【作者】 于源； 牟海津；

【Author】 Yu Yuan;Mou Haijin;College of Food Science and Engineering,Ocean University of China;

【机构】 中国海洋大学食品科学与工程学院；

【摘要】 为提升来源于Zobellia sp.ZM-2菌株的κ-卡拉胶酶的酶活力与功能,本文通过毕赤酵母表达系统对κ-卡拉胶酶目的基因cgkZ进行表达。设计并表达截除C-分泌末端结构域(Pst)的截短基因cgkZ△PSt与截除酶与底物结合域(CBM)的cgkZ△CBM;在含截短卡拉胶酶基因cgkZ△Pst的重组酵母菌株基础上,通过联合使用诱导型启动子AOX1与组成型启动子GAP,使卡拉胶酶表达水平得以提升。根据重组菌株培养条件的优化的结果,选取22℃,pH6.0,诱导剂甲醇添加量1%(v/v)的条件摇瓶培养96 h。重组菌株发酵期间,截短基因cgkZ△Pst与cgkZ△CBM的转录水平显著高于原目的基因cgkZ;重组酶cgkZ、cgkZ△PSt与cgkZ△CBM的酶活力分别为4.68、5.70和3.02 U/mL,分子量分别为65,45和40 kDa左右,K_m值分别为2.07、1.85和1.04 mg/mL,重组酶酶解终产物以κ-新卡四糖与六糖为主。联合使用诱导型启动子AOX1与组成型启动子GAP,使得目的基因cgkZ△Pst在无甲醇诱导的条件下得到表达,在22℃下摇瓶培养酶活力为2.73 U/mL。更多还原

【Abstract】 In order to enhance the enzymatic activity and function of K-carrageenase derived from Zobellia sp.ZM-2,the expression of κ-carrageenase gene in Pichia pastoris was studied in this paper.The C-terminal Por secretion tail-truncated gene cgkZ△PSt,and carbohydrate binding domain-truncated gene cgkZ△CBM were designed and amplified.Based on the truncated gene cgkZ△Pst contained recombinant strain,the combined using of inducible promoter AOX1 and constitutive promoter GAP enhanced expression level of k-carrageenase.According to the optimization results of culture conditions,recombinant strains were cultured under 22 ℃,pH 6.0,and 1%(v/v) methanol addition in shake flask for 96 h.During the fermentation of recombinant strains,the transcription levels of truncated genes cgkZ△Pst and cgkZ△CBM were significantly higher than that of cgkZ.The enzyme activities of recombinant enzymes cgkZ,cgkZ△PSt and cgkZ△CBM were 4.68,5.70 and 3.02 U/mL,their molecular weights were 65,45 and 40 kDa,and with K_m values of 2.07,1.85,and 1.04 mg/mL,respectively.The end products of enzymatic hydrolysis were mainly composed of tetrasaccharide and hexasaccharide.Under the combined using of inducible promoter AOX1 and constitutive promoter GAP.The target gene cgkZ△Pst could be expressed in the absence of methanol,with an enzymatic activity of 2.73 U/mL at 22℃ shake flask culturing.更多还原