Production and applications of Bacillus sp. CL14 crude protease: approaches for biomass valorization and silver recovery

Keywords

Agro-industrial by-products / Bioconversion / Enzyme production / Silver recovery / Protein hydrolysates / Antioxidant activity

References

[1] Liguori R, Amore A, Faraco V. Waste valorization by biotechnological conversion into added value products. Appl Microbiol Biotechnol, 2013, 97: 6129-47

[2] Kee SH, Chiongson JBV, Saludes JP, Vigneswari S, Ramakrishna S, Bhubalan K. Bioconversion of agro-industry sourced Biowaste into biomaterials via microbial factories—a viable domain of circular economy. Environ Pollut, 2021, 271: 116311

[3] Nargotra P, Ortizo RGG, Wang JX, Tsai ML, Dong CD, Sun PP, Bajaj BK, Kuo CH, Sharma V. Enzymes in the bioconversion of food waste into valuable bioproducts: a circular economy perspective. Syst Microbiol Biomanuf, 2024, 4: 850-68

[4] Mora-Villalobos V, González-Vargas M, Cortés-Herrera C, Velázquez-Carrillo C, Koschny ME, Barboza N. Cellulase production using a combination of Carrot Peel and corn husk (tusa) residues under solid-state fermentation. Syst Microbiol Biomanuf, 2025, 5: 843-53

[5] Shafique A, Shakir HA, Irfan M, Shakir HA, Irfan M, Ali S, Khan M, Ara C, Khan N. Optimization and partial purification of Tannase from Bacillus licheniformis AS1 under submerged fermentation and its application. Syst Microbiol Biomanuf, 2025, 5: 647-66

[6] Jagadeesan Y, Meenakshisundaram S, Pichaimuthu S, Balaiah A. A scientific version of Understanding why did the chickens cross the road?—a guided journey through Bacillus spp. Towards sustainable agriculture, circular economy and biofortification. Environ Res, 2024, 244: 117907

[7] Herrmann LW, Letti LAJ, Penha RO, Soccol VT, Rodrigues C, Soccol CR. Bacillus genus industrial applications and innovation: first steps towards a circular bioeconomy. Biotechnol Adv, 2024, 70: 108300

[8] Bindal S, Kadyan S, Saini M, Gupta R. In–situ and cell–free goat hair hydrolysis by a consortium of proteases from Bacillus licheniformis strain ER–15: hair hydrolysate valorization by melanin extraction. Waste Biomass Valoriz, 2022, 13: 3265-82

[9] Emran MA, Ismail SA, Hashem AM. Production of detergent stable thermophilic alkaline protease by Bacillus licheniformis ALW1. Biocatal Agric Biotechnol, 2020, 26: 101631

[10] Latafat S, Ashish MH, Vimal A, Bhargava P. Isolation and screening of keratinolytic bacteria from feather dumping soil near in Lucknow and Kanpur city, North region of Indian. Biocatal Biotransform, 2024, 42: 68-76

[11] Xie F, Feng F, Liu D, Quan S, Liu L, Zhang X, Chen G. Bacillus amyloliquefaciens 35 M can exclusively produce and secrete proteases when cultured in soybean-meal-based medium. Colloids Surf B Biointerfaces, 2022, 209: 112188

[12] Bhavikatti JS, Bodducharl SM, Kamagond RS, Desai SV, Shet AR. Statistical optimisation of protease production using a freshwater bacterium Chryseobacterium Cucumeris SARJS–2 for multiple industrial applications. 3 Biotech, 2020, 10: 279

[13] Borrajo P, Pateiro M, Barba FJ, Mora L, Franco D, Toldrá F, Lorenzo JM. Antioxidant and antimicrobial activity of peptides extracted from meat by-products: a review. Food Anal Methods, 2019, 12: 2401-15

