the enzymes after immobilization were characterized by the tryptophan fluorescence measurements using the Microtitre plate reader (Infinit M200 Pro Tecan, Austria) compared to the free Ldh and Pk. The excitation wavelength was set at 270 nm and the emission spectrum was recorded from 300 to 390 nm. Further temperature dependent enzymes conformation changes were recorded in the temperature range (25-42 oC).

3.4.4.2.5 Storage, and operational stability study. The storage stability was monitored by measuring the activity of enzyme stored at 4 oC in 0.1 M phosphate buffer saline. For control experiments, assay for free enzymes were run in parallel. Reusability of the immobilized enzymes Pi, Li and PL (for Pk and Ldh activity separately) consecutive operation cycles of enzymatic reaction for 1 min, conjugates were obtained by centrifugation and reconstituted in 0.1 M PBS buffer. The procedure was then repeated using fresh aliquot of substrate.

3.4.4.2.6 Application of the dual conjugates PL and PiLi. The Ak initial velocity was probed by discontinuous coupled assay using dual Pk- Ldh enzyme conjugates (PL and PiLi). The reaction buffer consists of a 1 mM acetate, 2 mM glutathione, 2 mM MgCl2, 1 mM KCl, 0.4 mM ATP, 1 mM PEP, 0.4 mM NADH, 10 µL Ak (in 1 mL reaction buffer) in 100mM PBS buffer pH 7.4. The Ak reaction was followed by addition of Pk, PL and PiLi respectively, in 100 µL assay medium. The ATP formation experiment was carried with different amounts of PL and PiLi in assay buffer containing 1mM MgCl2, 1mM KCl, 0.1 mM ADP, 1 mM PEP, 0.4 mM NADH, in 100mM PBS buffer (pH 7.4). The ATP

105 3.4 Sequential enzymes reactions on thermo-responsive microgels formation was stoichiometrically correlated to the amount of NADH oxidized at 340 nm using standard plot.

106 3. Results and discussions.

107

4 Summary

Polyketides are natural products with complex chemical structures and immense pharmaceutical potential that are synthesized via secondary metabolic pathways. The in-vitro synthesis of these molecules requires high supply of building blocks such as acetyl Co-enzyme A, Malonyl Co-enzyme A etc. Also many of the intermediate steps for polyketide synthesis are highly cofactor (adenosine triphosphate (ATP), nicotinamide dinucleotide (NADH)) dependent. Generally acetyl CoA and ATP-dependent enzymatic reaction are accomplished by either their direct addition in stoichiometric amounts or by the inclusion of an enzymatic cofactor regenerating system. Immobilization of enzymes improves the process economics by enabling multiple uses of catalyst and improving overall productivity and robustness. The aim of the thesis thus was to study biochemical reactions on the smart microgels support using single (acetyl CoA synthetase (Acs)) and multi (dual) pyruvate kinase (Pk) and L-lactic dehydrogenase (Ldh) enzymes systems. The microgels were designed and synthesized to obtain core shell morphology with cationic properties. In this respect two methods were used to synthesize different microgels. The poly-(N-isopropylacrylamide-aminoethylmethacrylate) (PNIPAm-AEMA) microgels was synthesized using two-step free radical polymerization and poly (N-isopropylacrylamide-ethyleneimine) PNIPAm-PEI microgels were synthesized via one pot grafting-from co-polymerization of PEI. Both the methods yielded narrowly size distributed microgel particles as indicated by SEM images. The DLS measurement proved the temperature sensitive nature of PNIPAM-AEMA, PNIPAm-PEI 1 and PNIPAm-PEI 2 microgels having hydrodynamic size of 320, 320 and 736 nm respectively at 24 oC in solution. Zeta potential measurement confirmed that the microgel possessed cationic shell. The immobilization of the acetyl CoA synthetase (Acs) was carried out on both the microgels with different immobilization strategies (figure 4.1). In the first approach Acs was covalently bound to PNIPAm-AEMA microgels via 1-ethyl-3-

(3- NN- dimethylaminopropyl) carbodiimide (EDC) coupling. Michaelis-Menten kinetics of the immobilized Acs indicates identical Km (Michaelis constant) but decrease in the conversion rate compared to free enzyme. Immobilized Acs shows an improvement in activity at wide temperature and pH range and also demonstrated good thermal, storage, and operational stability. The Acs-microgel bio-conjugate has been successfully reused for 4 consecutive operation cycles with more than 50% initial activity.

