扩展青霉脂肪酶基因在酿酒酵母中的表达及分子突变
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摘要
本论文报道了扩展青霉碱性脂肪酶(Penicillium expansum lipase，PEL)成熟肽基因lip及带有自身信号肽的基因lip07在酿酒酵母中的表达，对酿酒酵母表达脂肪酶的发酵条件进行了优化，并运用定点突变技术对PEL基因第227位氨基酸残基进行分子突变。
将碱性脂肪酶基因lip、lip07分别连接到酿酒酵母表达载体，构建了重组质粒pVT102U／α-lip、pVT102U／α-L-lip07，并分别电转入酿酒酵母Saccharomycescerevisiae S78，筛选重组菌株S78-pVT102U／α-lip及S78-pVT102U／α-lip07。结果表明lip及lip07均获得了分泌表达，表达产物分泌到培养基中，分子量约为28kDa，与天然扩展青霉脂肪酶基本一致。其中，重组酵母S78-pVT102U／α-lip摇瓶发酵72h，酶活可达20U／ml；S78-pVT102U／α-L-lip07表达的脂肪酶很少，表达产物仅在三丁酸甘油酯平板上形成透明圈。
从移种量、培养基pH、碳源及培养基组成等方面对S78-pVT102U／α-lip进行初步的发酵条件优化，结果表明：适宜的培养基组成为3％酵母抽提物、3％胰化蛋白胨、2％蔗糖；移种量为1.3％；起始pH为7.0，28℃摇瓶发酵72h，上清液酶活达56U／ml，为优化前的2.8倍。
采用重叠延伸PCR方法将脂肪酶第227位丝氨酸突变为脯氨酸，将突变后的基因lip-S227P克隆到毕赤酵母表达载体，将构建的重组质粒pPPIC3.5K-lip-S227P电转化到毕赤酵母GSI15，摇瓶发酵72h，上清液用加有底物的平板检测酶活。与野生型相比，突变体脂肪酶的酶活很低，不能在橄榄油平板上形成透明圈，但可以在以三丁酸甘油酯为底物的平板上形成透明圈，表明第227位丝氨酸可能对脂肪酶酶酶活具有重要影响。
Expression of two genes which encode mature PEL(Penicillium expansum lipasc) and pro-PEL, optimization of fermentation conditions were studied in this research. Meanwhile, the site-directed mutagenesis technology was used to introduce the mutation into PEL.
The genes lip, lip07 encoding mature PEL and pro-PEL were cloned into expression vector respectively, and the rccombinant plasmids pVT102U/a-lip, pVT102U/a -L-lip07 were then transformed into the Saccharomyces cerevisiae S78 competent cell by eleclToporalion, the recombinant strain S78-pVT102U/a -lip, S78-pVT102U/a-L-lip07 was obtained.The active lipase were secreted into the culture after fermentation. The expression product had a molecular mass of 28kDa,which was similar to that of the PEL from Penicullium expansion PF898. The activity of lipasc from S78- pVT102U/a -lip was up to 20U/ml after 72h fermentation; The product of S78-pVT102U/a-L-lip07 was little, only formed the halo on the tributyrin plate.
Different strategies (different volume of inoculation, different initial pH, different carbon sources, different medium concentration ) was applied to optimized the S78-pVT102U/a -lip fermentation condition, the optimum conditions as follow: The optimum culture medium composition was 3% yeast extraction, 3% tryptone, 2% sucrose;The volume of inoculation was about 1.3%; the initial pH was 7.0. The activity of lipase was up to 56U/ml under the optimal cultivation conditions.
The 227th Serine were replace with proline using overlap extension PCR method. The mutated gene lip-S227P was ligated into the pPPIC3.5K, the recombinants plasmid pPPIC3.5K-lip-S227P was transformed into the Pichia pastoris GS115 competent cell by electroporation. We use plate to check the lipase activity ,the lipase activity is very low, Compared to the wild type , the mutant could not form the clear halo on the olive oil plate but tributyrin plate, it showed that the 227 th Serine was essential for the lipase activity.
将碱性脂肪酶基因lip、lip07分别连接到酿酒酵母表达载体，构建了重组质粒pVT102U／α-lip、pVT102U／α-L-lip07，并分别电转入酿酒酵母Saccharomycescerevisiae S78，筛选重组菌株S78-pVT102U／α-lip及S78-pVT102U／α-lip07。结果表明lip及lip07均获得了分泌表达，表达产物分泌到培养基中，分子量约为28kDa，与天然扩展青霉脂肪酶基本一致。其中，重组酵母S78-pVT102U／α-lip摇瓶发酵72h，酶活可达20U／ml；S78-pVT102U／α-L-lip07表达的脂肪酶很少，表达产物仅在三丁酸甘油酯平板上形成透明圈。
从移种量、培养基pH、碳源及培养基组成等方面对S78-pVT102U／α-lip进行初步的发酵条件优化，结果表明：适宜的培养基组成为3％酵母抽提物、3％胰化蛋白胨、2％蔗糖；移种量为1.3％；起始pH为7.0，28℃摇瓶发酵72h，上清液酶活达56U／ml，为优化前的2.8倍。
采用重叠延伸PCR方法将脂肪酶第227位丝氨酸突变为脯氨酸，将突变后的基因lip-S227P克隆到毕赤酵母表达载体，将构建的重组质粒pPPIC3.5K-lip-S227P电转化到毕赤酵母GSI15，摇瓶发酵72h，上清液用加有底物的平板检测酶活。与野生型相比，突变体脂肪酶的酶活很低，不能在橄榄油平板上形成透明圈，但可以在以三丁酸甘油酯为底物的平板上形成透明圈，表明第227位丝氨酸可能对脂肪酶酶酶活具有重要影响。
Expression of two genes which encode mature PEL(Penicillium expansum lipasc) and pro-PEL, optimization of fermentation conditions were studied in this research. Meanwhile, the site-directed mutagenesis technology was used to introduce the mutation into PEL.
The genes lip, lip07 encoding mature PEL and pro-PEL were cloned into expression vector respectively, and the rccombinant plasmids pVT102U/a-lip, pVT102U/a -L-lip07 were then transformed into the Saccharomyces cerevisiae S78 competent cell by eleclToporalion, the recombinant strain S78-pVT102U/a -lip, S78-pVT102U/a-L-lip07 was obtained.The active lipase were secreted into the culture after fermentation. The expression product had a molecular mass of 28kDa,which was similar to that of the PEL from Penicullium expansion PF898. The activity of lipasc from S78- pVT102U/a -lip was up to 20U/ml after 72h fermentation; The product of S78-pVT102U/a-L-lip07 was little, only formed the halo on the tributyrin plate.
Different strategies (different volume of inoculation, different initial pH, different carbon sources, different medium concentration ) was applied to optimized the S78-pVT102U/a -lip fermentation condition, the optimum conditions as follow: The optimum culture medium composition was 3% yeast extraction, 3% tryptone, 2% sucrose;The volume of inoculation was about 1.3%; the initial pH was 7.0. The activity of lipase was up to 56U/ml under the optimal cultivation conditions.
The 227th Serine were replace with proline using overlap extension PCR method. The mutated gene lip-S227P was ligated into the pPPIC3.5K, the recombinants plasmid pPPIC3.5K-lip-S227P was transformed into the Pichia pastoris GS115 competent cell by electroporation. We use plate to check the lipase activity ,the lipase activity is very low, Compared to the wild type , the mutant could not form the clear halo on the olive oil plate but tributyrin plate, it showed that the 227 th Serine was essential for the lipase activity.
引文
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