Abstract
Objectives
Proteases have gained great attention due to their enormous applications in food, tannery, detergent, photography and many other industries. Proteases rank third position in the production of enzymes. This paper targets to isolate a bacterium with high alkaline protease activity and optimization of its production conditions using Response Surface Methodology (RSM).
Results
A bacterium isolated from soil contaminated with detergent exhibited clearance zone on skim milk agar medium with a protease activity of 22 U/ml. The bacterial strain was identified as Bacillus cereus KM05 and optimization of its production conditions were performed using statistical methods. Further optimization with Box Behnken design resulted in an increase in protease activity by 1.5-fold (28.6 U/ml). The protease enzyme was thermotolerant up to 70 °C with stability towards alkaline pH (pH 9). The enzyme was not affected by most of the metal ions and solvents. Moreover, the protease was also compatible with six commercial detergents tested. Densitometric analysis of the destained fabric materials following the detergent-enzyme treatment, revealed a stain removal efficiency of 97%.
Conclusion
The alkaline protease enzyme obtained was stable at different conditions with stain removal efficacy. Hence, the present alkaline protease could be used for detergent formulations.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
All data available on request.
Code availability
Not applicable.
References
Adetunji AI, Olaniran AO (2020) Statistical modelling and optimization of protease production by an autochthonous Bacillus aryabhattai Ab15-ES: A response surface methodology approach. Biocatal Agric Biotechnol 24:101528. https://doi.org/10.1016/j.bcab.2020.101528
Adinarayana K, Ellaiah P (2002) Response surface optimization of the critical medium components for the production of alkaline protease by a newly isolated Bacillus sp.. J Pharmacy Pharma Sci 5(3):272–278
Akçay FA, Avcı A (2020) Screening and selection of media components for protease production by Bacillus sp. EBTA6 using Plackett-Burman design. Turkish J Agri-Food Sci Technol 8(6):1250–1255. https://doi.org/10.24925/turjaf.v8i6.1250-1255.2986
Akhavan Sepahy A, Jabalameli L (2011) Effect of culture conditions on the production of an extracellular protease by Bacillus sp. isolated from soil sample of Lavizan Jungle Park. Enzyme Res. https://doi.org/10.4061/2011/219628
Asha B, Palaniswamy M (2018) Optimization of alkaline protease production by Bacillus cereus FT 1 isolated from soil. J Appl Pharma Sci 8(02):119–127. https://doi.org/10.7324/JAPS.2018.8219
Babu NR, Aji AM (2020) Molecular characterization of alkaline protease gene isolated from Aeromonas hydrophila strain AH10. Int J Appl Sci Biotechnol 8(2):154–160. https://doi.org/10.3126/ijasbt.v8i2.29583
Banerjee UC, Sani RK, Azmi W, Soni R (1999) Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process Biochem 35(1–2):213–219. https://doi.org/10.1016/S0032-9592(99)00053-9
Banik RM, Prakash M (2004) Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol Res 159(2):135–140. https://doi.org/10.1016/j.micres.2004.01.002
Baweja M, Nain L, Kawarabayasi Y, Shukla P (2016) Current technological improvements in enzymes toward their biotechnological applications. Front Microbiol 7:965. https://doi.org/10.3389/fmicb.2016.00965
Bayoudh A, Gharsallah N, Chamkha M, Dhouib A, Ammar S, Nasri M (2000) Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1. J Indus Microbiol Biotech 24(4):291–295. https://doi.org/10.1038/sj.jim.2900822
Beg QK, Sahai V, Gupta R (2003) Statistical media optimization and alkaline protease production from Bacillus mojavensis in a bioreactor. Process Biochem 39(2):203–209. https://doi.org/10.1016/S0032-9592(03)00064-5
Deepa T, Gangwane AK, Sayyed RZ, Jadhav HP (2020) Optimization and scale-up of laccase production by Bacillus sp. BAB-4151 isolated from the waste of the soap industry. Environ Sustai 3(4):471–479. https://doi.org/10.1007/s42398-020-00135-9
Devi MK, Banu AR, Gnanaprabhal GR, Pradeep BV, Palaniswamy M (2008) Purification, characterization of alkaline protease enzyme from native isolate Aspergillus niger and its compatibility with commercial detergents. Indian J Sci Tech 1(7):1–6. https://doi.org/10.17485/ijst/2008/v1i7/29599
Fujiwara N, Masui A, Imanaka T (1993) Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp.. J Biotech 30(2):245–256. https://doi.org/10.1016/0168-1656(93)90117-6
Gessesse A, Gashe BA (1997) Production of alkaline protease by an alkaliphilic bacteria isolated from an alkaline soda lake. Biotech Lett 19(5):479