Figure 4.1: Schematic representation of acetyl CoA synthetase (Acs) immobilized on microgel particle and membrane reactor for acetyl CoA synthesis.

Adsorption studies of Acs on PNIPAM-PEI 1 microgel indicated high binding of enzymes, with a maximum binding capacity of 286 µg/mg of microgel for Acs was achieved. The immobilized enzymes showed improved biocatalytic efficiency over free enzymes, beside this, the reaction parameters and circular dichroism (CD) spectroscopy studies indicated no significant changes in the enzyme structure after immobilization. This thoroughly characterized enzyme bioconjugate was further immobilized on an ultrathin membrane to assess the same reaction in flow through condition. Bioconjugate was covalently immobilized on a thin layer of pre-formed microgel support upon polyethylene terephthalate (PET) track etched membrane. The prepared membrane was used in a dead end filtration device to monitor the bioconversion efficiency and operational stability of cross-linked bioconjugate. The membrane reactor showed consistent operational stability and maintained >70% of initial activity after 7 consecutive operation cycles.

ii 4. Summary

Figure 4.2: Schematic representation of sequential reaction catalyzed by pyruvate kinase and lactate dehydrogenase on same and different microgel particles.

The sequential reactions catalyzed by pyruvate kinase (Pk) and L-lactic dehydrogenase (Ldh) was studied by covalent immobilization of two enzymes on the PNIPAm-PEI 2 microgels (figure 4.2). The amino groups on the microgel were activated with glutaraldehyde (GA) and enzymes were immobilized under optimized condition to obtain single and dual enzyme/s-microgel bioconjugates. For co-immobilized enzymes on same microgel particles, sequential and simultaneous additions of the Pk and Ldh in ratio-metric approach were undertaken. The bioconjugate prepared by simultaneous addition (PL) in 1:1 ratio of Pk and Ldh showed highest sequential activity. Similarly for single- enzyme bioconjugates (Pi and Li), 1:4 (PiLi) ratio of Pi to Li was found to be optimum combination. The kinetic studies indicated improved activity values for Pi, Li and PL but significant decrease in case of PiLi. The conjugates were subjected to reaction parameters studies, where shift in optimum temperature and pH was observed compared to the free enzymes. The stability studies indicated nearly similar profile for Pk bioconjugates but improved shelf life in case of Ldh bioconjugates with respect to the soluble enzymes. The temperature dependent fluorescence study also confirmed the improvement in thermal stability of the bio-conjugates. The co-factor dependent applications of dual enzymes conjugates proved superiority in performance of co-immobilized enzymes (PL) over enzymes bound to separate microgel particles (PiLi) nearly by factor of 2.

iii

The study indicated that the enzyme immobilization significantly depends on the enzyme, conjugation strategy and the support. The covalent immobilization provides rigidity to the enzyme structure as in case of Acs immobilized on PNIPAm-AEMA microgels but at the same time leads to loss in enzyme activity. Whereas for covalent immobilization of Ldh on microgel showed improved activity- On the other hand adsorption of the enzyme via ionic interaction provide better kinetic profile of enzymes hence the membrane reactor was prepared using PNIPAm-PEI conjugates for acetyl CoA synthesis. The better outcome of work with PNIPAm-PEI resulted in its further evaluation for dual enzyme system. This work is unique in the view that the immobilization strategies were well adapted to immobilize single and dual enzymes to achieve stable bioconjugates for their respective applications in precursor biosynthesis (Acetyl Co enzyme A) and co-factor dependent processes (ACoA and ATP). The positive end results of microgels as the support (particles in solution and as the thin film (membrane)) opens further prospective to explore these systems for other precursor biomolecule production.

iv 5. Future outlook

5 Future outlook

In the development of clean processes the enzymes play crucial role. Particularly in the field of pharmaceuticals and fine chemicals, optically pure compounds are most vital requirement.12 Diverse immobilization techniques have been developed to improve structural stability of the enzymes to ultimately achieve improved bioconversions.33 Polymeric materials are found to be most desirable candidates for bio-mimicry of the cellular counterparts.35 In this direction designing and synthesis of core shell polymeric particles possessing extensive surfaces for biocatalyst immobilization e.g. polymer brush on spherical particles can be utilized (figure 5.1.1).41 The improvement in carrier design must also be considered with respect to the ease in recovery of the bio-conjugates, achieving high load of enzymes and minimizing the adverse effects of the immobilization. One of the approaches could be to introduce magnetic nanoparticles into the core of the particles. This would help in easy recovery of the bioconjugates under the magnetic force. Furthermore, thin film of such particles can be casted in the form of membrane or a microfluidic surface to improve the efficiency of the process.257–260 Such systems can also be well adapted in non-aqueous biocatalysis application.