[14] Görgüç A, Gençdağ E, Yılmaz FM. Bioactive peptides derived from plant origin by-products: biological activities and techno-functional utilizations in food developments – A review. Food Res Int, 2020, 136: 109504

[15] Keshapaga UR, Jathoth K, Singh SS, Gogada R, Burgula S. Characterization of high–yield Bacillus subtilis cysteine protease for diverse industrial applications. Braz J Microbiol, 2023, 54: 739-52

[16] Brandelli A, Daroit DJ. Unconventional microbial proteases as promising tools for the production of bioactive protein hydrolysates. Crit Rev Food Sci Nutr, 2024, 64: 4714-45

[17] Lermen AM, Clerici NJ, Maciel DB, Daroit DJ. Characterization and application of a crude bacterial protease to produce antioxidant hydrolysates from Whey protein. Prep Biochem Biotechnol, 2023, 53: 12-21

[18] Kopplin BW, Bernardo BS, Moraes GP, Goettems TL, Clerici NJ, Lermen AM, Daroit DJ. Production of antioxidant hydrolysates from bovine caseinate and soy protein using three non-commercial bacterial proteases. Biocatal Biotransform, 2024, 42: 826-34

[19] Cavello IA, Hours RA, Cavalitto SF. Enzymatic hydrolysis of gelatin layers of X-ray films and release of silver particles using keratinolytic Serine proteases from Purpureocillium lilacinum LPS # 876. J Microbiol Biotechnol, 2013, 23: 1133-9

[20] Silva VG, Castro RJS. Enzymatic hydrolysis of proteins from chicken viscera in the presence of an ionic liquid enhanced their antioxidant properties. Waste Biomass Valoriz, 2020, 11: 3183-93

[21] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem, 1951, 193: 265-75

[22] Hoffmann RG, Moraes GP, Silva CB, Daroit DJ. Enzymatic processing of animal by–products: production of antioxidant hydrolysates with Bacillus sp. CL18 crude protease. Environ Sci Pollut Res, 2024, 31: 26737-46

[23] Khetkorn W, Phonlamai A, Sakdapetsiri C, Kitpreechavanich V, Lomthong T. A low-cost production, characterization, and application of Raw starch degrading enzyme from the thermophilic filamentous bacterium, Laceyella Sacchari P43. J Appl Biol Biotechnol, 2024, 12: 153-61

[24] Kim TI, Lim DH, Baek KS, Jang SS, Park BY, Mayakrishnan V. Production of chitinase from Escherichia fergusonii, chitosanase from Chryseobacterium indologenes, Comamonas koreensis and its application in N-acetylglucosamine production. Int J Biol Macromol, 2018, 112: 1115-1121

[25] Gomes TA, Santos LB, Nogueira A, Spier MR. Increase in an intracellular β-galactosidase biosynthesis using L. reuteri NRRL B-14171, inducers and alternative low-cost nitrogen sources under submerged cultivation. Int J Food Eng, 2018, 14: 20170333

[26] Associação Brasileira de Reciclagem Animal (ABRA). Anuário ABRA 2023 – Setor de reciclagem animal. Brasília: ABRA. 2024 [in Portuguese]. https://abra.ind.br/abra/wp-content/uploads/2024/10/anuario-2023.pdf. Accessed 02 Sep 2025.

[27] Downing DJ. US Market Rep Render, 2024, 53(210-8

[28] Selge B, Gusovius HJ, Ouagne P. Processing of linseed straw: state-of-the-art and further perspectives. Ind Crops Prod, 2024, 222: 119518

[29] Wongsirichot P, Gonzalez-Miquel M, Winterburn J. Recent advances in rapeseed meal as alternative feedstock for industrial biotechnology. Biochem Eng J, 2022, 180: 108373

[30] Alahyaribeik S, Sharifi SD, Tabandeh F, Honarbakhsh S, Ghazanfari S. Bioconversion of chicken feather wastes by keratinolytic bacteria. Process Saf Environ Prot, 2020, 135: 171-8

