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| Expression of Bacillus lipases in Pichia pastoris and characterization of its enzymatic properties |
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Received:June 13, 2025 Revised:March 09, 2026 Accepted:October 14, 2025
Published：July 20, 2026
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| DOI：10.19902/j.cnki.zgyz.1003-7969.250282 |
| KeyWord:Bacillus Pichia pastoris lipase recombinant expression enzymatic property |
| FundProject:国家重点研发计划（2022YFC2104905） |
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| Abstract: |
| To promote the application of Bacillus lipases, the Pichia pastoris X-33 strain was used for heterologous expression of the lipases LIPBST (derived from Bacillus stratosphericus L1) and BSLA (derived from Bacillus subtilis strain 168). After screening for the colonies with the highest protein expression and enzyme activity, the induction conditions were optimized. The recombinant proteins were purified by Strep-Tactin XT Sepharose chromatography, and their enzymatic properties were characterized after removal of impurities by dialysis. The optimal induction conditions for LIPBST were 2% methanol with an initial OD600 of 3, while those for BSLA were 1% methanol with an initial OD600 of 1 Under these optimal conditions, the production of purified LIPBST and BSLA were 230 μg/mL and 125 μg/mL, with corresponding enzyme activities of 195 U/mL and 126 U/mL, respectively. Enzymatic property studies showed that both recombinant lipases exhibited typical characteristics of alkaline lipases, with an optimal reaction pH of 9.0 and an optimal temperature of 30 ℃. Notably, LIPBST demonstrated superior thermostability （20-30 ℃）and alkaline tolerance(pH 10-11) compared to BSLA. Li+ and Mg2+ activated both lipases, while surfactants, inhibitors (except EDTA), and organic solvents (except DMSO) inhibited their activities. The two lipases showed similar substrate preferences, favoring medium-to-short-chain substrates, which was consistent with the structural characteristics of Bacillus lipases. In summary, a high-level secretory expression of LIPBST and BSLA in Pichia pastoris is successfully achieved, and LIPBST possesses better thermostability and alkaline tolerance, providing a potential for the industrial application. |
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