A rapid and sensitive fluorometric assay method for the determination of nitrilase activity

Banerjee, Anirban; Sharma, Rohit K.; Banerjee, Uttam Chand

doi:10.1042/ba20020106

Cited by 54 publications

(26 citation statements)

References 12 publications

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“…The reaction was stopped by adding 0.1 N HCl. The reaction mixture was centrifuged at 10,000 g for 10 min, and the clear supernatant obtained was utilized for ammonia estimation by fluorimetric method [11].…”

Section: Enzyme Assaymentioning

confidence: 99%

Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study

Singh

Banerjee

Kaul

et al. 2005

Bioprocess Biosyst Eng

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Nitrilases constitute an important class of hydrolases, however, cheap and ready availability of enzyme sources limit their practical synthetic applications. The present investigation was directed to compare the applicability of various physical cell disintegration methods namely, solid shear, liquid shear and sonication, for the release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126. Different parameters associated with each method were optimized in order to ensure maximal release of active nitrilase. The methods were also compared under optimal conditions for their efficiency of nitrilase release and extent of cell disruption, and enzyme release were visualized under a differential interference contrast microscope (DIC) and SDS-PAGE, respectively. Maximum release of the enzyme protein from the cells was observed in case of liquid shear method employing high-pressure homogenization, however, the specific activity of nitrilase was highest in cell-free extract (CFE) generated by sonication. Both the solid shear and liquid shear proved to be equally effective for maximum release of intracellular enzymes, however, from the specific activity point of view, sonication was found to be a better one compared to other two methodologies. The generated cell-free extract can be further employed for the production of enantiopure chiral carboxylic acids, which are important chiral building blocks.

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Section: Enzyme Assaymentioning

confidence: 99%

Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study

Singh

Banerjee

Kaul

et al. 2005

Bioprocess Biosyst Eng

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“…A nitrilase assay has been reported in which the released ammonia reacts with o-phthaldialdehyde and 2-mercaptoethanol to form a fluorescent isoindole derivative [37]. The method is simple and uses commercially available reagents, however, the signal requires very high concentrations of substrate (100 mM) to be detectable, implying that only very potent enzymes can be detected.…”

Section: Other Chromophoresmentioning

confidence: 99%

Enzyme assays for high-throughput screening

Goddard

Reymond

2004

Current Opinion in Biotechnology

242

123

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“…[31] Several analytical methods have been developed and used for the determination of PA, including chromatographic, [31][32][33] spectrophotometric [34,35] and fluorometric methods. [36,37] Although all of these methods have shown sensitivity and specificity for the detection of PA, they are for the most part cumbersome, time-consuming, expensive, and require complicated clean-up procedures or sophisticated technical equipment. As a result, there is considerable interest in the development of alternative technologies for rapid, sensitive, and inexpensive detection of PA, with the ideal situation being that detection could be visualized with the naked eye by using a pH-like test paper.…”

Section: Introductionmentioning

confidence: 99%

Hydrogel Photonic Sensor for the Detection of 3‐Pyridinecarboxamide

Yuan

Liu

et al. 2011

Chemistry A European J

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Molecular imprinting is recognized as a powerful technique for preparing polymeric structures that contains tailor-made recognition sites for certain molecules. By combining a responsive hydrogel photonic crystal and molecular imprinting of 3-pyridinecarboxamide, polyacrylamide (PAM) imprinted photonic crystals (IPCs) with an inverse opal structure were prepared. They showed a rapid, recoverable, and selective response to 3-pyridinecarboxamide, which was detected by measuring the diffraction peak. The position of the diffraction peak could be tuned by copolymerizing acrylamide (AM) with acrylic acid (AA), by changing the ratio of AM to AA, by adding N,N'-methylene bisacrylamide to the monomers, or by imprinting molecules to monomers. More interestingly, the change in the Bragg diffraction of the IPCs can be directly converted into a readable optical signal that is visible to the naked eye without any labeling treatment.

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A rapid and sensitive fluorometric assay method for the determination of nitrilase activity

Cited by 54 publications

References 12 publications

Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study

Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study

Enzyme assays for high-throughput screening

Hydrogel Photonic Sensor for the Detection of 3‐Pyridinecarboxamide

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