Figure 5.1.1: (A) Sketch of core-shell particles with polymer brush shell and magnetic core; (B) Thin films of the particles.

Alternatively, enzymes can be stabilized using conjugation techniques like the cross-linked enzyme aggregates (CLEA) or polymer coating (figure 5.1.2).184,261 v

These techniques can be employed in direction to minimize the adverse effects of immobilization like loss of the enzyme activity and leakage and also to achieve reactivation of enzyme.

(A)$ (B)$

Figure 5.1.2: (A) cross linked enzyme aggregates CLEA, and (B) Polymer stabilized enzymes.

Considering all the above points, the biochemical reactions of interest to be studied on such core-shell system would be multi enzymatic synthesis of precursors. One such sequential reactions is the synthesis of acetyl CoA using acetate kinase (Ak) and phosphotransacetylase (Pta) system (figure 5.1.3).262

ATP ADP CoA Pi O O O O

O P O- H C C CoA H3C C O- H3C C 3 Acetate kinase Phosphotransacetylase O- Acetyl-CoA Acetate Acetyl phophate

Figure 5.1.3: Schematic representation of Ak-Pta sequential reactions

The advantages of using these enzymes are that, (1) they are commercially available, (2) the specific activities of these enzymes are very high Ak (150 U/mg) and Pta (3000 U/mg) (Sigma Aldrich) and (3) higher expected stability as these enzymes are extracted from thermophilic bacteria.

vi 5. Future outlook

vii

viii 6. Appendix

6 Appendix

120

Adsorption 100 EDC coupling

40 Relative absorbance (%) Relative 20

0 PNIPAm-AEMA 6 PNIPAm-PEI 6 PNIPAm-AEMA 8 PNIPAm-PEI 8

Figure A1: The conjugation standardization of acetyl CoA synthetase with respect to the activity on PNIPAm-AEMA and PNIPAm-PEI microgels at different pH in buffer (0.1 M

MES buffer pH 6) and (0.1 M borate buffer pH 8).

8 20 (a) (b)

6 15

4 10

y = 0.0493x y = 1.0151x R² = 0.98628 R² = 0.94616 Pk activity (U/mL) Pk activity 2 Ldh activity(U/mL) 5

0 0 0 50 100 150 0 5 10 15 20 Pk protein (µg/mL) Ldh protein (µg/mL)

Figure A2: The standard plot of protein concentration verses activity of (a) Pk, and (b) Ldh.

x 6. Appendix

6.1 Abbreviations

EDC 1-ethyl-3-(3-NN-dimethylaminopropyl) carbodiimide ACoA Acetyl Co enzyme A Acs Acetyl Co enzyme A synthetase ATP Adenosine tri phosphate ADP Adenosine di phosphate AMP Adenosine mono phosphate Pk Pyruvate kinase CoA Co enzyme A Ldh Lactate dehydrogenase PEP Phosphoenol pyruvate PNIPAm poly-(N-isopropylacrylamide AEMA aminoethyl methacrylate PEI poly ethyleneimine LCST Lower critical solution temperature UCST Upper critical solution temperature VPTT Volume phase transition temperature UV-Vis Ultra violet- visible spectroscopy spectroscopy SANS Small angle neutron scattering PAA poly acrylic acid Cs Citrate synthase ATRP Atom-transfer radical polymerization RAFT Reversible Addition-Fragmentation chain Transfer HRP horse radish peroxidase tBuOOH tert- butyl hydroperoxidase MBA N,N' -Methylenebis(acrylamide) TEMED N,N,N',N'- Tetramethylethylenediamine APS Ammonium persulfate NADH β-Nicotinamide adenine dinucleotide, reduced

