Abstract
The acid protease structural gene was amplified from the genomic DNA of Saccharomycopsis fibuligera A11. When the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in the cells of Yarrowia lipolytica, the cells displaying the acid protease could form clear zone on the plate-containing milk indicating that they had extracellular acid protease activity. The cells displaying the acid protease can be used to effectively clot skimmed milk. The highest clotting milk activity (1,142.9 U/ml) was observed under the conditions of pH 3.0, 40 °C, 20 mM of CaCl2, and 10% skimmed milk powder. We found that the acid protease displayed on the cells of Y. lipolytica which has generally regarded as safe status could be easily isolated and concentrated compared to the free acid protease. Therefore, the displayed acid protease may have many potential applications in food and cheese industries. This is the first report that the yeast cells displaying the acid protease were used to clot milk.
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References
Abdelal TH, Kennedy H, Ahearn G (1970) Purification and characterization of a neutral protease from Saccharomycopsis lipolytica. J Dairy Sci 53:253–261
Adams A, Gottschling DE, Kaiser CA, Stearms T (1998) Yeast immunofluorescence. In: Burke D, Dawson D (eds) Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory, New York, p 100
Ahmed IAM, Morishima I, Babiker EE, Mori N (2009) Characterisation of partially purified milk-clotting enzyme from Solanum dubium Fresen seeds. Food Chem 116:395–400
Berridge NJ (1952) Some observation on the determination of the activity of rennet. Anal London 77:57–62
Chi ZM, Liu J, Zhang W (2001) Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu. Enzy Microb Technol 28:240–245
Chi ZM, Chi Z, Liu GL, Wang F, Ju L, Zhang T (2009) Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnol Adv 27:423–431
Davies DR (1990) The structure and function of the aspartic proteinases. Ann Rev Biophy Biophy Chem 19:189–215
Jolivalt C, Madzak C, Brault A, Caminade E, Malosse C, Mougin C (2005) Expression of laccase lllb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications. Appl Microbiol Biotechnol 66:450–456
Kamada M, Oda K, Murao S (1972) The purification of the extracellular acid protease of Rhodotorula glutinis K-24 and its general properties. Agr Biol Chem 36:1095–1101
Kitano H, Kataoka K, Furukawa K, Hara S (2002) Specific expression and temperature-dependent expression of the acid protease-encoding gene (PepA) in Aspergillus oryzae in solid-state culture (rice-koji). J Biosci Bioeng 93:563–567
Kocabiyik S, Ozel H (2007) An extracellular-pepstatin in sensitive acid protease produced by Thermoplasma volcanium. Bioresour Technol 98:112–117
Kumar S, Sharma NS, Saharan MR, Singh R (2005) Extracellular acid protease from Rhizopus oryzae: purification and characterization. Proc Biochem 40:1701–1705
Larson MK, Whitaker JR (1970) Endothia parasitica protease. Parameters affecting activity of the rennin-like enzyme. J Dairy Sci 53:253–261
Li J, Chi ZM, Liu ZQ, Yue LX, Peng Y, Wang L (2009) Cloning and characterization of a novel aspartic protease gene from marine-derived Metschnikowia reukaufii and its expression in E. coli. Appl Biochem Biotechnol 159:119–132
Li J, Chi ZM, Chi Z, Wang XH (2010) Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli. Microbiol Res 165:173–182
Liu GL, Yue LX L, Chi Z, Yu WG, Chi ZM, Madzak C (2009) The surface display of the alginate lyase on the cells of Yarrowia lipolytica for hydrolysis of alginate. Mar Biotechnol 11:619–626
Maddox IS, Hough JS (1970) Proteolytic enzymes of Saccharomyces carlsbergensis. Biochem J 117:843–852
Madzak C, Gaillardin C, Beckerich JM (2004) Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol 109:63–81
Nelson G, Young TW (1987) Extracellular acid and alkaline proteases from Candida olea. J Gen Microbiol 133:1461–1469
Ni XM, Yue LX, Chi ZM, Li J, Wang XH, Madzak C (2009) Alkaline protease gene cloning from the marine yeast Aureobasidium pullulans HN2-3 and the protease surface display on Yarrowia lipolytica for bioactive peptide production. Mar Biotechnol 11:81–89
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Beijing, pp 367–370 (Chinese translating ed.)
Shieh CJ, Thi LAP, Shih IL (2009) Milk-clotting enzymes produced by culture of Bacillus subtilis natto. Biochem Eng J 43:85–91
Strauss A, Gotz F (1996) In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus. Mol Microbiol 21:491–500
Tobe S, Takami T, Ikeda S, Mitsuzi K (1976) Production and some enzymatic properties of alkaline proteinase of Candida lipolytica. Agr Biol Chem 40:1087–1092
Xuan JM, Fournier P, Gaillardin C (1988) Cloning of the LYS5 gene encoding saccharopine dehydrogenase. Curr Genet 14:15–21
Yue LX, Chi ZM, Wang L, Liu J, Madzak C, Li J, Wang XH (2008) Construction of a new plasmid for surface display on cells of Yarrowia lipolytica. J Microbiol Methods 72:116–123
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This research was supported by grant 30771645 from National Natural Science Foundation of China.
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Yu, XJ., Madzak, C., Li, HJ. et al. Surface display of acid protease on the cells of Yarrowia lipolytica for milk clotting. Appl Microbiol Biotechnol 87, 669–677 (2010). https://doi.org/10.1007/s00253-010-2549-8
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DOI: https://doi.org/10.1007/s00253-010-2549-8