Skip to main content
Log in

Cloning and characterization of a novel thermostable amidase, Xam, from Xinfangfangia sp. DLY26

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

Objective

Identification and characterization of a novel thermostable amidase (Xam) with wide pH tolerance and broad-spectrum substrate specificity.

Results

Xam was identified from non-thermophilic Xinfangfangia sp. DLY26 and its acyl transfer activity was investigated. Recombinant Xam was optimally active at 60 °C and pH 9.0. The enzyme had a half life of 18 h at 55 °C and maintained more than 60 % of its maximum activity in the range of pH 3.0–11.0. Additionally, Xam exhibited broad substrate specificity towards aliphatic, aromatic, and heterocyclic amides.

Conclusions

These unique properties make Xam a promising biocatalyst for production of important hydroxamic acids at elevated temperatures.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Baek DB, Song JJ, Lee SG, Kwon SJ, Asano Y, Sung MH (2003) New thermostable d-methionine amidase from Brevibacillus borstelensis BCS-1 and its application for d-phenylalanine production. Enzym Microb Technol 32:131–139

    Article  CAS  Google Scholar 

  • Banerjee A, Sharma R, Banerjee UC (2002) The nitrile-degrading enzymes: current status and future prospects. Appl Microbiol Biotechnol 60:33–44

    Article  CAS  Google Scholar 

  • Bhatia RK, Bhatia SK, Kumar V, Bhalla TC (2015) Bi-substrate kinetic analysis of acyl transfer activity of purified amidase from Pseudomonas putida BR-1. Catal Lett 145:1033–1040

    Article  CAS  Google Scholar 

  • d’Abusco AS, Ammendola S, Scandurra R, Politi L (2001) Molecular and biochemical characterization of the recombinant amidase from hyperthermophilic archaeon Sulfolobus solfataricus. Extremophiles 5:183–192

    Article  Google Scholar 

  • Dakshina J, Mark B, Paul RM, Trevor SB, Richard CW (2003) CyD the cyanide dihydratase from Bacillus pumilus: gene cloning and structural studies. Appl Environ Microbiol 69:4794–4805

    Article  CAS  Google Scholar 

  • Egorova K, Trauthwein H, Verseck S, Antranikian G (2004) Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila. Appl Microbil Biotechnol 65:38–45

    CAS  Google Scholar 

  • Guo FM, Wu JP, Yang LR, Xu G (2015) Soluble and functional expression of a recombinant enantioselective amidase from Klebsiella oxytoca KCTC 1686 in Escherichia coli and its biochemical characterization. Process Biochem 50:1264–1271

    Article  CAS  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    Article  CAS  Google Scholar 

  • Labahn J, Neumann S, Buldt G, Kula MR, Granzin J (2002) An alternative mechanism for amidase signature enzymes. J Mol Biol 322:1053–1064

    Article  CAS  Google Scholar 

  • Liu DJ, Xi LJ, Han DY, Dou K, Su SJ, Liu JG (2019) Clong, expression, and characterization of a novel nitrilase, PaCNit, from Pannonibacter carbonis Q4.6. Biotechnol Lett 41:583–589

    Article  CAS  Google Scholar 

  • Nawaz MS, Khan AA, Bhattacharayya D, Siitonen PH, Cerniglia CE (1996) Physical, biochemical, and immunological characterization of a thermostable amidase from Klebsiella pneumonia NCTR 1. J Bacteriol 178:2397–2401

    Article  CAS  Google Scholar 

  • Ruan LT, Zheng RC, Zheng YG (2016a) A novel amidase from Brevibacterium epidermidis ZJB-07021: gene cloning, refolding and application in butyrylhydroxamic acid synthesis. J Ind Microbiol Biotechnol 43:1071–1083

    Article  CAS  Google Scholar 

  • Ruan LT, Zheng RC, Zheng YG (2016b) Mining and characterization of two amidase signature family amidases from Brevibacterium epidermidis ZJB-07021 by an efficient genome mining approach. Protein Expr Purif 126:16–25

