蔗糖异构酶基因在Escherichia coli BL21(DE3)中的表达及重组菌的细胞固定化  

Expression of Sucrose Isomerase in Escherichia coli BL21 (DE3) and the Immobilization of Recombinant

作　　者：任贲[1] 李莎[1] 徐虹[1] 蔡恒[1] 

机构地区：[1]材料化学工程国家重点实验室,南京工业大学食品与轻工学院,江苏南京210009

出　　处：《食品与发酵工业》2010年第6期1-6,共6页Food and Fermentation Industries

基　　金：国家自然科学基金项目(20906050);江苏省自然科学基金项目(BK2009357);江苏省高校自然科学研究计划项目(08KJB530004)

摘　　要：分别利用IPTG和乳糖两种诱导物诱导蔗糖异构酶(SIase)基因在E.coliBL21(DE3)中实现表达,对诱导温度、诱导时机、诱导物浓度、诱导持续时间进行比较分析并优化,确定了二者的最佳诱导条件,在E.coli培养3h后(OD600约为0.9)添加终浓度为0.8mmol/L的IPTG(0.5mmol/L乳糖)在20℃(24℃)条件下诱导14h(12h)能获得最高的蛋白表达量及SIase酶活。在最优条件下以IPTG为诱导物时目的蛋白占总蛋白的41.6%,单位体积培养液中SIase酶活为12.37U/mL,以乳糖为诱导物时分别为27.2%,14.72U/mL,从收获酶活角度考虑可见乳糖作为诱导物的优势;而后利用海藻酸钠包埋法固定化重组菌,转化初始浓度为500g/L的蔗糖溶液,转化10～11h后异麦芽酮糖平均得率在83%以上,蔗糖平均转化率大于99%,固定化细胞能够连续稳定转化25批次,转化效率相对于原始菌提高了近55%。The expression of sucrose isomerase （SIase） gene in recombinant E . coli BL21 （DE3） induced by lactose and IPTG was investigated. We optimized the inducing conditions such as the culture temperature after induction, the point of induction, the inducer concentration and the induction time. The results showed that the optimal inducing conditions were to add 0.8 mmol/L （final concentration） IPTG（0.5 mmol/L lactose） after cell growth for 3 h, then incubate at 20℃ （24℃ for lactose）for 14 h（12 h for lactose）. After induction, the expression level of recombinant protein induced by lactose was about 27.2% of the total cellular protein, which was less than that induced by IPTG （41.6%）. However, the SIase activity induced by lactose （ 14.72 U/mL） was higher than that induced by IPTG （ 12.37 U/mL） , these results indicated the advantages of lactose as inducer in yield of SIase activity. Then sucrose with initial concentration of 500 g/L was converted to isomaltulose by the recombinant E . coli cells which was immobilized using sodium-alginat, the average isomaltulose yield and conversion rate for sucrose after 10 - 11 h was above 83% and 99% respectively. The activity of immobilized cells was stable after 25 batchs of conversion. The conversion efficiency was increased by nearly 55% compared with the original strain.

关 键 词：蔗糖异构酶(SIase) 乳糖诱导 蛋白表达 IPTG 固定化 

分 类 号：Q814[生物学—生物工程]