扩展青霉脂肪酶基因在酿酒酵母中的表达及分子突变-生物化学与分子生物学专业论文.docxVIP

内容提要本论文报道了扩展青霉碱性脂肪酶(PeniciUium 内容提要 本论文报道了扩展青霉碱性脂肪酶(PeniciUium 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 酶的发孵条仆进行丁优化，并运用定点突变技术对PEL基因第227位氨基酸残基进 行分予突变。 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 pVTl02U／a-lip、pVTl02U／a-L．1ip07，并分别电转入酿酒酵母Saccharomyces cerevlsiae$78，筛选重组菌株$78-pVTl02U／Q—lip及S78-pVTl02U／a-L—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$78．pVTl02U／d—lip摇瓶发酵72h， 酶活可达20U／ml；$78．pVTl02U／d．L．1ip07表达的脂肪酶很少，表达产物仅在三丁 酸甘油酯平扳上形成透明圈。 从移种量、培养基口H、碳源及培养基组成等方面对$78一pVTl02U／o．1ip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。C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一$227P克隆到毕赤酵母缎达载体，将构建的重组质粒pPPIC3．5K-lip-$227P电 转化到毕赤酵母GSll5，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r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。 关键词：扩展青霉碱性脂肪酶；酿酒酵母：发酵优化：表达；突变 AbstractExpression Abstract Expression two which encode mature PEL(Penicillium lipase) E露硫 and pro-PEL，optimization of fermentation conditions were studied in this research Meanwhile，tim site—directed mutagenesis technology used introduce the mutation into The lip、lip07 encoding matu_re PEL and pro-P respectively,and the recombinant plasmids pVTl02U／d—lip、pVTl02U／Ⅱ -L-lip07 were then transformed into the Saccharomyces cerevisiae$78 competent cell by electroporation，the recombinant strain$78-pVTl02U／Ⅱ -Iip、S78-p110!里!：些￡!k矽镜 obtained The active lipase were secreted into the 。№。毹忑忑i忑磊hf。 expression product had molecular mass of 28kDa，which was similar that of the PEL from Penicullium expansum PF898．The activity of lipase from$78·pVTl02U／Ⅱ-lip 20U／ml after 72h fermentation；The product of$78-pVTl02U／a·L-lip07 was little，only formed the halo file tributyrin plate culture medium composition was 3％yeast extraction，3％tryptonz，2％sucrose；The volulne of inoculation about I．3％：the initial pH 7 The activity of lipasc 56UhnI under the optimal cultivation conditions． ．／一∥卵姐“ The 227 th Serine were replace阳th proline usi