Constitutive Expression Of Feruloyl Esterase From Aspergillus Niger In Yeast

As a subclass of the carboxylic acid esterase, feruloyl esterase(E.C 3.1.1.73) are able to hydrolyze the ester bonds between ferulic acid(or other hydroxycinnamic acids) and polysaccharides in the plant cell walls. This hydrolysis reaction can break the tight and complex structure of lignocelluloses, therefore feruloyl esterase have a broad potential applications in food, pulp and paper, feed industries fields. Several reports of feruloyl esterases from microorganisms were published. However, the low expression quantities of original strains can not meet the requirements of industrial production. Constructing heterologous expression system of feruloyl esterase gene is an efficient way to solve this problem. In this study, feruloyl esterase from Aspergillus niger were constitutively expressed in different yeast hosts. Then, the fermentation conditions of the best recombinant strain were optimized. It was helpful to study the constitutive expression system in yeast and improve the fermentation production of feruloyl esterase.The signal peptide of feruloyl esterase gene(AnfaeA) was predicted and the AnfaeA gene without its native signal sequence was amplified from A. niger by overlap extension PCR. The length of the AnfaeA gene was 783 bp. The expression vector pET28 aAnfaeA was constructed and transformed into Escherichia coli. The target protein was expressed as inactive inclusion bodies when induced by IPTG.The recombinant vectors pKLAC1 AnfaeA, pRACTHα1AnfaeA, pRACTHα2AnfaeA, pGAP9 KAnfaeA, pRGCTHα1AnfaeA and p RGCTHα2AnfaeA were constructed and transformed into Kluyveromyces lactis GG799, Pichia pastoris GS115, Saccharomyces cerevisiae separately. The following recombinant yeast strains were constructed: the recombinant K. lactis GG799: K. lactis/pKLAC1 AnfaeA, K. lactis/pRACTHα1AnfaeA, K. lactis/pRACTHα2AnfaeA; the recombinant P. pastoris GS115: P. pastoris/pGAP9 KAnfaeA, P. pastoris/pRGCTHα1AnfaeA, P. pastoris/pRGCTHα2AnfaeA; the recombinant S. cerevisiae: S. cerevisiae/pRACTHα1AnfaeA, S. cerevisiae/pRACTHα2AnfaeA. The feruloyl esterase activity of these recombinant yeast strains were compared and the result showed that feruloyl esterase activity of the recombinant P. pastoris GS115(P. pastoris/pGAP9KAnfaeA) was highest. The feruloyl esterase activity reached its peak(5.72±0.10 U?m L-1) after fermentation of 84 h.In shake flasks, the fermentation conditions of P. pastoris/pGAP9 KAnfaeA was optimized by single factor test, and the key medium components glucose, peptone and yeast extract was optimized by response surface methodology. The optimal fermentation conditions can be recognized as follows: glucose 42 g?L-1, peptone 16 g?L-1, yeast extract 28 g?L-1, CaCO3 0.2 g?L-1, inoculum age 28 hours, inoculation level 3%(v/v), medium volume 40 mL in 250 m L flask. Under the conditions, the feruloyl esterase activity of fermentation supernatant was 15.67±0.17 U?m L-1 with 200 r?min-1 for 84 hours at temperature of 30°C, which was 2.74 times than before optimization.The recombinant feruloyl esterase was purified by ammonium sulfate precipitation(60%) and ion-exchange chromatography with a DEAE-Sepharose Fast Flow column. The specific activity of recombinant feruloyl esterase was 59.75 U?mg-1 and the molecular weight of recombinant feruloyl esterase was about 40 k Da by SDS-PAGE. The optimum feruloyl esterase activity was achieved at 50°C and pH 5.0 and was stable at temperature from 30°C to 50°C, pH from 4 to 7.