Gene Polymorphisms, Expression And Characterization Of Rutin Degrading Enzyme From Tatary Buckwheat

Tatary buckwheat(Fagopyrum tataricum)seeds have markedly high rutin degrading enzyme(RDE) activity, which belongs to β-D-glucosidase family and hydrolyzes rutin releasing quercetin and rutinose, RDE activity is the main cause of bitterness in buckwheat. Based on obtained cDNA sequence of RDE previously, PCR primes were designed to amplify RDE genome DNA and products were sequenced. The results shows that rutin degrading enzymes have three copies in the genome, tentatively named rde1, rde2 and rde3, rde1 is intronless, however rde2 and rde3 have introns, rde3 is composed of 12 exons, a new intron is inserted at the 8th exon of rde3 to form rde2. Both rde2 or rde3 exists many single base mutations in exons, intron length polymorphisms is main difference between rde2 and rde3. AT content in introns of rde2 and rde3 are up to 70%, but they have conserved splice site and follow the GT-AG rules. We inferred that rde2 and rde3 may be homologous genes.Based on gene structure analysis, RDE gene was inserted into p PICZα A vector, and expressed extracellularly in Pichia pastoris strain SMD1168 H. After 1% methanol induction for 48 h, fermentation was precipitated by 80% ammonium sulfate, then washed twice with 80% acetone. The deposition was dissolved in pH5.5 sodium acetate, purfied by one step DEAE-Sepharose CL 6B chromatography, SDS-PAGE electrophoresis demonstrated that a recombinant protein band of approximately 75 KDa was far greater than expected. After PNGase F treated, the mass of recombinant protein migrated down to 54 KDa, results showed that the recombinant protein was a N-linked glycoprotein.The present study also invented a method for rapid purification RDE from buckwheat seeds. Based on different concentrations of methanol effect on extracting rutin degrading enzymes and its activity, Native-PAGE and UV-spectrophotometry showed that the optimum extraction concentration of methanol on RDE was 40%, so after Na OAc extracting, pre-cooled methanol was added to a final concentration of 40% for removing insoluble impurities in pH4.0. Extracted crude enzyme was purified by cation exchange chromatography SP-Sepharose HP and obtained a single band of 58 KDa on SDS-PAGE, purified yield up to 70%.Comparison of biochemical properties of recombinant RDE(rRDE)and native RDE(nRDE)by UV spectrophotometer determination, both enzymes showed optimal activity at pH 5.0, however, rRDE optimum temperature is 50 ℃, while nRDE is 40 ℃, both of Km for rutin were 5.375 Km mmol/L and 11.333 mmol/L, the maximum reaction rate of nRDE is 10 times higher than rRDE, it suggests that different sources of RDE have difference post-translational modifications which result in the discrepancy in catalytic properties.