Stepwise synthesis of chitooligosaccharides through a transition-state analogue substrate catalyzed by mutants of chitinase A1 from Bacillus circulans WL-12

Michinari Kohri; Atsushi Kobayashi; Masato Noguchi; Shin Ichi Kawaida; Takeshi Watanabe; Shin Ichiro Shoda

doi:10.1515/HF.2006.080

Stepwise synthesis of chitooligosaccharides through a transition-state analogue substrate catalyzed by mutants of chitinase A1 from Bacillus circulans WL-12

Michinari Kohri, Atsushi Kobayashi, Masato Noguchi, Shin Ichi Kawaida, Takeshi Watanabe, Shin Ichiro Shoda

Research output: Contribution to specialist publication › Article

14 Citations (Scopus)

Abstract

Several mutants of chitinase A1 from Bacillus circulans WL-12 have been prepared by site-directed mutagenesis of tryptophan 433. The hydrolytic activity of these mutants toward p-nitrophenyl chitobioside was lower than that of wild-type chitinase A1. The mutants were found to catalyze the transglycosylation reaction of the 1,2-oxazoline derivative of N-acetyllactosamine (Gal-GlcNAc-oxa) to N,N′-diacetylchitobiose (GlcNAc-Glc-NAc), giving rise to a transglycosylated tetrasaccharide product (Gal-GlcNAc-GlcNAc-GlcNAc) in high yield. Based on these results, a one-pot synthesis of chitooligosaccharides by combined use of mutant W433Y and β-galactosidase has been demonstrated. By controlling the reaction conditions, chitotriose and chitotetraose were obtained in 74% and 40% yield (determined by HPLC), respectively, without isolating any intermediate products.

Original language	English
Pages	485-491
Number of pages	7
Volume	60
No.	5
Specialist publication	Holzforschung
DOIs	https://doi.org/10.1515/HF.2006.080
Publication status	Published - 2006 Aug 1
Externally published	Yes

Keywords

Bacillus circulans WL-12
Chitinase A1
One-pot synthesis
Site-directed mutagenesis
Sugar oxazoline
Transglycosylation
Transition-state analog substrate

ASJC Scopus subject areas

Biomaterials

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10.1515/HF.2006.080

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title = "Stepwise synthesis of chitooligosaccharides through a transition-state analogue substrate catalyzed by mutants of chitinase A1 from Bacillus circulans WL-12",

abstract = "Several mutants of chitinase A1 from Bacillus circulans WL-12 have been prepared by site-directed mutagenesis of tryptophan 433. The hydrolytic activity of these mutants toward p-nitrophenyl chitobioside was lower than that of wild-type chitinase A1. The mutants were found to catalyze the transglycosylation reaction of the 1,2-oxazoline derivative of N-acetyllactosamine (Gal-GlcNAc-oxa) to N,N′-diacetylchitobiose (GlcNAc-Glc-NAc), giving rise to a transglycosylated tetrasaccharide product (Gal-GlcNAc-GlcNAc-GlcNAc) in high yield. Based on these results, a one-pot synthesis of chitooligosaccharides by combined use of mutant W433Y and β-galactosidase has been demonstrated. By controlling the reaction conditions, chitotriose and chitotetraose were obtained in 74% and 40% yield (determined by HPLC), respectively, without isolating any intermediate products.",

keywords = "Bacillus circulans WL-12, Chitinase A1, One-pot synthesis, Site-directed mutagenesis, Sugar oxazoline, Transglycosylation, Transition-state analog substrate",

author = "Michinari Kohri and Atsushi Kobayashi and Masato Noguchi and Kawaida, {Shin Ichi} and Takeshi Watanabe and Shoda, {Shin Ichiro}",

note = "Funding Information: This work was partly supported by a Grant-in-Aid for Scientific Research and JSPS Fellowships from the Ministry of Education, Culture, Sports, Science and Technology. We thank Yaizu Sui-sankagaku Industry for providing us with N-acetyllactosamine and di-N,N′-acetylchitobiose.",

year = "2006",

month = aug,

day = "1",

doi = "10.1515/HF.2006.080",

language = "English",

volume = "60",

pages = "485--491",

journal = "Holzforschung",

issn = "0018-3830",

publisher = "Walter de Gruyter GmbH & Co. KG",

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AU - Kohri, Michinari

AU - Kobayashi, Atsushi

AU - Noguchi, Masato

AU - Kawaida, Shin Ichi

AU - Watanabe, Takeshi

AU - Shoda, Shin Ichiro

N1 - Funding Information: This work was partly supported by a Grant-in-Aid for Scientific Research and JSPS Fellowships from the Ministry of Education, Culture, Sports, Science and Technology. We thank Yaizu Sui-sankagaku Industry for providing us with N-acetyllactosamine and di-N,N′-acetylchitobiose.

PY - 2006/8/1

Y1 - 2006/8/1

N2 - Several mutants of chitinase A1 from Bacillus circulans WL-12 have been prepared by site-directed mutagenesis of tryptophan 433. The hydrolytic activity of these mutants toward p-nitrophenyl chitobioside was lower than that of wild-type chitinase A1. The mutants were found to catalyze the transglycosylation reaction of the 1,2-oxazoline derivative of N-acetyllactosamine (Gal-GlcNAc-oxa) to N,N′-diacetylchitobiose (GlcNAc-Glc-NAc), giving rise to a transglycosylated tetrasaccharide product (Gal-GlcNAc-GlcNAc-GlcNAc) in high yield. Based on these results, a one-pot synthesis of chitooligosaccharides by combined use of mutant W433Y and β-galactosidase has been demonstrated. By controlling the reaction conditions, chitotriose and chitotetraose were obtained in 74% and 40% yield (determined by HPLC), respectively, without isolating any intermediate products.

AB - Several mutants of chitinase A1 from Bacillus circulans WL-12 have been prepared by site-directed mutagenesis of tryptophan 433. The hydrolytic activity of these mutants toward p-nitrophenyl chitobioside was lower than that of wild-type chitinase A1. The mutants were found to catalyze the transglycosylation reaction of the 1,2-oxazoline derivative of N-acetyllactosamine (Gal-GlcNAc-oxa) to N,N′-diacetylchitobiose (GlcNAc-Glc-NAc), giving rise to a transglycosylated tetrasaccharide product (Gal-GlcNAc-GlcNAc-GlcNAc) in high yield. Based on these results, a one-pot synthesis of chitooligosaccharides by combined use of mutant W433Y and β-galactosidase has been demonstrated. By controlling the reaction conditions, chitotriose and chitotetraose were obtained in 74% and 40% yield (determined by HPLC), respectively, without isolating any intermediate products.

KW - Bacillus circulans WL-12

KW - Chitinase A1

KW - One-pot synthesis

KW - Site-directed mutagenesis

KW - Sugar oxazoline

KW - Transglycosylation

KW - Transition-state analog substrate

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JF - Holzforschung

ER -

Stepwise synthesis of chitooligosaccharides through a transition-state analogue substrate catalyzed by mutants of chitinase A1 from Bacillus circulans WL-12

Abstract

Keywords

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