l-Selective Amidase with Extremely Broad Substrate Specificity fromOchrobactrum anthropiNCIMB 40321

Abstract

An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60°C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn 2؉ (to 80%), Mn 2؉ (to 400%), and Mg 2؉ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of ␣-hydrogen-and (bulky) ␣,␣-disubstituted ␣-amino acid amides, ␣-hydroxy acid amides, and ␣-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, ␤-amino and ␤-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/ formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.Enantiomerically pure ␣-amino acids, both natural and unnatural, form an important class of chiral building blocks for the pharmaceutical and agrochemical industries. Examples of important ␣-hydrogen-␣-amino acids are D-phenylglycine (DPhg) and D-p-hydroxyphenylglycine, which are produced as side chains for the manufacture of semisynthetic ␤-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin (14, 53), and D-valine, an intermediate for the pyrethroid insecticide fluvalinate (28). Another frequently used ␣-hydrogen-␣-amino acid is L-tert-leucine, which is used as a building block for a variety of antiviral (e.g., anti-human immunodeficiency virus), antiarthritis, and anticancer drugs under development and as a chiral auxiliary in chemical asymmetric synthesis (5, 10). Besides ␣-hydrogen-␣-amino acids, ␣,␣-disubstituted ␣-amino acids also constitute a group of compounds of increasing importance, as exemplified by the use of L-␣-methyl-3,4-dihydroxyphenylalanine (L-␣-methyldopa) as an antihypertensive drug (34, 42), ␣-methylvaline as an intermediate for the herbicide Arsenal and related herbicides (47, 49), and L-␣-methylphenylglycine as a building block for the new fungicide fenamidone (27).Enantiomerically pure ␣-amino acids can be produced on a commercial scale by a number of different processes, both chemical and enzymatic. Biocatalytic processes that have been commercialized include the acylase process operated by Degussa (8) a...

Hydrolysis of Amides

“…Finally, substituting the hydroxyl group with an amino group, giving to 3,3,3-trifluoro-2-amino-2-methylpropanamide as substrate, led to a 28-fold higher hydrolysis rate than for the corresponding a-hydroxy compound 108 [335]. A similar positive effect of an a-amino over an a-hydroxy group was observed for LamA from O. anthropi [309].…”

“…The solubility problem was overcome by turning to O. anthropiL-amidase as biocatalyst, which enabled execution of this resolution reaction at pH 6.5 and 50 C. By using this amidase both the (R)-amide and the (S)-acid were obtained in over 90% isolated yield and greater than 98% e.e. The (R)-amide 107 was subsequently hydrolyzed under mild conditions into the corresponding (R)-acid with the nonselective amidase from Rhodococcus erythropolis NCIMB 11540 (see Scheme 15.14 for The most important L-amidase in O. anthropi NCIMB 40321 was purified from the cell-free extract by ammonium sulfate fractionation, anion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography [309]. This Lamidase, which was named LamA, converts a broad range of a-hydrogen-and (bulky) a,a-disubstituted a-amino acid amides.…”

“…The activity of the EDTA-treated enzyme could be restored by the addition of Zn 2 þ (to 80%), Mn 2 þ (to 400%), and Mg 2 þ (to 560%). The gene encoding this Lamidase was cloned via reverse genetics and efficiently expressed in E. coli under control of the trc or aro promoter [309,310]. It encodes a polypeptide of 314 amino acids with clear homology to the acetamidase/formamidase family of proteins, including the stereoselective amidases from Enterobacter cloacae N-7901 [311], Thermus sp.…”

“…Sequencing revealed that the gene encoding this amidase (sad) coded for a polypeptide of 328 amino acid residues with a calculated molecular weight of 36 344 [313] and homology to the acetamidase/formamidase family of proteins, including the O. anthropi NCIMB 40321 LamA (28% full-length sequence identity) [309]. Efficient overexpression of this amidase in E. coli was possible by placing the gene downstream of the strong lac promoter.…”

Use of Enzymes in the Synthesis of Amino Acids

“…Recently, the purification, cloning, and heterologous expression in E. coli of the most important amidase (LamA) from O. anthropi NCIMB 40 321 has been reported [70]. LamA displays activity toward a broad range of substrates consisting of a-hydrogen-and (bulky) a,a-disubstituted a-amino acid amides, a-hydroxy acid amides, and a-N-hydroxyamino acid amides, and is thus responsible on its own for the extremely broad substrate specificity of the O. anthropi whole cells.…”