源自大黄鱼肠道柠檬酸杆菌的外切几丁质酶的特性分析

Characterization of an exo-chitinase from a Citrobacter strain isolated from the intestine content of large yellow croakers

摘要:

[目的] 从饲喂富含几丁质饲料的大黄鱼肠道分离具有几丁质分解功能的菌株并分离鉴定新的几丁质酶。[方法] 利用胶体几丁质平板分离饲喂杂鱼的大黄鱼肠道中的几丁质分解菌。对几丁质酶基因chi-X进行了克隆并在大肠杆菌中表达。对CHI-X的酶学性质进行了分析。[结果] 从饲喂杂鱼的大黄鱼肠道内容物中分离出1株具有几丁质分解功能的费氏柠檬酸杆菌,其中的几丁质酶基因编码1个含493个氨基酸残基的蛋白,其中包含一个糖苷水解酶18家族催化域。CHI-X对胶体几丁质具有分解功能。最适pH和温度分别是4.0和60 ℃。 CHI-X具有很强的pH稳定性,在pH 3.0-11.0的范围培育1 h仍保留90% 左右的活性。Mn²⁺, Li⁺和K⁺可促进CHI-X酶活,Ag⁺对CHI-X有抑制作用。CHI-X对蛋白酶和石斑鱼肠道内容物有较强的抗逆性。CHI-X可分解胶体几丁质为N-乙酰葡萄糖胺和N-乙酰葡萄糖胺二聚体,表明它是一个几丁质外切酶。最后,CHI-X和另一个几丁质酶Chi565表现出酶活性的加和效应。[结论] 分离自肠道菌的CHI-X能很好适应海水鱼类的肠道环境,可以作为温水海水养殖鱼类的饲料添加剂使用。

Abstract:

[Objective] We isolated bacterial strains with chitin-degrading activity from the digesta of large yellow croakers (Pseudosciaena crocea) fed with chitin-enriched trash fish, and characterized potential chitinases thereof. [Methods] Chitin-degrading strains were screened with colloidal chitin agar from the digesta of P. crocea fed with trash fish. The chitinase gene (chi-X) was cloned and expressed in Escherichia coli, and the enzymatic properties of the chitinase (CHI-X) were characterized. [Results] A Citrobacter freundii strain with chitin-degrading activity was isolated. The chitinase gene encodes a protein containing 493 amino acid residues, with a proposed glycoside hydrolase family-18 catalytic domain. CHI-X could hydrolyze colloidal chitin. The optimal pH for CHI-X was 4.0 at optimal temperature (60 ℃). CHI-X was active over a broad pH range, with around 90% of the activity maintained after incubation at pH between 3.0 and 11 for 1 h. The enzymatic activity of CHI-X was stimulated by Mn²⁺, Li⁺, and K⁺, but inhibited by Ag⁺. The enzyme was stable after treatment by proteases and grouper intestinal juice. CHI-X hydrolyzes colloidal chitin into GlcNAc and (GlcNAc)₂. Furthermore, an synergic effect was observed between CHIX and ChiB565 (a chitinase from Aeromonas veronii B565) on colloidal chitin. [Conclusion] CHI-X from intestinal bacterium may be potentially used as feed additive enzyme for warm water marine fish.