Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34

Prasad, Shreenath; Raj, Jog; Bhalla, Tek Chand

doi:10.1007/s12088-009-0033-x

Cited by 24 publications

(21 citation statements)

References 18 publications

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“…Since this is a heterodimeric enzyme, the elevated temperature may adversely affect enzyme stability, causing the two subunits to dissociate. Generally, the nitrile hydratases of mesophilic bacteria exhibit maximum activity at ambient temperatures between 20 and 40°C (59). Pseudomonas sp.…”

Section: -H-e-g-r-c-f-v-vmentioning

confidence: 99%

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Duca

Rose

Glick

2014

Appl Environ Microbiol

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Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.

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Section: -H-e-g-r-c-f-v-vmentioning

confidence: 99%

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Duca

Rose

Glick

2014

Appl Environ Microbiol

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“…by producing surfactant on hydrophobic substrate demonstrated high resistance towards cell lysing. There is even a published report previously stating that protein extraction using ultrasonification method of Rhodococcus strain seems to be less effective due to high rigidity and odd chemical composition of the cell membrane (Prasad et al 2009). Considering these, a comparison study to determine effective cell lysing method was esteemed to be necessary.…”

Section: Resultsmentioning

confidence: 99%

Rhodococcus UKMP-5M, an endogenous lipase producing actinomycete from Peninsular Malaysia

2013

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Escalation in food industries unctuous wastes has led to serious anthropogenic problems to the environment. Parallel to "green strategy", growing awareness in biological treatment emphasizes efficacy of enzymatic technology for bioremediation. Pertinently, researchers are in search for new lipase-lipid interaction for improved outcome. Rhodococcus species have documented inadequate evidences on lipase enzyme production. Consequent assessments on Rhodococcus isolates from Peninsular Malaysia have identified twelve promising strains as lipase producer. Interestingly, apart from usual lipolytic behaviour, Rhodococcus sp. exhibited significant level of lipase endogenously, while cryogenic grinding method effectively ruptured the cell. An isolate from petroleum-contaminated site, namely Rhodococcus UKMP-5M, projected the highest level of lipase specificity and has further been optimized. It was found out that the best specificity was apparent in acidic condition (pH 5) with 6% inoculum at 30• C for 72 hours of incubation. Due to high level of mycolic cell-surfactant developed in triacylglycerol supplements, cell lysis was employed with Triton X-100 detergent solubilisation. As a result, oil blend composed of various carbon-chain length fatty acids (composite 2) induces enzyme production extensively. Remarkably, R. UKMP-5M found to cater enzyme production without aid of inducer by nature, but additional carbon source like glucose represses lipase production. Further ability for biological treatment was revealed when the optimized R. UKMP-5M whole cell degraded waste cooking oil significantly by solubilizing fatty acids and commencing conversion into biomass. These qualities resemble practical new lipid-lipase biological lipid rich on-site treatment.

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“…The specific activity of RZ44 nitrilase was about 616 and 1383 U.mg -1 for acetonitrile and acetamide, respectively. The specific activities for R. rhodochrous J1 (115 U.mg -1 dcw) (17), R. pyridinovorans MW3 (97 U.mg -1 dcw) (46), B. imperialis CBS 498-74 (47 U.mg -1 dcw) (47), and R. rhodochrous PA-34 (55.0 U.mg -1 dcw) (25) have been reported. These results indicated that RZ44 has high substrate tolerance and higher nitrilase activity.…”

Section: Discussionmentioning

confidence: 99%

Production and Characterization of a Nitrilase from Pseudomonas aeruginosa RZ44 and its Potential for Nitrile Biotransformation

Badoei-dalfard

Ramezani-pour

Karami

2016

Iran. J. Biotechnol.

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Background The conversion of nitriles into amides or carboxylic acids by nitrilase has taken its application into consideration, as the scope of its applications has recently been extended. Objectives In this study, P. aeruginosa RZ44 was isolated from sewage in the Kerman which has Nitrile-degradation activity. In order to improve the nitrilase production, several optimization were done on environmental condition. Nitrilase activity was characterized against different pHs, temperatures, ions, and substrates. Materials and Methods Enzyme activity was evaluated by determining the production of ammonia following to the modification of the phenol/hypochlorite method. Different factors that affect production of the enzyme by P. aeruginosa RZ44 were optimized and evaluated in the culture mediums. Results The results showed that degradation of the acetonitrile by P. aeruginosa RZ44 increased the pH of the growth medium from the initial pH 7.0 to 9.37. Optimizing the medium for P. aeruginosa RZ44, it was found that glucose and starch (5 g.L-1) have strongly supported nitrilase production, compared to the control. As well, urea (5 g.L-1) and yeast extract (15 g.L-1) have favored an increased biomass and nitrilase production, as the nitrogen sources. These results show that nitrilase production increases in the pH range 5.0 to 7.0 and then start decreasing. Addition of the Mg2+, Fe2+ and Na+ has supported the biomass and nitrilase production. Co2+, Mn2+ and Cu2+ were confirmed to inhibit cell growth and enzyme production. Enzyme characterization results show that, P. aeruginosa RZ44 nitrilase exhibits comparatively high activity and stability at pH 7.0 and 40°C. Nitrilase was completely inhibited by CoCl2 and CaCl2, whereas, the inhibition in the presence of MnSO4 and CuSO4 was about 60%. Time course analysis of the nitrile conversion by the resting P. aeruginosa RZ44 cells showed that nitrile substrates (i.e. acetonitrile) was hydrolyzed within 8 h. Conclusions these results indicate that P. aeruginosa RZ44 has the potential to be applied in the biotransformation of nitrile compounds.

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Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34

Cited by 24 publications

References 18 publications

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Rhodococcus UKMP-5M, an endogenous lipase producing actinomycete from Peninsular Malaysia

Production and Characterization of a Nitrilase from Pseudomonas aeruginosa RZ44 and its Potential for Nitrile Biotransformation

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