Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction

Abstract

BackgroundA nitrilase-mediated pathway has significant advantages in the production of optically pure (R)-(−)-mandelic acid. However, unwanted byproduct, low enantioselectivity, and specific activity reduce its value in practical applications. An ideal nitrilase that can efficiently hydrolyze mandelonitrile to optically pure (R)-(−)-mandelic acid without the unwanted byproduct is needed.ResultsA novel nitrilase (BCJ2315) was discovered from Burkholderia cenocepacia J2315 through phylogeny-based enzymatic substrate specificity prediction (PESSP). This nitrilase is a mandelonitrile hydrolase that could efficiently hydrolyze mandelonitrile to (R)-(−)-mandelic acid, with a high enantiomeric excess of 98.4%. No byproduct was observed in this hydrolysis process. BCJ2315 showed the highest identity of 71% compared with other nitrilases in the amino acid sequence. BCJ2315 possessed the highest activity toward mandelonitrile and took mandelonitrile as the optimal substrate based on the analysis of substrate specificity. The kinetic parameters Vmax, Km, Kcat, and Kcat/Km toward mandelonitrile were 45.4 μmol/min/mg, 0.14 mM, 15.4 s-1, and 1.1×105 M-1s-1, respectively. The recombinant Escherichia coli M15/BCJ2315 had a strong substrate tolerance and could completely hydrolyze mandelonitrile (100 mM) with fewer amounts of wet cells (10 mg/ml) within 1 h.ConclusionsPESSP is an efficient method for discovering an ideal mandelonitrile hydrolase. BCJ2315 has high affinity and catalytic efficiency toward mandelonitrile. This nitrilase has great advantages in the production of optically pure (R)-(−)-mandelic acid because of its high activity and enantioselectivity, strong substrate tolerance, and having no unwanted byproduct. Thus, BCJ2315 has great potential in the practical production of optically pure (R)-(−)-mandelic acid in the industry.

Exploring the Potential of Fungal Arylacetonitrilases in Mandelic Acid Synthesis

“…A number of nitrilases suitable for the production of (R)-mandelic acid were also found by screening a metagenomic library [5,6]. The process was recently improved using recombinant strains [1,2,[7][8][9][10] or enzyme mutants [2]. A similar process was also developed for the production of (R)-o-chloromandelic acid, a precursor of the anti-coagulant drug Clopidogrel Ò [11].…”

“…Recently, the arylacetonitrilases from Alcaligenes sp., Alcaligenes faecalis, and Burkholderia cepacia were found to be promising for this purpose, being able to hydrolyze up to 0.5-1 M of mandelonitrile into (R)-mandelic acid with high yields [1,2,8,9].…”

Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of ( R )- o -Chloromandelic Acid

“…BCJ2315 and the mutants were expressed as His-tagged proteins in E. coli M15 and purified using Ni-nitrilotriacetic acid (NTA) Superflow columns (Qiagen, Hilden, Germany) as described previously (9).…”

“…In our previous study, we identified and characterized a novel arylacetonitrilase, BCJ2315, from Burkholderia cenocepacia strain J2315 (9). BCJ2315 demonstrated high activity toward o-chloromandelonitrile and tolerated up to 50 mM o-chloromandelonitrile, though the o-chloromandelonitrile was highly toxic to the enzyme, demonstrating great potential for production of (R)-ochloromandelic acid under high o-chloromandelonitrile concentrations (4).…”

Isolation and Characterization of a Nitrile-Hydrolysing Bacterium Isoptericola variabilis RGT01

“…Nitriles are toxic in nature, but can be hydrolysed to corresponding less-toxic and valuable carboxylic acids either by nitrilase (EC 3.5.5.1), or by nitrile-hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4). Nitrilases have been reported in a variety of microbes including Alcaligenes [1], Bacillus [2], Burkholderia [3], Labrenzia [4], Pseudomonas [5], Pyrococcus [6] and Rhodococcus [7]. In spite of many nitrilases reported in literature, the quest for their new sources has never ended.…”