Improvement Of The Thermal And Acidic Stability And Efficient Expression Of The Alpha Amylase BLA

The amylase is one of the most wildely used enzymes. There are lots of studies that have been carried out on the amylase. The gene bla isolated from the strain Bacillus licheniformis, has been employed in lots of the commercial applications. But in the practical application, the enzyme BLA also has some defects. For example: ⑴Although the BLA can tolerate the relative high temperature, its thermostability is limited in many industrial processes;(2)The optimal pH of the BLA is about the 6.0. The enzyme did not have few activity in the acidic condition;(3)The expression amount of the enzyme could be improved. Therefore, it is useful to construct the thermal and acidic BLA with highly expression.This study mainly contained the three parts, as shown in the followings.(1) The secondary structure and sequence of BLA were analyzed. As the glycine is the terminator of the alpha helix, there are fewchances that the amino acid located in the middle of the alpha-helix. However, there are four glycines(G81, G216, G268 and G364) in the middle of the alpha-helix of BLA. The four glycine residues were mutated into alanine by the site-specific mutagenesis. The thermal stability of the WT and mutantions were determined. The Tm values of the G81 A, G216 A and G268 A were increased 1.2 oC, 2.4 oC and 2.2 oC than the wild type. After the mutant of G216 A was incubated in 70 oC for 5 min, the residual enzyme activity was increased by 20% to the wild type.(2) The amino acid sequence of BAA, BLA and PFA were aligned. Here, the amylase PFA is a stable enzyme at the acidic condition. But the amylases(BAA and BLA) are not stabe at the acidic condition. There are six regions that are conservative in the protein BLA and BAA. But they are greatly distinct in the PFA. Based on the sequenc of PFA, the mutants were constructed by the site directed mutagenesis, expressed in the E. coli and purified. The results indicated that the Q264 could affect acidic stability of BLA. The acidic stability of the mutants Q264 D and Q264 S were higher than that of the wild type. After incubated in pH4.5 for 20 min, residual enzyme activity of Q264 S and Q264 D was 100% and 80%. However, the residual enzyme acitivy of the wild type is only 60%.(3) A random mutantion library of BLA was constructed and screened the mutation with high soluble expression in the E. coli. Two interesting mutations(A390I and D401V) were identified, which are located at the interaction surface between the A and C domains of BLA. The A390 I mutation enhanced soluble BLA expression by 2.0-fold compared to the wild type, while D401 V decreased soluble expression 160-fold. Structural analysis revealed that A390 and D401 residues could affect the interaction between the A and C domains of BLA analyzed. Therefore, soluble expression of the target protein in E. coli could be affected by introduction of a mutation in the protein sequence.In conclusion, this study was started from an excellent enzyme(BLA). Based on the methods of the molecular improvement, the thermal and acidic stability of BLA has been imporved and some interesting resides related to its soluble expression were detected. The study on BLA can help us understand the relationship between the molecular structure and function of alpha amylase and provide the theoretical basis for further improvement and application of alpha amylase.