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Saccharification of β-Chitin From Squid Pen by a Fermentation Method Using Recombinant Chitinase-Secreting Escherichia coli

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Abstract

Two strains [BL21(DE3) and HMS174(DE3)] of Escherichia coli harboring the recombinant chitinase expression plasmid pVP-Chi, which contains Vibrio parahaemolyticus chitinase gene with an attached signal sequence, were prepared. These E. coli transformants produced a large amount of recombinant chitinase, which hydrolyzes chitin to yield di-N-acetylchitobiose (GlcNAc)2, under the presence of isopropyl-1-thio-β-D-galactopyranoside (IPTG), and secreted the enzyme into their culture fluid with the aid of the signal peptide. Cultivation of these E. coli transformants in Luria-Bertani medium containing squid pen β-chitin and IPTG gave rise to the decomposition of this polysaccharide and the accumulation of (GlcNAc)2 in the culture fluid. Through these experiments, we confirmed that the use of strain HMS174(DE3) was preferable for the stable accumulation of (GlcNAc)2 in the culture fluid during cultivation owing to lower (GlcNAc)2 assimilation compared to BL21(DE3). Next, using E. coli HMS174(DE3) transformants, we conducted saccharification of different forms (fluffy fiber, flake, and powder) of β-chitin samples prepared from squid pens in Bacterion-N-KS(B)K medium containing 2 % of each sample under the presence of IPTG. In these experiments, (GlcNAc)2 was isolated with a more than 20 % stoichiometric yield from each culture supernatant through charcoal column chromatography followed by recrystallization.

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Acknowledgments

We thank Kenichi Koseki and Dr. Kazuhide Totani, Department of Chemical Engineering, Ichinoseki National College of Technology, for providing pens of Japanese common flying squid. This study was supported by a grant for Creative Scientific Research (C) of the Ministry of Education, Science, Sports and Culture (Japan) from 2009 to 2011. To perform this study, grant from College of Bioresearch Sciences of Nihon University was also used.

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Authors and Affiliations

  1. Department of Chemistry and Life Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa, 252-8510, Japan

    Takamitsu Minamoto, Narumi Takahashi, Sayaka Kitahara, Yuko Shinozaki, Takako Hirano, Wataru Hakamata & Toshiyuki Nishio

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  1. Takamitsu Minamoto
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  2. Narumi Takahashi
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  3. Sayaka Kitahara
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  4. Yuko Shinozaki
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  5. Takako Hirano
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  6. Wataru Hakamata
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  7. Toshiyuki Nishio
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Correspondence to Toshiyuki Nishio.

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Minamoto, T., Takahashi, N., Kitahara, S. et al. Saccharification of β-Chitin From Squid Pen by a Fermentation Method Using Recombinant Chitinase-Secreting Escherichia coli . Appl Biochem Biotechnol 175, 3788–3799 (2015). https://doi.org/10.1007/s12010-015-1547-9

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