【作者】 张银波；

【导师】 王汉中；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2009， 博士

【摘要】 本研究建立了纯培养和非培养微生物脂肪酶基因克隆的有效方法,并分别利用该方法克隆了葡枝根霉YF6脂肪酶基因和宏基因组脂肪酶基因Lip42,构建了高通量的毕赤酵母表面展示表达载体pAMB129,实现了葡枝根霉脂肪酶基因LipRs在毕赤酵母中的展示表达,研究了葡枝根霉全细胞催化合成生物柴油的反应条件,对宏基因组脂肪酶基因Lip42在毕赤酵母中的分泌表达进行了系统研究。具体摘要如下:1.筛选并鉴定了1株高产脂肪酶菌株葡枝根霉YF6,并采用简并PCR等分子技术从中克隆到1个新的脂肪酶基因LipRs及其对应的全长cDNA,序列分析表明该基因编码区全长1 173 bp,不含内含子序列,编码1段由26个氨基酸残基组成的信号肽和1个由365个氨基酸残基组成的成熟蛋白,有5个可能的N-糖基化位点,含有脂肪酶特征序列GHSLGGA,与来自米根霉的脂肪酶(AAF32408)同源性最高,一致性为83%。基因序列提交GenBank,登录号为DQ139862。2.构建了基于啤酒酵母絮凝蛋白Flo1的新型高通量毕赤酵母表面展示表达载体pAMB129,为了验证其有效性,葡枝根霉脂肪酶基因LipRs被克隆到絮凝蛋白N-端编码序列的下游,转化毕赤酵母,研究其表达情况。结果表明,重组的毕赤酵母菌株可以在橄榄油-罗丹明平板上表现出脂肪酶活性,并经过激光共聚焦显微镜观察,验证了构建的毕赤酵母展示表达载体的有效性,同时实现了葡枝根霉脂肪酶基因在毕赤酵母中的展示表达。3.以聚氨酯为葡枝根霉(Rhizopus stolonifer)YF6固定化培养载体,制成干燥的固定化YF6全细胞催化剂。用它进行酯化反应,油醇解反应最适醇油比为3:1,甲醇分3次加入,在反应温度35℃条件下,其酯化率最高达到89%左右。它不仅能使菜籽油、大豆油等油料直接经转酯化反应,生成脂肪酸甲酯,而且也能催化高酸值的废弃油的酯化反应形成生物柴油。4.采用简并PCR等分子技术从餐馆灶台油污宏基因组中克隆到脂肪酶基因Lip42及其对应的全长cDNA,序列分析表明脂肪酶基因的cDNA编码区序列全长为1692bp,编码1段由19个氨基酸残基组成的信号肽和1段由544个氨基酸残基组成的成熟肽,有2个可能的N-糖基化位点,BLAST比对分析表明该序列与已发表的白地霉(Geotrichum geotrichum)脂肪酶基因( U02541)在核苷酸水平的一致性为99%。该基因序列提交GenBank,登录号为DQ313172。将该脂肪酶基因的开放阅读框克隆到毕赤酵母表达载体,转化毕赤酵母GS115,经罗丹明B平板功能筛选得到具脂肪酶活性的重组毕赤酵母菌株[GS115(pAMB768)]。利用3种培养基(MM、BMM和BMMY)4种甲醇诱导浓度(0.5%、1.0%、1.5%和2.0%)在22℃恒温摇床中对重组菌株[GS115(pAMB768)]进行诱导表达,研究发现BMMY培养基在甲醇浓度为0.5%条件下表达量最高,纯化后计算表明粗酶液最高脂肪酶酶活为50.60 U/mL,重组脂肪酶的最高产量为147.93 mg/L培养基。重组脂肪酶经3步法纯化后,SDS-PAGE电泳表明该脂肪酶的表观分子量为68 kd,而以橄榄油为底物时其最适反应温度为30°C,最适反应pH值为pH 8.0。金属离子和去污剂对脂肪酶活性有较大影响,研究结果表明Mg2+对重组脂肪酶活性有促进作用,而Ca2+、K+、Fe2+、Cu2+和Co2+则抑制其活性, Tween 20、Triton-X100和SDS可以强烈抑制重组脂肪酶活性。更多还原

