地衣芽孢杆菌(Bacillus licheniformis)外切葡聚糖酶CelB基因的发掘及功能鉴定

Exploring and Function Characteristics of Exo-1,4-β-D-glucanase CelB Gene of Bacillus licheniformis

从地衣芽孢杆菌的基因组数据中寻找与外切葡聚糖酶相似性高的DNA序列,发现在地衣芽孢杆菌基因组中CelB DNA片段具有很高的序列相似性。在大肠杆菌中对来自地衣芽孢杆菌的CelB基因DNA片段进行表达并纯化蛋白质。CelB蛋白质对纤维素底物具有活性。根据糖基水解酶家族48的保守活性位点设计突变策略对CelB进行突变,突变体丧失对底物的活性,说明它具有糖基水解酶家族48的外切葡聚糖酶的典型活性位点。

Screening of DNA sequence similar with exo-1,4-β-D-glucanase from the genome data of Bacillus licheniformiswith bioinformatic techniques,one DNA fragmentCelBshowed high similarity was found.This DNA was cloned,expressed,and the result protein was purified.The CelB showed activity to cellulose substrate.Based on the conserved activity sites of glycoside hydrolase family 48 protein,amino acid sites of CelB were choosed for mutation and the mutant of CelB lost the activity,thus it showed that CelB contain the typical active sites of family 48.In this work,a exo-1,4-β-D-glucanase gene was successfully found and cloned from B.licheniformis,it establishes the foundation for the further study of the cellulase system and their working mechanism ofB.licheniformis.