Browse > Article
http://dx.doi.org/10.4014/jmb.1112.12026

Synthesis and High Expression of Chitin Deacetylase from Colletotrichum lindemuthianum in Pichia pastoris GS115  

Kang, Lixin (Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University)
Chen, Xiaomei (State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University)
Zhai, Chao (Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University)
Ma, Lixin (Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University)
Publication Information
Journal of Microbiology and Biotechnology / v.22, no.9, 2012 , pp. 1202-1207 More about this Journal
Abstract
A gene, ClCDA, encoding chitin deacetylase from Colletotrichum lindemuthianum, was optimized according to the codon usage bias of Pichia pastoris and synthesized in vitro by overlap extension PCR. It was secretorily expressed in P. pastoris GS115 using the constitutive expression vector pHMB905A. The expression level reached the highest with 110 mg/l culture supernatant after 72 h of methanol induction, which comprised 77.27 U/mg chitin deacetylase activity. SDS-PAGE, mass spectrometry, and deglycosylation assays demonstrated that partial recombinant protein was glycosylated with an apparent molecular mass of 33 kDa. The amino acid sequences of recombinant proteins were confirmed by mass spectrometry.
Keywords
Chitin deacetylase; Colletotrichum lindemuthianum; high expression; Pichia pastoris; synthesis;
Citations & Related Records

