Abstract
A novel enantioselective amidase screening system was developed and proved to be efficient and accurate. This screening system employed acyl transfer activity of amidase in the presence of hydroxylamine, leading to the formation of hydroxamic acids, followed by spectrophotometric quantification of hydroxamic acid/iron(III) complexes. The enantioselectivities of amidase were evaluated by employing (R, S)-2, 2-dimethyl cyclopropanecarboxamide (1), (S)-2, 2-dimethyl cyclopropanecarboxamide and their mixture as substrates concurrently under the same conditions. To prove the accuracy of the screening system, enantioselectivity of acyl transfer reaction (E T) and that of hydrolytic reaction (E H) was compared. With this method, we obtained eight microorganism strains with enantioselective amidase from 523 isolates, two of which showed R-stereospecific avtivity for (R, S)-1.
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References
Asano Y, Tachibana M, Tani Y, Yamada H (1982) Purification and characterization of amidase which participates in nitrile degradation. Agric Biol Chem 46:1175–1181
Banerjee A, Sharma R, Banerjee UC (2002) The nitrile-degrading enzymes: current status and future prospects. Appl Microbiol Biotechnol 60:33–44
Baumann M, Sturmer R, Bornscheuer UT (2001) A high-throughput-screening method for the identification of active and enantioselective hydrolases. Angew Chem Int Ed 40:4201–4203
Bornscheuer UT (2004) High-throughput-screening systems for hydrolases. Eng Life Sci 4:539–542
Buckles RE, Thelen CJ (1950) Qualitative determination of carboxylic esters. Anal Chem 22:676–678
Cawston TE (1996) Metalloproteinase inhibitors and the prevention of connective tissue breakdown. Pharmacol Ther 70:163–182
Chen CS, Fujimoto Y, Girdaukas G, Sih CJ (1982) Quantitative analyses of biochemical kinetic resolutions of enantiomers. J Am Chem Soc 104:7294–7299
Dean KES, Klein G, Renaudet O, Reymond JL (2003) A green fluorescent chemosensor for amino acids provides a versatile high-throughput screening (HTS) assay for proteases. Bioorg Med Chem Lett 13:1653–1656
Duchateau ALL, Hillemans-Crombach MG, van Duijnhoven A, Reiss R, Sonke T (2004) A colorimetric method for determination of amino amidase activity. Anal Biochem 330:362–364
Eichhorn E, Roduit J-P, Shaw N, Heinzmann K, Keiner A (1997) Preparation of (S)-piperazine-2-carboxylic acid, (R)-piperazine-2-carboxylic acid, and (S)-piperdine-2-carboxylic acid by kinetic resolution of the correspondence racemic carboxamides with stereoselective amidase in whole bacterial cells. Tetrahedron Asymmetry 8:2533–2536
Fawcett JK, Scott JE (1960) A rapid and precise method for the determination of urea. J Clin Pathol 13:156–159
Fournand D, Pirat JL, Bigey F, Arnaud A, Galzy P (1997) Spectrophotometric assay of aliphatic monohydroxamic acid and α-, β-, and γ-aminohydroxamic acid in aqueous medium. Anal Chim Acta 353:359–366
Fournand D, Bigey F, Arnaud A (1998a) Acly transfer activity of an amidase from Rhodococcus sp. strain R312: formation of a wide range of hydroxamic acids. Appl Environ Microbiol 64:2844–2852
Fournand D, Arnaud A, Galzy P (1998b) Study of the acyl transfer activity of a recombinant amidase overproduced in an Escherichia coli strain. Application for short-chain hydroxamic acid and acid hydrazide synthesis. J Mol Catal B Enzym 4:77–90
Friedmen M (1999) Chemistry, nutrition, and microbiology of d-amino acids. J Agric Food Chem 47:3475–3479
Graham DW, Ashton WT, Barash L, Brown JE, Brown RD, Canning LF, Chen A, Springer JP, Rogers EF (1987) Inhibition of the mammalian β-lactamase renal dipeptidase (dehydropeptidase-1) by (Z)-2-(acylamino)-3-substitude-propenoic acids. J Med Chem 30:1074–1090
Henke E, Bornscheuer UT (2003) Fluorophoric assay for the high-throughput determination of amidase activity. Anal Chem 75:255–260
Hirrlinger L, Stolz A (1997) Formation of a chiral hydroxamic acid with an amidase from Rhodococcus erythropolis MP50 and subsequent chemical Lossen Rearrangement to a chiral amine. Appl Environ Microbiol 63:3390–3393
Hongpattarkere T, Komeda H, Asano Y (2005) Purification, characterization, gene cloning and nucleotide sequencing of d-stereospecific amino acid amidase from soil bacterium: Delftia acidovorans. J Ind Microbiol Biotechnol 32:567–576
Klein G, Reymond J-L (2001) An enzyme assay using pM. Angew Chem Int Ed 40:1771–1773
Komeda H, Asano Y (2000) Gene cloning, nucleotide sequencing, and purification and characterization of the d-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3. Eur J Biochem 267:2028–2035
Krieg L, Ansorge-Schumacher MB, Kula MR (2002) Screening for amidase: isolation and characterization of a novel d-amidase from Variovorax paradoxus. Adv Synth Catal 344:965–973
Nawaz M, Khan A, Bhattacharya D, Sittoneu P, Cerniglia C (1996) Physical, biochemical and immunological characterization of a thermostable amidase from Klebsiella pneumoniae. J Bacteriol 178:2397–2401
Ozaki A, Kawasaki H, Yagasaki M, Hashimoto Y (1992) Enzymatic production of d-alanine from d,l-alaninamide by novel d-alaninamide specific amide hydrolase. Biosci Biotechnol Biochem 56:1980–1984
Reetz MT (2002) New methods for the high-throughput screening of enantioselective catalysts and biocatalysts. Angew Chem Int Ed 41:1335–1338
Shaw NM, Naughton A, Robins K, Tinschert A et al (2002) Selection, purification, characterisation, and cloning of a novel heat-stable stereo-specific amidase from Klebsiella oxytoca, and its application in the synthesis of enantiomerically pure (R)- and (S)-3,3,3-Trifluoro-2-hydroxy-2-methylpropionic acids and (S)-3,3,3-Trifluoro-2-hydroxy-2-methylpropionamide. Org Process Res Dev 6:497–504
Yamamoto K, Otsubo K, Matsuo A, Hayashi T, Fujimastu I, Komatsu K-I (1996) Production of R-(-)-ketoprofen from an amide compound by Comamonas acidovorans KPO-2771-4. Appl Environ Microbiol 62:152–155
Acknowledgements
This work was supported by the Major Basic Research Development Program of China (No. 2003CB716005), Doctor Program of High Education of China (No.20051033701), and Project of Department of Education of Zhejiang Province of China (No.20051384).
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Zheng, RC., Zheng, YG. & Shen, YC. A screening system for active and enantioselective amidase based on its acyl transfer activity. Appl Microbiol Biotechnol 74, 256–262 (2007). https://doi.org/10.1007/s00253-006-0642-9
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DOI: https://doi.org/10.1007/s00253-006-0642-9