Expanding the repertoire of nitrilases with broad substrate specificity and high substrate tolerance for biocatalytic applications

Enzymatic conversion of nitriles to carboxylic acids by nitrilases has gained significance in the green synthesis of several pharmaceutical precursors and fine chemicals. Although nitrilases from several sources have been characterized, there exists a scope for identifying broad spectrum nitrilases exhibiting higher substrate tolerance and better thermostability to develop industrially relevant biocatalytic processes. Through genome mining, we have identified nine novel nitrilase sequences from bacteria and evaluated their activity on a broad spectrum of 23 industrially relevant nitrile substrates. Nitrilases from Zobellia galactanivorans, Achromobacter insolitus and Cupriavidus necator were highly active on varying classes of nitriles and applied as whole cell biocatalysts in lab scale processes. Z. galactanivorans nitrilase could convert 4-cyanopyridine to achieve yields of 1.79 M isonicotinic acid within 3 h via fed-batch substrate addition. The nitrilase from A. insolitus could hydrolyze 630 mM iminodiacetonitrile at a fast rate, effecting 86 % conversion to iminodiacetic acid within 1 h. The arylaliphatic nitrilase from C. necator catalysed enantioselective hydrolysis of 740 mM mandelonitrile to (R)-mandelic acid in 4 h. Significantly high product yields suggest that these enzymes would be promising additions to the suite of nitrilases for upscale biocatalytic application.     

腈转化酶在精细化学品生产中的应用

腈化合物是一类重要的用于合成多种精细化学品的化合物,它们容易制备,并且可以合成多种化合物。传统化学水解方法将腈化合物转化为相应的羧酸或酰胺通常需要高温、强酸、强碱等相对苛刻的条件,腈转化酶(腈水解酶、腈水合酶和酰胺酶)由于其生物催化过程具有高效、高选择性、条件温和等特点,在精细化学品的合成中越来越受到人们的关注。许多腈转化酶已经被开发出来并用于精细化学品的生产。以下介绍了腈转化酶在医药及中间体、农药及中间体、食品与饲料添加剂等精细化学品生产中的应用。随着研究的不断深入,将会有更多的腈转化酶被开发出来并用于精细化学品的生产。     

Pseudomonas syringae pv. syringae B728a hydrolyses indole-3-acetonitrile to the plant hormone indole-3-acetic acid

Nitrilase enzymes catalyse the hydrolysis of nitrile compounds to the corresponding carboxylic acid and ammonia, and have been identified in plants, bacteria and fungi. There is mounting evidence to support a role for nitrilases in plant–microbe interactions, but the activity of these enzymes in plant pathogenic bacteria remains unexplored. The genomes of the plant pathogenic bacteria Pseudomonas syringae pv. syringae B728a and Pseudomonas syringae pv. tomato DC3000 contain nitrilase genes with high similarity to characterized bacterial arylacetonitrilases. In this study, we show that the nitrilase of P. syringae pv. syringae B728a is an arylacetonitrilase, which is capable of hydrolysing indole-3-acetonitrile to the plant hormone indole-3-acetic acid, and allows P. syringae pv. syringae B728a to use indole-3-acetonitrile as a nitrogen source. This enzyme may represent an additional mechanism for indole-3-acetic acid biosynthesis by P. syringae pv. syringae B728a, or may be used to degrade and assimilate aldoximes and nitriles produced during plant secondary metabolism. Nitrilase activity was not detected in P. syringae pv. tomato DC3000, despite the presence of a homologous nitrilase gene. This raises the interesting question of why nitrilase activity has been retained in P. syringae pv. syringae B728a and not in P. syringae pv. tomato DC3000.     

Enantioselective chemoenzymatic synthesis of (R)-γ-valerolactone from levulinic acid

A number of chemicals with high industrial value can be synthesized from levulinic acid, a feasible building block readily available from cellulosic biomass. Among them, γ-valerolactone is a versatile chemical precursor for the synthesis of value-added products including bio-active molecules, bio-fuels, and carbon-based chemicals. In this study, a novel two-step chemoenzymatic conversion of levulinic acid to (R)-γ-valerolactone via 4-hydroxyvaleric acid was investigated. For that purpose, an engineered 3-hydroxybutyrate dehydrogenase (e3HBDH) with improved catalytic activity toward levulinic acid was employed in the first-step reaction, and dehydration with 1 % (v/v) sulfuric acid was applied for the lactonization of 4-hydroxyvaleric acid to γ-valerolactone in the second step. As a result, enantiomerically pure (R)-γ-valerolactone (>99 % ee) was successfully produced from the free acid form of levulinic acid with the maximum yield of approximately 100 %.     

