Optimization of protease production using Bacillus velezensis through response surface methodology and investigating its applications as stain remover

Abstract

The aim of this research work was to evaluate the potential of Bacillus velezensis for proteases production by employing eggshells and egg membrane powder as substrate followed by statistical optimization via RSM. While, optimizing the physical parameters maximum protease production was observed at incubation period of 72-h, inoculum size of 2% and substrate concentration of 1.5%. Optimization of nutritional parameters was done through PBD by running 12 experiments, among 7 screened factors (Glucose, peptone, KH₂PO₄, MgSO₄, NaCl, CaCl₂ and yeast extract), 2 factors (Glucose and CaCl₂) was found significant. Significant factors were further optimized through CCD and statistical analysis of results were also performed by running 2-Factor ANOVA. Maximum protease activity of 301.0010 U/mlU/ml was observed. Then, enzyme characterization revealed maximum protease activity at 45 ˚C, pH-8, with highest thermal stability at 60 ˚C. Furthermore, effects of metal ions (Ca^{+ 2}, Cu^{+ 2}, Mn^{+ 2}, Mg^{+ 2}, K⁺, Na⁺) on protease activity was also evaluated and results have shown Cu^{+ 2} ions increase protease activity up-to 100-folds while Na⁺, Mg^{+ 2} and Mn^{+ 2} ions enhanced protease activity by 103.40, 115.42 and 106.01% respectively. The produced protease could be investigated further for potential application as excellent stain remover.

Keywords

Protease / Egg-shell waste / Bacillus velezensis fermentation / Response surface methodology / Destaining

References

[1] Shakir HA, Mahmood R, Irfan M, Deeba F, Javed I, Qazi JI. Protease production from Bacillus safensis in submerged fermentation using response surface methodology. Rev Mex Ing Quim, 2019, 18(1): 375-85

[2] Razzaq A, Shamsi S, Ali A, Ali Q, Sajjad M, Malik A, Ashraf M. Microbial proteases applications. Front Bioeng Biotechnol, 2019, 7: 110

[3] Furhan J, Sharma S. Microbial alkaline proteases: findings and applications. Int J Invent Pharm Sci, 2014, 2(4): 823-34

[4] Porter JL, Rusli RA, Ollis DL. Directed evolution of enzymes for Industrial Biocatalysis. Chem Bio Chem, 2016, 17: 197-203

[5] Singh R, Mittal A, Kumar M, Mehta PK. Microbial proteases in commercial applications. J Pharm Chem Biol Sci, 2016, 4(3): 365-74

[6] Haile G, Gessesse A. Properties of alkaline protease C45 produced by alkaliphilic Bacillus Sp. isolated from Chitu, Ethiopian Soda Lake. J Biotechnol Biomater, 2012, 2(4): 136

[7] Haile G. (2009). Alkaline proteaseproduction by an alkaliphilic bacterial isolate under solid state fermentation [thesis]. Addis Ababa: Addis Ababa University.

[8] Palazzini JM, Dunlap CA, Bowman MJ, Chulze SN. Bacillus velezensis RC 218 as a Biocontrol Agent to reduce Fusarium Head Blight and Deoxynivalenol Accumulation: genome sequencing and secondary metabolite cluster profiles. Microbiol Res, 2016, 192: 30-6

[9] Cai X-C, Liu C-H, Wang B-T, Xue Y-R. Genomic and metabolic traits endow Bacillus velezensis CC09 with a potential Biocontrol Agent in Control of Wheat Powdery Mildew Disease. Microbiol Res, 2017, 196: 89-94

[10] Khalid M, Ye M, Wei C, Dai B, Yang R, Huang S, Wang Z. Production of β-glucanase and protease from Bacillus velezensis strain isolated from the manure of piglets. Pre Biochem Biotech, 2020

[11] Mumecha TK, Mustefa SB, Prabhu VS, Zewde FT. Alkaline protease production using eggshells and membrane-based substrates: process modeling, optimization, and evaluation of detergent potency. Eng Appl Sci Res, 2021, 48(2): 171-80

[12] Hincke MT, Nys Y, Gautron J. The role of matrix protein in eggshell formation. J Poult Sci, 2010, 47: 208-19

[13] Ahmad TAE, Suso H-P, Hinckle MT. In-depth comparative analysis of the chicken eggshell membrane proteome. J Proteom, 2017, 155: 49-62

[14] Jafari Z, Najafpour GD, Zare H. Growth Media Optimization for Production of Alkaline Proteases from Industrial Wastewater using Bacillus subtilis PTCC 1254. Int J Eng, 2023, 36(3): 513-22

[15] Kanwar BS, Bhattacharya S, Tanvir MQ, John PJ. Characterization and optimization of alkaline protease producing thermostable bacteria. Int J Biosci, 2019, 14(5): 130-40

[16] Dashpute R, Bandal J, Bholay A, Kunde B, Kulkarni S, Barwant M. Isolation, identification, screening and optimization of parameters of protease producing Halophilic bacteria. Int J Res Anal Rev, 2021, 8(1): 2349-5138

[17] Zare H, Meiguni F, Najafpour GD. Production of Alkaline protease using Industrial Waste Effluent as low-cost fermentation substrate. Iran J Ene Env, 2021, 12(3): 264-72

[18] Ayantunji YJ, Omole RK, Olojo FO, Awojobi KO. (2020). Optimization of Alkaline Protease Production in Submerged Fermentation Using Bacillus cereus Isolated from an Abattoir Wastewater in IIe-Ife, Nigeria. J. Adv. Biol. Biotech, 23 (3): 1–15. https://doi.org/JABB/2020/V23i330143

