Skip to main content
SpringerLink
Log in

Cloning, expression, and characterization of an alkaline protease, AprV, from Vibrio sp. DA1-1

  • Research Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

A novel alkaline protease (named AprV) gene from Vibrio sp. DA1-1 was cloned and expressed in Escherichia coli BL21 (DE3) pLysS. The sequence analysis showed the highest homology of 68% with the characterized protease from Alkalimonas collagenimarina AC40T. The recombinant AprV was purified with the molecular weight of 28 kDa. The optimum temperature and pH were determined to be 55 °C and 10.0, respectively. The enzyme activity was slightly enhanced by Ca2+, Mg2+, Zn2+, Ba2+, and, however, was highly inhibited by Sn2+ and EDTA. The AprV was stable in the presence of some surfactants and oxidizing agents, such as 1% Tween 20–80, 1% JFC-2, and 5% JFC-2. Casein was found to be the ideal substrate with specific activity of 1139 U/mg. Moreover, we found that AprV (10,000 U), together with commercial detergent, could completely remove the blood on the cotton. Furthermore, AprV also demonstrated dehairing activity on goat and bull skin. These results indicated that the alkaline protease AprV might be a potential candidate for applications in the detergent and leather industries.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Guleria S, Walia A, Chauhan A, Shirkot CK (2016) Purification and characterization of detergent stable alkaline protease from Bacillus amyloliquefaciens SP1 isolated from apple rhizosphere. J Basic Microbiol 56(2):138–152

    Article  CAS  PubMed  Google Scholar 

  2. Abdel-Naeem HHS, Mohamed HMH (2016) Improving the physico-chemical and sensory characteristics of camel meat burger patties using ginger extract and papain. Meat Sci 118:52–60

    Article  CAS  PubMed  Google Scholar 

  3. Ha M, Bekhit AEA, Carne A, Hopkins DL (2012) Characterisation of commercial papain, bromelain, actinidin and zingibain protease preparations and their activities toward meat proteins. Food Chem 134(1):95–105

    Article  CAS  Google Scholar 

  4. Stenn KS, Link R, Moellmann G, Madri J, Kuklinska E (1989) Dispase, a neutral protease from Bacillus Polymyxa, Is a powerful fibronectinase and type IV collagenase. J Investig Dermatol 93(2):287–290

    Article  CAS  PubMed  Google Scholar 

  5. Seite S, Colige A, Piquemal-Vivenot P, Montastier C, Fourtanier A, Lapiere C, Nusgens B (2000) A full-UV spectrum absorbing daily use cream protects human skin against biological changes occurring in photoaging. Photodermatol Photoimmunol Photomed 16(4):147–155

    Article  CAS  PubMed  Google Scholar 

  6. Senthilvelan T, Kanagaraj J, Mandal AB (2012) Application of enzymes for dehairing of skins: cleaner leather processing. Clean Tchnol Envi 14(5):889–897

    Article  CAS  Google Scholar 

  7. Hu J, Xiao Z, Zhou R, Deng W, Wang M, Ma S (2011) Ecological utilization of leather tannery waste with circular economy model. J Clean Prod 19(2–3):221–228

    Article  CAS  Google Scholar 

  8. Ganesh Kumar C, Hiroshi T (1999) Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Adv 17:561–594

    Article  Google Scholar 

  9. Niyonzima FN, More S (2015) Detergent-compatible proteases: microbial production, properties, and stain removal analysis. Prep Biochem Biotechnol 45(3):233–258

    Article  CAS  PubMed  Google Scholar 

  10. Hmidet N, El-Hadj Ali N, Haddar A, Kanoun S, Alya S-K, Nasri M (2009) Alkaline proteases and thermostable α-amylase co-produced by Bacillus licheniformis NH1: Characterization and potential application as detergent additive. Biochem Eng J 47(1–3):71–79

    Article  CAS  Google Scholar 

  11. Haddar A, Agrebi R, Bougatef A, Hmidet N, Sellami-Kamoun A, Nasri M (2009) Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: purification, characterization and potential application as a laundry detergent additive. Bioresour Technol 100(13):3366–3373

    Article  CAS  PubMed  Google Scholar 

  12. Beg QK, Rani Gupta (2003) Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme Microb Tech 32:294–304

    Article  CAS  Google Scholar 

  13. Taylor MM, Bailey DG, Feairheller SH (1987) A review of the uses of enzymes in the tannery. J Am Leather Chem Assoc 82(6):153–164

