Saccharification of β-chitin from squid pen by a fermentation method using recombinant chitinase-secreting Escherichia coli.
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.
Department of Chemistry and Life Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa, 252-8510, Japan.
Takamitsu
T
Minamoto
Narumi
N
Takahashi
Sayaka
S
Kitahara
Yuko
Y
Shinozaki
Takako
T
Hirano
Wataru
W
Hakamata
Toshiyuki
T
Nishio
D000818:Animals; D002686:Chitin / Q000737:chemistry* / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049832:Decapodiformes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005285:Fermentation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases