CALB脂肪酶在毕赤酵母中的表达及酶学性质研究
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摘要
本研究利用基因工程技术将CALB脂肪酶基因分别与穿梭载体pGAPZαA和pPIC9K连接并在大肠杆菌DH5α中扩增,然后分别在毕赤酵母中进行了组成型表达和诱导型表达,并优化了表达过程。其中,组成型表达经发酵优化后确定其最佳表达条件为:初始pH值为6.0,温度为30℃,最佳碳源为甘油。诱导型最佳摇瓶发酵条件为初始pH5.0,甲醇诱导浓度为2% ,诱导最佳温度为22℃,最佳表达培养基为甘油。经过优化,组成型表达出的CALB脂肪酶酶活达到了14.5U/ml,诱导性表达的CALB脂肪酶酶活达到了58.72U/ml。并对表达后的蛋白质进行了纯化,经纯化后的蛋白含量分别达到了0.22mg/ml和0.45mg/ml。对发酵所得到的蛋白进行了酶学性质分析,发现该酶液在pH4.0~pH6.0范围内较稳定,在30℃~60℃的温度范围内具有较好的稳定性,在60℃保存1h后残留酶活达到50%。其最适反应温度是50℃~60℃,最适反应pH值是6.0~8.0;这些性质与商品化的游离CALB基本一致。
With the availability of gene recombination technology, the synthetic calB gene was linked with vector pGAPZαA-calB and pPIC9K-calB, then the reconbinant expression vectors were transferred into E. coli DH5α. The recombinant plasmids were extracted and transformed into Pichia pastoris GS115 via elecoporation. The constitutive expression and inductive expression of CALB in Pichia pastoris had been received.What’more, the fermentation proc- ess in shake-flask was optimized and we explored the optimizing parameters separately. The optimizing parameters of constitutive expression was pH=5.0, T=30℃and the optimum carbon resource was glycerol. And the optimizing parameters of inductive expression was pH =5.0, 2% methanol(V/V), the temperature of induction was 22℃and the optimum carbon resource was glycerol. Through optimization and purification, the concentration of protein in constitutive expression was 0.22mg/ml, while the concentration of protein in inductive expression achieved 0.45mg/ml. Then we studied the property of CALB that we got. The optimum temperature for maintaining CALB activity was 30℃~60℃, pH4.0 ~ pH6.0.and the optimum temperature and pH for CALB hydrolysis reactions was 50℃~60℃, pH8.0. And the property of CALB that we got and commercial CALB were similar.
With the availability of gene recombination technology, the synthetic calB gene was linked with vector pGAPZαA-calB and pPIC9K-calB, then the reconbinant expression vectors were transferred into E. coli DH5α. The recombinant plasmids were extracted and transformed into Pichia pastoris GS115 via elecoporation. The constitutive expression and inductive expression of CALB in Pichia pastoris had been received.What’more, the fermentation proc- ess in shake-flask was optimized and we explored the optimizing parameters separately. The optimizing parameters of constitutive expression was pH=5.0, T=30℃and the optimum carbon resource was glycerol. And the optimizing parameters of inductive expression was pH =5.0, 2% methanol(V/V), the temperature of induction was 22℃and the optimum carbon resource was glycerol. Through optimization and purification, the concentration of protein in constitutive expression was 0.22mg/ml, while the concentration of protein in inductive expression achieved 0.45mg/ml. Then we studied the property of CALB that we got. The optimum temperature for maintaining CALB activity was 30℃~60℃, pH4.0 ~ pH6.0.and the optimum temperature and pH for CALB hydrolysis reactions was 50℃~60℃, pH8.0. And the property of CALB that we got and commercial CALB were similar.