    CAS  Google Scholar 

  14. Sundararajan S, Kannan CN, Chittibabu S (2011) Alkaline protease from Bacillus cereus VITSN04: potential application as a dehairing agent. J Biosci Bioeng 111(2):128–133

    Article  CAS  PubMed  Google Scholar 

  15. Muhammad Nadeem JI, Qazi S Baig (2010) Enhanced production of alkaline protease by a mutant of Bacillus licheniformis N-2 for dehairing. Braz Arch of biol techn 53:1015–1025

    Article  Google Scholar 

  16. Pillai P, Archana G (2008) Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate. Appl Microbiol Biotechnol 78(4):643–650

    Article  CAS  PubMed  Google Scholar 

  17. Fang Z, Yong YC, Zhang J, Du G, Chen J (2017) Keratinolytic protease: a green biocatalyst for leather industry. Appl Microbiol Biotechnol 101(21):7771–7779

    Article  CAS  PubMed  Google Scholar 

  18. Orkin S, Sambrook J, Fritsch E, Maniatis T (1990) Molecular cloning a laboratory manual, 2nd edn Nature 343(6259):604–605

    Article  Google Scholar 

  19. Choi HK, Choi KH, Kramer KJ, Muthukrishnan S (1997) Isolation and characterization of a genomic clone for the gene of an insect molting enzyme, chitinase. Insect Biochem Mol Biol 27(1):37–47

    Article  CAS  PubMed  Google Scholar 

  20. Engleberg NC, Drutz DJ, Eisenstein BI (1984) Cloning and expression of legionella pneumophila antigens in Escherichia coli. Infect Immun 44(2):222–227

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Yamamura S, Morita Y, Hasan Q, Rao SR, Murakami Y, Yokoyama K, Tamiya E (2002) Characterization of a new keratin-degrading bacterium isolated from deer fur. J Biosci Bioeng 93(6):595–600

    Article  CAS  PubMed  Google Scholar 

  22. 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 72:248–254

    Article  CAS  PubMed  Google Scholar 

  23. Jankiewicz U, Larkowska E, Swiontek Brzezinska M (2016) Production, characterization, gene cloning, and nematocidal activity of the extracellular protease from Stenotrophomonas maltophilia N4. J Biosci Bioeng 121(6):614–618

    Article  CAS  PubMed  Google Scholar 

  24. Bouacem K, Bouanane-Darenfed A, Laribi-Habchi H, Elhoul MB, Hmida-Sayari A, Hacene H, Ollivier B, Fardeau ML, Jaouadi B, Bejar S (2015) Biochemical characterization of a detergent-stable serine alkaline protease from Caldicoprobacter guelmensis. Int J Biol Macromol 81:299–307

    Article  CAS  PubMed  Google Scholar 

  25. Kurata A, Uchimura K, Shimamura S, Kobayashi T, Horikoshi K (2007) Nucleotide and deduced amino acid sequences of a subtilisin-like serine protease from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T). Appl Microbiol Biotechnol 77(2):311–319

    Article  CAS  PubMed  Google Scholar 

  26. Kurata A, Uchimura K, Kobayashi T, Horikoshi K (2010) Collagenolytic subtilisin-like protease from the deep-sea bacterium Alkalimonas collagenimarina AC40T. Appl Microbiol Biotechnol 86(2):589–598

    Article  CAS  PubMed  Google Scholar 

  27. Miyamoto K, Nukui E, Itoh H, Sato T, Kobayashi T, Imada C, Watanabe E, Inamori Y, Tsujibo H (2002) Molecular analysis of the gene encoding a novel chitin-binding protease from Alteromonas sp. strain O-7 and its role in the chitinolytic system. J Bacteriol 184(7):1865–1872

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nancy George S, Sondhi, Sanjeev Kumar Soni (2014) Lime and sulphide-free dehairing of animal skin using collagenase-free alkaline protease from Vibrio metschnikovii NG155. Indian J Microbiol 54(2):139–142

    Article  CAS  PubMed  Google Scholar 

  29. Wani AH, Sharma M, Salwan R, Singh G, Chahota R, Verma S (2016) Cloning, expression, and functional characterization of serine protease Aprv2 from virulent isolate dichelobacter nodosus of indian origin. Appl Biochem Biotechnol 180(3):576–587

    Article  CAS  PubMed  Google Scholar 

  30. Fu X, Inouye M, Shinde U (2000) The inhibitory and chaperone functions of the subtilisin propeptide are not obligatorily linked. J Biol Chem 275:16871–16878

