Surface modification of polyacrylonitrile with nitrile hydratase and amidase from Agrobacterium tumefaciens

A new strain of Agrobacterium tumefaciens (BST05) was found to grow on polyacrylonitrile (PAN; ¹³C labelled) converting the polymer to polyacrylic acid as shown by solid state NMR. When cultivated in a medium containing acetonitrile the bacterium produced nitrile hydratase and amidase activity. Activity recovery after lyophilisation and enzyme stability was significantly enhanced in the presence of 5% sorbitol leading to half life times of 12, 72 and 154 days at 25°C, 4°C and -20°C. The enzymes were able to convert 1.1% of the nitrile groups of PAN-powder to the corresponding acids. PAN fabrics were mainly converted to the amides as shown by an 80% increase of the O/C ratio in ESCA analysis. These data were confirmed by cationic dyeing and FTIR-ATR analysis.     

Surface modification of polyacrylonitrile with nitrile hydratase and amidase from Agrobacterium tumefaciens

A new strain of Agrobacterium tumefaciens (BST05) was found to grow on polyacrylonitrile (PAN; ¹³C labelled) converting the polymer to polyacrylic acid as shown by solid state NMR. When cultivated in a medium containing acetonitrile the bacterium produced nitrile hydratase and amidase activity. Activity recovery after lyophilisation and enzyme stability was significantly enhanced in the presence of 5% sorbitol leading to half life times of 12, 72 and 154 days at 25°C, 4°C and –20°C. The enzymes were able to convert 1.1% of the nitrile groups of PAN-powder to the corresponding acids. PAN fabrics were mainly converted to the amides as shown by an 80% increase of the O/C ratio in ESCA analysis. These data were confirmed by cationic dyeing and FTIR-ATR analysis.     

Nitrile hydratase and amidase from Rhodococcus rhodochrous hydrolyze acrylic fibers and granular polyacrylonitriles

Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein(-1)) and amidase activity (38.4 nkat mg of protein(-1)) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C. I. Basic Blue 9.     

Nitrile Hydratase and Amidase from Rhodococcus rhodochrous Hydrolyze Acrylic Fibers and Granular Polyacrylonitriles

Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein⁻¹) and amidase activity (38.4 nkat mg of protein⁻¹) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C.I. Basic Blue 9.     

Acrylamide biodegradation ability and plant growth-promoting properties of Variovorax boronicumulans CGMCC 4969

Species of the genus Variovorax are often isolated from nitrile or amide-containing organic compound-contaminated soil. However, there have been few biological characterizations of Variovorax and their contaminant-degrading enzymes. Previously, we reported a new soil isolate, Variovorax boronicumulans CGMCC 4969, and its nitrile hydratase that transforms the neonicotinoid insecticide thiacloprid into an amide metabolite. In this study, we showed that CGMCC 4969 is able to degrade acrylamide, a neurotoxicant and carcinogen in animals, during cell growth in a mineral salt medium as well as in its resting state. Resting cells rapidly hydrolyzed 600 mg/L acrylamide to acrylic acid with a half-life of 2.5 min. In in vitro tests, CGMCC 4969 showed plant growth-promoting properties; it produced a siderophore, ammonia, hydrogen cyanide, and the phytohormone salicylic acid. Interestingly, in soil inoculated with this strain, 200 mg/L acrylamide was completely degraded in 4 days. Gene cloning and overexpression in the Escherichia coli strain Rosetta (DE3) pLysS resulted in the production of an aliphatic amidase of 345 amino acids that hydrolyzed acrylamide into acrylic acid. The amidase contained a conserved catalytic triad, Glu59, Lys 134, and Cys166, and an “MRHGDISSS” amino acid sequence at the N-terminal region. Variovorax boronicumulans CGMCC 4969, which is able to use acrylamide for cell growth and rapidly degrade acrylamide in soil, shows promising plant growth-promoting properties. As such, it has the potential to be developed into an effective Bioaugmentation strategy to promote growth of field crops in acrylamide-contaminated soil.     

Nitrile hydrolysis activity of Rhodococcus erythropolis NCIMB 11540 whole cells

The nitrile hydrolyzing properties of the bacterium strain Rhodococcus erythropolis NCIMB 11540 have been investigated. Using whole cells of the microorganism, a wide variety of aromatic and aliphatic cyanide-containing substrates was successfully hydrolyzed to the corresponding amide or acid. In the case of dicyanides, selective monohydrolysis took place, which was further explored in the desymmetrization of malononitriles resulting in the corresponding cyano amides in enantiomeric excesses of up to 98%.     

