Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi

Mueller, Patrick; Egorova, Ksenia S.; Vorgias, Constantinos E.; Boutou, Effrossyni; Trauthwein, Harald; Verseck, Stefan; Antranikian, Garabed

doi:10.1016/j.pep.2006.01.006

Cited by 69 publications

(41 citation statements)

References 46 publications

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“…A sharp activity decrease was observed at slightly alkaline pH values, only trace activity being observed at pH 8.5. This pH optimum was not like the pH optima of other nitrilases reported in the range of 7.0 to 9.0 (Mueller et al, 2006;Vejvoda et al, 2010;Kim et al, 2009). However, it was similar to that of the nitrilase from Rhodococcus rhodochrous K22, the pH optimum of which was reported at 5.5 (Kobayashi et al, 1990).…”

Section: Effects Of Temperatures and Ph Valuescontrasting

confidence: 74%

Characterization of a nitrilase from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid

Cai

Zhu

et al. 2014

J. Gen. Appl. Microbiol.

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A nitrilase gene cyc705 from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid (IDA) was cloned. This gene contained a 930 bp ORF, which encoded a polypeptide of 310 amino acids. A recombinant Escherichia coli BL21(DE3)/ pET28a-cyc705 was constructed to achieve the heterologous expression of cyc705. This recombinant nitrilase was purified to homogeneity with a molecular weight of 36.7 kDa on SDS-PAGE and mass spectrometry, and characterized to be an oligomer of 14 subunits by gel permeation chromatography. Using iminodiacetonitrile (IDAN) as the substrate, the V max , K m , k cat and k cat / K m were 9.05 U mg 1 , 43.17 mM −1 , 94.1 min −1 and 2.18 10 3 min −1 M −1 , respectively. The optimum temperature and pH were 25 C and 5.8. The suitable substrates for the purified nitrilase were short-chain aliphatic dinitriles. High concentration of IDAN could be hydrolyzed to IDA in a shorter time.Key words: Arthrobacter aurescens; characterization; iminodiacetic acid; nitrilase Introduction Nitrilase (EC 3.5.5.1), as an important industrial enzyme, belongs to the nitrilase superfamily (Brenner, 2002). Nitrilases are widely derived from various organisms, including bacteria, fungi and plants. Initial investigations suggested nitrilases to be specific for aromatic nitriles. But now, depending on the substrate spectrum, nitrilases are reported from different sources (Banerjee et al., 2002), which can be active on aromatic or heterocyclic nitriles (Vejvoda et al., 2010) and aliphatic nitriles (Bayer et al., 2011) as well as arylacetonitriles (Sosedov et al., 2010). Nitrilases can convert nitriles directly into the corresponding carboxylic acids and ammonia. Compared with chemical methods of producing carboxylic acids, nitrilases are widely used due to the mild reaction conditions, regioselectivity (Wang et al., 2014) and enantioselectivity (Vejvoda et al., 2010). Therefore, they are used to catalyze the production of various valuable carboxylic acids.IDA is an important fine chemical intermediate. It has wide applications and is mainly used in producing glyphosate herbicides, chelating agents and surfactants (Ni et al., 2009). Currently, the production of IDA mainly uses chemical methods, including the hydrocyanic acid method (Rmon, 1991), diethanolamine method (Bornscheuer and Kazlauskas, 2006), and chloroacetic acid method (Farbwerke Hoechst, 1969). These methods produce a large amount of waste water and byproducts, which are unfriendly to the environment (Duan et al., 2007). Enzymatic production of IDA has some advantages, such as the mild reaction conditions, environmental friendliness and low cost. So far, only a few papers have reported nitrilase-catalyzed production of IDA. A nitrilase-produced strain named Alcaligenes faecalis ZJB-09133 was screened by using IDAN as the carbon source, and the whole cells were used to catalyze IDAN to IDA, which showed the conversion only reached 65.3% at 8 h after optimization of the reaction conditions (Liu et al., 2011). The Acidovorax facilis nitrilase was cloned ...

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Section: Effects Of Temperatures and Ph Valuescontrasting

confidence: 74%

Characterization of a nitrilase from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid

Cai

Zhu

et al. 2014

J. Gen. Appl. Microbiol.

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“…It has been reported that nitrilases produced from thermophilic sources have substantially higher thermal stability than those produced from mesophillic sources 14,15 . The purification of nitrilase was carried by various protocols, since the enzyme is intracellular in nature and hence, the release of enzyme to the extra cellular environment with minimum activity loss is very crucial and that can be done using high pressure homogenizer 16 or an ultrasonicator 17 .…”

Section: Fig 1: the Representation Of Two Pathways Involved In The Dmentioning

confidence: 99%

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2017

IJPSR

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A thermostable bacterial 3-cyanopyridinase enzyme has been identified that catalyze 3-cyanopyridine to nicotinic acid and ammonia. Nicotinic acid is mainly used in food, pharmaceutical and biochemical industries. This study was undertaken to optimize the purification of 3-cyanopyridinase enzyme and characterization of its various kinetic properties. The partial purification of enzyme has been carried out in five steps that results in approximately 2.3 fold increased purification with 46.5 % of enzyme recovery. The partially purified enzyme was observed approximately 43 kDa of maximum activity at 50 °C temperature and pH 7.0. The partially purified enzyme showed the K m of 0.91 mM and maximum velocity (V max ) of 0.59 U. The half-life (t 1/2 ) of partially purified enzyme was about 60 min at optimal conditions. The enzyme has broad substrate specificity, hydrolyzing toxic compounds such as benzonitrile and propionitrile. Thus this novel enzyme has potential in bioremediation process.

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“…ions (required for the active expression of nitrile-hydratase enzyme) in minimal medium showed no significant effect on the nitrile-hydrolysing activity. It might be due to the presence of nitrilase enzyme in the I. variabilis RGT01 which is not a metalloprotein [6].…”

Section: Selection and Identification Of A Nitrile-utilizing Strainmentioning

confidence: 99%

“…Nitriles are toxic in nature, but can be hydrolysed to corresponding less-toxic and valuable carboxylic acids either by nitrilase (EC 3.5.5.1), or by nitrile-hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4). Nitrilases have been reported in a variety of microbes including Alcaligenes [1], Bacillus [2], Burkholderia [3], Labrenzia [4], Pseudomonas [5], Pyrococcus [6] and Rhodococcus [7]. In spite of many nitrilases reported in literature, the quest for their new sources has never ended.…”

Section: Introductionmentioning

confidence: 99%

Isolation and Characterization of a Nitrile-Hydrolysing Bacterium Isoptericola variabilis RGT01

et al. 2014

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A nitrile-hydrolysing bacterium, identified as Isoptericola variabilis RGT01, was isolated from industrial effluent through enrichment culture technique using acrylonitrile as the carbon source. Whole cells of this microorganism exhibited a broad range of nitrile-hydrolysing activity as they hydrolysed five aliphatic nitriles (acetonitrile, acrylonitrile, propionitrile, butyronitrile and valeronitrile), two aromatic nitriles (benzonitrile and m-Tolunitrile) and two arylacetonitriles (4-Methoxyphenyl acetonitrile and phenoxyacetonitrile). The nitrile-hydrolysing activity was inducible in nature and acetonitrile proved to be the most efficient inducer. Minimal salt medium supplemented with 50 mM acetonitrile, an incubation temperature of 30°C with 2 % v/v inoculum, at 200 rpm and incubation of 48 h were found to be the optimal conditions for maximum production (2.64 ± 0.12 U/mg) of nitrile-hydrolysing activity. This activity was stable at 30°C as it retained around 86 % activity after 4 h at this temperature, but was thermolabile with a half-life of 120 min and 45 min at 40°C and 50°C respectively.

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Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi

Cited by 69 publications

References 46 publications

Characterization of a nitrilase from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid

Characterization of a nitrilase from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid

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Isolation and Characterization of a Nitrile-Hydrolysing Bacterium Isoptericola variabilis RGT01

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