[重组β-葡萄糖苷酶生产龙胆寡糖]

[Production of gentiooligosaccharide by recombinant beta-glucosidase].

作者信息

Liu Lingling, Zhu Song, Zhu Ting, Zhang Min, Wu Jing, Chen Jian

机构信息

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.

出版信息

Wei Sheng Wu Xue Bao. 2009 May;49(5):597-602.

PMID:19637566

Abstract

OBJECTIVE

Beta-glucosidase can be used to prepare gentiooligosaccharide from glucose. The purpose of this study is to obtain beta-glucosidase through DNA recombinant technology as well as to optimize the production of gentiooligosaccharide by the recombinant beta-glucosidase.

METHODS

We cloned bgl, the gene encoding beta-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 beta-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 beta-glucosidase is optimized.

RESULTS

A. niger beta-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 degrees C, pH 4.5, 1 mmol/L K+, 60 U beta-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 beta-glucosidase.

摘要

目的

β-葡萄糖苷酶可用于从葡萄糖制备龙胆寡糖。本研究的目的是通过DNA重组技术获得β-葡萄糖苷酶，并优化重组β-葡萄糖苷酶生产龙胆寡糖的工艺。

方法

我们将编码黑曲霉（CMI CC 324626）β-葡萄糖苷酶的基因bgl克隆到表达载体pPIC9K中，构建重组质粒pPIC9K-bgl。然后将该载体转化到毕赤酵母KM71中，用于细胞外过量生产β-葡萄糖苷酶。通过转糖基化反应测定表达酶的活性，并通过HPLC和LC-MS鉴定转糖基化产物。此外，对利用该重组β-葡萄糖苷酶制备龙胆寡糖的条件进行了优化。