Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization

Nitrilase was purified from an extract of isovaleronitrile-induced cells of Rhodococcus rhodochrous J1 in seven steps. In the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous by polyacrylamide electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 78 kDa and consists of two subunits identical in molecular mass. The purified enzyme exhibits a pH optimum of 7.6 and a temperature optimum of 45 degrees C. The enzyme catalyzed stoichiometrically the hydrolysis of benzonitrile to benzoic acid and ammonia, and no formation of amide was detected. The enzyme required thiol compounds such as dithiothreitol, L-cysteine or reduced glutathione to exhibit maximum activity. The enzyme was specific for nitrile groups attached to an aromatic or heteroaromatic ring, e.g. benzonitrile, 3-chlorobenzonitrile, 4-tolunitrile, 2-furonitrile and 2-thiophenecarbonitrile. The comparison of the properties of the enzyme with other nitrilases and nitrile hydratases has been also discussed.     

Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association.

Nitrilase-containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mM benzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light-scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80-kDa enzyme to its active 410-kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations.     

Occurrence of Amidases in the Industrial Microbe Rhodococcus rhodochrous J1

Rhodococcus rhodochrous J1, of which the high-M_r nitrile hydratase has been used for the industrial manufacture of acrylamide from acrylonitrile, produced at least two amidases differing in substrate specificity, judging from the effects of various amides on amidase activity in this strain. These amidases seemed to be inducible enzymes depending on amide compounds.     

红球菌低分子量型腈水合酶的异源激活及激活子的结构域功能

腈水合酶激活子具有亚基自身交换伴随子或者金属离子伴随子的功能,能够辅助腈水合酶摄取金属离子,对于腈水合酶的活性表达必不可少。与腈水合酶自身相比,激活子的序列保守性低,研究其激活作用的特点,探索其结构与功能之间的关系,对于理解腈水合酶的成熟机制具有重要意义。将红球菌Rhodococcus rhodochrous J1低分子量型腈水合酶L-NHase分别与4种异源激活子组合共表达,测定异源激活子对L-NHase的激活作用,进一步对激活子进行序列分析和结构模拟,并研究关键结构域的功能。结果表明,4种异源激活子均能激活L-NHase,但激活后L-NHase的比酶活存在差异,激活子A对L-NHase的激活程度最高,激活后的L-NHase比酶活为出发酶的97.79%;激活子G对L-NHase的激活程度最低,激活后的L-NHase比酶活为出发酶的23.94%。激活子E和激活子G具有保守结构域TIGR03889,缺失其中部分序列会使两者的激活作用基本丧失。将激活子G的N端序列替换为激活子E的N端序列,并将激活子E的C端序列添加至激活子G的C端,能够使L-NHase的比酶活提高178.40%。激活子的激...     

Nitrile Hydratase-Catalyzed Production of Nicotinamide from 3-Cyanopyridine in Rhodococcus rhodochrous J1

Nitrile hydratase, which occurs abundantly in cells of Rhodococcus rhodochrous J1 isolated from soil samples, catalyzes the hydration of 3-cyanopyridine to nicotinamide. By using resting cells, the reaction conditions for nicotinamide production were optimized. Under the optimum conditions, 100% of the added 12 M 3-cyanopyridine was converted to nicotinamide without the formation of nicotinic acid, and the highest yield achieved was 1,465 g of nicotinamide per liter of reaction mixture containing resting cells (1.48 g as dry cell weight) in 9 h. The nicotinamide produced was crystallized and then identified physicochemically. The further conversion of the nicotinamide to nicotinic acid was due to the low activity of nicotinamide as a substrate for the amidase(s) present in this organism.     

Nitrilase-Catalyzed Production of Nicotinic Acid from 3-Cyanopyridine in Rhodococcus rhodochrous J1

The nitrilase which occurs abundantly in cells of Rhodococcus rhodochrous J1 catalyzes the direct hydrolysis of 3-cyanopyridine to nicotinic acid without forming nicotinamide. By using resting cells, the reaction conditions for nicotinic acid production were optimized. Under the optimum conditions, 100% of the added 3-cyanopyridine could be converted to nicotinic acid, the highest yield achieved being 172 mg of nicotinic acid per 1.0 ml of reaction mixture containing 2.89 mg (dry weight) of cells in 26 h.     

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.     

Rhodococcus rhodochrous J1腈水合酶在大肠杆菌中的表达策略

针对红球菌低分子量腈水合酶（L—NHase）在重组茵中难以表达这一问题,通过对其d亚基及调控蛋白NhlE基因的核糖体结合位点和0c,口亚基间隔序列的长度进行改造,构建了重组表达载体,实现了L．NHase及其调控蛋白NhlE在E．coliB121（DE3）中过量表达。通过培养条件优化,得到最佳表达条件为：37℃培养茵体浓度（DD600）到1．0时,加入终浓度为0．1g／L的CoCl2·6H,0,0．6mmol／L的IPTG,然后在24℃下诱导表达24h。最终得到的重组蛋白粗酶液的活性为（109．9-I-5．5）U／rag。采用Strep．tag／Strep—Tactin亲和层析简化了L-NHase的纯化方法,本研究结果为一些难于异源重组表达的多亚基蛋白质的表达具有一定的借鉴意义。     