[31] Ramalingum N, Bhagwat P, Permaul K, Pillai S. Production, characterization, and application of Pseudomonas aeruginosa S–04 keratinase for feather utilization. Biomass Convers Biorefin, 2024, 14: 11683-95

[32] Saravanan K, Vijayaveeran A, Kathirvel P. Biodegradation of keratin waste by Bacillus velezensis HFS_F2 through optimized keratinase production medium. Curr Microbiol, 2024, 81: 179

[33] Freitas AC, Baleeiro FCF, Fonseca RF, Bertucci Neto V, Pinto GAS, Farinas CS. Bioprocess development to add value to Canola cake used as substrate for proteolytic enzyme production. Food Bioprod Process, 2015, 95: 173-82

[34] López-Trujillo J, Mellado-Bosque M, Ascacio-Valdés JA, Prado-Barragán LA, Hernández-Herrera JA, Aguilera-Carbó AF. Temperature and pH optimization for protease production fermented by Yarrowia lipolytica from agro-industrial waste. Fermentation, 2023, 9: 819

[35] Boratyński F, Szczepańska E, Grudniewska A, Gniłka R, Olejniczak T. Improving of hydrolases biosynthesis by solid-state fermentation of Penicillium Camemberti on rapeseed cake. Sci Rep, 2018, 8: 10157

[36] El–Shazly AI, Wahba MI, Abdelwahed NAM, Shehata AN. Immobilization of alkaline protease produced by Streptomyces rochei strain NAM–19 in solid state fermentation based on medium optimization using central composite design. 3 Biotech, 2024, 14: 161

[37] Jin H, Zhang X, Li K, Niu Y, Guo M, Hu C, Wan X, Gong Y, Huang F. Direct bio-utilization of untreated rapeseed meal for effective Iturin a production by Bacillus subtilis in submerged fermentation. PLoS ONE, 2014, 9: e111171

[38] Mukhtar H, Haq I. Comparative evaluation of agroindustrial byproducts for the production of alkaline protease by wild and mutant strains of Bacillus subtilis in submerged and solid state fermentation. Sci World J, 2013, 2013: 538067

[39] Elumalai P, Lim JM, Park YJ, Cho M, Shea PJ, Oh BT. Agricultural waste materials enhance protease production by Bacillus subtilis B22 in submerged fermentation under blue light–emitting diodes. Bioprocess Biosyst Eng, 2020, 43: 821-30

[40] Nadeem F, Tayyab M, Mehmood T, Naseer R, Iqbal S. Optimization of fermentative parameters for hyperproduction of protease from Aspergillus viridi using lignocellulosic byproducts as sole substrate. Waste Biomass Valoriz, 2024, 15: 3761-71

[41] Sisa A, Sotomayor C, Buitrón L, Gómez-Estaca J, Martínez-Alvarez O, Mosquera M. Evaluation of by-products from agricultural, livestock and fishing industries as nutrient source for the production of proteolytic enzymes. Heliyon, 2023, 9: e20735

[42] 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, 2016, 10: 94-104

[43] He F, Chao J, Yang D, Zhang X, Yang C, Xu Z, Jiewei T, Yongqiang T. Optimization of fermentation conditions for production of neutral metalloprotease by Bacillus subtilis SCK6 and its application in goatskin-dehairing. Prep Biochem Biotechnol, 2022, 52: 789-99

[44] Yuan L, Zhang B, Dai H, Miao Y, Xu Z, Yang Z, Jiao X. Starvation-induced metabolic adaptation of Bacillus cereus during biofilm formation: phenotypic characterization and proteomic analysis. Food Res Int, 2025, 220: 117167

[45] The Silver Institute. World Silver Survey. Washington: The Silver Institute and Metals Focus. 2024. https://silverinstitute.opt-wp.cloud.bosslogics.com/wp-content/uploads/2024/07/World-Silver-Survey-2024.pdf. Accessed 10 Sep 2025.

