Fukamizo Tamo, Sato Hirokazu, Mizuhara Mamiko, Ohnuma Takayuki, Gotoh Takeshi, Hiwatashi Kazuyuki, Takahashi Saori
Department of Advanced Bioscience, Kinki University.
Biosci Biotechnol Biochem. 2011;75(9):1763-9. doi: 10.1271/bbb.110300. Epub 2011 Sep 7.
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.
苜蓿银纹夜蛾多核多角体病毒（AcMNPV）几丁质酶参与被感染宿主幼虫的最终液化过程。我们通过使用胃蛋白酶抑制剂 - 氨基己基 - 琼脂糖柱的简单程序，从感染AcMNPV的Sf - 9细胞中快速纯化几丁质酶至同质状态。在过去的研究中，通过使用带有荧光探针的人工底物进行分析，发现重组AcMNPV几丁质酶同时具有外切和内切几丁质酶活性。然而，在本研究中，我们通过使用天然寡糖和多糖底物对酶产物进行高效液相色谱分析，获得了关于几丁质酶作用模式的更准确信息。AcMNPV几丁质酶从几丁质寡糖底物[（GlcNAc）（n），n = 4、5和6]的非还原端水解第二个β-1,4糖苷键，产生（GlcNAc）₂的β-异头物。其作用模式与粘质沙雷氏菌几丁质酶A（SmChiA）相似，SmChiA的氨基酸序列与AcMNPV酶的氨基酸序列同源性为60.5％。该酶还水解固体β-几丁质，仅产生（GlcNAc）₂。AcMNPV几丁质酶以类似于SmChiA的方式连续水解固体β-几丁质。该酶的连续作用机制似乎有利于被感染宿主幼虫的液化。
