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D-(+)-lactate dehydrogenase from Lactobacillus murinus.

A M Strasser de Saad, A A Pesce de Ruiz Holgado, G Oliver

    Biotechnology and Applied Biochemistry
    |October 1, 1986
    PubMed
    Summary

    Researchers purified D-(+)-Lactate dehydrogenase from Lactobacillus murinus, finding optimal activity at 25°C and pH 6.0. The enzyme showed competitive inhibition by pyruvate analogs and noncompetitive inhibition by L-lactate.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Microbiology

    Background:

    • Lactate dehydrogenases (LDHs) are crucial enzymes in microbial metabolism, catalyzing the interconversion of pyruvate and lactate.
    • Understanding the properties of LDHs from specific bacterial species, like Lactobacillus murinus, is important for metabolic engineering and biotechnological applications.

    Purpose of the Study:

    • To purify and characterize the D-(+)-Lactate dehydrogenase enzyme from Lactobacillus murinus.
    • To determine the kinetic properties, optimal conditions, and inhibitor profiles of the purified enzyme.

    Main Methods:

    • Purification of D-(+)-Lactate dehydrogenase using biochemical techniques, achieving a 670-fold increase in purity.
    • Determination of molecular weight (Mr) via gel filtration.
    • Enzyme activity assays conducted at varying temperatures and pH levels.
    • Kinetic analysis to determine apparent Km values for pyruvate and NADH.
    • Inhibition studies using pyruvate analogs and related compounds.

    Main Results:

    • The purified D-(+)-Lactate dehydrogenase had a molecular weight of 140,000 Da.
    • Optimal enzymatic activity was observed at 25°C and pH 6.0.
    • The enzyme exhibited high thermal instability, becoming completely inactive within 5 minutes at 60-65°C.
    • Apparent Km values for pyruvate and NADH were determined to be 4.7 x 10⁻⁴ M and 1 x 10⁻⁵ M, respectively.
    • Pyruvate analogs (oxalate, oxamate, 2-oxobutyrate, malonate) acted as competitive inhibitors, while L-lactate and L-malate were noncompetitive inhibitors.

    Conclusions:

    • The D-(+)-Lactate dehydrogenase from Lactobacillus murinus is a distinct enzyme with specific kinetic and inhibition characteristics.
    • The enzyme's optimal activity and substrate affinities provide insights into its physiological role in Lactobacillus murinus.
    • The observed thermal instability and inhibition patterns suggest potential limitations and specific applications for this enzyme in biotechnological processes.

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