Enzymological Properties Of Native And Recombinant Pyruvate Decarboxylase From Monascus Anka CICC 5031

Objective:Pyruvate decarboxylase (PDC, EC4.1.1.1) is a key enzyme in the metabolism of pyruvate, and it can also catalyze the biotransformation of aromatic amino acid precursors into optically activeα-hydroxy ketones. In this study, Monascus cDNA library was constructed firstly, and then the pyruvate decarboxylase gene was screened from it. The recombinant plasmid pET-PDC was constructed, and the recombinant protein was expressed in prokaryotic cell. The dynamic test and enzymological properties of Monascus native pyruvate decarboxylase were compared with the recombinant PDC.Methods:Total Monascus RNA was extracted using the Trizol reagent. Monascus cDNA library was constructed using Creator SMART cDNA Library Construction Kit. The pyruvate decarboxylase gene was screened from the library, and amplified using polymerase chain reaction (PCR) from the cloning vector DNA. The amplified fragment and the plasmid were ligated after restriction endonuclease reaction, the products were transformed into E. coli DH5α, ampicillin resistant coating on the plate, 37℃overnight train, a number of monoclonal was picked to PCR test, positive clones were sent for DNA sequencing. The recombinant plasmid pET-PDC was transformed into E. coli BL21 (DE3), ampicillin resistant coating on the plate, 37℃overnight train, a number of monoclonal was picked to PCR test. Transformed host cells were induced with IPTG (100μM) and expressed the recombinant protein. The protein was detected by SDS-PAGE, purified by by Ni²⁺-NTA agarose affinity chromatography, and then refolded by dilution.Monascus anka was grown 7 days in potato extract glucose medium at 30°C. Cells were harvested and disrupted by sonication. The intact cells and debris were removed by centrifugation. The crude enzyme was obtained by ammonium sulfate salting-out, and then purified by gel chromatography SephadexG-25 and DEAE anion exchange column, respectively. Its purity was detected by SDS-PAGE.The activity and dynamic properties of recombinant and native PDC were measured, respectively, to compare the similarities and differences.Results: Monascus cDNA library was constructed, and its pyruvate decarboxylase gene was screened successfully. The entire open reading frame of pdc gene was 1713bp, encoding a 570 amino acid protein, and the sequence was deposited in GenBank, and the accession number is HM191265. The pdc gene was succussfully heterologously expressed in E.coli BL21(DE3), accounting for 32.7% of total cellular proteins. The recombinant protein was purified by affinity chromatography, and its purity reached 95 %. PDC activity was measured by a coupled enzyme assay with minor modification. The results of the kinetic analysis indicated that recombinant and native PDC had the same optimum conditions: pH 6.0, 30℃. The specific activity of recombinant and native PDC was 20.2 and 30.11 U/mg. the Km value of the former was 2.6 mmol/L and the later was 0.56 mmol/L, separately.Conclution: The high activity and stable PDC from Monascus accounts for the new candidate resources of fuel ethanol production.