Lee Y S, Yoo J S, Chung S Y, Lee Y C, Cho Y S, Choi Y L
Division of Biotechnology, Faculty of Natural Resources and Life Science, Dong-a University, Busan, 604-714, South Korea.
Appl Microbiol Biotechnol. 2006 Nov;73(1):113-21. doi: 10.1007/s00253-006-0444-0. Epub 2006 Apr 28.
A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 degrees C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca(2+). However, Hg(2+) and Ni(+) ions strongly inhibited the enzyme. The enzyme produced, GlcN(2-4), were the major products from a soluble chitosan.
从韩国传统食品中分离出一株产壳聚糖酶的芽孢杆菌属菌株DAU101。基于16S rDNA序列、gyrA基因的系统发育分析以及表型分析对该菌株进行了鉴定。克隆并测定了编码壳聚糖酶（csn）的基因序列。csn基因由一个837个核苷酸的开放阅读框组成，编码279个氨基酸，推导的分子量为31420 Da。芽孢杆菌属菌株DAU101的壳聚糖酶推导氨基酸序列与枯草芽孢杆菌和假单胞菌属的壳聚糖酶分别具有88%和30%的相似性。通过谷胱甘肽S-转移酶融合纯化系统对壳聚糖酶进行了纯化。纯化酶的分子量约为27 kDa，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果表明其信号肽已被去除。该酶的最适pH和温度分别为7.5和50℃。添加5或10 mM Ca(2+)可使酶活性提高约1.6倍。然而，Hg(2+)和Ni(+)离子强烈抑制该酶。该酶作用于可溶性壳聚糖产生的主要产物是GlcN(2-4)。
