New thermostable d-methionine amidase from Brevibacillus borstelensis BCS-1 and its application for d-phenylalanine production

Abstract

A new thermostable d-methionine amidase was found in a cell-free extract of Brevibacillus borstelensis BCS-1. After five steps of purification, the specific activity increased approximately 207-fold and the purity was more than 98%. The molecular weight of the enzyme was estimated to be 199 kDa by gel permeation chromatography and 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the thermostable d-methionine amidase was a homo-hexamer consisting of a single subunit. The purified enzyme was stable up to 65 • C within a broad pH range from 6.5 to 10.0, and its maximum activity was measured at pH 9.5 and 70 • C. The enzyme activity increased about five-fold with the addition of Co 2+ , yet was strongly inhibited by Hg 2+ , 2-mercaptoethanol, dithiothreitol, and ethylenediaminetetracetic acid.The thermostable d-methionine amidase exhibited a high amidase activity and d-stereospecificity toward d-amino acid amides and esters, yet did not hydrolyze d-peptides. The catalytic efficiencies (k cat /K m , mM −1 s −1 ) of the enzyme for d-methioninamide and d-alaninamide were 3086 and 21.5, respectively, and the enantiomeric excess (ee) and enantiomeric ratio of d-phenylalanine produced from dl-phenylalaninamide were 97.1 and 196%, respectively.

Amidases: versatile enzymes in nature

“…Amidase from thermophiles are generally active over wide pH range (5-9.5) (Egorova et al 2004;Makhongela et al 2007). Most of the mesophiles produce amidases in temperature range 25-30°C (Banerjee et al 2002), while optimum production temperatures for amidase by thermophilic bacteria lies in range of 50-65°C (Baek et al 2003;Egorova et al 2004;Makhongela et al 2007).…”

“…Amidases are either constitutively produced in the cells grown in rich medium (Nawaz et al 1996;Baek et al 2003;Kamphuis et al 1992;Kotlova et al 1999;Krieg et al 2002) or induced by the presence of amides in the growth medium (Bhalla et al 1997;Ciskanik et al 1995;Gilligan et al 1993;Joeres and Kula 1994). The inoculum size for amidase production as well as time coarse of incubation differs (0.5-8% v/v) in various organisms (Cramp et al 1997;Linardi et al 1996;Bhalla et al 1997).…”

Hydrolysis of Amides

“…Two highly thermostable D-amino acid amidases have been identified in the thermophile Brevibacillus borstelensis BCS-1. The first, a D-methionine amidase, efficiently hydrolyzed a broad range of D-amino acid amides with highest activity toward the amides of D-methionine (100%), D-norvaline (78%), D-norleucine (77%), D-lysine (60%), and D-leucine (59%) [291]. The enzyme also displayed activity toward different D-amino acid esters [e.g., D-alanine methyl ester (25%) and D-alanine benzyl ester (22%)] and arylamides [e.g., D-alanine-b-naphthylamide (33%) and D-leucine-pnitroanilide (40%)].…”

“…was produced from the racemic amide (E ¼ 196). The gene encoding this D-methionine amidase has not been reported yet, although the NH 2 -terminal amino acid sequence was already published in 2003 [291].…”

Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities

“…They have been reported to biosynthesize p-aminobenzoic acid, acrylic acid, nicotinic acid, pyrazinoic acid, and 3-indole acetic acid (Fournand et al 1998;Banerjee et al 2002). Amidases that exhibit enantioselectivity can be applied to produce optically pure compounds, including L-selective amidase from Ochrobactrum anthropi, D-amidase from Variovorax paradoxus, and D-alanine amidase from Brevibacillus borstelensis BCS-1 (Sonke et al 2005;Krieg et al 2005;Baek et al 2003). In addition, amidases play an important role in the biodegradation of toxic chemicals, such as acrylamide and acetonitrile.…”