Production and Immobilization of Alpha Amylase Using Biotechnology Techniques for Use in Biological and Medical Applications

Home , Glycosylase, Invertase, Isoamylase

Mansoura University Faculty of Science (Damietta) Chemistry Department

Production and Immobilization of alpha amylase using biotechnology techniques for use in biological and medical applications

Enass Esmail Fathi Mobasher B.Sc. (Chemistry/Biochemistry), Zagazig University, 2003

Supervisors

Prof. Dr. Mostafa. A. Shousha Prof. Dr. Mohamed. M. El-Sharabasy Professor of Biochemistry Professor of Biochemistry Biological Application Department Faculty of Science (Damietta), Nuclear Research Center Mansoura University. Atomic Energy Authority

A Thesis Submitted for partial Fulfillment of the Requirements for Master Degree of Science in Biochemistry

2009

Mansoura University Faculty of Science (Damietta) Chemistry Department

SUPERVISORS

Title of Thesis: Production and Immobilization of alpha amylase using biotechnology techniques for use in biological and medical application

Candidate Name: Enass Esmail Fathi Mobasher.

SupSupervisors:ervisors: NO Name Position Sign. 1 Prof. Dr. Mohamed. M. El- Professor of Biochemistry, Sharabasy Faculty of Science at Damietta, Mansoura University. 2 Prof. Dr. Mostafa . A. Shousha Professor of Biochemistry, and Head of Biological Application Department. Atomic Energy Authority

Head of Chemistry Department Vice-Dean of Post Graduate Dean of the Faculty Studies & Research

Prof. Dr. Mohammad. M. Mashaly Prof. Dr. Mohamed. O. Wady Prof. Dr. Mohamed. R. Mostafa

Mansoura University Faculty Of Science (Damietta) Chemistry Department

Supervisors and Examiners

Title of Thesis: Production and Immobilization of alpha amylase using biotechnology techniques for use in biological and medical application

Candidate Name : Enass Esmail Fathi Mobasher.

Supervisors Name Position NO 1 Prof. Dr. Mohamed. M. El-Sharabasy Professor of Biochemistry, Faculty of Science at Damietta, Mansoura University. Prof. Dr. Mostafa . A. Shousha Professor of Biochemistry, 2 and Head of Biological Application Department. Atomic Energy Authority Examiners:

No Name Position 111 Prof. Dr. Mostafa. A. Shousha Professor of Biochemistry in Atomic Energy Authority 2 Prof. Dr. Abd El-aziz. F. El- Assistant Professor of Biochemistry. aziz Faculty of Science. Mansoura University 3 Prof.Dr. Yousif M. Shehata Professor of Biochemistry. Veterinary medicine. Zagazig University

Head of Chemistry Department Vice-Dean of Post Graduate Dean of the Faculty Studies & Research

Prof. Dr. Mohammad. M. Mashaly Prof. Dr. Mohamed. O. Wady Prof. Dr. Mohamed. R. Mostafa

ا ــ رة آامط ا ـــــــ ء اجوااقاااات اوا ر اسا رسم آءوآء از ٢٠٠٣ إاف /د.أ ا /د.أ را أذاءا أذ اءا اتا آا مط آاثاو ارة هاار وذآءتالدرام اءا ٢٠٠٩

ا ــ رة آامط ا ـــــــ ء ااف

انا : اجوااقااا اتاوا .

اا : اسا.

ااف : م ا ا ا أذاءا ١ /د.أ را . آامط ارة. أذاءا ٢ /د.أ ا . ور اتا هاار . راء وآا رات ا اواث

/د.أ /د.أ ري /د.أ انوادي

ا ــ رة آامط ا ـــــــ ء او ا انا: اجوااقا اا اتاوا . اا: اسا .

ا اف : م ا ا ١ /د.أ را . أذاءا آامط ارة. ٢ /د.أ ا . أذاءا وراتا هاار . اوا : م ا ا ١ /د.أ ا اذاءااار ٢ /د.أ ا ح ا أذ اءاام. ارة ٣ /د.أ أذاءاىاز راء وآارات ا اواث /د.أ /د.أانوادي /د.أ ري

ACKNOWLEDGEMENT I would like to express my sincere gratitude and appreciation to my supervisor Prof. Dr. Mohamed Mansour El-Sharabasy , Professor of Biochemistry, Faculty of Science at Damietta, Mansoura University for direct supervision , careful advice and overall assistance throughout the present work.

My deepest obligation and profound gratitude to my supervisor Prof. Dr. Mostafa Abdalla Shousha , Professor of Biochemistry, Head of Biological Applications Department, Nuclear Research Center, Atomic Energy Authority for direct supervision , careful advice, help, and encouragement throughout the work.

Many thanks and sincere gratitude to Dr. Khaled Shalah Atia , Assistant Professor of Biochemistry, Biological Applications Department, Nuclear Research Center, Atomic Energy Authority for careful advice, help, encouragement, and overall assistance throughout the work.

Many thanks and deepest obligation to Dr. Sahar Ahmed Ismail , Assistant Professor of Chemistry, National Center For Radiation Research and Technology, Atomic Energy Authority for her help, support, encouragement throughout the work.

Special thanks and sincere gratitude to prof. Dr. Magdy Hassn Gamal professor of Endocrinology, Head of Endocrinology Research Unit, Biological Applications Department, Nuclear Research Center, Atomic Energy Authority for careful advice, help and support throughout the work.

Many thanks to Prof. Dr. Camelia Adly professor of Biochemistry, Faculty of Science at Damietta, Mansoura University and Prof. Dr. EL-Shahat Toson Assistant Professor of Biochemistry, Faculty of Science (Damietta), Mansoura University for their help in this work.

Many thanks to Chemistry Department in faculty of science at Damietta, Mansoura University and Physiology and Biochemistry Unit, Biological Applications Department in Nuclear Research Center.

Enass Esmail Mobasher.

Contents

Title Page List of abbreviations i List of figures iii List of tables iv INDRODUCTION AND AIM OF WORK. 1 I. LITERATURE REVIEW. I.1-enzyme. 3 I.1.1-Mechanism of enzymatic reaction. 5 I.1.2- Enzyme nomenclature. 6 I.1.3-Enzyme classification. 7 I.1.4-Uses of enzyme. 7 I.2- Enzyme immobilization. 10 I.2.1-Enzyme immobilization methods. 12 I.3- Polymers as carriers for enzyme immobilization. 20 I.3.2- Chitosan 22 I.3.3- Alginate 25 I.3.4- Thermoresponsive polymer 28 I.4- Amylases 32 I.5- α–amylase 34 I.6- Immobilization of α–amylase 39 II. Materials and Methods. 45 II.1- Production of alpha amylase 46 II.2-Immobilization of alpha enzyme 48 II.2.2.1- Preparation of immobilized enzyme on Natural 50 polymer II.2.2.2- Preparation of immobilized enzyme synthetic 50 polymer II.2.3- Determination of the amount of immobilized 51 enzyme II.2.4-Assay of enzyme activity 53 II.2.5- Physico-chemical properties of free and 54 immobilized α-amylase III. Results. 59 IV. Discussion. 81 V. Summary. 90

References. 92 ١ .Arabic Summary

LIST OF ABBREVATIONS

LIST OF ABBREVIATIONS

A. niger Aspergillus niger AAc Acrylic acid. ALAB Amylolytic lactic acid bacteria. bisacrylamide N,N, methylenebisacrylamide BSA Bovine Serum Albumin BuA Butylacrylate copolymer. C1 Carbon atom number 1 CDI Carbodiimide. CFF Cell free filtrate DEAE-cellulose Diethylaminoethylcellulose DNSA 3, 5–Dinitrosalicylic acid E Enzyme. EC 3.2.1.1 Alpha amylase commission number EC. Enzyme commission numbers EDA Ethylenediamine. EGDMA Ethylene glycol dimethacrylate. ES Enzyme substrate complex. FAD Flavin adenine dinucleotide G α-l-guluronic acid GDA Glutaricdialdehyde. HEMA Hydroxyethylmethacrylate. HMDA Hexa methylene diamine. IPNs Semi –interpenetrating polymer networks.

i LIST OF ABBREVATIONS

KDa Kilo Dalton. KPS Potassium persulfate. Km Michaelis constant. L. Lactobacilli species. LCST Lower critical solution temperature. M 1, 4-β-d-mannuronic acid. NAD Nicotinamide adenine dinucleotide. NAS N-acryloxy succinimide. NIPAM N-isopropylacrylamide. nm Nanometer. PNIPAAm Poly(N-isopropylacrylamide). rpm Round per minute. S Substrate. SDS Sodium dodecyl sulphate. SH Mercapto group. TEMED N, N, N, N tetramethylethylenediamine. Tris (Hydroxy methyl) amino methane. V Velocity. VIM Vinyl imidazole. Vmax Maximum velocity

ii LIST OF FIGUR

LIST OF FIGURES No of Title of figure No of figure page figure 1 Mechanism of enzyme action. 5 figure 2 Enzyme immobilization methods. 13 figure 3 Structure of chitin and chitosan. 23 figure 4 Structure of amylase and amylopectin. 32 figure 5 Standard curve for protein. 52 figure 6 Standard curve for maltose. 54 figure 7 Starch standard curve. 57 figure 8 Gel filtration of crude α-amylase produced from A. 63 niger . figure 9 The effect of temperature on the free enzyme and 65 immobilized enzyme activities figure 10 The effect of pH on the free enzyme and immobilized 66 enzyme activities figure 11 Reusability of immobilized α-amylase 68 figure 12 The storage stability plotted as % relative activity vs. 69 time, for the free and the immobilized α-amylase at 25 oC figure 13 The storage stability plotted as % relative activity vs. 70 time, for the free and the immobilized α-amylase at 4oC. figure 14 The thermal inactivation kinetics of the free and the 71 immobilized α-amylase at 40C. figure 15 The thermal inactivation kinetics of the free and the 72 immobilized α-amylase at 60 oC. figure 16 The effect of γ- radiation on both free and 73 immobilized α-amylase activity. figure 17 Michaelis-Menten direct fit (Free enzyzme) 75 figure 18 Lineweaver-Burk plot for the free enzyme 76 figure 19 Michaelis-Menten direct fit (chitosan-alginate) 77 figure 20 Lineweaver-Burk plot for (chitosan-alginate) 78 preparation figure 21 Michaelis-Menten direct fit (Acrylamide) 79 figure 22 Lineweaver-Burk plot for Acrylamide preparation 80 figure 23 Starch hydrolysis using entrapped alpha-amylase 81

iii LIST OF TABLES

LIST OF TABLES

No. of Title of table No. of table page Table (1) Classification of enzymes. 7 Table (2) Characteristics of immobilized enzyme methods 17 Table (3) Fundamental considerations in selecting support 21 Table (4) Gel filtration of the crude alpha amylase produced 62 from A. niger Table (5) Partial purification steps of α-amylase produced from 63 A. niger. Table (6) Kinetic Parameters of Free and Immobilized α- 74 amylase

iv Introduction & aim of work

Introduction and aim of work

Enzymes, which are structurally complex protein molecules, are the biochemical equivalent of conventional catalysts of most chemical reactions processes. They have advantages over their high degree of specificity and the ability to operate in ambient conditions.

Enzymes isolation is relatively expensive and loses their catalytic properties by any change in their conditions such as heating, which denature protein.

Investigations were therefore carried out at an early period on the possibilities of repeated use of these catalysts and the possibilities of using gamma rays for enzyme sterilization, since sterilization by heat is completely inapplicable in this case.

The above aims can be achieved with enzymes immobilization. This is because numerous applications of the immobilized enzymes in analysis, industrial applications, biotechnology, biomedical engineering and other applications.

Since the immobilized α-amylase has gained considerable attention owing to its widespread use in medical and industrial fields, this enzyme will be purified from Aspergillus niger to be immobilized on two different polymers by entrapment methods. One of them is natural consist of chitosan and sodium alginate while the other is synthetic and consists of N-isopropylacrylamide and sodium alginate.

The efficiency of the free enzyme and the immobilized α-amylase will be determined by estimating some physico-chemical properties such as reuse

1 Introduction & aim of work efficiency, optimum temperature, optimum pH, storage as well as thermal stabilities. The effect of radiation on the free and the immobilized enzyme will be estimated.

2 Chapter I: Review of literature

Review of literature

I.1- Enzymes:

Enzymes are very efficient catalysts which serve to accelerate the biochemical reactions of living cells and catalyze a variety of chemical reactions. They speed up biochemical reactions by lowering the energy of activation without themselves appearing in the reaction products (Emel et al., 2006).

Enzymes are proteins, and their function is determined by their complex structure. The reaction takes place in a small part of the enzyme called the active site, while the rest of the protein acts as "scaffolding". This makes the enzyme specific for one reaction. This specificity allows them to discriminate not only between reactions but also between substrates (substrate specificity), similar parts of molecules (regiospecificity) and between optical isomers (stereospecificity) (Palomo et al., 2004).These specificities warrant that the catalyzed reaction is not perturbated by side-reactions, resulting in the production of one wanted end-product, whereas production of undesirable by- products is minimized. This provides substantially higher reaction yields, thus, reducing material costs (Barbara , 2004).

