Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50 °C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187 g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170 U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli.     

The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli

We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the α- and β-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328–2 (ReNHase) was expressed with (ReNHase^+Act) and without (ReNHase^?Act) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4 Å resolution revealing an αβ heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.     

Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in Escherichia coli

The nitrile hydratase (NHase) of Rhodococcus species N-774, which is composed of two subunits, alpha and beta, catalyzes the hydration of various nitrile compounds to the corresponding amides. The amino acid sequences of the NH2 termini and the fragments obtained by digesting each of the two subunits with lysyl endopeptidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 4.4-kb SphI fragment which contained DNA sequences hybridizing to several of the probes was cloned in pBR322 in Escherichia coli. The nucleotide sequences together with the determined amino acid sequences indicated that the alpha and beta subunits of NHase consisted of 207 amino acids (Mr, 22918) and 212 amino acids (Mr, 23428), respectively. The open reading frame for the alpha subunit includes that for the beta subunit with a short interval of only 26 base pairs; the two genes are probably translated in a polycistronic manner. Although large amounts of the alpha- and beta-subunit proteins were produced as insoluble forms in E. coli when the cloned genes were placed under the control of the lac promoter, no enzymatic activity was detected. The activity of the enzyme was restored, to some extent, by solubilization of the proteins with 8 M urea and subsequent dialysis for refolding at pH 10 in the presence of Fe2+ and pyrroloquinoline quinone.     

Chaperone-assisted maturation of the recombinant Fe-type nitrile hydratase is insufficient for fully active expression in Escherichia coli

Nitrile hydratase (NHase) has attracted substantial attention for industrial applications to produce large-scale amides. Several NHases have been investigated for functional expression in Escherichia coli (E. coli). A Fe-type NHase was obtained from an acetamiprid-degrading bacterium, Pseudoxanthomonas sp. AAP-7 and functionally expressed in E. coli BL21 (DE3). No significant NHase activity was detected from the E. coli expressing either the NHase gene alone or NHase and P46K genes transcribed as one unit. Purified recombinant NHase, co-expressed with P46K on two separate plasmids, exhibited the maximal enzyme activity. Furthermore, a GST tag attached to the N-terminus of α subunit resulted in a slight increase in the solubility and stability of NHase compared with a His tag at the C-terminus of β subunit. When co-expressed with the chaperones GroEL-GroES, the yield of the soluble recombinant NHase was improved substantially, while a small decrease in NHase activity was observed. The putative activator P46K was strictly required for production of the recombinant NHase for full enzyme activity, although the chaperones GroEL-GroES appeared to assist NHase to fold properly. This study of the expression of a fully active Fe-type NHase would provide another example to enhance our understanding of NHase biosynthesis.     

Addition of Co2+ to culture medium decides the functional expression of a recombinant nitrile hydratase in Escherichia coli

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans, cloned and expressed in Escherichia coli, gave an enzyme that efficiently hydrated 3-cyanopyridine to nicotinamide with high thermal stability. We have now found that adding Co²⁺ at 0.1 mM to LB medium was essential for production of an active enzyme. However, ≥0.3 mM Co²⁺ inhibited the growth of host cells in LB medium and decreased the production of the recombinant NHase. Furthermore, β-mercaptoethanol promoted regeneration of the Co²⁺-defective apoenzyme in vitro possibly by breaking a key disulfide bond thereby promoting the incorporation of Co²⁺ into the apoenzyme.     

高分子量腈水合酶在大肠杆菌中的表达策略及重组菌的细胞催化

【目的】实现红球菌Rhodococcus rhodochrous J1来源的高分子量腈水合酶H-NHase在Escherichia coli BL21(DE3)中过量表达,以提高菌体总酶活,缩短菌体发酵周期,提高生产效率。【方法】将H-NHase中编码α亚基的基因nhh A和调控基因nhh G上游的核糖体结合位点(Shine-Dalgarno sequence,SD)替换成翻译起始强度更高的异源SD,同时优化各亚基之间间隔序列的长度,并根据大肠杆菌密码子偏好性对目的基因进行优化。通过E.coli重组表达系统过表达优化后的腈水合酶基因。采用离子交换层析对H-NHase进行纯化,并通过凝胶过滤层析确定重组酶的相对分子量。优化了全细胞催化条件,并建立底物恒速流加的细胞催化工艺,模拟了烟酰胺的生产工艺。【结果】H-NHase在E.coli中实现了过量表达。重组蛋白粗酶液的活性为85.5±4.3 U/mg,纯酶比活为234.0±11.7 U/mg,H-NHase相对分子质量为504.5±9.8 k D。细胞催化最适p H为7.5,最适温度为25°C,底物浓度为400 mmol/L。在此条件下,重组菌细胞酶活为256.0±10.4 U/m L,流加工艺最终的产物转化率可达99.9%。【结论】重组H-NHase大肠杆菌细胞生长迅速,发酵周期短,应用此重组菌进行细胞催化可以提高酰胺类物质的生产效率,具有潜在的工业价值。     

Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli

A moderate thermophile, Bacillus sp. BR449 was previously shown to exhibit a high level of nitrile hydratase (NHase) activity when growing on high levels of acrylonitrile at 55 degrees C. In this report, we describe the cloning of a 6.1 kb SalI DNA fragment encoding the NHase gene cluster of BR449 into Escherichia coli. Nucleotide sequencing revealed six ORFs encoding (in order), two unidentified putative proteins, amidase, NHase beta- and alpha-subunits and a small putative protein of 101 amino acids designated P12K. Spacings and orientation of the coding regions as well as their gene expression in E. coli suggest that the beta-subunit, alpha-subunit, and P12K genes are co-transcribed. Analysis of deduced amino acid sequences indicate that the amidase (348 aa, MW 38.6 kDa) belongs to the nitrilase-related aliphatic amidase family, and that the NHase beta- (229 aa, MW 26.5 kDa) and alpha- (214 aa, MW 24.5 kDa) subunits comprise a cobalt-containing member of the NHase family, which includes Rhodococcus rhodochrous J1 and Pseudomonas putida 5B NHases. The amidase/NHase gene cluster differs both in arrangement and composition from those described for other NHase-producing strains. When expressed in Escherichia coli DH5alpha, the subcloned NHase genes produced significant levels of active NHase enzyme when cobalt ion was added either to the culture medium or cell extracts. Presence of the P12K gene and addition of amide compounds as inducers were not required for this expression.     

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.     

Over-expression in Escherichia coli of a thermally stable and regio-selective nitrile hydratase from Comamonas testosteroni 5-MGAM-4D

The genes encoding a thermally stable and regio-selective nitrile hydratase (NHase) and an amidase from Comamonas testosteroni 5-MGAM-4D have been cloned and sequenced, and active NHase has been over-produced in Escherichia coli. Maximal activity requires co-expression of a small open reading frame immediately downstream from the NHase beta subunit gene. Compared to the native organism, the E. coli biocatalyst has nearly threefold more NHase activity on a dry cell weight basis, and this activity is significantly more thermally stable. In addition, this biocatalyst converts a wide spectrum of nitrile substrates to the corresponding amides. Such versatility and robustness are desirable attributes of a biocatalyst intended for use in commercial applications.     

Efficient inducible expression of nitrile hydratase in Corynebacterium glutamicum

Nitrile hydratases are important industrial catalysts to produce valuable amides. In this study, we describe a comprehensive and systematic approach to the development of an inducible expression system for enhanced nitrile hydratase expression in Corynebacterium glutamicum. Through promoter engineering, codon optimization and design of ribosome binding site sequences, the nitrile hydratase activity toward 3-cyanopyridine was improved from 0.33 U/mg DCW to 12.03 U/mg DCW in shake-flask culture. By introduction of the novel inducible mmp expression system, the nitrile hydratase activity was further elevated to 14.97 U/mg DCW. Finally, a high nitrile hydratase yield of 1432 U/mL was achieved in a fed-batch fermentation process and used for nicotinamide production. These results provide new insights for the development of heterologous protein expression systems in C. glutamicum.     

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.     

Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli.

The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells.     

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.     

Functional expression of a Drosophila antifungal peptide in Escherichia coli

Drosomycin is a key effector molecule involved in Drosophila innate immunity against fungal infection. This peptide is composed of 44 residues stabilized by four disulfide bridges. As the first step towards the understanding of the molecular basis for its specific antifungal activity, rapid and efficient production of the wild-type peptide and its mutants is needed. Here, we report a pGEX system for high-level expression of recombinant Drosomycin. The fusion Drosomycin protein with a carrier of Glutathione S-transferase (GST) was initially purified by affinity chromatography followed by Enterokinase cleavage. The digested product was separated by gel filtration and reverse phase HPLC. Mass spectrometry and circular dichroism spectroscopy analysis revealed that the recombinant peptide has identical molecular weight and correct structural conformation to native Drosomycin. Classical inhibition assay showed clear antifungal activity against Neurospora crassa with the IC(50) of 1.0muM. Successful expression of the CSalphabeta-type antifungal peptide in E. coli offers a basis for further studying its functional surface by alanine scanning mutagenesis strategy. Also, our work should be helpful in developing this peptide to an antifungal drug.     

Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterisation

The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, M_r 23 kDa) and 218 amino acids (β subunit, M_r 24 kDa) and the NHase activator of 413 amino acids (M_r 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest stability at 4°C in thermal inactivation studies between 4°C and 50°C.     

An over expression and high efficient mutation system of a cobalt-containing nitrile hydratase

A superior novel recombinant strain, E. coli BL21(DE3)/pETNH^M, containing the start codon mutation of the subunit, was constructed and selected as an overexpression and high efficient mutation platform for the genetic manipulation of the nitrile hydratase (NHase). Under optimal conditions, the specific activity of the recombinant strain reached as high as 452 U/mg dry cell. Enzymatic characteristics studies showed that the reaction activation energy of the recombinant NHase^M was 24.4 ± 0.5 kJ/mol, the suited pH range for catalysis was 5.5–7.5, and the K_m value was 4.34 g/L (82 mM). To assess the feasibility of the NHase improvement by protein rational design using this E. coli, site-directed mutagenesis of S122A, S122C, S122D and βW47E of the NHase^M were carried out. The NHase^M (S122A) and NHase^M (S122D) mutants were entirely inactive due to the charge change of the side-chain group. The product tolerance of the NHase^M (S122C) mutant was enhanced while its activity decreased by 30%. The thermo-stability of the NHase^M (βW47E) mutant was significantly strengthened, while its activity reduced by nearly 50%. These results confirmed that the specific activity of the mutant NHase expressed by the recombinant E. coli BL21(DE3)/pETNH^M can reasonably change with and without mutations. Therefore, this recombinant E. coli can be efficiently and confidently used for the further rational/random evolution of the NHase to simultaneously improve the activity, thermo-stability and product tolerance of the target NHase.     

Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe³⁺ or Co³⁺ center with two modified Cys ligands: cysteine sulfininate (Cys-SO₂ ⁻) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, αS113A and βY72F. The αS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV–vis absorption spectra indicated that the electronic state of the Fe center was altered by the αS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the αS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV–vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The βY72F mutant exhibited no activity. The structure of the βY72F mutant was highly conserved but was found to be the inactivated state, with αCys114-SO(H) oxidized to Cys-SO₂ ⁻, suggesting that βTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.     

cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta

Two nearly full-length cDNAs for placental plasminogen activator inhibitor (PAI) have been isolated from a human placenta lambda gt11 cDNA library. One positive (lambda PAI-75.1) expressed a protein that could adsorb and purify anti-PAI antibodies. The expressed protein inhibited the activity of human urokinase in a fibrin autography assay, and formed a 79-kDa (reduced) covalent complex with 125I-urokinase that could be immunoprecipitated with anti-PAI. The cDNA insert of the longer isolate (lambda PAI-75.15) consisted of 1909 base pairs, including a 5'-noncoding region of 55 base pairs, an open reading frame of 1245 base pairs, a stop codon, a 3'-noncoding region of 581 base pairs, and a poly(A) tail. The size of the mRNA was estimated to be 2.0 kilobases by Northern blot analysis. The translated amino acid sequence consisted of 415 amino acids, corresponding to a 46.6-kDa protein. The sequence was related to members of the serpin gene family, particularly ovalbumin and the chicken gene Y protein. Like these avian proteins, placental PAI appears to lack a cleavable NH2-terminal signal peptide. Residues 347-376 were identical to the partial sequence reported recently for a PAI isolated from the human monocytic U-937 cell line. Placental PAI mRNA was apparently expressed at low levels in human umbilical vein endothelial cells, but was not detectable in HepG2 hepatoma cells. It was present in U-937 cells and was inducible at least 10-fold by phorbol 12-myristate 13-acetate. Thus placental PAI is a unique member of the serpin gene family, distinct from endothelial-type PAI. It is probably identical to monocyte-macrophage PAI.     

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。