Characterization and partial purification of an enantioselective arylacetonitrilase from Pseudomonas fluorescens DSM 7155

doi:10.1016/S1381-1177(98)00075-7

Journal of Molecular Catalysis B: Enzymatic

Volume 5, Issues 5–6, 15 October 1998, Pages 467-474

https://doi.org/10.1016/S1381-1177(98)00075-7 Get rights and content

Abstract

Pseudomonas fluorescens DSM 7155 after growth on phenylacetonitrile as sole nitrogen source contained an inducible nitrilase which consists of two different functional subunits (40 and 38 kDa). The nitrilase catalysed the exclusive hydrolysis of arylacetonitrile substrates into the equivalent carboxylic acids plus ammonia as major products. The corresponding amides were formed at low levels (<5%) during nitrile hydrolysis but were not substrates for the purified enzyme. The native enzyme, which had a pH optimum of 9 and a temperature optimum of 55°C, was activated (140–160%) by the thiol protectant 2-mercaptoethanol (50–100 mM). The purified nitrilase catalysed the hydrolysis of the two enantiomers of racemic 2-(methoxy)-mandelonitrile to the corresponding acid at significantly different rates: at 50% overall conversion the predominant product was the (R)-acid (enantiomeric excess=92%) whereas at 85% overall conversion the ee% of the (R)-acid had decreased to 27%.

Introduction

Nitrilases are the subject of much attention because of their potential as biocatalysts for the production of higher-value acids [1]. Resting cells from Rhodococcus rhodochrous J1 have been used successfully for the production of both p-aminobenzoic acid and nicotinic acid from the corresponding nitriles 2, 3. Nitrilases have also been applied successfully to catalyse the regiospecific hydrolysis of dinitrile compounds into corresponding cyano-carboxylic acids 4, 5. With the exception of the nitrilase from Rh. rhodochrous PA-34 [6]and an Arthrobacter sp. [7]which consist of a single polypeptide acting as a monomer, all other nitrilases characterised to data are homopolymers often containing substantial numbers (6–16) of the relevant component subunit [8].

In this paper, we report the purification and properties of a novel hetero-oligomeric enantioselective arylacetonitrilase from Pseudomonas fluorescens DSM 7155.

Section snippets

Materials

Phenyl-Sepharose FF, Mono Q, Superose 12 and the reference proteins used to determine molecular weight were purchased from Pharmacia (Sweden). All other chemicals used were from commercial sources except phenylacetamide which was prepared by T. Stock (Chemistry, Exeter) and 2-(methoxy)-mandelonitrile plus 2-(methoxy)-mandelic acid which were provided by J. Parratt (Chiroscience, Cambridge).

Microorganisms and culture conditions

P. fluorescens DSM 7155 which was previously isolated from soil (Synonym: P. fluorescens EBC191; [9]) was

Results

Nitrilase from P. fluorescens DSM 7155 was only present after growth on defined media containing phenylacetonitrile as sole nitrogen source. The use of either ammonia as a replacement nitrogen source in the minimal medium or a complex medium such as nutrient broth caused a substantially higher cell yield (approximately 4-fold), but no nitrile hydrolysing activity could be detected in such cells. Interestingly, the presence of both ammonia plus phenylacetonitrile in minimal media also resulted

Discussion

A nitrilase from P. fluorescens DSM 7155 was purified 259-fold with a yield of 10% from cell-free extract of the bacterium after growth on phenylacetonitrile as the sole nitrogen source. The presence of the nitrile compound was essential for the induction of nitrilase. The co-presence of ammonium ions repressed the enzyme induction indicating some form of N-catabolite repression as observed with various other enzymes 19, 20. This suggests that the natural role of the nitrilase in this bacterium

References (31)

M. Kobayashi et al.
Eur. J. Biochem.
(1989)
M. Kobayashi et al.
FEMS Microbiol. Lett.
(1994)
M. Bradford
Anal. Biochem.
(1976)
M. Kobayashi et al.
Trends Biotech.
(1992)
S. Levy-Schil et al.
Gene
(1995)
D.B. Harper
Int. J. Biochem.
(1985)
K. Yamamoto et al.
J. Ferment. Bioeng.
(1992)
R.H. Hook et al.
J. Biol. Chem.
(1964)
J.E. Rothman
Cell
(1989)
M. Kobayashi et al.
Biochim. Biophys. Acta
(1991)

D.M. Stalker et al.