Many enzymes need cofactors (or coenzymes) to work properly. These can be metal ions (e.g. Fe 2+, Mg 2+, and Cu2+) or organic molecules such as haem, biotin, FAD, NAD or coenzyme A. Many of these are derived from dietary vitamins, which is why they are so important. The complete active enzyme with its cofactor is called a holoenzyme, while just the protein part without its cofactor is called the apoenzyme (Devlet et al., 2004).

Like all catalysts, enzymes take part in the reaction. That is how they provide an alternative reaction pathway. But they do not undergo permanent

Chapter I: Review of literature changes and so remain unchanged at the end of the reaction. They can only alter the rate of reaction, not the position of the equilibrium. Enzymatic reaction can be favored over chemical catalysis under such circumstances where thermal degradation of labile compounds is minimized and use of chemicals with a potential for pollution can be avoided. Enzymes are extremely effective, as biological catalysts. Most of the reactions they catalyze would not proceed in their absence in a reasonable time without extremes of temperature, pressure, or pH. Enzyme catalyzed reactions, or enzymatic reactions, are 10 3 to 10 17 times faster than the corresponding non catalyzed reactions. In addition, enzymes generally operate at mild conditions of temperature, pressure and pH with reaction rates of the order of those achieved by chemical catalysts at more extreme conditions. This makes for substantial process energy savings and reduced manufacturing costs (Tarik et al., 2004).

The efficiency of enzymes not only saves energy for the living cell but also precludes buildup of potentially toxic metabolic by-products. Some enzyme- catalyzed reactions function as control points in metabolism. Metabolism is regulated in a variety of ways, including alteration in the concentrations of enzymes, substrates, enzyme inhibitors, and modulation of the activity levels of certain enzymes. Even the simplest living organisms contain multiple copes of nearly a thousand different enzymes. In multicellular organisms, it is the particular complement of enzymes present that differentiates one cell type from another.

Chapter I: Review of literature

I.1.1-The general mechanism of enzyme's action is illustrated below:

S + E <==> ES <==> EP <==> E + P

Where: S= substrate, E= enzyme, P = product, ES= enzyme –substrate Complex and EP= enzyme –product complex.

Figure 1. Mechanism of enzyme action.

Chapter I: Review of literature

I.1.2- Enzyme nomenclature: All enzymes known to date have individual enzyme commission numbers (EC) based on (Schmid et al ., 2001).The first digit number denotes the main class, which specifies the catalyzed reaction type. There are six main classes of enzymes namely oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase. The second number (sub class number) indicates the substrate type, the type of transferred functional group or the nature of one specific bond involved in the catalyzed reaction. The third digit number (sub-sub class number) expresses the nature of substrate or co-substrate. The forth digit number is an arbitrary number. For example, alpha amylase has commission number (EC 3.2.1.1). The number 3 indicates that it belong to hydrolases enzymes, number 2 indicates that it belong to the sub-class glycosylase enzymes, number 1 indicates that it belong to the sub-sub class glycosidase enzymes which cleave O- or S- glycosiyl bonds, and the last number 1 is serial number and means alpha type.

Chapter I: Review of literature

I.1.3- Enzyme classification: Enzyme classification is based on two points, the reaction catalyzed and the substrate specificity (Robert et al., 2003 ). Enzymes are grouped into six main classes according to the type of reaction catalyzed as shown in table 1. Table 1: Classification of enzymes.

Classes of Enzymes Class Chemical Reaction Catalyzed Examples EC 1 Oxidoreductase Oxidation-reduction in which Cytochrome oxidase, oxygen and hydrogen are gained lactate dehydrogenase or lost. EC 2 Transferase Transfer of groups, such as Acetate kinase, alanine phosphate group, an amino group deaminase. or acetyl group. EC 3 Hydrolase Hydrolysis amylases, cellulase EC 4 Lyase Removal of groups or atoms Oxalate decarboxylase, without hydrolysis. isocitrate lyase Ec 5 Isomerase Rearrangement of atoms within a Glucose –phosphate molecule. isomerase EC 6 Ligase Joining of two molecules. DNA ligase

I.1.4-Uses of enzyme:

• Therapeutic use of enzymes:

Enzymes have a wide variety of biotechnological, biomedical, and pharmaceutical applications. They are used as biosensors, in bioengineering, clinically as therapeutic agents, in modern diagnostics, and as catalysts for chemical or biochemical reactions (Chui and Wan, 1987). In the medical field, enzymes are playing an increasing role in the therapy (Naeher and Thum, 1974).

Chapter I: Review of literature

The local application of proteinases, ribonuclease, and deoxyribonuclease brings about a hydrolysis of mucopolysaccharides tissue, masses of pus, secretions, or hematomas. Some enzymes play important role in the treatment of thrombolysis such as urokinase and streptokinase. Also, there is possibility of using enzymes in tumor therapy. For example, there are good results obtained in the treatment of some leukemias with asparaginase. There is also possibility for enzymatic therapy in cases suffering from metabolic disturbances resulting from genetic origin.

• Enzymatic analysis:

The determination of metabolites (glucose, cholesterol, urea, etc…) by enzyme catalyzed reactions plays an important role in clinical chemistry and the food stuffs industry (mono- and disaccharides organic acids, alcohols, pyrophosphate, etc...).

• Industrial application of enzymes:

An important application of the enzymes is in the industrial processes. This is due to their non-toxicity, good rate of reaction, water solubility which are major advantages for the enzymatic reactions over that of the inorganic catalysts (Betigeri and Neau, 2002). Of the about 3000 enzymes known at the present time, one comparatively few are produced in the large scale and used industrially. These are mainly extracellular hydrolytic enzymes, which degrade naturally occurring polymers such as starch, proteins, pectin and cellulose.

Also enzymes have been used in laundry detergents since the 1960s, where their main environmental benefit is that they help to save energy. Calculations showed that in Denmark, with a population of five million, a reduction of wash temperature by 20 oC, would lead to an energy saving which can be used to produce 40.000 tones of coal a year (Naeher and Thum, 1974). In comparison,

Chapter I: Review of literature the energy used to produce enzymes less than 300 tones of year. Enzymes work best at mild temperatures and in mild conditions. They can be used to replace harsh conditions and harsh chemicals, thus saving energy and preventing pollution. They are also highly specific, which means fewer unwanted side effects and by-products in the production process. Enzymes can also be used to treat the waste which is consisting of biological material.

Also, enzymes practically do not present disposal problems, since being mostly proteins and peptides; they are biodegradable and easily removed from contaminated streams. This unique set of advantageous features of enzymes as catalysts has been exploited since the 1960s and several enzyme-catalyzed processes have been successfully introduced to industry, e.g. in the production of certain foodstuffs, pharmaceuticals and agrochemicals, but now also increasingly to organic chemical synthesis (Barbara, 2004).

In addition to the unquestionable advantages, there are number of practical problems in the use of enzymes. To these belong: the high cost of isolation and purification of enzymes, the instability of their structures once they are isolated from their natural environments (Peng et al., 2006). Also, the practical problems may be due to their sensitivity both to process conditions such as a particular pH and temperature and to the trace levels of substances that can act as inhibitors. The latter two result in enzymes' short operational lifetimes (Chui and Wan, 1987).

In addition, unlike conventional heterogeneous chemical catalysts, most enzymes operate dissolved in water in homogeneous catalysis systems, which are why they contaminate the product and as a rule cannot be recovered in the active form from the reaction mixtures for reuse (Barabra, 2004). Therefore, several methods have been proposed to overcome these limitations, one of the most successful being enzyme immobilization (Senay and Oztop, 2003 ).

Chapter I: Review of literature

I.2- Enzyme immobilization: Enzyme immobilization has attracted a wide range of interest from fundamental academic research to many different industrial applications. Immobilized biocatalysts are freely used in the production of medicines, chemicals, food, beverages and wastewater treatments (Saleem et al., 2005). The basic idea behind enzyme immobilization is to restrict the freedom of the enzyme by fixing it to solid supports or within a semi-permeable support material, which prevents the enzyme from leaving while allowing substrates, products, and co-factors to pass through (Abdel-Fattah et al., 1997).

Although the exact requirements for the immobilizing matrix are dictated by the type of enzyme and the intended application, it is clear that the material should at least be non-degradable and compatible with the enzymes. The process for immobilization should also be mild enough so as not to denature the enzyme during the steps of its preparation (Taqieddin and Amiji, 2004).

As a result of immobilization process heterogeneous immobilized enzyme systems are obtained. The heterogeneity of the immobilized enzyme systems allows easy recovery of enzyme and product, multiple reuses of enzymes, continuous operation of enzymatic processes (Yang et al., 2004). In addition, immobilized enzymatic systems improve storage, pH operational, thermal (Gawande et al ., 1998), conformational stabilities (Nascimento et al ., 2002), and rapid termination of the reactions (Aksoy et al., 1998). They also enhance the enzyme activity over a broader range of pH and temperature (Pencreach et al ., 1997), easier prevention of microbial growth (Bornscheuer, 2003), and increased shelf life (Gilles et al., 2005).

Immobilized enzymes are currently used in industrial, analytical, therapeutic, and for structural applications. Most large-scale industrial

Chapter I: Review of literature applications are in food industry and in column reactors. Among these applications, the use of immobilized glucose isomerase for producing high fructose corn syrup (Sonnur et al., 2003).

Analytical tests using immobilized enzymes fall into two groups, the first as biochemical sensors, which consist of those instruments in which the immobilized enzymes have contact with the sensors. The second as partitioned- enzyme sensor, in which the immobilized enzyme is in column or reactor separated from the sensor (Tzu-Chien et al., 2006).

In therapeutic applications, micro-encapsulated enzymes are protected from inactivation by external proteases and have reduced immune responses compared to the free enzyme. In addition to in the Vivo applications, immobilized enzymes have potential uses in extracorporeal treatments and in the local applications (Rauf et al., 2006).

Chapter I: Review of literature

I.2.1-Enzyme immobilization methods: Enzymes may be immobilized by a variety of methods, as shown in figure 2, which may be broadly classified as Physical methods; where weak interactions between enzyme and support material exist and to Chemical methods; where covalent bonds are formed between the enzyme and the supporting material (Arıca and Bayram ŏglu, 2004).

The physical methods include: Adsorption on a water-insoluble matrix. Gel entrapment. Micro encapsulation with a solid membrane.

The chemical methods include: Ionic binding Covalent attachment to a water-insoluble solid support (carrier binding method). Cross-linking by using a multifunctional and low molecular weight reagent.

However, no single method and support is best for all enzymes and their applications. This is because of the widely different chemical characteristics and composition of the enzymes, the different in the properties of their substrates and products, and the different uses to which the product can be applied.

Chapter I: Review of literature

Figure 2: enzyme immobilization methods

I.2.1.1- Adsorption method:

This immobilization procedure is very simple in which, enzyme solution is added to the support, mixed, and then surplus enzyme is removed by washing (Bickerstaff, 1995).

This method involves reversible surface interactions between the enzyme and the supporting material. Adsorption forces are of different types (Vander Waals, ionic interactions and hydrogen bonding) and are relatively weak. Although losses in enzyme activity are usually low, desorption (leakage) from the carrier is caused by minor changes in the reaction parameters, such as a variation of substrate concentrations, temperature or pH. Desorption can be turned to

Chapter I: Review of literature advantage if regeneration of support is carried out to allow rapid expulsion of exhausted biocatalyst and replacement with fresh one.

Numerous inorganic and organic materials including activated charcoal, aluminum oxide, cellulose and synthetic resins have been used as carriers (Bickerstaff, 1995).

The disadvantages of external adsorption are: • A relatively low surface area for bonding. • The enzyme is more subject to physical abrasion, inhibitory effects and the turbulence of the bulk solution. 1. The enzyme is more exposed to microbial attack. • Smaller particles result in high pressure drops in a continuous packed bed reactor and are difficult to retain in fluidized beds or continuous-stirring.

I.2.1.2- Ionic binding:

Ion exchange resins readily adsorb proteins and have been widely employed for enzyme immobilization. Both cation exchange resins such as carboxy methylcellulose or amberlite IRA and anion exchange resins, e.g. diethylaminoethyl cellulose (DEAE-cellulose) and sephadex are used industrially. Although being stronger in nature compared to the forces involved in simple physical adsorption, ionic binding forces are particularly susceptible to the presence of other ions.

As a consequence, proper maintenance of ion concentrations and pH is important for continued immobilization by ionic binding and for prevention of desorption of the enzymes. When the biocatalytic activity exhausted, reloading it with fresh biocatalyst may reuse the carrier. Although renewal of the carrier and recovery of enzyme from the carrier are easy and the conditions of

Chapter I: Review of literature immobilization are mild, this binding is affected by the buffer used, pH, ionic strength, and by the reaction temperature.

I.2.1.3- Covalent attachment method:

This method of immobilization involves the formation of a covalent bond between an enzyme and a support material. The bond is normally formed between functional groups present on the surface of the support and the functional groups belonging to the amino acid residues on the surface of the enzyme (Kabral and Kennedy, 1991).

In most cases, the immobilization procedure is conducted in two steps. The first step involves activation of the support by using a specific reagent then attachment to the enzyme. Covalent binding of an enzyme to carriers leads to the formation of stable linkages thus inhibiting leakage completely. A disadvantage of this method is the partial loss of the activity due to conformational change of the enzyme is always observed. The functional groups of the enzyme, which are commonly, involved in covalent binding, are the nucleophilic species such as amino groups, carboxy-, sulfydryl-, hydroxyl- and phenolic functions (Chibata, 1987).

