Kim Pyoung Il, Kang Tae Heung, Chung Kyoung Jin, Kim In Seon, Chung Ki-Chul
Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, USA.
FEMS Microbiol Lett. 2004 Nov 1;240(1):31-9. doi: 10.1016/j.femsle.2004.09.006.
A chitosanase produced constitutively by Bacillus sp. MET 1299 was purified by SP-Sephadex column chromatography. The molecular weight was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal enzyme activity was observed at a pH of 5.5 and temperature of 60 degrees C. The purified chitosanase showed high activity on 90% deacetylated colloidal chitosan and beta-glucan, but not on hydrolyzed colloidal chitin, CMC, or their derivatives. The N-terminal amino acid sequence of the enzyme was determined. The cloned full length gene, 1362 bp in size, encoded a single peptide of 453 amino acids and had a conserved amino acid sequence of glycosyl hydrolase family 8. A search of the cDNA sequence with NCBI BLAST showed homology with chitosanase of Bacillus sp. KTCC 0377BP and Bacillus sp. No. 7-M. The recombinant protein was expressed in Escherichia coli, purified using affinity chromatography and characterized.
由芽孢杆菌属MET 1299组成型产生的壳聚糖酶通过SP-葡聚糖凝胶柱色谱法进行纯化。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估计其分子量为52 kDa。在pH 5.5和温度60℃时观察到最佳酶活性。纯化的壳聚糖酶对90%脱乙酰化的胶体壳聚糖和β-葡聚糖具有高活性，但对水解的胶体几丁质、羧甲基纤维素（CMC）或其衍生物没有活性。测定了该酶的N端氨基酸序列。克隆的全长基因大小为1362 bp，编码一个由453个氨基酸组成的单一肽段，并且具有糖基水解酶家族8的保守氨基酸序列。使用NCBI BLAST对cDNA序列进行搜索，结果显示其与芽孢杆菌属KTCC 0377BP和芽孢杆菌属7-M的壳聚糖酶具有同源性。该重组蛋白在大肠杆菌中表达，通过亲和色谱法进行纯化并对其特性进行了表征。
