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Applied and Environmental Microbiology, December 2008, p. 7596-7599, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00677-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Purification and Gene Cloning of {alpha}-Methylserine Aldolase from Ralstonia sp. Strain AJ110405 and Application of the Enzyme in the Synthesis of {alpha}-Methyl-L-Serine{triangledown}

Hiroyuki Nozaki,* Shinji Kuroda, Kunihiko Watanabe, and Kenzo Yokozeki

Aminoscience Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan

Received 22 March 2008/ Accepted 13 October 2008

By screening microorganisms that are capable of assimilating {alpha}-methyl-DL-serine, we detected {alpha}-methylserine aldolase in Ralstonia sp. strain AJ110405, Variovorax paradoxus AJ110406, and Bosea sp. strain AJ110407. A homogeneous form of this enzyme was purified from Ralstonia sp. strain AJ110405, and the gene encoding the enzyme was cloned and expressed in Escherichia coli. The enzyme appeared to be a homodimer consisting of identical subunits, and its molecular mass was found to be 47 kDa. It contained 0.7 to 0.8 mol of pyridoxal 5'-phosphate per mol of subunit and could catalyze the interconversion of {alpha}-methyl-L-serine to L-alanine and formaldehyde in the absence of tetrahydrofolate. Formaldehyde was generated from {alpha}-methyl-L-serine but not from {alpha}-methyl-D-serine, L-serine, or D-serine. {alpha}-Methyl-L-serine synthesis activity was detected when L-alanine was used as the substrate. In contrast, no activity was detected when D-alanine was used as the substrate. In the {alpha}-methyl-L-serine synthesis reaction, the enzymatic activity was inhibited by an excess amount of formaldehyde, which was one of the substrates. We used cells of E. coli as a whole-cell catalyst to express the gene encoding {alpha}-methylserine aldolase and effectively obtained a high yield of optically pure {alpha}-methyl-L-serine using L-alanine and formaldehyde.

* Corresponding author. Mailing address: Fine Chemical & Pharmaceutical Industrialization Center, Ajinomoto Co., Inc., 1730 Hinaga-cho, Yokkaichi 515-0885, Japan. Phone: 81-593-46-0121. Fax: 81-593-46-0127. E-mail: hiroyuki_nozaki{at}ajinomoto.com

{triangledown} Published ahead of print on 24 October 2008.

Applied and Environmental Microbiology, December 2008, p. 7596-7599, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00677-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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