Improved Recombinant Expression of Maltogenic α-Amylase AmyM in <i>Bacillus subtilis</i> by Optimizing Its Secretion and NADPH Production

Abstract

The maltose α-amylase AmyM from Bacillus stearothermophilus can be used for flour modification, baked goods preservation, and maltose production. Here, we optimized the recombinant expression of AmyM in Bacillus subtilis WB800 via several strategies. By screening the optimal promoter, a double promoter combination (P43 and PamyL) could improve the expression level of AmyM by 61.25%, compared with the strong promoter P43. Then, we optimized the secretion efficiency of recombinant AmyM by over-expressing the molecular chaperone prsA gene. SDS-PAGE results suggested that over-expression of the prsA could improve the secretion efficiency of AmyM to the extracellular environment. The extracellular enzyme activity of AmyM was increased by 101.58% compared to the control strain. To further improve the expression of AmyM, we introduced the hemoglobin gene of Vitreoscilla (vgb) into the AmyM recombinant strain. The results revealed that the introduction of vgb could promote the transcription and translation of AmyM in B. subtilis. This may be due to the increasing level of intracellular NADPH and NADP+ caused by the expression of vgb. By this strategy, the expression level of AmyM was increased by 204.08%. Finally, we found the recombinant AmyM showed an optimal temperature of 65 °C and an optimal pH of 5.5. Our present results provided an effective strategy for increasing the heterologous expression level of AmyM in B. subtilis.

Keywords

• maltogenic α-amylase AmyM
• recombinant expression
• molecular chaperone prsA
• hemoglobin gene of Vitreoscilla (<i>vgb</i>)