毕赤酵母不同甲醇利用表型Mut^+和Mut^s表达FsGLUm基因的比较  

Evaluation of Mut^+ and Mut^s Pichia pastoris phenotypes for expression of Fibrobacter succinogenes β-glucanase gene

作　　者：杨玉霞[1] 罗艳丽[2] 张慧玲[1] 汪艳[1] 陈勇[1] 裴建华[1] 

机构地区：[1]新疆农业大学新疆肉乳用草食动物营养与饲料重点实验室,新疆乌鲁木齐830052 [2]新疆农业大学草业与环境科学学院,新疆乌鲁木齐830052

出　　处：《食品工业科技》2015年第6期220-224,239,共6页Science and Technology of Food Industry

基　　金：新疆维吾尔自治区科技支疆项目(201191138);新疆维吾尔自治区高技术研究发展项目(201211104);自治区重点产业紧缺人才专业大学生创新项目(JQRCP32010023)

摘　　要：将来源于产琥珀酸丝状杆菌的β-葡聚糖酶基因根据毕赤酵母的偏好性进行密码子优化,通过全基因合成该优化基因(Fs GLUm)并构建重组表达载体p PIC9K-Fs GLUm。将p PIC9K-Fs GLUm分别用SalⅠ和BglⅡ酶切线性化后电击转化入毕赤酵母GS115染色体DNA中,经过表型筛选和抗性筛选获得不同甲醇利用表型Mut+和Muts阳性菌株。经刚果红平板检测,在摇瓶水平下,Mut+型菌株表达产物产生的水解透明圈明显大于Muts型菌株表达产物。在发酵罐水平下,Mut+型菌株各时间段表达产物酶活性明显高于Muts型菌株。Mut+型菌株表达的酶活性在甲醇诱导96h达到最大值,为6424U/m L,比活性为2607U/mg,菌体干重为123.6g/L。Muts型菌株表达的酶活性在诱导后108h达到最大值,为119U/m L,比活性为1867U/mg,菌体干重为113.5g/L。以上结果表明,Mut+型毕赤酵母更有利于产琥珀酸丝状杆菌β-葡聚糖酶基因的表达。According to the codon optimization of Pichia pastoris, β-glucanase gene from Fibrobacter succinogenes（FsGLUm） was synthesized and the recombinant expression vector,named pPIC9K-FsGLUm, was constructed, With Sal Ⅰ and Bgl Ⅱ ,the pPIC9K-FsGLUm was linearized and the β-glucanase gene was electroporated into chromosome DNA of Pichia pastoris GS115. The different methanol utilization phenotype positive strains,Mut^＋ and Mut^s,were obtained after phenotype and resistance screening. In the shake flask level,the halo zones on Congo red plate produced by expression products of Mut^＋ strain were significantly larger than that of Mut^s strain. In the fermenter level,β-glucanase activity expressed by Mut^＋ strain was significantly higher than those of Muts strain in each time period, Enzyme activity,specific enzyme activity,and dry cell weight of Mut^＋ strain reached the maximum of 6424U/mL,2607U/mg,and 123.6g/L,respectively at 96h after methanol induction. While,that of Muts strain reached the maximum of 119U/mL, 1867U/mg,and 113.5g/L, respectively at 108h after induction. The above results showed that,Mut^＋ strain of Pichia pastoris GS115 was more conducive for expression of Fibrobacter succinogenes β-glucanase gene.

关 键 词：产琥珀酸丝状杆菌 Β-葡聚糖酶 毕赤酵母 甲醇利用表型 

分 类 号：TS201.3[轻工技术与工程—食品科学]