牛凝乳酶原基因的合成及其在乳酸克鲁维酵母中的表达

凝乳酶在奶酪加工中应用广泛,为获得高活性的凝乳酶制剂,采用乳酸克鲁维酵母为宿主,首次对经密码子优化的牛凝乳酶原基因进行表达。利用DNAWorks3.0软件辅助设计,用两步PCR法合成了小牛凝乳酶原基因(GenBank Accession No.AA30448)。将该基因插入酵母表达载体pKLAC1,构建了重组载体pKLAC1-Prochy,并用电脉冲法将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,PCR鉴定,最后获得了一株多拷贝整合的基因工程菌chy1。该菌株可分泌表达牛凝乳酶原,经SDS-PAGE分析,证明重组牛凝乳酶原的分子量约为41kDa,符合预期大小,酸化处理后为36kDa,证明可以正确自我剪切。液体培养96h后,酶活最高达到99.67SU/mL。分别以半乳糖和葡萄糖为碳源的条件下表达,其酶活性差异不大,说明在发酵期间,可以不经过半乳糖诱导即可产生高水平的牛凝乳酶原产物。该工程菌的获得为进一步优化产酶条件及放大工艺提供了条件,并为凝乳酶的工业化生产奠定了基础。

Gene synthesis of the bovine prochymosin gene and high-level expression in Kluyvermyces lactis

Chymosin is an important industrial enzyme widely used in cheese manufacture. To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene (GenBank Accession No. AA30448) by using optimized codons. The synthesized prochymosin gene was amplified by two-step PCR method, and then cloned into the expression vector pKLAC1, resulting in pKLAC1-Prochy. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain chy1 with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. The activities of chymosin were not prominent increased when galactose was used as carbon source instead of glucose, which proved that the fermentation of recombinant strain does not need galactose inducing. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.