【作者】 江雪青；

【导师】 闫云君； 徐莉；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 近年来,脂肪酶的工业化应用领域越来越宽,获取更多具有不同特性的脂肪酶及其基因资源,并实现规模化生产,成为生物技术的研发热点。皱褶假丝酵母(Candida rugosa,原名Candida cylindracea)脂肪酶(CRL)是脂肪酶中较为重要的品种之一,可以催化脂水解、酯交换、酯合成等反应,广泛应用于油脂加工、食品、医药、日化等工业领域中,其含有多个同工酶,性质特异,并均能在异源宿主中实现一定程度的表达、分泌,具有较好的发展前景。本文从皱褶假丝酵母(ATCC 14830)菌株出发,以总DNA基因组为模板扩增出六个脂肪酶基因(lip1-lip5和lipJ08)。其中,基因lipJ08的全长序列为首次报道。同时,通过置换17个非常规丝氨酸密码子,实现了lipJ08基因在巴斯德毕赤酵母(GS115)中的异源表达。主要工作如下:(1)借助生物信息学,对已克隆的皱褶假丝酵母脂肪酶全长基因序列进行同源比对,根据保守序列设计引物,在基因组DNA水平上,从皱褶假丝酵母(ATCC 14830)菌株中克隆得到六个脂肪酶基因(lip1-lip5和lipJ08),其中lip1-lip5序列与已报道的皱褶假丝酵母脂肪酶同工酶相同, lipJ08基因为皱褶假丝酵母脂肪酶新的同工酶基因。序列分析表明,lipJ08基因全长为1650 bp,编码549个氨基酸,包括一个含15个氨基酸残基的信号肽,成熟脂肪酶由534个氨基酸组成。(2)由于C.rugosa脂肪酶基因中存在非通用密码子CTG,编码Ser,而在毕赤酵母中则编码Leu。因此,使得以Ser为脂肪酶活性中心的皱褶假丝酵母脂肪酶异源表达时得到的是没有催化活性的表达产物。利用重叠延伸PCR(Over-lap extension PCR)技术对lipJ08中的17个CTG位点进行了突变改造,转换为在毕赤酵母中编码Ser的TCT,突变基因命名为MlipJ08。(3)将lipJ08和MlipJ08分别亚克隆到表达载体pPIC9K中,成功构建了表达质粒pPIC9K-lipJ08和pPIC9K-MlipJ08,并将表达载体电转导入巴斯德毕赤酵母(GS115)中,成功地得到分泌表达。SDS-PAGE结果显示,表达的LIPJ08和MLIPJ08的分子量大小约为60 kDa。酶学特性研究表明,经密码子改造后获得的重组脂肪酶MLIPJ08对橄榄油的水解活力达4.65 U/mL,而LIPJ08活力几乎检测不到;MLIPJ08的最适温度为37℃,最适pH为7.0,对长碳链(C16)和短碳链(C4)pNp酯的水解活力高于对中碳链C8、C10和C12 pNp酯。更多还原

【Abstract】 In recent years, the industrialized application of lipases has attracted an increasing attention, it is becoming a hot spot in biotechnological field to get more and more the versatile lipases and their gene resources. C. rugosa (formerly C. cylindracea) lipase (CRL) is an important industrial enzyme capable of catalyzing lipolytic, transesterification, esterification reaction, has been applied in many fields such as oil processing, food, pharmaceuticals, and refined chemical manufacture. The CRL family includes several lipase isozymes, and they have different properties from each other, some of them have been successfully expressed in heterologous hosts. The lipase exhibits a good prospect in industrialized application.In this paper, six lipase genes (lip1-lip5 and lipJ08) were amplified from the total genomic DNA of the C. rugosa ATCC 14830, among which the full sequence of lipJ08 was reported for the first time. Moreover, lipJ08 gene was functionally expressed in Pichia pastoris GS115 system after substituting 17 CTGser triple codons with TCTser universal codons. The main work was as follows:(1) By means of bioinformatics, we aligned nucleotide sequences of reported lipase isoforms from C. rugosa. Primers were designed based on the conservative nucleotide sequences, and six lipase genes (lip1-lip5 and lipJ08) of C.rugosa ATCC 14830 were cloned. The nucleotide sequencing revealed that the lipJ08 gene with an ORF of 1650 bp without any intron, was a novel gene of CRL isoform, encoding a protein of 534 amino acid residues, including a potential signal sequence of 15 amino acid residues. The deduced amino acid sequence of the lipase showed an overall identity of 66 % to lip1-lip5 from C.rugosa. The nucleotide sequences of the other 5 isoforms of lipase genes cloned from C. rugosa ATCC 14830 were identical to CRL isoforms, lip1-lip5, as previously reported, respectively.(2) The deviations from the universal genetic code for the asporogenic yeast C.rugosa, in which the universal codon for leucine, CUG, is used to code for serine, including the catalytic Ser-209, has hindered the use of recombinant CRL isoenzymes (isoforms). The conversion of most or all CTG codons is required for the expression of functional lipase proteins in heterologous system. To overcome the difficulty, in this work, 17 of the non-nuniversal serine codons (CTG) of lipJ08 were converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis, and the optimized gene was termed as MlipJ08.(3) The native and codon-optimized MlipJ08 genes were subcloned into pPIC9K vector, and expressed in P.pastoris GS115, respectively. SDS-PAGE analysis of LipJ08 produced by P. pastoris harboring the plasmid pPIC9K-lipJ08 or pPIC9K-MlipJ08 respectively showed a single protein band about 60 kDa. The assays of emzyme characteristics demonstrated that the hydrolysis active of MLIPJ08 toward olive oil from the supernatant was 4.65 U/mL, whereas the native lipase LIPJ08 has no hydrolysis activity after methanol induction for 120 hours in shake flasks. Furthermore, the observation of the MLIPJ08 hydrolyzing pNp esters with variety of carbon chain length showed that it exhibited a preference for both long-chain (C16 acyl group) and short-chain (C4 acyl group) pNp esters to medium-chain cholesteryl esters (C8, C10 and C12 acyl groups), and its optimal reaction temperature and pH were 37℃and pH 7.0, respectively.更多还原