【作者】 林琳；

【导师】 谢联辉； 吴祥甫；

【作者基本信息】 福建农林大学 ， 植物病理学， 2001， 博士

【摘要】 扩展青霉PF898可产生一种具有重要工业生产价值的碱性脂肪酶，该产生菌是从我国土壤中分离并经多代的诱变育种选育的脂肪酶高产菌株。为阐明扩展青霉PF898碱性脂肪酶（PEL）的基因和蛋白结构，我们做了如下几方面工作：1．PEL的纯化和N-端氨基酸序列分析 扩展青霉PF898的发酵液经硫酸铵沉淀、DE52纤维素离子交换柱、凝胶过滤等步骤纯化了79.3倍，回收率约15%，比活为76.9U／mg。纯化蛋白经SDS-PAGE和HPLC TSK-GEL G3000SW凝胶过滤分析显示其分子量为28-29kDa。纯化蛋白经SDS-PAGE电泳并电转移至PVDF膜上，通过氨基酸测序仪分析表明，其N-端12个氨基酸的序列为：A-T-A-D-A-A-A-F-P-D-L-H。该序列与扩展青霉DSM1994产生的碱性脂肪酶的N-端12个氨基酸序列的同源性为75％。2．PEL cDNA及基因组DNA的克隆和序列分析 我们利用PCR、RT-PCR、RACE等技术扩增了PEL的全长cDNA和基因组DNA。将扩增产物与载体pBluescript Ⅱ SK（+）连接，通过T7和T3引物测序获得了该脂肪酶的cDNA全序列（GenBank登陆号AF284064）和DNA全序列（GenBank登陆号AF330635）。该脂肪酶cDNA全长由855个碱基组成；DNA全长由1135个碱基组成，含有5个内含子，大小分别为58bp、47bp、50bp、56bp和69bp。与其它来源脂肪酶的基因组DNA序列同源性为40％左右。3．PEL蛋白质一级结构分析和空间结构模建 我们通过cDNA序列推导出了该脂肪酶蛋白完整的氨基酸序列（GenBank protei_id AAG22769.1），该蛋白由285个氨基酸组成，信号肽（或信号肽和前肽）部分由27个氨基酸组成，成熟肽部分由258个氨基酸组成。该脂肪酶与其它几种真菌来源脂肪酶的氨基酸序列的同源性为33-39％左右，序列中含有各类脂肪酶中普遍存在的保守序列G-X-S-X-G。成熟肽氨基酸序列中含有4个Cys，无N-糖基化位 博士学位论文－点。该脂肪酶有着与其它脂肪酶类似的的空间结构。Ser刁、Asp－ 188、His－241组成其催化三分体。成熟肽的分子量为 27．3 kDa，稍 大于德国人发表的扩展青霉 DSM1994 碱性脂肪酶的分子量（25 kDa）。 4．PEL的基因表达 将克隆在 PBluescriPt 11 SK什）中的 PEL成熟肽基因片段酶切，插 人到表达质粒pET－22b（＋）和pET－41b（＋）中，构建了表达质粒pET－ 22b（＋）lipase和pET－4fo（＋）lipase，将该两种表达质粒分另转化大肠 杆菌 BLZI（DE3），筛选表达菌株 BL－pET－22b（＋）lipase和 BL－pET－ 41b卜）lipase。表达菌株用lmmol／L IPTG诱导表达4h后，产生大量 的表达蛋白。SDS－PAGE 电泳、计算机扫描灰度分析表明，两种表 达蛋自分别约占菌株可溶性蛋白的25％和18％，但表达蛋白在以橄揽 油为底物的酶活测定中不显示脂肪酶活性。二 将克隆在 pBluescript 11 SK（＋冲的 PEL成熟肽基因片段酶切，插 入至大肠杆菌－酵母菌穿梭质粒pPIC干和pPIC今 中，构建表达质 粒 pPICg－lipase和 pPICgK－lipase。利用电转化法将重组质粒转化进 入毕赤甲醇酵母OSlls，通过甲醇利用缓慢型MUf和 ＊4m抗性筛 选阳性转化子和具有高拷贝外源基因的转化菌株GS－pPICg1ipase和 GS－pPICgKdipase，表达菌经0．5％的甲醇诱导培养实现了脂肪酶蛋 白的分泌表达。发酵液在橄榄油平板酶活测定中显示出水解圈， NaOH滴定法测定表明发酵液脂肪酶酶活为20U／mL。同时，我们发 现，在无Ca‘”存在时表达产物不能分解橄榄油，必需有Ca‘”存在表 达产物才能显示活性，因此认为该脂肪酶是一种Ca‘”激活蛋白。在 10－30 mM Ca‘”存在的条件下，表达产物可分解橄榄油，适宜的 Ca‘” 作用浓度为 10 mM左右。更多还原

