Improve The Secretion Of Lipase By Genetic Modification In Bacillus Subtilis

The extracellular lipase(LipA) of B. subtilis, which is an alkaline lipase with the minimum molecular weight, has important value in application. In this work, some critical factors that influenced the secretion of the lipase were studied, including the host strains, deficiencies of a number of extracelluar proteins and the overexpression of tatAD-tatCD operon in B. subtilis.First, the extracellular neutral protease(NprE) and the alkaline protease(AprE) were deleted in B. subtilis 168 and obtained recombinant B. subtilis BNA. Then the plasmid pHP13 L for expression of lipsae was transformed into B. subtillis 168, BNA and DB104, respectively. The activity and stability of lipase in the medium of B. subtillis BNA were significantly higher than them of B. subtillis 168 and DB104. The results showed that the B. subtillis 168 was more suitable for lipase fermentation and the absences of NprE and AprE were beneficial to keep the stability of the secreted lipase.The extracellular proteins YolA, YolB, XynA, YncM and Csn were orderly deleted in B. subtilis BNA and DB104. Then the derivative strains BNAY2-5/pHP13 L and BDY2-5/pHP13 L were obtained by pHP13 L transformed, respectively. The results of fermentation indicated that the deletion of extracellular proteins contributed to increasing the secretion and stability of lipase. Among them, strains BNAY5/pHP13 L and BDY5/pHP13 L that had the highest lipase enzyme activity which reached 230 and 170 U/m L respectively. In addition, the lipase activities in the fermentation broth were significantly stable for 60 h. It was presumed that the significant increase of the secretion of lipase was due to the lack of these extracellular proteins which stopped Sec pathway from staying at the saturated state to some extent.The overexpressed tatAD-tatCD operon was integrated into gene sacB on the chromosome in B. subtilis BNA and BNAY5 to construct B. subtilis BNAT/pHP13 L and BNATY5/pHP13 L. The results of fermentation displayed that the overexpression of TatAdCd transposase made the fermentation unit of the lipase reach 290 U/mL in the strain BNAT/pHP13 L which was as 1.35 times as the original strain. But the lipase activity had no significant difference compared with the control strain in BNATY5/pHP13 L. This phenomenon might indicate that the effect of overexpression of TatAdCd transposase on the secretion of lipase was equivalent to the lack of five extracellular proteins.