Abstract

Plant raw materials are an accessible and efficient substrate for multipurpose enzymes and multi-enzyme complexes obtaining. Thermophilic bacteria are reliable producer strains for industrial enzymes with valuable physicochemical properties, e.g., heat resistance, stability at extreme pH, and chemical stability. Food scientists are on the lookout for new thermophilic strains capable of producing high yields of industrially valuable enzymes from cheap plant raw materials. In this research, various plant cultivation media affected the complex hydrolytic enzymes produced by a thermophilic strain of Bacillus subtilis Kb.12.Gl.35. The research objective was to select the optimal raw materials for the synthesis of thermostable hydrolases, i.e., proteases, amylases, and hemicellulases.
The thermophilic strain was isolated from a compost sample, identified by 16S rRNA sequencing, and tested using standard microbiological and biochemical methods. The strain grew on different media with plant flour combined with yeast or corn extract. The activity of extracellular enzymes made it possible to study the composition of the hydrolase complex. The proteolytic activity was determined using casein as the substrate and by zymography. The 3,5-dinitrosalicylic acid (DNS) method revealed the carbohydrase activity.
When culturing the hydrolase producer on nutrient media with plant flour (1%) and corn extract (0.5%), the highest level of protease synthesis (≥ 40 units/mL) was detected in the samples with amaranth, oat, and rice flour. The highest level of amylase (≥ 1,300 units/mL) belonged to the chickpea, oat, and rice flour samples. The biggest yields of galactomannanase (≥ 200 units/mL) and xylanases (≥ 60 units/mL) was found in the samples with amaranth, pea, and chickpea samples. The biggest amount of arabinogalactanase (≥ 35 units/mL) belonged to the amaranth, chickpea, and rice flour. Based on the zymographic analysis, the enzymes of 24.7–28.2 kDa and the proteases of 62.7–75.0 kDa appeared to be the most efficient proteolytic agents. Their activity was obvious on plant raw materials, in contrast to the standard LB medium. The secretion level of 15.1 kDa proteases was the same on the plant media as on the standard medium.
Bacillus subtilis Kb.12.Gl.35 proved to be an effective producer of proteases, amylases, and hemicellulases with plant flour and corn extract in the medium. Further studies are needed to optimize the composition of nutrient media and cultivation conditions to increase the yield of the target hydrolytic enzymes.

REFERENCES

1. Mokrani S, Nabti EH. Recent status in production, biotechnological applications, commercial aspects, and future prospects of microbial enzymes: A comprehensive review. International Journal of Agricultural Science and Food Technology. 2024;10(1):006–020. https://doi.org/10.17352/2455-815x.000202
2. Tripathi P, Sinha S. Industrial biocatalysis: An insight into trends and future directions. Current Sustainable/Renewable Energy Reports. 2020;7:66–72. https://doi.org/10.1007/s40518-020-00150-8
3. Meghwanshi GK, Kaur N, Verma S, Dabi NK, Vashishtha A, et al. Enzymes for pharmaceutical and therapeutic applications. Biotechnology and Applied Biochemistry. 2020;67(4):586–601. https://doi.org/10.1002/bab.1919
4. Shukla P. Synthetic biology perspectives of microbial enzymes and their innovative applications. Indian Journal of Microbiology. 2019;59(4):401–409. https://doi.org/10.1007/s12088-019-00819-9
