Obtaining Feruloyl Esterase Gene And Heterologous Expression

Feruloyl esterase is a hydrolase that hydrolyse the ester bond associated ferulicacid and lignin or arabinogalactan which exsit in plant cell wall. There is a synergisticeffect with feruloyl esterase and other cellulase enzymes, such as xylanase enzymesand arabinosidase, efficient degradation can be made in the plant cell walls whencombined these enzymes. Feruloyl esterase reduced a lot of environmental issueswhich brought by chemical production processes and widly used in paper、textile、feed、food and other light industries. Other more, ferulic acid produced by thehydrolysis of feruloyl enzyme is a natural antioxidant, has a great potential in thefood、health products and cosmetics applications.A bacterial strain named A216, with feruloyl esterase producing capability, wasisolated from forest soil after enrichment. The strain was identified at the species levelby morphology, physiological-biochemical tests and16S rRNA gene sequencing assay.The results identified the strain A216as a Burkholderia fungorum. The16S rRNA andITS sequence of the species was submitted to the NCBI with the accession numberKJ026454and KJ130021. Given the current few research on B. fungorum, genomiclibrary was constructed in order to get their genetic information. The genomic DNA ofB. fungorum A216was partially digested by Sau3A Ⅰ, and the fragment of300-4000bp were obtained. The linear vector fragment was recycled when Vector pUC19wasdigested by BamHⅠand treated with SAP. At a molar ratio of10:1, genomic fragmentand the vector were linked and transformed into E. Coli DH5α competent cells. Toobtain B. fungorum genetic information，the library was randomly sequenced. Theresult showed that the library contained32000clones, insert rate was96%with anaverage insert2000bp. The present study successfully constructed a high quality B.fungorum genomic library, it can be used for sequencing or specific screening in thefuture research. But the gene of B. fungorum A216feruloyl esterase were not obtainedsuccessfully by this transparent circle screening method.In this paper, overlap extension PCR method was used to obtain the feruloylesterase encoding sequence of the Aspergillus terreus strain which was pre-screened inour laboratory. Then the heterologous expression experiment were carried out inprokaryotic host Escherichia coli and eukaryotic host Pichia pastoris. In theheterologous expression of E. coli, the feruloyl esterase gene and pET-22b (+) vector were digested by the two enzymes EcoRⅠand NotⅠ r espectively,then the fragmentwere connected, the expression vector pET22b-fae was obtained. This expressionvector was successfully transformed into E. coli expression strain BL21(DE3). Theenzyme activity result of transformants fermentation suggested that the feruloylesterase was expressed successfully. SDS-PAGE showed that the size of theheterologous expression feruloyl esterase is32kDa. The fermentation conditions of theE. Coli transformant were optimized in the medium LB. The highest enzyme activity420mU/ml was obtained when1%seed inoculated in medium,37℃incubated for4hours, and IPTG was added to a final concentration of0.01mM, induced nine hours.The enzyme activity is about28times higher than the starting strain of A. terreus. Theoptimum reaction temperature of the recombinant feruloyl esterase of the E. colitransformant is50℃when4-NPF as a substrate was used, the temperature stabilityexperiment showed that the half time is6min,15min,4h and18h in60℃,55℃,50℃and45℃, respectively. It has good stability below40℃. The optimum pH is6.8,in the range of pH6-10the enzyme activity is greater than70%.In the heterologous expression of P. pastoris, the feruloyl esterase gene andpPICZαA vector were digested by the two enzymes EcoR Ⅰa ndNotⅠ r espectively,andthe fragment connected to obtained the expression vector pPICZαA-FAE. Thisexpression vector was linearized by SacⅠand successfully transformed into P. pastorisstrain GS115. The transformants screened by Zocin plate and verified by PCR, and theresult suggested that the insertion of the feruloyl esterase was successed. However,SDS-PAGE and enzyme activity experiments showed the protein was not expressed.The possible reason is that the RNA spatial structure of target genes hindered thetranslation progress, the transformants can be detected by RT-PCR in the futurestudies.