NAD+ β-Nicotinamide adenine dinucleotide

Km Michaelis constant kcat Turn over number DLS Dynamic light scattering SEM Scanning electron microscopy CD spectroscopy Circular dichroism spectroscopy PPi Pyrophosphate SFEP Surfactant free emulsion polymerization NaCl Sodium chloride PET membrane Polyethylene terephthalate membranes FE Free enzyme IE Immobilized enzyme CB Conjugate in batch mode CM Conjugate on membrane BCA assay Bicinchoninic acid assay Mdh Malate dehydrogenase Cs Citrate synthase GA Glutaraldehyde Ak Acetate kinase PL dual enzyme conjugate of Pk and Ldh on PNIPAm-PEI 2 microgel PiLi combination of single enzyme conjugates of Pk and Ldh on PNIPAm-PEI 2 microgel

xii 6. Appendix

6.2 List of figures

Figure 1.1. The chart shows the World enzyme market in 2010 for different sectors (The Freedonia Group Inc.).13,14 ...... 1 Figure 1.2: The chart represents the contribution of different classes of enzymes in industrial bio-transformations.15 ...... 2 Figure 1.3: Sketch representing core shell microgel-enzyme(s) bio-conjugate for (a) synthesis of acetyl CoA using S acetyl CoA synthetase (Acs); and (b) Sequential reaction of pyruvate kinase (Pk) and lactate dehydrogenase (Ldh)...... 6 Figure 2.1.1: Schematic representation showing different applications of the smart polymer hydrogel.66,69 ...... 9 Figure 2.1.2: Figure showing chemical structures of the different thermo- responsive polymers.78 ...... 11 Figure 2.1.3: Phase diagrams of the thermo-responsive polymer with the characteristic (a) UCST and (b) LCST where the transition temperature is indicated by red arrow.80,82 ...... 12 Figure 2.1.4: Schematic representation of volume phase transition temperature (VPTT exhibited by PNIPAM microgels due to hydrophobic- .hydrophilic balance at different temperature conditions.37,85 ...... 12 Figure 2.1.5: pH responsive swelling and de-swelling of (a) anionic and (b) cationic microgels.100 ...... 16 Figure 2.1.6: The chemical structure of the branched poly (ethyleneimine) (PEI)...... 17 Figure 2.1.7: The classification of the core shell hydrogels depending on the material used for core preparation while the shell is of hydrogel like material.66,112 ...... 18 Figure 2.1.8: Scheme representing the different approaches to synthesize core shell particles.113 ...... 19 Figure 2.1.9: Two-step reaction of PNIPAm-AEMA core shell microgel synthesis...... 21

xiii

Figure 2.1.10: Schematic for PNIPAm-PEI microgel synthesis in one step graft co polymerization method...... 22 Figure 2.2.1: Two step reaction for acetyl CoA synthesis catalyzed by acetyl CoA synthetase (Acs); Step I: Acetate is activated to Acetyl-AMP by ATP hydrolysis at lysine residue of enzyme active site with release of pyrophosphate (PPi). Step II: Marks the synthesis of Acetyl CoA from CoA and Acetyl AMP...... 25 Figure 2.2.2: The structure of yeast ACS (ribbon diagram), the trimer consists of large domains (yellow, cyan, and green,) and small domains (grey) respectively.144 ...... 26 Figure 2.2.3: The flowchart shows anabolic and catabolic pathways of acetyl CoA metabolism.152,153 ...... 27 Figure 2.2.4: Represent sequential reaction catalyzed by pyruvate kinase and lactate dehydrogenase.157 ...... 28 Figure 2.2.5: The structure of Ldh (a) tetrameric structure, and (b) binding of the substrates pyruvate and NADH in the active site.163,164 ...... 29 Figure 2.2.6: The crystal structure of Pk tetramer containing domains and interfaces.166 ...... 30 Figure 2.3.1: Scheme representing different types of immobilization techniques ...... 31 Figure 2.3.2: Schematic representation of EDC coupling of enzyme and carrier (cationic microgel).177 ...... 32 Figure 2.3.3: Schematic representation of glutaraldehyde coupling of enzyme and carrier (cationic microgel)...... 33 Figure 2.3.4: (a) Michaelis-Menten plot; (b) Lineweaver Burk plot ...... 37 Figure 2.4.1: The Rayleigh and Mei theory of scattering by spherical particles.198 ...... 39 Figure 2.4.2: Optical configurations of the PCS instrument for DLS measurements...... 41 Figure 2.4.3: Structure of the electrochemical double layer on a negative particle surface.80 ...... 42 Figure 2.4.4: Schematic representation of the SEM instrumentation.209 ...... 44

