Identification Of Hyperthermic Ferulic Acid Esterase And Its Heterogenic And High Expression Technique

Feruloyl esterases [E.C.3.1.1.73; also known as ferulic acid esterases,FAE] are a subclass of the carboxylic acid esterases. FAEs have received much attention in recent years, for their potential uses over a broad range of applications. FAEs hydrolyse ester linkages between ferulic acid, lignin and hemicelluloses present in the plant, so that partially disrupt and loosen the cell wall structure, make the lignin-carbohydrate complex be more susceptible to enzymatic attack and its degradation products be more diffusible. At the same time, FAEs have the ability to release ferulic acid which has physiological activity. Besides, FAEs have bright future over abroad range of applications in many industries. For example, the cosmetic industry, food and pharmaceutical industry, feed and paper-making industry and so on.However, there are several problems of FAEs’ industrialized application:(1) FAEs are a less well-known group of esterases in microorganisms, and only a small number of microbial FAEs have been identified and characterized; (2) to date the enzyme activity of the existing FAEs are insufficient to meet the needs of the industry; (3) lack the tailor-made esterases with novel functionalities, such as the thermostable enzyme, the high pH stability enzyme and so on.The aim of this experiment is to find a thermostable FAE from Thermotoga maritima MSB8(ATCC 43589, Tma), and try to realize its over-expression in recombinant bacteria,In this study, a gene from Tma which homologous to the feruloyl esterase gene fae, was amplified and ligated into pHsh, and transformed to E.coli DH10B. The recombinant bacteria was cultured in the solid medium which contain the substrate of ferulic acid ethyl ester. Screening results show that this new enzyme has the feruloyl esterase activity.Through structural optimization on this recombinant plasmid, the ultimate plasmid, pHsh-faeI, which possessed the most appropriate structure and free energy of mRNA, was obtained. However, the SDS-PAGE result showed that there’s no significant increase in its expression level. Therefore, "irrational design method" was used for protein directed evolution—using a novel method for constructing mutagenesis libraries via in situ error-prone PCR, coupling with high-throughput screening, in order to find the positive transformants.A PCT patent of our lab described this novel method that by adding a ligation step mediated by thermostable Thermotoga maritima (Tma) DNA ligase to form the repeated cycles of "denaturation-annealing-elongation-ligation", which allows exponential amplification of circular DNA. When performed under an error-prone condition with Taq DNA polymerase, random mutations are introduced into the target gene, and the products are the circular plasmids DNA that can be directly transformed. PCR production of circular plasmids (PPCP) allows one-step construction of mutagenesis libraries based on in situ error-prone PCR. In this method, circular PCR products are generated by using a long dsDNA primer pair, which is the same as the template vector except for missing the DNA segment to be mutagenized. If the dsDNA primer pair is designed to carry a selection marker different from the original selection marker of the template plasmid, the template plasmid carrying the original marker is eliminated when transformed host cells are cultured under the new selection pressure. However, there’s no positive mutation be discovered in the library. This result maybe due to the improper mutation frequency, or the undersized mutation library. Additionally, the accumulation of FAE in E.coli may be toxic to cells, this give explain to the lower cell density after 42℃ heat shock induction.Constructing the FAE gene into the Yeast expression system. The cpy signal peptide and the fae gene were subcloned into the vector pADH-GFAT with gfaA as new selectable marker gene, resulting the recombinant plasmid pADH-GFAT-cpy-fae with signal peptide. The expression plasmid were transformed and expressed in the Schizosaccharomyces pombe[YHL6381(S.pombe)]△gfaA competent cells. Collecting the extracellular enzyme, and the detection results showed that the enzyme expression and enzyme activity both improved slightly. This may suggested that cpy is not the optimal signal peptide for fae gene.