神马作文网英语翻译嗜热腾冲菌脂肪酶是来源于腾冲菌的一种耐高温脂肪酶,这类来源于极端嗜热环境嗜热菌的酶类具有极好的高温反应活性和稳定
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英语翻译
嗜热腾冲菌脂肪酶是来源于腾冲菌的一种耐高温脂肪酶,这类来源于极端嗜热环境嗜热菌的酶类具有极好的高温反应活性和稳定性,在能源、化工、食品和医药等行业展现出越来越广阔的应用潜力.本课题对腾冲菌耐热性的脂肪酶基因进行定点突变.
参考腾冲菌的脂肪酶序列,设计两对脂肪酶基因表达片断的PCR引物,以构建好的腾冲菌基因质粒为模板进行定点突变,对281位（脯氨酸变为天冬氨酸）、316位（亮氨酸变为组氨酸）进行定点突变.将成功突变后的质粒热激转入大肠杆菌表达宿主DH10B中,当重组菌OD600至0.0.6时,用终浓度1 mmol/L IPTG进行诱导表达,收集菌体、洗涤、超声波破壁,得到粗酶液.将粗酶经热处理,Ni-his NTA(Novagen)进行纯化,对脂肪酶进行跑蛋白胶验证,并测定该重组酶的酶学性质.
此酶在12% SDS-PAGE凝胶电泳显示相对分子量为44kDa.对硝基苯十二酯(pNP-C12)为底物在80℃,pH为6.3的条件下,重组脂肪酶活达到最大,为376.49U/mL,在80℃保温2h残余酶活大于75%.其米氏常数Km值为1.229 mol/L.该酶在终浓度5 mmol/LNi2+、Mg2+、Ba2+、Mn2+、Co2+、Ca2+、和终浓度0.25% Tween 20作用下对酶活没较大影响.然而,在终浓度5 mmol/LZn2+、Cu2+、和终浓度0.005%的SDS作用下相对酶活的下降至32.97%、54.97%和54.97%,且在终浓度5 mmol/LAl3+作用下相对酶活的明显降低,降至9.76%.
嗜热腾冲菌脂肪酶是来源于腾冲菌的一种耐高温脂肪酶,这类来源于极端嗜热环境嗜热菌的酶类具有极好的高温反应活性和稳定性,在能源、化工、食品和医药等行业展现出越来越广阔的应用潜力.本课题对腾冲菌耐热性的脂肪酶基因进行定点突变.
参考腾冲菌的脂肪酶序列,设计两对脂肪酶基因表达片断的PCR引物,以构建好的腾冲菌基因质粒为模板进行定点突变,对281位（脯氨酸变为天冬氨酸）、316位（亮氨酸变为组氨酸）进行定点突变.将成功突变后的质粒热激转入大肠杆菌表达宿主DH10B中,当重组菌OD600至0.0.6时,用终浓度1 mmol/L IPTG进行诱导表达,收集菌体、洗涤、超声波破壁,得到粗酶液.将粗酶经热处理,Ni-his NTA(Novagen)进行纯化,对脂肪酶进行跑蛋白胶验证,并测定该重组酶的酶学性质.
此酶在12% SDS-PAGE凝胶电泳显示相对分子量为44kDa.对硝基苯十二酯(pNP-C12)为底物在80℃,pH为6.3的条件下,重组脂肪酶活达到最大,为376.49U/mL,在80℃保温2h残余酶活大于75%.其米氏常数Km值为1.229 mol/L.该酶在终浓度5 mmol/LNi2+、Mg2+、Ba2+、Mn2+、Co2+、Ca2+、和终浓度0.25% Tween 20作用下对酶活没较大影响.然而,在终浓度5 mmol/LZn2+、Cu2+、和终浓度0.005%的SDS作用下相对酶活的下降至32.97%、54.97%和54.97%,且在终浓度5 mmol/LAl3+作用下相对酶活的明显降低,降至9.76%.
Thermophilic bacteria tengchong lipase is a kind of high temperature resistant lipase from tengchong bacteria,this class is derived from the extreme environment heat-loving thermophiles enzymes has excellent high temperature reactivity and stability,in energy,chemical,food and pharmaceutical industries show more and more wide application potential.This topic to tengchong,bacterium of heat resistance lipase gene point mutation.
Lipase sequence reference tengchong bacteria,design two clips of PCR primers,the expression of lipase gene to construct good tengchong gene plasmid as a template for fixed point mutation,on 281 (proline into aspartic acid),316 (leucine into histidine) fixed point mutation.Will succeed after mutation plasmid heat shock into e.coli expression host DH10B,when OD600 of recombinant strains to 0.3 0.6,with a final concentration of 1 tendency/L IPTG induction expression,collecting thalli,washing,ultrasonic wall-breaking,crude enzyme liquid.The crude enzyme after heat treatment,Ni - ihs NTA (Novagen) to purify,to run fibrin glue validation of lipase,and determining the nature of the recombinant enzyme enzymology.
This enzyme at 12% sds-page gel electrophoresis showed the relative molecular weight of 44 kda.P-nitrophenyl twelve ester (pNP - C12) as the substrate in the 80 ℃,under the condition of pH 6.3,recombinant lipase reached maximum,376.49 U/mL,80 ℃ heat preservation 2 h residual enzyme activity is greater than 75%.The michaelis constant k value is 1.229 mol/L.The enzyme in the final concentration of 5 tendency LNi2 +,magnesium 2 +,Ba2 + and Mn2 +,Co2 +,Ca2 +,and the final concentration of 0.25% Tween 20 under the action of no big impact for enzyme activity.,however,the final concentration of 5 tendency LZn2 +,Cu2 +,and the final concentration of 0.005% SDS under the action of relative enzyme activity dropped to 32.97%,54.97% and 54.97%,and the final concentration of 5 tendency LAl3 + under the action of relative enzyme activity decreased obviously,dropped to 9.76%.
Lipase sequence reference tengchong bacteria,design two clips of PCR primers,the expression of lipase gene to construct good tengchong gene plasmid as a template for fixed point mutation,on 281 (proline into aspartic acid),316 (leucine into histidine) fixed point mutation.Will succeed after mutation plasmid heat shock into e.coli expression host DH10B,when OD600 of recombinant strains to 0.3 0.6,with a final concentration of 1 tendency/L IPTG induction expression,collecting thalli,washing,ultrasonic wall-breaking,crude enzyme liquid.The crude enzyme after heat treatment,Ni - ihs NTA (Novagen) to purify,to run fibrin glue validation of lipase,and determining the nature of the recombinant enzyme enzymology.
This enzyme at 12% sds-page gel electrophoresis showed the relative molecular weight of 44 kda.P-nitrophenyl twelve ester (pNP - C12) as the substrate in the 80 ℃,under the condition of pH 6.3,recombinant lipase reached maximum,376.49 U/mL,80 ℃ heat preservation 2 h residual enzyme activity is greater than 75%.The michaelis constant k value is 1.229 mol/L.The enzyme in the final concentration of 5 tendency LNi2 +,magnesium 2 +,Ba2 + and Mn2 +,Co2 +,Ca2 +,and the final concentration of 0.25% Tween 20 under the action of no big impact for enzyme activity.,however,the final concentration of 5 tendency LZn2 +,Cu2 +,and the final concentration of 0.005% SDS under the action of relative enzyme activity dropped to 32.97%,54.97% and 54.97%,and the final concentration of 5 tendency LAl3 + under the action of relative enzyme activity decreased obviously,dropped to 9.76%.