Advertisement

Applied Microbiology and Biotechnology

, Volume 98, Issue 17, pp 7491–7499 | Cite as

Purification of an amide hydrolase DamH from Delftia sp. T3-6 and its gene cloning, expression, and biochemical characterization

  • Fei Wang
  • Ying Hou
  • Jie Zhou
  • Zhoukun Li
  • Yan Huang
  • Zhongli Cui
Biotechnologically relevant enzymes and proteins

Abstract

A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, diethylaminoethyl anion exchange, hydrophobic interaction chromatography, and Sephadex G-200 gel filtration. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The sequence of the N-terminal 15 amino acid residues was determined to be Gly-Thr-Ser-Pro-Gln-Ser-Asp-Phe-Leu-Arg-Ala-Leu-Phe-Gln-Ser. Based on the N-terminal sequence and results of peptide mass fingerprints, the gene (damH) was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5,036 U/mg for 2′-methyl-6′-ethyl-2- chloroacetanilide (CMEPA) (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with K m and k cat values of 0.197 mM and 2,804.32 s−1, respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35 °C, respectively; the enzyme was activated by Mn2+ and inhibited by Cu2+, Zn2+, Ni2+, and Fe2+. DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.

Keywords

Delftia sp. T3-6 Amide hydrolase Purified Cloning Characterization 

Notes

Acknowledgments

Grants from the Natural Science Foundation of Jiangsu Province, China (No. BK2012029), the Natural Science Foundation of China (31270095), and the National Science and Technology Support Program (2012BAD14B02) supported this work.