[46] Qamar SA, Asgher M, Bilal M. Immobilization of alkaline protease from Bacillus brevis using Ca–alginate entrapment strategy for improved catalytic stability, silver recovery, and dehairing potentialities. Catal Lett, 2020, 150: 3572-83

[47] Shankar S, More SV, Laxman RS. Recovery of silver from waste X-ray film by alkaline protease from Conidiobolus coronatus. Kathmandu Univ J Sci Eng Technol, 2010, 6: 60-9

[48] Vaniushenkova AA, Poberezhniy DY, Panyukova NS, Morozov AN, Kalenov SV, Belov AA. The co-use of various forms of silver and proteases in the development of new dressing biomaterials for wound healing. Biointerface Res Appl Chem, 2024, 14: 119

[49] Hamza TA. Isolation of protease producing bacteria from soil for polyester and silver recovery from waste X-ray film. Am J BioSci, 2017, 5: 74-9

[50] Masui A, Fujiwara N, Takagi M, Imanaka T. Feasibility study for decomposition of gelatin layers on X-ray films by thermostable alkaline protease from alkaliphilic Bacillus Sp. Biotechnol Tech, 1999, 13: 813-5

[51] Instituto de Pesquisa Econômica Aplicada (IPEA). Diagnóstico dos resíduos orgânicos do setor agrossilvopastoril e agroindústrias associadas. Brasília: IPEA. 2012 [in Portuguese]. https://repositorio.ipea.gov.br/bitstream/11058/7687/1/RP_Diagn%c3%b3stico_2012.pdf. Accessed 12 Aug 2025.

[52] Kerr BJ, Jha R, Urriola PE, Shurson GC. Nutrient composition, digestible and metabolizable energy content, and prediction of energy for animal protein byproducts in finishing pig diets. J Anim Sci, 2017, 95: 2614-26

[53] Cunha IC, Brandelli A, Braga ARC, Sala L, Kalil SJ. Feather meal as a source of peptides with antioxidant activity from enzymatic hydrolysis. Waste Biomass Valoriz, 2023, 14: 421-30

[54] Hussain SM, Khurram F, Naeem A, Shah SZH, Sarker PK, Naeem E, Arsalan MZH, Riaz D, Yousaf Z, Faisal M, Amjad M. A review on the prospects and potentials of fishmeal replacement with different animal protein sources. Int Aquat Res, 2024, 16: 7-16

[55] Jeampakdee P, Puthong S, Srimongkol P, Sangtanoo P, Saisavoey T, Karnchanatat A. The apoptotic and free radical-scavenging abilities of the protein hydrolysate obtained from chicken feather meal. Poult Sci, 2020, 99: 1693-704

[56] Alves FESB, Carpiné D, Teixeira GL, Goedert AC, Scheer AP, Ribani RH. Valorization of an abundant slaughterhouse by–product as a source of highly technofunctional and antioxidant protein hydrolysates. Waste Biomass Valoriz, 2021, 12: 263-79

[57] Doan CT, Tran TN, Nguyen MT, Nguyen HK, Tran TKT, Nguyen TH, Tran TPH, Nguyen VB, Nguyen AD, Wang SL. Potential of the liquid fermentation of fishery waste by Paenibacillus elgii for metalloprotease production. Polymers, 2022, 14: 741

[58] Moritz JS, Latshaw JD. Indicators of nutritional value of hydrolyzed feather meal. Poult Sci, 2001, 80: 79-86

[59] Machado GS, Correa APF, Pires PGS, Marconatto L, Brandelli A, Kessler AM, Trevizan L. Determination of the nutritional value of diet containing Bacillus subtilis hydrolyzed feather meal in adult dogs. Animals, 2021, 11: 3553

