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（zlipolytica Bohaisea-9145，LipYp）基因坳rp（1116bp）克隆并在Pichiapastoris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pH7．0—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某苯基酯类和甘油三酯类rC10-C12、有较强的水解能力。%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 expressio6 vector pPIC9K and successfully integrated into a heterologous fungal host Pichia pastoris GS 115. 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/mi 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 （C 10--C 12） and toward p-nitrophenyl esters.
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