A novel D-stereoselective amino acid amidase from Brevibacterium iodinum: Gene cloning, expression and characterization

作者：Komeda Hidenobu; Asano Yasuhisa^*

来源：Enzyme and Microbial Technology, 2008, 43(3): 276-283.

DOI：10.1016/j.enzmictec.2008.03.008

• D-amino acid
• amidase
• D-phenylalanine
• Brevibacterium iodinum
• class A beta-lactamase

The gene encoding D-stereoselective amino acid amide amidohydrolase (D-amino acid amidase, named DaaA(Bi)) was cloned from a chromosomal DNA library of Brevibacterium iodinum TPU 5850 and sequenced. The gene, daaA(Bi), encoded a protein composed of 266 amino acids with a M(r) of 30035. The deduced amino-acid sequence of the daaA(Bi) gene did not exhibit any similarity with any other previously reported D-amino acid amidases. but did show similarity with hypothetical class A beta-lactamases. DaaA(Bi) protein was produced in Escherichia coli, purified to electrophoretic homogeneity, and characterized. The purified enzyme was about 290,000 based on gel filtration chromatography and about 30,000 based on SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is active as a decamer with identical subunits. DaaA(Bi) showed maximum activity at pH 7.2 and 35 degrees C. It exhibited strict D-Stereoselective hydrolyzing activity towards a broad range Of D-amino acid amides including D-methioninamide, D-lysinamide, D-glutaminamide, and D-phenylalaninamide, while L-amino acid amides. peptides composed of L- or D-amino acids, and beta-lactam compounds could not serve as substrates for the enzyme. Almost complete hydrolysis Of D-phenylalaninamide with highly strict D-stereoselectivity was achieved in 3 h from 180 mM Of DL-phenylalaninamide using the purified DaaA(Bi) enzyme.

• 出版日期2008-9-5