Immobilization of Lactobacillus acidophilus β-galactosidase on chitosan obtained from the shells of the African giant snail, Achatina achatina
Article Sidebar
Main Article Content
Abstract
High enzyme activity and reusability are the major factors that limit enzyme application in the industry. This study explored the properties of Lactobacillus acidophilus β-galactosidase immobilized on Achatina chitosan, for improved enzyme reusability in industry. β-Galactosidase was produced from environmentally well-adapted Lactobacillus acidophilus. The enzyme was purified by ion exchange chromatography using DEAE-cellulose and had a molecular weight of 43kDa. Mg2+ was a major positive effector of the β-galactosidase activity. Chitin was extracted from Achatina shells by demineralization and deproteination, and deacetylated to chitosan. The chitin and chitosan yields were 74.64% and 58.60%. However, a hypochlorite-decolorized chitin, deacetylated to chitosan, gave a yield of 71%. FTIR spectra of chitin showed major bands at 711 cm-1, 855 cm-1, 1082 cm-1, and 1438 cm-1 for chitin and for chitosan at 6778 cm-1, 711 cm-1, 851 cm-1, 1082 cm-1 and 1436 cm-1. The β-galactosidase was immobilized on chitosan beads by adsorption and covalent linkage using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), and glutaraldehyde, separately. The enzyme had optimal temperature and pH of 70°C and 5.5. The Michaelis-Menten constant (KM) and maximal velocity (Vmax) of the free and immobilized β-galactosidase were 0.262/0.251mM, and 270.27/290 μmol/min using p-NPG as substrate, and 0.53/10.02 mM and 250/275 μmol/min for lactose as substrate, respectively. Covalent immobilization by glutaraldehyde improved the β-galactosidase activity more than adsorption, in comparison to EDC/NHS. The results show that extracellular β-galactosidase from Lactobacillus acidophilus, isolated from dairy wastewater, can be immobilized on chitosan support produced using cheaply available Achatina shell chitosan for greater reusability.
Downloads
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
How to Cite
References
Schulz P, Rizvi SSH. Hydrolysis of lactose in milk: Current status and future products. Food Rev Int. 2021; 2021:1-20.
Movahedpour A, Ahmadi N, Ghalamfarsa F, Ghesmati Z, Khalifeh M, Maleksabet A, Shabaninejad Z, Taheri-Anganeh M, Savardashtaki A. β-Galactosidase: From its source and applications to its recombinant form. Biotechnol. Appl Biochem. 2022; 69(2):612-628.
Forsgard RA. Lactose digestion in humans: intestinal lactase appears to be constitutive whereas the colonic microbiome is adaptable. Am J Clin Nutr. 2019; (110):273-279.
Cai Y, Zhou H, Zhu Y, Su Q, Ji Y, Xue A, Wang Y, Chen W, Yu X, Wang L, Chen H, Li C, Luo T, Deng H. Elimination of senescent cells by β-galactosidase-targeted prodrug attenuates inflammation and restores physical function in aged mice. Cell Res. 2020; (30):574-589.
Vidya B, Palaniswamy M, Angayarkanni J, Nawaz KA, Thandeeswaran M, K. K. Chaithanya KK, Tekluu B, Muthusamy K, Gopalakrishnan VK. Purification and characterization of β-galactosidase from newly isolated Aspergillus terreus (KUBCF1306) and evaluating its efficacy on breast cancer cell line (MCF-7). Bioorg Chem. 2020; (94): 103442.
Liburdi K, Esti M. Galacto-Oligosaccharide (GOS) Synthesis during Enzymatic Lactose-Free Milk Production: State of the Art and Emerging Opportunities. Beverages. 2022; 8(2):21.
Xavier JR, Ramana KV, Sharma RK. β-Galactosidase: Biotechnological applications in food processing. J Food Biochem. 2018; (e12564): 1-15.
Saji R, Balakrishnan S. β-Galactosidase: Application in Dairy and Food Industry. Intl J Ferment Food. 2021; 10(01): 51-59.
