Biotransformation of nitriles by rhodococci

Rhodococci have been shown to be capable of a very wide range of biotransformations. Of these, the conversion of nitriles into amides or carboxylic acids has been studied in great detail because of the biotechnological potential of such activities. Initial investigations used relatively simple aliphatic nitriles. These studies were quickly followed by the examination of the regio- and stereoselective properties of the enzymes involved, which has revealed the potential synthetic utility of rhodococcal nitrile biotransforming enzymes. Physiological studies on rhodococci have shown the importance of growth medium design and bioreactor operation for the maximal conversion of nitriles. This in turn has resulted in some truly remarkable biotransformation activities being obtained, which have been successfully exploited for commercial organic syntheses (e.g. acrylamide production from acrylonitrile).The two main types of enzyme involved in nitrile biotransformations by rhodococci are nitrile hydratases (amide synthesis) and nitrilases (carboxylic acid synthesis with no amide intermediate released). It is becoming clear that many rhodococci contain both activities and multiple forms of each enzyme, often induced in a complex way by nitrogen containing molecules. The genes for many nitrile-hydrolysing enzymes have been identified and sequenced. The crystal structure of one nitrile hydratase is now available and has revealed many interesting aspects of the enzyme structure in relationship to its catalytic activity and substrate selectivity.     

Nitrile Hydratase and Its Application to Industrial Production of Acrylamide

Nitrile hydratase (NHase) was discovered in our laboratory. This enzyme was purified and characterized from various microorganisms. NHases are roughly classified into two groups according to the metal involved: Fe-type and Co-type. NHases are expected to have great potential as catalysts in organic chemical processing because they can convert nitriles to the corresponding higher-value amides under mild conditions. We have used microbial enzymes for the production of useful compounds; NHase has been used for the industrial production (production capacity: 30,000 tons/year) of acrylamide from acrylonitrile. This is the first successful example of a biotransformation process for the manufacture of a commodity chemical. This review summarizes the history of NHase studied not only from a basic standpoint but also from an applied point of view.     

Enzymatic hydrolysis of nitriles by an engineered nitrile hydratase (papain Gln19Glu) in aqueous-organic media

A mutant of the cysteine protease papain, displaying nitrile hydratase and amidase activities, was expressed in Pichia pastoris and used for the hydrolysis of peptide nitriles in aqueous-organic media. The rate of hydrolysis of these nitriles is lowered by increasing acetone concentration. This is caused by an increase of the Michaelis constant, and a variation of Vmax proportional to the amount of water in the mixture. The hydrolysis of the amide is less affected by the increase in co-solvent, which results in lower accumulation of this intermediate product. With the peptide nitrile tested, high nitrile concentrations could be used to promote the production of the amide and prevent its hydrolysis to the acid by diminishing the relative rate of amide hydrolysis. A number of non-peptidyl nitriles were also tested as potential substrates but activity was detected for only one compound with structural resemblance to peptide nitriles.     

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。     

对羟基苯乙腈水解酶产生菌的筛选及产酶条件研究

以对羟基苯乙腈为唯一氮源,从土壤中筛选到5株腈水解酶产生菌。在初筛的过程中,用薄板层析（TLC）对细胞转化液定性检测,高效液相色谱法（HPLC）定量分析。考察了培养时间、不同碳源对细胞酶产量的影响及在反应过程中金属离子对细胞酶产量的影响,实验结果发现,培养时间在70h左右时细胞酶产量最高,以葡萄糖为碳源质量,质量浓度在12．5～15g／L附近酶产量最高。Cu^2＋对细胞酶活力具有非常强烈的抑制作用,Co^2＋、Hg^2＋、Ni^2＋等对细胞酶活力具有明显的抑制,而Ba^2＋、Mg^2＋具有较明显的促进作用,但在高浓度下促进作用减弱。     

Comparative characterisation of two Rhodococcus species as potential biocatalysts for ammonium acrylate production

Two Rhodococcal isolates, one possessing a nitrile hydratase and an amidase enzyme, the other an aliphatic nitrilase enzyme have been isolated. The kinetic constants for the enzymes in each isolate have been determined. This data coupled with stability tests indicate that Rhodococcus ruber NCIMB 40757, the nitrilase containing organism, should be an excellent biocatalyst for the commercial production of ammonium acrylate. This is confirmed by a fed-batch bioconversion to produce 5.7 M ammonium acrylate.     

Enzymatic hydrolysis of cyanohydrins with recombinant nitrile hydratase and amidase from hodococcus erythropolis

Nitrile hydratase and amidase from Rhodococcus erythropolis CIMB11540 were both cloned and expressed in Escherichia coli.Crude cell free extracts were used for the hydrolysis of different aromatic cyanohydrins. Nitrile hydratase expression was increased up to 5-fold by redesign of the expression cassette. The recombinant enzymes were successfully used for the conversion of several cyanohydrins to the corresponding α-hydroxy amides and acids while retaining enantiopurity.     

