Studies On Production Of Gentiooligosaccharide From Glucose With β-glucosidase

Gentiooligosaccharides are one of functional oligosaccharides, composed of glucose units linked byβ-1,6-xylosidete bonds. They are highly-effective Bifid bacteria proliferation factor and have many excellent physiological activities and broad markert prospect.The research and application of Gentiooligosaccharides started late, but developing rapidly. The industrial production of Gentiooligosaccharides is mainly through the enzymatic production, but currently not been able to product in large scale in china.In this paper, the enzymatic production process of Gentiooligosaccharides has been systemically studied. The Gentiooligosaccharide syrup, producted from high concentrations of glucose by using highly active recombinantβ-glucosidase, was then purified to obtain high-purity products. The main research contents are as follows:1. Aspergillus niger (CMI CC 324626)β-glucosidase encoding gene, bgl, was cloned into the expression vector pPIC9K to construct the recombinant plasmid pPIC9K- bgl.The plasmid was then transformed into Pichia pastoris KM71 to achieve the secretive extracellular overproduction ofβ-glucosidase.The activity of the expressed protein was performed by the assay of transglucosidation reaction and the transglucosidation product was identified by HPLC and MS.The results showed that this recombinant enzyme can be used in the production of Gentiooligosaccharides.2. The required nutrients forβ-glucosidase production by recombinant P. pastoris in shaking flasks were investigated.The optimized ferment condition of the recombinant P. pastoris has been determined: initial concentration of glycerol 30g/L, inoculating amount 10%, initial pH 6.0, growth temperature 30℃, initial methanol concentration 4%, methanol supplementation quantity 0.5%(in every 24h), duration of induction 120h initial pH 7.5, inducing temperature 30℃,liquid volume 50mL in 250mL flask, ,and the maximum yield of BGL was achieved to 245U/L. This study laid a solid foundation for large-scale production of Gentiooligosaccharides. 3. In addition, the main operation parameters of this enzymatic conversion were optimized.At 80% glucose,60℃,pH 4.5,1 mmol/L K+,60 Uβ-glucosidase per gram substrate, and 48 h reaction time, the gentiooligosaccharide produced reached its maximum of 50 g/L.4. Compared with crystallization and and cation exchange chromtography ,we found the second way was better.The purification of gentiooligosaccharide were studied with the resin DTF-01.and correponding operat techniques were researched too.The experiments date showed that the optimal purifiration result could be acieved on the resin column ofф15mm×600mm with eluting rate 2mL/min at 50℃,when 5mL syrup used.The content of active component would be increased from 9.3% to 73% and recovery were 43%. It would be possible to produce high-purity Gentiooligosaccharides for further separation.