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 (2006)
JOURNAL OF BIOTECHNOLOGY 125(4): 503-512.

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Abstract
We constructed the high-expression system of the alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli BL21 (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 78 kDa. The Alr required pyridoxal 5'-phosphate (PLP) as a coenzyme and contained 2 mol of PLP per mol of the enzyme. The holoenzyme showed maximum absorption at 420 nm, while the reduced form of the enzyme showed it at 3 10 nm. 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 26 min. The K-m and V-max values were determined as follows: L-alanine to D-alanine, K-m (L-alanine) 5.01 mM and V-max 306 U/Mg; D-alanine to L-alanine, K-m (D-alanine) 5.24 mM and V-max 345 U/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. (c) 2006 Elsevier B.V. All rights reserved.
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Oikawa T, Tauch A, Schaffer S, Fujioka T. Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase. JOURNAL OF BIOTECHNOLOGY. 2006;125(4):503-512.
Oikawa, T., Tauch, A., Schaffer, S., & Fujioka, T. (2006). Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase. JOURNAL OF BIOTECHNOLOGY, 125(4), 503-512.
Oikawa, T., Tauch, A., Schaffer, S., and Fujioka, T. (2006). Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase. JOURNAL OF BIOTECHNOLOGY 125, 503-512.
Oikawa, T., et al., 2006. Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase. JOURNAL OF BIOTECHNOLOGY, 125(4), p 503-512.
T. Oikawa, et al., “Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase”, JOURNAL OF BIOTECHNOLOGY, vol. 125, 2006, pp. 503-512.
Oikawa, T., Tauch, A., Schaffer, S., Fujioka, T.: Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase. JOURNAL OF BIOTECHNOLOGY. 125, 503-512 (2006).
Oikawa, Tadao, Tauch, Andreas, Schaffer, Steffen, and Fujioka, Toru. “Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase”. JOURNAL OF BIOTECHNOLOGY 125.4 (2006): 503-512.
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