Isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes, exemplified by purification of glucoamylase.
A highly purified preparation of glucoamylase G1 from Aspergillus niger was found, by isoelectric focusing in immobilized pH gradients, to contain a major form with a pI of 3.50 and a number of minor, more acidic and more basic contaminants. The major isoform was purified to homogeneity by recycling isoelectric focusing in a multicompartment electrolyzer, by confining this form in between two zwitterionic membranes, with pI 3.49 at the anodic side and pI 3.52 at the cathodic side. Recoveries were high (90%) and, notwithstanding the rather low operational pH, the electrosmotic flow was minimal and no protein precipitation occurred up to concentrations of 2.5 mg/ml (at the isoelectric point). The forms resolved in an analytical focusing gel were subjected to two types of in situ enzyme detections, by the glucose oxidase peroxidase (GOP) test and by the starch-iodine test. By both criteria all resolved zones exhibited enzyme activity, the GOP assay, however, following more closely the Coomassie blue stained protein profile. By computer modelling, it is shown that it is impossible to obtain linear pH gradients at such low pH values (pH 2.5-4.5 intervals) when the mixture has a low buffering power (beta = 2.0 mequiv.l-1 pH-1). When the beta power was gradually raised (beta = 4, beta = 6, beta = 8) the pH gradient became progressively linear until, in a recipe with beta = 10 mequiv.l-1 pH-1 full linearity of the pH gradient could be obtained. This is shown to be due to the substantial buffering power of bulk water in the pH 2.5-3.5 region.
Wenisch
E
E
Institute of Applied Microbiology, School of Food and Biotechnology, University of Agriculture and Forestry, Vienna, Austria.
Schneider
P
P
Hansen
S A
SA
Rezzonico
R
R
Righetti
P G
PG
D001234:Aspergillus niger / Q000201:enzymology; D003198:Computer Simulation; D004563:Electrochemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007525:Isoelectric Focusing / Q000379:methods; D008567:Membranes, Artificial; D008956:Models, Chemical
D005656:Fungal Proteins; D007477:Ions; D008567:Membranes, Artificial; D005087:Glucan 1,4-alpha-Glucosidase