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

Abstract

Background: A 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.

Results: A 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 1.1×10(5) M(-1)s(-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.

Conclusions: PESSP 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.

Figure 1

Unrooted neighbor-joining tree based on the amino acid sequences of the organisms from Table 1(accession numbers are in parentheses). A consensus tree was constructed using a bootstrap test with 1000 replications. Bootstrap values greater than 50% are shown at the branch points. The organisms harboring defined nitrilase activity are shadowed.

Figure 2

SDS-PAGE analysis of the nitrilase BCJ2315. Lane 1: protein marker; Lane 2; whole cell lysates of E. coli M15/pQE30; Lane 3: whole cell lysates of E. coli M15/BCJ2315; Lane 4: insoluble fractions of the whole cell lysates of E. coli M15/BCJ2315; Lane 5: soluble fractions of the whole cell lysates of E. coli M15/BCJ2315; Lane 6: purified BCJ2315.

Figure 3

Effect of temperature and pH on the activity of purified BCJ2315 toward mandelonitrile. (a) Optimum temperature: enzyme activity was measured at various temperatures (18°C to 70°C) in 100 mM of sodium phosphate buffer (pH 7.0). (b) Optimum pH: enzyme activity was measured in different buffers (pH 4.0 to 10.6) at 30°C. The relative activity was expressed as a percentage of the maximum activity under the experimental condition used.

Figure 4

Time course of the mandelonitrile hydrolysis mediated by the recombinant E. coli M15/BCJ2315. The reaction mixture (10 ml) containing wet cells (100 mg) and mandelonitrile (100 mM) suspended in phosphate buffer (100 mM, pH 8.0) was incubated in a rotary shaker (30°C, 200 rpm). Samples were taken every 10 min and quenched by the addition of 10% (v/v) 2 M HCl.