Enhanced The Secretion Level Of Lipase A In Bacillus Subtilis Through A Combined Strategy

Lipases hydrolyze triglycerides into glycerol and free fatty acids,it can also catalyze esterification and transesterification.It is widely used in detergent industry,food industry,chemical synthesis and medicine field.Lipase A?LipA?derived from Bacillus subtilis 168 has the following characteristics compared to other lipases,small molecular weight,lack of lid domain,no interfacial activation phenomenon,preference for C8 substrates and excellent alkali tolerance.LipA has been deeply studied with its remarkable characteristics,many mutants with different characteristics have been reported,but the secretion expression level is still low.This study aims to use a combined strategy to improve LipA secretory expression level in B.subtilis WB600,which is deficient in six extracellular protease,to facilitate LipA subsequent researches.In order to achieve effective secretory expression of LipA,we compared the secretion efficiency of different signal peptides and the intensity of different promoters and double promoters.The maximum lipase activity in the culture supernatant was identified as 64.9 U/m L after 12 h fermentation at 37 ^oC in Luria-Bertani medium by the strain B.subtilis L25 containing signal peptide SP_lipA and dual promoter P₄₃-P_hag.Through the exploration of fermentation temperature,incubation time and different commercial media,this study determined that the most suitable conditions for the recombinant strain B.subtilis L25 to secrete lipase A were pre-culture in Terrific-Broth medium at 37 ^oC for3 hours then transferred to 30 ^oC for 19 h,the lipase A yield increased to 710.3 U/m L,which is10.9 times higher than before optimization.Furthermore,the fermentation conditions of lipase A accumulation using glycerol and chicken feather hydrolysates as cheap carbon-nitrogen sources were explored.The results showed that a certain proportion of glycerol?3.6%,v/v?or chicken feather hydrolysate?7.5%,v/v?could increase the accumulation of lipase A to 960.8 U/ml and 956.1 U/ml,respectively.Nevertheless,too much addition will inhibit the accumulation of lipase A,presumably due to the imbalance of carbon and nitrogen.After exploring the ratio of carbon to nitrogen,the lipase yield increased to 1164.9 U/ml at a C/N ratio of 0.5?v/v?,which is the highest extracellular lipase A yield reported in the current literature.This study combined genetic engineering and fermentation optimization to achieve efficient secretory expression of LipA in Bacillus subtilis,which provides a theoretical basis for the application of LipA.Meanwhile,it demonstrates that B.subtilis L25 can use glycerol and microbes treated chicken feather hydrolysates as cheap carbon and nitrogen source,which has certain potential for industrial lipase production.