I.2.1.4- Gel entrapment method: In this method enzymes are physically enclosed in suitable matrix. The entrapment method differs from the adsorption and the covalent binding in that the enzyme molecules are free in solution, but restricted in movement by the lattice structure of the gel (Bickerstaff, 1995). The porosity of the gel lattice is controlled to ensure that the structure is tight enough to prevent leakage of enzymes, yet at the same time allow free movement of substrate and product.

Chapter I: Review of literature

With optimal pore size and proper configuration, the support matrices can keep firm hold of intact enzymes, while permit selective passage of small substrate molecules. Enzyme activity and stability are retained to a satisfactory extent under favorable microenvironmental conditions (Gonzalez-Saiz and Pizarro, 2001).

I.2.1.5- Encapsulation method : Encapsulation of enzymes can be achieved by enveloping the biological components within various forms of semi permeable membranes (Oboillot et al., 1994).

It is similar to the entrapment method in that enzymes are free in solution, but restricted in space. Many materials have been used to construct microcapsules, for example, nylon and cellulose nitrate. The problems associated with diffusion are more acute and may result in rupture of the membrane if products from a reaction accumulate rapidly. A further problem is that the immobilized enzyme particle may have a density fairly similar to that of the bulk solution with consequent problems in reactor configuration, flow dynamics, and so on (Bickerstaff, 1995).

I.2.1.6- Cross-linking method : In this method the enzymes crosslink through their amino or carboxylic groups using different cross-linkers. Immobilization of enzymes has been achieved by intermolecular cross-linking of the protein, either to other protein molecules or to functional groups on an insoluble support matrix (Kabral and Kennedy, 1991).

Cross-linking of enzyme to itself is both expensive and insufficient, as some of the protein material will certainly be acting mainly as a support. This

Chapter I: Review of literature will result in relatively low enzymatic activity. Generally, cross-linking is best used in conjunction with one of the other methods. Cross-linking reactions are carried out under harsh conditions which can change the conformation of the active center of the enzyme and so may lead to significant loss of activity (Groboillot et al., 1994).

The reagents used in this method include glutaric dialdehyde, toluene diisocyante, and dimethyl adipimidate are bifunctional reagents that most widely used for this method. However, the toxicity of such reagents is a limiting factor in applying this method to many enzymes (Ckerstaff, 1995).

Each immobilization method has its advantages and disadvantages since it is generally accompanied by changes in enzymatic activity, optimum pH, temperature and stability. Depending on the type of support and type of immobilization process, these changes vary to a large extent (Aksoy and Tumturk, 1998). The comparisons between the different types of immobilization are listed in table 2

Table 2: Characteristics of methods of the immobi8lization (Tadashi and Tetsuya, 1999).

Characteristic Physical Ionic Covalent Cross- Entrapping adsorption binding binding linking method method Preparation Easy Easy Difficult Difficult Difficult Enzyme activity Low High High Moderate High Binding forces Weak Moderate Strong Strong Strong regeneration Low Moderate Moderate Low High Immobilization Low Low High Moderate Low cost

Chapter I: Review of literature

Among of these methods, the entrapment methods were recently and -more widely used. All of these methods based on the physical occlusion of the enzyme molecules within caged gel structure such that the diffusion of enzyme molecules to the surrounding medium is severely limited, if not rendered totally impossible (Yang, 2008).

In the entrapment method, the enzyme is added to a solution of synthetic monomers or natural polymers before gel formation. Gel formation is then initiated by changing the temperature, by adding a gel-inducing chemical or by using radiation of high or low energy (Bickerstaff, 1995).

Cross-linking in the gel can be achieved by mixing an enzyme with a polyionic synthetic or natural polymer and then cross-linking the polymer with multivalent cations in an ion-exchange reaction to form a lattice structure that traps the enzymes (ionotropic gelation) . A highly cross-linked gel can more effectively hold enzymes (Fadnavis et al., 2003).

Many natural polymers are used. They include agar gel, chitosan and alginate gel. The most common synthetic polymer used in the entrapment method is the polyacrylamide gel. Monomer and cross-linking agent proportions are responsible for both the porous structure and pore size of the gel. A correct selection of these variables and the use of the suitable synthesis conditions lead to an increase in the activity retained by the gel (Pizarro et al., 2000).

In this method to ensure catalytic activity, it is necessary that substrate and product molecules can pass into and out of the macroscopic structure unhindered.

Chapter I: Review of literature

Entrapment method differs from adsorption and covalent binding in that enzyme molecules are free in solution, but restricted in movement by the lattice structure of the gel (Bickerstaff, 1995).

Due to the lack of covalent binding, entrapment is a mild immobilization method, which is also applicable to the immobilization of viable cells (Yang, 2008). In this study, entrapment method was used in immobilization of α- amylase on natural polymer (containing chitosan and sodium alginate) and synthetic Thermoresponsive copolymer of N-isopropyl acrylamide and sodium alginate.

Chapter I: Review of literature

I.3- Polymers as carriers for enzyme immobilization:

The versatile chemical compositions and physical properties coupled with widely available structural forms of polymers have made them excellent candidates as supports for enzyme immobilization.

The choice of matrix is very important for the good performance of an immobilized enzyme system. It is then desirable that an enzyme carrier possesses large surface area, permeability, hydrophilic character, insolubility, chemical stability, mechanical stability, thermal stability, high rigidity, suitable shape, particle size, resistance to microbial attack, and regenerability (Qing- Ling and Yuan-Xun, 1998).

The physico-chemical properties of the polymeric support, therefore, directly affect the choice of a suitable coupling procedure for immobilization and, consequently, the biochemical and biophysical behavior of the immobilized enzymes. Enzymes immobilized on insoluble polymers are gaining importance in many industrial and biomedical applications (Nilsson, 1987).

A large number of natural and synthetic polymers have been used as solid supports for the attachment of enzymes. The selection of the supports depends on the method of immobilization. Owing to the importance of existence of suitable reactive groups, hydrophilic polymers were selected as enzymes immobilized supports.

3.I .1-Fundamental considerations in selecting a support and the method of imm obilization : The first consideration is to decide on the support material, then the main method of immobilization taking into account the intended used and application. Some of the points consider when making a decision are listed in table 3.

Chapter I: Review of literature

Table 3: Fundamental considerations in selection of the supporting material in enzyme immobilization.

Property Points for consideration

Physical Strength, noncompression of particles, available surface area, shape (beads/sheets), degree of porosity, pore volume, permeability, and density Chemical Hydropholicity (water binding by the support), inertness toward enzyme, available functional groups for modification, and regeneration/reuse of support Stability Storage residual enzyme activity, regeneration of enzyme activity, and mechanical stability of support material Resistance Disruption by chemicals, pH, temperature, and organic solvents. Safety Biocompatibility, toxicity of component reagents, health and safety for process workers and end product users, specification of immobilization preparation for food, pharmaceutical, and medical application. Economic Availability and cost of support, chemicals, special equipment, reagents, technical skill required, environmental impact, industrial-scale chemical preparation, feasibility for scale-up, continuous processing, effective working life, and reusable support. Reaction Flow rate, enzyme loading and catalytic productivity, reaction kinetics, side reactions

Natural polymers including polysaccharides (cellulose and its derivatives, dextran and chitosan) and proteins as well as synthetic polymers, such as polystyrene and polyacrylates, have been studied to immobilize enzymes. The selection of polymer depends on the method of immobilization. With most polymers, the major barrier is the lack of highly reactive functional groups on the surfaces for direct covalent bonding (Mohy Eldin et al., 1999). Often, surface modification and reactions are needed to fulfill this particular task.

Chapter I: Review of literature

I.3.2-Chitosan:

Chitosan [poly-β (1-4)-2-amino-2-deoxy-D-glucose], a cationic biopolymer, is a deacetylated derivative from chitin, the second most abundant polysaccharide in nature next to cellulose (Amorim et al., 2003).

The structures of chitin, chitosan and are shown in figure (3). Commercially available chitosan is obtained from crustacea and has been used in a wide variety of applications. Its membrane has several uses including food processing, protein purification, and skin replacement technology (Muzzarelli, 1983). Recently, chitosan has attracted great attention, which has been reported to be a promising polymer in medical and biotechnology areas.

Chitosan is known as an ideal support material for enzyme immobilization because of its many characteristics like hydrophilicity, biocompatibility, and low biodegradability (Martino et al., 1995), form versatility (powder, gel beads, fibers, capsules and membranes) high permeability toward water, good adhesion (Noda et al., 2001) and high affinity towards proteins. In addition to these, chitosan is an inexpensive, inert, non-toxic, high mechanical strength support, and is thus attractive for enzyme immobilization (Kumar, 2000 and Ibahim et al., 2002).

Chapter I: Review of literature

Figure 3: Structure of chitin and chitosan.

Chitosan amino and hydroxyl groups are reacted to modify the structural units under conditions mild enough to prevent glycosidic linkage hydrolysis. Also the amino groups of chitosan facilitate the immobilization of enzyme, either by physical or chemical reaction (Desai et al., 2006).

An important aspect is the uniformity of reaction when heterogeneous conditions have to be used, along with the degree of substitution of the modified samples. A number of reactions useful to enhance the chitosan properties and to impart new ones are reported in the literature (Peter, 1995).

For instance, the chemical modifications of chitosan yield more soluble polymers. The latter have higher biodegradability in animal bodies and physical properties of interest for applications in the solid state or in solution.

Enzymes such as proteases (Hideo and Atsuhito, 1991), catalase (Pifferi et al., 1993), proteinase (Min-Liang et al ., 1996), dextranase (Cetinus Oztop, 2003), peroxidase (Yuqing and Swee, 2000), tyrosinase (Carvalho et al., 2000), papain (Hong et al., 2002), β-galactosidase (Ghanem and

Chapter I: Review of literature

Skonberg, 2002), trypsin (Jianmin et al., 2006), urease (Tzonka et al., 2007), and pepsin (Gamze and Senay, 2007) have been immobilized on commercial crustacean chitosan.

Highly swollen chitosan beads, were prepared from cuttlefish wastes and cross-linked with glutaraldehyde or glyoxal, were used as support material for the immobilization of acid phosphatase and β-glucosidase (Ruey-Shin et al., 2002).

Glucose oxidase was immobilized on novel porous gels of chitosan-SiO 2 which were prepared by coupling chitosan with tetramethoxy silicane through the sol-gel approach at ambient conditions. Also chitosan exhibited a considerable affinity for β-glucosidase and was used as a carrier for it through different means such as adsorption and cross-linked with glutaraldehyde (Gallifuoco et al., 1998).

Furthermore, chitosan was used for the immobilization of glutamate oxidase and hence forming a biosensing element for the development of glutamate biosensors (Maogen et al., 2005). Natural polymer chitosan, in the form of beads, was successfully used for immobilization of lipase (Magnin et al., 2001).

Chapter I: Review of literature

I.3.3- Alginate:

Alginate is another type of the natural polymers which can be used in enzyme immobilization. Alginate is a natural anionic polysaccharide obtained by extraction from marine brown algae (Smidsrd, 1970). Alginate is a linear binary copolymer consisting of (1, 4) β-d-mannuronic acid (M) and α-l- guluronic acid (G) residues.

The uronic acid residues are distributed along the polymer chain in a pattern of blocks, where homopolymeric blocks of G residues (G-blocks), homopolymeric blocks of M residues (M-blocks) and blocks with alternating sequence of M and G units (MG-blocks) coexist. Alginate polymers isolated from different alginate sources vary in properties. Different algae, or for that matter different part of the same algae, yield alginate of different monomer composition and arrangement.

Different types of alginate are selected for each application on the basis of the molecular weight and the relative composition of mannuronic and guluronic acids. For example, the thickening function (viscosity property) depends mainly on the molecular weight of the polymer; whereas, gelation (affinity for cation) is closely related to the guluronic acid content. Thus, high guluronic acid content results in a stronger gel (Ertesvag and Valla, 1998).

Alginic acid can be either water soluble or insoluble depending on the type of the associated salt. The salts of sodium, other alkali metals, and ammonia are soluble, whereas the salts of polyvalent cations, e.g., calcium, are water insoluble, with the exception of magnesium. The alginate polymer itself is anionic (i.e., negatively charged) overall Martinsen et al ., 1989).

Polyvalent cations bind to the polymer whenever there are two neighboring guluronic acid residues. Thus, polyvalent cations are responsible for

Chapter I: Review of literature the cross-linking of both different polymer molecules and different parts of the same polymer chain. The process of gelation, simply the exchange of calcium ions for sodium ions, is carried out under relatively mild conditions. Because the method is based on the availability of guluronic acid residues, the molecular permeability does not depend on the immobilization conditions. Rather, the pore size is controlled by the choice of the starting material (Ertesvag and Valla, 1998). The ionically linked calcium alginate gel structure is thermostable over the range of 0-100ºC; therefore heating will not liquefy the gel (Ohlson et al., 1979).

++ + 2 Na (Alginate) + Ca ------> Ca (Alginate) 2 + 2 Na

The alginate beads have been characterized with respect to mechanical resistance and size distribution immediately after production and as a function of storage conditions. The beads remain stable in the presence of acetic acid, hydrochloric acid, water, basic water, and sodium ions. The latter stability applies when the ratio of sodium: calcium is less than 1:5. Complexing agents such as sodium citrate result in the rapid solubilization of the beads due to calcium removal (Serp et al ., 2000).

