335 / 2018-07-30 16:38:33

Gene Cloning and Characterization of An Organic Solvent Stimulate β-Glucosidase and Its Application for Ethanol and Succinic Acid Co-Production

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A novel β-glucosidase(Bgl) gene from Hypocrea sp.W63 was cloned into pPIC9K vector and expressed in Pichia pastoris. The specific purity activity of recombinant epB-BGL could attain to 194.25 IU/mg with p-nitrophenyl-β-D-glucoside (pNPG) as substrate. The optimum pH and temperature for epB-BGL were 5.0 and 70 °C, respectively. With adding of 10% (v/v) methanol and 15% (v/v) ethanol, the activity of epB-BGL would be improved 144% and 183%, respectively. Other alcohols also showed contribution to activate enzyme activity either in the concentration of 1% or 10% (v/v). Furthermore, epB-BGL activity is stimulated to the inhibitors existed at the bioenergy procedure such as 5-(hydroxyl methyl)-2-furaldehyde (HMF)(127.9%), DL-Dithiothreitol (DTT)(114.14%) and formic acid(110.8%). Due to its distinctive performance, epB-BGL was applied in co-production of ethanol and succinic acid, using Actinobacillus succinogenes ATCC55618 and Clostridium autoethanogenum. In this work, a flexible gas-cultivation system was used to co-fermentation the both two strains from which could efficiently utilize the sugarcane bagasse hydrolysate with Bgl. In the experimental range, succinic acid was reached to 13.3g/L with a higher yield of 91.56% and the ethanol production was about 1.33g/L. What’s more, a by-product of acetate was also produced at 9.745g/L which could be potentially converting to ethanol. This work represents the first report of a Bgl gene performance under overcome inhibitors from sugarcane bagasse hydrolysate and fermentation process, application on ethanol and succinic acid co-production, suggests that this enzyme is in great application potential on lignocellulosic bioconvertion.