Purification and Characterization of a Thermally Tolerant Pectin Methylesterase from a Commercial Valencia Fresh Frozen Orange Juice

A thermally tolerant form of pectin methylesterase (TT-PME) has been purified from a commercially available Valencia fresh frozen orange juice by gel filtration, heparin, and concanavalin A chromatography. TT-PME represented 8.3% of total PME activity in the solubilized dialysis precipitate. This TT-PME retained 83.3 ± 1.1% relative activity after a 2 min incubation in an 80 °C water bath and 49.2 ± 4.1% relative activity after a 60 s incubation in a 95 °C water bath. It also retained 3.3 ± 0.6% and 8.3 ± 1.5% relative activity at pH 3.5 and 4.5, respectively. It had a native molecular mass of 40.1 kDa (gel filtration chromatography) and a denatured M r of 42.7 ± 0.1. Binding to concanavalin A and treatment with PNGase F suggests it is an N-linked glycoprotein. After extended deglycosylation with PNGase F, polypeptide bands at M r 41.7 ± 0.6 ( n = 4) and 40.1 ± 0.4 ( n = 4) were observed.