Solvent Effects on Circular Dichroism of Colominic Acid

To produce acrylamide from acrylonitrile by use of a new enzyme, nitrile hydratase, a number of nitrile-utilizing microorganisms were screened for the enzyme activity by an intact cell system. An isobutyronitrile-utilizing bacterium, strain B23, showed the best productivity among 186 strains tested. The strain was identified taxonomically as Pseudomonas chlor or aphis. The culture and reaction conditions for the production were studied for the strain. Under the optimum conditions, 400 grams/liter of acrylamide was produced in 7.5 hr. The yield was nearly 100% with a trace amount of acrylic acid. The cell-free extract of the strain showed strong activity of nitrile hydratase toward acrylonitrile and extremely low activity of amidase toward acrylamide.     

A chemoenzymatic synthesis of aromatic carboxylic acid vinyl esters

The practical synthesis of vinyl p-coumarate (vinyl 4-hydroxycinnamate) and vinyl ferulate (vinyl 4-hydroxy-3-methoxycinnamate) was accomplished via a transesterification to the corresponding aromatic acid using vinyl acetate and a catalytic amount of PdCl₂, followed by the lipase-catalyzed regioselective alcoholysis in EtOH.     

Using a nitrilase for the surface modification of acrylic fibres

The surface of an acrylic fibre was modified with a commercial nitrilase (EC 3.5.5.1). The effect of fibre solvents and polyols on nitrilase catalysis efficiency and stability was investigated. The nitrilase action on the acrylic fabric was improved by the combined addition of 1 M sorbitol and 4% N, N-dimethylacetamide. The colour levels for samples treated with nitrilase increased 156% comparing to the control samples. When the additives were introduced in the treatment media, the colour levels increased 199%. The enzymatic conversion of nitrile groups into the corresponding carboxylic groups, on the fibre surface, was followed by the release of ammonia and polyacrylic acid. A surface erosion phenomenon took place and determined the "oscillatory" behaviour of the amount of dye uptake with time of treatment. These results showed that the outcome of the application of the nitrilase for the acrylic treatment is intimately dependent on reaction media parameters, such as time, enzyme activity and formulation.     

Utilization of aliphatic nitriles under haloalkaline conditions by Bacillus alkalinitrilicus sp. nov. isolated from soda solonchak soil

Enrichment with isobutyronitrile as the sole carbon, energy and nitrogen source at pH 10, using soda solonchak soils as an inoculum, resulted in the selection of a binary culture consisting of two different spore-forming phenotypes. One of them, strain ANL-iso4, was capable of growth with isobutyronitrile as a single substrate, while the other phenotype only utilized products of isobutyronitrile hydrolysis, such as isobutyroamide and isobutyrate. Strain ANL-iso4 is an obligate alkaliphile and a moderately salt-tolerant bacterium. Apart from isobutyronitrile, it grew on other (C3-C6) aliphatic nitriles at pH 10. Resting cells of ANL-iso4 actively hydrolyzed a number of aliphatic and arylaliphatic nitriles and their corresponding amides. The latter, together with the intermediate formation of amides during nitrile hydrolysis, indicated the presence of a nitrile hydratase/amidase system in the novel bacterium. Although present in an alkaliphilic bacterium, both nitrile- and amide-hydrolyzing activities had a pH optimum within the neutral range, probably due to their intracellular localization. On the basis of phenotypic and phylogenetic analyses, strain ANL-iso4 is proposed as a new species Bacillus alkalinitrilicus sp. nov.     

Isolation of a novelAgrobacteriun spp capable of degrading a range of nitrile compounds

Summary Using soil enrichment techniques we have isolated micro-organisms capable of degrading simple nitrile compounds. One species identified as anAgrobacterium spp. was examined in detail. This isolate was capable of utilising a range of aliphatic and aromatic nitriles as sole sources of carbon and nitrogen. Assays for enzymes involved in nitrile degradation and growth/production studies with this organism growing on acetonitrile indicated that breakdown occurred via a two step mechanism firstly to the amide and then via the production of the corresponding acid with the subsequent release of ammonia. Our studies indicate that the system of nitrile breakdown is inducible and levels of the amidase are 170 times that of the nitrile hydratase in crude extracts.     

