宏基因组中新角蛋白酶基因KerA的克隆与鉴定  

Cloning and analysis of the novel keratinase( KerA) gene from soil metagenome

作　　者：马建[1] 许诺[2] 王英哲[3] 王云鹏[3] 刘艳芝[3] 

机构地区：[1]吉林农业大学农学院,长春130118 [2]温州大学生命科学研究院,浙江温州325035 [3]吉林省农业科学院农业生物技术研究所,长春130124

出　　处：《黑龙江畜牧兽医》2017年第3期37-40,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：现代农业产业技术体系建设专项资金项目(CARS-35-02)

摘　　要：皮革鞣制过程中产生大量有害物质,对环境造成了严重污染。角蛋白酶是一类水解角蛋白的酶,是皮革鞣制中理想的鞣制剂替代品。为了发掘角蛋白酶重要的基因资源,试验从土壤中克隆KerA基因,构建了原核表达载体p ET-22b-KerA,通过热激法转入BL21(DE3)中进行表达并在表达时间上进行了优化,对表达的产物进行Western-blot鉴定,通过Ni2+Sepharose Fast Flow分离纯化了KerA,并通过水解角蛋白验证其活性。结果表明:克隆的KerA基因可以在原核中表达,并具有水解角蛋白的活性。说明基因工程重组手段生产KerA具有可行性。Leather tanning process produces a large amount of harmful substances, causing serious environment pollution. Keratinase is a kind of enzyme that hydrolyzes keratin, which is an ideal substitute of leather tanning agents. To explore important genetic resources of keratinase, keratinase （KerA） gene was cloned from the soil, and was used to construct prokaryotic expression vector pET - 22b - KerA. The expression vector was transformed into BL21 （DE3） for expression by the heat shock method, while the time of expression was optimized. The expression product was identified by Western - blot, and then the KerA was isolated and purified by Ni^2＋ Sepharose Fast Flow, while its activity was verified by the hydrolyzed keratin. The results showed that the cloned KerA gene can be expressed in the prokaryotic system, and has the activity of hydrolyzed keratin. The results indicate that it is feasible to produce KerA by genetic engineering restructuring.

关 键 词：宏基因组 皮革鞣制 角蛋白酶 KerA基因 基因工程重组 

分 类 号：S859.81[农业科学—临床兽医学] Q55[农业科学—兽医学]