Luan S, Duan XA. Novel thermal-activated β-galactosidase from Bacillus aryabhattai GEL-09 for Lactose Hydrolysis in Milk. Foods. 2022; (11): 372-387.
Asadpoor M, Peeters C, Henricks PAJ, Varasteh S, Pieters RJ, Folkerts G, Braber S. Anti-Pathogenic Functions of Non-Digestible Oligosaccharides In Vitro. Nutrients. 2020; 12(6):1789.
Di Lodovico S, Gasparri F, Di Campli E, Di Fermo P, D'Ercole S, Cellini L, et al. Prebiotic Combinations Effects on the Colonization of Staphylococcal Skin Strains. Microorganisms. 2020; 9(1):37.
Vera C, Guerrero C, Illanes A. Trends in lactose-derived bioactives: synthesis and purification. Syst Microbiol Biomanuf. 2022; (2): 393-412.
Prazdnova EV, Gorovtsov AV, Vasilchenko NG, Kulikov MP, Statsenko VN, Bogdanova AA, Refeld AG, Brislavskiy YA, Chistyakov VA, Chikindas ML. Quorum-Sensing Inhibition by Gram-Positive Bacteria. Microorganisms. 2022; 10(2):350.
Shi X, Wu D, Xu Y, Yu X. Engineering the optimum pH of β-galactosidase from Aspergillus oryzae for efficient hydrolysis of lactose. J Dairy Sci. 2022; 105(6):4772-4782.
Bintsis T. Lactic acid bacteria: their applications in foods. J Bacteriol Mycol. 2018; 6(2):89-94.
Gomes TA, Santos LB, Nogueira A, Spier MR. Increase in an intracellular β-galactosidase biosynthesis using L. reuteri NRRL B-14171, inducers and alternative low-cost nitrogen sources under submerged cultivation. Int J Food Eng. 2018; 14(3):20170333.
He X, Luan M, Han N, Wang T, Zhao X, Yao Y. Construction and analysis of food-grade Lactobacillus kefiranofaciens β-galactosidase overexpression system. J. Microbiol Biotechnol. 2021; 31(4):550-558.
Bilal M, Iqba HMN. State-of-the-art strategies and applied perspectives of enzyme biocatalysis in food sector - current status and future trends. Crit Rev Food Sci Nutr 2020; (60): 2052-2066.
Verma ML, Kumar S, Das A, Randhawa JS, and Chamundeeswari M. Enzyme Immobilization on Chitin and Chitosan-Based Supports for Biotechnological Applications. Environ Chem Lett. 2019; 18(2):1-9.
Lyu X, Gonzalez R, Horton A, Li T, Immobilization of Enzymes by Polymeric Materials. Catalysts. 2021; 11:1-15.
Prokopijevic M. Natural polymers: suitable carriers for enzyme immobilization Biologia Serbica. 2021; 43(1): 43-49.
Iber BT, Kasan NA, Torsabo D, Omuwa JW. A review of various sources of chitin and chitosan. J Renew Mater. 2022; 10(4): 42-49.
Elieh-Ali-Komi D, Hamblin MR. Chitin and Chitosan: Production and Application of Versatile Biomedical Nanomaterials. Int J Adv Res (Indore). 2016; 4(3):411-427.
Rubini D, Banu SF, Subramani P, Hari BNV, Gowrishankar S, Pandian SK, Wilson A, Nithyanand P. Extracted chitosan disrupts quorum sensing mediated virulence factors in Urinary tract infection causing pathogens. Pathog Dis. 2019; 77(1):ftz009.
Shramko M, Berezueva E, Alieva L, Lodygin A, Evdokimov I. Influence of oligochitosans and highly molecular chitosan on Lactobacillus bulgaricus cultivation. Nutri Food Sci Int J. 2020; 9(5): 555773.
Nag M, Lahiri D, Mukherjee D, Banerjee R, Garai S, Sarkar T, et al. Functionalized Chitosan Nanomaterials: A Jammer for Quorum Sensing. Polymers. 2021; 13:2533.