Fungal phenol oxidase was immobilized by entrapment in copper alginate beads (Palmieri et al ., 1994). Also ß-fructofuranosidase (Busto et al., 1995), inulase, glucose oxidase (Kierstan and Buke, 2000), catalase (Canh et al ., 2004) and α-amylase (Etran et al ., 2007) were successfully immobilized onto calcium alginate beads.

Alginate can be modified or activated to increase its stability and rigidity by adding some activators such as sodium periodate which oxidatively converting 2,3-dihydroxy groups into dialdehyde residues before gelation. Nisin which considered as strong bacteriocin was covalently immobilized on these

Chapter I: Review of literature activated alginate beads surface (Canh et al ., 2004). Immobilized nisin when submitted to proteolysis conserved its bacteriocin activity, strongly inhibiting the growth of Lactobacillus, whereas free nisin totally lost its activity.

Furthermore calcium alginate was copolymerized with poly (acrylamide-co- acrylic acid) to be used in immobilization of tyrosinase (Yahsi et al ., 2005) to improve its storage and thermal stability.

In addition, carbonyl groups of calcium alginate gal beads were activated by using (CDI) to be used in acetylcholiestrase immobilization (Tumturk et al., 2007). It was found that, this method of immobilization increased reuse number, and thermal stability of acetylcholiestrase.

It has been known that the net pores of Ca-alginate gel beads are so large that enzymes can sometimes leak out from the gel (Won et al., 2005). In order to overcome leakage of entrapped enzyme, coating of the alginate gel surface with chitosan has been attempted in the present work. Furthermore chitosan increases rigidity and protects alginate beads from solubilization in acetate and phosphate buffer.

Therefore alginate and chitosan can be copolymerized to form chitosan- alginate gel beads. This system can be used in enzyme immobilization (by entrapment method) to improve stability of the enzyme and control permeability.

Lipase (Betigeri et al., 2002), ß-galactosidase (Taqieddin et al., 2004), invertase (Gomez et al., 2006), and many enzymes were immobilized onto alginate–chitosan copolymer beads by entrapment method to increase their catalytic activities

Chapter I: Review of literature

In addition chitosan alginate beads have a wide range of medical and biological applications. For example Chitosan-alginate beads were used in antibodies immobilization after activation by glutaraldehyde (Albarghouthi et al ., 2000). Such a system can be applied for the development of immunoaffinity purifications and immunoassay.

Due to nontoxicity of alginate and chitosan, alginate-chitosan beads were used in cells immobilization. Such as Saccharomyces cerevisiae (Li, 1996) and Candida tropicalis (Jamai et al., 2001) were entrapped in these gel beads to increase their ethanol production.

Also lactic acid bacteria were immobilized on alginate-chitosan beads by entrapment after functionalization of these beads by succinylation (increasing the anionic charges) or by acylation (improving matrix hydropholicity) before gelation with calcium chloride (Le-Tien et al ., 2004). All these modifications were performed to increase the beads stability in acidic media and increase production of lactic acid.

I.3.4- Thermoresponsive polymer:

There is another type of the synthetic polymers, these thermoresponsive polymers belong to the stimuliresponsive polymers which can change its swelling behavior and other properties in response to environmental stimuli such as temperature, pH, solvent composition and electric fields. They have attracted great interest not only because of their unique properties but also because of their potential for significant technological and biomedical applications (Hoffman, 2002).

Chapter I: Review of literature

These thermoresponsive polymers include hydrogels. Among all hydrogels, temperature and pH-responsive hydrogels are the most favorable members, and have been widely investigated during the last decade, because temperature and the pH value are important environmental factors in biomedical and other systems (Miyata et al ., 2006).

In this study, thermoresponsive polymer was used in the enzyme immobilization. The thermoresponsive polymers have lower critical solution temperature (LCST). Below this critical temperature, water is a good solvent for the polymer, the polymer–solvent interactions are stronger than the polymer– polymer interactions so the gel expands and reswells. Above this critical temperature, water becomes a poor solvent for the polymer. Here, the hydrogen bonds are disrupted, and water is expelled from the polymer coils, which start to collapse and this process results in aggregation and formation of compact globules (Qiu et al., 1997).

It was reported the LCST behavior of a series of copolymers of NIPA with relatively hydrophilic monomers such as acrylic and propenoic acids, acrylamide, N-methylacrylamide, or with relatively hydrophobic monomers such as N-butylacrylamide and N-tertbutylacrylamide (Katime et al., 2001).

Poly(N-isopropylacrylamide) (PNIPAAm) hydrogel is the most studied temperature-sensitive hydrogel, having a critical temperature or lower critical solution temperature (LCST) around 33 oC .the polymer precipitates when heated above the (LCST) but become soluble when cooled below that temperature (Chen et al., 1998). This process is fully reversible. Owing to this unique property, PNIPAAm hydrogels have been widely used in biomedical fields, including protein–ligand recognition, immobilization of enzymes and cells and drug controlled release (Zhang et al., 2002).

Chapter I: Review of literature

In enzyme immobilization , the enzyme reaction can occur in a solution , after which the enzyme and the product can be recovered in an insoluble state by a small change of solution temperature then the immobilized enzyme can be recycled and utilized for further reaction (Miyata et al ., 1994).

The activities of immobilized enzymes depend on the degree of the gel swelling which influences the diffusion rates of substrates in the gel and products out of the gel matrix, which in turn would control the overall kinetic properties of the immobilized enzymes (Miller, 2001).

Poly (N-isopropylacrylamide) (PNIPAAm) hydrogel is prepared by polymerization of N-isopropylacrylamide with a suitable bifunctional cross- linking agent, most commonly, is bisacrylamide. Gel polymerization is usually initiated with potasium persulphate and the reaction rate is accelerated by the addition of a catalyst such as N,N,N ',N '-tetramethylethylenediamine (TEMED) (Hoare and Pelton, 2004).

Novel Thermoresponsive polymer was prepared by copolymerization of methacrylic acid and N-isopropylacrylamide, which was used to immobilize amylase (Hoshino et al., 1994). ß-galactosidase was entrapped into a thermally reversible hydrogel. This hydrogel was prepared from copolymer of N- isopropylacrylamide and acrylamide cross-linked by bisacrylamide (Park and Hoffman, 1998).

Furthermore, many enzymes immobilized into Thermoresponsive hydrogel copolymer such as isoamylase into NIPAAm-NAS, chymotrypsin into alkaline phosphatase into NIPAAm-NAS (Park and Hoffman, 1993), lipase into NIPAAm-N,N, dimethylacrylamide (DMAA) (Matsukata et al ., 1994),

Chapter I: Review of literature

NIPAAm-NAS (Chen et al., 1997), and thermolysin into NIPAAm-GMA- methacrylamide (Mam ) (Hoshino et al., 1997).

Composite membrane was prepared by casting hydrogel onto polyester support and used for α-amylase immobilization. This hydrogel consist of N- isopropylacrylamide, cross-linker (bisacrylamide), 2-hydroxyethyl methacrylate, soluble starch and N-acryloxy succinimide (NAS). This composite membrane is temperature sensitive with lower critical solution temperature (LCST) around 35 oC. The immobilized was more thermally stable than free enzyme (Chen et al., 1998).

Interpenetrated polymer networks (IPN) of poly (N-isopropylacrylamide) (PNIPA) and chitosan were prepared by free radical polymerisation and cross- linking of PNIPA with bisacrylamide (Alvarez-Lorenzo et al ., 2005). This interpenetration developed drug delivery systems with improved drug loading capacity (owing to chitosan) and sustained release behaviour (owing to PNIPA).

Chapter I: Review of literature

I.4- Amylases enzymes: Amylases are a class of hydrolases widely distributed in microbes, plants and animals. They can specifically cleave the O-glycosidic bonds in starch. Starch is a storage polysaccharide present in plants. Starch consists of two components, a linear glucose polymer, amylose, which contains α-1,4 linkages and a branched polymer, amylopectin in which linear chains of α-1,4 glucose residues are interlinked by α -1,6 linkages . Starch depolymerization by amylases is the basis for several industrial processes such as the preparation of glucose syrups, bread making and brewing. Amylases also play a significant role in seed germination and maturation and are instrumental in starch digestion in animals resulting in the formation of sugars, which are subsequently used for various metabolic activities.

amylose

amylopectin. Figure 4: Structure of amylase and amylopectin.

Chapter I: Review of literature

I.4.1- Types of amylases: Amylases can be classified into three main groups based on their mode of action: endoamylases, exoamylases, and debranching enzymes.

1- Endoamylases Endoamylases are known as liquefying enzymes. Theses enzymes hydrolyze the bonds located in the inner region of the substrate. Endoamylases are termed as α-amylase which cleave α-1, 4 glycosidic linkages in amyloses, amylopectin and related polysaccharides as glycogen resulting in rapid decrease of viscosity of starch solution as well as decrease in iodine staining power.

The products of hydrolysis are oligosaccharides with varying chain lengths and have α configuration at the C1 of the reducing glucose units (Jin et al ., 1999).

2- Exoamylases Exoamylases are known as saccharifying enzymes. These enzymes cleave α-1,4 glycosidic linkages in amyloses, amylopectin and glycogen from non-reducing ends by successive removal of maltose/glucose in step wise manner (Hill and McGregor, 1988).

Cereal and bacterial ß-amylases and fungal glucoamylases come under this category. The product of hydrolysis have ß configuration at the C1 of the reducing glucose units due to inversion of the product. In contrast to the action of endoamylase, this result in slow decrease in viscosity and iodine staining power of starch (Banks and Greenwood, 1977).

Chapter I: Review of literature

3- debranching enzymes: The branching points containing α-1,6glycosidic linkages are resistant to attack by α- and ß- amylases. Pullulanase specifically attack α-1,6 glycosidic linkages and can be produced by Aureobasidium pullulans (Muralikrishna and Nirmala, 2005). Glucoamylase can also attack a-1, 6 linkages but the reaction proceeds slowly compared to pullulanase action.

1.5-Alpha amylase:

α -Amylases (EC 3.2.1.1 endo-1, 4-α glucohydrolase) are extra cellular enzymes that randomly cleave the 1, 4- α -D-glucosidic linkages between adjacent glucose units in the linear amylase chain. They belong to the class of endoenzymes that split the substrate in the interior of the molecules and are classified according to their action and properties. For examples, amylases that produce free sugars are termed saccharogenic (dextrinogenic) and those that liquefy starch without producing free sugars are known as starch-liquefying (Pandy et al., 2000).

α- amylases are small proteins with a molecular weight of 20–55 kDa. They are calcium-containing enzymes with a single polypeptide chain (Vander et al., 2002). Amino acid composition of α- amylase from different sources is quite different, yet catalytic function is the same.

I.5.1-Role of calcium in α-amylase: Calcium ions protect α-amylase from denaturation and proteolytic enzymes. There is no evidence that calcium ions participate in enzyme substrate complex (ES) formation but calcium ions stabilize the tertiary structure of the enzyme to keep the enzyme in correct configuration for activity (Agarwal and Henkin, 1987).

Chapter I: Review of literature

I.5.2-Application of α- amylase: These enzymes are of great significance in present day biotechnology with applications ranging from food, fermentation, and textile to paper industries (Pandy et al., 2000). α- amylases are one of the most popular and important form of industrial amylases and the present review highlights the various aspects of microbial a-amylases. α- amylases are of particular interest because these enzymes are inexpensive and commercially available, and the assay is fast, simple, and inexpensive (Patcharin et al., 2003).

α- amylase is an important industrial enzyme, particularly those of bacterial and fungal origin. There are many applications for α–Amylases which can be summarized as follows: • Application in pharmaceutical industries (Arica et al., 2004). • Application in paper manufacture, food and fermentation industry (Pandy et al., 2000), baking and textile industries. • Application in detergents. • Application in starch liquefaction, starch degradation, synthetic chemistry for the production of oligosaccharides (Terashima and Katoh, 1996), in addition to biological approaches to obtain useful biochemicals from the starch. • Application in biotechnology for the determination of serum or urine, α– amylase activity for the diagnosis of acute pancreatitis in clinical chemistry and clinical medicine (Panteghini et al., 2002).

Chapter I: Review of literature

I.5.3-Existence of α-amylase in organisms:

α- amylase has been found in plant, animals and microorganisms.

• Plant α- amylase:

The enzyme is reported to occur in wheat, rye, barley, rice, maize, peanut and many plants (Qadeer et al., 1980).

• Animal α- amylase:

Among animals, α- amylases have been reported in the pancreas and saliva of animals. Pancreatic amylases hydrolyze cooked starch and uncooked one into dextrin then to maltose. They have (SH) groups which rapidly inactivated by heavy metal ions. In the presence of chloride ions, optimal activity is observed at pH 7.0, and calcium and chloride ions are necessary for stability (Granger et al., 1975). Salivary amylase can hydrolyze cooked starch into dextrin and maltose (optimum pH 6.7 and activated by chloride ions).

• Microbial α- amylase:

Although amylases can be derived from several sources, including plants, animals and microorganisms, microbial enzymes generally meet industrial demands. The major advantage of using microorganisms for the production of amylases is the economical bulk production capacity and microbes are easy to manipulate to obtain enzymes of desired characteristics (Lonsane and Ramesh, 1990).

Today a large number of microbial amylases are available commercially and they have almost completely replaced chemical hydrolysis of starch in starch processing industry (Najafi and Deobogkar, 2005).

