【作者】 柳辉；

【导师】 闫云君；

【作者基本信息】 华中科技大学 ， 生物化工， 2007， 硕士

【摘要】 α-淀粉酶是一种重要的工业用酶,一般采取筛选高产α-淀粉酶菌株或利用基因工程技术构建α-淀粉酶分泌型工程菌规模生产,以满足工业生产的需求。本文利用低温显色法和摇瓶发酵法,从样品中筛选出一株产α-淀粉酶菌株XL-15,初测酶活力达到30.0U/mL。通过菌落形态、菌体特征观察以及16S rDNA序列比对鉴定为枯草芽孢杆菌（Bacillus subtilis XL-15）。发酵粗酶液经硫酸铵初步沉淀、透析脱盐后,对其基本酶学性质进行了初步分析。结果表明所产α-淀粉酶最适反应pH和温度分别为6.5和50℃,Ca²⁺、Mn²⁺对该酶有激活作用,而Cu²⁺、Zn²⁺和EDTA对其有抑制作用,酶动力学实验测得的米氏常数Km值为1.726mg/mL。以B. subtilis XL-15基因组DNA为模板,运用PCR方法成功克隆了α-淀粉酶基因。该扩增片断中含α-淀粉酶基因自身信号肽序列,开放式阅读框（ORF）大小为1980bp,编码659个氨基酸残基。将该结构基因分别转入大肠杆菌表达载体pET-28a（+）和毕赤酵母表达载体pPIC9K中,构建重组表达质粒pET28-Amy和pPIC9K-Amy,分别转化大肠杆菌BL21（DE3）和毕赤酵母GS115感受态细胞,并进行诱导培养表达。结果表明:大肠杆菌菌体破碎上清液中未检测出酶活性,SDS-PAGE电泳分析显示表达产物均以无活性的包涵体形式存在;而毕赤酵母在α-Factor及AOX1基因启动子和终止信号的调控下,利用甲醇进行诱导,经高密度培养后表达产物分泌至胞外,发酵液酶活力检测为4.3U/mL,实现了B. subtilisα-淀粉酶基因在毕赤酵母中的分泌表达,为α-淀粉酶基因在毕赤酵母中表达做了有益探索。更多还原

【Abstract】 Screeningα-amylase-producing strains or constructing gene engineering strains may produce abundantα-amylase thus settle for the demand in the industry. In this paper, anα-amylase-producing strain was isolated from soil samples by initial screening under the low temperature and flasks fermentation, and theα-amylase activity measured was 30.0 U/ml. Based on partial 16S rDNA gene sequence aligned with the GenBank database and its morphological characteristics, the strain was identified as Bacillus subtilis XL-15. The basic properties of theα-amylase produced by the strain were examined after ammonium sulfate fraction and dialysis. The results showed that the optimum pH and temperature of the enzyme were 6.5 and 50℃respectively; the enzyme activity was obviously inhibited by Cu²⁺, Zn²⁺and EDTA, while stimulated by Ca²⁺ and Mn²⁺, and its measured Km was 1.726mg/mL.Subsequently, theα-amylase gene was amplified by PCR method by using B. subtilis XL-15 genomic DNA as template. The amplified DNA includedα-amylase gene signal sequences, and the Open Reading Frame （ORF） was composed of 1980bp, encoding 659 amino acid. The coding gene was cloned into the vectors of pET-28a（+） and pPIC9K respectively, then transformed into Escherichia coli BL21（DE3） and Pichia pastoris GS115 respectively. The results showed thatα-amylase activity was not tested in the supernatant of E.coli derupted by sonication, and the expressed protein were all present with inclusion body. But theα-amylase could secreted from the recombinant strain of Pichia pastoris GS115 withα-Factor singal peptide, promoter of AOX1 gene and termination signal of yeast genomic, and the activity reached 4.3U/mL, when the P. pastoris GS115 was fermented with high density liquid media and induced by 1.0%methanl, which brings a strong foundation for further expression in large scale.更多还原