Abstract
Chymosin can specifically break down the Phe105–Met106 peptide bond of milk κ-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was obtained for the purified enzyme, which appeared as a single band in SDS–PAGE having a molecular mass of approximate 36 kDa. Proteolysis assay showed that it specifically hydrolyzed κ-casein. It was stable at 25–50°C and had optimal activity at 37°C and pH 4.0. The activity of the recombinant chymosin was activated by cations such as Mn2+, Fe3+, Mg2+ and Na+, but inhibited by K+, Co2+, Zn2+, Ni2+, and to a lesser extent by Cu2+. These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe and efficient enzyme suitable for use in cheese production.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ageitos JM, Vallejo JA, Sestelo ABF, Poza M, Villa TG (2007) Purification and characterization of a milk-clotting protease from Bacillus licheniformis strain USC13. J Appl Microbiol 103:2205–2213
Beldarraín A, Acosta N, Montesinos R, Mata M, Cremata J (2000) Characterization of Mucor Pusillus rennin expressed in Pichia pastoris: enzymic, spectroscopic and calorimetric studies. Biotechnol Appl Biochem 31:77–84
Bitter GA, Egan KM (1984) Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehydes-3-phosphate dehydrogenase gene promoter. Gene 32:263–274
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66
Ciofalo V, Barton N, Kreps J, Coats I, Shanahan D (2006) Safety evaluation of a lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil. Regul Toxicol Pharm 45:1–8
Cregg JM, Vedvick TS, Raschke WC (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology 11:905–910
Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechol 16:23–52
Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T (2009) Expression in the yeast Pichia pastoris. Methods Enzymol 463:169–189
Davis FL, Law BA (1984) Advances in the microbiology and biochemistry of cheese and fermented milk. Elsevier Applied Science, London
Egito AS, Girardet JM, Laguna LE, Poirson C, Molle D, Miclo L, Humbert JL (2007) Milk-clotting activity of enzyme extracts from sunflower and albizia seeds and specific hydrolysis of calf κ-casein. J Int Dairy 17:816–825
Elagamy EI (2000) Physicochemical, molecular and immunological characterization of camel calf rennet: a comparison with buffalo rennet. J Dairy Res 67:73–81
Emtage JS, Angal S, Doel MT, Harris TJ, Jenkins B, Lilley G, Lowe PA (1983) Synthesis of calf prochymosin (prorennin) in Escherichia coli. Proc Natl Acad Sci USA 80:3671–3675
Jana S, Deb JK (2005) Strategies for efficient production of heterologous protein in Escherichia coli. Appl Microbiol Biotechnol 67:289–298
Kaprálek F, Tichý PJ, Fábry M, Sedlácek J (1998) Effects of temperature and novobiocin on the expression of calf prochymosin gene and on plasmid copy number in recombinant Escherichia coli. Folia Microbiol (Praha) 43:63–67
Kumar A, Sharma J, Mohanty AK, Grover S, Batish VK (2006) Purification and characterization of milk clotting enzyme from goat (Capra hircus). Comp Biochem Physiol B Biochem Mol Biol 145:108–113
Kumar A, Grover S, Sharma J, Batish VK (2010) Chymosin and other milk coagulants: source and biotechnological interventions. Crit Rev Biotechnol 30:243–258
Makino T, Skretas G, Georgiou G (2011) Strain engineering for improved expression of recombinant proteins in bacteria. Microb Cell Fact 10:32
McMahon DJ, Brown RJ, Ernstrom CA (1984) Enzymic coagulation of milk casein micelles: a review. J Dairy Sci 67:745–748
Mezina MN, Lavrenova GI, Prokofev MI, Starovoitova VV, Ermolaev VI, Chernyh VY, Balandina GN, Demidovich SS (2001) Isolation of milk-clotting enzyme from transgenic sheep milk and its comparison with calf chymosin. Biochemistry 66:378–383
Mohanty AK, Mukhopadhyay UK, Grover S, Batish VK (1999) Bovine chymosin: production by rDNA technology and application in cheese manufacture. Biotechnol Adv 17:205–217
Mohanty AK, Mukhopadhyay UK, Kaushik JK, Grover S, Batish VK (2003) Isolation, purification and characterization of chymosin from riverine buffalo (Bubalos bubalis). J Dairy Res 70:37–43
Moschopoulou EE, Kandarakis IG, Alichanidis E, Anifantakis EM (2006) Purification and characterization of chymosin and pepsin from kid. J Dairy Res 73:49–57
Nouani A, Dako E, Morsli A, Belhamiche N, Belbraouet S, Bellal MM, Dadie A (2009) Characterization of the purified coagulant extracted derived from artichoke flowers (Cynara scolymus) and from the fig tree latex (Ficus carica) in light of their use in the manufacture of traditional cheeses in Algeria. J Food Technol 7:20–29
Pedersen VB, Christensen KA, Foltmann B (1979) Investigations on the activation of bovine prochymosin. Eur J Biochem 94:573–580
Petsch D, Anspach FB (2000) Endotoxin removal from protein solutions. J Biotechnol 76:97–119
Poza M, Prieto-Alcedo M, Sieiro C, Villa TG (2004) Cloning and expression of clt genes encoding milk-clotting proteases from Myxococcus xanthus 422. Appl Environ Microbiol 70:6337–6341
Puhan Z (1969) Protease composition of a rennet substitute from Bacillus subtilis and properties of its component proteases. J Dairy Sci 52:1372–1378
Rosenberg S, Coit D, Tekamp-Olson P (1990) Glyceraldehyde-3-phosphate dehydrogenase-derived expression cassettes for constitutive synthesis of heterologous proteins. Methods Enzymol 185:341–351
Vallejo JA, Ageitos JM, Poza M, Villa TG (2008) Cloning and expression of buffalo active chymosin in Pichia pastoris. J Agric Food Chem 56:10606–10610
Waterham HR, Digan ME, Koutz PJ, Lair SV, Cregg JM (1997) Isolation of the Pichia pastoris glyceraldehydes-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186:37–44
Zhang Y, Wei Zhou, Liu N, Yang K (1991) Expression of calf prochymosin gene in Escherichia coli. Chin J Biotechnol 7:169–175
Acknowledgments
This work is supported by the National Key Project for Basic Research (2010CB126102), the National Natural Science Foundation of China (31070715, 81102378), the National High Technology Research and Development Program of China (2011AA10A204), the National Key Technology R&D Program(2011BAE06B05), Scientific Research Foundation for the Returned Overseas Chinese Scholars, the Fundamental Research Funds for the Central Universities (DUT11SM02). The authors thank Dr Alan K. Chang for checking and revising the language of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Jiang, X.P., Yin, M.L., Chen, P. et al. Constitutive expression, purification and characterization of bovine prochymosin in Pichia pastoris GS115. World J Microbiol Biotechnol 28, 2087–2093 (2012). https://doi.org/10.1007/s11274-012-1012-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11274-012-1012-7