摘要
为了促进家畜对纤维素饲料的消化吸收,提高饲料利用率,改善畜产品品质,本研究基于海栖热袍菌(Thermotoga maritima)的基因组信息,通过筛选获得一段具有潜在耐热特性的葡聚糖内切酶基因(EG12B)序列。经删除预测信号肽和优化密码子偏好性,运用基因工程手段将成熟肽EG12B基因克隆至原核表达载体pET-28a中,成功构建了重组表达质粒pET28-EG12B。转化至大肠杆菌BL21(DE3)感受态细胞中,经重组菌发酵培养及IPTG诱导表达,实现了EG12B在大肠杆菌系统中的胞内高效表达,表达量约占全细胞总蛋白的36%。通过细胞超声破碎、镍离子亲和层析以及热处理分离纯化,获得了电泳纯重组酶EG12B。酶学特性分析表明,该重组酶的最适反应温度为90℃,最适反应pH为5.2,在70℃下保温1 h,酶活力基本维持不变。表明葡聚糖内切酶EG12B具有优越的耐热性和良好的热稳定性,为进一步作为饲料添加剂用于纤维素饲料关键技术的开发和畜产品品质的提升奠定了理论基础。
In order to promote digestion and absorption of livestock for cellulose feed, improve feed conversion rate and quality of livestock, a potential thermostable endoglucanase gene( EG12B ) was screened based on Thermotoga maritima genome information. After deletion of predicted signal peptide and optimization of codon preference, mature peptide EG12B gene was cloned into prokaryotic expression vectors of pET-28a by gene engineering technology. The recombinant expression plasmid of pET28-EG12B was successfully constructed, and then transformed into E.coli BL21 (DE3) competent cells. Recombinant strain was cultured and induced by IPTG to achieve intracellular efficient expression of EG12B in E.coli system, and the expression level was about 36% of total cell proteins. Recombinant enzyme EG12B with electrophoretic purity was obtained by separation and purification including ultrasonic cell disruption, nickel column affinity chromatography and heat treatment. The analysis of enzymatic properties showed that the optimum reaction temperature was 90 ℃ and the optimum reaction pH was 5.2. The enzyme activity basically remained unchanged under 70 ℃ for 1 h. It suggested that endoglucanase EG12B has excellent thermal resistance and good thermostability, which provids a preliminary foundation for further development of the key technology of cellulose feed and improvement of livestock quality as feed additive.
作者
王珊珊
任艳艳
张涛
路宏朝
王令
WANG Shanshan;REN Yanyan;ZHANG Tao;LU Hongzhao;WANG Ling(School of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong 723001, China;Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Xi'an 710069, China)
出处
《家畜生态学报》
北大核心
2019年第4期20-26,共7页
Journal of Domestic Animal Ecology
基金
陕西省教育厅专项科研计划项目(17JK0154)
陕西省科技厅重点研发计划项目(2017NY-162)
国家自然科学基金项目(21506171)
博士后科学基金项目(2015M572593)
陕西理工大学科研基金项目(SLGQD16-05)
