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

β-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 β-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 β-1,3-1,4-glucanase production by Aspergillus awamori CAU33 were optimized using single-factor experiment and response surface method.The β-1,3-1,4-glucanase was extracted and purified from the liquid-state fermentation.The characterization and application of the β-1,3-1,4-glucanase were also studied.The main conclusions are as follows:(1)A fungus with high yield of β-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 highestβ-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 β-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 β-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 β-1,3-1,4-glucan,while could not hydrolyze β-1,3-glucan and β-1,4-glucan,hydrolysis of barley β-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 β-1,3-1,4-glucanase(70 U/g of malt)was added.