Asymmetric hydrolysis of α-aminonitriles to optically active amino acids by a nitrilase of Rhodococcus rhodochrous PA-34

Summary Rhodococcus rhodochrous PA-34 isolated from soil as a propionitrile-utilizing microorganism, hydrolysed several -aminonitriles to optically active amino acids. The hydrolysis of -aminonitriles was found to be catalysed by a nitrilase. The characteristics of the purified enzyme revealed that this is a new nitrilase as it has a molecular mass of 45 kDa and acts as a monomer. The optimum pH and temperature for the activity of the purified enzyme were 7.5 and 35° C, respectively. Thiol-specific reagents caused inhibition whereas chelators did not significantly alter the activity of this enzyme. The amino acids produced were of L-form, except for alanine. In the case of leucine production from -aminoisocapronitrile, the enantiomeric ratio of L-leucine to D-leucine was about 60.     

Bioconversion of butyronitrile to butyramide using whole cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of β-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10°C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10°C for 6 h.     

Characterization and functional cloning of an aromatic nitrilase from Pseudomonas putida CGMCC3830 with high conversion efficiency toward cyanopyridine

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.     

Cloning and expression of a gene encoding cyanidase from Pseudomonas stutzeri AK61

The gene coding for cyanidase, which catalyzes the hydrolysis of cyanide to formate and ammonia, was cloned from chromosomal DNA of Pseudomonas stutzeri AK61 into Escherichia coli. The cyanidase gene consisted of an open reading frame of 1004 bp, and it was predicted that cyanidase was composed of 334 amino acids with a calculated molecular mass of 37 518 Da. The amino acid sequence of cyanidase showed a 35.1% and 26.4% homology to aliphatic nitrilase from Rhodococcus rhodochrous K22 and cyanide hydratase from Fusarium lateritium, respectively. A unique cysteine residue of aliphatic nitrilase, which was suggested to play an essential role in the catalytic activity, was conserved in cyanidase. The active form of cyanidase was successfully expressed by a DNA clone containing the cyanidase gene in E.coli. Its productivity was approximately 230 times larger than that of P. stutzeri AK61. The characteristics of the expressed cyanidase, including optimum pH, optimum temperature, Michaelis constant (K _m) for cyanide and specific activity, were similar to those of the native enzyme from P. stutzeri AK61. Received: 24 October 1997 / Received last revision: 17 March 1998 / Accepted: 20 March 1998     

Dimethylformamide is a novel nitrilase inducer in Rhodococcus rhodochrous

Nitrilases are of commercial interest in the selective synthesis of carboxylic acids from nitriles. Nitrilase induction was achieved here in three bacterial strains through the incorporation of a previously unrecognised and inexpensive nitrilase inducer, dimethylformamide (DMF), during cultivation of two Rhodococcus rhodochrous strains (ATCC BAA-870 and PPPPB BD-1780), as well as a closely related organism (Pimelobacter simplex PPPPB BD-1781). Benzonitrile, a known nitrilase inducer, was ineffective in these strains. Biocatalytic product profiling, enzyme inhibition studies and protein sequencing were performed to distinguish the nitrilase activity from that of sequential nitrile hydratase-amidase activity. The expressed enzyme, a 40-kDa protein with high sequence similarity to nitrilase protein Uniprot Q-03217, hydrolyzed 3-cyanopyridine to produce nicotinic acid exclusively in strains BD-1780 and BD-1781. These strains were capable of synthesising both the vitamin nicotinic acid as well as β-amino acids, a compound class of pharmaceutical interest. The induced nitrilase demonstrated high enantioselectivity (> 99%) in the hydrolysis of 3-amino-3-phenylpropanenitrile to the corresponding carboxylic acid.

     

Cloning and Nucleotide Sequence of the Gene Encoding Dimethyl Sulfoxide Reductase from Rhodohacter sphaeroides f. sp. denitrijicans

The gene encoding dimethyl sulfoxide (DMSO) reductase, which contains a molybdenum cofactor, of the phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans was isolated using an oligonucleotide probe, which was synthesized based on a internal amino acid sequence of the purified enzyme. The DMSO reductase gene coded for 822 amino acids (2466 base pairs, M_r = 89,206) as a precursor form having a signal peptide of 42 amino acids. The deduced amino acid sequence had high homology with those of some enzymes containing a molybdenum cofactor: trim ethyl amine N-oxide reductase (48%), biotin sulfoxide reductase (44%), and DMSO reductase (29%) of Escherichia coli.     

