| 蔡明洁， 盖新双， 王永华， 陈文.芽孢杆菌属脂肪酶在毕赤酵母中的表达及酶学性质研究[J].中国油脂,2026,51(7):.[CAI Mingjie，GE Xinshuang，WANG Yonghua，CHEN Wen.Expression of Bacillus lipases in Pichia pastoris and characterization of its enzymatic properties[J].China Oils and Fats,2026,51(7):.] |
| 芽孢杆菌属脂肪酶在毕赤酵母中的表达及酶学性质研究 |
| Expression of Bacillus lipases in Pichia pastoris and characterization of its enzymatic properties |
|
投稿时间：2025-06-13 修订日期：2026-03-09 录用日期：2025-10-14
出版日期：2026-07-20
|
| DOI：10.19902/j.cnki.zgyz.1003-7969.250282 |
| 中文关键词: 芽孢杆菌 毕赤酵母 脂肪酶 重组表达 酶学性质 |
| 英文关键词:Bacillus Pichia pastoris lipase recombinant expression enzymatic property |
| 基金项目:国家重点研发计划（2022YFC2104905） |
|
|
| 摘要点击次数: 408 |
| 全文下载次数: 5 |
| 中文摘要: |
| 为促进芽孢杆菌属脂肪酶的应用，利用毕赤酵母X-33菌株对芽孢杆菌属脂肪酶LIPBST（来源于Bacillus stratosphericus L1）和BSLA（来源于Bacillus subtilis strain 168）进行异源表达，筛选出蛋白表达量及酶活最高的菌株进行诱导表达，并对诱导表达条件优化，进一步利用Strep-Tactin XT Sepharose层析进行纯化，透析除杂后分析其酶学性质。结果表明：筛选得到的 LIPBST的最佳诱导表达条件为甲醇体积分数2%、诱导起始 光密度（OD600 ） 为 3， BSLA的最佳诱导表达条件为甲醇体积分数1%、诱导起始 OD600 为 1， 在此条件下诱导表达的酶液经纯化后LIPBST和BSLA产量分别为230 μg/mL和125 μg/mL，酶活分别为195 U/mL和126 U/mL；酶学性质研究表明，LIPBST和BSLA均表现出典型的碱性脂肪酶特征，最适反应pH均为9.0，最适反应温度均为30 ℃，且LIPBST的热稳定性（20~30 ℃）和耐碱性(pH 10~11)均优于BSLA，金属离子Li+和Mg2+对2种脂肪酶的酶活均具有激活效果，表面活性剂、抑制剂（除EDTA）、有机溶剂〔除二甲基亚砜（DMSO）〕对2种脂肪酶的酶活具有抑制作用，2种脂肪酶底物偏好性较为一致，均偏好中短碳链底物，符合芽孢杆菌属脂肪酶的结构特点。综上，成功实现了LIPBST和BSLA在毕赤酵母中的高效分泌表达，且LIPBST具有更优的热稳定性和耐碱性，具有工业化应用潜力。 |
| 英文摘要: |
| 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. |
| 查看全文 查看/发表评论 下载PDF阅读器 |
| 关闭 |
