American Journal of Advanced Drug Delivery

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Original Article Novel Approach of Alkaline Protease Mediated Biodegradation Analysis of Mustard Oil Driven Emulsified Bovine Serum Albumin Nanospheres for Controlled Release of Entrapped Pennisetum glaucum (Pearl Millet) Amylase Kirti Rani*, Shreya Goyal and Chanchal Chauhan

Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, Sec-125, Noida-201303 (UP), India

Date of Receipt- 10/01/2015 ABSTRACT Date of Revision- 05/03/2015 Date of Acceptance- 10/03/2015 Amylase is known for starch saccharification and catalyses the starch hydrolysis producing glucose and limit dextrin which has wide range of applications in fermentation, textile, leather, detergents, food and paper industries. In the present work, amylase was extracted from Pennisetum glaucum (pearl millet) was entrapped in bovine serum albumin (BSA) nanoparticles in which emulsification was done by mustard oil and covalently coupled by using well known cross linking agents, glutaraldehyde. Bovine serum albumin is considered the most biodegradable, biocompatible and non-toxic biological matrix material for the preparation of enzyme bound nanospheres to enhance thermal stability, storage stability and suitability of bound enzymes as compared to free enzymes toward persisting ambient conditions. Formation of nanoparticles was confirmed by Dynamic Light scattering (DLS) and determination of exact size was observed Address for by Scanning Electron Microscopy (SEM). Thermal stability of Correspondence entrapped Pennisetum glaucum amylase at 70ºC and storage stability Amity Institute of at 4ºC was enhanced for 3 hours 30 minutes and 12 months Biotechnology, Amity respectively as compared to free enzyme which was observed for 50 University Uttar minutes and 1 day only. Studied optimal temperature of entrapped Pradesh, Noida,, Sec- amylase was found to be 70ºC and it was significantly higher as 125, Noida-201303 compared to free enzyme (40ºC) which increased the industrial (UP), India. efficacy and catalytic viability of amylase after entrapment. Their E-mail: biodegradation was carried out by using different units of alkaline [email protected] protease (10U, 15U, 20U, 25U, 30U, 35U, 40U, 45U, 50U) to study sustained and controlled release of entrapped Pennisetum glaucum.

American Journal of Advanced Drug Delivery www.ajadd.co.uk Rani et al______ISSN 2321-547X

Characterization of kinetic parameters were also performed for Pennisetum glaucum extracted amylase and entrapped amylase for their comparative study for their optimal pH, incubation time, substrate concentration, CaCl2 concentration and temperature for maximal enzyme activity which was tested by dinitrosalicylic acid method.

Keywords: Pearl millet, Pennisetum glaucum, Amylase, Bovine serum albumin, Mustard oil, Nanospheres, Glutaraldehyde, Encapsulated, Emulsified, Dynamic light scattering, Scanning electron microscopy.

INTRODUCTION Amylases (EC 3.2.1.1) are very aroused innovative industrial interest for popular ubiquitous enzymes produced by using supporting nanomaterials to provide plants, animals and microbes, where they high surface area/volume ratio, low mass play a dominant role in carbohydrate transfer limitation and excellent particle metabolism.1 The increased industrial use of mobility in enzyme catalysed reactions. biocatalysts is coined to be emphasized by Hence, various cross-linked enzyme the increasing demand of commercially aggregates, microwave-assisted immobili- enzymes worldwide approximately to US $ zation, click chemistry technology, 3 billion. Globally, 45% of the enzymes mesoporous supports and most recently produced are being used in the food industry nanoparticle-based immobilization of and the rest % is shared by detergent enzymes.6 Enzymes immobilized into (34.4%), textile (11 %), leather (2.8%), nanoparticles are showed to have broader paper and pulp (1.2%) and other industries operational range of pH and temperature as including their diagnostics and therapeutics well as having higher thermal stability than implications (5.6%).2 Yet, despite of the native enzymes.7 Nanoparticles are well industrial applications of enzymes, their uses known, these days, for offering a wide has been limited due to several factors variety of biomedical applications including broadly categorised into two categories e.g. cancer diagnosis, radiotherapy, drug and high cost of the biological catalysts and their gene delivery, sensors and biosensors , labile nature.3,4 Immobilized enzymes have environment remediation and water several advantages over free enzymes such purification etc.8,9 Researchers are still as more convenient handling of the enzyme, working to improve current technologies to facile separation from the product, develop new advanced applications that minimizing or eliminating protein favour immobilization of industrial contamination of the product and efficient important enzymes into desired recovery and reuse of the biocatalyst.5 nanoparticles to make them more feasible Immobilization of enzymes is done by and convenient in the point of their handling entrapment, covalent Binding, cross-linking and resistivity.10,11 Amylases have and adsorption. Advanced nanotechnology significance biotechnological approach inspired researchers to immobilize the which has already encased the class of industrial important enzymes in chemically industrial enzymes which has been grabbed modified nanopreparation which have approximately 25-30% of the world enzyme