5. Singh RS, Singh T, Pandey A. Microbial enzymes–An overview. In: Singh RS, Singhania RR, Pandey A, Larroche C, editors. Advances in Enzyme Technology. MA: Elsevier; 2019. pp. 1–40. https://doi.org/10.1016/B978-0-444-64114-4.00001-7
6. Zhang Y, Geary T, Simpson BK. Genetically modified food enzymes: A review. Current Opinion in Food Science. 2019;25:14–18. https://doi.org/10.1016/j.cofs.2019.01.002
7. Burkhardt C, Baruth L, Meyer-Heydecke N, Klippel B, Margaryan A, et al. Mining thermophiles for biotechnologically relevant enzymes: Evaluating the potential of European and Caucasian hot springs. Extremophiles. 2023;28:5. https://doi.org/10.1007/s00792-023-01321-3
8. Thapa S, Li H, OHair J, Bhatti S, Chen F-C, et al. Biochemical characteristics of microbial enzymes and their significance from industrial perspectives. Molecular Biotechnology. 2019;61:579–601. https://doi.org/10.1007/s12033-019-00187-1
9. Atalah J, Cáceres-Moreno P, Espina G, Blamey JM. Thermophiles and the applications of their enzymes as new biocatalysts. Bioresource Technology. 2019;280:478–488. https://doi.org/10.1016/j.biortech.2019.02.008
10. Kumar S, Dangi AK, Shukla P, Baishya D, Khare SK. Thermozymes: Adaptive strategies and tools for their biotechnological applications. Bioresource Technology. 2019;278:372–382. https://doi.org/10.1016/j.biortech.2019.01.088
11. Han H, Ling Z, Khan A, Virk AK, Kulshrestha S, et al. Improvements of thermophilic enzymes: From genetic modifications to applications. Bioresource Technology. 2019;279:350–361. https://doi.org/10.1016/j.biortech.2019.01.087
12. Ajeje SB, Hu Y, Song G, Peter SB, Afful RG, et al. Thermostable cellulases / Xylanases from thermophilic and hyperthermophilic microorganisms: Current perspective. Frontiers in Bioengineering and Biotechnology. 2021;9:794304. https://doi.org/10.3389/fbioe.2021.794304
13. Verma J, Sourirajan A, Dev K. Bacterial diversity in 110 thermal hot springs of Indian Himalayan Region (IHR). 3 Biotech. 2022;12:238. https://doi.org/10.1007/s13205-022-03270-8
14. Finore I, Feola A, Russo L, Cattaneo A, di Donato P, et al. Thermophilic bacteria and their thermozymes in composting processes: A review. Chemical and Biological Technologies in Agriculture. 2023;10:7. https://doi.org/10.1186/s40538-023-00381-z
15. Romanova MV, Dolbunova AN, Epishkina YuM, Evdokimova SA, Kozlovskiy MR, et al. A thermophilic L-lactic acid producer of high optical purity: Isolation and identification. Foods and Raw Materials. 2024;12(1):101–109. https://doi.org/10.21603/2308-4057-2024-1-591
16. Kambourova M. Thermostable enzymes and polysaccharides produced by thermophilic bacteria isolated from Bulgarian hot springs. Engineering in Life Sciences. 2018;18(11):758–767. https://doi.org/10.1002/elsc.201800022
17. Liang Q, Zhan Y, Yuan M, Cao L, Zhu C, et al. Improvement of the catalytic ability of a thermostable and acidophilic β-mannanase using a consensus sequence design strategy. Frontiers in Microbiology. 2021;12:722347. https://doi.org/10.3389/fmicb.2021.722347
18. Tan S, Tao X, Zheng P, Chen P, Yu X, et al. Thermostability modification of β-mannanase from Aspergillus niger via flexibility modification engineering. Frontiers in Microbiology. 2023;14:1119232. https://doi.org/10.3389/fmicb.2023.1119232
19. Solanki P, Putatunda C, Kumar A, Bhatia R, Walia A. Microbial proteases: Ubiquitous enzymes with innumerable uses. 3 Biotech. 2021;11:428. https://doi.org/10.1007/s13205-021-02928-z
20. Herrera-Márquez O, Fernández-Serrano M, Pilamala M, Jácome MB, Luzón G. Stability studies of an amylase and a protease for cleaning processes in the food industry. Food and Bioproducts Processing. 2019;117:64–73 https://doi.org/10.1016/j.fbp.2019.06.015