Chapter I: Review of literature

Two major classes of amylases have been identified in microorganisms, namely α -Amylase and Glucoamylase. In addition, β -amylase which is generally of plant origin, has also been reported from a few microbial sources (Siedenberg et al., 1997).

Several industries employ microbial amylolytic enzymes and a growing industrial application for them is the enzymatic conversion of starch into a variety of sugar solution . Many species of microorganisms have already been identified as good amylase producers (Pandy et al., 2000). α -Amylase has been derived from several fungi, yeasts, bacteria and actinomycetes, however, enzymes from fungal and bacterial sources have dominated applications in industrial sectors fungi (Gupta et al ., 2003).

Bacillus species are considered to be the important sources of α-amylase and have been used for enzymes production . Even the most common species, B. subtilis was used in production of heat stable α- amylase (Najafi and Deobogkar, 2005).

In addition, α- amylase was purified from Amylolytic lactic acid bacteria (ALAB) which were used in lactic acid production. The studies on purified enzymes from different ALAB indicated that they generally are of high molecular weight, in contrast to that from Bacillus species (Olympia et al., 1995). For instance α-amylase from L. plantarum, L. amylovorus and L. amylophilus were reported to be 230 KDa, 140 KDa, and 100 KDa respectively (Aguilar et al., 2000).

Furthermore, another species of bacteria excreted α- amylase such as Clostridium acetobutylicum (Paquet et al., 1991), Clostridium perfringens type A (Shih and Labbe, 1995), and Nacardiopsis species (Stamford et al., 2001)

Chapter I: Review of literature

Several workers showed that many fungi exhibited α- amylase activity. These fungi include Aspergillus falvus , Aspergillus oryzae , Rhizopus delmer , Pencillium italicum , and Thermomyces lanuginosus (Sidkey et al., 1997).

Fungal α- amylases are less resistant to heat than bacterial α- amylases and become inactivated before starch gelatinizes. This is important in the making of bread and many industries.

In this study we concern with production of α- amylase from Aspergillus niger which is a filamentous ascomycete fungus and the most common species of the genus Aspergillus . A. niger is ubiquitous in soil and is commonly reported from indoor environment (Perfect et al., 2001).

A. niger was used in the industrial production of many substances. Various strains of A. niger are used in preparation of organic acids such as citric acid and gluconic acid. It is the most widely known for its role as a citric acid producer (Baker, 2006).

In addition, A. niger was used in production of many enzymes. A variety of these enzymes are important in the biotechnology industry. These enzymes include cellulose, lactases, invertases, pectinases and acid proteases (Schuster et al., 2002) elastase, proteinase and phospholipase (Alp and Arikan, 2008).

A. niger is important model organism for several important research areas including the study of eukaryotic protein secretion in general (Baker, 2006).

Chapter I: Review of literature

I.6- Immobilization of α–amylase: α–Amylase is an enzyme used in the catalysis of the hydrolysis of polysaccharides and converts them mainly to maltose and larger oligosaccharides. In the literature there are numbers of reports about α–amylase immobilization on various supports using different processes.

Organic polymeric carriers are the most widely studied supports because of the presence of rich functional groups, which provide essential interactions with the enzymes. One of the most important polymeric supports for α–amylase immobilization is the natural ones.

Many natural polymers were used in α–amylase immobilization. Dextran and carboxymethylcellulose are examples of natural polymers which were used (Wykes et al., 1971) for α–amylase immobilization. The bounded enzyme retained 67% from its initial activity.

In addition α-amylase was immobilized on collagen membrane. The immobilized enzyme resisted thermal inactivation better than the soluble α- amylase. Raising the temperature from 30°C to 50°C increases the rate of the activity of the bound enzyme (Strumeyer et al., 1974).

Also, gelatin was found to be a very efficient natural polymer, due to its extraordinary diffusion characteristics for immobilization as a carrier. immobilization of α–amylase into photographic gelatin was by chemical cross- linking with chromium (III) acetate and chromium (II) sulfate (Bayramoglu et al., 1992).The immobilized enzyme by chromium acetate and chromium sulphate retained 35.6 and 41.2% of their activities even after 20 assays during 200 minutes, respectively.

Chapter I: Review of literature

Another example for natural polymers is chitosan which was used widely as enzyme immobilization support. α–amylase was immobilized on chitosan micro beads with glutaraldehyde (GDA) as a cross-linker (Gonzalez et al., 1997).

A composite bead was prepared by mixing an equal weight of activated clay and chitosan and cross-linked with (GDA), were used as the supports for immobilization of α–amylase, β–amylase, and glucoamylase. In general, immobilized enzymes had broader pH and temperature ranges of high activity than free enzymes (Juang et al., 2005).

Cellulose and its derivatives are very attractive natural polymer supports for enzyme immobilization. Porous nitrocellulose was prepared to immobilize α– amylase (Tanyolac et al., 1998). Up to the third use, immobilized enzyme showed higher activity than that of free one mainly due to higher enzyme concentration in the membrane structure, and then the apparent activity decreased gradually.

Furthermore, α–amylase and glucoamylase, were immobilized on hydrophilic silica gel and DEAE–cellulose which entrapped in alginate beads (Seungjoo et al., 2005). When performing the hydrolysis of starch, the shifts of optimum temperature from 50°C to 60°C and pH from 5.0 to 5.5 after immobilization were due to covalent bond formation and polyionic characteristics of carriers, respectively. The remaining activities of the immobilized enzymes on hydrophilic silica gel and DEAE-cellulose entrapped in alginate beads were found to be 92.3% and 88.9% respectively, even after 10 times of reuse.

Acrylated soy bean was deoxidized to immobilize α-amylase by covalent binding and entrapment method. The catalytic properties of the enzyme were not

Chapter I: Review of literature considerably changed as compared with that of the non immobilized enzyme, but the immobilized enzyme has higher stability than the free enzyme (Kahraman et al., 2006).

While a thermostable α–amylase produced from Bacillus subtilis was immobilized by entrapment in calcium alginate gel capsules. Capsules prepared from 2% (w/v) sodium alginate and 5% (w/v) CaCl 2 were suitable for up to 20 repeated uses, losing only 30% of their initial efficiency (Maria et al., 2006). These calcium alginate capsules Increase of the enzyme reaction temperature from 50°C to 70°C resulted in higher starch hydrolysis rates by the immobilized α–amylase without affecting the capsules stability.

α–amylase was covalently immobilized on new isocyanate, acid chloride and carboxylic acid-activated plastic supports (Roig et al., 1993). The pH curves for the immobilized enzyme were in general found not to be shifted from the soluble enzyme's pH optimum, but the immobilized enzyme's temperature activity profiles were shifted to a lower temperature range when compared to the soluble enzyme.

Poly (methyl methacrylate –acrylic acid), is an example of synthetic polymer, which was prepared in the form of microspheres and its acid groups were activated by using either carbodiimide (CDI) or thionyl chloride (SOCl 2) (Hasirci et al., 1998). α–Amylase was covalently bound on these activated microspheres. The relative activities were found to be 80.4% and 67.5% for

(CDI) and (SOCl 2) bound enzymes, respectively. Maximum activities were obtained at lower pHs and higher temperatures upon immobilization compared to free enzyme.

Chapter I: Review of literature

Also, composite membrane was prepared by casting hydrogel onto a nonwoven polyester support and used for enzyme immobilization (Chen et al., 1998). The hydrogel consists of N –isopropylacrylamide, cross-linker bisacrylamide), HEMA, soluble starch, and N – (acryloxy) succinimide (NAS). α–Amylase was immobilized to the membrane by covalent bonds through reacting with the high reactive ester groups in NAS. The membrane – immobilized enzyme retained 32% of specific activity toward soluble starch when compared with that of free enzyme, and its properties were characterized and compared with those of the free enzyme. The immobilized enzyme was more thermally stable than the free one.

On the other hand, semi –interpenetrating polymer networks (IPNs) of poly (ethylene glycol), poly (vinyl alcohol) and polyacrylamide were prepared (Bajpai et al., 2003) as a support for α-amylase immobilization. The composition of IPN affected on the extent of immobilization.

Furthermore, poly (ethylene glycol dimethacrylate –n–vinyl imidazole) p(EGDMA –VIM) hydrogel was prepared by copolymerizing (EGDMA) with (VIM). Then these beads were used in the adsorption of α–amylase from Aspergillus oryzae in batch system. Storage stability was found to increase with immobilization. It was observed that enzyme could be repeatedly adsorbed and desorbed without significant loss in adsorption capacity or enzyme activity (Denizli et al., 2005).

α–amylase was entrapped into butylacrylate –acrylic acid copolymer (BuA – co –AAc) using γ–irradiation and found that, covering of α-amylase with bis-(2- ethylhexyl)sulfosuccinate sodium salt made the enzyme more stable than the uncovered form (Atia et al., 2005). Numerous publications have appeared

Chapter I: Review of literature describing the immobilization of α–amylase on some inorganic carriers and it will be summarized as following:

Also, a thermostable α–amylase was immobilized on controlled-pore glass beads (Emneus et al., 1990). The optimization of the hydrolytic effect of the immobilized enzyme on glycogen and native starch as substrates was investigated. The optimum pH was 6.0 for hydrolysis of the substrates.

Moreover, purified α- amylase was isolated from sugar cane juice then they have been immobilized on tricalcium phosphate gel (Gurmukh et al., 1990). The pH and temperature profiles of free and immobilized amylase did not show much alteration. The heat stability in the immobilized enzyme was found to be 83% compared to the initial value.

Specific activity of α–amylase immobilized in ordered mesoporous silicas for hydrolysis of starch has been studied (Raksh et al., 2005). When the enzyme was immobilized only on the external surface, the observed specific activity is low, but when enzyme immobilized inside the pores, the immobilized enzyme had high specific activity. It was further observed that, thermal and pH stability of the immobilized enzyme is higher compared to free α–amylase.

Immobilized α-amylase was prepared on malodorous silica. It was further observed that, thermal and pH stability of immobilized enzyme is higher compared to free enzyme (Pandya et al., 2005). This immobilization include three steps through alkyl amine was covalently bondedto silanol groups present on the surface of malodorous silica followed by reaction of aldehyde with alkylamine then the enzyme was immobilized on the modified silica through reaction of CHo with NH 2 of the enzyme.

Chapter I: Review of literature

Poly (dimer acid -co- alkyl polyamine) particles were prepared then activated by various chemicals such as carbodiimide (CDI), ethyl diamine (EDA), and hexamethylenediamine (HMDA) for covalent immobilization of α- amylase (Hasicri et al., 2006). It is found that HMDA activated poly (dimer acid -co- alkyl polyamine) particles demonstrated the highest stability and efficient reusability for the immobilized α-amylase.

α-amylase was covalently immobilized on phthaloyl chloride – containing amino group functionalized glass beads (Kahraman et al., 2007). The immobilized enzyme was stable up to 5 days and lost only 20% of activity in 25 days but the free enzyme lost its all activity within 15 days.

Chapter II: Materials and Methods Materials and Methods II.1- Production of alpha amylase: II.1.1- Cultural media for Aspergillus niger : • Dox’s agar medium (Dox, 1910): The contents of the Dox’s agar medium are as follow (g/L): Sodium nitrate 3.0

Potassium dihydrogen phosphate 1.0 Potassium chloride 0.5 Magnesium sulphate 0.5 Ferrous sulphate 0.001

Calcium chloride 0.001 Sucrose 2 All these contents dissolved into 1000 ml tap water and pH was adjusted c at 6.5 (by using 1M NaOH and concentrated HCl), then 20 g agar was added before sterilization.

II.1.2 -production medium • (ISP9 medium) (Shirling and Gottlieb, 1966): These contents of (ISP9 medium) are as follow (g/L):

Ammonium sulphate 2.64

Dipotassium phosphate 5.65

Potassium dihydrogen phosphate 2.38 Magnesium sulphate 1 Starch 10

All these contents were dissolved in 1000 ml distilled water then pH was adjusted (by using 1M NaOH and concentrated HCl) at 7 before sterilization

45 Chapter II: Materials and Methods II.1.3 - Purification of alpha amylase from Aspergillus niger : The following steps were performed for purification of α-amylase form Aspergillus niger .

1- Amylase production and preparation of Cell Free Filtrate (CFF): Aspergillus niger was cultivated in 2 liters capacity Erlenmeyer flasks each containing 1 liter of the ISP media. After sterilization, the flasks were inoculated with A. niger . The inoculated flasks were incubated into incubator at 30 oC for 72 hr. At the end of the incubation period, the culture was filtered through 0.2 µm pore size Millipore (Vivas et al., 2004), to obtain the cell free filtrate (crude enzyme preparations). These preparations were used immediately for extraction and purification of enzymes in various ways.

2- Crude enzyme fractional precipitation by ammonium sulphate: Solid ammonium sulphate was added to the crude enzyme preparation to reach 80 % saturations (Mitidieri et al ., 2005). This mixture was stored overnight at 4o C. The precipitated protein was obtained by centrifugation for 15 min at 15000 rpm under cooling. The obtained protein pellets were dissolved in known volume of 50 mM tris HCl (pH 8). This precipitate was used for further determination of both enzyme activity and protein content.

