Optimization Of Fermentation Conditions For ?-1,3-1,4-glucanases Production From Aspergillus Awamori And Purification,Characterization And Application Of The Enzyme

?-1,3-1,4-glucanase has broad application prospects in the fields of beer brewing,feed production and biochemical control.In the present investigation,a ?-1,3-1,4-glucanase from fungus was isolated from soil sample and identified as Aspergillus awamori CAU33.The conditions of solid-state and liquid-state fermentation for ?-1,3-1,4-glucanase production by Aspergillus awamori CAU33 were optimized using single-factor experiment and response surface method.The ?-1,3-1,4-glucanase was extracted and purified from the liquid-state fermentation.The characterization and application of the ?-1,3-1,4-glucanase were also studied.The main conclusions are as follows:(1)A fungus with high yield of ?-1,3-1,4-glucanase was screened from the soil.The strain was identified as Aspergillus awamori by colony morphology,sporogenous structure and ITS gene sequences and named Aspergillus awamori CAU33.The optimum solid-state fermentation conditions were as follows: beer grains was carbon source,water content 80%,Tween 60 10 g/L,soybean peptone 25 g/L,pH natural,the optimal condition was cultured at 35 ? for 6 days.Under the conditions,the highest?-1,3-1,4-glucanase production of 38452 U/g dry substrate.The optimal conditions were as follows: corncob 55 g/L,soybean peptone 25 g/L,Triton X-114 23g/L,initial pH of 4.5 and culture temperature of 35?.Under the optimized conditions,the highest ?-1,3-1,4-glucanase activity of 8446.9 U/mL was achieved after 6 days of cultivation,which is about 17.6 times that of the activity before optimization.This all were the highest yield for ?-1,3-1,4-glucanase production ever reported.(2)The crude enzyme was purified to homogeneity subsequently use ammonium sulfate precipitation,Q-Sepharose Fast Flow and DEAE Sepharose Fast Flow,a purification fold of 10.5 and the recovery of 22.1 %,molecular weights was 27.7 kDa,a specific activity increased from 1432.19 U/mg to 15084.86 U/mg.The results of enzymatic properties showed that the optimal pH and temperature of the enzyme were pH 5.5 and 55 ?,respectively,with more stronger acid resistance and wide pH stability.The enzyme showed strict substrace specificity on ?-1,3-1,4-glucan,while could not hydrolyze ?-1,3-glucan and ?-1,4-glucan,hydrolysis of barley ?-glucan,the hydrolyzate is trisaccharide and tetrasaccharide;hydrolysis of lichenan,the product istrisaccharide.It turned out that the decrease of mash viscosity and filtration time were5.25% and 28% when the ?-1,3-1,4-glucanase(70 U/g of malt)was added.