21. Velázquez-De Lucio BS, Hernández-Domínguez E, Villa-García M, Díaz-Godínez G, Mandujano-González V, et al. Exogenous enzymes as zootechnical additives in animal feed: A review. Catalysts. 2021;11(7):851. https://doi.org/10.3390/catal11070851
22. Nagarajan DR, Krishnan C. Use of a new catabolite repression resistant promoter isolated from Bacillus subtilis KCC103 for hyper-production of recombinant enzymes. Protein Expression and Purification. 2010;70(1):122–128. https://doi.org/10.1016/j.pep.2009.09.020
23. Xiao F, Li Y, Zhang Y, Wang H, Zhang L, et al. A new CcpA binding site plays a bidirectional role in carbon catabolism in Bacillus licheniformis. iScience. 2021;24(5):102400. https://doi.org/10.1016/j.isci.2021.102400
24. Sun B, Zou K, Zhao Y, Tang Y, Zhang F, et al. The fermentation optimization for alkaline protease production by Bacillus subtilis BS-QR-052. Frontiers in Microbiology. 2023;14:1301065. https://doi.org/10.3389/fmicb.2023.1301065
25. Naik B, Kumar V, Rizwanuddin S, Chauhan M, Gupta AK, et al. Agro-industrial waste: A cost-effective and eco-friendly substrate to produce amylase. Food Production, Processing and Nutrition. 2023;5:30. https://doi.org/10.1186/s43014-023-00143-2
26. Bhange K, Chaturvedi V, Bhatt R. Simultaneous production of detergent stable keratinolytic protease, amylase and biosurfactant by Bacillus subtilis PF1 using agro industrial waste. Biotechnology Reports. 2016;10:94–104. https://doi.org/10.1016/j.btre.2016.03.007
27. Su Y, Liu C, Fang H, Zhang D. Bacillus subtilis: A universal cell factory for industry, agriculture, biomaterials and medicine. Microbial Cell Factories. 2020;19:173. https://doi.org/10.1186/s12934-020-01436-8
28. 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. Frontiers in Microbiology. – 2016;7:01195. https://doi.org/10.3389/fmicb.2016.01195
29. Abd-Elhalim BT, Gamal RF, El-Sayed SM, Abu-Hussien SH. Optimizing alpha-amylase from Bacillus amyloliquefaciens on bread waste for effective industrial wastewater treatment and textile desizing through response surface methodology. Scientific Reports. 2023;13:19216. https://doi.org/10.1038/s41598-023-46384-6
30. Devi S, Dwivedi D, Bhatt AK. Utilization of agroresidues for the production of xylanase by Bacillus safensis XPS7 and optimization of production parameters. Fermentation. 2022;8(5):221. https://doi.org/10.3390/fermentation8050221
31. Srivastava PK, Kapoor M. Cost-effective endo-mannanase from Bacillus sp. CFR1601 and its application in generation of oligosaccharides from guar gum and as detergent additive. Preparative Biochemistry & Biotechnology. 2014;44(4):392–417. https://doi.org/10.1080/10826068.2013.833108
32. Romanova MV, Kuznetsov AYe, Beloded AV. Molecular biological and biochemical characteristics of extracellular proteases of thermophilic bacterial strains. Butlerov Communications. 2021;68(12):103–111. (In Russ.) https://elibrary.ru/LBSMNK
33. de Vos P, Garrity GM, Jones D, Krieg NR, Ludwig W, et al. Bergey’s Manual of Systematic Bacteriology, Second Edition, Vol. 3. NY: Springer; 2009. pp. 21–128. https://doi.org/10.1007/978-0-387-68489-5_3
34. Tamura K, Stecher G, Kumar S. MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution. 2021;38(7):3022–3027. https://doi.org/10.1093/molbev/msab120
35. Oliver GW, Stetler-Stevenson WG, Kleiner DE. Zymography, casein zymography, and reverse zymography: Activity assays for proteases and their inhibitors. In: Sterchi EE, Stöcker W, editors. Proteolytic enzymes. Berlin: Springer Lab Manual; 1999. pp. 63–76. https://doi.org/10.1007/978-3-642-59816-6_5
