Submitted to: European Congress on Biotechnology
Publication Type: Abstract Only
Publication Acceptance Date: August 29, 2003
Publication Date: August 29, 2003
Citation: SAHA, B.C. A NOVEL MANNITOL DEHYDROGENASE FROM LACTOBACILLUS INTERMEDIUS. 11TH EUROPEAN CONGRESS ON BIOTECHNOLOGY. 2003. PAPER NO. P-291. Technical Abstract: Mannitol 2-dehydrogenase (MDH) catalyzes the pyridine nucleotide dependent reduction of fructose to mannitol. Recently, Lactobacillus intermedius (NRRL B-3693), a heterofermentative lactic acid bacterium (LAB), was found to be an excellent producer of mannitol. The objective of this study is to purify and characterize the properties of the enzyme from this strain in order to more clearly understand the fermentative production of mannitol. The bacterium was grown in a pH-controlled fleaker system with 15% fructose as carbon source at 37 deg C with pH controlled at 5.0 for 15 h. The cell extract was prepared by breaking the cells with glass beads. It was dialyzed against 50 mM immidazole buffer, pH 6.8 containing 1 mM DTT, 1 mM PMSF, and 10% glycerol. MDH was purified from the dialyzed crude enzyme preparation to homogeniety by DEAE Bio-Gel column chromatography, gel filtration on Bio-Gel A-0.5 m gel, octyl-Sepharose hydrophobic interaction chromatography, and Bio-Gel hydroxyapatite HT column chromatography. The purified enzyme was a glycoprotein with a molecular weight (MW) of about 170,000 and subunit MWs of 43,000 and 35,000. The isoelectric point of the enzyme was 4.7. The optimum temperature for the action of the purified MDH was at 35 deg C with 51% activity at 50 deg C and only 15% activity at 60 deg 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. The purified enzyme was quite stable at pH 5.5-6.5 and temperature up to 35 deg C. The activity of the enzyme on fructose was 4.5 times greater with NADPH than NADH as cofactor. The substrate specificity and detailed kinetics of the action of purified MDH on fructose as well as on mannitol will be described. The N-terminal amino acid sequence up to 40 amino acids of the enzyme (both subunits have the same sequence) and its comparison with other MDHs will be presented. This is the first NADPH dependent MDH (EC 126.96.36.199) from LAB. The MDH has great potential to be used in a single step synthesis of mannitol from fructose if the cofactor NADPH can be regenerated in an efficient way as the enzyme converts fructose to mannitol completely.