Co-production of Amylase and Protease by Locally Isolated Thermophilic Bacterium Anoxybacillus rupiensis T2 in Sterile and Non-sterile Media Using Waste Potato Peels as Substrate

  • 40 Accesses


The present study investigated the potential of thermophilic bacteria isolated from hot springs to simultaneously produce protease and amylase enzymes. Among ten isolates, the strain T2 was found to be more favorable for amylase and protease. This strain was identified as Anoxybacillus rupiensis (GenBank number: MN252572). Potato peel powder (PPP) was used as a substrate for co-production of amylase and protease from A. rupiensis T2. Experiments were performed under sterile and non-sterile culture conditions. The optimal parameters for co-production of these enzymes were a PPP concentration of 60 g/L, temperature of 50 °C, initial pH of 7.0 and incubation time of 48 h. Under these culture conditions, the amylase and protease activities were determined as 64.9 and 26.2 U/mL in sterile medium. Relatively lower amylase (41.1 U/mL) and protease (14.2 U/mL) activities were attained in non-sterile medium.

Graphic Abstract

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

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 99

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    Salem, R.B., Abbassi, M.S., Cayol, J.L., Bourouis, A., Mahrouki, S., Fardeau, M.L., Belhadj, O.: Thermophilic Bacillus licheniformis rbs 5 isolated from hot tunisian spring co-producing alkaline and thermostable [alpha]-amylase and protease enzymes. J. Microbiol. Biotechnol. Food Sci. 5, 557 (2016)

  2. 2.

    Mala, B., Rao, A.M., Deshpande, V.V.: Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev. 62, 597–635 (1998)

  3. 3.

    Singh, G., Bhalla, A., Kaur, P., Capalash, N., Sharma, P.: Laccase from prokaryotes: a new source for an old enzyme. Rev. Environ. Sci. Biotechnol. 10, 309–326 (2011)

  4. 4.

    Bhalla, A., Bansal, N., Kumar, S., Bischoff, K.M., Sani, R.K.: Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresour. Technol. 128, 751–759 (2013)

  5. 5.

    Dettmer, A., dos Anjos, P.S., Gutterres, M.: Special review paper: Enzymes in the leather industry. J. Am. Leather Chem. Assoc. 108, 146–158 (2013)

  6. 6.

    Bhange, K., Chaturvedi, V., Bhatt, R.: Simultaneous production of detergent stable keratinolytic protease, amylase and biosurfactant by Bacillus subtilis PF1 using agro industrial waste. Biotechnol. Rep. 10, 94–104 (2016)

  7. 7.

    Hmidet, N., Ali, N.E.H., Zouari-Fakhfakh, N., Haddar, A., Nasri, M., Sellemi-Kamoun, A.: Chicken feathers: a complex substrate for the co-production of α-amylase and proteases by B. licheniformis NH1. J. Ind. Microbiol. Biotechnol. 37, 983–990 (2010)

  8. 8.

    Corrêa, T.L.R., Moutinho, S.K.D.S., Martins, M.L.L., Martins, M.A.: Simultaneous α-amylase and protease production by the soil bacterium Bacillus sp. SMIA-2 under submerged culture using whey protein concentrate and corn steep liquor: compatibility of enzymes with commercial detergents. Food Sci. Technol. 31, 843–848 (2011)

  9. 9.

    Qureshi, A.S., Khushk, I., Ali, C.H., Chisti, Y., Ahmad, A., Majeed, H.: Coproduction of protease and amylase by thermophilic Bacillus sp. BBXS-2 using open solid-state fermentation of lignocellulosic biomass. Biocatal. Agric. Biotechnol. 8, 146–151 (2016)

  10. 10.

    Kavitha, R.: Production of amylase and protease from fruit peels using Bacillus subtilis by solid-state fermentation. IJSRR 7, 652–663 (2018)

  11. 11.

    Mukherjee, R., Paul, T., Soren, J.P., Halder, S.K., Mondal, K.C., Pati, B.R., Mohapatra, P.K.D.: Acidophilic α-amylase production from Aspergillus niger RBP7 using potato peel as substrate: a waste to value added approach. Waste Biomass Valoriz. 10, 851–863 (2019)

  12. 12.

