Stain Breeding And Fermentation Condition For β-glucosidase Production

Development and application of bio-energy is the important way to realize China’s commitment on carbon emission reduction. Since China is a country with great population and less arable land, utilization of non-grain raw materials especially lignocelluloses are the mainstream of the modern bio-energy production. β-glucosidase is an important component of cellulase which plays an important role in cellulose degradation, it is also the speed-limiting enzyme in degradation of lignocelluloses. Improve the activity of β-glucosidase is favorable to enhance the hydrolysis ability of cellulase system.In this study, Aspergillus niger (Aspergillus niger, provided by Henan Agricultural University)H12 was used as the starting strain, a mutant with high β-glucosidase activity was obtained after mutation. The fermentation conditions, crude enzyme hydrolysis properties and the optimal hydrolysis conditions of this mutant were studied. The results were as follows:(1) Using strain Aspergillus Niger H12 as the starting strain, prepared 1×107spores/mL spore suspension of H12, two mutants A104, A179 were obtained after UV mutagenesis, the mutants’ activities of β-glucosidase increased 25.6% and 23.1% respactivly compared with that of the starting strain H12 10.42IU/mL. Subsequently, using strain A104、A179 as the starting strain, the mutant B135 with the activity of 12.66IU/mL was obtained after being mutagenized by N+ implantation. At last, using strain B135 as the starting strain, after UV and N+ implantation compound mutation, mutant X06 was obtained with the enzyme activity of 16.12IU/mL, its β-glucosidase activity increased 52.9% compared with that of the starting strain H12. All of the mutants showed stable genetic character after they were cultured for 10 generations.(2) The fermentation process of X06 were studied by single factor and orthogonal experiments. The obtained optimize medium was, corn cobs 4%, yeast 1.0%, (NH4)2SO4 0.4%, KH2PO4 0.4%, MgSO4·7H2O 0.08%, CaCl2 0.05%,Tween 80 0.03% and trace elements fluids 0.1%. The optimized cultivation conditions were as follows, liquid volume in flask 40mL/250mL, inoculum 6%, initial pH 5.0, fermentation temperature 30℃, rotating speed of rocking incubator 220r/min and fermentation time 7d. β-glucosidase enzyme activity of X06 reached 43.66IU/mL under optimized cultivation condition, increased 170.1% compared with the previously enzyme activity of 16.21IU/mL.(3) Component analyses of crude enzyme produced by strain X06 showed that it was consisted of β-glucosidase, xylanase and cellulase, the activity level were 42.50IU/mL, 853.2IU/mL and 23.68IU/mL respectively. The optimum temperature for β-glucosidase was 70 □.β-glucosidase showed better temperature stability, the enzyme still had 70% relative activity after kept at 55 □ for 24h. The optimum pH for P-glucosidase was pH 4.5 and was stable at a pH range of pH 3-8, the enzyme still had 80% relative activity at this pH range.5mM of Fe2+and Mn2+above could promote the enzyme’s activity, the relative activity reached 137.44% and 138.34% respectively.