Kurchenko V, Lodygin A, Halavach T, Evdokimov I, Shramko M. Application of Chitosan for Fermented Dairy Products with Lactobacillus delbrueckii subsp. Bulgaricus Manuf. Intelligent Biotechnol. Nat Syn Biol Active Sub. 2022; (408):165-175.
Oparaji EH, Okwuenu PC, Onosakponome I, Eze SOO, Chilaka FC. Optimization of Culture Conditions for Production of β-galactosidase from Lactobacillus acidophilus Isolated from Dairy Industrial Effluent. Enzyme Eng. 2022b; 11(2): 1-5.
Ezeonu M, Okafor J, Ogbonna J. Laboratory Exercises in Microbiology. (1st ed). Nsukka: Ephrata Publishing and Printing Company, 2013; Pp 100-117.
Oparaji EH, Eze CG, Okwuenu P C, Onosakponome I, Eze SOO, Chilaka FC. Purification and Enzymatic Properties of β-galactosidase Produced from Lactobacillus acidophilus isolated from Dairy Waste-Water. J Enzyme Eng. 2022a; (1): 16-38.
Chilaka FC, Nwachukwu A, Uvere P. Thermal stability studies of β –galactosidase from germinating seeds of the brown beans, Vigna unguiculata. Nig J Biochem Mol Biol. 2002; (17): 51-56.
Lowry O, Roseburg N, Farr A, Randall R. Protein Measurement with Folin- Phenol Reagents. J Biol Chem. 1951; (93): 265-275.
Chilaka FC, Anosike EO, Egbuna PC. Purification and properties of polyphenol oxidase from oil bean (Pentaclethra macrophylla Benth) seeds. J Sci Food Agric. 1993; (61):125-127.
Santos VP, Maia P, de-Sá-Alencar N, Farias L, Andrade RFS, Souza D, Ribaux DR., de Oliveira-Franco L, Campos-Takaki GM. Recovery of chitin and chitosan from shrimp waste with microwave technique and versatile application. Arq Inst Biol. 2019; (86): 1-7.
Kumirska J, Czerwicka M, Kaczyński Z, Bychowska A, Brzozowski K, Thöming J, Stepnowski P. Application of spectroscopic methods for structural analysis of chitin and chitosan. Mar Drugs. 2010; 8(5):1567-636.
Kamburov M, Lalov I. Preparation of chitosan beads for trypsin immobilization. Biotechnol Biotechnol Equip. 2014; 26(1):156-163.
Kazenwadel F, Wagner H, Rapp BE, Franzreb M. Optimization of enzyme immobilization on magnetic microparticles using 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent Anal. Methods. 2015; (7):10291-10298.
Cheng Z, Sun Z, Wei F, Yu J, Zhao J, Chen J , Wang J, Zhang Y. Immobilization of the crude enzyme extracted from Stenotrophomonas sp. GYH within modified zeolitic imidazolate framework (ZIF-8-NH2) and its application in trichloromethane remova. EFM. 2023; 2: 36-45.
Chilaka FC, Nwamba CO. Kinetic analysis of urea-inactivation of beta-galactosidase in the presence of galactose. J Enzyme Inhib Med Chem. 2008; 23(1):7-15.
Batra N, Singh J, Banerjee UC, Patnaik PR, Sobti RC. Production and characterization of a thermostable beta-galactosidase from Bacillus coagulans RCS3. Biotechnol Appl Biochem. 2002; 36(1):1-6.
Xu Z, Li C, Ye Y, Wang T, Zhang S, Liu X. The β-galactosidase LacLM plays the major role in lactose utilization of Lactiplantibacillus plantarum. LWT Food Sc. Technol. 2022; 153: 112481.
Bhalla TC, Arpita D, Kunzes A, Neerja T, Anila K. “β-Galactosidase from Lactobacillus brevis PLA28: Purification, Characterization and Synthesis of Galacto-oligosaccharides.” (2018). J food ind mirobiol. 2018; 1(1): 1-5.
Kittibunchakul S, Pham ML, Tran AM, Nguyen TH. β-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry. Int J Mol Sci. 2020; 20(4):947.
