Vibrio parahaemolyticusが生産するキチン分解酵素の精製，諸性質解析，クローニングおよびリコンビナント酵素のオリゴ糖生産への利用

門倉 一成; 坂本 裕輔; 六谷 明子; 池上 孝紀; 平野 貴子; 山本 真広; 齋藤 香織; 袴田 航; 糸井 史朗; 杉田 治男; 奥 忠武; 西尾 俊幸

doi:10.5458/jag.55.157

Proceedings of the Symposium on Amylases and Related Enzymes, 2007

Purification, Characterization and Cloning of Vibrio parahaemolyticus Chitinolytic Enzymes and Application to Oligosaccharide Production

Kazunari Kadokura, Yusuke Sakamoto, Akiko Rokutani, Takanori Ikegami, Takako Hirano, Mahiro Yamamoto, Kaori Saito, Wataru Hakamata, Shiro Itoi, Haruo Sugita, Tadatake Oku, Toshiyuki Nishio

Author information

Kazunari Kadokura
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University
Yusuke Sakamoto
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University
Akiko Rokutani
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University
Takanori Ikegami
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University
Takako Hirano
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University
Mahiro Yamamoto
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University
Kaori Saito
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University
Wataru Hakamata
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University
Shiro Itoi
Department of Marine Sciences and Resources, College of Bioresource Sciences, Nihon University
Haruo Sugita
Department of Marine Sciences and Resources, College of Bioresource Sciences, Nihon University
Tadatake Oku
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University
Toshiyuki Nishio
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University

Keywords: Vibrio parahaemolyticus, chitinase, chitin oligosaccharide deacetylase, heterodisaccharide, recombinant enzyme

JOURNAL FREE ACCESS

2008 Volume 55 Issue 2 Pages 157-164

DOI https://doi.org/10.5458/jag.55.157

Details

Abstract

Chitinase (Pa-Chi) and chitin oligosaccharide deacetylase (Pa-COD) are involved in the production of a heterodisaccharide, β-D-N-acetylglucosaminyl-(1,4)-D-glucosamine (GlcNAc-GlcN). These enzymes were recovered from the supernatant of Vibrio parahaemolyticus KN1699 cell culture and purified and characterized. For each enzyme, an ORF encoding gene and its signal sequence were cloned from genomic DNA of strain KN1699. In addition, the expression plasmid was constructed for each enzyme gene and inserted into Escherichia coli cells, and recombinant Pa-Chi and Pa-COD (Pa-rChi and Pa-rCOD) were secreted into the culture medium with the aid of signal peptides. Di-N-acetylchitobiose [(GlcNAc)₂] was produced in 60% (w/w) yield by cultivating the Pa-rChi-secreting E. coli cells in 2% (w/v) β-chitin-containing medium. Moreover, GlcNAc-GlcN was produced in high yield by treating (GlcNAc)₂ with the culture supernatant of Pa-rCOD-secreting E. coli cells.

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