Gupta R, Beg Q, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotech 59(1):15–32. https://doi.org/10.1007/s00253-002-0975-y
Gupta A, Joseph B, Mani A, Thomas G (2008) Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus. World J Microbiol Biotech 24(2):237–243. https://doi.org/10.1007/s11274-007-9462-z
Hammami A, Bayoudh A, Hadrich B, Abdelhedi O, Jridi M, Nasri M (2020) Response-surface methodology for the production and the purification of a new H2O2-tolerant alkaline protease from Bacillus invictae AH1 strain. Biotech Prog 36(3):e2965. https://doi.org/10.1002/btpr.2965
Huang Q, Peng Y, Li X, Wang H, Zhang Y (2003) Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. Curr Microbiol 46(3):0169–0173. https://doi.org/10.1007/s00284-002-3850-2
Jadhav HP, Sayyed RZ, Shaikh SS, Bhamre HM, Sunita K, El Enshasy HA (2020) Statistically designed bioprocess for enhanced production of alkaline protease in Bacillus cereus HP_RZ17. J Scientific Industrial Res. 79(06):491–498
Jayashree S, Annapurna B, Jayakumar R, Sa T, Seshadri S (2014) Screening and characterization of alkaline protease produced by a pink pigmented facultative methylotrophic (PPFM) strain, MSF 46. J Genetic Eng Biotech 12(2):111–120. https://doi.org/10.1016/j.jgeb.2014.11.002
Kalaiarasi K, Sunitha PU (2009) Optimization of alkaline protease production from Pseudomonas fluorescens isolated from meat waste contaminated soil. Afr J Biotechnol 8(24):7035–7041
Kumar S, Divya G, Jain A (2021) Substantial improvement of protease production using statistical optimization and Downstream approach from the strain Bacillus toyonensis VKB5 sp., isolated from the host Actinidia delicosa. J Microbiol Biotechnol Food Sci. https://doi.org/10.15414/jmbfs.3721
Limkar MB, Pawar SV, Rathod VK (2019) Statistical optimization of xylanase and alkaline protease co-production by Bacillus spp using Box-Behnken Design under submerged fermentation using wheat bran as a substrate. Biocat Agric Biotech 17:455–464. https://doi.org/10.1016/j.bcab.2018.12.008
Maal KB, Emtiazi G, Nahvi I (2009) Production of alkaline protease by Bacillus cereus and Bacillus polymixa in new industrial culture mediums and its immobilization”. Afr J Microbiol Res 3(9):491–497
Maurer KH (2004) Detergent proteases. Curr Opinion Biotech 15(4):330–334. https://doi.org/10.1016/j.copbio.2004.06.005
Mechri S, Berrouina MBE, Benmrad MO, Jaouadi NZ, 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 Biological Macromol 94:221–232. https://doi.org/10.1016/j.ijbiomac.2016.09.112
Mechri S, Khelifa B, Nadia ZJ, Hatem R, Mouna BE, Maroua OB, Hocine H, Samir B, Amel BD, Bassem J (2019) Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive. Extremophiles 23(6):687–706. https://doi.org/10.1007/s00792-019-01123-6
Mehta VJ, Thumar JT, Singh SP (2006) Production of alkaline protease from an alkaliphilic actinomycetes. Biores Techn 97(14):1650–1654. https://doi.org/10.1016/j.biortech.2005.07.023
Moon SH, Parulekar SJ (1991) A parametric study of protease production in batch and fed-batch cultures of Bacillus firmus. Biotech Bioeng 37(5):467–483. https://doi.org/10.1002/bit.260370509
Qureshi AS, Simair AA, Ali CH, Khushk I, Khokhar JA, Ahmad A, Danish M, ChangruiLu C (2018) Production, purification and partial characterization of organo-solvent tolerant protease from newly isolated Bacillus sp. BBXS-2. Ferment Technol. https://doi.org/10.4172/2167-7972.1000151
Rajeswari VD, Jayaraman G, Sridharan TB (2012) Purification and characterization of extracellular protease from halotolerant bacterium Virgibacillus dokdonensis VITP14. Asian J Biochem 7:123–132. https://doi.org/10.3923/ajb.2012.123.132
Rekik H, Jaouadi NZ, Gargouri F, Bejar W, Frikha F, Jmal N, Bejar S, Jaouadi B (2019) Production, purification and biochemical characterization of a novel detergent-stable serine alkaline protease from Bacillus safensis strain RH12. Int J Biological Macro 121:1227–1239. https://doi.org/10.1016/j.ijbiomac.2018.10.139
Saeki K, Ozaki K, Kobayashi T, Ito S (2007) Detergent alkaline proteases: enzymatic properties, genes, and crystal structures. J Biosci Bioeng 103(6):501–508. https://doi.org/10.1263/jbb.103.501
Sandhya T, Tambekar D (2013) Optimization of the production and partial characterization of an extracellular alkaline protease from thermohalo-alkalophilic Lonar lake bacteria. Biosci Discov 4(1):30–38
Sathishkumar R, Gnanakkan A, Chelladurai R (2015) Production and characterization of haloalkaline protease from ascidian-associated Virgibacillus halodenitrificans RSK CAS1 using marine wastes. Annals Microbiol 65(3):1481–1493. https://doi.org/10.1007/s13213-014-0987-8
