【作者】 王希菊；

【导师】 邵蔚蓝；

【作者基本信息】 江南大学 ， 发酵工程， 2005， 硕士

【摘要】 本文通过DNA分析程序,对海栖热袍菌Thermotoga maritimaα-淀粉酶A基因的序列进行分析,找出可能影响该基因在大肠杆菌中表达的各种因素。从最主要因素着手,逐步提高了海栖热袍菌α-淀粉酶A在E.coli中的表达水平。用PCR方法扩增出α-淀粉酶A的完整基因amyA,插入pET-20(b)中构建成质粒pET-amyA;运用基因工程手段将amyA基因富含稀有密码子的信号肽进行切除,将不含信号肽的amyAⅠ基因分别插入pET-20(b)和pHsh中构建成质粒pET-amyAⅠ、pHsh-amyAⅠ;用PCR法从大肠杆菌基因组中扩增出argU基因,插入pET-amyAⅠ中构建成质粒pET-R-amyAⅠ。将重组质粒分别转化不同的宿主。通过诱导,测酶活,得到结果:重组菌株E.coli JM109(DE3)/pET-amyA、pET-amyAⅠ、pHsh-amyAⅠ、pET-R-amyAⅠ和重组菌株E.coliBL21-CodonPlus(DE3)-RIL/ pET-amyAⅠ、pHsh-amyAⅠ经诱导后表达的酶活分别是1658.0 U/ mL、6721.7 U/mL、7420.0 U/mL、8904.5 U/mL、13867.7 U/mL、14860.7 U/mL。与原始重组菌株E.coli JM109(DE3)/ pET-amyA单位体积菌液的最高酶活相比,重组菌株E.coli JM109(DE3)/ pET-amyAⅠ、pHsh-amyAⅠ、pET-R-amyAⅠ和重组菌株E.coliBL21-CodonPlus(DE3)-RIL/ pET-amyAⅠ、pHsh-amyAⅠ单位体积菌液的最高酶活分别是原始重组菌株E.coli JM109(DE3)/ pET-amyA单位体积菌液的最高酶活的4.05、4.48、5.37、8.38、8.97倍。对重组菌株E.coli BL21-CodonPlus(DE3)-RIL/ pET-amyAⅠ进行诱导条件优化,结果为:IPTG浓度1.0 mmol/L,对数生长期即OD600为0.8时开始诱导,诱导4小时效果最好。对重组菌株E.coli BL21-CodonPlus(DE3)-RIL/ pHsh-amyAⅠ进行诱导条件优化,结果为:OD600为0.8时开始热激诱导,诱导8小时效果最好。重组菌株E.coli BL21-CodonPlus(DE3)-RIL/ pHsh-amyAⅠ在最佳诱导条件下诱导表达后进行细胞破碎,其上清经过70℃30 min的热处理后,再通过一步Ni2+亲和层析柱可使酶纯化,达到电泳均一。SDS-PAGE测得酶的分子质量为63.11 kD,与理论推算值63.44 kD相吻合。重组酶酶学性质研究表明,重组α-淀粉酶A最适反应温度为85～90℃,最适反应pH在6.5～7.0之间,在pH 5.0～9.0范围内酶活都比较稳定。热稳定性试验表明,该重组酶在90℃的半衰期可达17小时以上,具有很高的热稳定性,在工业化应用方面具有非常可观的开发潜力。动力学参数测定表明,重组酶以可溶性淀粉为底物,米氏常数Km为1.5459 g/L,Vmax为23.430 mmol/min.mg。更多还原

【Abstract】 The gene α-amyA from Thermotoga maritima was analyzed by using DNA analysisprograms. Some factors were found to affect the expression of α-amyA in E. coli. Thecomplete gene encoding extracellular α-amylase was amplified from the genomic DNA ofThermotoga maritima by polymerase chain reaction. The recombinant plasmid pET-amyAwas constructed by inserting the amplified segment into the expression vector pET-20(b). Thesignal peptide of amyA gene bound of unoptimal codon was cut off to form amyAⅠ. Therecombinant plasmid pET-amyAⅠand pHsh-amyAⅠwas obtained by inserting the amyAⅠinto the vector pET-20(b) and pHsh. The complete argU gene was amplified from the genomeof E. coli by polymerase chain reaction and was inserted into the plasmid pET-amyAⅠtoform pET-R-amyAⅠ.The recombinant plasmids pET-amyA, pET-amyAⅠ, pHsh-amyAⅠ, pET-R-amyAⅠwere transformed into the E.coli JM109(DE3) respectively , The recombinant plasmidspET-amyAⅠ, pHsh-amyAⅠwere transformed into the E.coli BL21-CodonPlus(DE3)-RIL.The recombinant strains produced amylase activities of 1658.0 U/ ml, 6721.7 U/ml, 7420.0U/mL, 8904.5 U/ml, 13867.7 U/ml, 14860.7 U/ml through induction, respectively. Incomparison, enzyme activity produced by E.coli BL21-CodonPlus(DE3)-RIL harbouringpHsh-amyAⅠwas 8.97 times higher than that by E.coli JM109(DE3) harbouring pET-amyAat their optimized induction conditions.The recombinant protein was purified by the heat treatment and immobilized metalaffinity chromatography, purified enzyme presented as a single protein band on SDS-PAGEwith molecular weight of 63.11 kD. The optimum activity of a α-amyA was found to be at pH6.5~7.0 and 85~90℃. In a pH range of 5.0~9.0, the enzyme was stable, and had a half-life of17 h at 90℃. The apparent Michaelis constant of the α-amyA was 1.5459 g/L for amylum,and Vmax was 23.430 mmol/min.mg protein.更多还原