[19] Abdullah R, Asim M, Nadeem M, Nisar K, Kaleem A, Iqtedar M. Production, optimization, purification, kinetic analysis, and applications of alkaline proteases produced from Bacillus subtilis through solid-state fermentation of agricultural by-products. Kuwait J Sci, 2022, 49(4): 1-15

[20] Shafique T, Shafique J, Zahid S, Kazi M, Alnemer O, Ahmad A. Screening, selection and development of Bacillus subtilis apr-IBL04for hyper production of macromolecule alkaline protease. Saudi J Biol Sci, 2020

[21] Lima LA, Cruz FRF, Santos DJG, Silva WC. Protease with collagenolytic activity produced by Bacillus sp. DPUA 1728 from amazonian soil. Braz J Microbiol, 2015, 46(4): 1217-23

[22] Iqbalsyah TM, Febriani, Oesman F, Zuliani R. (2008). Specific Activity of Thermostable Protease from Local Isolate of Bacillus Sp Purified by Acetone Fractionation. Chem Env Frie Ind

[23] Hammami A, Bayoudh A, Hadrich B, Abdelhedi O, Jridi M, Nasri M. Response-surface methodology for the production and purification of a new H₂O₂- tolerant alkaline protease from Bacillus invictae AH1 strain. Biotecnol Prog, 2020, 36(3): e2965

[24] Jadhav HP, Sayyed RZ, Shaikh SS, Bhamre HM, Sunita K, El-Enshasy HA. Statistically designed Bioprocess for enhanced production of Alkaline protease in Bacillus cereus HP_RZ17. J Sci Ind Res, 2020, 79: 491-8

[25] Baweja M, Tiwari R, Singh PK, Nain L, Shukla P. An Alkaline protease from Bacillus pumilus MP 27: functional analysis of its binding model toward its applications as detergent additive. Font Microbiol, 2016, 7: 1195

[26] Jeevan SC, Pallavi P, Anuradha BS, Ram SR. Optimization of bioprocess for enhanced production of alkaline protease by a Bacillus subtilis SHmIIIa through Plackett-Burman design. Afri J Microbiol Res, 2015, 9(28): 1738-44

[27] Paqueno ACL, Arruda AA, Silva DF, Duarte JMW, Silveira VMF, Converti A, et al. Production and characterization of collagenase from a new amazonian Bacillus cereus strain. Prep Biochem Biotechnol, 2019

[28] Shafique T, Shafique J, Zahid S, Kazi M, Alnemer O, Ahmad A. Screening, selection and development of Bacillus subtilis apr-IBL04for hyper production of macromolecule alkaline protease. Sau J Biol Sci, 2020

[29] Mahakhan P, Apiso P, Srisunthorn K, Vichitphan K, Vichitphan S, et al. Alkaline protease production from Bacillus gibsonii 6BS15-4 using dairy Effluent and Detergent Additive. J Microbiol Biotechnol, 2023, 33(2): 195-202

[30] Tekin A, Uzuner U, Sezen K. Homology modeling and heterologous expression of highly alkaline subtilisin-like serine protease from Bacillus halodurans C-125. Biotechnol Lett, 2020

[31] Cui H, Wang L, Yu Y. Production and characterization of Alkaline proteases from a high yielding and moderately halophilic strain of SD11 marine Bacteria. J Chem. 2015;798304. https://doi.org/10.1155/2015/798304.

[32] Ibrahim ASS, Elbadawi YB, El-Tayeb MA, Al-aary KS, Abdel DFM, Salah SSI. Alkaline serine protease from the new halotolerant alkaliphilic salipaludibacillus agaradhaerens strain AK-R: purification and properties. 3 Biotech, 2019, 9(11): 391

[33] Abdollah P, Ghane M, Babaeekhou L. Isolation and characterization of thermophilic Bacteria from Gavmesh Goli Hot Spring in Sabalan Geothermal Field, Iran: Thermomonas hydrothermalis and Bacillus altitudinis isolates as a potential source of Thermostable protease. Geomicrobiol J, 2020

[34] Saberianpour S, Abkhooie L, Elaysifar B, Dilmaghani A. Screening and optimization of protease enzyme produced by strains of Alkalihalobacillus specie and Bacillus sp. Curr Biotech, 2021, 10(1): 40-5

[35] Olawande AO, Olosegun VO, Juwon DA. Purification and characterization of a dtergent compatible alkaline protease produced by Bacillus ruris isolated from Vegetable Oil Factory Effluent in Owo, Ondo State, Nigeria. Jord J Biol Sci, 2020, 14(4): 629-35

[36] Merin SL, Umesh M, Nag A, Chinnathambi A, Ali SA, Jhanani GK, et al. Optimized production of keratinolytic proteases from Bacillus tropicus LS27 and its application as a sustainable alternative for dehairing, destaining and metal recovery. Env Res, 2023, 221: 115283

[37] David A, Singh PC, Kumar A, Angural S, Kumar D, Puri N, Gupta N. Coproduction of protease and mannanase from Bacillus nealsonii PN-11 in solid state fermentation and their combined application as detergent additives. Int J Biomac, 2018, 108: 1176-84

[38] Thazeem B, Primo GB, Umesh M. A comparative study on alkaline protease production from Bacillus spp. and their biodegradative, dehairing and destaining activity. Int J Aca Res Dev, 2017, 2(2): 74-9