Expression Of Ferulic Acid Esterase Gene In Pichia Pastoris And Its Application In Beer

Ferulic acid esterase（FAE）,a subclass of carboxylesterase,can hydrolyze the ester bond of ferulic acid and polysaccharide in plant cell wall.By cooperation with xylanase,cellulase and arabinosidase,FAE can degrade plant hemicellulose.Therefore,FAE has broad application prospects in textile,feed,food and bio-energy and other fields.Due to the low expression level of FAE in wild strains,and its low catalytic activity,the application and promotion of ferulic acid esterase in industry are severely restricted.In this study,recombinant FAE was over expressed in the host strain Pichia pastoris,and purified for use in beer brewing process to improve the filtration performance and antioxidant ability of wort,and to increase the content of wheat beer flavor substances 4-vinyl guaiacol（4-VG）in beer.The ferulic acid esterase gene was firstly amplified by overlapping extension PCR using the genome of Aspergillus niger as the template,and inserted in the plasmid pPICZαA to construct the Pichia pastoris expression vector pPICZαA-AnfaeA.To increase the expression level and activity of recombinant FAE,the AnfaeA gene was then optimized using “one by one optimization” and "random optimization" strategies according to coden preference of Pichia pastoris.The activities of coden optimized recombinant FAE was 5.2±0.1 U·mL^-1 and39.9±0.1 U·mL^-1,respectively,which was 76.5% and 6 times of the un-optimized recombinant FAE.The optimum fermentation conditions were determined by single factor experiment and orthogonal experiment,the fermentation trials of host Pichia pastoris for FAE expression were optimized as follows,3%（v/v）inoculation amount,30°C temperature,pH7.0,1%（v/v）methanol complementation,inducing period of 4.5 d.The FAE activity in fermentation liquid was 49.8 U·mL^-1with the optimized induction trials,which was 24.8%higher than that before optimization.After purification,the recombinant FAE was characterized.The FAE was most active at pH 5.5 and 50°C.It was stable when stored at pH4.5⁷.0,and 40°C⁶0°C.The metal ions Na⁺,K⁺,Ca²⁺,Mg²⁺ and Zn²⁺ could improve its activity,while Fe²⁺,Co²⁺,Mn²⁺and EDTA had negative effects on its activity.The purified recombinant FAE was supplemented in the saccharification stage of beer brewing process.And it could effectively hydrolyze the bounded ferulic acid,inhibit the oxidative crosslinking of water-soluble arabinoxylans,and improve the wort filtration rate by14.5%.As a result,the content of free ferulic acid in wort increased from 1.3 mg·L^-1to 9.8mg·L^-1,which could effectively improve the anti-oxidant ability of wort and therefore postpone beer gaining.Furthermore,with the addition of recombinant FAE,the content of4-VG in final beer product increased from 3.1 mg·L^-1 to 9.3 mg·L^-1,which significantly enhanced the typical flavor of beer.