【作者】 李安娜；

【导师】 李多川；

【作者基本信息】 山东农业大学 ， 植物病理学， 2007， 博士

【摘要】 蛋白水解酶催化蛋白质中肽链的裂解,是一类在生理学和商业领域中具有广泛应用价值的酶。蛋白酶执行大量的不同的生理功能,负责包括在正常和非正常状态下复杂的病理、生理功能。它们也存在于致病生物体的生活史中,这就使它们成为一个潜在的发展治疗因子的对象,来治愈如癌症和艾滋病等可怕疾病。蛋白酶在食品和清洁剂工业中的应用具有悠久的历史。微生物来源的蛋白酶,由于具有培养简便,产量丰富等特点,适于工业化生产而得以广泛应用。其中,热稳定性蛋白酶的研究和开发具有重要的商业价值。嗜热毛壳菌(Chaetomium thermophilum)是一种广泛分布的,生长上限温度较高的嗜热真菌,从该菌中已分离了多种嗜热酶,但未见该菌嗜热蛋白酶的报道。本研究中C. thermophilum在以酪蛋白为唯一碳源的合成培养基中诱导产生了蛋白酶,通过硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose疏水层析等步骤获得了两种凝胶电泳均一的蛋白酶。SDS-PAGE测得所分离纯化酶蛋白的分子量分别约为33kD和63kDa。两种酶都可以被PMSF所抑制,但不能被iodoacetamide和EDTA抑制。PRO33和PRO63的最适反应pH分别为10.0和5.0。两种酶的最适反应温度均为65℃,在60℃以下较稳定,Ca2+对酶的的热稳定性具有显著的增强作用。根据真菌热稳定蛋白酶的同源保守序列设计兼并引物,通过RT-PCR及快速扩增cDNA末端(RACE)的方法,克隆了该蛋白酶的编码基因pro,全长cDNA为2007bp,包含一个由532个氨基酸组成的开放阅读框。该基因已在Genbank中注册,登录号为DQ839520,DNA序列的登陆号为EF100880。由核苷酸序列推导的相应氨基酸序列前15个氨基酸为信号肽序列,比较蛋白酶的催化区序列,发现与丝氨酸蛋白酶S8家族的枯草杆菌蛋白酶subtilisin的同源性很高,在催化区中有两个基序GHGTHV和GTSMASPH非常保守。这些保守基序与丝氨酸蛋白酶家族催化活性有关。将经EcoRⅠ和NotⅠ双酶切的pro基因和原核表达载体pET-22b(+)进行连接,构建成原核表达载体pET/pro,并转化大肠杆菌E. coli BL21。经IPTG诱导培养4~5h,目的蛋白得到大量表达,SDS-PAGE电泳检测,在约41kDa处有一条明显的蛋白带,蛋白大小与从该蛋白酶氨基酸序列估计的蛋白分子量相符;而空质粒pET-22b(+)转化BL21经诱导培养后无相应蛋白产生。可溶性分析结果显示,表达的蛋白以包涵体形式存在。pro基因和酵母分泌型表达载体pPIC9K双酶切后体外连接,构建酵母重组表达载体pPIC9K/pro并测序,保证正确的阅读框。将重组表达质粒pPIC9K/pro和pPIC9K空质粒分别用限制性内切酶SacⅠ(位于5’AOX1区内)线性化后,采用电击法转化Pichia pastoris GS115酵母感受态细胞,于MD/MM平板上筛选His+Mut+表型的酵母转化子。经过PCR检测及G418抗性筛选多拷贝整合子,进行甲醇诱导培养。通过检测各转化子每隔24h的蛋白表达情况来筛选高效表达目的蛋白酶的工程菌株。筛选到表达酶活性最高的菌株GS-PRO-16,甲醇诱导6d酶活性达到最高,达16.73 U/mL,酶蛋白表达量为0.77 mg/mL。检测目的蛋白的表达量及遗传稳定性,并测定表达蛋白酶的酶学性质。酵母工程菌株GS- PRO-16在YPD平板上划线,连续传代10次后,PCR检测呈阳性,重组蛋白酶的表达量基本保持稳定。表达蛋白酶PRO的最适反应温度和pH分别为60℃和8.0,该酶在60℃以下稳定;70℃的半衰期为60min;在pH值为5.0～12.0之间表达蛋白酶保持稳定的酶活性。C.thermophilum蛋白酶能在毕赤酵母中分泌出具生物活性的目的蛋白,而且表达产物同样具有原始菌株C. thermophilum蛋白酶的优良特性,这就预示了表达蛋白酶在工业和生物学领域的广阔应用前景。因此,期望能对该基因进行改造,或进一步优化表达条件,获得真正有工业应用价值的酵母工程菌株。更多还原