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market.12,13 These are ubiquitous enzymes nanospheres was done with alkaline protease which are produced by plants, animals and (10U, 15U, 20U, 25U, 30U, 35U, 40U, 45U, microbes where they have prominent role in 50U) for controlled release of entrapped carbohydrate metabolism [14]. Amylases Pennisetum glaucum amylase. Successful saccharifiy starch, glycogen and sustained release of enzyme from emulsified oligosaccharides in a random manner, chemically modified bovine serum albumin liberating reducing sugars as glucose, is widened the application of immobilized maltose and limit dextrins which have amylase by increased sustained and potential industrial applications.15-17 controlled usability.30,31 Kinetic parameters Recently, amylase was immobilized onto of free and immobilized enzymes was silica, alumina, chitin, tannin sepharose, carried out to study the effect of pH (1.5- ionic binding on Amberlite IR-120, Dowex 11.5), effect of temperature (5°C-100°C), 50W, DEAE-Cellulose DE-52 by either effect of incubation time (20mins- 4 hours), physical adsorption or entrapment in effect of substrate concentration (0.50%- 18,19 calcium alginate beads. It was also found 1.5%) and effect of CaCl2 (1%-10%) was to be entrapped within a semi-permeable studied for their activity by dinitrosalicyclic polymeric membrane and gel matrix too.20 acid method (DNS method).24,23 In our present study, entrapment of Pennisetum glaucum amylase was achieved MATERIALS AND METHODS into bovine serum albumin nanospheres by chemical modification done by butanol, Extraction amylase from Pennisetum emulsification done by mustard oil and glaucum seeds enzyme was loaded by using glutaraldehyde Cotyledons of 3-day old seedlings of as cross-linking agent into nanospheres in germinating seeds of Pennisetum glaucum which the bound active gradient was were homogenized in 0.05M sodium dispersed in a hydrophilic or non-polymer phosphate buffer (pH 7.0) in the ratio of layer aqueous medium.21,23,24 Hence, this 6ml: 1grams of seeds. It was centrifuged for immobilization approach was coined 15min at 4°C at 3000rpm. The supernatant was collected for crude enzyme and stored reasonably safe, simple, cheap and offering 10,22,23 good mechanical strength, high porosity for at 4°C. substrate/ product diffusion used for immobilization of enzymes.25 Bovine serum Amylase assay by DNS method albumin was chosen biocompatible, Amylase activity was estimated by biodegradable, non-toxic and echo-friendly determining the amount of reducing sugars matrix in which emulsified entrapment or released from starch. 0.5 ml of the enzyme encapsulation of amylase was done where extracts were added to 1 ml of the starch sustained release of bound enzyme form solution and the mixture incubated at 37ºC prepared nanospheres was achieved for 20 min. After incubation, 2 ml of successfully by alkaline protease.21,24-27 For dinitrosalicylic acid (DNS) was added and the determination of size and the reaction mixture was boiled at 100ºC for characterization of prepared enzyme loaded 5 minutes. The amount of reducing sugars in nanospheres was done by Dynamic light the final mixture was determined Scattering (DLS) and Scanning electron spectrophotometrically at 570 nm. One unit Microscope was used.28,29 Their of enzymatic activity is defined as the amount of enzyme that produces 1 μmol of biodegradation of mustard oil driven 32, 34 emulsified bovine serum albumin maltose per minute.

