| 摘要: |
| 采用基因重组和分子生物学相关技术, 对Yarrowia lipolytica Bohaisea-9145海洋低温碱性脂肪酶(Y. lipolytica Bohaisea-9145, LipYp)基因lipYp (1116bp)克隆并在Pichia pastoris中进行了异源真核表达研究。通过MD和MM平板及PCR扩增, 筛选和鉴定了重组子。结果表明, 阳性重组子经摇瓶发酵54h后上清液酶活力达到1956U/ml。发酵液经两步纯化得到在SDS-PAGE上显示为单一条带的重组脂肪酶。实验同时研究了不同反应温度、pH值对重组LipYp活力和稳定性的影响, 结果显示重组LipYp最适反应温度和pH分别为35℃和8.5, 在pH 7.0—10.5之间以及45℃以下有较好稳定性。另外, 重组脂肪酶对中长链对硝基苯基酯类和甘油三酯类(C10—C12)有较强的水解能力。 |
| 关键词: Y. lipolytica Bohaisea-9145, 毕赤酵母GS115, 基因克隆, 异源表达, 性质 |
| DOI：10.11693/hyhz201202005005 |
| 分类号: |
| 基金项目:国际科技合作与交流专项, 2011DFB30250 号；国家“863”计划资助项目, 2011AA090703 号；中央级公益性科研院所基本科研业务费专项目, 20603022011011 号 |
附件 |
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| AN ALKALINE LIPASE FROM YARROWIA LIPOLYTICA BOHAISEA-9145: GENE CLONING, HETEROLOGOUS EXPRESSION, AND CHARACTERIZATION |
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LI Zhong-Lei1,2, WANG Yue-Jun1, SHENG Jun1, LIU Jun-Zhong1, SUN Mi1
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1.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;2.College of Food Science & Technology, Shanghai Ocean University
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| Abstract: |
| A pair of primers were designed according to the nucleotide sequence of the alkaline lipase gene (lipYp, 1116bp in length) from psychrotrophic marine yeast Yarrowia lipolytica BohaiSea-9145. The lipYp sequence was subcloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host Pichia pastorisGS115. Recom-binant yeast was obtained by minimal dextrose and minimal methanol plates and confirmed by PCR. In shake-flask culti-vation, the enzyme activity was 1956U/ml after induction by methanol for 54h. After fermentation, the recombinant lipase was purified, and SDS-PAGE analysis showed the purified lipase was of high purity. The recombinant lipase showed high lipolytic activity in the range of pH 7.0—10.5. The optimal temperature and pH value for the lipases activity were 35℃ and 8.5, respectively. Additionally, the enzyme hydrolyzed p-nitrophenyl caprate (C10) at the highest hydrolysis efficiency among the other p-nitrophenyl esters. The lipase showed high activity toward long-chain fatty acid esters (C10—C12) and toward p-nitrophenyl esters. |
| Key words: Yarrowia lipolytica Bohaisea-9145, Pichia pastoris GS115, Gene clone, Heterologous expression, Characterization |
