Purification and Gene Cloning of α-Methylserine Aldolase from Ralstonia sp. Strain AJ110405 and Application of the Enzyme in the Synthesis of α-Methyl- l -Serine

Abstract

ABSTRACT By screening microorganisms that are capable of assimilating α-methyl- dl -serine, we detected α-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 α-methyl- l -serine to l -alanine and formaldehyde in the absence of tetrahydrofolate. Formaldehyde was generated from α-methyl- l -serine but not from α-methyl- d -serine, l -serine, or d -serine. α-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 α-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 α-methylserine aldolase and effectively obtained a high yield of optically pure α-methyl- l -serine using l -alanine and formaldehyde.