Eur. J. Biol. Chem.

(1988)

Faber, K. Biotransformations in Organic Chemistry, 2nd edn., Springer-Verlag, Berlin,...

C.D. Mathew et al.

Appl. Environ. Microbiol.

(1988)

C. Bengis-Gerber et al.

Appl. Microbiol. Biotech.

(1989)

M. Kobayashi et al.

Appl. Microbiol. Biotech.

(1988)

Cited by (57)

Biotransformation of 4-hydroxyphenylacetonitrile to 4-hydroxyphenylacetic acid using whole cell arylacetonitrilase of Alcaligenes faecalis MTCC 12629
2018, Process Biochemistry
Citation Excerpt :
A number of nitrilases have been reported for their substrate specificity as aromatic, aliphatic, heterocyclic, but only a few of the nitrilases are reported to degrade arylacetonitriles. The nitrilase of A. faecalis MTCC 12629 in the present study preferably hydrolyzed arylacetonitriles similar to that of P. fluorescens DSM 7155 [34], Bacillus subtilis ZJB-063 [27], P. fluorescens EBC191 [35] and Alcaligenes sp. MTCC 10675 [16].
The whole cell arylacetonitrilase of Alcaligenes faecalis MTCC 12629 was employed to develop a bioprocess for the synthesis of 4-hydroxyphenylacetic acid (4-HPAA) from 4-hydroxyphenylacetonitrile (4-HPAN). Isobutyronitrile emerged as the most appropriate inducer with a 1.2-fold enhanced nitrilase production in inducer feeding approach. A. faecalis MTCC 12629 nitrilase preferably hydrolyzed 4-HPAN, 4-aminoyphenylacetonitrile, mandelonitrile and phenylacetonitrile to their corresponding acids. Thermostability profile revealed the stability of nitrilase at 40, 45 and 50°C with the half-life of 45 h, 36 h and 13 h, respectively. Michaelis constant (K_m) and reaction velocity (V_max) of nitrilase were calculated as 20 mM and 2 U/mg dcw, respectively. This nitrilase exhibited complete conversion of 4-HPAN to 4-HPAA at 45°C in 50 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.0) in 30 min. A fed batch process designed at 500 ml with five feedings of substrate resulted in 150 mM product formation using 18 U/ml whole cells nitrilase (16 mg/ml dcw) at 45°C. This is the first report on the production of 4-HPAA through nitrilase mediated pathway with the volumetric and catalytic productivity of 23 g/l and 4.13 g/g dcw/h, respectively.
Enzymatic cascade synthesis of (S)-2-hydroxycarboxylic amides and acids: Cascade reactions employing a hydroxynitrile lyase, nitrile-converting enzymes and an amidase
2015, Journal of Molecular Catalysis B: Enzymatic
Citation Excerpt :
Early reactions (see Fig. 2) were performed with CLEA biocatalysts [28] of MeHnl [19] and PfNLase [29], taking the optimum reaction conditions for hydrocyanation – diisopropyl ether containing 10% aqueous buffer pH <5 – as a starting point. The PfNLase CLEA maintained its activity in the biphasic medium – in contrast with free nitrilases [21] – but the preferred low pH was more problematic. As a suitable compromise between maintaining the enantiomeric purity of the HCN adduct (2) and NLase activity, the reactions were performed at pH 5.5 [30,31].
Enantiomerically pure 2-hydroxycarboxylic acids are valuable synthetic building blocks. This review summarises the development of bienzymatic cascades to convert aldehydes, via hydrocyanation and subsequent hydration or hydrolysis, into the corresponding (S)-2-hydroxycarboxylic amides and acids. The biocatalysts comprised an (S)-specific hydroxynitrile lyase combined with a nonselective nitrile hydratase or nitrilase. The key to success was preventing racemisation of the intermediate (S)-2-hydroxynitrile while adequately protecting the nitrile-converting enzyme, either in a cross-linked enzyme aggregate (CLEA) or in resting cells.