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Preparation of Pennisetum glaucum amylase Alkaline mediated biodegradation of loaded mustard oil driven emulsified bovine prepared bovine serum albumin nanospheres serum albumin nanospheres for controlled release of entrapped Mustard oil bath was prepared by Pennisetum glaucum amylase adding 25% glutaraldehyde and 2.6ml of n- Entrapped Pennisetum glaucum butanol into 50ml of almond oil in glass Amylase was incubated at 37°C with different beaker. 50U Pennisetum glaucum amylase concentrations of alkaline Protease (10U, was mixed with 8ml of bovine serum 15U, 20U, 25U, 30U, 35U, 40U, 45U, 50U). albumin. This solution filled in a 10 guage Alkaline protease was already reported syringe and was dispersed in the prepared excellent enzyme to combat high alkaline mustard oil bath. It was continuously stirred reaction conditions and has suitability for overnight on the magnetic stirrer. Next day, biodegradation.34,38 The enzyme assay of it was centrifuged at 5000rpm at 4°C for entrapped enzyme was performed for 20mins and the supernatant was discarded. consecutive twelve days by DNS The pellet was washed with chilled diethyl method.11,24,27,34 ether and acetone. The pellet was re- dispersed into acetone in bath sonicator for Study of kinetic properties 12mins.11 Enzyme assay of supernatant was The free enzyme and immobilized performed by DNS method to know % of enzyme are characterized for their different entrapment of enzyme in mustard oil driven kinetic properties i.e. effect of pH, effect of bovine serum albumin nanospheres. temperature, effect of incubation time, effect of CaCl2 concentration and effect of substrate % of entrapment concentration.11,24 The effect of pH on activity % of entrapment of enzyme was of free and immobilized enzymes was studied calculated by determining the residual by performing enzyme assay at different pH enzyme activity from reaction mixture in using acetate and phosphate buffer by varying which enzyme entrapment was done into pH from 1.5 to 11.5. The effect of time on the bovine serum albumin nanospheres. activity of free and immobilized enzyme was Amylase assay was performed by using studied by performing the enzyme assay at DNS.31,32 different time (20 minutes to 4 hours). % of encapsulated enzyme = Optimal substrate concentration for maximal Specific activity of encapsulated / Specific enzyme activity for free and immobilized activity of free enzyme X 100. enzymes which were estimated by incubating the reaction mixture at different Characterization of amylase loaded bovine concentrations of starch solution (0.50%- serum albumin nanospheres 1.50%). The effect of CaCl2 on activity for Prepared enzyme loaded bovine free and entrapped enzymes was studied by serum albumin nanoparticles were confirmed performing the enzyme assay at different by Dynamic Light Scattering Technique. The concentrations (1%-10%). Optimal size and morphologies of the prepared temperature for free and encapsulated enzyme nanospheres were further studied by scanning for maximal activity was studied by electron microscopy.11,28 incubating the reaction mixture for 15minutes at different1 temperature (5°C – 100°C). These kinetic properties of enzyme were determined by performing DNS method.33-37

