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Title: CLONING, EXPRESSION, PURIFICATION, AND ANALYSIS OF MANNITOL DEHYDROGENASE OF MTLD AND MTLK GENES FROM LACTOBACILLUS PLANTARUM AND LACTOBACILLUS BREVIS

Author
item Liu, Siqing
item Saha, Badal
item Cotta, Michael

Submitted to: Biotechnology for Fuels and Chemicals Symposium Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 5/12/2004
Publication Date: 5/12/2004
Citation: Liu, S., Saha, B.C., Cotta, M.A. 2004. Cloning, expression, purification, and analysis of mannitol dehydrogenase of mtlD and mtlK genes from Lactobacillus plantarum and Lactobacillus brevis [abstract]. Biotechnology for Fuels and Chemicals. Paper No. 2-24.

Interpretive Summary:

Technical Abstract: 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 E. coli BL21 cells after IPTG induction. Both mannitol-1-phosphate 5-dehydrogenase and mannitol-2-dehydrogenase proteins were purified using Ni**2+ matrix column. Further characterization of these enzymes is in progress.