Monascus anka Pyruvate Decarboxylase Accounts for The New Candidate Resources of Fuel Ethanol Production

摘要

In order to study the nature and function of Pyruvate decarboxylase (PDC, E.C.4.1.1.1), which is the key enzyme to produce ethanol by fermentation; full-length cDNA library was constructed with SMART technique from Monascus anka CICC 5031. The pdc gene, including a 1713-bp open reading frame, encoding a 570 amino acid protein, was obtained by screening the constructed M. anka cDNA library. The pdc gene was successfully heterologously expressed in E.coli BL21(DE3), accounting for 32.7% of total cellular proteins. Recombinant PDC was expressed in prokaryotic cells and purified by affinity chromatography, and native PDC was extracted and purified from M. anka through Sephadex G-25 and DEAE-anion exchange resin. The enzymatic characterization of both recombinant and native PDC were studied, respectively. The specific activity of recombinant and native PDC was 20.2 and 30.11U/mg respectively. Kinetic analysis indicated that recombinant and native PDC had the same optimum conditions: pH6.0, 30°C, the Km value for pyruvate of recombinant PDC was 2.6 mmol/L and native PDC was 0.56 mmol/L. The high activity and stable PDC from M. anka accounts for the new candidate resources of fuel ethanol production.