Regiospecific hydrolysis of dinitrile compounds by nitrilase fromRhodococcus rhodochrous J1

Summary Nitrilase fromRhodococcus rhodochrous J1 catalyses the hydrolysis of nitriles to acids without the formation of amides. TheRhodococcus nitrilase exhibited regiospecificity for dicyanobenzenes. Three- and 4-cyanobenzoic acids were synthesized from isophthalonitrile and terephthalonitrile, respectively, with conversion ratios of more than 90% using theRhodococcus nitrilase.     

Bench scale conversion of 3-cyanopyidine to nicotinamide using resting cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

The nitrile hydratase (NHase, EC 3.5.5.1) activity of Rhodococcus rhodochrous PA-34 was explored for the conversion of 3-cyanopyridine to nicotinamide. The NHase activity (∼18 U/mg dry cell weight, dcw) was observed in 0.1 M phosphate buffer, pH 8.0 containing 1M 3-cyanopyridine as substrate, and 0.75 mg of resting cells (dry cell weight) per ml reaction mixture at 40°C. However, 25°C was more suitable for prolonged batch reaction at high substrate (3-cyanopyridine) concentration. In a batch reaction (1 liter), 7M 3-cyanopyridine (729 g) was completely converted to nicotinamide (855 g) in 12h at 25°C using 9.0 g resting cells (dry cell weight) of R. rhodochrous PA-34.     

Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.     

Identification and characterization of a novel nitrilase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Pf-5

Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0°C and 45°C, respectively, with a specific activity of 1.7 and 1.9 μmol min⁻¹ mg protein⁻¹ for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K _m = 17.9 mM and k _cat/K _m = 0.5 mM⁻¹ s⁻¹ for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids.     

Biotransformation of benzonitrile herbicides via the nitrile hydratase–amidase pathway in rhodococci

The aim of this work was to determine the ability of rhodococci to transform 3,5-dichloro-4-hydroxybenzonitrile (chloroxynil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and 2,6-dichlorobenzonitrile (dichlobenil); to identify the products and determine their acute toxicities. Rhodococcus erythropolis A4 and Rhodococcus rhodochrous PA-34 converted benzonitrile herbicides into amides, but only the former strain was able to hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid, and produced also more of the carboxylic acids from the other herbicides compared to strain PA-34. Transformation of nitriles into amides decreased acute toxicities for chloroxynil and dichlobenil, but increased them for bromoxynil and ioxynil. The amides inhibited root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification.     

Pep5, a new lantibiotic: structural gene isolation and prepeptide sequence

A wobbled 14-mer oligonucleotide was derived from the amino acid sequence of the 34-residue propeptide of the lantibiotic Pep5 (Kellner et al. 1989). Using this hybridization probe, the structural gene of Pep5, pepA, was located on the 18.6 kbp plasmid pED503. The nucleotide sequence of pepA codes for a prepeptide with 60 residues and proves that Pep5 is ribosomally synthesized. The N-terminus of the prepeptide has a high -helix probability and a characteristic proteolytic cleavage site precedes the C-terminal 34-residue propeptide. Our present theory is that maturation of Pep5 involves (a) enzymic conversion of Thr, Ser and Cys into dehydrated amino acids and sulfide bridges, (b) membrane translocation and cleavage of the modified prepeptide.Dedicated to Prof. H. Zähner on the occasion of his sixtieth birthday     

Primary structure of an aliphatic nitrile-degrading enzyme, aliphatic nitrilase, from Rhodococcus rhodochrous K22 and expression of its gene and identification of its active site residue.

Peptides obtained by cleavage of a Rhodococcus rhodochrous K22 nitrilase, which acts on aliphatic nitriles such as acrylonitrile, crotonitrile, and glutaronitrile, have been sequenced. The data allowed the design of oligonucleotide probes which were used to clone a nitrilase encoding gene. Plasmid pNK21, in which 2.05-kb sequence covering the region encoding the nitrilase was was placed under the control of the lac promoter, directed overproduction of enzymatically active nitrilase in response to addition of isopropyl beta-D-thiogalactopyranoside in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell extract showed that the amount of nitrilase was about 40% of the total soluble proteins, leading to the establishment of a simple purification of the nitrilase. The nucleotide sequence of the nitrilase gene predicts a protein composed of 383 amino acids (M(r) = 42,275), including only one cysteine. The amino acid sequence homology between the Rhodococcus nitrilase and the Klebsiella ozaenae bromoxynil nitrilase [Stalker et al. (1988) J. Biol. Chem. 263, 6310-6314] was 38.3%, and a unique cysteinyl residue (Cys-170) in the former nitrilase was conserved at the corresponding position in the latter nitrilase. Cys-170 of the Rhodococcus nitrilase was replaced by Ala or Ser by site-directed mutagenesis. Both mutations resulted in the complete loss of nitrilase activity, clearly indicating that this cysteinyl residue is essential for the catalytic activity.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Random mutagenesis of the arylacetonitrilase from Pseudomonas fluorescens EBC191 and identification of variants,which form increased amounts of mandeloamide from mandelonitrile