Protein engineering of nitrilase for chemoenzymatic production of glycolic acid

A key step in a chemoenzymatic process for the production of high-purity glycolic acid (GLA) is the enzymatic conversion of glycolonitrile (GLN) to ammonium glycolate using a nitrilase derived from Acidovorax facilis 72W. Protein engineering and over-expression of this nitrilase, combined with optimized fermentation of an E. coli transformant were used to increase the enzyme-specific activity up to 15-fold and the biocatalyst-specific activity up to 125-fold. These improvements enabled achievement of the desired volumetric productivity and biocatalyst productivity for the conversion of GLN to ammonium glycolate.     

Microbial nitrilases: versatile, spiral forming, industrial enzymes

The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a αββα–αββα sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.     

Efficient lactic acid production from high salt containing dairy by-products by Lactobacillus salivarius ssp. salicinius with pre-treatment by proteolytic microorganisms

Lactic acid bacteria have an inefficient proteolytic system. Therefore, cultivation media which may have high protein content are usually supplemented with yeast extract or protein lysates (peptones). These additives might be conveniently replaced by in situ treatment of the cultivation medium with proteolytic enzymes or proteolytic microbes. Lactobacillus salivarius ssp. salicinius, a lactic acid bacterium species that can grow at high salt concentration, was used to ferment lactic acid in cheese whey (with 3 gl(-1) whey protein content) and lactose mother liquor (90 gl(-1) lactose, 9 gl(-1) proteins, 30 gl(-1) minerals). The contribution of protease enzymes or proteolytic microbes to acid production by lactobacilli was examined. Efficient conversion of lactose to lactic acid was obtained in the presence of additional proteolytic activity. Fastest acid production was obtained with the addition of protease enzymes. However, almost equally efficient acid production was obtained by treating the medium with Bacillus megaterium. The results show that fast and complete conversion of lactose to lactic acid can be obtained in dairy by-products without expensive additives.     

Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue.

The amino acid sequences of the NH2 terminus and internal peptide fragments of a Rhodococcus rhodochrous J1 nitrilase were determined to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 750-base DNA fragment thus amplified was used as the probe to clone a 5.4-kilobase PstI fragment coding for the whole nitrilase. The nitrilase gene modified in the sequence upstream from the presumed ATG start codon was expressed to approximately 50% of the total soluble protein in Escherichia coli. The predicted amino acid sequence of the nitrilase gene showed similarity to that of the bromoxynil nitrilase from Klebsiella ozaenae. The 5,5'-dithiobis(2-nitrobenzoic acid) modification of the nitrilase from R. rhodochrous J1 resulted in inactivation with the loss of one sulfhydryl group/enzyme subunit. Of 4 cysteine residues in the Rhodococcus nitrilase, only Cys-165 is conserved in the Klebsiella nitrilase. Mutant enzymes containing Ala or Ser instead of Cys-165 did not exhibit nitrilase activity. These findings suggest that Cys-165 plays an essential role in the function of the active site.     

Efficient production of methionine from 2-amino-4-methylthiobutanenitrile by recombinant Escherichia coli harboring nitrilase

Methionine as an essential amino acid has been attracting more attention for its important applications in food and feed additives. In this study, for efficient production of methionine from 2-amino-4-methylthiobutanenitrile, a codon-optimized nitrilase gene was newly synthesized and expressed, and the catalytic conditions for methionine production were studied. The optimal temperature and pH for methionine synthesis were 40 °C and 7.5, respectively. The recombinant nitrilase was thermo-stable with half-life of 5.52 h at 40 °C. The substrate loading was optimized in given amount of catalyst and fixed substrate/catalyst ratio mode to achieve higher productivity. Methionine was produced in 100 % conversion within 120 min with a substrate loading of 300 mM. The production of methionine with the immobilized resting cells in packed-bed reactor was investigated. The immobilized nitrilase exhibited good operation stability and retained over 80 % of the initial activity after operating for 100 h. After separation, the purity and the total yield of methionine reached 99.1 and 97 %, respectively. This recombinant nitrilase could be a potential candidate for application in production of methionine.     

