Fig 1.
Chemical formulae for compounds 1–3.
2,3-dihydro-1,4-benzodioxin-2-carboxylic acid (1), 2,3-dihydro-1,4-benzodioxin-2-carboxamide (2) and 2,3-dihydro-1,4-benzodioxin-2-carbonitrile (3)
Fig 2.
Effect of isopropanol on activity and enantioselectivity of A. faecalis nitrilase.
(A) The effect of isopropanol concentration (0–18% v/v) on e.e and % conversion. The reaction was done in phosphate buffer, pH 7.0 at 30°C in presence of varying amounts of isopropanol. (B) Conversion rates and e.e. in presence of isopropanol 18% (v/v) at various time points (0 to 27 hr). (C) HPLC trace of reaction in presence of isopropanol 18% (v/v) at 50% conversion. Column: Chiracel OJ, 10 μm, 250×4.6 mm; detection UV at 254 nm; elution hexane/isopropanol/trifluoroacetic acid in ratio of 90:10:0.1; temperature 25°C; flow rate 0.5 ml/min.
Fig 3.
Effect of cyclohexyl amine (CHA) on activity and e.e. of A. faecalis nitrilase.
(A) Chiral HPLC trace (Chiracel OJ, 10 μm, 250×4.6 mm; detection UV at 254 nm; elution hexane/isopropanol/trifluoroacetic acid in ratio of 90:10:0.1; temperature 25°C; flow rate 0.5 ml/min) showing inversion in configuration and appearance of unidentified peak (marked?) in reaction of rac-nitrile 3 in phosphate buffer, pH 7.0 at 30°C in presence of 3% CHA. (B) Chiral HPLC trace of reaction done under similar conditions as in A, but in absence of biocatalyst (control reaction).
Fig 4.
Effect of reaction conditions on A. faecalis catalysed conversion of rac-amide 2.
Effect of pH (A), temperature (B) and cell mass (wet cell basis) (C) on A. faecalis catalysed conversion of rac-amide 2. (D) Conversion of rac-amide 2 in 50 mM bicarbonate buffer, pH 11.4 at 30°C at various time points (0 to 12 hr).
Fig 5.
A. faecalis catalysed asymmetric resolution of rac-2.
Chiral HPLC trace (Chiracel OJ, 10 μm, 250×4.6 mm; detection UV at 254 nm; elution hexane/isopropanol/trifluoroacetic acid in ratio of 90:10:0.1; temperature 25°C; flow rate 0.5 ml/min) of reaction of rac-2 in sodium carbonate buffer, pH 11.4 at 30°C at ~50% conversion (A) and 53% conversion (B).
Table 1.
Summary of purification steps of the IaaH from Alcaligenes faecalis subsp. parafaecalis
Fig 6.
SDS-PAGE of IaaH at various purification steps and cloning and expression of IaaH in E. coli.
(A) Protein fractions were run under reducing conditions on 12% SDS-PAGE. Lane 1, molecular weight markers; lane 2, cell-free extract; lane 3, ammonium sulphate fractionation (40–65%); lane 4, phenyl sepharose chromatography; lane 5, size exclusion chromatography. (B) Electrophoresis was done on 1.2% agarose gel. Lane 1, 1 Kb ladder; lane 2, Nhe1-Xho1 digested pET23(a); lane 3, Nhe1-Xho1 digested 1.42 Kb IaaH in pET23(a); lane 4, Nhe1-Xho1 digested 1.42 Kb PCR amplified IaaH; lane 5, 100 bp ladder.
Fig 7.
Molecular mass of recombinant IaaH.
(A) SDS PAGE of IaaH was run under reducing conditions on 12% SDS-PAGE. Lane 1, MW Marker and Lane 2, Ni-NTA agarose beads purified IaaH. (B) Elution profile of purified enzyme on TSKgel G2000SWxL column.
Table 2.
A. faecalis IaaH homologs in bacteria and plants.
Fig 8.
Homology and evolutionary analysis of IaaH of Alcaligenes faecalis subsp. parafaecalis MTCC 12564.
(A) Amino acid sequence alignment of IaaH of this study (GenBank accession number: KX352469, dotted box) with other indole-3-acetamide hydrolases of bacterial and plant origin around the amidase signature motif. The alignment was generated with CLUSTALW and viewed with Jalview. Identical residues present in all aligned sequences are marked in black and identical residues present in more than 50% of the aligned sequences are marked in grey. (B) Phylogenetic analysis of IaaH of this study (in bold letters) with other indole acetamide hydrolases of bacterial and plants. Phylogenetic tree was constructed using neighbour-joining method with CLUSTALW in MEGA 6.0. Bootstrap values were calculated by taking 1000 replicates are shown next to the branches. 0.2 substitution per amino acid position is represented by bar. Nitrilase from Alcaligenes faecalis was taken as an out-group. The NCBI accession numbers of enzymes are represented in parentheses.
Fig 9.
Substrate spectrum of A. faecalis IaaH.
All activities are expressed in per cent relative to activity against indole-3-acetamide, which was taken as 100%. Values are average of experiments done in triplicate.
Fig 10.
Amides that showed no activity in A. faecalis catalysed reaction (Entry 11, Fig 9).