The nitrilase from Pseudomonas fluorescens EBC191 was modified by introducing random mutations via error-prone PCR techniques in order to obtain nitrilase variants, which form increased amounts of mandeloamide from racemic mandelonitrile. A screening system was established and experimentally optimized, which allowed the screening of nitrilase variants with the intended phenotype. This system was based on the simultaneous expression of nitrilase variants and the mandeloamide converting amidase from Rhodococcus rhodochrous MP50 in recombinant Escherichia coli cells. The formation of increased amounts of mandeloamide from mandelonitrile by the nitrilase variants was detected after the addition of hydroxylamine and ferric iron ions by taking advantage of the acyltransferase activity of the amidase, which resulted in the formation of coloured iron(III)–hydroxamate complexes from mandeloamide. The system was applied for the screening of libraries of nitrilase variants and 30 enzyme variants identified, which formed increased amounts of mandeloamide from racemic mandelonitrile. The increase in amide formation was quantified by high-performance liquid chromatography and the genes encoding the relevant nitrilase variants sequenced. Thus, different types of mutations were identified. One group of mutants carried different deletions at the carboxy-terminus. The other types of variants carried amino acid exchanges in positions that had not been related previously to an increased amide formation. Finally, a nitrilase variant was created by combining two independently obtained point mutations. This enzyme variant demonstrated a true nitrile hydratase activity as it formed mandeloamide and mandelic acid in a ratio of about 19:1 from racemic mandelonitrile.     

Biotransformation of benzonitrile to benzohydroxamic acid by Rhodococcus rhodochrous in the presence of hydroxylamine

Whole cells of the bacterium Rhodococcus rhodochrous LL100-21, which had been grown on benzonitrile to induce the nitrilase enzyme, converted benzonitrile to benzohydroxamic acid in the presence of hydroxylamine.     

Sequence of a cDNA encoding nitrite reductase from the tree Betula pendula and identification of conserved protein regions

Summary The sequence of an mRNA encoding nitrite reductase (NiR, EC 1.7.7.1.) from the tree Betula pendula was determined. A cDNA library constructed from leaf poly(A)⁺ mRNA was screened with an oligonucleotide probe deduced from NiR sequences from spinach and maize. A 2.5 kb cDNA was isolated that hybridized to an mRNA, the steady-state level of which increased markedly upon induction with nitrate. The nucleotide sequence of the cDNA contains a reading frame encoding a protein of 583 amino acids that reveals 79% identity with NiR from spinach. The transit peptide of the NiR precursor from birch was determined to be 22 amino acids in size by sequence comparison with NiR from spinach and maize and is the shortest transit peptide reported so far. A graphical evaluation of identities found in the NiR sequence alignment revealed nine well conserved sections each exceeding ten amino acids in size. Sequence comparisons with related redox proteins identified essential residues involved in cofactor binding. A putative binding site for ferredoxin was found in the N-terminal half of the protein.These sequence data appear in the EMBL/GenBank/DDBJ nucleotide sequence data bases under the accession number X60093     

编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交(suppression subtractive hybridization,SSH)的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。     

Purification of chloroplast elongation factor Tu and cDNA analysis in tobacco: the existence of two chloroplast elongation factor Tu species

We have purified a chloroplast elongation factor Tu (EF-Tu) from tobacco (Nicotiana tabacum) and determined its N-terminal amino acid sequence. Two distinct cDNAs encoding EF-Tu were isolated from a leaf cDNA library of N. sylvestris (the female progenitor of N. tabacum) using an oligonucleotide probe based on the EF-Tu protein sequence. The cDNA sequence and genomic Southern analyses revealed that tobacco chloroplast EF-Tu is encoded by two distinct genes in the nuclear genome of N. sylvestris. We designated the corresponding gene products EF-Tu A and B. The mature polypeptides of EF-Tu A and B are 408 amino acids long and share 95.3% amino acid identity. They show 75–78% amino acid identity with cyanobacterial and chloroplast-encoded EF-Tu species.     

编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交(suppression subtractive hybridization,SSH)的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。