Two biocatalyst systems for the synthesis of (S)-mandelic acid were developed: a combined crosslinked enzyme aggregate (combi-CLEA) of an (S)-hydroxynitrile lyase and a nitrilase, as well as a whole-cell Escherichia coli biocatalyst expressing both enzymes. The nitrilase formed large amounts of mandelic amide, which was remedied by including an amidase in the combi-CLEA as well as by using nitrilase variants obtained by directed mutagenesis in the whole-cell biocatalyst. Eventually, excellent results – >95% conversion of benzaldehyde into (S)-mandelic acid with near-quantitative enantiomeric purity – were obtained with both biocatalyst systems.
Directed mutagenesis of the nitrilase provided an amide-selective whole-cell biocatalyst, which produced (S)-mandelic amide in near-stoichiometric yields. (S)-2-hydroxylalkanoic carboxamides were synthesised in the presence of CLEAs of hydroxynitrile lyase and nitrile hydratase.
The combi-CLEA approach: Enzymatic cascade synthesis of enantiomerically pure (S)-mandelic acid
2013, Tetrahedron Asymmetry
Citation Excerpt :
Intermediates and products were characterised by comparison with an authentic sample. Full spectral data are available in the literature: mandelonitrile (2),23 mandelic acid (3) 1H and 13C NMR,24 MS,25 mandelic amide (4),26 2-hydroxy-4-phenyl-trans-3-butenenitrile.27 The progress of all of the reactions was monitored by HPLC, using either a Waters 590 pump and a Waters 486 Tunable Absorbance Detector at 215 nm or a Waters Alliance 2695 Separation Module and a Waters 2487 Dual Wavelength Absorbance Detector at 215 nm.
Enantiomerically pure (S)-mandelic acid was synthesised from benzaldehyde by sequential hydrocyanation and hydrolysis in a bienzymatic cascade at starting concentrations up to 0.25 M. A cross-linked enzyme aggregate (CLEA) composed of the (S)-selective oxynitrilase from Manihot esculenta and the non-selective nitrilase from Pseudomonas fluorescens EBC 191 was employed as the biocatalyst. The nitrilase produces approx. equal amounts of (S)-mandelic acid and (S)-mandelic amide from (S)-mandelonitrile under standard conditions, but we surprisingly found that high (up to 0.5 M) concentrations of HCN induced a marked drift towards amide production. By including the amidase from Rhodococcus erythopolis in the CLEA we obtained (S)-mandelic acid as the sole product in 90% yield and >99% enantiomeric purity.
Characterization and functional cloning of an aromatic nitrilase from Pseudomonas putida CGMCC3830 with high conversion efficiency toward cyanopyridine
2013, Journal of Molecular Catalysis B: Enzymatic
Nitrilases have long been considered as an attractive alternative to chemical catalyst in carboxylic acids biosynthesis due to their green characteristics and the catalytic potential in nitrile hydrolysis. A novel nitrilase from Pseudomonas putida CGMCC3830 was purified to homogeneity. pI value was estimated to be 5.2 through two-dimensional electrophoresis. The amino acid sequence of NH₂ terminus was determined. Nitrilase gene was cloned through CODEHOP PCR, Degenerate PCR and TAIL-PCR. The open reading frame consisted of 1113 bp encoding a protein of 370 amino acids. The predicted amino acid sequence showed the highest identity (61.6%) to nitrilase from Rhodococcus rhodochrous J1. The enzyme was highly specific toward aromatic nitriles such as 3-cyanopyridine, 4-cyanopyridine, and 2-chloro-4-cyanopyridine. It was classified as aromatic nitrilase. The nitrilase activity could reach up to 71.8 U/mg with 3-cyanopyridine as substrate, which was a prominent level among identified cyanopyridine converting enzymes. The kinetic parameters K_m and V_max for 3-cyanopyridine were 27.9 mM and 84.0 U/mg, respectively. These data would warrant it as a novel and potential candidate for creating effective nitrilases in catalytic applications of carboxylic acids synthesis through further protein engineering.
