cbh1重组菌的构建、产酶条件及酶特性分析  

Recombinant construction,optimization and enzymatic properties analysis of cellobiohydrolase 1

作　　者：谢志皓 张庆庆[1,2] 汤斌[1,2] 李松[1] 党同学 

机构地区：[1]安徽工程大学生物与化学工程学院,安徽芜湖241000 [2]微生物发酵安徽省工程技术研究中心,安徽芜湖241000

出　　处：《食品工业科技》2015年第18期224-228,243,共6页Science and Technology of Food Industry

基　　金：国家自然科学基金项目(31270135);安徽省自然科学青年基金资助项目(1408085QC61)

摘　　要：从匍枝根霉TP-02中克隆得到cbh1的c DNA,序列测定分析表明,该基因编码526个氨基酸的蛋白质。通过DS2.5同源模拟分析,CBHI酶的活性部位是一个＂桶状＂的隧道结构。将cbh1基因的c DNA序列克隆到大肠杆菌表达载体p ET22b（＋）中,在大肠杆菌BL21（DE3）中进行诱导表达。采用两步层析法对重组的CBHI蛋白进行纯化,对纯酶的特性进行分析。结果表明：在异丙基-β-D-硫代吡喃半乳糖苷诱导终浓度为0.5 mmol/L,诱导温度为20℃,诱导时间为14 h,经过纯化后获得比活力为3.57 IU/mg。重组酶的最适反应温度和p H分别为55℃和5.5,在p H4.5~6.5的范围内稳定性较好,Fe3＋和Co2＋对其具有较强的激活作用;以微晶纤维素为底物,Km值为6.1 mg·m L-1。In this study,the c DNA sequence was cloned from the total RNA of the strain Rhizopus stolonifer TP-02.Sequence analysis showed that this gene encoded a polypeptide with 526 amino acids. Homology modeling was established by DS2.5 software to study cbh1 catalytic hydrolysis and the active site of CBHI was a tunnel structure of ＂barrel. The cbh1 c DNA sequence of the gene was inserted into expression vector p ET22b（＋） and expressed in E.coli BL21（DE3）. Using two-step chromatography column to purification of CBHI. Enzymatic properties of the recombinant CBHI were also investigated. Results showed that the recombinant CBHI activity could reach 3.57 IU/mg at the IPTG concentration of 0.5 mmol/L at 20 ℃ for induction 14 h. The optimum reaction temperature and p H were 55 ℃ and 5.5,and it was stable over a range of p H 4.5~6.5. Fe3＋and Co2＋were promoted to CBHI activity and the Kmwas 6.1 mg·m L-1with avicel.

关 键 词：匍枝根霉 cbh1 克隆和表达 纯化 酶学性质 

分 类 号：TS201.3[轻工技术与工程—食品科学]