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RESULT AND DISCUSSION minutes for encapsulated enzyme whose results were also comparable with previous Percentage of entrapment observations (Fig. 6).11,24,33 Optimal substrate Entrapment of Pennisetum glaucum concentrations of free enzyme and extracted amylase in to mustard oil driven encapsulated enzyme was found to be 1.5% chemically modified bovine serum albumin for both by varying starch concentration from nanospheres was observed to be 74% which 1%-1.5% whose results were also pretty was found to be comparable with previous comparable to previous finding (Fig. studies done.11,12,31,32,36 11,24,33,35,36 7). Optimal CaCl2 concentration for maximal amylase activity for free and bound Characterization of Pennisetum glaucum enzyme was found to be 5% and 4% amylase loaded bovine serum albumin respectively whose results were similar to nanospheres previous results (Fig. 8).11,24,33-35 Optimal The prepared enzyme loaded bovine temperature for maximal activity for free serum albumin nanospheres was confirmed enzyme and bound enzyme was observed by using Dynamic Light Spectroscopy that 40⁰C and 70⁰C respectively whose results average particle size were in range of 1 to were also pretty comparable to previous 10nm with a observed sharp peak in between 11,33-36 11,28 results (Fig. 4). It was also found that (DLS) (Fig 1). The nanospheres was after encapsulation, thermal stability (Fig. 5) found to be spherical in shape and and storage stability (Fig. 9) were enhanced fluroresence also observed under Scanning for bound enzyme (at 70ºC for 2 hours 30 Electron Microscope (SEM) (Fig. 2) which minutes and 12 months respectively) as confirmed the formation of diene adduct that compared to free enzyme (at 70ºC for 50 leads of exposure of tryptophan residue minutes and one day only respectively) which during the encapsulation by using were also sharply comparable to previous glutaraldehyde which acts as free acid and has observations (Table 1).34-36,39,40 much stronger fluroresence as compared to 12 phenylalanine and tyrosin. Observed size Alkaline mediated biodegradation Study for prepared amylase loaded bovine serum Biodegradation of mustard oil driven albumin nanospheres under Scanning emulsified bovine serum albumin Electron Microscope was found to be in the nanospheres of encapsulated Pennisetum range of 76.3nm to 111.5nm which were glaucum amylase was performed by pretty lesser than the previous study (Fig. 11,40 incubating 2 mg of encapsulated amylase 2). loaded nanosphere with alkaline protease (10U, 15U, 20U, 25U, 30U, 35U, 40U, 45U, Studied kinetic properties 50U) overnight at 4°C. The study was carried The free and encapsulated enzyme out for consecutive 14 days and 45U of were characterized for their different kinetic alkaline protease was found to achieve properties i.e. effect of pH, effect of controlled and sustained release of temperature, effect of incubation time, effect encapsulated Pennisetum glaucum amylase of CaCl2 concentration and effect of substrate form almond oil driven emulsified bovine concentration. Optimal pH was found to be serum albumin nanoparticles (Fig. 10). First 4 similar which was 11.5 for encapsulated days, the release of the enzyme was negligible amylase and free enzyme whose results were while it increased over the next 2 days. From slightly comparable to previous studies (Fig th 11,24 6 day onwards, the enzyme activity was 3). Optimal incubation time for free stable and not much change was observed enzyme was found to 50 minutes and 30

