一种几丁质酶基因的克隆表达及酶解产物分析

Cloning and Expression of a Novel Chitinase Gene and the Analysis of Its Hydrolysis Products

摘要:

【目的】克隆耐冷菌假交替单胞菌(Pseudoalteromonas sp. DL-6)的几丁质酶基因并进行原核表达，纯化重组蛋白并研究其酶解产物。【方法】采用PCR扩增法从Pseudoalteromonas sp. DL-6中克隆几丁质酶基因(chiA)，连接到表达载体pET28a，导入Escherichia coli BL21(DE3)进行诱导表达。SDS-PAGE检测几丁质酶ChiA的分子量与纯度，4-甲基伞形酮荧光底物4MU-(GlcNAc)2测定酶活，电喷雾质谱(ESI-MS)检测酶解产物。【结果】chiA基因(GenBank登录号KF234015)在大肠杆菌中高效表达，Ni-NTA亲和层析柱纯化几丁质酶chiA的总活力可达168.68 U。ESI-MS检测结果表明重组蛋白酶解1%胶体几丁质的产物为几丁寡糖。【结论】利用内切几丁质酶ChiA水解几丁质生产几丁寡糖，为其在食品、医药和农业等领域的潜在应用提供有利参考。

Abstract:

[Objective] A novel chitinase gene was cloned from the genomic DNA of psychrophilic bacterium, Pseudoalteromonas sp. DL-6 and heterologously expressed in Escherichia coli BL21(DE3). The recombinant protein was purified and the enzymatic hydrolysates were analyzed. [Methods] The chitinase gene, chiA, was amplified from Pseudoalteromonas sp. DL-6 by using a PCR protocol, linked to pET28a, and finally expressed in E. coli BL21(DE3). The molecular weight and purity were determined by SDS-PAGE, and enzymatic activity was detected by 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside (4MU-(GlcNAc)2). The hydrolysis products were identified by electrospray ionisation mass spectrometry (ESI-MS). [Results] The chiA (GenBank: KF234015) was efficiently expressed in E. coli BL21(DE3), and the total activity of purified ChiA was 168.68 U using Ni-NTA resin. ESI-MS analysis showed that the hydrolysis products of 1% colloidal chitin after ChiA digestion consisted of a series of chitin oligomers. [Conclusion] The research provided a reference for endo-type ChiA used in the food, pharmaceutical and agricultural industry.