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 degrees C water bath and 49.2 +/- 4.1% relative activity after a 60 s incubation in a 95 degrees 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.