重组β-葡萄糖苷酶生产龙胆低聚糖的工艺条件优化

Production of gentiooligosaccharide by recombinant β-glucosidase

摘要:

摘要：【目的】β-葡萄糖苷酶可用于酶法生产龙胆低聚糖。为了给龙胆低聚糖的生产提供大 量的酶来源，构建基因工程菌表达黑曲霉(CMI CC 324626)β-葡萄糖苷酶基因（bgl）并研究重组酶生产龙胆低聚糖的工艺条件。【方法】将bgl克隆到表达载体pPIC9K，转化毕赤酵母（Pichia pastoris）KM71。表达产物通过HPLC和LC-MS鉴定了其可用于生产龙胆低聚糖的转苷活性，并对酶转化葡萄糖生产龙胆低聚糖的反应条件进行了优化。【结果】实现了β-葡萄糖苷酶的过量表达。当底物葡萄糖浓度为80%，反应pH4.5，温度为60℃，加酶量为每克葡萄糖60 U，添加1 mmol/L的K+，转化周期为48 h，龙胆低聚糖累计达到最大为50 g/L。【结论】本研究是国内外首次利用重组酶酶法生产龙胆低聚糖的报道。

Abstract:

Abstract: [Objective] β-glucosidase can be used to prepare gentiooligosaccharide from glucose. The purpose of this study is to obtain β-glucosidase through DNA recombinant technology as well as to optimize the production of gentiooligosaccharide by the recombinant β-glucosidase. [Methods] We cloned bgl, the gene encoding β-glucosidase from Aspergillus niger (CMI CC 324626) into the expression vector pPIC9K to construct the recombinant plasmid pPIC9K- bgl. The vector was then transformed into Pichia pastoris KM71 for extracellular overproduction of β-glucosidase. The activity of the expressed enzyme was measured by the assay of transglucosidation reaction and the transglucosidation product was identified by HPLC and LC-MS. Furthermore, the condition for prepare gentiooligosaccharide by this recombinant β-glucosidase is optimized.[Results] A. niger β-glucosidase was successfully expressed in P. pastoris and the recombinant produced gentiooligosaccharide from glucose. In addition, the main operation parameters of this enzymatic conversion were optimized. At 80% glucose, 60℃, pH 4.5, 1 mmol/L K+, 60 U β-glucosidase per gram substrate, and 48 h reaction time, the gentiooligosaccharide produced reached 50 g/L. [Conclusion] This is the first report of producing gentiooligosaccharide by recombinant β-glucosidase.