36. Goldshtein V, Lukin N, Radin O. Corn extract in feeds. Compound Feeds. 2022;(3):45–46. (In Russ.) https://doi.org/10.25741/2413-287X-2022-03-3-170
37. Harwood CR, Kikuchi Y. The ins and outs of Bacillus proteases: Activities, functions and commercial significance. FEMS Microbiology Reviews. 2022;46(1):fuab046. https://doi.org/10.1093/femsre/fuab046
38. Al-Attar H, Ahmed J, Thomas L. Rheological, pasting and textural properties of corn flour as influenced by the addition of rice and lentil flour. LWT. 2022;160:113231. https://doi.org/10.1016/j.lwt.2022.113231
39. Filipović J, Ivkov M, Košutić M, Filipović V. Ratio of omega-6/omega-3 fatty acids of spelt and flaxseed pasta and consumer acceptability. Czech Journal of Food Sciences. 2016;34(6):522–529. https://doi.org/10.17221/384/2015-CJFS
40. Agume ASN, Njintang NY, Mbofung CMF. Effect of soaking and roasting on the physicochemical and pasting properties of soybean flour. Foods. 2017;6(2):12. https://doi.org/10.3390/foods6020012
41. Malik M, Sindhu R, Dhull SB, Bou-Mitri C, Singh Y, et al. Nutritional composition, functionality, and processing technologies for amaranth. Journal of Food Processing and Preservation. 2023;2023(1):1753029. https://doi.org/10.1155/2023/1753029
42. Noguchi M, Hasegawa Y, Suzuki S, Nakazawa M, Ueda M, et al. Determination of chemical structure of pea pectin by using pectinolytic enzymes. Carbohydrate Polymers. 2020;231:115738. https://doi.org/10.1016/j.carbpol.2019.115738
43. Karki B, Maurer D, Kim TH, Jung S. Comparison and optimization of enzymatic saccharification of soybean fibers recovered from aqueous extractions. Bioresource Technology. 2011;102(2):1228–1233. https://doi.org/10.1016/j.biortech.2010.08.004
44. Hsiao H-Y, Anderson DM, Dale NM. Levels of β-mannan in soybean meal. Poultry Science. 2006;85(8):1430–1432. https://doi.org/10.1093/ps/85.8.1430
45. Ray S, Paynel F, Morvan C, Lerouge P, Driouich A, et al. Characterization of mucilage polysaccharides, arabinogalactanproteins and cell-wall hemicellulosic polysaccharides isolated from flax seed meal: A wealth of structural moieties. Carbohydrate Polymers. 2013;93(2):651–660. https://doi.org/10.1016/j.carbpol.2012.12.034
46. Yan F, Tian S, Chen H, Gao S, Dong X, et al. Advances in xylooligosaccharides from grain byproducts: Extraction and prebiotic effects. Grain & Oil Science and Technology. 2022;5(2):98–106. https://doi.org/10.1016/j.gaost.2022.02.002
47. Xie F, Feng F, Liu D, Quan S, Liu L, et al. Bacillus amyloliquefaciens 35M can exclusively produce and secrete proteases when cultured in soybean-meal-based medium. Colloids and Surfaces B: Biointerfaces. 2022;209(Part 2):112188. https://doi.org/10.1016/j.colsurfb.2021.112188
48. Wu XC, Nathoo S, Pang A, Carne T, Wong S. Cloning, genetic organization, and characterization of a structural gene encoding bacillopeptidase F from Bacillus subtilis. The Journal of Biological Chemistry. 1990;265(12):6845–6850. https://doi.org/10.1016/s0021-9258(19)39225-7
49. Ning Y, Yang H, Weng P, Wu Z. Analysis and identification of the extracellular proteases from Bacillus velezensis SW5. Applied Biochemistry and Microbiology. 2021;57:S27–S37 https://doi.org/10.1134/S0003683821100082
50. Yoo JY, Yao Z, Lee SJ, Jeon HS, Kim JH. Cloning and expression of a fibrinolytic enzyme gene, aprECJ1, from Bacillus velezensis CJ1 isolated from Myeolchi Jeotgal. Microbiology and Biotechnology Letters. 2021;49(3):289–297. https://doi.org/10.48022/mbl.2104.04012