Page Banner

United States Department of Agriculture

Agricultural Research Service

Title: Purification and Characterization of a Novel Mannitol Dehydrogenase from Lactobacillus Intermedius

Author
item Saha, Badal

Submitted to: Biotechnology Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 28, 2003
Publication Date: December 2, 2003
Citation: Saha, B.C. 2004. Purification and characterization of a novel mannitol dehydrogenase from Lactobacillus intermedius. Biotechnology Progress. 20:537-542.

Interpretive Summary: A lactic acid bacterium was found to be an excellent producer of mannitol by fermentation. A novel enzyme (mannitol dehydrogenase) responsible for converting fructose to mannitol from this bacterium has been purified and characterized in order to more clearly understand the fermentation process and also to develop an enzymatic method to produce pure mannitol using the enzyme. The enzyme possessed very specific substrate specificity and was able to convert fructose to mannitol with a 100% yield. It has great potential to be used in a single step synthesis of mannitol from fructose if the cofactor required for such conversion can be regenerated in an efficient way.

Technical Abstract: 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 homogeniety 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 per mg protein) had 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 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 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 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 5.5-6.5 and temperature up to 35 deg C. The Km and Vmax values of the enzyme for the reduction of fructose to mannitol was 20 mM and 396 micromole per min per 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.

Last Modified: 7/28/2014
Footer Content Back to Top of Page