Expression,Characterization And Application Of β-1,3-1,4-glucanase AaBglu12A From Aspergillus Awamori

β-1,3-1,4-glucanase is one of the most important glycoside hydrolases in food and feed industries.It is widely used in beer saccharification,oligosaccharide preparation and other processes.It is also a common feed additive.Because of its important application value,more and more attention has been paid to the development ofβ-1,3-1,4-glucanase.Therefore,based on the previous research results,the expression,properties and application of theβ-1,3-1,4-glucanase gene AaBglu12A from Aspergillus awamori CAU33 were studied in this study.The main conclusions are as follows:1.AaBglu12A,a glucanase gene encoding 239 amino acids,was cloned from Aspergillus awamori CAU33.The coding region contains two introns,with the length of 48 bp and 66 bp,respectively.The molecular weight of the mature protein encoded by AaBglu12A is predicted to be 24.17 kDa with isoelectric point of 4.63.It is inferred that AaBglu12A is a newβ-1,3-1,4-glucanase of GH 12 family by amino acid sequence alignment.Subsequently,AaBglu12A was successfully expressed in P.pastoris expression system.Clones with high activity ofβ-1,3-1,4-glucanase were obtained by high-copy screening.By high-density fermentation in a 5-L fermentation system,the enzyme activity in the supernatant reached 159500 U/mL,the protein content reached31.7 mg/mL,and the wet cell weight reached 404 g/L.It is the high level reported forβ-1,3-1,4-glucanase at present.2.The crude enzyme of AaBglu12A was purified after one-step purification by Q-Sepharose FF anion exchange column.The specific enzyme activity reached 7049U/mg.The molecular weight of AaBglu12A determined by SDS-PAGE gel electrophoresis and gel filtration chromatography was 23.6 kDa and 23.2 kDa,respectively,indicating that AaBglu12A was a monomer.The optimum pH and temperature of AaBglu12A were 5.0 and 55 ~oC,respectively,and it was sTab.at pH in the range from 2.0 to 9.0,and below 60 ~oC.AaBglu12A had strict substrate specificity towardsβ-1,3-1,4-glucanase.The hydrolysates of barley and oatβ-glucan contained mainly trisaccharide and tetrasaccharide,and the hydrolysates of lichenin were mainly oligosaccharides of DP 2-4.3.The purified AaBglu12A was used in beer saccharification process.When 70U/g malt of AaBglu12A was added,the viscosity of wort was reduced by 9.61%and the filtration time was shortened by 34.5%.The results were superior to most reportedβ-1,3-1,4-glucanases,indicating that AaBglu12A was a good candidate for the industrial application in beer brewing process.For the preparation of gluco-ligosaccharides from oat bran,the enzymatic hydrolysis conditions of AaBglu12A were optimized by single factor experiments.When the solid-liquid ratio was 1:20,the temperature of enzymatic hydrolysis was 50 ~oC,the amount of enzymatic hydrolysis was 100 U/g oat bran and the time was 4 h,the yield of glucoligosaccharides reached90%,which indicated that AaBglu12A had excellent industrial application potential.