Screening, Cultivation Conditions Optimizing Of A Producing FAE Strain And Enzymatic Properties Of FAE

Feruloyl esterase(FAE) is one type of hemicellulose degrading enzymes,it can hydrolyze the crosslinking feruloyl ester bond between hemicelluloses,hemicellulose and lignin. It can promote the separation between hemicelluloses,hemicellulose and lignin,and increase the efficiency of degrading cell-wall. Meanwhile,it can also promote the separation of ferulic acid that is one kind of very important healthy food materials. Feruloyl esterase has a widespread application value in bioenergy,food,pulping papermaking and the biosynthesis industries.In this paper,we studied on isolating and identification of FAE producing strains,fermentation condition for FAE production,enzymatic characteristics of FAE and TLC of FAE hydrolysis products.In the paper,more than 100 strains producing FAE were screened using ethyl ferulate as sole carbon source in medium in the plates. Through the secondary screening, we found that G3-7 had the highest FAE activity among the screened bacterial strains. Then through 16SrDNA analysis of G3-7, its colony and individual shape observation,physiological and biochemical characteristics,we preliminarily determined that this strain belongs to Burkhoderia.We optimized the liquid shaking fermentation condition of strain G3-7 that produces FAE. The adaptive culture conditions were as follows:The strain G3-7 precultured 3d in the medium containing glucose10g/L,KH2PO40.37 g/L,MgSO4·7H2O 0.25 g/L,CaCl2·2H2O 0.07 g/L,FeCl3 0.02 g/L, yeast extract 0.4g/L and then added 1%WB to induce and produce FAE with initial pH6.5, inoculation amount 1%,culture temperature 37℃, liquid volume in flask 50mL(250mL triangular flask). After optimizing the medium and culture condition,the FAE producing level of this strain had been raised 37%.Analyzing the properties of crude enzyme produced by strain G3-7,when 4-nitrophenyl ferulate(4-NPF) was the reaction substrate,it showed that:the optimum pH was7.0, the optimum temperature was 40℃and the optimum concentration of substrate was 7.5mM. At the same time,we found the crude enzyme produced by strain G3-7 did not have catalytic activities to 4-Methylumbelliferyl-Acetate,4-nitrophenyl acetate. However,this enzyme had high catalytic activities to long-chain carbon substrates which contain ester bond,such as 4-Nitrophenyl laurate and 4-Nitrophenyl myristate.The qualitative-analysis method of ferulic acid (FA) by the use of TLC was set up. The resultes showed that FA was found by the method of TLC after degrading the substrates of DSWB,methyl ferulate and ethyl ferulate by extracellular,intracellular and cell wall enzyme. Among these cases, extracellular enzyme of FAE had the best activity on degrading substrate DSWB.