References

  1. Akutsu-Shigeno Y, Adachi Y, Yamada C, Toyoshima K, Nomura N, Uchiyama H, Nakajima-Kambe T (2006) Isolation of a bacterium that degrades urethane compounds and characterization of its urethane hydrolase. Appl Microbiol Biotechnol 70(4):422–429PubMedCrossRefGoogle Scholar
  2. Arpigny J, Jaeger K (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343:177–183PubMedCentralPubMedCrossRefGoogle Scholar
  3. Bano S, Qader SAU, Aman A, Syed MN, Azhar A (2011) Purification and characterization of novel α-amylase from Bacillus subtilis KIBGE HAS. AAPS PharmSciTech 12(1):255–261PubMedCentralPubMedCrossRefGoogle Scholar
  4. Bornscheuer UT (2002) Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 26(1):73–81PubMedCrossRefGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1):248–254PubMedCrossRefGoogle Scholar
  6. Capel PD, Ma L, Schroyer BR, Larson SJ, Gilchrist TA (1995) Analysis and detection of the new corn herbicide acetochlor in river water and rain. Environ Sci Technol 29(6):1702–1705PubMedCrossRefGoogle Scholar
  7. Cheng Q, Thomas SM, Kostichka K, Valentine JR, Nagarajan V (2000) Genetic analysis of a gene cluster for cyclohexanol oxidation in Acinetobacter sp. strain SE19 by in vitro transposition. J Bacteriol 182(17):4744–4751PubMedCentralPubMedCrossRefGoogle Scholar
  8. Coleman S, Linderman R, Hodgson E, Rose RL (2000) Comparative metabolism of chloroacetamide herbicides and selected metabolites in human and rat liver microsomes. Environ Health Perspect 108(12):1151–1157PubMedCentralPubMedGoogle Scholar
  9. Crump D, Werry K, Veldhoen N, Van Aggelen G, Helbing CC (2002) Exposure to the herbicide acetochlor alters thyroid hormone-dependent gene expression and metamorphosis in Xenopus Laevis. Environ Health Perspect 110(12):1199–1205PubMedCentralPubMedCrossRefGoogle Scholar
  10. Dagnac T, Jeannot R, Mouvet C, Baran N (2002) Determination of oxanilic and sulfonic acid metabolites of acetochlor in soils by liquid chromatography–electrospray ionisation mass spectrometry. J Chromatogr A 957(1):69–77PubMedCrossRefGoogle Scholar
  11. Dictor M-C, Baran N, Gautier A, Mouvet C (2008) Acetochlor mineralization and fate of its two major metabolites in two soils under laboratory conditions. Chemosphere 71(4):663–670PubMedCrossRefGoogle Scholar
  12. Dowd JE, Riggs DS (1965) A comparison of estimates of Michaelis–Menten kinetic constants from various linear transformations. J Biol Chem 240(2):863–869PubMedGoogle Scholar
  13. El-Dib MA, Abdel-Rahman MO, Aly OA (1975) 4-Aminoantipyrine as a chromogenic agent for aromatic amine determination in natural water. Water Res 9(5):513–516CrossRefGoogle Scholar
  14. Grube A, Donaldson D, Kiely T, Wu L (2011) Pesticides industry sales and usage: 2006 and 2007 market estimates. In: Office of Chemical Safety and Pollution Prevention. US EPA, WashingtonGoogle Scholar
  15. Hua N (2011) Amide herbicides formulations and their progress of R&D. Mod Agrochem 10(1):8–15Google Scholar
  16. Ihara F, Kageyama Y, Hirata M, Nihira T, Yamada Y (1991) Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species. J Biol Chem 266(27):18135–18140PubMedGoogle Scholar
  17. Jablonkai I (2000) Microbial and photolytic degradation of the herbicide acetochlor. Int J Environ Anal Chem 78(1):1–8CrossRefGoogle Scholar
  18. Janknecht R, de Martynoff G, Lou J, Hipskind RA, Nordheim A, Stunnenberg HG (1991) Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A 88(20):8972–8976PubMedCentralPubMedCrossRefGoogle Scholar
  19. Kaiser P, Raina C, Parshad R, Johri S, Verma V, Andrabi KI, Qazi GN (2006) A novel esterase from Bacillus subtilis (RRL 1789): purification and characterization of the enzyme. Protein Expr Purif 45(2):262–268PubMedCrossRefGoogle Scholar
  20. Khersonsky O, Roodveldt C, Tawfik DS (2006) Enzyme promiscuity: evolutionary and mechanistic aspects. Curr Opin Chem Biol 10(5):498–508PubMedCrossRefGoogle Scholar
