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 CC324626)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60U,添加1mmol/L的K+,转化周期为48h,龙胆低聚糖累计达到最大为50g/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。
[ 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 AspergiUus 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 gentiooligosacharide produced reached 50 g/L. [ Conclusion] This is the first report of producing gentiooligosaccharide by recombinant β-glucosidase.
出处
《微生物学报》
CAS
CSCD
北大核心
2009年第5期597-602,共6页
Acta Microbiologica Sinica
基金
国家杰出青年基金(20625619)
食品科学与技术国家重点实验室科研基金(SKLF-MB-200802)~~
关键词
龙胆低聚糖
酶转化
Β-葡萄糖苷酶
表达
毕赤酵母
gentiooligosaccharide
enzymatic conversion
β-glucosidase
expression
Pichia pastoris, AspergiUus niger
