title: Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase.
abstract: Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc(n), with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc(n). On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.
authors.affiliation: a Biochemistry-Electrochemistry Research Unit , Schools of Chemistry and Biochemistry, Suranaree University of Technology , Nakhon Ratchasima , Thailand.
authors.forename: Paknisa
authors.initials: P
authors.lastname: Sirimontree
authors.forename: Wipa
authors.initials: W
authors.lastname: Suginta
authors.forename: Natchanok
authors.initials: N
authors.lastname: Sritho
authors.forename: Yuka
authors.initials: Y
authors.lastname: Kanda
authors.forename: Shoko
authors.initials: S
authors.lastname: Shinya
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006868:Hydrolysis; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis*; D015202:Protein Engineering; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D001224:Aspartic Acid; D001216:Asparagine; D002688:Chitinases; D000409:Alanine; D000117:Acetylglucosamine
keywords: Vibrio harveyi; chitooligosaccharides; family GH18 chitinase; site-directed mutagenesis; transglycosylation