High Level Secretion Expression Of A Lipase From Stenotrophomonas Maltophilia In Bacillus Subtilis

Bacillus subtilis has many advantages on the expression of lipase, such as good safety performance and high secretory expression efficiency. It has become another important prokaryotic expression system f1r lipase apart from E. coli. The lipase LipS is anorganic solvent tolerant lipase, it can catalize hydrolysis reaction, esterification and transesterification reaction. The lipase has important industrial application potential. A very important application of the lipase LipS is that it can prepare L-menthol with high selectivity.As a secretory expression strain, there are many problems affecting the level of secretion. The efficiency of the signal peptide to the lipase, transcriptional level and translational level of lipase are three main bottlenecks for the secretory expression of the lipase LipS.First, we screened the signal peptides of Bacillus subtilis according to the difference of structures of signal peptides. In the Sec pathway, we fixed the number of charged residues of N-domain to be 2, and changed the length of H-domain to find a suitable signal peptide. In the Tat pathway, we screened the signal-peptide(SP for short) phoD and SP-lipA which were researched sufficiently. The SP-phoD’s secretion level was the best of all, its secretion efficiency reached 24.29%, and the activity of extracellular lipase reached 9.82 U/L. In the Sec pathway, the SP-NprB had the highest secretion level, its secretion efficiency reached 19.26%, and the activity of extracellular enzyme reached 7.68 U/L.Second, we mutated the SP-phoD and SP-NprB to increase the secretion level of lipase LipS. For the SP-phoD, the mutation SP-phoDDFR had the highest secretion efficiency, reaching 31.70% and the extracellular lipase’s acitvity reached 12.57 U/L. For the SP-NprB, the mutation SP-NprB3K had the highest secretion efficiency, reaching 19.26% and the extracellular lipase’s acitvity reached 7.68 U/L.Then, we changed the vectors with different promoters to enhance the transcriptional level We found the recombinant strain WB800N(pHBphoDDFRL) was the most outstanding of these strains. The extracellular lipase’s acitvity of it reached 40.12 U/L and its culture time was much shorter.To enhance the translaonal level, we changed the sequence of mRNA to increase the flexibility of the mRNA, and raised the translation initiation rate of the lipase. The recombinant strain WB800N(pHBRCphoDDFRL) was the most efficient of all strains. The extracellular lipase’s acitvity reached 62.18 U/L. Compared with WB800N (pHBphoDDFRL), it had increased by 53.83%.In the end, we optimized the culture conditions of the recombinant bacteria WB8 OON(pHBRCphoDDFRL), and the extracellular enzyme’s acitvity reached 91.17 U/L with the optimum growth conditions, and we measured the stability of the vector pHB RCphoDDFRL.