xiv 6. Appendix

Figure 2.4.5: Interaction volume is the depth range probed by various types of scattered electrons and X-rays. 210 ...... 45 Figure 2.4.7: White light spectra in visible range (E is energy and λ is the wavelength) and color of the samples absorbing respective wavelength.215 ...... 47 Figure 2.4.8: Schematic representation of instrumentation of UV-visible setup in microtiter plated reader (Tecan Manual)...... 48 Figure 2.4.9: Structures of aromatic amino acids (Tryptophan, tyrosine and phenylalanine)...... 49 Figure 2.4.10: CD spectroscopy (A) CD spectra of different protein secondary structures; (B) table depicting wavelength maxima for respective electron transitions.220 ...... 50 Figure 2.4.11: Schematic representation of instrumentation of CD spectroscopy.221,222 ...... 51 Figure 3.1.1: Schematic representation of the two steps synthesis of PNIPAm- AEMA core-shell microgel; Step 1: PNIPAm core synthesis and Step 2 cationic shell synthesis...... 54 Figure 3.1.2: Temperature dependent dynamic light scattering measurement of PNIPAm-AEMA microgels in water (0.01 mg/mL) ...... 55 Figure 3.1.3: SEM image of PNIPAm-AEMA microgels dried under vacuum and sputter coated with 3 nm platinum...... 55 Figure 3.1.4: Zeta potential measurement with respect to pH of microgel in water (0.01 mg/mL) ...... 56 Figure 3.1.5: Schematic representation of PNIPAm-PEI core shell microgel synthesis...... 57 Figure 3.1.6: Dynamic light scattering measurement of (a) PNIPAm-PEI1 and (b) PNIPAm-PEI2 microgels (0.01mg/mL in water) sizes with respect to the temperature...... 58 Figure 3.1.7: SEM images of PNIPAm-PEI microgels (a) PNIPAm-PEI 1 and (b) PNIPAm-PEI 2 dried under vacuum and sputter coated with 3 nm platinum...... 59 Figure 3.2.1: Schematic representation of bio-conjugation of Acs on PNIPAm- AEMA microgels using EDC coupling...... 63

Figure 3.2.2: Standardization of immobilization conditions with respect to; (a) effect of EDC, (b) microgel, and (c) salt on activity of Acs...... 64 Figure 3.2.3: Effect of different temperatures on the activities of free and immobilized Acs was studied in borate buffer pH 8. (Left figure: the specific activity, and Right figure: the normalized specific activities of the respective enzymes) ...... 66 Figure 3.2.4: Effect of different pH on the activities of free and immobilized Acs was studied in borate buffer at 37 oC. (Left figure: the specific activity, and Right figure: the normalized specific activities of the respective enzymes)...... 66

Figure 3.2.5: Fluorescence emission spectroscopy (Ex280/Em 310-390)) at different temperatures of free (a) and immobilized (b) Acs in 0.01 M phosphate buffer (protein concentration = 0.05 mg/mL) ...... 68 Figure 3.2.6: Thermal stability studies of free and immobilized Acs (Left figure: the specific activity, and Right figure: the normalized specific activities of the respective enzymes)...... 69 Figure 3.2.8: Operation stability of immobilized Acs was checked for 5 cycles at standard reaction conditions (FE=12mU/mL)...... 71 Figure 3.3.1: Schematic representation of preparation of (a) Acs-PNIPAm PEI 1 conjugate, and (b) microgel-PET (polyethylene terephthalate) membrane for bioconversion study...... 77 Figure 3.3.2: Adsorption isotherm study of Acs on PNIPAm-PEI 1 microgels. (a) the plot of adsorbed amount of Acs on microgel vs Acs in solution. (b) Linearized Langmuir plot...... 78 Figure 3.3.3: CD spectra of free enzymes and enzymes desorbed from PNIPAm- PEI at pH 4 (protein concentration (0.05mg/mL) ...... 80 Figure 3.3.4: Effects of different temperature on the activities of free and immobilized Acs was studied in tris buffer pH 8. (Left figure: the specific activity, and Right figure: the normalized specific activities of the respective enzymes)...... 82 Figure 3.3.5: Effect of different pH on the activities of free and immobilized Acs was studied in tris buffer at 37 oC. (Left figure: the specific activity,

