The Primary Structure Identification,Expression And Purification Of Rutin-Hydrolyzing Enzyme

In the food processing process,foods prepared from buckwheat,especially tartary buckwheat,have a certain bitter taste,which seriously reduces the use value of buckwheat.The quercetin,which is from the hydrolysis of rutin by the rutin hydrolase,is the main cause of its bitter astringency.Therefore,if the fagothrum tartaricum rutin-hydrolyzing enzyme gene(FtRHE)is knocked out in buckwheat,it is possible to obtain buckwheat without bitterness and astringency,which is benificial for the utilization and promotion of buckwheat.Although a lot of studies have been made on the properties and substrate specificity of rutin hydrolase at home and abroad,the DNA sequence of RHE has not been reported so far.Our group has cloned the FtRHE gene from the transcriptome of tartary buckwheat and recombined it in insect baculovirus systems for expression.mainly completed the following research:1.Sequence identification of FtRHE gene.In order to obtain the FtRHE gene in tartary buckwheat,the natural RHE was obtained from the seed of tartary buckwheat No.1.The natural RHE sequence information was identified by MALDI-TOF/TOF-MS and the FtRHE was screened in the transcriptome data.The results showed that the molecular weight of natural RHE wasapproximately 62 kD.The peptide fingerprinting showed that it was a ?-glucosidase and the peptide FSISWSR with a mass-to-charge ratio of914.4286 was its characteristic peptide.Based on the FtRHE gene sequence obtained from the transcriptome data of tartary buckwheat seeds,the FtRHE gene was obtained by reverse transcription PCR.The open reading frame of FtRHE consists of 1539 bases and encodes 512 amino acids.The 1-29 amino acids was its signal peptide.Multiple sequence alignments showed that the amino acid conservation of RHE with species-specific ?-glucosidase was not particularly high,and homology was between 54% and 62%.The biochemical,molecular biological methods combined with software analysisshowed that FtRHE may have mainly four hydrophobic regions;its secondary structure consisted of 48.44% random coils,37.30% ?-helices,and14.26% ?-sheets;the domain was found to have glycosides.The typical(?/?)8 TIM barrel domain of hydrolase family 1 predicts its active site based on the related structure of rutin hydrolase and found that its active site may be Glu200 and Glu412 in the(?/?)8TIM barrel domain.2.Construction and Expression of FtRHE gene expression system.In order to obtain recombinant RHE with the same biological activity as natural RHE,it lays the foundation for subsequent functional studies,the first is to construct the prokaryotic expression vector of FtRHE,which includes the vectors expressing PRSETC,pGEX-4T-1 and pET-32 a in E.coli.The construction is completed and the expression is induced after sequencing.However,there is no specific bands.We tried to optimize the sequence based on the expression characteristics of E.coli,but we failed.Therefore,an insect baculovirus expression system was used to construct an eukaryotic expression vector first,and Bacmid-FtRHE recombinant bacmid was constructed by transforming DH10 Bac competent cells.After transfection of insect cell Sf9,a higher hydroquinone hydrolase activity was successfully expressed.The recombinant FtRHE.The results of enzyme activity assay showed that the FtRHE we expressed was similar to the RHE activity extracted from natural buckwheat.The research lays a good foundation for the application of high-content rutin,no-bitter taste buckwheat breeding,and the application of rutin biotransformation to produce quercetin.