【Abstract】 Protease was an important group of enzymes both in physiology and commerce fields. Microbial proteases dominate commercial applications. There has been an increasing interest in proteases from thermophiles, which were expected to produce thermostable proteases. Two thermostable extracellular proteases from culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by fractional ammonium sulphate precipitation, ion exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular weight of the two purified enzymes was estimated to be 33 kDa and 63 kDa respectively. The two proteases were found to be inhibited by PMFS, while not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63 kDa protease (PRO63) at pH 5.0. The optimum temperature for the two proteases was 65oC. The PRO33 had a Km value of 6.6 mM and a Vmax value of 10.31μmol/L/min. The PRO63 was 17.6 mM and 9.08μmol/L/min with casein as substrate. They were thermostable at 60oC. The protease activity of PRO33 and PRO63 remained 67.2% and 17.31% respectively after incubation at 70oC for 1h. The thermal stability of the two enzymes was significantly enhanced by Ca2+. The residual activity of PRO33 and PRO63 at 70oC after 60 min was approximately 88.59% and 39.2% respectively when kept in the buffer containing Ca2+. These properties make them applicable for many biotechnological purposes.Degenerate primers designed on the conserved domain of other reported serine proteases, and a cDNA fragment encoding the protease gene was obtained through RT-PCR. The RACE was used to generate full-length cDNA clones. The full length of pro cDNA gene is 2007bp, which contained an ORF of 1596bp encoding 532 amino acids. The cDNA and DNA sequence of gene pro has been registered in Genbank with accession number DQ839520 and EF100880 respectively.The alignment results of putative amino acids sequence showed the catalytic domain of pro was high homology with the catalytic domains of the subtilisin serine proteases. The pro gene and expression vector pET-22b(+) were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant plasmid pET/pro was generated, and transformed into E. coli BL21. The recombinant protein was produced in large amount after IPTG induction, approximately 41kDa protein was determined by SDS-PAGE, and this size was coincident with the protein molecular weight from the putative amino acid sequence; no interest protein was produced by inducing with IPTG after the pET-22b(+) was transformed into BL21. The solubility analysis showed the recombinant protein was presented in a fusion form. The high induce temperature results in E. coli BL21 rapid growth and over expression of recombinant protein in host cell maybe the reason.The pro gene and expression vector pPIC9K were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant expression plasmid pPIC9K/pro was constructed and sequenced to confirm the correct reading frame. The constructed plasmid pPIC9K/pro was linearized with a restriction enzyme SacI (insertion at 5’AOX1), and transformed into Pichia pastoris GS115 competent cell by electroporation methods, and Screened for His+Mut+ transformants on MD and MM plates. The parent vector was linearized with the same restriction enzyme and transformed GS115 as a control. PCR analysis of P. pastoris integrants and G418 screening determined the multicopy integrants to induce by methanol. These integrants were used to analyze expression levels of interest protein every 24 hours. The engineering strain with highest expression level was called GS-PRO-16. The genetic and protease expression stability of recombinant P. pastoris GS-PRO-16 was tested and characterized.After streak culture for single colony of His+ transformant GS-PRO-16 on YPD plate for 10 generations, PCR analysis showed the interest gene was integrated in P. pastoris genome, and the expression level was also kept stable. The C. thermophilum protease was secreted into the culture medium by the yeast P. pastoris in a functionally activity form, and some specific properties of expressed protease were similar to those of the native strain C. thermophilum. It implied the expressed protease could be widely applied in the industry and biological fields. Therefore, we hope to reconstruct the pro gene and optimize the expression condition to obtain the yeast engineering strains suitable for industrial applications.更多还原