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from day 6 till day 12th and then increased till Hence, the spectrum of encapsulated amylase day 14th. Enzymatic functionality was in alkaline protease mediated biodegradation observed even after 2 weeks of protease of mustard oil driven emulsified bovine serum incubation owing to highly stable albumin form of nanospheres is coined nanoparticle formation whose results are significant scientific approach due to which found to be comparable with previous storage stability, thermal stability and findings.11,34,36,38-40 reusability of industrial important amylase are increased which, in turn, whose application CONCLUSION may be expanded to automatic dishwashing detergents as well as textile desizing, In our study, Pennisetum glaucum pharmaceutical, paper industries and also may (Pearl millet) amylase was entrapped into be used as bio-robotic tool for drug targeted mustard oil driven emulsified bovine serum delivery system to combat myriad of lethal albumin nanospheres via glutaraldehyde epidemics and diseases as most advanced bio- coupling with 74% of % of retention activity. clinical appliances for betterment of mankind The Characterization of prepared amylase too. loaded nanoparticles was performed by Dynamic Light Spectroscopy (DLS) showed REFERENCES the presence of nanoparticles and the exact size was confirmed by observing under the 1. Sivaramakrishnan S, Gangadharan D, Scanning Electron Microscope (SEM). The Nampoothiri K M, Soccol C R and observed size of prepared nanoparticles was Pandey A, Alpha -Amylase from found to be 56.2nm to 84.3nm and prepared microbial sources- an overview on enzyme loaded bovine serum albumin recent developments. Food Technol nanopsheres were spherical in shape as well Biotechnol. 2006; 44: 173-184. fluroresence was also observed due to diene 2. D'Souza S.F. Trends in Immobilized adduct formation by having tryptophan Enzyme and Cell Technology. Indian residue after the glutaraldehyde coupling. The Journal of Biotechnology, 2002; 1: 321- result of biodegradation study were 338. performed for consecutive 14 days which 3. D’Souza S.F. Immobilised enzymes in showed that 45U of alkaline protease was Bioprocess. Current Science, 1999; found efficient in sustained and more 77(1). sustained release of bound enzyme from 4. Roger A. S. Enzyme Immobilization: almond oil driven emulsified bovine serum The Quest for Optimum Performance. albumin nanoparticles. It was observed that in Adv. Synth. Catal, 2007; 349: 1289 – the initial 7 days, the release of amylase was 1307. negligible. From the 11th day onwards, there 5. Bernfeld P, Amylases α and β. In: was an increase in amylase activity and at 13th Methods in Enzymology, ed. I, By day the release of bound enzyme was noticed Colowick S.P. and Kalpan N. O. (1955), at its highest. The bound amylase was found Academic Press: New York. to have increase storage stability for 12 6. Shakeel A, Qayyum H. Potential months when stored at 4°C and excellent applications of enzymes immobilized reproducibility and thermal stable up to 70ºC on/in nano materials: A review. for 3 hours 30 minutes which was remarkably Biotechnology Advances, 2012; 30(3): higher as compared to free enzyme with 512–523. storage stability for one day only and having 7. Xu J, Ju C. Synthesis and thermal stability at 70ºC for 50 minutes only. Characterization of Magnetic

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Nanoparticles and Its Application in 15. Sivaramakrishnan S, Gangadharan D, Lipase Immobilization. Bull. Korean Nampoothiri K M, Soccol C R and Chem. Soc, 2013; 34(8). Pandey A. Alpha -Amylase from 8. Petkova GA Záruba K, Žvátora P and microbial sources- an overview on Král V. Gold and silver nanoparticles for recent developments. Food Technol biomolecule immobilization and Biotechnol, 2006; 44: 173-184. enzymatic catalysis. Nanoscale Research 16. Rani K. A novel comparative assessment Letters, 2012; 7:287. of extracted amylase acitivity in 9. Xu J, Sun J, Wang Y, Sheng J, Wang F germinating and germinated seeds of and Sun M. Application of Iron Cicer arietinum, Ceci neri and Pisum Magnetic Nanoparticles in Protein sativum. Int J Pure &Applied Sci, 2014; Immobilization, Molecules 2014; 19(8): 2(2): 191-193. 11465-11486. 17. Van der Maarel MJEC, van der Veen B, 10. Wang J, Meng G, Tao K, Feng M, Zhao Uitdehaag JCM, Leemhuis H, X. Immobilization of Lipases on Alkyl Dijkhuizen L. Properties and Silane Modified Magnetic applications of starch converting Nanoparticles: Effect of Alkyl Chain enzymes of the α-amylase family, J. Length on Enzyme Activity, PLoS ONE, Biotechnol, 2002; 94: 137-155. 2012; 7(8). 18. Thippeswamy S, Girigowda K and 11. Rani K, Chauhan C. Biodegradation of Mulimani V. H, Isolation and Cicer Arietinum Amylase loaded identification of α-amylase producing Coconut oil driven Emulsified Bovine Bacillus sp. from dhal industry waste, Serum Albumin Nanoparticles and their Indian Journal of Biochemistry & application in Washing Detergents as Biophysics, 2006; 43: 295-298. Eco-Friendly Bio-Active Addictive, 19. Morais R. R, Pascoal A. M, Caramori S. World Journal of Pharmacy and S, Flavio M. Lopes, and Kátia F. Pharmaceutical Sciences, 2014; 3. Fernandes, Immobilization of -Amylase 12. Sharma KR. Preparation of emulsified onto Luffa operculata Fibers, Enzyme encapsulated nanoparticles of bovine research, 2013; 13(2): 134-140. serum albumin of bound glucose oxidase 20. Kennedy J F. Enzyme Technology,in and their application in soft drinks/non- Biotechnology, Kennedy J F and Cabral alcoholic beverages, Biotechnol & J M S (Eds.), VCH Publ, 1987; 7. Biomaterials, 2012; 2(2): 1-5. 21. Elcin A. E. and Y.M. Elcin. Polycation- 13. Azad MA, Bae JH, Kim JS, Lim JK, coated polyanion microspheres of urease Song KS, Shin BS, Kim HR, Isolation for urea hydrolysis. Artif Cells Blood and characterization of a novel Substit Immobil, Biotechnol, 2000; 28: thermostable alpha-amylase from 95-111. Korean pine seeds. N Biotechnol, 2009; 22. Rani K. Aqueous two phase purification 26: 143–149. of Vigna radaita amylase and its 14. Deb P, Talukdar S A, Mohsina K, characterization, Int J Cur Pharm Rev & Sarker P K, and Sayem S M. Production Res, 2012; 3(3): 47-53. and partial characterization of 23. Rani K. Immobilization of Vigna extracellular amylase enzyme from radiate, Vigna mungo, Cicer arietinum Bacillus amyloliquefaciens P-001. (white) and Cicer arietinum (Black) Springer Plus 2013; 2:154. amylases onto variety of activated