Optimum culture conditions for the production of benzonitrilase by Rhodococcus rhodochrous J1

We sought the optimum conditions for the production of benzonitrilase by Rhodococcus rhodochrous J1. The use of isovaleronitrile or isobutyronitrile as an inducer greatly enhanced benzonitrilase formation. When Rhodococcus rhodochrous J1 was cultivated at 28°C for 96 h in a medium consisting of 0.1 ml of isovaleronitrile, 0.5 g of polypeptone, 0.3 g of malt extract, 0.3 g of yeast extract and 1 g of glycerol per 100 ml of tap water (pH 7.2), and isovaleronitrile was fed twice at the concentrations of 0.1% (v/v) and 0.2% (v/v) at 55 h and 77 h, respectively, during the course of cultivation, the enzyme activity in the culture broth reached approximately 3,100-times higher than the initially obtained level.     

Occurrence of a cobalt-induced and cobalt-containing nitrile hydratase in Rhodococcus rhodochrous J1

The formation of nitrile hydratase required cobalt ions in Rhodococcus rhodochrous J1. No other transition-metals could replace the cobalt ion. The Rhodococcus nitrile hydratase was purified to homogeneity and found to contain a cobalt atom. The occurrence of a cobalt-induced and cobalt-containing nitrile hydratase, different from the nitrile hydratases in Pseudomonas chlororaphis B23 and Brevibacterium R312 containing a ferric ion in their active center, has been demonstrated here for the first time.     

Isolation and primary characterization of an amidase from Rhodococcus rhodochrous

Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q. The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8. The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity. Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R. rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme. The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312.     

Production of acrylic acid and methacrylic acid using Rhodococcus rhodochrous J1 nitrilase

Summary -Caprolactam-induced Rhodococcus rhodochrous J1 cells containing abundant nitrilase were used in the production of acrylic acid and methacrylic acid from acrylonitrile and methacrylonitrile, respectively. Under a periodic substrate feeding system, the highest accumulations, 390 g acrylic acid/l and 260 g methacrylic acid/l, were attained. Offprint requests to: T. Nagasawa     

Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1

A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (alpha subunit 26 kDa, beta subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.     

Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1

Summary We sought the optimum conditions for production of nitrile hydratase by Rhodococcus rhodochrous J1. The addiiion of both cobalt ions and an aliphatic nitrile or amide as an inducer was indispensable for the appearance of nitrile hydratase activity in R. rhodochrous J1 cells. Crotonamide was an efficient inducer and, moreover, urea was found to be the most powerful inducer for the production of nitrile hydratase. When R. rhodochrous J1 was cultivated under optimal conditions, the enzyme activity in the culture broth and the specific activity was approximately 32,000 and 512 times higher than the initially obtained levels, respectively. The nitrile hydratase formed corresponded to more than 45% of the total soluble protein in urea-induced cells, as judged by quantitative evaluation of the gel track.Offprint requests to: T. Nagasawa     

Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1.

Rhodococcus rhodochrous J1 produces two kinds of cobalt-containing nitrile hydratases (NHases); one is a high molecular mass-NHase (H-NHase) and the other is a low molecular mass-NHase (L-NHase). Both NHases are composed of two subunits of different sizes (alpha and beta subunits). The H- and L-NHase genes were cloned into Escherichia coli by a DNA-probing method using the NHase gene of Rhodococcus sp. N-774, a ferric ion-containing NHase producing strain, as the hybridization probe and their nucleotide sequences were determined. In each of the H- and L-NHase genes, the open reading frame for the beta subunit was located just upstream of that for the alpha subunit, which probably belongs to the same operon. The amino acid sequences of each subunit of the H- and L-NHases from R. rhodochrous J1 showed generally significant similarities to those from Rhodococcus sp. N-774, but the arrangement of the coding sequences for two subunits is reverse of the order found in the NHase gene of Rhodococcus sp. N-774. Each of the NHase genes was expressed in E. coli cells under the control of lac promoter, only when they were cultured in the medium supplemented with CoCl2.     

Cloning, Sequencing, and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from Rhodococcus rhodochrous

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.     

Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form

Nitrilases convert nitriles to the corresponding carboxylic acids and ammonia. The nitrilase from Rhodococcus rhodochrous J1 is known to be inactive as a dimer but to become active on oligomerization. The recombinant enzyme undergoes post-translational cleavage at approximately residue 327, resulting in the formation of active, helical homo-oligomers. Determining the 3D structure of these helices using electron microscopy, followed by fitting the stain envelope with a model based on homology with other members of the nitrilase superfamily, enables the interacting surfaces to be identified. This also suggests that the reason for formation of the helices is related to the removal of steric hindrance arising from the 39 C-terminal amino acids from the wild-type protein. The helical form can be generated by expressing only residues 1-327.     

Essential tyrosine residues in 3-ketosteroid-delta(1)-dehydrogenase from Rhodococcus rhodochrous.

Tetranitromethane treatment of 3-ketosteroid-Delta(1)-dehydrogenase of Rhodococcus rhodochrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0. 014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.