骆驼凝乳酶原基因在乳酸克鲁维酵母中的重组表达

Recombinant Expression of Camel-prochymosin Gene in Kluyvermyces lactis

骆驼凝乳酶具有独特的凝乳特性,在干酪加工制作中具有潜在应用前景。为获得高活性的骆驼凝乳酶制剂,采用乳酸克鲁维酵母GG799为宿主,首次对经密码子优化的骆驼凝乳酶原(c PC)基因进行表达。将人工合成cPC基因插入乳酸克鲁维酵母表达载体pKLAC1,构建重组载体pKLAC1-c PC,并用电脉冲法将SacⅡ线性化的重组质粒转化到乳酸克鲁维酵母GG799中。通过含1%酪蛋白的YEPD平板活性筛选,全细胞PCR鉴定,最后获得一株多拷贝整合的基因工程菌cPC 1。该菌株可分泌表达cPC,经SDS-PAGE分析,重组cPC的分子质量约为41 ku,符合预期;酸化处理后cPC的分子质量约为36 ku,可正确自我剪切。摇瓶发酵培养120h,酶活最高达230 SU/m L。分别以牛乳、骆驼乳和牦牛乳作为底物检测酶活性,结果重组骆驼凝乳酶对3种动物乳均具有凝乳活性。

Camel chymosin with the unique milk-clotting characterizations has potential applications in cheese manufacture. To improve expression efficiency of recombinant camel prchymosin gene in Kluyveromyces lactis strain GG799, a full-length camel prochymosin gene was synthesized based on the preference of Kluyveromyces yeast codon. The synthesized camel prochymosin gene was cloned into the expression vector p KLAC1, resulting in p KLAC1-c PC. The p KLAC1-c PC was linearized by SacⅡ and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YPD plates containing 1% casein. A recombinant strain c PC 1 with highest activities and multi-copy integration,which was detected by using specifical integration primers, was chosen and then fermented in flask. SDS-PAGE revealed that the molecular mass of the recombinant camel prochymosin was approximately 41 ku. After acid treatment, molecular weight of the chymosin was about 36 ku, the same as native camel chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 230 SU/m L after 120 hours cultivation. Milk-clotting test confirmed that bovine milk, camel milk and yak milk could be coagulated completely by recombinant camel chymosin.