Times Cited By Web Of Science : 0  (Related Records In Web of Science)
연도 인용수 순위
  • Reference
1 Alfonso, C., O. M. Nuero, F. Santamaría, and F. Reyes. 1995. Purification of a heat stable chitin deacetylase from Aspergillus nidulans and its role in cell wall degradation. Curr. Microbiol. 30: 49-54.   DOI   ScienceOn
2 Araki, Y. and E. A. Ito. 1975. Pathway of chitosan formation in Mucor rouxii. Eur. J. Biochem. 55: 71-78.   DOI   ScienceOn
3 Blair, D. E., O. Hekmat, A. W. Schuttelkopf, B. Shrestha, K. Tokuyasu, S. G. Withers, and D. M. F. van Aalten. 2006. Structure and mechanism of chitin deacetylase from the fungal pathogen Colletotrichum lindemuthianum. Biochemistry 45: 9416-9426.   DOI   ScienceOn
4 Carr, P. A., J. S. Park, Y. J. Lee, T. Yu, S. Zhang, and J. M. Jacobson. 2004. Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Res. 32: e162.   DOI   ScienceOn
5 Caufrier, F., A. Martinou, C. Dupont, and V. Bouriotis. 2003. Carbohydrate esterase family 4 enzymes: Substrate specificity. Carbohydr. Res. 338: 687-692.   DOI   ScienceOn
6 Chen, G. Q., I. Choi, B. Ramachandran, and J. E. Gouaux. 1994. Total gene synthesis: Novel single step and convergent strategies applied to the construction of a 779 base pair bacteriorhodopsin gene. J. Am. Chem. Soc. 11: 8799-8800.
7 Christodoulidou, A., P. Briza, A. Ellinger, and V. Bouriotis. 1999. Yeast ascospore wall assembly requires two chitin deacetylase isozymes. FEBS Lett. 460: 275-279.   DOI   ScienceOn
8 Dutta, P. K., J. Dutta, and V. S. Tripathi. 2004. Chitin and chitosan: Chemistry, properties and applications. J. Sci. Ind. Res. 63: 20-31.
9 Gao, X. D., T. Katsumoto, and K. Onodera. 1995. Purification and characterization of chitin deacetylase from Absidia coerulea. J. Biochem. 117: 257-263.   DOI
10 Gauthier, C., F. Clerisse, J. Dommes, and M. F. Jaspar-Versali. 2008. Characterization and cloning of chitin deacetylases from Rhizopus circinans. Protein Expr. Purif. 59: 127-137.   DOI   ScienceOn
11 Hekmat, O., K. Tokuyasu, and S. G. Withers. 2003. Subsite structure of the endo-type chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum: An investigation using steadystate kinetic analysis and MS. Biochem. J. 374: 369-380.   DOI   ScienceOn
12 Jaworska, M. M., J. Bryjak, and J. Liesiene. 2009. A search of an optimal carrier for immobilization of chitin deacetylase. Cellulose 16: 261-270.   DOI   ScienceOn
13 Kafetzopoulos, D., A. Martinou, and V. Bouriotos. 1993. Bioconversion of chitin to chitosan: Purification and characterization of chitin deacetylase from Mucor rouxii. Proc. Natl. Acad. Sci. USA 90: 2564-2568.   DOI   ScienceOn
14 Kauss, H., W. Jeblick, and D. H. Young. 1983. Chitin deacetylase from the plant pathogen Colletotrichum lindemuthianum. Plant Sci. 28: 231-236.
15 Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.   DOI   ScienceOn
16 Tsigos, I. and V. Bouriotis. 1995. Purification and characterization of chitin deacetylase from Colletotrichum lindemuthianum. J. Biol. Chem. 270: 26286-26291.   DOI   ScienceOn
17 Matsuo, Y., K. Tanaka, H. Matsuda, and M. Kawamukai. 2005. Cda1+, encoding chitin deacetylase is required for proper spore formation in Schizosaccharomyces pombe. FEBS Lett. 579: 2737-2743.   DOI   ScienceOn
18 Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68: 850-858.   DOI   ScienceOn
19 Shrestha, B., K. Blondeau, W. F. Stevens, and F. L. Hegarat. 2004. Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: Purification and characterization. Protein Expr. Purif. 38: 196-204.   DOI   ScienceOn
20 Sreekrishna, K., R. G. Brankamp, K. E. Kropp, D. T. Blankenship, J. T. Tsay, P. L. Smith, et al. 1997. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190: 55-62.   DOI   ScienceOn
21 Tokuyasu, K., S. Kaneko, K. Hayashi, and Y. Mori. 1999. Production of recombinant chitin deacetylase in the culture medium of Escherichia coli cells. FEBS Lett. 458: 23-26.   DOI   ScienceOn
22 Tokuyasu, K., M. Ohnishi-Kameyama, and K. Hayashi. 1996. Purification and characterization of extracellular chitin deacetylase from Colletotrichum lindemuthianum. Biosci. Biotechnol. Biochem. 60: 1598-1603.   DOI   ScienceOn
23 Tokuyasu, K., M. Ohnishi-Kameyama, K. Hayashi, and Y. Mori. 1999. Cloning and expression of chitin deacetylase gene from a Deuteromycete, Colletotrichum lindemuthianum. J. Biosci. Bioeng. 87: 418-423.   DOI   ScienceOn
24 Win, N. N. and W. F. Stevens. 2001. Shrimp chitin as substrate for fungal chitin deacetylase. Appl. Microbiol. Biotechnol. 57: 334-341.   DOI   ScienceOn
25 Tokuyasu, K., M. Mitsutomi, I. Yamaguchi, K. Hayashi, and Y. Mori. 2000. Recognition of chitooligosaccharides and their Nacetyl groups by putative subsites of chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum. Biochemistry 39: 8837-8843.   DOI   ScienceOn
26 Tsigos, I., A. Martinou, D. Kafetzopoulos, and V. Bouriotis. 2000. Chitin deacetylases: New, versatile tools in biotechnology. Trends Biotechnol. 18: 305-312.   DOI   ScienceOn
27 Wang, Y., J. Z. Song, Q. Yang, Z. H. Liu, X. M. Huang, and Y. Chen. 2010. Cloning of a heat-stable chitin deacetylase gene from Aspergillus nidulans and its functional expression in Escherichia coli. Appl. Biochem. Biotechnol. 162: 843-854.   DOI
28 Zhang, G. M., Y. Hu, and Y. H. Zhuang. 2006. Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris. Biocatal. Biotransform. 24: 371-379.   DOI   ScienceOn
29 Zhao, Y., R. D. Park, and R. A. Muzzarelli. 2010. Chitin deacetylases: Properties and applications. Mar. Drugs 8: 24-46.   DOI   ScienceOn