    Article  CAS  PubMed  Google Scholar 

  31. Jalkute CB, Waghmare SR, Nadaf NH, Dhanavade MJ, Jadhav DB, Pendhari SI, Patil RS, Sonawane KD (2017) Purification and characterization of SDS stable protease from Bacillus safensis strain CK. Biocatal Agric Biotechnol 10:91–95

    Google Scholar 

  32. Salihi A, Asoodeh A, Aliabadian M (2017) Production and biochemical characterization of an alkaline protease from Aspergillus oryzae CH93. Int J Biol Macromol 94:827–835

    Article  CAS  PubMed  Google Scholar 

  33. Mashayekhi Mazar F, Shahbaz Mohammadi H, Ebrahimi-Rad M, Gregorian A, Omidinia E (2012) Isolation, purification and characterization of a thermophilic alkaline protease from Bacillus subtilis BP-36. J Sci Islam Repub Iran 23(1):7–13

    Google Scholar 

  34. Hammami A, Hamdi M, Abdelhedi O, Jridi M, Nasri M, Bayoudh A (2017) Surfactant and oxidant-stable alkaline proteases from Bacillus invictae: characterization and potential applications in chitin extraction and as a detergent additive. Int J Biol Macromol 96:272–281

    Article  CAS  PubMed  Google Scholar 

  35. Vojcic L, Pitzler C, Korfer G, Jakob F, Ronny M, Maurer KH, Schwaneberg U (2015) Advances in protease engineering for laundry detergents. N Biotechnol 32(6):629–634

    Article  CAS  PubMed  Google Scholar 

  36. Anwar A, Saleemuddin M (1998) Alkaline proteases: a review. Bioresour Technol 64:175–183

    Article  CAS  Google Scholar 

  37. Gao R, Shi T, Liu X, Zhao M, Cui H, Yuan L (2017) Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum. J Sci Food Agric 97(5):1412–1419

    Article  CAS  PubMed  Google Scholar 

  38. Souza PM, Werneck G, Aliakbarian B, Siqueira F, Ferreira Filho EX, Perego P, Converti A, Magalhaes PO, Junior AP (2017) Production, purification and characterization of an aspartic protease from Aspergillus foetidus. Food Chem Toxicol 109(Pt 2):1103–1110

    Article  CAS  PubMed  Google Scholar 

  39. Uttatree S, Charoenpanich J (2016) Isolation and characterization of a broad pH and temperature active, solvent and surfactant stable protease from a new strain of Bacillus subtilis. Biocatal Agric Biotechnol 8:32–38

    Google Scholar 

  40. Salwan R, Sharma V, Pal M, Kasana RC, Yadav SK, Gulati A (2018) Heterologous expression and structure-function relationship of low-temperature and alkaline active protease from Acinetobacter sp. IHB B 5011(MN12). Int J Biol Macromol 107(Pt A):567–574

    Article  CAS  PubMed  Google Scholar 

  41. Mhamdi S, Bkhairia I, Nasri R, Mechichi T, Nasri M, Kamoun AS (2017) Evaluation of the biotechnological potential of a novel purified protease BS1 from Bacillus safensis S406 on the chitin extraction and detergent formulation. Int J Biol Macromol 104(Pt A):739–747

    Article  CAS  PubMed  Google Scholar 

  42. Pessoa TBA, Rezende RP, Marques ELS, Pirovani CP, Dos Santos TF, Dos Santos Goncalves AC, Romano CC, Dotivo NC, Freitas ACO, Salay LC, Dias JCT (2017) Metagenomic alkaline protease from mangrove sediment. J Basic Microbiol 57(11):962–973

    Article  CAS  PubMed  Google Scholar 

  43. Ben Elhoul M, Zarai Jaouadi N, Rekik H, Omrane Benmrad M, Mechri S, Moujehed E, Kourdali S, El Hattab M, Badis A, Bejar S, Jaouadi B (2016) Biochemical and molecular characterization of new keratinoytic protease from Actinomadura viridilutea DZ50. Int J Biol Macromol 92:299–315

    Article  CAS  PubMed  Google Scholar 

  44. Bouacem K, Bouanane-Darenfed A, Zarai Jaouadi N, Joseph M, Hacene H, Ollivier B, Fardeau ML, Bejar S, Jaouadi B (2016) Novel serine keratinase from Caldicoprobacter algeriensis exhibiting outstanding hide dehairing abilities. Int J Biol Macromol 86:321–328