3- Dialysis: This purification step was carried out to remove the traces of ammonium sulphate. The dissolved precipitated pellets obtained from ammonium sulphate fractionation, which was dissolved in 50 mM tris HCl (pH 8), was introduced inside a dialysis membrane to be dialyzed against the same buffer for 24 hr at 4oC Finally, the enzyme preparation was concentrated against pure sucrose crystals (Tohamy and Shindia, 2001). The dialyzed concentrated enzyme kept at 4oC. The enzyme activity and protein content were also estimated.

46 Chapter II: Materials and Methods 4- Purification of α- amylase by applying on Sephadex G-100 gel chromatography techniques: The dialyzed crude enzyme preparation was applied on column packed with Sephadex G-100 (mesh, 100 µ). That was equilibrated with 50 mM tris HCl (pH 8) then eluted with the same buffer at pH 8. The dialyzed enzyme preparation was applied carefully to the top of the gel.

It was allowed to pass into the gel by running the column. Then the buffer was added without disturbing the gel surface and then the column was connected to the reservoir. A number of fractions were collected (each contain ~3 ml).

Both enzyme activity and protein content were determined for each separate fraction. The protein by Lowry method (Lowry et al ., 1951) and enzyme activity by Bernfield method (Bernfield, 1955) were measured after each step to determine enzyme specific activity. Sharp peak fractions was obtained, lyophilized and stored at -20°C until use for subsequent studies.

47 Chapter II: Materials and Methods II.2-Immobilization of alpha enzyme: II.2.1- Materials Enzyme: • α-Amylase (EC 3.2.1.1) was purified from A. niger as mentioned above. Monomers and polymers: • Chitosan flakes have 85% degree of deacetylation (Sigma). • Sodium alginate (Aldrich). • N-isopropylacrylamide (Aldrich). Amino acids and proteins: • Bovine serum albumin (Aldrich). Cross-linkers: • N, N methylenebis-acrylamide (Aldrich). Substrates: • Starch (British Drug Houses) (BDH). Buffer solution: • Sodium dihydrogen phosphate anhydrous (Fluka). • Sodium phosphate dibasic anhydrous (El–Gomhouria Co.). • Boric acid (Laboratory reagents). • Borax (Laboratory reagents). Sugars: • Maltose (British Drug Houses) (BDH). Other Chemicals: • Potassium sodium tartrate (British Drug Houses) (BDH). • Potassium persulfate (KPS) (Laboratory Rasyan). • Sodium carbonates anhydrous (Laboratory Rasyan). • Sodium chloride (British Drug Houses) (BDH). • Calcium chloride (Laboratory reagents). • Sodium hydroxide (Laboratory Rasyan).

48 Chapter II: Materials and Methods • 3, 5–Dinitrosalicylic acid (DNSA) (Panreac). • N, N, N, N tetramethylethylenediamine (TEMED) (Sigma). • 1% acetic acid (prepared by diluting 1ml of glacial acetic acid to 100 ml by distilled water).

Reagents for protein determination (Lowry et al ., 1951):

(1)- 2X Lowry concentrate was prepared from three main solutions as follow:

(a)-Copper reagent consist of 20 g sodium carbonate was dissolved in 260

ml distilled H 2O, 0.4 g copper sulphate penta hydrate were dissolved in 20

ml distilled H 2O and 0.2 g sodium potassium tartarate were dissolved in

20 ml distilled H 2O then all contents were mixed well.

(b)- 1% (w/v) sodium dodecyl sulphate (SDS).

(c)- 1M sodium hydroxide solution.

2X Lowry concentrate was prepared with ratio 3 (copper reagent):1(1% SDS):1(1M sodium hydroxide).

(2)- 0.2 N folin reagent.

49 Chapter II: Materials and Methods II.2. 2-Methods: II.2.2.1- Preparation of immobilized enzyme on chitosan and sodium alginate (Natural polymer): 0.5 g chitosan was dissolved in 50 ml glacial acetic acid (1%), 0.5 g sodium alginate was dissolved in 50 ml distilled H 2O at room temperature, then chitosan solution was added to sodium alginate solution and blended with magnetic stirrer for 6 hr.

6 ml of α-amylase solution (contain 3 mg α-amylase) was added to this solution and magnetically stirred for 1 hr. Then the homogeny mixture was dropped into 0.27 M CaCl2 to form immobilized enzyme beads.

II.2.2.2 - Preparation of immobilized enzyme on thermo-responsive polymer (synthetic polymer): The continuous medium was prepared by dissolving 400 mg potassium persulphate (KPS) (polymerization initiator) and 1200 mg CaCl2 in 40 ml of distilled water. The medium was purged by bubbling nitrogen (for get off oxygen) for (1 hr) before the injection of the disperse phase (including monomer, cross linker, stabilizer and enzyme).

For preparation of disperse phase, 480 mg N-isopropylacrylamide (NIPAM) (monomer), 20 mg N, N – methylenebisacrylamide (cross-linker), 30 mg Na- alginate were dissolved in 2.6 ml cold distilled water. One ml of enzyme solution including 0.36 mg α-amylase and 1 ml tetramethylethylenediamine (TEMED) (accelerator) were added to resulting solution.

This mixture was dropped by a system including syringe and dosage pump into the continuous medium which was kept at 20 o C and stirred magnetically with 250 rpm. The polymerization was conducted at 20 oC with the same stirring

50 Chapter II: Materials and Methods rate for 4 hr. The uniform and thermally reversible enzyme - gel beads 3 mm in size were obtained. The gel beads were filtered and washed extensively by cold distilled water and with cold borate buffer (pH 7.8), respectively.

The washed gel beads were collapsed in borate buffer medium (pH 7.8) at 35 o C for 30 min. Then the enzyme-gel beads were stored in the refrigerator at +4o C until use.

II.2.3- Determination of the amount of immobilized enzyme: The amount of immobilized enzyme was determined by subtracted amount of protein determined in supernatants and washing solutions from the amount of initial protein used for immobilization.

After the immobilization process, the supernatant and the washing solutions were collected. The Lowry method was used to determine the enzymes amount in solution (Lowry et al ., 1951).

In Lowry method, 400 µl 2X Lowry concentrate were added to 400µl protein solution. Then this mixture was incubated at ambient (room temperature) for a minimum 10 min. 200 µl of Folin reagent were added and mixed well (because this reagent decomposes rapidly). After 30 min the optical density of the sample was measured colorimetrically at 750 nm wavelength using spectrophotometer.

The protein content was determined from the standard curve for solutions containing known amounts of bovine serum albumin (BSA).

51 Chapter II: Materials and Methods

Figure 5: Standard curve for protein.

52 Chapter II: Materials and Methods II.2.4-Assay of enzyme activity: The activities of free and immobilized α-amylase were assayed by the method of Bernfield (Bernfield, 1955). In this method, The reducing sugar liberated from starch hydrolysis by α- amylase reduce 3, 5-dinitrosalicylic acid, resulting in formation of a colored product which can be measured spectrophotometrically at 540 nm.

The activities were determined by incubating 1ml of the reaction mixture containing equal volume of 1% starch and α-amylase in phosphate buffer (0.02 M, pH 7) at 25°C for 3 min, and then the reaction was terminated by the addition of 1ml of (DNSA) then the mixture incubated in boiling water bath for 5min then cooling. The amount of reducing sugar (maltose) was measured at 540 nm with maltose as the standard.

The specific activity of free and immobilized α-amylase was calculated according to the following equation:

moles maltose released Units/ mg = mg of α-amylase in reaction mixture The retained activities yield of the immobilized α-amylase on the different polymeric supports was calculated according to the following equation:

Specific activity of immobilized α-amylase Retained activity yield (%) = X 100 Specific activity of free α-amylase

53 Chapter II: Materials and Methods

Figure 6: Standard curve for maltose.

II.2.5- Physico-chemical properties of the free and the immobilized α- amylase: The activities of the free and the immobilized α-amylase on the different previous mentioned polymers (via entrapment method) were calculated by measuring the absorbance of the librated maltose at (540 nm), taking into account that the retention activities of the bound α-amylase on the different polymeric materials after immobilization process would be the initial activities of them in the following parameters study (relative activity 100%). The studying reactions were carried out at various pH values and temperatures, the effects of these parameters as well as storage stabilities and repeated use capabilities were examined.

54 Chapter II: Materials and Methods II.2.5.1- Effect of temperature on activity of the free and the immobilized α- amylase: The effect of temperature on the enzymes activities was investigated at various temperatures in range (30-80°C) in thermostatically controlled water bath under standard assay conditions for both the free and the immobilized α- amylase.

II.2.5.2- Effect of pH on activity of the free and the immobilized α-amylase: The effect of pH on enzymes activity was investigated by measuring the specific activities at different pH in the range (4.0-9.0) for both the free and the immobilized α-amylase respectively.

II.2.5.3- Repeated batch operational stability of the immobilized α-amylase: To evaluate the reusability of the immobilized α-amylase, the immobilized α- amylase was washed with distilled water and phosphate buffer after use and then suspended again in a fresh reaction mixture to measure the enzymatic activity. This process was repeated.

II.2.5.4- Storage stability of the free and the immobilized α-amylase: This experiment was carried to determine the stabilities of the free and the immobilized α-amylase after storage in phosphate buffer (0.02 M, pH 7) at 4°C and at 25 oC for 30 days. Then the residual activities were determined as described above and the activity of each preparation was expressed as a percentage of its residual activity compared to its initial activity.

55 Chapter II: Materials and Methods II.2.5.5-Thermal Inactivation: Thermal stability of the free and the immobilized enzymes was estimated by ◦ ◦ measuring their activities after incubation at 40 C and 60 C in phosphate buffer

(0.02 M, pH 7) with change of incubation time.

II.2.5.6- Effect of γ -irradiation on both free and immobilized α-amylase activity: The effect of γ–irradiation on the activities of both the free and the immobilized α-amylase were estimated by determination of enzyme activity after exposure various doses (0–50 kGy).

A 60 Co γ -ray source with a dose rate of 1.22 k G/h was used as the γ -ray source.

II.2.5.7-Kinetic measurements:

The Km and the Vmax of the free and the immobilized enzyme were determined from the initial rates of the reaction of the enzyme with starch ◦ solution using different concentrations of the substrate at 25 C and pH 7, from the plot of the reciprocal of the reaction velocity against the reciprocal of the substrate concentration. The values for Km and Vmax were calculated from the intercepts on the axes.

IV.2.5.8- Determination of the starch hydrolysis efficiency of the free and the immobilized α-amylase: The starch hydrolysis efficiencies of the free and the immobilized enzyme were determined by measuring starch concentration after hydrolysis process. In this method, Solution of starch (10 mg/ ml) in phosphate buffer pH 7 was mixed

56 Chapter II: Materials and Methods with the free or with the immobilized enzyme. The samples were taken every 5 minute then the starch concentration was estimated.

The starch concentrations were determined by adding 200 l sample was to a 1 mL 0.5 N hydrochloric acid. From this solution, a 200 l sample was taken and added to 5 mL of iodine solution. The absorbance of each sample was measured after 15 minutes spectrophotometrically at 620 nm.

0.2

0.18 y = 0.0338x + 0.0033 R2 = 0.9999 0.16

0.14

0.12

0.1

0.08

0.06

Optical denisty at 620nm at denisty Optical 0.04

0.02

0 0 1 2 3 4 5 6

Starch concentration (mg/ml)

Figure 7: Starch standard curve.

57 Chapter II: Materials and Methods Equipments: • UV/ visible recording spectrophotometer (Spectronic® Gensystem 2). • Hotplate with magnetic stirrer (Jenway, Model 1000). • Electronic balance PM300 for weighting. • pH meter. • Shaker water bath (GFL) (Type 1083). • Incubator. • Autoclave. • Laminar flow (SLEE Mainz).

58 Chapter III: Results

RESULTS

III.1.Precipitation, Purification and Characterization α-amylase Produced by A. niger: In the present study, the crude enzyme preparations, were obtained from A. niger cultures was subjected to purification methods using ammonium sulphate precipitation, dialysis and gel filtration.

The crude α-amylase preparations were achieved by fermentation processes with 7 days old cultures of A. niger, under the optimum cultural conditions. At the end of the fermentation process of the tested organism, the culture broth from each fermented flask was filtered through whatman No. 1 filter paper to separate the mycelium pellet from the culture filtrates.

The specific activity of the crude enzyme was measured after filtration process and it was found to be was 16.5 U/mg then the crude enzyme was purified by the following steps:

III.1.1. Fractional precipitation with ammonium sulphate: The previous filtrates were purified by fractional precipitation with ammonium sulphate. The results showed that, a highly considerable amount of protein in the culture filtrate was recovered when ammonium sulphate was used for precipitation with 80% saturation (Mitidier et al., 2005).

The solutions were left overnight at 4°C until the complete precipitation occurred and then centrifuged at 15000 rpm for 15 min at 4oC. After precipitation, each of the obtained fractions was resuspended in a definite volume of 50mM tris-HCl at pH 8. The dissolved fractional precipitates were tested for both α-amylase activity and protein content.

59 Chapter III: Results

The results in table 5 showed that the precipitation with 80% saturated ammonium sulphate increased the specific activity of α-amylase from 16.5 to 20U/mg, with a purification fold 1.4

III.1.2- Dialysis:

After precipitation, the enzyme solution was dialyzed against the same buffer for 24 hr at 4oC then dialyzed against sucrose crystals for another 24 hr at 4o C. After that the specific activity was 28.2 U/mg and the purification fold was increased to 1.7

III.1.3- Purification by gel filtration chromatography:

After allowing the Sephadex gel column to be settled, the enzyme sample was applied to Sephadex G-100 column (2.5 x 45cm), equilibrated, and eluted with the same buffer. 50mM tris-HCl buffer pH 8 was used for elution. About 30 fractions were collected, each with 5ml aliquot. Enzyme activity and protein content of each fraction was evaluated.

