Poster Presentation 2-24

 

Cloning, Expression, Purification and Analysis of Mannitol Dehydrogenase of mtlD and mtlK Genes from Lactobacillus plantarum and Lactobacillus brevis

 

 

Siqing Liu,* Badal C. Saha and Michael A. Cotta

 

 

Fermentation Biotechnology Research Unit

National Center for Agriculture Utilization Research

USDA, ARS

1815 N. University St.

Peoria, IL 61604

Phone:  (309)681-6566

Fax:  (309)681-6427

E-mail:  lius@ncaur.usda.gov

 

 

 

The commercial production of mannitol involves high-pressure hydrogenation of fructose using a nickel catalyst, a fairly costly and inefficient process.  Mannitol can be produced through fermentation processes by microorganisms.  Currently, a few lactobacillus strains are being used to develop a mannitol production process.  However, Lactobacillus sp. produce products in addition to mannitol during fermentation, and the production efficiency could be improved.  An approach toward improving this process would be to genetically engineer lactobacillus strains to increase mannitol production efficiency.  The mannitol dehydrogenase genes mtlD, encoding mannitol-1-phosphate 5-dehydrogenase (EC 1.1.1.17) from L. plantarum, and mtlK, encoding mannitol-2-dehydrogenase (EC 1.1.1.67) from L. brevis, were isolated and characterized.  The mtlD and mtlK were cloned via PCR from genomic DNA using the GenBank sequence of L. plantarum (http://www.ncbi.nlm.nih.gov) and the partially assembled genomic DNA sequence of L. brevis (http://www.er.doe.gov).  The clone for mtlD of L. plantarum contains 1375 bp genomic DNA sequence.  This includes the full length open reading frame of 1158 nucleotides encoding mannitol-1-phosphate 5-dehydrogenase that consists of 385 amino acids with a predicted molecular mass of about 42 kDa.  The clone for mtlK gene in L. brevis contains 1328 bp genomic DNA sequence and includes the full length open reading frame of 1002 nucleotides encoding mannitol-2-dehydrogenase that consists of 333 amino acids with a predicted molecular mass of about 36 kDa.  The mtlD and mtlK genes were then moved into an expression vector and expressed in Escherichia coli BL21 cells after IPTG induction.  Both mannitol-1-phosphate 5-dehydrogenase and mannitol-2-dehydrogenase proteins were purified using Ni2+ matrix column.  Further characterization of these enzymes is in progress.