引文
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[4] Taipa M.A., Aires-Barros M.R., Cabral,J.M.S. Purification of lipases [J]. Biotechnol, 26: 11-142
[5] Jaeger K.E., Wohlfarth S. Bacterial lipases: biochemistry molecular genetics and appli- cations in biotechnology [J]. Bioengineering,1993, 9: 39-46
[6] Antonian E. Recent advances in the purification,characterization and structure determ- ination of lipases [J]. Lipids, 1988, 23:1101-1106
[7] Gilbert E.J. Pseudomonas lipases: biochemical properties and molecular cloning Enzy- me [J]. Microb Technol, 1993, 15: 634-645
[8] Huge-Jensen B.F., Andreasen, et al. Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae [J]. Lipids, 1989, 24(9): 781-785
[9] Odera M. et al. Ferment [J]. Technol, 1986, 64: 363
[10] Kuginiya W.,et al. Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi [J]. Biochem Biophys Res.Commun, 1986, 141(1):185-190
[11] Gotz F., et al. Complete nucleotide sequence of the lipase gene from Staphylococcus hyicus cloned in Staphylococcus carnosus [J]. Nucl Acids Res, 1985, 13: 5895-5906
[12] Zaks A., Klibanov A.M. Enzymatic catalysis in organic media at 100℃[J]. Science, 1984, 224: 1249
[13]曹淑桂.脂肪酶的底物特异性及其应用潜力[J].生物化学与生物物理进展,1995,22(1): 9-13
[14]高修功.微生物脂肪酶生产及非水相催化性质与应用研究[D].无锡轻工业大学博士学位论文
[15]许建军,张颖.脂肪酶的应用研究进展[J].江苏食品与发酵, 2002, 111(4): 19-21
[16] Stead D. Microbial lipase: their characteristic role in food spoilage and industrial uses [J]. Dairy Res, 1986, 53: 481-505
[17] McNeill G.P., Shimizu S., Yamane T. High yield enzymatic glycerolysis of fats and oils [J]. Am.Oil.Chem.Soc, 1991, 68: 1-5
[18] McNeill G.P., Yamane T. Further improvements in the yield of monoglycerides during enzymatic glycerolysis of fats and oils [J]. Am.Oil.Chem.Soc, 1991, 68: 6-10
[19] Osada K., Takahashi K., Hatano M. Polyunsaturated fatty acid glyceride syntheses by microbial lipases [J]. Am.Oil.Chem.Soc, 1990, 67: 921-922
[20] Babayan V.K. Medium chain trigylcerides and structured lipids [J]. Lipids, 1987, 22: 417-420
[21]马吉胜.枯草杆菌脂肪酶的基因克隆、定向进化与非水酶学研究[D].吉林大学博士学位论文, 2005
[22]李生强.扩展青霉脂肪酶基因在酿酒酵母中的表达及分子突变[D].福建师范大学硕士学位论文
[23] Macauley Patrick S., Fazenda M.L., McNeil B., et al. Heterologous protein production using the Pichia pastoris expression system [J]. Yeast, 2005, 22: 249-270.
[24] Daly Rachel, Hearn M.T.W. Expression of heterologous proteins in Pichia pastoris:a useful experimental tool in protein engineering and production [J]. Journal of Molecular Recognition, 2005, 18: 119-138.
[25] David R., Higgins James M., Cremes. Pichia Protocols [M]. New Jersey: Human Press Inc, 1998
[26] Shen S., Sulter G., Jeffries T.W., et al. Expression of functional mouse 52HT5A seroto- nin receptor in the methylotrophic yeast Pichia pastoris: pharmacological charcterizati- on and localization [J]. FEBS Lett, 1995, 377 (3): 451-456.
[27] Cereghino J.L., Cregg J.M. Heterlogous protein expression in the methylotrophic yeast Pichia pastoris [J]. FEMS Microbiology Reviews, 2000, 24: 45-66.
[28] Peng L, Zhong X, Ou J, et al. High-level secretory p roduction of recombinant bovine enterokinase light chain by Pichia pastoris [J]. Biotechnol, 2004, 108 (2): 185-192.
