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Oikawa, T. et al.

Oikawa, Tauch, Schaffer, Fujioka,

Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase.

We constructed the high-expression system of the alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli BL 21 (DE3) to characterize the enzymological and structural properties of the gene product, Alr. The Alr was expressed in the soluble fractions of the cell extract of the E. coli clone and showed alanine racemase activity. The purified Alr was a dimer with a molecular mass of 78kDa. The Alr required pyridoxal 5'-phosphate (PLP) as a coenzyme and contained 2mol of PLP per mol of the enzyme. The holoenzyme showed maximum absorption at 420nm, while the reduced form of the enzyme showed it at 310nm. The Alr was specific for alanine, and the optimum pH was observed at about nine. The Alr was relatively thermostable, and its half-life time at 60 degrees C was estimated to be 26min. The K(m) and V(max) values were determined as follows: l-alanine to d-alanine, K(m) (l-alanine) 5.01mM and V(max) 306U/mg; d-alanine to l-alanine, K(m) (d-alanine) 5.24mM and V(max) 345U/mg. The K(eq) value was calculated to be 1.07 and showed good agreement with the theoretical value for the racemization reaction. The high substrate specificity of the Alr from C. glutamicum ATCC 13032 is expected to be a biocatalyst for d-alanine production from the l-counter part.[1]

References

Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase. Oikawa, T., Tauch, A., Schaffer, S., Fujioka, T. J. Biotechnol. (2006) [Pubmed]

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