7.7 Hydrolysis and Reverse Hydrolysis: Selective Nitrile Hydrolysis using Nitrilase and its Related Enzymes
2012, Comprehensive Chirality
The ability to catalyze highly chemo, regio, and enantioselective transformations under mild reaction conditions has generated a huge interest in nitrile-hydrolyzing enzymes, nitrile hydratase, and nitrilase. Nitrilases have been found to be present in many fungi, yeast, plants, and to a much greater extent in microorganisms. After the initial discovery of the involvement of nitrilases in regulating plant growth, its applications have taken a huge leap forward into industrial biotechnology. The scope of synthetic application of nitrilases has further been significantly broadened by the discovery of enzymes capable of accepting a wide range of molecules as substrates using molecular approaches such as metagenome screening or genome databases, the ability to perform reaction engineering to overcome limitations, and approaches that engineer the enzyme and improve its characteristics as compared with what has been achieved by natural evolution.
Frontiers in catalytic nitrile hydration: Nitrile and cyanohydrin hydration catalyzed by homogeneous organometallic complexes
2011, Coordination Chemistry Reviews
Citation Excerpt :
The successful application of microbial NHases in the industrial production of acrylamide demonstrated the potential of biocatalysis in the synthesis of a variety of alkylacrylic monomers via the hydration of alkyl cyanohydrins (Scheme 3). In fact, the use of NHases to promote the hydration of selected cyanohydrins, like lactonitrile and p-chloromandelonitrile, has received much attention in the chemical literature [23–30]. A range of NHases (including those belonging to the genera Rhodococcus [27,28], Corynebacterium [23], Pseudomonas [26], Arthrobacter [23], Alcaligenes [30], Brevibacterium [23], and Nocardia [23]) have been used, often in combination with amidase, to prepare α-hydroxyamides and carboxylic acids.
Acrylic monomers are a significant part of the global economy, contributing to the manufacture of over a billion tons of diverse polymeric consumer products every year. The development of more efficient, greener methods to manufacture this highly demanded class of compounds is an important goal in the realization of a sustainable chemical industry. The pursuit of environmentally benign production processes has inspired a rich body of industrial and academic research on methods for the catalytic hydration of nitriles, and this review surveys both established and newer methods of generating acrylic amides, acids, and esters from nitrile and cyanohydrin substrates. The review also examines synthetic and mechanistic studies of homogeneously catalyzed nitrile hydration reactions with an emphasis on explicating the parameters that impact catalyst performance. The final section is a discussion of catalyst properties, gleaned from the mechanistic studies, that will be useful in designing the next generation of nitrile hydration catalysts.

View all citing articles on Scopus

View full text

Characterization and partial purification of an enantioselective arylacetonitrilase from Pseudomonas fluorescens DSM 7155

Abstract

Introduction

Section snippets

Materials

Microorganisms and culture conditions

Results

Discussion

Eur. J. Biochem.

FEMS Microbiol. Lett.

Anal. Biochem.

Trends Biotech.

Gene

Int. J. Biochem.

J. Ferment. Bioeng.

J. Biol. Chem.

Cell

Biochim. Biophys. Acta

Eur. J. Biol. Chem.

Appl. Environ. Microbiol.

Appl. Microbiol. Biotech.

Appl. Microbiol. Biotech.