Transformation of the herbicide 2,6-dichlorobenzonitrile to the persistent metabolite 2,6-dichlorobenzamide (BAM) by soil bacteria known to harbour nitrile hydratase or nitrilase

In soil the herbicide 2,6-dichlorobenzonitrile (dichlobenil) is degraded to the persistent metabolite 2,6-dichlorobenzamide (BAM) which has been detected in 19% of samples taken from Danish groundwater. We tested if common soil bacteria harbouring nitrile-degrading enzymes, nitrile hydratases or nitrilases, were able to degrade dichlobenil in vitro. We showed that several strains degraded dichlobenil stoichiometrically to BAM in 1.5–6.0 days; formation of the amide intermediate thus showed nitrile hydratase rather than nitrilase activity, which would result in formation of 2,6-dichlorobenzoic acid. The non-halogenated␣analogue benzonitrile was also degraded, but here the benzamide intermediate accumulated only transiently showing nitrile hydratase followed by amidase activity. We conclude that a potential for dichlobenil degradation to BAM is found commonly in soil bacteria, whereas further degradation of the BAM intermediate could not be demonstrated.     

Enantioselective biotransformations of racemic 2-aryl-3-methylbutyronitriles using Rhodococcus sp. AJ270

Rhodococcus sp. AJ270 is a useful biocatalyst for the synthesis of some enantiopure S-(+)-2-aryl-3-methylbutyric acids and R-(+)-2-aryl-3-methylbutyramides from the hydrolysis of 2-aryl-3-methylbutyronitriles under mild conditions. The nitrile-hydrolyzing enzymes involved in this novel microorganism are very sensitive to the steric effect of the para-substituent on the aromatic ring. While the nitrile hydratase displays a low S-enantioselectivity against nitriles, the amidase has a strict S-enantioselectivity against 2-aryl-3-methylbutyramides.     

Studies on utilization of acetonitrile by Rhodococcus erythropolis A10

A petrochemical wastewater isolate, capable of utilizing high concentrations of acetonitrile and acetamide as the sole source of carbon and nitrogen was identified as Rhodococcus erythropolis A10. Cell-free extracts of acetonitrile-grown cells exhibited activities corresponding to nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4), which mediate the two-step breakdown of acetonitrile into acetic acid and ammonia. Studies indicated that both these enzymes in R. erythropolis A10 are intracellular, inducible and capable of hydrolysing a wide range of nitriles, including simple (acetonitrile, propionitrile), branched-chain (isobutyronitrile) and dinitrile (succinonitrile). The specific activity of the amidase was found to be several-fold higher than nitrile hydratase.     

Nitrile-converting enzymes as a tool to improve biocatalysis in organic synthesis: recent insights and promises

Nitrile-converting enzymes, including nitrilase and nitrile hydratase (NHase), have received increasing attention from researchers of industrial biocatalysis because of their critical role as a tool in organic synthesis of carboxylic acids and amides from nitriles. To date, these bioconversion approaches are considered as one of the most potential industrial processes using resting cells or purified enzymes as catalysts for production of food additives, pharmaceutical, and agrochemical precursors. This review focuses on the distribution and catalytic mechanism research of nitrile-converting enzymes in recent years. Molecular biology aspects to improve the biocatalytic performance of microbial nitrilase and NHase are demonstrated. The process developments of microbial nitrilase and NHase for organic synthesis are also discussed.     

Cyanide Metabolism in Higher Plants: Cyanoalanine Hydratase is a NIT4 Homolog

Cyanoalanine hydratase (E.C. 4.2.1.65) is an enzyme involved in the cyanide detoxification pathway of higher plants and catalyzes the hydrolysis of β-cyano-l-alanine to asparagine. We have isolated the enzyme from seedlings of blue lupine (Lupinus angustifolius) to obtain protein sequence information for molecular cloning. In contrast to earlier reports, extracts of blue lupine cotyledons were found also to contain cyanoalanine-nitrilase (E.C. 3.5.5.4) activity, resulting in aspartic acid production. Both activities co-elute during isolation of cyanoalanine hydratase and are co-precipitated by an antibody directed against Arabidopsis thaliana nitrilase 4 (NIT4). The isolated cyanoalanine hydratase was sequenced by nanospray-MS/MS and shown to be a homolog of Arabidopsis thaliana and Nicotiana tabacum NIT4. Full-length cDNA sequences for two NIT4 homologs from blue lupine were obtained by PCR using degenerate primers and RACE-experiments. The recombinant LaNIT4 enzymes, like Arabidopsis NIT4, hydrolyze cyanoalanine to asparagine and aspartic acid but show a much higher cyanoalanine-hydratase activity. The two nitrilase genes displayed differential but overlapping expression. Taken together these data show that the so-called ‘cyanoalanine hydratase’ of plants is not a bacterial type nitrile hydratase enzyme but a nitrilase enzyme which can have a remarkably high nitrile-hydratase activity.     

Kinetic resolution of amino acid esters catalyzed by lipases

Racemic amino acids were resolved by lipase via hydrolysis of their esters. Lipases (Pseudomonas lipase from Amano PS, Rhizopus lipase from Serva, and porcine pancrease lipase from Sigma) could selectively hydrolyze the L-amino acid esters in aqueous solution with high reactivities and selectivities. The effect of the structural changes in the ester moiety on the stereoselectivity of the lipases was also investigated using D ,L -homophenylalanine as a model. Procedures were developed for the resolution of natural and unnatural amino acids. © 1996 Wiley-Liss, Inc.     

