title: Chitinase from Autographa californica multiple nucleopolyhedrovirus: rapid purification from Sf-9 medium and mode of action.
abstract: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase is involved in the final liquefaction of infected host larvae. We purified the chitinase rapidly to homogeneity from Sf-9 cells infected with AcMNPV by a simple procedure using a pepstatin-aminohexyl-Sepharose column. In past studies, a recombinant AcMNPV chitinase was found to exhibit both exo- and endo-chitinase activities by analysis using artificial substrates with a fluorescent probe. In this study, however, we obtained more accurate information on the mode of action of the chitinase by HPLC analysis of the enzymatic products using natural oligosaccharide and polysaccharide substrates. The AcMNPV chitinase hydrolyzed the second β-1,4 glycosidic linkage from the non-reducing end of the chitin oligosaccharide substrates [(GlcNAc)(n), n=4, 5, and 6], producing the β-anomer of (GlcNAc)₂. The mode of action was similar to that of Serratia marcescens chitinase A (SmChiA), the amino acid sequence of which is 60.5% homologous to that of the AcMNPV enzyme. The enzyme also hydrolyzed solid β-chitin, producing only (GlcNAc)₂. The AcMNPV chitinase processively hydrolyzes solid β-chitin in a manner similar to SmChiA. The processive mechanism of the enzyme appears to be advantageous in liquefaction of infected host larvae.
authors: Department of Advanced Bioscience, Kinki University. fukamizo@nara.kindai.ac.jp
authors: Tamo
authors: T
authors: Fukamizo
authors: Hirokazu
authors: H
authors: Sato
authors: Mamiko
authors: M
authors: Mizuhara
authors: Takayuki
authors: T
authors: Ohnuma
authors: Takeshi
authors: T
authors: Gotoh
authors: Kazuyuki
authors: K
authors: Hiwatashi
authors: Saori
authors: S
authors: Takahashi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007814:Larva / Q000821:virology*; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000821:virology*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D002686:Chitin; D002688:Chitinases