[60] Kaewsalud T, Yakul K, Insomphun C, Jantanasakulwong K, Rachtanapun P, Tapingkae W, Chuetor S, Watanabe M, Chaiyaso T. Hydrothermal-enzymatic process for the bio-valorization of keratin wastes by thermostable keratinase from Thermoactinomyces vulgaris TK1-21. J Chem Technol Biotechnol, 2023, 98: 1203-14

[61] Ketnawa S, Martínez-Alvarez O, Benjakul S, Rawdkuen S. Gelatin hydrolysates from farmed giant catfish skin using alkaline proteases and its antioxidative function of simulated gastro-intestinal digestion. Food Chem, 2016, 192: 34-42

[62] Munteanu IG, Apetrei C. Analytical methods used in determining antioxidant activity: a review. Int J Mol Sci, 2021, 22: 3380

[63] Zou TB, He TP, Li HB, Tang HW, Xia EQ. The structure-activity relationship of the antioxidant peptides from natural proteins. Molecules, 2016, 21: 72

[64] Amaral YMS, Castro RJS. Unraveling the biological potential of chicken viscera proteins: a study based on their enzymatic hydrolysis to obtain hydrolysates with antioxidant properties. Prep Biochem Biotechnol, 2024, 54: 809-18

[65] Hong C, Zhu JQ, Zhao YM, Ma H. Effects of dual-frequency Slit ultrasound on the enzymolysis of high-concentration hydrolyzed feather meal: biological activities and structural characteristics of hydrolysates. Ultrason Sonochem, 2022, 89: 106135

[66] Zheng Z, Zhang M, Fan H, Liu Y. Effect of microwave combined with ultrasonic pretreatment on flavor and antioxidant activity of hydrolysates based on enzymatic hydrolysis of bovine bone. Food Biosci, 2021, 44: 101399

[67] Cheng JH, Zhang XY, Wang Z, Zhang X, Liu SC, Song XY, Zhang YZ, Ding JM, Chen XL, Xu F. Potential of thermolysin-like protease A69 in Preparation of bovine collagen peptides with moisture-retention ability and antioxidative activity. Mar Drugs, 2021, 19: 676

[68] Li S, Tian Q, Meng T, Guan Z, Cai Y, Liao X. Production, purification and activity evaluation of three novel antioxidant peptides obtained from grass carp (Ctenopharyngodon idella) scale waste by microbial protease BaApr1 hydrolysis. Syst Microbiol Biomanuf, 2022, 2: 568-79

[69] Rathod BG, Poosarla VG, Kuppili SK, Chouhan KSYR, Shivshetty N. Production and characterization of a halotolerant protease from Bacillus siamensis F2 for chicken byproducts valorization. Biomass Convers Biorefin, 2024, 14: 1343-58

[70] Tacias-Pascacio VG, Morellon-Sterling R, Siar EH, Tavano O, Berenguer-Murcia Á, Fernandez-Lafuente R. Use of alcalase in the production of bioactive peptides: a review. Int J Biol Macromol, 2020, 165: 2143-96

[71] Badoei-Dalfard A, Karami Z. Screening and isolation of an organic solvent tolerant-protease from Bacillus sp. JER02: activity optimization by response surface methodology. J Mol Catal B Enzym, 2013, 89: 15-23

[72] Badoei-Dalfard A, Khankari S, Karami Z. One-pot synthesis and biochemical characterization of protease metal organic framework (protease@MOF) and its application on the hydrolysis of fish protein-waste. Colloids Surf B Biointerfaces, 2020, 196: 111318

[73] Badoei-Dalfard A, Monemi F, Hassanshahian M. One-pot synthesis and biochemical characterization of a magnetic collagenase Nanoflower and evaluation of its biotechnological applications. Colloids Surf B Biointerfaces, 2022, 211: 112302

Funding

Universidade Federal da Fronteira Sul(PES-2023-0080)

RIGHTS & PERMISSIONS

Jiangnan University