Production Of Feruloyl Esterase And Its Utilizition In The Degradation Of Lignocellulosic Materials

Ferulic acid is the most abundant, ubiquitous hydroxycinnamic acid distributed widely throughout the plant kingdom. It has a wide variety of applications in pharmaceutical, cosmetic and chemical industry because it has effects of erasing radical and active oxygen, absorbing UV, inhibiting pigment formation and inflammatory. Furthermore, ferulic acid is crosslinking with hemicellulose and lignin by ester, which strengthened the three-dimensional polysaccharide network and inhibiting the utilizaion of cellulose and bio-degradation of plant fiber material by microorganisms.Feruloyl esterase (FAE) is one type of hemicellulose degrading enzymes. FAE hydrolyzed the ester bond between the arabinose substitutions and ferulic acid, which is involved in crosslinking xylan to lignin, and destroyed three-dimensional network structures of plant cell walls. Feruloyl esterase has been widely used in the bio-ethanol production, pulping industry, animal feed production and the biological synthesis process.In the paper, Fungus 4-3-a was screened by selective screening using ethyl ferulate as sole carbon source in medium in the plates, and continuously culturing in liquid fermentation to produce FAE. This fungus has a good ability to produce Feruloyl esterase. The strain was identified to be Aspergillus flavus by analyzing 18S rDNA, ITS fragment and morphological properties. The strain was the second reported Aspergillus flavus strain with the ability to produce feruloyl esterase.Destarched wheat bran (DSWB) was used as substrate to determine FAE enzyme activity. The process of DSWB production was researched and DSWB with lower residual starch content was obtained by amylase treatment substitute for chemical treatment reported in many literatures. The DSWB was suitable for determining enzyme activity of FAE.Compositions of culture mediums in liquid fermentation of strain 4-3-a were optimized by using single factor experiments, orthogonal experiments and statistical methods, and the suitable culture medium was:carbon source of 6%, yeast extract of 0.4%, KH2PO4 0.05%, NaNO3 0.9%, MgSO4 0.05%. It was shown that carbon source has a significant influence on production of FAE. The mixture design method was used to optimize the composition and ratio of carbon sources to obtain high activity enzyme. It was found that enzyme with the highest activity of FAE can be produced when dosage of total carbon source was 6%, and ratio of corn stover to corn cob was 36:64. The culture conditions for liquid fermentation of strain 4-3-a were also optimized in the paper and shown as follows:initial pH 7.0, speed 210 rpm, culture temperature 32℃, inculation of 6%, and culture volume of 100 mL in 500 mL flask. Compared to original medium and culture conditions, the activity of FAE was increased to 2.5 folds when strain 4-3-a was cultured in the optimized cultures and conditions. The experimental method of mixture design was firstly used to optimize carbon source composition of the culture mediums.The properties of feruloyl esterase produced by strain 4-3-a were analyzed and shown that the optimum pH is 6.0 and the optimum temperature of 65℃. It is stable in the range of pH 5.0 to 7.0 and temperature below 50℃. It is also an enzyme with ethanol-tolerance when ethanol consistency was below 40% in enzyme solution to dissolve synthetic substrate for determining enzymatic activity.The lignin content, single sugars contents and morphology of wheat straw were analyzed by Kappa number test, HPLC and infrared spectroscopy after wheat straw was treated with the strain 4-3-a by liquid culture method. It was shown that the lignin and sugar contents were decreased after bio-treatment, especially for the arabinose content; and absorption of ester bond was decreased, which means the FAE produced by the strain 4-3-a breaks ester bond and promotes degradation of arabinoxylan. The saccharification of cellulose in wheat straw pretreated with hot water was improved by adding FAE to cellulase solution and conversion ratio of cellulose was increased. Also, the dosage of cellulase can be decreased by adding FAE to hydrolyzing system when conversion ratio was kept at a similar degree.