Nitrilase immobilization and transposition from a micro-scale batch to a continuous process increase the nicotinic acid productivity

In recent years, many biocatalytic processes have been developed for the production of chemicals and pharmaceuticals. In this context, enzyme immobilization methods have attracted attention for their advantages, such as continuous production and increased stability. Here, enzyme immobilization methods and a collection of nitrilases from biodiversity for the conversion of 3-cyanopyridine to nicotinic acid were screened. Substrate conversion over 10 conversion cycles was monitored to optimize the process. The best immobilization conditions were found with cross-linking using glutaraldehyde to modify the PMMA beads. This method showed good activity over 10 cycles in a batch reactor at 30 and 40°C. Finally, production with a new thermostable nitrilase was examined in a continuous packed bed reactor, showing very high stability of the biocatalytic process at a flow rate of 0.12 ml min^–1 and a temperature of 50°C. The complete conversion of 3-cyanopyridine was obtained over 30 days of operation. Future steps will concern reactor scale-up to increase the production rate with reasonable pressure drops.     

Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata

(R)-o-Chloromandelic acid is the key precursor for the synthesis of Clopidogrel?, a best-selling cardiovascular drug. Although nitrilases are often used as an efficient tool in the production of α-hydroxy acids, there is no practical nitrilase specifically developed for (R)-o-chloromandelic acid. In this work, a new nitrilase from Labrenzia aggregata (LaN) was discovered for the first time by genomic data mining, which hydrolyzed o-chloromandelonitrile with high enantioselectivity, yielding (R)-o-chloromandelic acid in 96.5% ee. The LaN was overexpressed in Escherichia coli BL21 (DE3), purified, and its catalytic properties were studied. When o-chloromandelonitrile was used as the substrate, the V(max) and K(m) of LaN were 2.53 μmol min?1 mg?1 protein and 0.39 mM, respectively, indicating its high catalytic efficiency. In addition, a study of substrate spectrum showed that LaN prefers to hydrolyze arylacetonitriles. To relieve the substrate inhibition and to improve the productivity of LaN, a biphasic system of toluene-water (1:9, v/v) was adopted, in which o-chloromandelonitrile of 300 mM (apparent concentration, based on total volume) could be transformed by LaN in 8 h, giving an isolated yield of 94.5%. The development of LaN makes it possible to produce (R)-o-chloromandelic acid by deracemizing o-chloromandelonitrile with good ee value and high substrate concentration.     

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.     

Development of a robust nitrilase by fragment swapping and semi-rational design for efficient biosynthesis of pregabalin precursor

Protein engineering is a powerful tool for improving the properties of enzymes. However, large changes in enzyme properties are still challenging for traditional evolution strategies because they usually require multiple amino acid substitutions. In this study, a feasible evolution approach by a combination of fragment swapping and semi-rational design was developed for the engineering of nitrilase. A chimera BaNIT harboring 12 amino acid substitutions was obtained using nitrilase from Arabis alpine (AaNIT) and Brassica rapa (BrNIT) as parent enzymes, which exhibited higher enantioselectivity and activity toward isobutylsuccinonitrile for the biosynthesis of pregabalin precursor. The semi-rational design was executed on BaNIT to further generate variant BaNIT/L223Q/H263D/Q279E with the concurrent improvement of activity, enantioselectivity, and solubility. The robust nitrilase displayed a 5.4-fold increase in whole-cell activity and the enantiomeric ratio (E) increased from 180 to higher than 300. Molecular dynamics simulation and molecular docking demonstrated that the substitution of residues on the A and C surface contributed to the conformation alteration of nitrilase, leading to the simultaneous enhancement of enzyme properties. The results obtained not only successfully engineered the nitrilase with great industrial potential for the production of pregabalin precursor, but also provided a new perspective for the development of novel industrially important enzymes.     

Induction of auxin biosynthetic enzymes by jasmonic acid and in clubroot diseased Chinese cabbage plants

Nitrilase (NIT) and myrosinase are important enzymes for auxin biosynthesis in Brassicaceae, which is increased during clubroot disease. Therefore, NIT and myrosinase levels during club development and possible regulation mechanisms were investigated. In addition, the occurrence of different nitrilase isoforms in Chinese cabbage has been shown. Nitrilase activity was enhanced in infected roots during later stages of club development (35–42 days after inoculation). However, no differences in nitrilase mRNA levels between infected and healthy roots were found during symptom development. Myrosinase expression was increased in clubbed roots at slightly earlier time points (28 days after inoculation) and also at later time points during infection. The activities of tryptophan oxidizing enzyme (TrpOxE), which catalyzes the first step in tryptophan-dependent auxin biosynthesis in Brassicaceae, and nitrilase were enhanced after treatment with jasmonic acid (JA) and methyl jasmonate. Similarly, the amount of myrosinase mRNA was increased by JA. During clubroot disease the endogenous concentration of JA increased in infected roots 3–5 weeks after inoculation. From our results it can be concluded that: (1) de novo indole-3-acetic acid (IAA) biosynthesis plays a role for symptom development of clubroot disease in Brassicaceae during later developmental stages; and (2) JA which increased during club development, may be involved in the up-regulation of three enzymes important for IAA synthesis.     