  21. Kolkenbrock S, Parschat K, Beermann B, Hinz H-J, Fetzner S (2006) N-Acetylanthranilate amidase from Arthrobacter nitroguajacolicus Rü61a, an α/β-hydrolase-fold protein active towards aryl-acylamides and-esters, and properties of its cysteine-deficient variant. J Bacteriol 188(24):8430–8440PubMedCentralPubMedCrossRefGoogle Scholar
  22. Kotresha D, Vidyasagar G (2008) Isolation and characterisation of phenol-degrading Pseudomonas aeruginosa MTCC 4996. World J Microbiol Biotechnol 24(4):541–547CrossRefGoogle Scholar
  23. Kourist R, Bartsch S, Fransson L, Hult K, Bornscheuer UT (2008) Understanding promiscuous amidase activity of an esterase from Bacillus subtilis. ChemBioChem 9(1):67–69PubMedCrossRefGoogle Scholar
  24. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685PubMedCrossRefGoogle Scholar
  25. Liu Y, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25(3):674–681PubMedCrossRefGoogle Scholar
  26. Liu Y, Zhang Y, Liu J, Huang D (2006) The role of reactive oxygen species in the herbicide acetochlor-induced DNA damage on Bufo raddei tadpole liver. Aquat Toxicol 78(1):21–26PubMedCrossRefGoogle Scholar
  27. Liu Z, Yang H, Huang Z, Zhou P, Liu S-J (2002) Degradation of aniline by newly isolated, extremely aniline-tolerant Delftia sp. AN3. Appl Microbiol Biotechnol 58(5):679–682PubMedCrossRefGoogle Scholar
  28. Mayaux JF, Cerebelaud E, Soubrier F, Faucher D, Pétré D (1990) Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase. J Bacteriol 172(12):6764–6773PubMedCentralPubMedGoogle Scholar
  29. Mirza IA, Yachnin BJ, Wang S, Grosse S, Bergeron H, Imura A, Iwaki H, Hasegawa Y, Lau PCK, Berghuis AM (2009) Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor. J Am Chem Soc 131(25):8848–8854PubMedCrossRefGoogle Scholar
  30. Nielsen TK, Hildmann C, Dickmanns A, Schwienhorst A, Ficner R (2005) Crystal structure of a bacterial class 2 histone deacetylase homologue. J Mol Biol 354(1):107–120PubMedCrossRefGoogle Scholar
  31. Norwitz G, Keliher PN (1980) Effect of acidity and alkalinity on the distillation of phenol: interferences of aromatic amines and formaldehyde with the 4-aminoantipyrine spectro-photometric method for phenol. Anal Chim Acta 119(1):99–111CrossRefGoogle Scholar
  32. Ohtaki A, Murata K, Sato Y, Noguchi K, Miyatake H, Dohmae N, Yamada K, Yohda M, Odaka M (2010) Structure and characterization of amidase from Rhodococcus sp. N-771: insight into the molecular mechanism of substrate recognition. BBA-Proteins Proteomics 1804(1):184–192PubMedCrossRefGoogle Scholar
  33. Patricelli MP, Lovato MA, Cravatt BF (1999) Chemical and mutagenic investigations of fatty acid amide hydrolase: evidence for a family of serine hydrolases with distinct catalytic properties. Biochemistry 38(31):9804–9812PubMedCrossRefGoogle Scholar
  34. Pohlenz H-D, Boidol W, Schüttke I, Streber WR (1992) Purification and properties of an Arthrobacter oxydans P52 carbamate hydrolase specific for the herbicide phenmedipham and nucleotide sequence of the corresponding gene. J Bacteriol 174(20):6600–6607PubMedCentralPubMedGoogle Scholar
  35. Rashamuse K, Magomani V, Ronneburg T, Brady D (2009) A novel family VIII carboxylesterase derived from a leachate metagenome library exhibits promiscuous β-lactamase activity on nitrocefin. Appl Microbiol Biotechnol 83(3):491–500PubMedCrossRefGoogle Scholar
  36. Saha S, Dutta D, Karmakar R, Ray DP (2012) Structure–toxicity relationship of chloroacetanilide herbicides: relative impact on soil microorganisms. Environ Toxicol Pharmacol 34(2):307–314PubMedCrossRefGoogle Scholar
  37. Sambrook J, Russell D (2001) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  38. Schmidt M, Henke E, Heinze B, Kourist R, Hidalgo A, Bornscheuer UT (2007) A versatile esterase from Bacillus subtilis: cloning, expression, characterization, and its application in biocatalysis. Biotechnol J 2(2):249–253PubMedCrossRefGoogle Scholar
  39. Shen W, Chen H, Jia K, Ni J, Yan X, Li S (2012) Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities. Appl Microbiol Biotechnol 94(4):1007–1018PubMedCrossRefGoogle Scholar
