右旋糖酐蔗糖酶工程菌株的构建及其培养条件的研究

Construction and Culture Conditions of Dextransucrase-secreting Engineered Strain

摘要:

【目的】右旋糖酐蔗糖酶是一种以蔗糖为底物, 催化转移D-葡萄糖基生成a-葡聚糖或低聚糖的葡萄糖基转移酶。【方法】利用PCR扩增技术, 将已获得的右旋糖酐蔗糖酶基因dexYG亚克隆到表达载体PET28a(+)上, 转化E.coli BL21(DE3), 经过卡那霉素抗性筛选和酶切验证后, 得到右旋糖酐蔗糖酶工程菌株BL21 (DE3)/pET28-dexYG。【结果】经IPTG诱导该基因在E.coli BL21(DE3)中能有效表达,在诱导过程中菌体生长受到抑制。通过对培养时间、IPTG浓度、培养温度、菌浓(OD600)和pH值等产酶因素的优化考察, 得到最佳培养条件为：培养时间5h、IPTG 浓度0.5mmol/L、25℃、OD600值1.0和pH6.0。酶活力由最初的5.39U/mL提高到35.62U/mL, 其中pH值对产酶活力影响最大, 在pH6.0时的最高产酶活力是LB原始pH条件下最高酶活的3.5倍, 并且pH值也是导致在诱导后期酶活迅速下降的主要原因之一。【结论】酶的表达和酶活的研究结果表明, 构建的工程菌株能够异源高效表达右旋糖酐蔗糖酶, 并且表现出较高的酶活力。

Abstract:

[Objective] Dextransucrase was a glucosyltransferases catalyzing the transfer of D-glucopyranosyl units from sucrose to synthesize α-glucans or oligosaccharides. [Methods] dexYG gene (GenBank Accession No. DQ345760), encoding the dextransucrase from Leuconstoc mesenteriodes 0326, was subcloned into expression plasmid pET28a(+). The recombinant plasmid was then transformed into E. coli BL21 (DE3). Kanamycin resistant transformants were selected and verified by restriction endonuclease assay. [Results] Dextransucrase could be efficiently expressed in engineered strain BL21 (DE3)/pET28-dexYG by Isopropyl b-D-thiogalactopyranoside (IPTG) induction, although the growth of E. coli host was inhibited during induction. Recombinant enzyme producing conditions such as induction time, IPTG concentration, incubation temperature, cell density (OD600) and pH value were studied．The optimum conditions for producing dextran-sucrase were as follows: incubation at 25℃, 0.5mmol/L Isopropyl b-D-thiogalactopyranoside (IPTG) induction at cell density (OD600) of 1.0 for 5h, pH 6.0. Under these conditions, the recombinant dextransucrase activity was increased from 5.39U/mL to 35.62 U/mL. The highest activity under the optimal culture conditions after 5h induction in medium with pH 6.0 was 3.5 times as that of in Luria-Bertani medium without pH-adjustment. Moreover, the pH value was one of the main reasons that caused the degradation of enzyme in the later stage of induction. [Conclusion] These results showed that dextransucrase could be efficiently heterologous expressed in E. coli and a strong dextransucrase activity had been de-tected.