    Article  CAS  Google Scholar 

  • Santoshkumar M, Ismailsab M, Nayak AS, Mashetty SB, Karegoudar TB (2017) Purification and characterization of amidase from Paracoccus sp. SKG: utilization of amidase-inhibited whole cells for bioconversion of acrylonitrile to acrylamide. Biocatal Agric Biotechnol 10:256–263

    Article  Google Scholar 

  • Sedmak JJ, Grossberg SE (1977) A rapid, sensitive and vertile assay for protein using Coomassia Brilliant Blue G250. Anal Biochem 79:544–552

    Article  CAS  Google Scholar 

  • Sharma M, Sharma NN, Bhalla TC (2009) Amidases: versatile enzymes in nature. Rev Environ Sci Biotechnol 8:343–366

    Article  CAS  Google Scholar 

  • Sharma M, Sharma NN, Bhalla TC (2013) Purification studies on thermo-active amidase of Geobacillus pallidus BTP-5x MTCC 9225 isolated from thermal springs of Tatapani (Himachal Pradesh). Appl Biochem Biotechnol 169:1–14

    Article  CAS  Google Scholar 

  • Shen WL, Chen HH, Jia KZ, Ni J, Yan X, Li SP (2012) Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities. Appl Microbiol Biotechnol 94:1007–1018

    Article  CAS  Google Scholar 

  • Sonke T, Ernste S, Tandler RF, Kaptein B, Peeters WPH, Assema FBJ, Wubbolts MG, Schoemaker HE (2005) L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321. Appl Environ Microbiol 71:7961–7973

    Article  CAS  Google Scholar 

  • Stelkes-Ritter U, Wyzgol K, Kula MR (1995) Purification and characterization of a newly screened microbial peptide amidase. Appl Microbiol Biotechnol 44:393–398

    Article  CAS  Google Scholar 

  • Suzuki Y, Ohta H (2006) Identification of a thermostable and enantioselective amidase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. Protein Expr Purif 45:368–373

    Article  CAS  Google Scholar 

  • Wu ZM, Zheng RC, Zheng YG (2016) Exploitation and characterization of three versatile amidase super family members from Delftia tsuruhatensis ZJB-05174. Enzym Microb Technol 86:93–102

    Article  CAS  Google Scholar 

  • Wu ZM, Zheng RC, Zheng YG (2017) Identification and characterization of a novel amidase signature family amidase from Parvibaculum lavamentivorans ZJB14001. Protein Expr Purif 129:60–68

    Article  CAS  Google Scholar 

Download references

Supporting information

Supplementary Fig. 1—Sequence alignment of Xam with other amidases. Conserved residues are marked with black arrow. (Xam: Xinfangfangia sp. DLY26; YP_145063.1: Thermus thermophilus HB8; NP_229077.1: Thermotoga maritima MSB8; BAA36596.1: Rhodococcus sp. N-771; AET98897.1: Paracoccus sp. M-1; CAD36560.1: Rhodococcus erythropolis; WP_045444860.1: Tepidicaulis marinus).

Supplementary Fig. 2—SDS-PAGE analysis of amidase samples. M, protein marker; I, soluble extract of induced recombinant strain; II, purified Xam.

Supplementary Fig. 3—Elution profile of the purified Xam on column Superose 6 10/300 GL. 1, Ferritin (440 kDa); 2, Glucose oxidase (160 kDa); 3, BSA (66.4 kDa); 4, Albumin (45 kDa); 5, α-chymotrypsin ogen A (25.7 kDa); 6, Lysozyme (14 kDa).

Funding

This work was supported by the National Natural Science Foundation of China (Nos. 21473256, 3140005), the Key Research and Development Project of Shandong Province (Nos. 2019GSF107077, 2019GGX102062) and the Fundamental Research Funds for the Central Universities of China (No. 18CX05015A).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jianguo Liu.

Ethics declarations

Conflict of interest

The authors declare no conflict of interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 554 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xi, L., Tan, W., Li, J. et al. Cloning and characterization of a novel thermostable amidase, Xam, from Xinfangfangia sp. DLY26. Biotechnol Lett 43, 1395–1402 (2021). https://doi.org/10.1007/s10529-021-03124-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-021-03124-y

Keywords

Navigation