耐热β-半乳糖苷酶基因在大肠杆菌中的表达及酶学性质研究  

Studies on expression and enzymatic properties of thermostable β-galactosidase gene in Escherichia coli

作　　者：龚月生[1] 刘锦妮[1] 王晶[1] 杨明明[1] 袁新宇[2] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]湖北工业大学生物工程学院,湖北武汉430068

出　　处：《西北农林科技大学学报（自然科学版）》2008年第10期59-66,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家重点新产品计划项目(2003ED760039)

摘　　要：【目的】研究耐热β-半乳糖苷酶基因在大肠杆菌中的表达,并检测其酶学性质,为耐热β-半乳糖苷酶的应用和工业化生产奠定基础。【方法】利用基因工程的原理和方法,将来源于嗜热脂肪芽孢杆菌的耐热β-半乳糖苷酶基因(bgaB)克隆到大肠杆菌pET表达系统(pET-28a(+)),并在大肠杆菌BL21(DE3)中进行表达。将构建的重组菌(pET28a-bgaB)在含硫酸卡那霉素的液体LB培养基中以IPTG为诱导剂,对表达产物的最适反应温度、最适反应时间I、PTG诱导浓度、热稳定性及酶促动力学进行检测。【结果】成功构建了表达载体pET28a-bgaB,并测得耐热β-半乳糖苷酶的最适反应温度为65℃,最适反应时间为6 h,IPTG的最佳诱导浓度为1 mmol/L,且具有良好的热稳定性;在诱导后6 h,耐热β-半乳糖苷酶的表达量占菌体总可溶性蛋白的20%,表达效率较高;对透析蛋白进行蛋白质定量,蛋白质量浓度为0.75 mg/mL,透析蛋白的耐热β-半乳糖苷酶活性为75 U/mL,比活性为100 U/mg;酶促动力学研究表明,耐热β-半乳糖苷酶的反应常数为0.398μmol/mL,最大反应速度为27.435μmol/(min.mL)。【结论】耐热β-半乳糖苷酶基因在大肠杆菌中得到成功表达,并且在很大程度上提高了耐热β-半乳糖苷酶的活性。【Objective】 For further industrial production,β-galactosidas was expressed in Escherichia coli and its enzymatic properties were detected.【Method】The β-galactosidas gene（bgaB）was amplified from the genome of Bacillus stearothermophilis through polymerase chain reaction（PCR） and then cloned into Escherichia coli expression vector pET-28a（＋）.Constructed recombinant bacteria（pET28a-bgaB）was cultured in kanamycin sulfate liquid medium by IPTG inducer.The optimum reaction temperature,reaction time,IPTG inducer concentration,temperature thermostable experiment and enzyme kinetics were studied in our research.【Result】 The optimum reaction conditions of β-galactosidas such as temperate,reaction time and IPTG inducer concentration were 65 ℃,6 h and 1 mmol/L respectively,inaintained good thermal stability.After been induced for 6 hours,the expression of β-galactosidase accounted for 20% of the cell total soluble protein and achieved a high efficiency.Dialysis protein was qualified and the concentration of protein was 0.75 mg/mL,dialysis protein β-galactosidase enzyme activity 75 U/mL,specific activity 100 U/mg,Enzyme kinetics demonstrated the response constant 0.398 μmol/mL,the maximum reaction velocity 27.435 μmol/（min·mL）.【Conclusion】 β-galactosidas gene was successfully expressed in Escherichia coli and the activity of β-galactosidas was improved to a large extent.

关 键 词：Β-半乳糖苷酶基因 大肠杆菌 酶学性质 

分 类 号：Q786[生物学—分子生物学]