Chitinase catalyzes β,4-glycosidic linkages in chitin and has attracted re- search interest due to it being a potential pesticide target and an enzymatic tool for preparation of N-acetyl-β D-glucosamine. An individu...Chitinase catalyzes β,4-glycosidic linkages in chitin and has attracted re- search 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, Of ChtS, the glycoside hydrolase family 18 chiti- nase 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 Of CHT5 consisted of an open reading frame of 1 665-bp nucleotides. Phy- logenic analysis suggested Of Cht5 belongs to the Group I insect chitinases. Expression of Of Cht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol. Through two steps of purification, consisting of ammonium sulfate pre- cipitation and metal chelating chromatography, about 7 mg of the recombinant Of Cht5 was purified to homogeneity from 1 L culture supernatant. Of Cht5 effectively converted colloidal chitin into chitobiose, but had relatively low activity toward a-chitin. When chi- tooligosaccharides [（GlcNAc）n, n = 3-6] were used as substrates, Of Cht5 was observed to possess the highest catalytic efficiency parameter toward （GlcNAc）4 and predominan- tely hydrolyzed the second glycosidic bond from the non-reducing end. Together with fl-N-acetyl-D-hexosaminidase OfHexl, Of Cht5 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 Of Cht5 versus Of Hexl in the range of 9 ： 1-15 ： 1. This work provides an alternative to existing preparation of chitinase for pesticides and other applications.展开更多
文摘Chitinase catalyzes β,4-glycosidic linkages in chitin and has attracted re- search 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, Of ChtS, the glycoside hydrolase family 18 chiti- nase 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 Of CHT5 consisted of an open reading frame of 1 665-bp nucleotides. Phy- logenic analysis suggested Of Cht5 belongs to the Group I insect chitinases. Expression of Of Cht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol. Through two steps of purification, consisting of ammonium sulfate pre- cipitation and metal chelating chromatography, about 7 mg of the recombinant Of Cht5 was purified to homogeneity from 1 L culture supernatant. Of Cht5 effectively converted colloidal chitin into chitobiose, but had relatively low activity toward a-chitin. When chi- tooligosaccharides [（GlcNAc）n, n = 3-6] were used as substrates, Of Cht5 was observed to possess the highest catalytic efficiency parameter toward （GlcNAc）4 and predominan- tely hydrolyzed the second glycosidic bond from the non-reducing end. Together with fl-N-acetyl-D-hexosaminidase OfHexl, Of Cht5 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 Of Cht5 versus Of Hexl in the range of 9 ： 1-15 ： 1. This work provides an alternative to existing preparation of chitinase for pesticides and other applications.