[29] Kout P, Davis, G.R., Stillman C.,et al. Structural comparison of the Pichia pastoris alcohol oxidase genes [J]. Yeast, 1999,5: 167-177
[30] Waterham H.R., Digan M.E., Koutz P.J., et al. Isolation of the Pichia pastoris glyceral dehydephosphate and regulation and use of its p romoter [J]. Gene, 1997, 186 (1): 37-44.
[31] Shen S., Sulter G., Jeffries T.W., et al. A strong nitrogen source regulated promoter for controlled expression of foreign genes in the yeast Pichia Pastoris [J]. Gene, 1998, 216: 93-102.
[32] Brierley R.A. Secretion of recombinant human insulin-like growth factor ( IGF21) [J]. Methods MolBiol, 1998, 103: 149-177.
[33] BEI J.L., WANG J.W., WANG X.Z., et al. High exp ression of LIP1 in Pichia pastoris [J]. Acta Biochimica at Biophysica Sinica, 2003, 35 (4) : 366-370.
[34] Tschopp J.F., Brush P.F., Cregg J.M., et al. Expression of the lacZ gene from two meth- anol regulated promoters in Pichia pastoris [J]. Nucleic Acid Res, 1987, 15 (9): 3859-3876
[35] Cregg J.M., Madden K.R., Barringer K.J., et al. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris [J]. Mol Cell Biol, 1989, 9 (3) : 1316-1323
[36] CHUNG J.D. Design of metabolic feed controllers: application to high-density fermen- tations of Pichia pastoris [J]. Biltechnol Bioeng, 2000, 68 (3): 298-303
[37] QUYEN D.T., SCHMIDT D.C. High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase [J]. Protein Expression & Purification, 2003, 28 (1): 102-110
[38] Vassileva A., Chugh D.A., Swaminathan S., et al. Effect of copymunber on the expressi- on levels of hepatitisB surface antigen in the methylotrophie yeast Pichia pastoris [J] . Protein ExprPurif, 2001, 21: 71-76
[39]杨莉莉. PHO1信号肽在毕赤酵母中引导分泌表达天然天然N端rBPT1 [J].吉林大学学报(医学版), 2005, 31 (6): 830-832
[40] Scorer C.A., Clare J.J., Mccombiew R., et al. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expressiom [J]. BioTechnology, 1994, 12 (2): 181-185
[41] Jahic M., Gustavsson M., Jansen A.K., et al. Analysia and control of p roteolysis of afusion p rotein in Pichia pastoris fed-batch processes [J]. Biotechnol, 2003, 102 (1): 45-53
[42] Loewen M.C., Daugulis A.J., Davies P.L. et al. Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris [J]. Appl Microbiol Biotechnol , 1997, 48: 480
[43] Fahnestock S.R., Bedzyk L.A. Production of synthetic spider dragline silk protein in Pichia pastoris [J]. Appl Microbio Biotechnol, 1997, 47: 33
[44] Clare J., Scorer C., Buckholz R.,et al. Expression of EGF and HIV envelope glycopro- tein [J]. Methods Mol Biol , 1998, 103: 209
[45] Sreekrishna K., Barr K.A., Hoard S.A., et al. Expression of human serum albumin in Pichia pastoris [J]. Yeast, 1990, 6(Special Issue) : S447
[46]张丞斌,傅正伟.影响外源蛋白在毕赤酵母中表达和分泌的研究进展[J].现代生物医学进展, 2008, 8(7): 1382-1384
[47] Frank D., Stephan T., Hannelore D. Expression and functional characterization of the mammalian intestinal peptide transporter pepT1 in the methylotropic yeast Pichia pasto- ris [J]. Biochem Biophys Res Commun, 1997, 232: 6561
[48] Shen S.G., Sulter G., Jeffris T.W., et al. A strong nitrogen source-regulated promoter f- or controlled expression of foreign genes in the yeast Pichia pastoris [J]. Gene, 1998, 216: 93-102
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