Shafee N, Aris SN, Rahman RNZA, Basri M, Salleh AB (2005) Optimization of environmental and nutritional conditions for the production of alkaline protease by a newly isolated bacterium Bacillus cereus strain 146. J Appl Sci Res 1(1):1–8
Shafique T, Shafique J, Zahid S, Kazi M, Alnemer O, Ahmad A (2021) Screening, selection and development of Bacillus subtilis apr-IBL04 for hyper production of macromolecule alkaline protease. Saudi J Biological Sci 28(2):1494–1501. https://doi.org/10.1016/j.sjbs.2020.11.079
Shakir HA, Mahmood R, Irfan M, Deeba F, Javed I, Qazi JI (2019) Protease production from Bacillus safensis in submerged fermentation using response surface methodology. Rev Mex Ing Quím 18(1):375–385. https://doi.org/10.24275/uam/izt/dcbi/revmexingquim/2019v18n1/Shakir
Silva CRD, Delatorre AB, Martins MLL (2007) Effect of the culture conditions on the production of an extracellular protease by thermophilic Bacillus sp. and some properties of the enzymatic activity. Braz J Microbiol 38:253–258. https://doi.org/10.1590/S1517-83822007000200012
Singh J, Vohra RM, Sahoo DK (2001) Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus. J Indus Microbiol Biotech 26(6):387–393. https://doi.org/10.1038/sj.jim.7000151
Smulders E, Rahse W, Von Rybinski W, Steber J, Sung E, Wiebel F (2002) Detergent Ingredients. In: Smulders E (ed) Laundry detergent. Wiley, Weinheim, pp 38–98
Subba RC, Sathish T, Ravichandra P, Reddy SP (2009) Characterization of thermo-and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications. Process Biochem 44(3):262–268. https://doi.org/10.1016/j.procbio.2008.10.022
Suganthi C, Mageswari A, Karthikeyan S, Anbalagan M, Sivakumar A, Gothandam KM (2013) Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments. J Genetic Eng Biotech 11(1):47–52. https://doi.org/10.1016/j.jgeb.2013.02.002
Takami H, Akiba T, Horikoshi K (1990) Characterization of an alkaline protease from Bacillus sp. no. AH-101. Appl Microbiol Biotech 33(5):519–523. https://doi.org/10.1007/BF00172544
Tambekar SD, Tambekar DH (2013) Optimization of the production and partial characterization of an extracellular alkaline protease from thermo-halo-alkalophilic Lonar lake bacteria. Biosci Discov 4:30–38
Vadlamani S, Parcha SR (2011) Studies on industrially important alkaline protease production from locally isolated superior microbial strain from soil microorganisms. Int J Biotech Application 3(3):102–105. https://doi.org/10.9735/0975-2943.3.3.102-105
Vishnupriya CS, Sunish KS, Rebello S (2016) Molecular characterisation of alkaline protease producing Bacillus subtilis from soil. Int J Res Pharm Chem 6:485–490
Wang HY, Liu DM, Liu Y, Cheng CF, Ma QY, Huang Q, Zhang YZ (2007) Screening and mutagenesis of a novel Bacillus pumilus strain producing alkaline protease for dehairing. Lett Appl Microbiol 44(1):1–6. https://doi.org/10.1111/j.1472-765X.2006.02039.x
Wang B, Wang ZQ, Jin J, Ren HJ, Liu LN, Cui J (2013) Cloning, expression and characterization of a Trichinella spiralis serine protease gene encoding a 35.5 kDa protein. Exp Parasitol 134(2):148–154. https://doi.org/10.1016/j.exppara.2013.03.004
Ward OP (1985) Proteolytic enzymes. In: Moo-Young M (ed) Comprehensive Biotechnology, 3. Pergamon Press, Oxford, UK, pp 789–818
Yadav VK, Singh V, Mishra V (2019) Alkaline protease: a tool to manage solid waste and its utility in detergent industry. In: Tripathi V, Kumar P, Tripathi P, Kishore A, Kamle M (eds) Microbial genomics in sustainable agroecosystems. Springer, Singapore, pp 231–254
Yang JK, Shih L, Tzeng YM, Wang SL (2000) Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes. Enzy Microb Tech 26(5–6):406–413. https://doi.org/10.1016/s0141-0229(99)00164-7
Acknowledgements
Authors acknowledge the help rendered from Maharajas College Ernakulam, Kerala, India and Unibiosys Biotech Research Laboratory to facilitate this research.
Funding
No funds, grants, or other support was received.
Author information
Authors and Affiliations
Contributions
Methodology and analysis—DJ, SKS & RKSM; Data curation—SKS, NVK & RKSM; Writing-original draft preparation, review and editing—DJ, SKA, NVK & RKSM.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Research involving human and animal rights
This article does not contain any studies with human participants or animals.
Informed consent
Informed consent is not required for this study.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Jayakumar, D., Sachith, S.K., Nathan, V.K. et al. Statistical optimization of thermostable alkaline protease from Bacillus cereus KM 05 using response surface methodology. Biotechnol Lett 43, 2053–2065 (2021). https://doi.org/10.1007/s10529-021-03172-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-021-03172-4