xvi 6. Appendix

and Right figure: the normalized specific activities of the respective enzymes)...... 83 Figure 3.3.6: (a & b) cross-section SEM image of ultrathin bioconjugate- membrane on PET track etched support. (c) Flux versus pressure relation of bioconjugate-membrane PET membrane...... 84 Figure 3.3.7: Acetyl CoA formation is given as percent yield with respect to time by free Acs (¾) and conjugate ( ● ) in batch condition and conjugate on the membrane (¿) ...... 86 Figure 3.3.8: Operational stability of Acs on PNIPAm-PEI microgel membrane. Relative activities are normalized activities of enzymes with respect to the highest activity of respective enzymes and experiment...... 87 Figure 3.4.1: The sequential enzymatic reactions catalyzed by pyruvate kinase and lactate dehydrogenase where phosphoenol pyruvate (PEP) is converted finally to lactate with generation of ATP in the first reaction and oxidation of NADH in the later...... 91 Figure 3.4.2: Schematic representation of preparation of Pk and Ldh bioconjugates; (i) single enzyme-microgel conjugates (Pi and Li) and dual enzymes systems PiLi (on different microgel particles),

(ii) PL (simultaneous addition) and (iii) P3Lo and L3Po (sequential addition) (on same microgel particles)...... 92 Figure 3.4.3: Screening of dual and single enzyme/s system: enzyme activities of Pk and Ldh in dual enzyme microgel particles prepared by different initial enzyme ratios (a) added simultaneously and (b) sequentially. (c) Pk activities of combinations of single enzyme conjugates (pk and ldh) added in respective ratios during the assay…………………………………….89 Figure 3.4.4: Temperature dependent fluorescent spectra of (a) free Ldh; (b) immobilized Ldh; (c) free Pk, (d) immobilized Pk, (e) PL and (f) PiLi ...... 98 Figure 3.4.5: Effect of pH (a) on activity of free and immobilized Ldh; (b) on activity of free and immobilized Pk, (a) on activity of PL and PiLi at 37 °C in buffers with different pH...... 99

xvii

Figure 3.4.6: Effect of temperature (a) on activity of free and immobilized Ldh; (b) on activity of free and immobilized Pk, (a) on activity of PL and PiLi at different temperature in phosphate buffer pH 7.4……..…….97 Figure 3.4.7: Storage stability of free and immobilized enzymes (a) Pk activity, (b) Ldh activity...... 100 Figure 3.4.8: Reusability of Pi and PL for Pk activity and Li and PL for Ldh activity (100% activity for Pk and Ldh were 1.1 and 5.5 U/mL respectively)...... 101 Figure 3.4.9: Application of Dual enzyme bioconjugates for (a) determining initial velocity of Ak activity. (b) Bioconjugate concentration dependent ATP formation ...... 102 Figure 4.1: Schematic representation of acetyl CoA synthetase (Acs) immobilized on microgel particle and membrane reactor for acetyl CoA synthesis...... ii Figure 4.2: Schematic representation of sequential reaction catalyzed by pyruvate kinase and lactate dehydrogenase on same and different microgel particles...... iii Figure 5.1.1: (A) Sketch of core-shell particles with polymer brush shell and magnetic core; (B) Thin films of the particles...... v Figure 5.1.2: (A) cross linked enzyme aggregates CLEA, and (B) Polymer stabilized enzymes...... vi Figure 5.1.3: Schematic representation of Ak-Pta sequential reactions ...... vi

