[Production of gentiooligosaccharide by recombinant beta-glucosidase].
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.


CONCLUSIONS
This is the first report of producing gentiooligosaccharide by recombinant beta-glucosidase.
Liu
Lingling
L
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China. linglingliuwuxi@yahoo.com.cn
Zhu
Song
S
Zhu
Ting
T
Zhang
Min
M
Wu
Jing
J
Chen
Jian
J
D001234:Aspergillus niger / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D019848:Gene Expression Regulation, Archaeal; D005822:Genetic Vectors; D009844:Oligosaccharides / Q000096:biosynthesis*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D009844:Oligosaccharides; D001617:beta-Glucosidase