Poly(ethylene oxide)-grafted thermoplastic membranes for use as cellular hybrid bio-artificial organs in the central nervous system

Poly(acrylonitrile-co-vinyl chloride) (PAN/VC) anisotropic membranes were chemically modified with poly(ethylene oxide) (PEO) (5000 and 20,000 g/mol) by one of two aqueous reactions: (a) acid hydrolysis of the nitrile group to a carboxylic acid with which amine-terminated PEO (PEO-NH(2)) reacted or (b) base reduction of the nitrile group to an amine with which PEO-succinimide (PEO-SC) reacted. Approximately 1.3% of the bulk material was modified with PEO-NH(2) whereas 1.8 to 3.5% was modified with PEO-SC as determined by proton nuclear magnetic resonance ((1)H NMR) and attenuated total reflectance Fourier transform infrared (ATR FTIR) spectra. Approximately 50 to 75% less bovine serum albumin (BSA) adsorbed to PEO-grafted single skin fibers than to unmodified PAN/VC. Transport properties of modified and unmodified fibers were compared by passive diffusion, convective nominal molecular weight cutoff, and hydraulic permeability. Neither hydraulic permeability nor nominal molecular weight cutoff of BSA changed appreciably after surface modification with PEO indicating that pore structure was not adversely affected by the chemistry involved in grafting poly(ethylene oxide). However, in the absence of any membrane conditioning, the apparent diffusion of alpha-chymotrypsinogen (24,000 g/mol) was enhanced in PEO-grafted PAN/VC fibers possibly as a result of reduced sorption of the permeating protein. In vivo biocompatibility in the brain tissue of rats was judged by histological assessment of the host's cellular response to fibers implanted for 30 days; biocompatibility of both PAN/VC and PAN/VC-g-PEO was satisfactory but improved slightly with PEO grafting. (c) 1994 John Wiley & Sons, Inc.     

The effect of additives and mechanical agitation in surface modification of acrylic fibres by cutinase and esterase

The surface of an acrylic fibre containing about 7% of vinyl acetate was modified using Fusarium solani pisi cutinase and a commercial esterase, Texazym PES. The effect of acrylic solvents and stabilising polyols on cutinase operational stability was studied. The half-life time of cutinase increased by 3.5-fold with the addition of 15% N,N-dimethylacetamide (DMA) and by 3-fold with 1M glycerol. The impact of additives and mechanical agitation in the protein adsorption and in the hydrolysis of vinyl acetate from acrylic fabric was investigated. The hydroxyl groups produced on the surface of the fibre were able to react specifically with Remazol Brilliant Blue R (cotton reactive dye) and to increase the colour of the acrylic-treated fabric. The best staining level was obtained with a high level of mechanical agitation and with the addition of 1% DMA. Under these conditions, the raise in the acrylic fabric colour depth was 30% for cutinase and 25% for Texazym. The crystallinity degree, determined by X-ray diffraction, was not significantly changed between control samples and samples treated with cutinase. The results showed that the outcome of the application of these enzymes depends closely on the reaction media conditions.     

Cloning of a nitrilase gene from the cyanobacterium Synechocystis sp. strain PCC6803 and heterologous expression and characterization of the encoded protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

Cloning the amidase gene from Rhodococcus rhodochrous M18 and its expression in Escherichia coli

The amidase gene from Rhodococcus rhodochrous M18 was cloned by PCR amplification with primers developed by use of peptide amino acid sequences obtained after treating amidase with trypsin. Nucleotide sequence analysis of this gene revealed high homology with aliphatic amidases from R. erythropolis R312 and Pseudomonas aeruginosa. Considering the substrate specificity and the results of DNA analysis, amidase from R. rhodochrous M8 was assigned to the group of aliphatic amidases preferentially hydrolyzing short-chain aliphatic amides. The amidase gene was expressed in cells of Escherichia coli from the self promoter and from the lac promoter. To clone a fragment of R. rhodochrous M8 chromosome (approximately 9 kb), containing the entire structural gene and its flanking regions, plasmid pRY1 that can be integrated into the chromosome via homology regions was used. No sequences of the nitrile hydratase gene, the second key gene of nitrile degradation in strain R. rhodochrous M8, were detected. Thus, genes encoding amidase and nitrile hydratase in strain R. rhodochrous M8 are not organized into a single operon despite their common regulation.     