The results of this step were recorded in table 5 and represented graphically in figure 4. These results showed that, the highest enzyme activity, protein content and specific α-amylase activity were found in the fractions numbers (7, 8, 9, 10, 11, and 12). These fractions were collected, pooled and dialyzed against the same buffer at 4°C, and then lyophilized and stored at -20 °C to be used in the immobilization steps.

After performing the gel filtration step of the obtained protein on Sephadex G-100 column, the specific activity of A. niger α- amylase was increased to be 34.9 U/mg protein, which is corresponding to 2.11 purification fold.

60 Chapter III: Results

Table 4: Gel - filtration of the crude α-amylase enzyme produced from A. niger.

No of Amount of enzyme Specific fraction protein (mg/ml) activity(U/ml) activity(U/mg) 1 0.01 0.00 0.00 2 0.029 0.026 0.89 3 0.024 0.4 1.6 4 0.013 0.26 2 5 0.059 0.26 4.4 6 0.019 0.16 8.4 7 0.0195 0.21 10.7 8 0.108 2.07 19.1 9 0.155 3.5 22.5 10 0.27 9.5 34.5 11 0.030 0.31 10.13 12 0.031 0.3 9.6 13 0.0139 0.13 9.35 14 0.0297 0.21 7.07 15 0.0255 0.18 7.05 16 0.032 0.22 6.87 17 0.045 0.25 5.55 18 0.0158 0.08 5.06 19 0.032 0.22 6.87 20 0.03 0.14 4.6 21 0.029 0.1 3.4 22 0.03 0.08 2.8 23 0.024 0.08 3.9 24 0.05 0.05 1.0 25 0.01 0.03 1.5 26 0.05 0.03 0.6 27 0.06 0.04 0.6 28 0.01 0.012 0.12 29 0.01 0.001 0.1 30 0.00 0.00 0.00

61 Chapter III: Results

Figure 8: Gel filtration of crude α-amylase produced from A. niger .

The results of partial purification steps of A. niger showed that, as the purification steps precede the specific activities of the enzyme were increased from (16.5 to 34.9 U/mg), while the recovery in each case decreased (from 100 to 20.7 %), and the corresponding purification folds were also increased up to 2.09 (table 5).

Table 5: The results of the partial purification steps of A. niger α-amylase. Purification Steps Enzyme Soluble Specific Folds Activity Protein Activity (U/ml) (mg/ml) (U/mg) Crude enzyme 0.297 0.018 16.5 1 Ammonium sulphate 0.64 0.032 20 1.4 precipitation Dialysis 1.4 0.049 28.57 1.7

Sephadex G-100 9.5 0.272 34.9 2.11

62 Chapter III: Results

III.2-Immobilization of α-amylase: In this study, α-amylase was immobilized by entrapment into a natural polymer and into one of the synthetic polymers. The natural polymer consists of chitosan and alginate. The synthetic polymer is Thermoresponsive hydrogel which was prepared by copolymerization of N-isopropylacrylamide and sodium alginate.

The steps of the entrapment of this enzyme inside these two polymers were discussed in the chapter of material and methods. After entrapping of the enzyme by the two methods, the immobilized α-amylase gel beads were formed and washed with distilled water and stored to determine their physicochemical properties.

III.2.1- Physicochemical properties of the immobilized and the free α- amylases: III.2.1.1- Optimum temperature: The activities of the free and the immobilized α-amylase were assayed at various temperatures (30 - 80°C) in a thermostatically controlled water bath under the standard assay conditions.

The results were depicted in figure 9 showed that, as the reaction temperature increase the activities of free, the enzyme immobilized on chitosan and alginate, and enzyme which was immobilized on N-isopropylacrylamide and chitosan were increased until reached to their optimum temperatures then the activities were decreased.

The optimum temperature of the free enzyme was 40 oC and that for the immobilized enzymes was slightly shifted toward 50 oC. Also, figure 9 showed

63 Chapter III: Results that, the activity of the free enzyme decreased to 37% at 80 oC but the activities of the immobilized enzyme onto the two polymers were the same (80%) at 80 oC.

Furthermore, it was observed that the temperature profile of the immobilized enzyme was slightly broader than that of the free one.

Figure 9: The effect of temperature on the enzyme activity of the free and the immobilized enzyme on chitosan and alginate as well as the immobilized enzyme on N-isopropylacrylamide and alginate.

64 Chapter III: Results

III.2.1.2- Optimum pH: The pH dependence of the immobilized α-amylase activity was compared with that of the free enzyme in the pH range 3.0–9.0 at 25°C.

Figure 10 showed that, the optimum pH of the immobilized enzyme on chitosan and alginate is similar to that of the free enzyme (pH 7.5) while the optimum pH for the immobilized enzyme on N-isopropylacrylamide and alginate was slightly shifted to higher pH.

Figure (10): The effect of pH on the enzyme activity of the free enzyme, immobilized enzyme on chitosan and alginate, and on that of immobilized enzyme on N-isopropylacrylamide and alginate.

65 Chapter III: Results

It was observed that, at pH 9 the retained activities of the free enzyme and that of the immobilized enzyme into N-isopropylacrylamide and alginate gel beads, and that of the immobilized enzyme into chitosan –alginate beads were 40%, 56%, and 70% respectively. While at the pH 4, the corresponding retained activities were 10%, 15%, 30%, respectively.

III.2.1.3-Repeated Batch Operational Stability of immobilized α-amylase: To investigate the ability of the immobilized enzyme for re-usability, after the reaction the immobilized enzyme was removed and washed with distilled water and phosphate buffer and added to another fresh reaction.

A repeated batch operational stability curve of the immobilized α- amylase on chitosan and alginate and on N-isopropylacrylamide and alginate was constructed (figure 11).

The results of 12 batches showed that, the activities of immobilized enzymes were 85% after 3 reuses then deceased gradually to 45% after 12 reuses.

66 Chapter III: Results

Figure 11: Reusability of the immobilized α-amylase on chitosan and alginate and N-isopropylacrylamide and alginate polymers.

III.2.1.4-Storage Stability:

The stability of the free and the immobilized α-amylase on chitosan and alginate and on N-isopropylacrylamide and chitosan was examined after they stored in the dry state for 30 days in phosphate buffer, pH 7.

The results of the storage stability were illustrated in figure 12. This figure showed the relative activity of the free and the immobilized enzymes stored at 25°C. It was observed that, the relative activity of the free enzyme dropped to 0% after a storage time of 20 days but the relative activity of the immobilized enzyme was decreased slowly to 80% after 30 days.

67 Chapter III: Results

Figure 12: The storage stability plotted as a % relative activity vs. time, for the free and the immobilized α-amylase at 25 oC.

68 Chapter III: Results

Figure 13: The storage stability plotted as % relative activity vs. time, for the free and the immobilized α-amylase at 4oC.

On the other hand, figure 13 showed the relative activity of the free and the immobilized enzymes stored at 4°C in which, the relative activity of the free and the immobilized enzymes decreased to 65% and 90%, respectively, after 30 days.

69 Chapter III: Results

III.2.1.5-Thermal Inactivation: The thermal inactivation kinetics at 40°C and at 60°C of the free and the immobilized enzymes showed remarkable achievement of thermostability by the immobilized form.

Firstly, figure 14 showed the thermal inactivation kinetics at 40°C in which, the relative activity of the free and the immobilized enzymes were decreased to 45% and 85%, respectively.

Figure 14: The thermal inactivation kinetics of the free and the immobilized α- amylase at 40 ˚C.

70 Chapter III: Results

Secondly, during incubation of the free at 60°C as lost its activity after 45 min but the immobilized enzymes activities were only decreased to 70% in each case after 60 min (figure 15).

Figure (15): Thermal inactivation kinetics of free and immobilized α-amylase at o 60 C.

71 Chapter III: Results

III.2.1.6- Effect of γ-irradiation on both the free and the immobilized α- amylase activities: Figure 16 showed that, the free α- amylase loses about 60% its relative activity at a dose of about 50 kGy. However, the immobilized α -amylase showed more radiation-resistance. The activity of the immobilized α -amylase, irradiated at a dose of 20 kGy, showed no significant change in its relative activity, while a reduction of about 25% was measured at a dose of 50 kGy.

Figure 16: Effect of γ -irradiation on both the free and the immobilized α- amylase activities.

72 Chapter III: Results

III.2.1.7-Kinetic Properties: The reaction kinetics were analyzed for the free and the immobilized α- amylase in phosphate buffer (pH 7.0) using different concentrations of starch (0.25- 25 mg/mL).

Figures 17 and 18 were used in determination of km and Vmax of the free enzyme. Figures 19 and 20 were used in determination of the km and the Vmax of immobilized enzyme on chitosan and alginate. Figures 21 and 22 were used in determination of the km and the Vmax of the immobilized enzyme on N- isopropylacrylamide and alginate.

The data in Table 6 showed the kinetic parameters of both free and immobilized α-amylase preparations. The increase of the Km and decrease of the Vmax values is as expected because of less availability of the substrate to the active site of the enzyme due to chemical boding, diffusion limitation, and confinement of enzyme molecules with polymeric support.

Table 6: Kinetic Parameters of Free and Immobilized α-amylase.

-1 -1 Enzyme form Km (mmol L ) Vmax (mmol min )

Free Enzyme 0267 02

Chitosan-aliginate preparation 0833 021

Acrylamide preparation 0909 019

73 Chapter III: Results

Figure 17: Michaelis-Menten direct fit for the Free enzyme.

74 Chapter III: Results

Figure 18: Lineweaver-Burk double reciprocal plot of 1/vi versus 1/[S] for the free enzyme

75 Chapter III: Results

Figure 19: Michaelis-Menten direct fit for the immobilized enzyme on chitosan- alginate.

76 Chapter III: Results

Figure 20: Lineweaver-Burk double reciprocal plot of 1/vi versus 1/[S] for the immobilized enzyme on chitosan-alginate.

77 Chapter III: Results

Figure 21: Michaelis-Menten direct fit for the immobilized enzyme on N- isopropylacrylamide and alginate.

78 Chapter III: Results

Figure 22: Lineweaver-Burk double reciprocal plot of 1/vi versus 1/[S] for the immobilized enzyme on N-isopropylacrylamide and alginate.

79 Chapter III: Results

III.2.1.8-Hydrolysis of starch using free and immobilized enzymes: Figure 23 demonstrated the time-course of the soluble starch hydrolysis using the free and the immobilized α-amylase preparations. In which the immobilized enzymes on alginate and chitosan and on alginate and N- isopropylacrylamide decreased the starch concentration from 10 mg/ml to 0.5 mg/ml after 25 min but the free enzyme decreased starch concentration from 10 mg/ml to 2 mg/ml. So the immobilized enzymes on alginate and chitosan and the immobilized enzyme on alginate and N- isopropylacrylamide had higher hydrolytic activity than the free enzyme because the immobilized enzymes increase the starch hydrolysis rate.

Figure (23): Starch hydrolysis using the entrapped alpha-amylase.

80 Chapter IV: Discussion

Discussion In the present study, the crude enzyme preparations obtained from A. niger cultures was subjected to purification methods using ammonium sulphate precipitation, dialysis and gel filtration. After fermentation of A. niger , all the culture broth was filtered to separate the mycelium pellet from the culture filtrates. This crude enzyme was with specific activity of 16.5 U/mg. The crude was then precipitated with 80% saturation of ammonium sulphate.

Most of the bacterial and of the fungal α-amylases were precipitated with 80% saturation of ammonium sulphate. Ammonium sulphate was the strongest precipitant since it is highly soluble in water, cheap and has no deleterious effect on the structure of proteins, so far all these reasons, precipitation with ammonium sulphate was selected as the former step of purification program (Mitidier et al., 2005).

The α-amylase precipitation was completely achieved at 80% saturation level of ammonium sulphate. Similar conditions of α-amylase from Lactobacillus plantrum and bacillus subtilis (Najafi et al ., 2005).

On the other hand, α-amylase precipitation was performed with 60% saturation of ammonium sulphate as in α-amylase purification from Nocardiopsis sp. (Stamford et al ., 2001).

Precipitation step increase the specific activity and the purification fold. As in this study, the specific activity of α-amylase increased after precipitation from 16.5 to 20 U/mg, with a purification fold of 1.4.

81 Chapter IV: Discussion

After precipitation, the resultant precipitate was dissolved in a least amount of 50 mM tris HCl buffer with pH 8, and then it was dialyzed against the same buffer for 24 hr then against sucrose crystals for another 24 hr. These steps decreased the total volume of the enzyme causing increase in the enzyme activity to 28.57 U/mg.

The dialyzed crude enzyme was concentrated by gel filtration step in which the dialyzed crude enzyme was applied on Sephadex G100 column chromatography. The first peak contains the highest activity and protein content. The most active fractions from the Sephadex column were pooled for further investigation. The gel filtration step of the obtained protein on Sephadex G-100 column increased the specific activity of α-amylase to 34.9 U/mg proteins, which is corresponding to 2.11 purification fold. This means that the enzyme was more purified than that of the first. Also, these results indicate the sephadex G 100 is suitable for purification of the enzyme.