    Article  CAS  PubMed  Google Scholar 

  45. Ellaiah P, Srinivasulu B, Adinarayana K (2002) A review on microbial alkaline proteases. J Sci Ind Res India 61:690–704

    CAS  Google Scholar 

  46. Boulkour Touioui S, Zarai Jaouadi N, Bouacem K, Ben Ayed R, Rekik H, Zenati B, Kourdali S, Boudjella H, Sabaou N, Bejar S, El Hattab M, Badis A, Annane R, Jaouadi B (2018) Biochemical and molecular characterization of a novel metalloprotease from Pseudomonas fluorescens strain TBS09. Int J Biol Macromol 107(Pt B):2351–2363

    Article  CAS  PubMed  Google Scholar 

  47. Bagliniere F, Salgado RL, Salgado CA, Vanetti MC (2017) Biochemical characterization of an extracellular heat-stable protease from Serratia liquefaciens isolated from raw milk. J Food Sci 82(4):952–959

    Article  CAS  PubMed  Google Scholar 

  48. Devi SG, Fathima AA, Sanitha M, Iyappan S, Curtis WR, Ramya M (2016) Expression and characterization of alkaline protease from the metagenomic library of tannery activated sludge. J Biosci Bioeng 122(6):694–700

    Article  CAS  PubMed  Google Scholar 

  49. Ghafoori H, Askari M, Sarikhan S (2016) Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis (X5B). Extremophiles 20(2):115–123

    Article  CAS  PubMed  Google Scholar 

  50. Jeong YJ, Baek SC, Kim H (2017) Cloning and characterization of a novel intracellular serine protease (IspK) from Bacillus megaterium with a potential additive for detergents. Int J Biol Macromol 27(5):1136–1140

    Google Scholar 

  51. Yildirim V, Baltaci MO, Ozgencli I, Sisecioglu M, Adiguzel A, Adiguzel G (2017) Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents. J Enzyme Inhib Med Chem 32(1):468–477

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Haddar A, Bougatef A, Agrebi R, Sellami-Kamoun A, Nasri M (2009) A novel surfactant-stable alkaline serine-protease from a newly isolated Bacillus mojavensis A21 purification and characterization. Process Biochem 44(1):29–35

    Article  CAS  Google Scholar 

  53. Mechri S, Ben Elhoul Berrouina M, Omrane Benmrad M, Zarai Jaouadi N, Rekik H, Moujehed E, Chebbi A, Sayadi S, Chamkha M, Bejar S, Jaouadi B (2017) Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest. Int J Biol Macromol 94(Pt A):221–232

    Article  CAS  PubMed  Google Scholar 

  54. Chung S-S, Shin Y-U, Kim H-J, Jin G-H, Rho H-M, Lee H-H (2000) Isolation of high yielding alkaline protease mutants of Vibrio metschnikovii strain RH530 and detergency properties of enzyme. J Microbiol Biotechnol 10(3):349–354

    CAS  Google Scholar 

  55. Jaouadi NZ, Rekik H, Badis A, Trabelsi S, Belhoul M, Yahiaoui AB, Ben Aicha H, Toumi A, Bejar S, Jaouadi B (2013) Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities. PLoS One 8(10):e76722

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Xu W, Zhang J, Hao L (2009) Hair-saving enzyme-assisted unhairing effects of sodium hydrosulfide and peroxide. Proc XXX IULTCS Congress 2009:1–6

    Google Scholar 

  57. Sivasubramanian S, Manohar BM, Rajaram A, Puvanakrishnan R (2008) Ecofriendly lime and sulfide free enzymatic dehairing of skins and hides using a bacterial alkaline protease. Chemosphere 70(6):1015–1024

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (no. 21676121) and the National High Technology Research and Development Program of China (2012AA022204).

Author information

Authors and Affiliations

  1. School of Pharmaceutical Science, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Xiujuan Chen & Jinsong Shi

  2. State Key Lab of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, China

    Cheng Zhou, Yanfen Xue & Yanhe Ma

  3. National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

    Cheng Zhou, Yanfen Xue & Yanhe Ma

Authors
  1. Xiujuan Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cheng Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yanfen Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jinsong Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yanhe Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Cheng Zhou or Jinsong Shi.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, X., Zhou, C., Xue, Y. et al. Cloning, expression, and characterization of an alkaline protease, AprV, from Vibrio sp. DA1-1. Bioprocess Biosyst Eng 41, 1437–1447 (2018). https://doi.org/10.1007/s00449-018-1972-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-018-1972-6

Keywords

Search

Navigation