Study On The Constructing Engineering Bacteria Of Feruloyl Esterase And Enzymatic Properties

Feruloyl esterases are essential accessory enzymes acting in synergy with main-chain degrading carbohydrases to allow an efficient degradation of plant cell. Feruloyl esterases (FAEs) act synergistically with xylanases to hydrolyze ester-linked ferulic (FA) and diferulic (diFA) acid from cell wall material and therefore play a major role in the degradation of plant biomass. Their potential application in the agrifood industries is driving increasing research. Several types of feruloyl esterases have been identified based on their substrate specificity, i.e. type A feruloyl esterases which have been reported to be preferentially active towards the methyl esters of ferulic and sinapic acids; and type B feruloyl esterases which showed a preference for the methyl esters of caffeic and p-coumaric acids. At present, the secreted FAE from microbes still can not reach the industrialized FAE production requirements, the FAE extraction method needs to be improved, so its industrial production is restricted.To obtain high quantities of FAE that was not available from the wild-type strain. In this study, we used a new method to achieve FAE cDNA library, alignment of the XM₀01393300, XM₀01217492, AB032760, Y09331 gene, found the conservative DNA sequence. Obtain the target FAE gene by using specific primer though the RT-PCR. Then, link with pET-28a(+) vector to express the protein by using pNPF as a substrate to screening high activity FAE. Also the enzyme can release ferulic acid from pNPF.FAE production was estimated to be 2805mU/mg activity under optimal conditions.The optimal temperature and pH values of the recombinant enzyme were determined to be 40℃and 6.4, respectively. The cloned gene encoding a protein with calculated molecular mass of 40 KDa. The optimal concentration of pNPF was 0.6mmol/L, Km=0.3×10-3,kcat=2.24×10-5.As shown above, FAE from engineering bacteria have some interesting characteristics in the primary structure, enzyme kinetics of their function. Although the tertiary structure and the enzyme reaction mechanism of FAE are unknown, the present study provides good insights to better understand the molecular evolution of FAE proteins. We obtain a new feruloyl esterase gene which could not get from a traditional method, while it has a high activity by using p-nitrophenyl ferulate (pNPF) as substrate.The contents of ferulic acid in corn bran which was hydrolysised by FAE were determinated and analysed by using gas chromatography.GC (column:DB1701,80℃-210℃, programmed temperature,15℃/min,80℃do not stop,210℃stay 3min. injector:temperature 240℃,The detectior temperature 240℃), establishment a method of ferulic acid by gas chromatography.