Partial purification and characterization of pectin methyl esterase in vegetable soybeans

This study was conducted to partially purify pectin methyl esterase (PME) of vegetable soybeans, to determine optimum pH and salt concentration for maximum PME activity, to ascertain heat stability and energy of activation of PME, and to determine the Michaelis-Menten constant (Km) and maximum velocity (Vmax) of PME of soybeans.

PME was obtained from soybeans which were raised on the University of Tennessee Plant Science Farm at Knoxville. Progressive purification of PME was accomplished by acetone powder, salt extraction, ammonium sulfate precipitation, dialysis, and pH precipitation in order to obtain partially purified PME which was used for assay of PME activity. The Beckman Model K automatic titrator was used for quantitative determination of PME activity.

The activity values for the effect of salt concentration and pH levels on PME activity were analyzed by analysis of variance using orthogonal comparisons. On the basis of the analysis of variance, a regression equation was chosen which described a three dimensional response surface of PME activity as affected by salt concentration and pH. Interaction between salt concentration and pH was significant at the 0.01 level of probability. No salt concentration or pH level was chosen as the optimum for PIE activity.

Partially purified PME in aqueous solution was completely inactivated after a five minute treatment at 80°C. Energy of activation was calculated to be 7,570 calories per mole. By use of the Lineweaver-Burk form of the Michaelis-Menten equation, Km was calculated to be 0.111 percent pectin and Vmax 20.60 PME units (x 10⁴).

After soybean PME was partially purified by the progressive purification procedures listed above, the PME containing material was.lyophilized and purified further by DEAE- and CM-cellulose column chromatography. Fractions were collected using an automatic fraction collector. There were at least two molecular forms of the soybean PME. Two activity peaks were detected after DEAE-cellulose column chromatography. The PME material from the first activity peak from the DEAE-cellulose column yielded two activity peaks upon CM-cellulose column chromatography. PME material from the second activity peak from the DEAE-cellulose column yielded one activity peak upon CMcellulose column chromatography. DEAE-cellulose column chromatography was an acceptable means of additional PME purification. CMcellulose column chromatography was unsatisfactory for additional PME purification due to retention of detectable PME activity by the CM-cellulose column until one-third of the elution volume had been collected.