One-step purification of glucoamylase by affinity precipitation with alginate.
It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens.
Teotia
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Chemistry Department, Indian Institute of Technology, Hauz Khas, New Delhi-110016, India.
Lata
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R
Khare
S K
SK
Gupta
M N
MN
D000464:Alginates / Q000737:chemistry*; D001234:Aspergillus niger / Q000201:enzymology; D001407:Bacillus / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000302:isolation & purification*; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D008024:Ligands; D008320:Maltose / Q000494:pharmacology; D000516:alpha-Amylases / Q000737:chemistry
D000464:Alginates; D006603:Hexuronic Acids; D008024:Ligands; D008320:Maltose; D020723:Glucuronic Acid; D000516:alpha-Amylases; D005087:Glucan 1,4-alpha-Glucosidase