Kinetic modeling of immobilized-lipase catalyzed transesterification of n-octanol with vinyl acetate in non-aqueous media

Organic esters are employed as solvents, fragrance, flavors, and precursors in a variety of industries. Particularly, aliphatic esters are greatly used in flavor industry, mainly as fixatives and modifiers, and aromatic esters in fragrance compositions. Esters are produced by a variety of methods among which esterification and transesterification with acid catalysts under reflux conditions are prominent. The use of biocatalysts provides an opportunity for carrying out reactions under milder conditions leading to better quality products suitable in fragrance and flavor industry. Transesterification of n-octanol with vinyl acetate was studied at 30 °C as a model reaction by employing different lipases as catalysts such as Psedomonas species lipase immobilized on diatomite, free Candida rugosa lipase. Novozym 435 (lipase B from Candida antarctica; immobilized on macro-porous polyacrylic resin beads) and Lipozyme IM 20 (Mucor miehei lipase immobilized on anionic resin). Novozym 435 was found to be the most active catalyst in heptane as a solvent. A conversion of 82% with 100% selectivity of n-octyl acetate was obtained at 30 °C in 90 min using equimolar quantities of the reactants with 0.833 g l⁻¹ of Novozym 435. Transesterification of other alcohols such as n-decanol, benzyl alcohol, cinnamyl alcohol, 2-ethyl-1-hexanol, 1-phenyl ethyl alcohol, and 2-phenyl ethyl alcohol was also studied with vinyl acetate. The analysis of the initial rate data and progress curve data showed that the reaction obeys the ternary complex bi–bi mechanism with inhibition by n-octanol. The experimental and theoretical values matched very well.

The order of transesterification reactivity of vinyl acetate with various alcohols in presence of Novozym 435 under otherwise identical conditions at 30 °C was found to be as follows:

n-octanol>n-decanol>benzylalcohol>cinnamylalcohol>2-ethyl-1-hexanol>2-phenylethylalcohol>1-phenylethylalcohol.

     

Cloning of a Nitrilase Gene from the Cyanobacterium Synechocystis sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

Cloning the amidase gene from Rhodococcus rhodochrous M8 and its expression in Escherichia coli

The amidase gene from Rhodococcus rhodochrous M8 was cloned by PCR amplification with primers developed by use of peptide amino acid sequences obtained after treating amidase with trypsin. Nucleotide sequence analysis of this gene revealed high homology with aliphatic amidases from R. erythropolis R312 and Pseudomonas aeruginosa. Considering the substrate specificity and the results of DNA analysis, amidase from R. rhodochrous M8 was assigned to the group of aliphatic amidases preferentially hydrolyzing short-chain aliphatic amides. The amidase gene was expressed in cells of Escherichia coli from the self promoter and from the lac promoter. To clone a fragment of R. rhodochrous M8 chromosome (approximately 9 kb), containing the entire structural gene and its flanking regions, plasmid pRY1 that can be integrated into the chromosome via homology regions was used. No sequences of the nitrile hydratase gene, the second key gene of nitrile degradation in strain R. rhodochrous M8, were detected. Thus, genes encoding amidase and nitrile hydratase in strain R. rhodochrous M8 are not organized into a single operon despite their common regulation.     

A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles

A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase, amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid intermediates. It has a strong bias for C₃–C₆ dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in 4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity. The strain has been deposited as ATCC 55746. Received: 26 January 1999 / Received revision: 10 June 1999 / Accepted: 27 June 1999     

Conversion of aliphatic nitriles by the arylacetonitrilase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> EBC191

The conversion of aliphatic nitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191 (NitA) was analyzed. The nitrilase hydrolysed a wide range of aliphatic mono- and dinitriles and showed a preference for unsaturated aliphatic substrates containing 5–6 carbon atoms. In addition, increased reaction rates were also found for aliphatic nitriles carrying electron withdrawing substituents (e.g. chloro- or hydroxy-groups) close to the nitrile group. Aliphatic dinitriles were attacked only at one of the nitrile groups and with most of the tested dinitriles the monocarboxylates were detected as major products. In contrast, fumarodinitrile was converted to the monocarboxylate and the monocarboxamide in a ratio of about 65:35. Significantly different relative amounts of the two products were observed with two nitrilase variants with altered reaction specifities. NitA converted some aliphatic substrates with higher rates than 2-phenylpropionitrile, which is one of the standard substrates for arylacetonitrilases. This indicated that the traditional classification of nitrilases as “arylacetonitrilases”, “aromatic” or “aliphatic” nitrilases might require some corrections. This was also suggested by the construction of some variants of NitA which were modified in an amino acid residue which was previously suggested to be essential for the conversion of aliphatic substrates by a homologous nitrilase.