Purification and characterization of a novel mannitol dehydrogenase from Lactobacillus intermedius.
Mannitol 2-dehydrogenase (MDH) catalyzes the pyridine nucleotide dependent reduction of fructose to mannitol. Lactobacillus intermedius (NRRL B-3693), a heterofermentative lactic acid bacterium (LAB), was found to be an excellent producer of mannitol. The MDH from this bacterium was purified from the cell extract to homogeneity by DEAE Bio-Gel column chromatography, gel filtration on Bio-Gel A-0.5m gel, octyl-Sepharose hydrophobic interaction chromatography, and Bio-Gel Hydroxyapatite HTP column chromatography. The purified enzyme (specific activity, 331 U/mg protein) was a heterotetrameric protein with a native molecular weight (MW) of about 170 000 and subunit MWs of 43 000 and 34 500. The isoelectric point of the enzyme was at pH 4.7. Both subunits had the same N-terminal amino acid sequence. The optimum temperature for the reductive action of the purified MDH was at 35 degrees C with 44% activity at 50 degrees C and only 15% activity at 60 degrees C. The enzyme was optimally active at pH 5.5 with 50% activity at pH 6.5 and only 35% activity at pH 5.0 for reduction of fructose. The optimum pH for the oxidation of mannitol to fructose was 7.0. The purified enzyme was quite stable at pH 4.5-8.0 and temperature up to 35 degrees C. The K(m) and V(max) values of the enzyme for the reduction of fructose to mannitol were 20 mM and 396 micromol/min/mg protein, respectively. It did not have any reductive activity on glucose, xylose, and arabinose. The activity of the enzyme on fructose was 4.27 times greater with NADPH than NADH as cofactor. This is the first highly NADPH-dependent MDH (EC 1.1.1.138) from a LAB. Comparative properties of the enzyme with other microbial MDHs are presented.
Saha
Badal C
BC
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U. S. Department of Agriculture, Peoria, Illinois 61604, USA. sahabc@ncaur.usda.gov
D002850:Chromatography, Gel / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D005632:Fructose / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000145:classification / Q000201:enzymology*; D008354:Mannitol Dehydrogenases / Q000096:biosynthesis* / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification*; D008970:Molecular Weight; D013045:Species Specificity; D013696:Temperature
D005632:Fructose; D008354:Mannitol Dehydrogenases