    Singh, S., Bajaj, B.K.: Agroindustrial/forestry residues as substrates for production of thermoactive alkaline protease from Bacillus licheniformis K-3 having multifaceted hydrolytic potential. Waste Biomass Valoriz. 8, 453–462 (2017)

  13. 13.

    Arapoglou, D., Varzakas, T., Vlyssides, A., Israilides, C.: Ethanol production from potato peel waste (PPW). Waste Manag. 30, 1898–1902 (2010)

  14. 14.

    Bretón-Toral, A., Trejo-Estrada, S.R., McDonald, A.G.: Lactic acid production from potato peel waste, spent coffee grounds and almond shells with undefined mixed cultures isolated from coffee mucilage from Coatepec Mexico. Ferment. Technol. 6, 1–6 (2016)

  15. 15.

    Wang, S.L., Chen, Y.H., Wang, C.L., Yen, Y.H., Chern, M.K.: Purification and characterization of a serine protease extracellularly produced by Aspergillus fumigatus in a shrimp and crab shell powder medium. Enzyme Microb. Technol. 36, 660–665 (2005)

  16. 16.

    Khosravi-Darani, K., Falahatpishe, H.R., Jalali, M.: Alkaline protease production on date waste by an alkalophilic Bacillus sp. 2–5 isolated from soil. Afr. J. Biotechnol. 7, 1536–1542 (2008)

  17. 17.

    Bora, L., Kalita, M.: Thermozymes: an area of potential research. Curr. Sci. 93, 593 (2007)

  18. 18.

    Lele, O.H., Deshmukh, P.V.: Isolation and characterization of thermophilic Bacillus sp. with extracellular enzymatic activities from hot spring of Ganeshpuri, Maharashtra. India. Int. J. Appl. Res. 2, 427–430 (2016)

  19. 19.

    Namsaraev, Z.B., Babasanova, O.B., Dunaevsky, Y.E., Akimov, V.N., Barkhutova, D.D., Gorlenko, V.M., Namsaraev, B.B.: Anoxybacillus mongoliensis sp. nov., a novel thermophilic proteinase producing bacterium isolated from alkaline hot spring, central Mongolia. Microbiology 79, 491–499 (2010)

  20. 20.

    Bekler, F.M., Güven, K.: Isolation and production of thermostable α-amylase from thermophilic Anoxybacillus sp. KP1 from Diyadin hot spring in Ağri, Turkey. Biologia 69, 419–427 (2014)

  21. 21.

    Ozdemir, S., Okumus, V., Ulutas, M.S., Dundar, A., Akarsubasi, A.T., Dumonted, S.: Isolation of a novel thermophilic Anoxybacillus flavithermus SO-13, production, characterization and industrial applications of its thermostable [alpha]-amylase. J. Bioprocess Biotech. 5, 1 (2015)

  22. 22.

    Acer, Ö., Bekler, F.M., Pirinççioğlu, H., Güven, R.G., Güven, K.: Purification and characterization of thermostable and detergent-stable α-amylase from Anoxybacillus sp. AH1. Food Technol. Biotechnol. 54, 70 (2016)

  23. 23.

    Mukhtar, H., Ikram-Ul-Haq: Concomitant production of two proteases and alpha-amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor. Braz. J. Microbiol. 43, 1072–1079 (2012)

  24. 24.

    Chugh, P., Soni, R., Soni, S.K.: Deoiled rice bran: a substrate for co-production of a consortium of hydrolytic enzymes by Aspergillus niger P-19. Waste Biomass Valoriz. 7, 513–525 (2016)

  25. 25.

    Kumar, V., Sankaranarayanan, M., Jae, K.E., Durgapal, M., et al.: Co-production of 3-hydroxypropionic acid and 1, 3-propanediol from glycerol using resting cells of recombinant Klebsiella pneumoniae J2B strain overexpressing aldehyde dehydrogenase. Appl. Microbiol. Biotechnol. 96, 373–383 (2012)

  26. 26.

    Ali, S.M., Omar, S.H., Soliman, N.A.: Co-production of cellulase and xylanase enzymes by thermophilic Bacillus subtilis 276NS. Int. J. Biotechnol. Wellness Ind. 2, 65–74 (2013)

  27. 27.