xviii 6. Appendix

6.3 List of tables

Table 2.1.1: The characteristics difference between various types of free radical polymerization...... 20 Table 2.1.2: The use of different PNIPAm based microgels for enzyme immobilization...... 24 Table 3.1.1: Zeta potential values of PNIPAm-PEI microgels...... 58 Table 3.2.1. Kinetic parameters of immobilized and free enzyme for acetate substrate at 37 °C...... 67 Table 3.3.1. The contributions of secondary structure elements of free and immobilized Acs...... 80 Table 3.3.2. The kinetic parameters of immobilized and free enzymes for acetate substrate at 37 °C...... 81 Table 3.4.1: Immobilization yield (from equation 3.4.1) and enzyme load for single and dual enzyme microgel bioconjugate...... 95 Table 3.4.2: Kinetic parameters of free enzyme and conjugates: (A) kinetic parameters for ADP at constant PEP concentration for free and immobilized Pk. (B) kinetic parameters for pyruvate at constant NADH concentration for free and immobilized Ldh...... 96

xix

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xlii 8. Acknowledgements

8 Acknowledgements

I would first like to thank Leibniz-Institut für Polymerforschung Dresden e.v (IPF) for providing the facilities, finance and opportunity to do the research that is core of this thesis. It is my profound privilege to verbalize my feelings of gratitude and deep respect to Professor Manfred Stamm for giving me the opportunity to pursue PhD under his kind guidance at the IPF. The interactions that I have had with him were very encouraging and helped me in achieving better perspective for science and life in general. I am very grateful to Dr. Leonid Ionov for his scientific guidance and support during the research work. He has always been there to discuss and find fitting solutions to the problems. He has also encouraged independent thinking and always given me the freedom to work.

I want to thank Dr. Petra Uhlmann, for her support and help for making special provisions to work in the lab during my pregnancy period. I must also acknowledge to Dr. Alla Synytska, for her kind words of appreciations and supports during the course of this study.

I am short in words to pay my gratitude to all the scientific staffs for extending their valuable assistance and their kind support for instrumental facilities, Dr. Dietmar Appelhans and Dr. Nikita Polikarpov for introducing with the basics of microgel synthesis, Dr Bellmann, and Ms. Anja Casperi for DLS and zeta potential, Dr. Peter Formanek for SEM, Dr. Martin Müller for CD spectroscopy, Dr. Manfred Maitz, and Dr. Mikhail Tsurkan for the instrument facilities in MBZ. I appreciate for all your contributions and kind suggestions during the difficulties.

I would like to express my heartfelt indebtedness to Dr. Lippmann, Ms. Janett Forkel, Ms. Nicole Krause and all technical staff members for all their contribution related to documentation, supplies and office related help.

I want to thank my most cooperative and helpful colleagues and friends Dr. Ulla König, Mr. Leonard Schelkof, Dr. Nikolay Puretskiy, Mr. Ivan Raguzin, Mr. Marcus

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Binner, Dr. Marion Fischer, Dr. Somyadip Chowdhary, Ms. Evmorfia Pssara Ms. Aruni Shaj Kumar and Mr. Jaganath Chanda for all their support and memorable time, which I will pursue throughout my life. I am thankful to Ms. Kristin Lorenz, my German language teacher, who made me acquainted with the basics of the language. A special thanks to all the lovely ladies of Studentenwerk Miniforscher kindergarten, for their care, love and support.

The part that a family plays in life cannot be expressed in gratitutes, but I would like to take this opportunity to thank my Parents, my Husband and my Son who are pillars of my life, and had always fill my life with positivity and jubilance. It was because of their love and care that I never realized the hard times. Amma- Babuji, I thank you for all your blessings, affection and support. Thanks to my siblings Niteesh and Shreya for all their innocent love and strength. Finally I owe for everything to the Almighty for his endless blessings.

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Curriculum vitae

First name: Nidhi Chandrama Surname: Dubey Mobile: +49-15143383982 Email: [email protected], [email protected]

Educational Qualification: 2011-2015 Ph.D. in Physical chemistry and Biochemistry, Technische Universität Dresden (continue) 2006-2008 M. Sc. Microbiology, Savitribai Phule Pune University, Maharashtra, India (71.50/ Ist/ Distinction, Obtained first rank) 2003-2006 B. Sc. Microbiology, Chemistry & Zoology, Savitribai Phule Pune University, Maharashtra, India (72.86/ Ist/ Distinction)

Fellowships & Academic Achievements: 2011 Scholarship for PhD at Leibniz Institute of Polymer Research Dresden, Germany 2009 - Junior Research Fellowship from Gov. of India (CSIR-UGC- NET-JRF, December2008) - Graduate Aptitude Test for Engineering (GATE 2009; 93.27 percentile) 2008 -National Eligibility Test for Lectureship from Gov. of India (CSIR-NET-LS) June 2008 -Graduate Aptitude Test for Engineering (GATE 2008, 95.96 percentile) 2004 Second Prize in College Chemiad and third prize in University Chemiad

