Purification and characterization of a novel lactonohydrolase, catalyzing the hydrolysis of aldonate lactones and aromatic lactones, from Fusarium oxysporum.
A novel lactonohydrolase, an enzyme that catalyzes the hydrolysis of aldonate lactones to the corresponding aldonic acids, was purified 10-fold to apparent homogeneity, with a 61% overall recovery, from Fusarium oxysporum AKU 3702, through a purification procedure comprising DEAE-Sephacel, octyl-Sepharose CL-4B and hydroxyapatite chromatographies and crystallization. The molecular mass of the native enzyme, as estimated by high-performance gel-permeation chromatography, is 125 kDa, and the subunit molecular mass is 60 kDa. The enzyme contains 15.4% (by mass) glucose equivalent of carbohydrate, and about 1 mol calcium/subunit. The enzyme hydrolyzes aldonate lactones, such as D-galactono-gamma-lactone and L-mannono-gamma-lactone, stereospecifically. Furthermore, it can catalyze the asymmetric hydrolysis of D-pantoyl lactone, which is a promising chiral building block for the chemical synthesis of D-pantothenate. These reactions are reversible, and the reaction equilibrium at pH 6.0 has a molar ratio of nearly 1:1 with D-pantoyl lactone and D-pantoic acid. The Km and Vmax for D-galactono-gamma-lactone are 3.6 mM and 1440 U/mg, respectively, and those for D-galactonate are 52.6 mM and 216 U/mg, respectively. The enzyme also irreversibly hydrolyzes several aromatic lactones, such as dihydrocoumarin and homogentisic-acid lactone.
Shimizu
S
S
Department of Agricultural Chemistry, Kyoto University, Japan.
Kataoka
M
M
Shimizu
K
K
Hirakata
M
M
Sakamoto
K
K
Yamada
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004795:Enzyme Stability; D005670:Fusarium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007783:Lactones / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013400:Sugar Acids / Q000378:metabolism*; D013696:Temperature
D000596:Amino Acids; D007783:Lactones; D013400:Sugar Acids; D002265:Carboxylic Ester Hydrolases; C077288:aldonate lactonohydrolase