【Abstract】 Penicillium expansum PF898, which is a mutant strain by many generation mutagenesis and breeding from the original strain isolated from soil in China, produced an alkaline lipase at a high level. The research on the gene and protein structure of the lipase from Penicillium expansum PF898 (designated PEL) that we have done is listed as follows:1.Purification and N-terminal amino acid sequence of PELThe lipase from P.expansum PF898 was purified 79.3-fold by ammonium-sulfated precipitation, DE52 ion exchange chromato-graph and Sephadex G 100 gel filtration to a final specific activity of 76.9U/mg. The molecular weight of the enzyme was 2 8-29 kDa determined by SDSPAGE and gel filtration. Purified protein was electrophoretically transfered to a PVDF membrane to analysis N-terminal amino acid sequence. The 12 aa of N-terminal were A-T-A-D-A-A-A-F-P-D-L-H and 3 of them are different from the N-terminal aa sequence of P.expansumDSM1994:V-A-A-S-A-A-F-P-D-L-X. The homologous is about 75% between the two N-terminal sequences.2.Gene cloning and sequence of PELThe cDNA and genomic DNA encoding PEL were amplified by PCR, RT-PCR, RACE. The amplified products were ligated to plasmid pBluescript H SK(+) and the sequences of them were analyzed. The whole cDNA sequence and genomic DNA sequence were obtained and submitted to GenBank and the accession numbers were AF284064 and AF330635 respectively. The whole cDNA of the enzyme consists of 855 bp and the genomic DNA of PEL is composed of 1135 bp and has six exons and five short introns (58 bp, 47 bp, 50 bp, 56 bp, and 69 bp). The homology is about 40% between the genomic DNA sequence of PEL and that of other lipases.3.Amino acid sequence analysis and model constraction of three-dimensional structure of PELThe whole amino acid sequence of PEL (GenBank protein_id AAG22769.1) was deduced according to the cDNA sequence which encodes a protein of 285 aa including a presumptive signal peptide or signal and prepro-sequence composed of 27 aa and a mature peptide composed of 258 aa. The homology of the mature peptide between PEL and several lipases of other fungi is 33-39%. The mature peptide of PEL has 4 Cys residues and has no N梘lycosylation site, and contains a pentapeptide G-X-S-X-G highly preserved among lipases. The threedimensional structure of PEL is similar with that of other lipases and the catalytic triad is composed of Ser條32~ Asp?88. His?41. The mature peptide has a molecular weight of 27.3 kDa which is larger than that of P.expansum DSM 1994 lipase (25 kDa).4.Gene expression of PELThe mature peptide gene of PEL was cloned to construct the expression vectors pET-22b(+)Iipase and pET-41b(+)lipase and expressed in E.coli BL21(DE3) using the T7 RNA polymerase proteins as a promotor. The products were accumulated at 25% and 18% of the soluble cell protein when expression strain BL-pET-22b(?lipase and BL-pET-41b(+)lipase induced by IPTG for 4 hours, but activities of the products were not detected.The expression vectors pPIC9-lipase and pPIC9k-lipase were constructed and transformed into methylotrophic yeast Pichia pastoris (GS 115) by electroporation. Transformant GS-pPIC9-lipase and GSpPIC9K-lipase can functionally expressed and secreted 20 U/mL lipase when 0.5% methanol were fed during the cultivition. 10-30 mM (the optimum 10 mM) Ca2~ was needed for the expression protein to hydrolyze olive oil on the olive oil plate. PEL need Ca2~ for enzyme activity.更多还原