Cellulose is the most abundant renewable energy material on the Earth, and the exploration of highly efficient cellulose-degrading bacteria and cellulose enzymes has been a focal point of research.In this study, a cellulose-degrading Bacillus strain, designated as I2, was isolated and the functionality of its cellulose genes was validated.I2 was isolated from the accumulated wheat straw in the Baotianman Nature Reserve in Nanyang, Henan Province, with a genome size of 4.2 Mbp containing 4 635 genes, capable of degrading a significant portion of filter paper within 48 hours.Through predictions from the Carbohydrate-Active enZymes (CAZy) carbohydrate database and enzyme activity assays, 182 genes detected in the database were analyzed, andit was established that four proteins in I2 exhibit cellulose enzyme activity.Among them, G2269 displayed the highest cellulase endoglucanase activity, reaching 49.02 U/mL under pH 5.0 and 55 ℃ conditions.Metal ions Ca2+ and Zn2+ had a significant enhancing effect on the activity of G2269, while Mn2+ and Cu2+ significantly inhibited the activity of G2269.Mg2+, Co2+, K+, Ni2+, and NH4+ had no significant effect on the activity of G2269.Furthermore, G2269 demonstrated good thermal stability and acid-alkali stability, retaining 70% of its activity after 30 minutes at 65 ℃, presenting promising application prospects, particularly in the fields of food processing and feed production.
[1] HARVEY D G.On the metabolism of some aromatic nitro compounds by different species of animal.III.The toxicity of the dinitrophenols, with a note on the effects of high environmental temperatures[J].Journal of Pharmacy and Pharmacology, 1959, 11:462-474.
[2] SHARMA A, TEWARI R, RANA S S, et al.Cellulases:Classification, methods of determination and industrial applications[J].Applied Biochemistry and Biotechnology, 2016, 179(8):1346-1380.
[3] AREESHI M Y.Microbial cellulase production using fruit wastes and its applications in biofuels production[J].International Journal of Food Microbiology, 2022, 378:109814.
[4] ŠUCHOVÁ K, FEHÉR C, RAVN J L, et al.Cellulose- and xylan-degrading yeasts:Enzymes, applications and biotechnological potential[J].Biotechnology Advances, 2022, 59:107981.
[5] 邹潇潇, 易子霆, 孙前光, 等.纤维素降解真菌DF14101的筛选与鉴定[J].微生物学杂志, 2016, 36(6):68-72.
ZOU X X, YI Z T, SUN Q G, et al.Screening and identification of a cellulose-degrading fungus DF14101[J].Journal of Microbiology, 2016, 36(6):68-72.
[6] KUMARI D, SINGH R.Pretreatment of lignocellulosic wastes for biofuel production:A critical review[J].Renewable and Sustainable Energy Reviews, 2018, 90:877-891.
[7] 张新杰, 周兴瑞, 孙慧颖, 等.拮抗植物病原真菌的纤维素/木质素降解菌株的筛选及鉴定[J].微生物学通报, 2023, 50(1):251-261.
ZHANG X J, ZHOU X R, SUN H Y, et al.Screening and identification of cellulose/lignin-degrading strains against plant pathogenic fungi[J].Microbiology China, 2023, 50(1):251-261.
[8] BEHERA B C, SETHI B K, MISHRA R R, et al.Microbial cellulases-diversity & biotechnology with reference to mangrove environment:A review[J].Journal of Genetic Engineering and Biotechnology, 2017, 15(1):197-210.
[9] AWASTHI M K, PANDEY A K, KHAN J, et al.Evaluation of thermophilic fungal consortium for organic municipal solid waste composting[J].Bioresource Technology, 2014, 168:214-221.
[10] 白长胜, 刘秋瑾, 尹珺伊, 等.产木质纤维素降解酶真菌的筛选及产酶特性[J].微生物学通报, 2023, 50(3):1098-1110.
BAI C S, LIU Q J, YIN J Y, et al.Screening and enzymatic characterization of the fungal strains producing lignocellulose-degrading enzymes[J].Microbiology China, 2023, 50(3):1098-1110.
[11] 李子婧, 刘帆, 汤胜, 等.纤维素降解菌长枝木霉菌(Trichoderma longibrachiatum)ZJ-10的筛选及产酶条件优化[J].浙江大学学报(农业与生命科学版), 2022, 48(5):614-624.
LI Z J, LIU F, TANG S, et al.Screening of cellulose-degrading fungus Trichoderma longibrachiatum ZJ-10 and optimization of enzyme production conditions[J].Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(5):614-624.
[12] CAWOY H, MARIUTTO M, HENRY G, et al.Plant defense stimulation by natural isolates of Bacillus depends on efficient surfactin production[J].Molecular Plant-Microbe Interactions, 2014, 27(2):87-100.
[13] JIN Q, JIANG Q Y, ZHAO L, et al.Complete genome sequence of Bacillus velezensis S3-1, a potential biological pesticide with plant pathogen inhibiting and plant promoting capabilities[J].Journal of Biotechnology, 2017, 259:199-203.
[14] 董自星, 李伟国, 佟新新, 等.黑曲霉内切β-1, 3(4)-葡聚糖酶的基因克隆与酶学特性分析[J].食品与发酵工业, 2016, 42(11):58-64.
DONG Z X, LI W G, TONG X X, et al.Gene cloning and biochemical characterization of endo-1, 3 (4)-β-glucanases from Aspergillus niger[J].Food and Fermentation Industries, 2016, 42(11):58-64.
[15] SONG X F, LI Y Z, WU Y Z, et al.Metabolic engineering strategies for improvement of ethanol production in cellulolytic Saccharomyces cerevisiae[J].FEMS Yeast Research, 2018, 18(8):pp620.
[16] AMRAINI S Z, ARIYANI L P, HERMANSYAH H, et al.Production and characterization of cellulase from E.coli EgRK2 recombinant based oil palm empty fruit bunch[J].Biotechnology and Bioprocess Engineering, 2017, 22(3):287-295.
[17] KIM D, KU S.Bacillus cellulase molecular cloning, expression, and surface display on the outer membrane of Escherichia coli[J].Molecules, 2018, 23(2):503.
[18] 许颖, 刘智磊, 詹晓北, 等.毕赤酵母中内切β-1, 4葡聚糖酶的表达及共培养制备低分子质量黄原胶[J].食品与发酵工业, 2023, 49(9):1-8.
XU Y, LIU Z L, ZHAN X B, et al.Endo-β-1, 4-glucanase expression in Pichia pastoris for coupled fermentation with Xanthomonas campestris for low-molecular-weight xanthan gum preparation[J].Food and Fermentation Industries, 2023, 49(9):1-8.
[19] 董茂林, 陈李栋, 黄六莲, 等.酸性助水溶剂制备木质纳米纤维素及功能应用研究进展[J].化工学报, 2023, 74(6):2281-2295.
DONG M L, CHEN L D, HUANG L L, et al.Research progress in preparation of lignonanocellulose by acid hydrotropes and their functional applications[J].CIESC Journal, 2023, 74(6):2281-2295.
[20] KALOGEROPOULOU A, PLIONI I, DIMITRELLOU D, et al.Biosynthesis of fuel-grade ethanol from cellobiose by a cell-factory of non-GMO Saccharomyces cerevisiae/starch-gel-cellulase[J].Fuel, 2022, 313:122986.
[21] WANG L, XIE Y J, CHANG J J, et al.A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger[J].Biotechnology for Biofuels and Bioproducts, 2023, 16(1):23.