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Research Experience: 2009-2011 UGC JRF: Topic “Diagnosis of invasive aspergillosis using (2 years) immunoassays”. Centre for Nanobioscience, Agharkar Research Institute, Pune 2008 - Project-JRF entitled “Climate change and adaptation of species 2009 complexes”. Department of Marine Biotechnology and Ecology, (5 months) at Central Salt and Marine Chemicals Research Institute, (CSIR) 2007 Master Dissertation: “Quantification of Antibacterial Effects of (3 months) Plant Extracts Using ELISA Reader & Characterization of the Active Principles”. Dept. Biotechnology, Savitribai Phule Pune University.

Publications, Patent, and Conferences: 1. NC Dubey, BP Tripathi, M Stamm, L Ionov. Sequential multi-enzymes reaction on thermo-responsive microgels and their co-factor dependent applications. (under preparation). 2. NC Dubey, BP Tripathi, M Mueller, M Stamm, L Ionov. Enhanced activity of acetyl CoA synthetase adsorbed on smart microgel: an implication for precursor biosynthesis. ACS Applied Materials & Interfaces, 2015,7, 1500. 3. NC Dubey, BP Tripathi, M Stamm, L Ionov. Smart Core–Shell Microgel Support for Acetyl Coenzyme A Synthetase: A Step Toward Efficient Synthesis of Polyketide-Based Drugs. Biomacromolecules, 2014, 15, 2776-2783. 4. BP Tripathi, NC Dubey, M Stamm. Hollow Microgel Based Ultrathin Thermoresponsive Membranes for Separation, Synthesis, and Catalytic Applications. ACS applied materials & interfaces, 2014, 6, 17702-17712.

5. BP Tripathi, NC Dubey, M Stamm. Polyethylene glycol cross-linked sulfonated polyethersulfone based filtration membranes with improved antifouling tendency. Journal of Membrane Science 2014, 453, 263-274 6. BP Tripathi, NC Dubey, F Simon, M Stamm. Thermo responsive ultrafiltration membranes of grafted poly (N-isopropyl acrylamide) via polydopamine. RSC Advances 2014, 4, 34073-34083. 7. BP Tripathi, NC Dubey, S Choudhury, F Simon, M Stamm. Antifouling and antibiofouling pH responsive block copolymer based membranes by selective surface modification. Journal of Materials Chemistry B, 2013, 1 (27), 3397- 3409

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8. BP Tripathi, NC Dubey, M Stamm. Functional polyelectrolyte multilayer membranes for water purification applications. Journal of hazardous materials, 2013, 252, 401-412 9. B.P. Tripathi, N.C. Dubey, S Choudhury, M Stamm. Antifouling and tunable amino functionalized porous membranes for filtration applications. Journal of Materials Chemistry, 2012, 22 (37), 19981-19992 Patent: 1. NC Dubey, L Ionov, BP Tripathi, M Stamm, Enzyme reactor system. EP 14 160 765.5 Conference & Symposia: 1. NC Dubey, BP Tripathi, M Stamm, L Ionov. Thermo-responsive core-shell microgels as matrix for enzyme immobilization and biosynthesis. 8th ECNP International Conference on Nanostructured Polymers and Nanocomposites, Dresden, Germany, September 16 to 19, 2014

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Erklärung

Hiermit versichere ich, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfmittel angefertigt habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Die Arbeit wurde bisher weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.

Die Dissertation wurde in der Zeit von Februar 2011 bis März 2015 unter der Betreuung von Herrn Prof. Dr. Manfred Stamm am Leibniz-Institute für Polymerforschung Dresden e. V. in der Abteilung Nanostrukturierte Materialien angefertigt. Es haben keine früheren erfolglosen Promotionsverfahren stattgefunden. Ich erkenne die Promotionsordnung der Technischen Universität Dresden der Fakultät Mathematik und Naturwissenschaften vom 23.02.2011 in geänderter Fassung vom 15.06.2011 und 18.06.2014 an.

………………………………. ………………………………….

Datum Unterschrift

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