Keywords
Precipitation
Purification
Enzyme kinetics.
How to Cite
Abstract
The search for rennet substitutes such as microbial rennet has increased fold due to increase in the demand for cheese products. Microbial rennet covers about one-third of the cheese consumption worldwide. Hence it is important to develop commercially viable and cost efficient method for purification of rennet from microbial sources. Hence the present work was focused on the production and purification of microbial rennet from Aspergillus candidus. The rennet was purified using a two step purification process involving solvent precipitation and chromatographic separation. The purity of the milk clotting enzyme was increased by 7.43 fold by solvent precipitation using equal mixture of 150% (v/v) ethanol-acetone. Then the enzyme was further purified using Diethylaminoethyl (DEAE) cellulose chromatography and 10.45 fold increase in enzyme activity was obtained after purification. The temperature of 35°C and substrate concentration of 0.25 mg/ml were found as optimum for maximum enzyme activity. The kinetics of the purified enzyme was studied and the Michaelis-Menten parameters such as rate constant (Km) and the maximum reaction rate (Vmax) were found as 0.059 mg/ml and 8.59 x 10-3 mmol/ml/sec respectively.
References
Ruchi R. Cheese-Global Consumption. Worldwide Cheese Consumption Patterns. 2010.
Bernardinelli SE, Bottaro Castilla HR, Waehner, RS, Muse J, Fraile ER. Production and properties of the milk-clotting enzyme. Rev Argent Microbiol 1983; 15: 95-104.
Fox PF, Paul M, Timothy MC and Timothy PG. Diversity of cheese varieties: An overview. Cheese: Major cheese groups 2004; 1-23.
Poza M, Sieiro C, Carreira L, Barros VJ, Villa TG. Production and characterization of the milk-clotting protease of Myxococcus xanthus strain 422. J Ind Microbiol Biotechnol 2003; 30: 691-98. http://dx.doi.org/10.1007/s10295-003-0100-y
Osman HG, Abdel-Fattah AF, Abdel-Samie M and Souhair SM. Production of a Milk-clotting Enzyme Preparation by Aspergillus niger and the Effect of Various Factors on its Activity. J Gen Microbiol 1969; 59:125-29. http://dx.doi.org/10.1099/00221287-59-1-125
Abdel-Fattah F, Souhair SM and El-Hawwary NM. Production and Some Properties of Rennin-like Milk-clotting Enzyme from Penicillium citrinum. J Gen Microbiol 1971; 70:151-55. http://dx.doi.org/10.1099/00221287-70-1-151
Bernardinelli SE, Bottaro Castilla HR, Waehner, RS, Muse J and Fraile ER. Production and properties of the milk-clotting enzyme, Rev Argent Microbiol 1983; 15 (2): 95-104.
Preetha S, Boopathy R. Purification and characterization of a milk clotting protease from Rhizomucor miehei. World J Microb Biotechnol 1997; 13: 573-578. http://dx.doi.org/10.1023/A:1018525711573
Poza M, Sieiro C, Carreira L, Barros VJ and Villa TG. Production and characterization of the milk-clotting protease of Myxococcus xanthus strain 422. J Ind Microbiol Biotechnol 2003, 30: 691–98. http://dx.doi.org/10.1007/s10295-003-0100-y
Lebedeva GV and Proskuryakov MT. Purification and Characterization of Milk-Clotting Enzymes from Oyster Mushroom (Pleurotus ostreatus (Fr.) Kumm.). App Biochem Microbiol 2009; 45 (6): 623–25. http://dx.doi.org/10.1134/S0003683809060088
Carolina MD, Eleni G, Mauricio B and Roberto S. Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31. Food Chem 2010; 120: 87–3. http://dx.doi.org/10.1016/j.foodchem.2009.09.075
Xiaoling H, Fazheng R, Huiyuan G, Weibing Z, Xi S and Bozhong. Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4. Korean J Chem Eng 2011; 28(1): 203-208. http://dx.doi.org/10.1007/s11814-010-0347-8
Rashbehari T, Binita S, Rintu B. Purification and characterization of a protease from solid state cultures of Aspergillus parasiticus. Process Biochem 2003; 38: 1553-58. http://dx.doi.org/10.1016/S0032-9592(03)00048-7
Arima K, Iwasaki S, Tamura G. Milk clotting enzyme from microorganisms. 1. Screening tests and identification of the potent fungus. Agric Boil Chem 1967; 31: 540-54. http://dx.doi.org/10.1271/bbb1961.31.540
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-75.
Lineweaver, H and Burk, D. The Determination of Enzyme Dissociation Constants. J Am Chem Soc 1934; 56 (3): 658–66. http://dx.doi.org/10.1021/ja01318a036
Hayakawa K, Guo L, Terentyeva EA, Li XK; Kimura H, Hirano M, Yoshikawa K, Nagamine T, Katsumata N, Ogata T, Tanaka T. Determination of specific activities and kinetic constants of biotinidase and lipoamidase in LEW rat and Lactobacillus casei (Shirota). J Chromatogr B Analyt Technol Biomed Life Sci 2006; 844(2): 240–50. http://dx.doi.org/10.1016/j.jchromb.2006.07.006
Baskar G, Arulluca Sundarsingh S, Bharath Kumar S, Muthukumaran C, Renganathan S. Optimization and Kinetics of Enzymatic Hydrolysis of Cassava Starch by Cross-Linked Fungal α–Amylase. Asian J Microbiol Biotechnol Environ Sci 2009; 11(2): 323-26.
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Copyright (c) 2014 G. Baskar, S. Babitha Merlin, D.V. Sneha , J. Angeline Vidhula