  40. Shin S, Lee T-H, Ha N-C, Koo HM, Kim S-Y, Lee H-S, Kim YS, Oh B-H (2002) Structure of malonamidase E2 reveals a novel Ser-cisSer-Lys catalytic triad in a new serine hydrolase fold that is prevalent in nature. EMBO J 21(11):2509–2516PubMedCentralPubMedCrossRefGoogle Scholar
  41. Syrén P-O, Hult K (2011) Amidases have a hydrogen bond that facilitates nitrogen inversion, but esterases have not. Chemcatchem 3(5):853–860CrossRefGoogle Scholar
  42. Terauchi R, Kahl G (2000) Rapid isolation of promoter sequences by TAIL-PCR: the 5′-flanking regions of Pal and Pgi genes from yams (Dioscorea). Mol Gen Genet 263(3):554–560PubMedCrossRefGoogle Scholar
  43. Van Beilen JB, Mourlane F, Seeger MA, Kovac J, Li Z, Smits TH, Fritsche U, Witholt B (2003) Cloning of Baeyer–Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers. Environ Microbiol 5(3):174–182PubMedCrossRefGoogle Scholar
  44. Wang J-L, Xu J-M, Wu Q, Lv D-S, Lin X-F (2009) Promiscuous enzyme-catalyzed regioselective Michael addition of purine derivatives to α, β-unsaturated carbonyl compounds in organic solvent. Tetrahedron 65(12):2531–2536CrossRefGoogle Scholar
  45. Weatherburn M (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39(8):971–974CrossRefGoogle Scholar
  46. Wei Y-L, Kurihara T, Suzuki T, Esaki N (2003) A novel esterase from a psychrotrophic bacterium, Acinetobacter sp. strain no. 6, that belongs to the amidase signature family. J Mol Catal B Enzym 23(2):357–365CrossRefGoogle Scholar
  47. Xiao N, Jing B, Ge F, Liu X (2006) The fate of herbicide acetochlor and its toxicity to Eisenia fetida under laboratory conditions. Chemosphere 62(8):1366–1373PubMedCrossRefGoogle Scholar
  48. Xu J, Qiu X, Dai J, Cao H, Yang M, Zhang J, Xu M (2006) Isolation and characterization of a Pseudomonas oleovorans degrading the chloroacetamide herbicide acetochlor. Biodegradation 17(3):219–225PubMedCrossRefGoogle Scholar
  49. Xu J, Yang M, Dai J, Cao H, Pan C, Qiu X, Xu M (2008) Degradation of acetochlor by four microbial communities. Bioresour Technol 99(16):7797–7802PubMedCrossRefGoogle Scholar
  50. Yasmin S, D'Souza D (2010) Effects of pesticides on the growth and reproduction of earthworm: a review. Appl Environ Soil Sci 2010(2010):1–9CrossRefGoogle Scholar
  51. Ye C, Wang X, Zheng H (2002) Biodegradation of acetanilide herbicides acetochlor and butachlor in soil. J Environ Sci China 14(4):524–529PubMedGoogle Scholar
  52. Zafeiridou G, Geronikaki A, Papaefthimiou C, Tryfonos M, Kosmidis EK, Theophilidis G (2006) Assessing the effects of the three herbicides acetochlor, 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T) and 2, 4-dichlorophenoxyacetic acid on the compound action potential of the sciatic nerve of the frog (Rana ridibunda). Chemosphere 65(6):1040–1048PubMedCrossRefGoogle Scholar
  53. Zhang J, Sun J-Q, Yuan Q-Y, Li C, Yan X, Hong Q, Li S-P (2011) Characterization of the propanil biodegradation pathway in Sphingomonas sp. Y57 and cloning of the propanil hydrolase gene prpH. J Hazard Mater 196:412–419PubMedCrossRefGoogle Scholar
  54. Zhang J, Yin J-G, Hang B-J, Cai S, He J, Zhou S-G, Li S-P (2012) Cloning of a novel arylamidase gene from Paracoccus sp. strain FLN-7 that hydrolyzes amide pesticides. Appl Environ Microbiol 78(14):4848–4855PubMedCentralPubMedCrossRefGoogle Scholar
  55. Zhou Q-X, Zhang Q-R, Liang J-D (2006) Toxic effects of acetochlor and methamidophos on earthworm Eisenia fetida in phaiozem, northeast China. J Environ Sci-China 18(4):741–745Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Fei Wang
    • 1
    • 2
  • Ying Hou
    • 3
  • Jie Zhou
    • 1
  • Zhoukun Li
    • 1
  • Yan Huang
    • 1
  • Zhongli Cui
    • 1
  1. 1.Key Laboratory of Agricultural Environmental Microbiology, Ministry of AgricultureNanjing Agriculture UniversityNanjingPeople’s Republic of China
  2. 2.College of Bioscience and BioengineeringJiangxi Agriculture UniversityNanchangPeople’s Republic of China
  3. 3.College of Food and BioengineeringHenan University of Science and TechnologyLuoyangPeople’s Republic of China

Personalised recommendations