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Table 1. Kinetic Parameters of free and mustard oil driven emulsified bovine serum albumin nanospheres of encapsulated Pennisetum glaucum amylase

Kinetic Parameters Free amylase Encapsulated amylase Optimal pH 11.5 11.5 Temperature Optima 40°C 70°C Thermal Stability at 70°C Up to 50 minutes Up to 2 hours 30 minutes Optimal time of incubation 50 minutes 30 minutes Optimal Substrate concentration 1.5% 1.5%

Optimal CaCl2 concentration 5% 4% Storage stability at 4ºC Up to 1 day Up to 12 months

Figure 1. DLS of Mustard oil driven emulsified bovine serum albumin nanoparticles of encapsulated Pennisetum glaucum (Pearl millet) amylase

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Figure 2. SEM of mustard oil driven emulsified bovine serum albumin nanospheres of encapsulated Pennisetum glaucum (Pearl millet) amylase

Figure 3. Effect of pH on free amylase and mustard oil driven emulsified bovine serum albumin nanospheres of encapsulated Pennisetum glaucum amylase

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Figure 4. Effect of temperature on free amylase and mustard oil driven

emulsified bovine serum albumin nanospheres of encapsulated Pennisetum glaucum amylase

Figure 5. Study of thermal stability at 70°C of free amylase and mustard oil driven emulsified bovine serum albumin nanospheres of encapsulated Pennisetum glaucum amylase

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Figure 6. Effect of incubation time on activity of free amylase and mustard

oil driven emulsified bovine serum albumin nanospheres of encapsulated Pennisetum glaucum amylase

Figure 7. Effect of substrate concentration on activity of free amylase and mustard oil driven emulsified bovine serum albumin nanospheres of encapsulated Pennisetum glaucum amylase

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Figure 8. Effect of CaCl2 concentration on activity of free amylase and

mustard oil driven emulsified bovine serum albumin nanospheres of encapsulated Pennisetum glaucum amylase

Figure 9. Study of storage stability at 4°C of mustard oil driven emulsified bovine serum albumin nanospheres of encapsulated Pennisetum glaucum amylase

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Figure 10. Biodegradation analysis of mustard oil driven emulsified bovine serum albumin nanospheres to study controlled release of encapsulated Pennisetum glaucum amylase with alkaline protease (10U, 15U, 20U, 25U, 30U, 35U, 40U, 45U, 50U)*EU-Enzyme Units

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