【Abstract】 Lipase genes, LipRs from Rhizopus stolonifer YF6 and Lip42 from metagenome of oil dirt on the hearth of the restaurant were cloned by using degenerate PCR. A nevol highthroughput Pichia pastoris cell-surface display expression vector was constructed based on Flo1 from Saccharomyces cerevisiae and the display expression of RSL was detected. At the same time, preparing biodesiel by cells of R. Stolonifer YF6 immobilized, overexpression and characterization of Lip42 in P. pastoris were conducted. Details as following:A novel lipase-encoding cDNA from R. stolonifer YF6 was cloned by using degenerate PCR, rapid amplification of cDNA ends (RACE) and RT-PCR, and its structural gene was amplified by PCR. No intron was found in this lipase gene by nucleotide sequences alignment between the structural gene and its corresponding cDNA. The cDNA of the putative lipase gene (LipRs) consisted of 1 173 bp, including an open reading frame encoding a 26-amino acid signal peptide at the N-terminal end and a 365-amino acid mature protein with a predicted molecular mass of 39 268, a pI value of 7.66 and 5 potential N-glycosylation sites (N-X-T/S). The 7 conserved amino acid residues, GHSLGGA, required to the active site of the lipase from Rhizopus sp. were all found in this lipase, RSL. The amino acid sequences alignment result showed that the RSL had a high degree of identity with other Rhizopus lipases, and the highest one from Rhizopus oryzae (AAF32408) was 83%. This lipase gene sequence has been submitted to GenBank and its accession number is DQ139862.A novel system based on Flo1 from S. cerevisiae was developed for cell-surface display of heterologous proteins in P. pastoris. In order to test the vector, R. oryzae lipase RSL was fused to the C-terminal of the truncated peptide of Flo1p. The expression of fusion protein Flo1p-RSL was detected throughout the P. pastoris cell surface by confocal laser scanning microscopy. And lipase specific activity was detected on the MM agar medium containing olive oil and Rodanmine B. The results indicated that a Flo1p-based system could express proteins on the surface of P. pastoris and that the fusion proteins did not affect the function of which expressed protein.And also, the methanolysis of plant oils was investigated by cells of R. Stolonifer YF6 immobilized within polyurethane material. In order to enhance the methanolysis activity of whole cell, substrate-related compounds, such as 0.5% rapeseed oil and 0.1 % soybean oil, were added to the culture medium. The lyophilized immobilized whole cell was used as biocatalyst to prepare biodiesel. The molar ratio of methanol/oil was 3:1. Methanol should be added 3 times stage by stage to avoid reducing the specific activity of the immobilized whole cells. At optimized condition, reaction yield reached 89%. Various plant oils could be used as stuff to prepare biodiese fuel, not only vegetable oil, such as rapeseed oils and soybean oils, but also high acidic value wasted oil.A lipase gene was cloned by degenerate PCR from metagenome of the oil dirt on the hearth of the restaurant and designated as Lip42. Its sequence was submitted to GenBank with the accession number DQ313172 obtained. The cDNA of the putative lipase gene (Lip42) consisted of 1 692 bp, including an open reading frame encoding a 19-amino acid signal peptide at the N-terminal end and a 544-amino acid mature protein with a predicted molecular mass of 61 541.51 Dal, a pI value of 5.482 and 2 potential N-glycosylation sites (N-X-T/S). The open reading frame was transformed into P. pastoris strain GS115 under control of the AOX1 promoter by using the vector pHBM906. Secretion expression of Lip42 was detected from P. pastoris recombinant strain [GS115(pAMB768)]. Furthermore the expression was carried out at three different mediums (MM\BMM\BMMY), four different methanol concentration (0.5%/1.0%/1.5%/2.0%) to optimize the expression for shake-flask cultures at 22℃. The maximum yield of lipase is about 147.93 mg/L of culture medium. The recombinant lipase was purified in a three-step procedure involving ammonium sulfate fractionation and ion exchange. Characterizations of the purified enzyme revealed a molecular mass of 68 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, maximum activity at 30°C and pH 8.0 for hydrolysis of olive oil. The highest recombinant lipase activity, 50.60 U/mL, was got under the optimum reaction conditions. The metal ions Mg2+ can activate the recombinant lipase, whereas Ca2+, K+, Fe2+, Cu2+, and Co2+ inhibited it, and Tween 20,Triton-X100, SDS inhibited it strongly.更多还原