Sizing of theRhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation

The two restriction enzymesAsnI andDraI were found to produce DNA fragment sizes that could be used for mapping theRhodococcus sp. R312 (formerlyBrevibacterium sp. R312) genome by pulsed-field gel electrophoresis.AsnI produced 24 fragments (4 to 727 kb) andDraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on theAsnI andDraI fragments.     

Reductive biotransformations of organic compounds by saccharomyces cerevisiae: study of oxidoreductases involved using electrophoretic zymograms

The role of oxidoreductases in reduction of carbonyl compounds was investigated by application of zymogram techniques. Eight bands were observed using ethanol with nicotinamide adenine dinucleotide (NAD) as coenzyme. Bands observed with lactic acid and (R)-(-)-phenyl-1,2-ethanediol with nicotinamide adenine dinucleotide phosphate (NADP) had similar R(m) values. 2-Hydroxyvalerate and malate manifested bands having similar R(m) values and were active with both NAD and NADP. Based on their structural similarity and identical R(m) values, oxidation of 1,4-cyclooctanediol (band #2) and cis-1,5-cyclooctanediol may be due to a common enzyme. The PAGE-zymogram technique may be used on a preparative scale to facilitate purification and full characterization on the observed stained bands.     

对羟基苯乙腈水合酶高产菌株的选育

从全省各处采集的50多份土样筛选到一株产腈水合酶能力较高的菌株E10a,该菌株产生的腈水合酶为非诱导酶,产酶条件优化实验结果表明,产酶培养基组成:麦芽糖20 g/L,酵母膏5 g/L,尿素7.5 g/L,味精0.75 g/L,K2HPO40.5 g/L,KH2PO40.5 g/L,MgSO40.5 g/L,FeSO4.7H2O 10 mg/L,CoCl210 mg/L,微量元素母液0.8 ml/L;最佳培养条件为:培养温度28℃,摇床转速150 r/min,培养基起始pH 7.0,培养时间5 d,在优化培养条件下,1 h可将1 g/L质量浓度的底物对羟基苯乙腈全部转化为对羟基苯乙酰胺。     

Improved product-per-glucose yields in P450-dependent propane biotransformations using engineered Escherichia coli

P450-dependent biotransformations in Escherichia coli are attractive for the selective oxidation of organic molecules using mild and sustainable procedures. The overall efficiency of these processes, however, relies on how effectively the NAD(P)H cofactors derived from oxidation of the carbon source are utilized inside the cell to support the heterologous P450-catalyzed reaction. In this work, we investigate the use of metabolic and protein engineering to enhance the product-per-glucose yield (Y(PPG)) in whole-cell reactions involving a proficient NADPH-dependent P450 propane monooxygenase prepared by directed evolution [P450(PMO)R2; Fasan et al. (2007); Angew Chem Int Ed 46:8414-8418]. Our studies revealed that the metabolism of E. coli (W3110) is able to support only a modest propanol: glucose molar ratio (YPPG ~ 0.5) under aerobic, nongrowing conditions. By altering key processes involved in NAD(P)H metabolism of the host, considerable improvements of this ratio could be achieved. A metabolically engineered E. coli strain featuring partial inactivation of the endogenous respiratory chain (Δndh) combined with removal of two fermentation pathways (ΔadhE, Δldh) provided the highest Y(PPG) (1.71) among the strains investigated, enabling a 230% more efficient utilization of the energy source (glucose) in the propane biotransformation compared to the native E. coli strain. Using an engineered P450(PMO)R2 variant which can utilize NADPH and NADH with equal efficiency, we also established that dual cofactor specificity of the P450 enzyme can provide an appreciable improvement in Y(PPG). Kinetic analyses suggest, however, that much more favorable parameters (K(M), k(cat)) for the NADH-driven reaction are required to effectively compete with the host's endogenous NADH-utilizing enzymes. Overall, the metabolic/protein engineering strategies described here can be of general value for improving the performance of NAD(P)H-dependent whole-cell biotransformations in E. coli.     

Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus sp. AJ270

Nitrile hydratase (NHase, EC 4.2.1.84) from Rhodococcus sp. AJ270 was purified with 23.96% yield after sonication, ammonium sulfate fractionation, ion exchange, hydrophobic and gel-filtration column chromatography. The enzyme showed intriguing characteristics: it hydrated not only aliphatic and heterocyclic nitriles but also aromatic ones. Some substrates were also hydrated enantioselectively to the corresponding amides. The enantiomeric excess (ee) value of the enzyme hydrating trans-2,2-dimethyl-3-phenylcyclopropanecarbonitrile was 84.7. The enzyme is composed of two subunits: an alpha subunit and beta subunit of 22 975 Da and 23 493 Da, respectively. The optimal temperature and pH for the catalytic reaction of the enzyme was 25 degrees C and pH 7.6. The enzyme activity of the purified NHase was strongly inhibited by some oxidizing agents and heavy metals.     

Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture

Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present.The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems.Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.