    Liu, D., Chen, Y., Ding, F., Guo, T., et al.: Simultaneous production of butanol and acetoin by metabolically engineered Clostridium acetobutylicum. Metabol. Eng. 27, 107–114 (2015)

  28. 28.

    García-Pérez, T., López, J.C., Passos, F., Lebrero, R., Revah, S., Muñoz, R.: Simultaneous methane abatement and PHB production by Methylocystis hirsuta in a novel gas-recycling bubble column bioreactor. Chem. Eng. J. 334, 691–697 (2018)

  29. 29.

    Taskin, M., Ortucu, S., Unver, Y., Tasar, O.C., Ozdemir, M., Kaymak, H.C.: Invertase production and molasses decolourization by cold-adapted filamentous fungus Cladosporium herbarum ER-25 in non-sterile molasses medium. Process Saf. Environ. Protect. 103, 136–143 (2016)

  30. 30.

    Taskin, M., Ortucu, S., Aydogan, M.N., Arslan, N.P.: Lipid production from sugar beet molasses under non-aseptic culture conditions using the oleaginous yeast Rhodotorula glutinis TR29. Renew. Energy 99, 198–204 (2016)

  31. 31.

    Adiguzel, A., Ozkan, H., Baris, O., Inan, K., Gulluce, M., Sahin, F.: Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey. J. Microbiol. Method 79, 321–328 (2009)

  32. 32.

    Miller, G.L.: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428 (1959)

  33. 33.

    Takami, H., Akiba, T., Horikoshi, K.: Production of extremely thermostable alkaline protease from Bacillus sp. no. AH-101. Appl. Microbiol. Biotechnol. 30, 120–124 (1989)

  34. 34.

    Erdal, S., Taskin, M.: Production of alpha-amylase by Penicillium expansum MT-1 in solid-state fermentation using waste Loquat (Eriobotrya japonica Lindley) kernels as substrate. Romanian Biotechnol. Lett. 15, 5342–5350 (2010)

  35. 35.

    Mohammad, B.T., Al Daghistani, H.I., Jaouani, A., Abdel-Latif, S., Kennes, C.: Isolation and characterization of thermophilic bacteria from Jordanian hot springs: Bacillus licheniformis and Thermomonas hydrothermalis isolates as potential producers of thermostable enzymes. Int. J. Microbiol. (2017).

  36. 36.

    Taskin, M., Saghafian, A., Aydogan, M.N., Arslan, N.P.: Microbial lipid production by cold-adapted oleaginous yeast Yarrowia lipolytica B9 in non-sterile whey medium. Biofuels Bioprod. Bioref. 9, 595–605 (2015)

  37. 37.

    Tasar, O.C., Erdal, S., Taskin, M.: Chitosan production by psychrotolerant Rhizopus oryzae in non-sterile open fermentation conditions. Int. J. Biol. Macromol. 89, 428–433 (2016)

  38. 38.

    Smerilli, M., Neureiter, M., Wurz, S., Haas, C., Frühauf, S., Fuchs, W.: Direct fermentation of potato starch and potato residues to lactic acid by Geobacillus stearothermophilus under non-sterile conditions. J. Chem. Technol. Biotechnol. 90, 648–657 (2015)

  39. 39.

    Kivistö, A., Santala, V., Karp, M.: Non-sterile process for biohydrogen and 1, 3-propanediol production from raw glycerol. Int. J. Hydrog. Energy 38, 11749–11755 (2013)

Download references

Author information

Correspondence to Hakan Ozkan.

Ethics declarations

Conflict of interest

All authors declare that there is no conflict of interest.

Additional information

Publisher's Note

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tuysuz, E., Gonul-Baltaci, N., Omeroglu, M.A. et al. Co-production of Amylase and Protease by Locally Isolated Thermophilic Bacterium Anoxybacillus rupiensis T2 in Sterile and Non-sterile Media Using Waste Potato Peels as Substrate. Waste Biomass Valor (2020) doi:10.1007/s12649-020-00936-3

Download citation


  • Anoxybacillus rupiensis T2
  • Thermophilic bacteria
  • Potato peel
  • Protease
  • Amylase
  • Co-production