Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes: starch synthase and branching enzyme from potato tubers.
Soluble starch-synthesizing enzymes, starch synthase (SSS) and starch-branching enzyme (SBE), were isolated, fractionated, and purified from white potato tubers (Solanum tuberosum) on a large scale. Five steps were used: potato tuber extract from 2 kg of peeled potatoes, two acetone precipitations, and two fractionations on a large ultrafiltration polysulfone hollow fiber 100 kDa cartridge. Three kinds of fractions were obtained: (1) mixtures of SSS and SBE; (2) SSS, free of SBE; and (3) SBE, free of SSS. Contaminating enzymes (amylase, phosphorylase, and disproportionating enzyme) and carbohydrates were absent from the 2nd acetone precipitate and from the column fractions, as judged by the Molisch test and starch triiodide test. Activity yields of 122% (300,000-400,000 units) of SSS fractions and 187% (40,000-50,000 units) of SBE fractions were routinely obtained from the cartridge. Addition of 0.04% (w/v) polyvinyl alcohol 50K and 1 mM dithiothreitol to the glycine buffer (pH 8.4) gave long-term stability and higher yields of SSS and SBE, due to activation of inactive enzymes. Several SSS and SBE fractions from the two fractionations had very high specific activities, indicating high degrees of purification. Polyacrylamide gel electrophoresis of selected SSS and SBE fractions gave two to five SSS and/or SBE activity bands, corresponding to the one to five protein bands present in the 2nd acetone precipitate.
Mukerjea
Rupendra
R
Laboratory of Carbohydrate Chemistry and Enzymology, Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
Falconer
Daniel J
DJ
Yoon
Seung-Heon
SH
Robyt
John F
JF
D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000096:Acetone / Q000737:chemistry; D002240:Carbohydrate Sequence; D005591:Chemical Fractionation / Q000379:methods*; D011232:Chemical Precipitation; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D035281:Plant Tubers / Q000201:enzymology*; D011108:Polymers / Q000737:chemistry; D011198:Solanum tuberosum / Q000201:enzymology*; D012995:Solubility; D013213:Starch / Q000096:biosynthesis*; D013214:Starch Synthase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013450:Sulfones / Q000737:chemistry; D014462:Ultrafiltration
D011108:Polymers; D013450:Sulfones; D000096:Acetone; C017662:polysulfone P 1700; D013213:Starch; D015061:1,4-alpha-Glucan Branching Enzyme; D013214:Starch Synthase