环糊精葡萄糖基转移酶(cyclodextrin glucosyltransferase,CGTase)制备环糊精(cyclodextrin,CD)时,部分淀粉底物无法被利用,导致淀粉原料利用率以及生产效率低,为解决上述问题,该研究用Escherichia coli BL21(DE3)克隆表达了一种新型淀粉脱支酶,来源于光滑念珠菌(Candida glabrata)的糖原脱支酶(glycogen debranching enzyme,GDE)CgGDE,当摇瓶发酵温度为25 ℃,诱导剂IPTG终浓度为0.1 mmol/L,诱导时间为36 h时,CgGDE的酶活力达69.3 U/mL。酶学性质结果表明,CgGDE的最适pH值为6.0,最适温度为35 ℃。利用CgGDE与β-CGTase复配制备β-CD,结果表明,在非溶剂条件下,当马铃薯淀粉质量浓度为50 g/L,温度40 ℃,反应pH值为6.0,CgGDE添加量为750 U/g干淀粉,β-CGTase添加量为7 U/g干淀粉,转化周期24 h时,β-CD转化率为53.1%,比单独使用β-CGTase转化率提高了93.8%,与异淀粉酶β-CGTase联用相比,转化率提高了50.9%,为β-CD在非溶剂条件下的转化奠定了基础。
When cyclodextrin (CD) is produced by cyclodextrin glucosyltransferase (CGTase), some starch substrates cannot be used, which leads to low utilization rate of starch raw materials and low production efficiency.To solve the above problems, a new starch debranching enzyme, glycogen debranching enzyme CgGDE from Candida glabrata, was cloned and expressed by Escherichia coli BL21(DE3).When the fermentation temperature was 25 ℃, the final concentration of the inducer IPTG was 0.1 mmol/L, and the induction time was 36 h, the activity of CgGDE reached 69.3 U/mL.The results of enzymatic properties showed that the optimum pH of CgGDE was 6.0, the optimum temperature was 35 ℃.β-CD was prepared by combining CgGDE with β-CGTase.Results showed that under non-solvent conditions, when the concentration of potato starch was 50 g/L, at 40 ℃, pH was 6.0, the CgGDE addition was 750 U/g dry starch, the β-CGTase addition was 7 U/g dry starch, the total yield of β-CD reached 53.1% after 24 h, which was 93.8% and 50.9% higher than that transformation with β-CGTase alone and combination of CGTase enzyme and isoamylase.This work provides a basis for the transformation of β-CD under non-solvent conditions.
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