【作者】 任艳丽；

【导师】 陈红歌；

【作者基本信息】 河南农业大学 ， 微生物学， 2004， 硕士

【摘要】 本研究以野油菜黄单胞菌的野生α-淀粉酶基因和采用 5-溴脱氧尿苷三磷酸（BrdUTP）在野生基因基础进行体外诱变后所得的酶活提高的基因为研究对象,利用基因重组技术,成功构建了重组表达载体 pXC8004N 和 pXCH21N,并在大肠杆菌中实现了高效表达,同时对重组菌破碎后所得的粗酶液的酶学特性进行了分析研究。 1．利用原核表达载体 pET-30a(+)构建重组表达载体 pXC8004 和 pXCH21，转化到宿主菌 BL21 中，利用 IPTG 诱导，使α-淀粉酶基因获得了高效表达。通过对表达条件的摸索，当菌体 OD600为 0.4～0.6 时，IPTG 终浓度为 1mmol/L时诱导结果最好，表达量约占菌体总蛋白的 11%，诱导表达温度以 26℃诱导培养时可减少包涵体的形成。 2．重组菌株诱导表达后，调整二者的菌体 OD600相同,利用超声波破碎细胞后，测定这两种粗酶液的酶活，比较二者之间的差别。在表达量相同的情况下，诱变后的高酶活基因重组菌的酶活性比初始基因重组菌的酶活性高30u/mL 。这说明突变位点处于酶活性部位，通过改变酶与底物的作用效果而提高酶的活性。 3. 本试验设计的一对引物由于在起始密码子前多了一个碱基,致使构建好的重组表达载体在翻译外源基因时发生了移码错误翻译,导致蛋白质的翻译在 616 个碱基处遇到了 TAG 终止密码子,提前终止了翻译,产生了两个大小为 24KDa 的错码蛋白质。测这两个蛋白的α－淀粉酶活性时发现其具有能够水解淀粉的能力，也就是说这两个蛋白能够切开α-1、4 糖苷键，经测定这两个蛋白的酶活性可达到 105U/mL。将这两个蛋白的氨基酸序列与野油菜黄单胞菌的野生α－淀粉酶基因的氨基酸序列比对发现它们之间同源性只有 8％左右。更多还原

【Abstract】 Based on alpha-amylase gene and mutant gene of Xanthomonas campestris k8004,pET-30a(+) plasmid ,The recombinant expression vector pXC8004 and pXCH21 wasconstructed by gene cloning technology. The mutant gene was gotten for the originalalpha-amylase by gene-directde mutagenesis method. The activity of enzyme which wasexpressed by the mutant gene was higher than the original gene. We have realized the tworecombinent vector pXC8004 and pXCH21 high level expression in E.coli BL21(DE3)PlusS.1.The recombinant expression vector pXC8004 and pXCH21 wan transformated into E.coliBL21 competent cell and induced by IPTG . We got the high-level expression of alpha-amylasegene and its mutant. From the study of references and experiment, we got the optimum inducedconditions. When the OD600 of bacterial solution reached to 0.4~0.6 and the dose of IPTGwas 1mmol/l , the induced result was the best. The target proteins amount to 11% of the totalproteins in cell.2.The induced cultivated E.coli BL21 was fragmentated by ultrasonic, then centrifugated andkept in the supernatant. Analyzed the enzyme activity of supernatant in the same expressionamount , the result indicated that the enzyme activity of mutant gene recombinant expressionvector pXCH21 was higher than the original gene recombinant expression vector pXC8004 by30U/L. So, we can conclude that it was the enzyme activity center that had been changed ,not theincreasing of enzyme expression amount which improved the activity of mutant alpha-amylasegene.3.We had designed a pair of pcr primers, but there was an extra base in its sense primer, whichresulted in terminating of translation of the target protein ahead of schedual. The molecular oferror-reading protein was 24 KDa. Although it had very low identity by comparing with theoriginal gene, the mutant gene had still the function of hydrolysis of α-1,4 glucosidicanlinkages.更多还原