title: Cloning, expression and biocharacterization of OfCht5, the chitinase from the insect Ostrinia furnacalis.
abstract: Chitinase catalyzes β-1,4-glycosidic linkages in chitin and has attracted research interest due to it being a potential pesticide target and an enzymatic tool for preparation of N-acetyl-β-D-glucosamine. An individual insect contains multiple genes encoding chitinases, which vary in domain architectures, expression patterns, physiological roles and biochemical properties. Herein, OfCht5, the glycoside hydrolase family 18 chitinase from the widespread lepidopteran pest Ostrinia furnacalis, was cloned, expressed in the yeast Pichia pastoris and biochemically characterized in an attempt to facilitate both pest control and biomaterial preparation. Complementary DNA sequence analysis indicated that OfCHT5 consisted of an open reading frame of 1 665-bp nucleotides. Phylogenic analysis suggested OfCht5 belongs to the Group I insect chitinases. Expression of OfCht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol. Through two steps of purification, consisting of ammonium sulfate precipitation and metal chelating chromatography, about 7 mg of the recombinant OfCht5 was purified to homogeneity from 1 L culture supernatant. OfCht5 effectively converted colloidal chitin into chitobiose, but had relatively low activity toward α-chitin. When chitooligosaccharides [(GlcNAc)n , n= 3-6] were used as substrates, OfCht5 was observed to possess the highest catalytic efficiency parameter toward (GlcNAc)4 and predominantely hydrolyzed the second glycosidic bond from the non-reducing end. Together with β-N-acetyl-D-hexosaminidase OfHex1, OfCht5 achieved its highest efficiency in chitin degradation that yielded N-acetyl-β-D-glucosamine, a valuable pharmacological reagent and food supplement, within a molar concentration ratio of OfCht5 versus OfHex1 in the range of 9 : 1-15 : 1. This work provides an alternative to existing preparation of chitinase for pesticides and other applications.
authors: Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China.
authors: Qingyue
authors: Q
authors: Wu
authors: Tian
authors: T
authors: Liu
authors: Qing
authors: Q
authors: Yang
mesh_terms: D000818:Animals; D055162:Biocatalysis; D002686:Chitin; D002688:Chitinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007915:Lepidoptera / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D002686:Chitin; D002688:Chitinases
keywords: N-acetyl-β-D-glucosamine; Ostrinia furnacalis; chitin; chitinase; glycoside hydrolase; pest control