Cloning and optimization of a nitrilase for the synthesis of (3S)-3-cyano-5-methyl hexanoic acid

In this paper we describe the cloning and optimization of a nitrilase for a regio- and stereo-specific synthesis of (3S)-3-cyano-5-methyl hexanoic acid (2) from isobutylsuccinonitrile (IBSN, 1). Ten representative plant and bacterial nitrilases have been cloned and their substrate specificity was studied using a fluorescent assay. The desired nitrilase AtNit1 from Arabidopsis thaliana was identified with high enantioselectivity (E > 150). This enzyme was then purified and characterized to be an oligomer of 12 subunits by size exclusion chromatography. AtNit1 was subsequently optimized to increase expression and engineered to improve activity. Preliminary screening of a small percentage (1%) of the mutant library shows that the mutant C236S has a nearly 3-fold increase in reactivity in the hydrolysis of IBSN.     

The cyclodextrins and their applications in biotechnology

Cyclodextrins and their derivatives enhance the solubility of complexed substrates in aqueous media, but do not damage the microbial cells or the enzymes. Therefore the enzymatic conversion of lipophilic substrates can be intensified (accelerated, or performed at higher substrate concentrations). Examples are the hydrolysis of triglycerides and lanatoside glycosides or the conversion of hydrocortisone to prednisolon and of cholesterol to androstenedione. In the presence of an appropriate cyclodextrin-derivative (e.g. 2,6-dimethyl-β-cyclodextrin). The lipid-like inhibitor substances are complexed, therefore the propagation of Bordatella pertussis and the production of pertussis toxin increases up to 100-fold. Cyclodextrins or their fatty acid complexes can substitute mammalian serum in tissue cultures. A highly soluble γ-cyclodextrin-nystatin complex can protect tissue cultures from fungal infections. The tolerance level to toxic compounds during biological detoxication of organic chemical industries sewage can be elevated by admixing small amounts of β-cyclodextrin to the system, because the complexed toxic substances do not kill the detoxicating microbes.     

Cross-linked enzyme aggregates (CLEAs): A novel and versatile method for enzyme immobilization (a review)

The key to obtaining optimum performance of an enzyme is often a question of devising an effective method for its immobilization. This review describes a novel, versatile and effective methodology for enzyme immobilization, namely, as cross-linked enzyme aggregates (CLEAs). The method is exquisitely simple – involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules – and amenable to rapid optimization. It is applicable to a wide variety of enzymes, including cofactor-dependent oxidoreductases and lyases, and affords stable, recyclable catalysts with high retention of activity, sometimes higher than that of the free enzyme it was derived from. The enzyme does not need to be of high purity. Indeed, the methodology is essentially a combination of purification and immobilization in one step. The technique is also applicable to the preparation of combi-CLEAs, containing two or more enzymes, for use in one-pot, multi-step syntheses. For example, an oxynitrilase/nitrilase combi-CLEA was used for the one-pot synthesis of (S)-mandelic acid from benzaldehyde, in high yield and enantiomeric purity.     

Cross-linked enzyme aggregates (CLEAs): A novel and versatile method for enzyme immobilization (a review)

The key to obtaining optimum performance of an enzyme is often a question of devising an effective method for its immobilization. This review describes a novel, versatile and effective methodology for enzyme immobilization, namely, as cross-linked enzyme aggregates (CLEAs). The method is exquisitely simple - involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules - and amenable to rapid optimization. It is applicable to a wide variety of enzymes, including cofactor-dependent oxidoreductases and lyases, and affords stable, recyclable catalysts with high retention of activity, sometimes higher than that of the free enzyme it was derived from. The enzyme does not need to be of high purity. Indeed, the methodology is essentially a combination of purification and immobilization in one step. The technique is also applicable to the preparation of combi-CLEAs, containing two or more enzymes, for use in one-pot, multi-step syntheses. For example, an oxynitrilase/nitrilase combi-CLEA was used for the one-pot synthesis of (S)-mandelic acid from benzaldehyde, in high yield and enantiomeric purity.     

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

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%.