摘要
采用易错PCR方法对来自于极耐热海栖热袍菌的内切葡聚糖酶Cel1B进行定向进化。携带有Cel12B基因的重组质粒pET-20b-Cel12B在固定Mn2+浓度下,通过使用不同浓度的Mg2+优化易错PCR条件,产物构建成pET-20b-Mu-Cel12B重组子,并建立突变体文库,重组子经改进的刚果红平板法筛选,通过透明圈的大小获得了酶活性大幅度提高的突变体3个,突变基因经诱导后的酶活力分别是在同样条件下诱导获得的亲本酶的2.1倍、3.2倍和3.7倍。
The key role and important value of β- glucanase has been revealed not only in biomass conversion but also in various industries, including food processing, textiles, laundry detergents, and paper manufacturing. A thermostable β-glucanase with high catalytic activity is a particularly attractive candidate for complete degradation of cellulose and brewery. In this study, the directed evolution was employed to increase the activity of β-glucanase from Thermotoga maritime by error-prone PCR. The cel12B gene encoding β-glucanase in recombinant vector pET-20b- Cel12B was amplified by error-prone PCR at a definite concentration of Mn^2＋ , different concentration of Mg^2＋ was used to optimize the PCR for enhancing specificity and mutation, and PCR products were cloned into pET-20b to form recombinant plasmid pET-20b-Mu-Cel12B, followed by generating a mutant molecular library in E. coli. The mutant variants of the encoded -glucanase with enhanced activity were selected by the change method of Congo Red staining. Three mutants （Mut1, Mut2 and Mut3 ） that showed increased enzyme activity compared to the wild type were select- ed according to the size of transparent circles on Congo Red plates. After the three mutants were induced under the same conditions as wild enzyme, the results showed the enzyme activity of Mutl, Mut2, Mut3 was 2. 1-fold, 3.2- fold, and 3.7-fold than that of wild -glucanase, respectively.
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2010年第5期1-4,共4页
Food and Fermentation Industries
基金
江苏省自然科学基金(20971050
BK2007067)
江苏省"六大人才高峰"
青蓝工程"项目支持
长江大学博士启动基金(801100010112)
江苏自教育厅自然科学基金(08KJ220003)