The purified α- amylase was immobilized by entrapment into two different polymers. The first polymer was natural copolymer from sodium alginate with chitosan. The other polymer was synthetic copolymer from N- isopropylacrylamide with sodium alginate.

during preparation of the immobilized α-amylase by entrapment into calcium alginate beads, α-amylase was added to a mixture of chitosan and soluble sodium alginate before gelation process. Then such mixture was dropped into calcium chloride solution in which the calcium sodium replacement was occurred to form insoluble calcium alginate chitosan beads entrapping α- amylase.

82 Chapter IV: Discussion

The immobilized α-amylase beads on chitosan and alginate had regular size of 3mm. This was similar to immobilized invertase and lipase by entrapment into chitosan alginate beads as was previously reported by Gomez et al. (2006).

In this study, the synthetic polymer was a copolymer of polyN- isopropylacrylamide and alginate. PolyN-isopropylacrylamide is a thermoresponsive hydrogel and responsible for lower critical solution temperature (LCST) of the polymer or copolymer which controls the activity of the immobilized enzyme. Alginate is natural, nontoxic and was used to give the immobilized enzyme in the beads form.

After preparation of the immobilized α-amylase, the physicochemical properties of the free and the immobilized enzymes into both polymers were investigated to determine their optimum conditions.

The upper temperature for enzyme activity is governed by the limits of enzyme stability. As well known immobilized enzyme is more resistant to heat and denaturing agents than the free form (Ohtsuka et al., 1984).

The temperature/activity profiles of the two immobilized enzymes on chitosan-alginate and polyN-isopropylacrylamide were remarkably different from that of the free enzyme. In this work, the optimum temperature of free enzyme was 40 oC and that for the immobilized enzymes was shifted slightly toward 50 oC. Also it was observed that the temperature profile of the immobilized enzyme was slightly broader than that of the free one.

A similar increase in the temperature optima had been found for the immobilized α-amylase by Yoshida et al .(1989), β-amylase (Emi et al., 1990),

83 Chapter IV: Discussion and α- chymotrypsin (Chen et al., 1997) but a reverse effect had been shown for the immobilized urease Atia et al.( 2005).

The increase in the optimum temperature for the activity of the immobilized enzyme could be due to the fact that the actual temperature in the microenvironment of the matrix was lower than that in the bulk solution and change the physical and chemical properties of the enzyme (Decordt and Saraiva, 1993).

For the immobilized α-amylase on chitosan and alginate, the increase in optimum temperature may be due to the presence of chitosan which can cause increase in the stability of the immobilized enzyme towards denaturizing by raising temperature.

Concerning the immobilized α-amylase on copolymer of N-isopropyl acrylamide and alginate, the increase in the optimum temperature was caused by PolyN-isopropylacrylamide which with LCST (33 oC). As the temperature was increased above the LCST, the immobilized beads became collapsed which increased the partition of the substrate near the enzyme active site causing an increase in the enzyme activity of the entrapped enzyme (Chen et al ., 1997).

Similar effect of the copolymers of N-isopropyl acrylamide as support was observed in the immobilization of trypsin, peroxidase, and β- galactosidase enzymes (Chen et al. , 1998). These results demonstrated the effectiveness of the carriers protecting the enzyme activity under higher temperature values (Tien and Chiang, 1999). Free enzyme has a different optimum pH than that of the immobilized enzyme on a solid matrix. This difference depends on the surface and residual charges on the solid matrix and the nature of the enzyme-bound pH value in the

84 Chapter IV: Discussion immediate vicinity of the enzyme activity. A change in the optimum pH normally accompanies the insolubilization of enzymes, depending upon the polymer used as support. Since the enzyme activity is markedly influenced by the environmental conditions, especially pH, the behavior of enzyme immobilization is useful for understanding the structure-function relationships of enzyme proteins.

Therefore, it is very useful to compare the activity of the free α-amylase and the immobilized α-amylase as a function of pH (Elcin, 1995). The activity of the free α-amylase was compared with that of immobilized preparation at various pH values (4.0-9.0), and the activity was measured under the standard assay conditions.

The chitosan-alginate immobilized preparation has the same optimum pH as the free one, but the pH profile is considerably widened. This may be due to diffusional limitations (Kim and Park, 2004). While the optimum pH for the immobilized enzyme on poly N-isopropyl acrylamide and alginate was slightly shifted to a higher pH value than the free enzyme. This result was consistent with the previous immobilization processes (De Cordt et al., 1993).

The enzymes are very sensitive biocatalysts against environmental conditions and may lose their activities quiet easily. Thus, it is meaningful to characterize their reusability and storage stability for preparative or industrial uses.

Reuse stability for the immobilized enzyme is very important in economics, and this stability can make the immobilized enzyme more advantageous than its free form (Peng et al., 2005).

85 Chapter IV: Discussion

In this study, the immobilized α-amylase on chitosan and alginate and on N- isopropylacrylamide and chitosan retained 85% of its activity after three reuses. After that, the immobilized enzyme activity decreased gradually. The significant fall in the activity of the immobilized enzymes in the repeated-batch operation could be due to gradual inactivation of the immobilized α-amylase (Turnturk et al., 2007).

One of the most important parameters to be considered in the enzyme immobilization is the storage stability. The stability of the free and the immobilized α-amylase on the polymeric supports was examined after they stored in the dry state.

It had been seen from the results that, the relative activity of the free enzyme decreased from 100% to 60% after a storage time of 30 days at 4o C but it dropped to 0% after 20 days incubation at room temperature. Unlike free enzyme, the immobilized enzymes retained 80% and 90% of the specific activity at 4o C and at room temperature, respectively.

The higher stability of the immobilized α-amylase can be attributed to preventation of autodigestion and thermal denaturation as a result of the entrapment of α-amylase molecules into chitosan and alginate and into N- isopropylacrylamide and alginate. As expected, the loss in the relative activities when the enzyme was stored at 25 °C was much greater than that when the enzyme was stored at 4 °C (Lee et al ., 2000)

The effect of incubation at various temperatures on the residual activities of the free and the immobilized enzymes was estimated.

86 Chapter IV: Discussion

The thermal inactivation kinetics at 40°C and 60°C of the free and the immobilized enzymes showed remarkable thermal stabilities after immobilization. This thermal stability was higher at 40°C than that at 60 °C under the same conditions.

The increased tolerance to thermal denaturation may be due to multipoint attachment of enzymes molecules to the polymer carrier through reduction in molecular mobility (Lee et al., 2000).

The improvement in the thermal stability of the immobilized a-amylase preparations is more clearly evident from the experiments conducted at 60°C than those in which preparations were exposed to a temperature of 40 °C.

The effect of γ–irradiation on the activities of the free and the immobilized α- amylase was determined by estimation of enzyme activity after exposure to various radiation doses (0–50 kGy). It was found that, the immobilization of the enzyme onto polymeric support results in a good radiation-resistance of the enzyme. These results indicated that, the immobilization of the enzyme increase its stability and rigidity against the destructive effect of radiation.

The determination of the difference in km between the free and the immobilized enzymes is always useful to understand the influence of the microenvironment on substrate diffusion to the immobilized enzymes.

E + S ES E + P Where E is the enzyme, S is the substrate, and P is the product.

The relation between substrate concentration and the rate of the reaction can be described by the Michaelis–Menten equation. Km is defined as the substrate

87 Chapter IV: Discussion concentration that gives a reaction velocity of 1/2 Vmax. This parameter reflects the effective characteristics of the enzyme and depends upon both partitioning and diffusional effects (Chase and Yuhui, 2005).

Where Vmax is the maximum rate of reaction and Km is the Michaelis constant (mg/mL).

Vmax defines the highest possible velocity when the enzyme is saturated with substrate. Therefore, this parameter reflects the intrinsic characteristics of the immobilized enzyme, but may be affected by diffusional constraints.

The km (Michaelis constant) values were estimated from Line weaver-Burk plots. In this study, Line weaver-Burk plots for the enzymatic hydrolysis were shown to be linear relationships and the apparent kinetic parameters obtained Km and V max for the two enzyme preparations were listed in table 6.

The immobilized α-amylase showed higher km value than that of the free enzyme because of the α-amylase concentration gradient that is established from the bulk of the solution to the immobilized form within the support. Therefore, the substrate saturation of the immobilized enzyme requires a higher concentration of α-amylase in solution.

In general, Km of an immobilized enzyme is higher than that of the free enzyme due to less availability of the substrate to the active sites of the enzyme due to chemical boding, diffusion limitation, and confinement of the enzyme

88 Chapter IV: Discussion molecules with the polymeric support. The change in the affinity for its substrate is also caused by structural changes in the enzyme introduced by the immobilization procedure and lower accessibility of the substrates to the active site of the immobilized enzyme (Arica et al., 2004). Similar results were obtained by immobilization of α-amylase on poly methyl methacrylate (Li et al ., 1996), alumina, and zirconia and immobilization of catalase on glyoxal- crosslinked chitosan (Cetinus and Oztop, 2003).

For the enzyme which was immobilized by entrapment techniques, a slight increase in both Km and Vmax were found. These reults demonstrated that, the immobilization procedure and the support did not markedly alter the microenvironment of the enzyme molecules probably because of the mild immobilization procedure.

The time-course of soluble starch hydrolysis using the free and the immobilized α-amylase preparations was estimated. In this work, it was found that, the immobilization process resulted in a high increase of the starch hydrolysis rate. These results showed that, the efficiency of the hydrolysis reaction using α-amylase can be greatly improved by previously covering the enzyme with the polymer that protected the enzyme during the hydrolysis process maintained its hydrolytic activity. This effect of immobilization is similar to immobilized lipase which had high catalytic activity than that of the free one ( Zaidi et al ., 2002).

89 Chapter V: Summary

SUMMARY

The immobilized enzymes on polymeric supports are prepared for purpose of repeated use and the possibilities of continuous reaction system. One of the most important properties is the stability of proteins when they are used in some medical and industrial applications. The immobilization of the enzymes improves this property as well as many other properties.

In this study, alpha amylase was purified and immobilized onto two different polymers. α- amylase was used in this study for its biological and industrial applications. It is used in paper textile, pharmaceutical applications, food, and detergent industries.

α- amylase was found in plants, animals, and microorganisms. Purification of α-amylase from microorganisms is the main source of α-amylase because it was excreted from many bacteria and fungi.

In this study, α-amylase was purified from Aspergillus niger. Fractional precipitation of the α-amylase produced by A. niger with 80% ammonium sulphate saturation.

The crude enzyme was applied on column chromatography packed with

Sephadex G 100 for purification. The active eluents containing partially purified enzyme were collected for further investigation. The specific activity of α- amylase was (34.9 U/mg) which was corresponding to 2.09 fold purification for the tested organism.

The purified α-amylase was immobilized by entrapment method into two types of polymers. One of them was natural consist of chitosan and alginate. The other polymer was synthetic consist of N- isopropyl acrylamide and alginate.

90 Chapter V: Summary

The temperature optimum and thermal inactivation showed a severe loss in the activity of the free enzymes, while the temperature profile of the immobilized enzymes was much broader at higher temperatures demonstrating the effectiveness of the polymer protecting the enzymes. Also, the immobilized enzymes (natural polymer and synthetic polymer) showed higher thermal stability.

Optimum pH and stability showed that immobilization of enzymes resulted in more stability for these enzymes and even to a pH profile considerably widened due to the diffusional limitations.

The reuse efficiency of the free and immobilized enzymes showed the immobilized enzymes showed decrease in the relative activity after being used 12 times.

Storage of the free and immobilized enzymes for a month showed that the free α-amylase lost most of its catalytic activity after storage. The storage of the immobilized enzymes Storage at room temperature showed much less stability of the immobilized enzymes than in 4o C.

Subjecting the free and immobilized enzymes to doses of γ- radiation (0-50 kGy) showed complete loss in the activity of free enzyme at 5 kGy, while the immobilized enzymes showed high resistance to γ- radiation. The kinetic studies of free and immobilized enzymes showed that the immobilization increased Km and decreased Vmax values of the enzyme.

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113 اا

اا اتااادافادةاا اتااواااهاوت اااتاواواتهة ا ا اصاىآة . وامهةاراأااا اأهات ا . أااماومآ اتاوا . وارقواتو ا وااتا . وااأأاتوااتواتا . و اتاهأرجااوذواج تآةواامااا اتا . وهةارااجااااا وذ اوتآة . أ اا اا اا اامآتام،أنأ امآتامآ ٨٠ ٪ . ( د) فآ دا اواامدآج ١٠٠ ،وااءا ا ا اات . ووأناطا ارا٣٤٩ وة/ ام .

١ اا

اااااتاهوا . واانازانواتوهات . زاناتااى. أات ا . أااناوواآاو ازان. ودراااواااات . وآدراارةاوااار ىأت تاةآندراارةتاآن اآادراتاارةالآءةاوااد اوتا . ودردراسارواو ،وأتاأن ات ااتأدىاأآاتوأ اساروآنأآأل Diffusional Limitation. أتدرااتاةوادر٤ و ٢٥ در ل أناتاةتوآنا تا ٤ دراا ٢٥ در . اتاتااو ٠( ٥٠ ) آاى،ثانآطاتا ةوات ااتطت ٢٠ آاى . ودراااصاآتاةوااات . وان Km اتااةأ Vmax .