Percival GC, Chamundeeswari M, Lovlyna FR, Seethalakshmi R, Sreekumar G. Production and partial purification of β-galactosidase enzyme from probiotic Bacillus subtilis SK09. Indian J Biotechnol. 2019; (18):139-144.
Zhu J, Sun J, Tang Y, Xie J, Wei D. Expression, characterization and structural profile of a heterodimeric -galactosidase from the novel strain Lactobacillus curieae M2011381, Process Biochem. (2020); 97:87-95. doi: https://doi.org/10.1016/j.procbio.2020.06.02
Zhou Z, He N, Han Q, Liu S, Xue R, Hao J, Li S. Characterization and Application of a New β-Galactosidase Gal42 From Marine Bacterium Bacillus sp. BY02. Front. Microbiol. 2021; (12): 1-10.
Abdel-Baky YM, Omer AM, El-Fakharany EM, Ammar YA, Abusaif MS, Ragab A. Developing a new multi-featured chitosan-quinoline Schiff base with potent antibacterial, antioxidant, and antidiabetic activities: design and molecular modeling simulation. Sci Rep 2023; 13: 22792.
Lavall RL, Assis OB, Campana-Filho SP. Beta-chitin from the pens of Loligo sp.: extraction and characterization. Bioresour Technol. 2007; 98(13): 2465-72.
Hou J, Aydemir BE, Dumanli AG. Understanding the structural diversity of chitins as a versatile biomaterial. Philos. Trans A Math Phys Eng Sci. 2021; 379(2206):20200331.
Fernando LD, Dickwella Widanage MC, Penfield J, Lipton AS, Washton N, Latgé JP, Wang P, Zhang L, Wang T. Structural Polymorphism of Chitin and Chitosan in Fungal Cell Walls From Solid-State NMR and Principal Component Analysis. Front Mol Biosci. 2021; 8:727053.
Al-Zahrani SS, Bora RS, AlGarni SM. Antimicrobial activity of chitosan nanoparticles, Biotechnol Biotechnol Equip. 2021; (35):1874-1880.
Aranaz I, Alcántara AR, Civera MC, Arias C, Elorza B, Caballero AH, Acosta N. Chitosan: An Overview of Its Properties and Applications. Polymers (Basel). 2021; 13(19):3256.
Ke CL, Deng FS, Chuang CY, Lin CH. Antimicrobial Actions and Applications of Chitosan. Polymers (Basel). 2021; 13(6):904.
Incili G K, Karatepe P, Akgöl M, Tekin A, Kanmaz H, Kaya B, Çalıcıoğlu, M, Hayaloğlu, AA. Impact of chitosan embedded with postbiotics from Pediococcus acidilactici against emerging foodborne pathogens in vacuum-packaged frankfurters during refrigerated storage. Meat Sci. 2022; (188):108786.
Ureta MM, Martins GN, Figueira O, Pires PF, Castilho PC, Gomez-Zavaglia A. Recent advances in β-galactosidase and fructosyltransferase immobilization technology. Crit Rev Food Sci Nutr. 2021; 61(16): 2659-2690.
Yushkova ED, Nazarova EA, Matyuhina AV, Noskova AO, Shavronskaya DO, Vinogradov VV, Krivoshapkina, EF. Application of Immobilized Enzymes in Food Industry. J Agric Food Chem. 2019; 67(42):11553-11567.
Maghraby YR, El-Shabasy RM, Ibrahim AH, Azzazy HME. Enzyme Immobilization Technologies and Industrial Applications. ACS Omega. 2023; 8(6): 5184-5196. doi: 10.1021/acsomega.2c07560.
Ahmed SA, Saleh SAA, Abdel-Hameed SAA, Fayad A M. Catalytic, kinetic and thermodynamic properties of free and immobilized caseinase on mica glass-ceramics. Heliyon. 2019;5(5): e01674
Damin BIS, Kovalski FC, Fischer J, Piccin JS, Dettmer A. Challenges and perspectives of the β-galactosidase enzyme. Appl Microbiol Biotechnol. 2021; 105(13):5281-5298.