枯草杆菌manA基因的克隆及定点突变

Cloning and Site-directed Mutation of manA Gene from Bacillus subtilis

摘要:

manA是编码β-甘露聚糖酶(β-1,4-mannan mannohydrolase EC 3.2.1.78)的基因。将枯草杆菌A33株的manA基因插入到pET-32a载体，并在大肠杆菌BL21(DE3)中实现了异源非融合表达，表达活力为41.58U/mL。为了提高酶的表达活力，当采用PCR介导的定点突变技术将该基因第2号密码子CUU 突变为GUU，构建成突变表达载体pET-32a-manA^*并转入大肠杆菌BL21(DE3)中表达，目标酶表达活力增加到138.65U/mL。说明当β-甘露聚糖酶N端第二号氨基酸由亮氨酸突变为缬氨酸后，酶在大肠杆菌中的表达活力大大提高。推测是由于突变后的β-甘露聚糖酶在大肠杆菌中的稳定性增强所致。突变表达的β-甘露聚糖酶最适作用温度和pH值并没有发生明显改变。

Abstract:

manA is a gene that encode β-mannanase (β-1,4-mannan mannohydrolase EC 3.2.1.78). ManA gene isolated from Bacillus subtilis strain A33 was cloned into an E.coli expression vector pET-32a and be transformed into E. coli strain BL21(DE3). An expression activity 41.58 U/ml was obtained after inducing. To get a better expression level that a site-directed mutation based on PCR was used to induce a mutant β-mannanase gene. The second code of the gene CUU was changed into GUU thus the second amino acid of β-mannanase of N-terminus changed from leucine to valine. The constructed mutated gene vector pET-32a-manA^* was transformed into strain E. coli BL21(DE3) and induced. The expression activity is increased to 138.65U/ml. It is predicted that a single change of amino acid enhancing the stability of expressed β-mannanase and greatly improve the expression level. The optimum temperature and pH of the enzyme is not changed observably.