作　　者： ; ; ; ; ; ;

机构地区： 中山大学

出　　处： 《生物化学与生物物理学报》 2003年第4期366-370,共5页

摘　　要： 根据褶皱假丝酵母脂肪酶CRL中LIP1的成熟多肽序列 ,人工合成了由毕赤酵母 (Pichiapastoris)中偏爱密码子组成的lip1基因序列。该基因以N端融合的方式分别正确插入毕赤酵母诱导型表达载体pPICZαA与组成型表达载体pGAPZαA中。通过电激将线性化的上述两种重组质粒分别转化毕赤酵母SMD116 8H细胞 ,筛选获得两株分别具有诱导型表达和组成型表达生物活性LIP1能力的高产菌株。其中 ,组成型表达菌株CHT II换液后 4 8h上清液中含脂肪酶活力为 2 .0 0× 10 5u/L ,表达产物在pH 4～ 8与温度 30～ 5 0℃范围内具有较高的脂肪酶活性 ;高密度发酵条件下 ,发酵 72h ,上清液中含脂肪酶活力可达 1.395× 10 6u/L ,表明构建的重组菌株具有更大的工业化生产优势。 The gene of LIP1, the most important isoenzyme of Candida rugosa lipase (CRL), was artificially synthesized according to its mature peptide sequence. It consisted of 20 codons of preference in Pichia pastoris. The artificial gene was cloned into methanol-inducible expression vector pPICZαA, and constitutive expression vector pGAPZαA, respectively. The linearized recombinant plasmids were transformed into chromosome of Pichia pastoris SMD1168H strain by electroporation. The abilities of expressing LIP1 in both transformed yeasts had been compared, and the yeast transformed with pGAPZαA was more efficient than pPICZαA. A recombinant yeast strain named CHT-II expressed LIP1 constitutively, and was the most efficient one. Some enzymetic properties of the recombinant LIP1 were also determined. CHT-II secreted LIP1 into supernate at a level of 2.00×10 5 u/L after 72 h (the cells had been transferred to fresh culture medium at the 24th h). After optimizing the conditions for high cell-density fermentation, the selected yeast strain could secrete LIP1 into supernate at a level of 1.395×10 6 u/L after 72 h. The results indicated that this modification of lip1 gene was successful.

关 键 词： 褶皱假丝酵母脂肪酶 脂肪酶 毕赤酵母 诱导型表达 组成型表达 高密度发酵 重组菌株

领　　域： [轻工技术与工程] [化学工程]