短双歧杆菌α-D-半乳糖苷酶基因aga1在大肠杆菌中的高效表达  

High expression of α-D-galactosidase gene (aga1) of Bifidobacterium breve 203 in Escherichia coli

作　　者：陆宇[1] 赵晗[1] 王勤鹏[1] 刘巍峰[1] 肖敏[1] 陈忠民 钱新民[1] 

机构地区：[1]山东大学微生物技术国家重点实验室,济南250100

出　　处：《微生物学报》2005年第2期241-246,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金 (3 0 170 0 0 8)~~

摘　　要：短双歧杆菌 (Bifidobacteriumbreve 2 0 3)α_D_半乳糖苷酶基因 (aga1)被克隆到大肠杆菌温度诱导表达质粒pBV2 2 0中 ,构建重组质粒pBVaga1,转入大肠杆菌进行温度诱导表达 ,得到的重组酶Aga1在大肠杆菌DH5α、DH10B和BL2 1中的比活分别为 2 8 0 8、19 4 4和 13 85U mg ,均高于短双歧杆菌α_D_半乳糖苷酶的比活 1 76U mg。重组质粒pBVaga1在E .coliBL2 1中稳定性较好。重组酶Aga1蛋白亚基分子量约 6 7kD ,最适反应温度为 4 5℃ ,酶在 4 0℃以下稳定 ,6 0℃仅剩余约 5 %的酶活性 ,70℃时酶全部失活 ;最适反应pH为 4 0～ 4 4 ,酶在pH 3 6～ 6 0范围内稳定 ;酶对p_硝基苯酚_α_半乳糖苷的Km =1 4 3mmol L ,Vmax=35 71μmol (L·min) ,对蜜二糖的Km =2 6 1mmol L ,Vmax=6 3 6 9μmol (L·min) ;酶在蜜二糖、棉子糖水解体系中不显示转糖基活性。结果说明Aga1与已经报道的一种短双歧杆菌的α_D_半乳糖苷酶不同 ,是新发现的一种短双歧杆菌的α_D_半乳糖苷酶。D-galactosidase gene (aga1) of Bifidobacterium breve 203 was cloned into temperature expression vector pBV220 and transformed into E. coli. The recombinant plasmid pBVaga1was induced to express with temperature. The specific activities of recombinant enzyme Aga1 in E. coli DH5α, E. coli DH10B and E. coli BL21 were 28.08 U/mg, 19.44 U/mg and 13.85 U/mg, respectively. The recombinant plasmid pBVaga1 is more stable in E. coli BL21. The molecular weight of Aga1 as determined by SDS-PAGE was about 67 kD. The optimum pH of Aga1 was pH 4.0～4.4, and it was stable between pH 3.6 and 6.0 (kept at 4℃ overnight). The optimum temperature of Aga1 was 45℃, and it was stable below 40℃ (incubated for 30min). Km-values for p-nitrophenyl-α-galactopyranoside( pNPGal) and melibiose were calculated with 1.43mmol/L and 261mmol/L, respectively. No transgalactosylation activity was found when Aga1 hydrolyzed melibiose or raffinose. The results suggest that Aga1 is much different from reported α-D-galactosidase from Bi. breve 203. Aga1 is another kind of α-D-galactosidase in the same bifidobacteria strain.

关 键 词：双歧杆菌 Α-D-半乳糖苷酶 温度诱导表达 

分 类 号：Q786[生物学—分子生物学]