Enzyme purification by immobilized metal ion affinity partitioning--application to D-hydroxyisocaproate dehydrogenase.
Extraction and purification of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei has been studied by means of immobilized metal ion affinity partitioning (IMAP) in aqueous two-phase systems. The partition of the enzyme can be influenced strongly by inclusion of iminodiacetic acid as chelating ligand coupled to polyethylene glycol and loaded with Cu2+ ions into the phase system. This applies to polyethylene glycol/dextran as well as polyethylene glycol/salt phase systems. An increase in enzyme partition coefficient of up to about 1000-fold was observed. Based on the mathematic model presented recently by Suh and Arnold (1990) approximately 6.4 histidine residues were calculated to be involved in the enzyme-metal chelate complex. Direct extraction of the enzyme from both cell homogenate and cell debris supernatant proved unsatisfactory due to disturbances caused by the presence of cell debris and low molecular weight cell components. A combination with a preceding prepurification by a fractional precipitation with polyethylene glycol resulted in a strong affinity effect accompanied by an efficient purification during IMAP (purification factor of 11 with a yield of approximately 90%). Based on this step, an efficient downstream process can be designed for D-hydroxyisocaproate dehydrogenase.
Schustolla
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D
Schering AG, Wolfenbüttel, Germany.
Deckwer
W D
WD
Schügerl
K
K
Hustedt
H
H
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D001665:Binding Sites; D001709:Biotechnology; D002614:Chelating Agents; D011232:Chemical Precipitation; D003300:Copper; D006863:Hydrogen-Ion Concentration; D007098:Imino Acids; D007780:Lacticaseibacillus casei / Q000201:enzymology; D011092:Polyethylene Glycols
D002614:Chelating Agents; D007098:Imino Acids; D011092:Polyethylene Glycols; D003300:Copper; D000429:Alcohol Oxidoreductases; C047961:2-hydroxyisocaproate dehydrogenase; C008109:iminodiacetic acid