D-Malic enzyme of Pseudomonas fluorescens.

Area of Science:

• Microbiology
• Enzymology

Background:

• Microorganisms capable of metabolizing D(+)-malic acid are crucial for biotechnological applications.
• Understanding the enzymes involved in D(+)-malic acid catabolism provides insights into microbial metabolic pathways.

Purpose of the Study:

• To isolate and identify microorganisms that can use D(+)-malic acid as a sole carbon source.
• To characterize the D-malic enzyme from a selected bacterial strain, focusing on its purification and kinetic properties.

Main Methods:

• Enrichment culture technique for microbial isolation.
• Bacterial identification using standard microbiological methods.
• Enzyme purification using chromatography and electrophoresis.
• Enzyme activity assays and kinetic analysis (Km, pH optimum).
• Determination of molecular weight using gel filtration and gradient gel electrophoresis.

Main Results:

• 14 gram-negative bacteria and 2 yeast strains were isolated using D(+)-malic acid.
• Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Klebsiella aerogenes were identified.
• D-malic enzyme (NAD-dependent) was purified from P. fluorescens, showing a specific activity of 4-5 U/mg.
• The enzyme requires divalent cations, has Km values of 0.3 mM for malate and 0.08 mM for NAD, and an optimal pH of 8.1-8.8.
• The molecular weight was determined to be approximately 175,000 Da.

Conclusions:

• Microbial isolation successfully identified several D(+)-malic acid utilizing bacteria and yeasts.
• The purified D-malic enzyme from P. fluorescens exhibits specific kinetic and stability characteristics.
• This study contributes to the understanding of D-malic acid metabolism and provides a characterized enzyme for potential applications.