Soybean seed pectinesterase

Methanol accumulates in axis tissues of maturing soybean seeds, correlating with preharvest seed deterioration. Accumulation of methanol appears to be associated with the enzymic demethylation of pectin methyl esters by pectinesterase (PE; EC 3.1.1.11). To characterize PE in developing and maturing soybean (Glycine max (L.) Merrill) seeds, enzyme activity was assayed in axis and cotyledon tissues. Activity per g fresh weight was 20-25 times higher in axes than in cotyledons with highest activities between 45 and 60 days after flowering (DAF). Twenty to 33% of the total PE activity was in the 'soluble' form (extracted with water, 0.5 M sucrose, 1 M sucrose and water). Soluble and cell-wall-bound PE (subsequently extracted with 1 M NaCl) were purified and characterized from axes of seeds at 45-60 DAF. Purification of PE was achieved through concentration of extracts by ultra-filtration, precipitation with ammonium sulfate (30-80% saturation), dialysis, gel filtration on Sephadex G-75 columns, and ion exchange chromatography on CM Sepharose CL-GB. Further purification of both soluble and bound PE was by isoelectric focusing (IEF) on ultrathin layers of polyacrylamide gel with simultaneous detection of protein and PE activity. It was possible to follow seven bands exhibiting PE activity with pi values between 6.0 and 9.5 in 1 M NaCl-extracts of total homogenates. Differences in the IEF patterns of bound and soluble PE were observed. Whereas the bound enzyme exhibited more basic PE bands (pI 8-9.5), the soluble enzyme had more active bands at pI 6.5, 7.0 and 7.5. The Mr was close to 33 000 and the pH optimum was 7.8 for both soluble and bound PE.