There is a wide range of applications for β-1,4-endoglucanase in the detergent industry, which with good low temperature and alkaline tolerance is an important prerequisite for its industrial application. In this thesis, a novel β-1,4-endoglucanase gene EgDC from Deinococcus cellulosilyticus with low-temperature tolerance was obtained by gene mining. It was expressed in Escherichia coli, with most of it existed in the periplasmic space and a small portion secreted to the extracellular space. The enzymatic properties of EgDC enzyme were characterized, and it was found that the optimum temperature and pH of the enzyme were 40 °C and pH6.5, respectively. It could maintain more than 70% of its activity at 20 °C, while its half-life reached 83.43 min and 53.79 min at pH8.0 and pH9.0. The β-1,4-endoglucanase EgDC had good catalytic properties with specific enzyme activity and kcat/Km values reaching (26.38±1.43) U/mg and (520.03±17.07) s-1/mg, respectively, which is also highly tolerant to metal ions and surfactants. Thus, the new β-1,4-endoglucanase EgDC has good catalytic ability and is stable at low temperature, alkaline environment and in the presence of surfactant. Therefore, it has potential industrial applications.
SHENG Diqing
,
NIU Chengtuo
,
YAN Yanan
,
ZHENG Feiyun
,
LIU Chunfeng
,
WANG Jinjing
,
LI Qi
. Gene mining, heterologous expression and characterization of enzymatic properties of low-temperature β-1,4-endoglucanase[J]. Food and Fermentation Industries, 2024
, 50(4)
: 17
-24
.
DOI: 10.13995/j.cnki.11-1802/ts.035378
[1] KYTE J, DOOLITTLE R F.A simple method for displaying the hydropathic character of a protein [J].Journal of Molecular Biology, 1982, 157(1):105-132.
[2] GAUR R, TIWARI S.Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07 [J].BMC Biotechnology, 2015, 1519.
[3] 温康文, 孙水裕, 舒生辉.嗜碱微生物及其工业应用的研究进展 [J].广东化工, 2007, 34(4):51-53.
WEN K W, SUN S Y, SHU S H.Progress of alcaliphilic microorganism and its industrial application [J].Guangdong Chemical Industry, 2007, 34(4):51-53.
[4] ITO S.Alkaline cellulases from alkaliphilic Bacillus:Enzymatic properties, genetics, and application to detergents [J].Extremophiles, 1997, 1(2):61-66.
[5] ALI S M, HALL J, SOOLE K L, et al.Targeted Expression of Microbial Cellulases in Transgenic Animals [M].Progress in biotechnology.Amsterdam:Elsevier, 1995:279-293.
[6] SELMER-OLSEN I, HENDERSON A R, ROBERTSON S, et al.Cell wall degrading enzymes for silage.1.The fermentation of enzyme-treated ryegrass in laboratory silos [J].Grass and Forage Science, 1993, 48(1):45-54.
[7] NIYONZIMA F N.Detergent-compatible bacterial cellulases [J].Journal of Basic Microbiology, 2019, 59(2):134-147.
[8] HADIKOLAEI K K, GHARECHAHI J, HAGHBEEN K, et al.A cold-adapted endoglucanase from camel rumen with high catalytic activity at moderate and low temperatures:An anomaly of truly cold-adapted evolution in a mesophilic environment [J].Extremophiles, 2018, 22(2):315-326.
[9] PATEL A, SHAH A.Purification and characterization of novel, thermostable and non-processive GH5 family endoglucanase from Fomitopsis meliae CFA 2 [J].International Journal of Biological Macromolecules, 2021, 182:1161-1169.
[10] SHIRAI T, ISHIDA H, NODA J-I, et al.Crystal structure of alkaline cellulase K:Insight into the alkaline adaptation of an industrial enzymel[J].Journal of Molecular Biology, 2001, 310(5):1079-1087.
[11] SCHWARZ W.The cellulosome and cellulose degradation by anaerobic bacteria [J].Applied Microbiology and Biotechnology, 2001, 56(5):634-649.
[12] SINGHANIA R R, RUIZ H A, AWASTHI M K, et al.Challenges in cellulase bioprocess for biofuel applications [J].Renewable and Sustainable Energy Reviews, 2021, 151:111622.
[13] ITO S, SHIKATA S, OZAKI K, et al.Alkaline cellulase for laundry detergents:Production by Bacillus sp.KSM-635 and enzymatic properties [J].Agricultural and Biological Chemistry, 1989, 53(5):1275-1281.
[14] FUKUMORI F, KUDO T, HORIKOSHI K.Purification and properties of a cellulase from alkalophilic Bacillus sp.no.1139 [J].Microbiology, 1985, 131(12):3339-3345.
[15] ZIEMERT N, ALANJARY M, WEBER T.The evolution of genome mining in microbes-A review [J].Natural Product Reports, 2016, 33(8):988-1005.
[16] SÉVIN D C, FUHRER T, ZAMBONI N, et al.Nontargeted in vitro metabolomics for high-throughput identification of novel enzymes in Escherichia coli[J].Nature Methods, 2017, 14(2):187-194.
[17] ZHANG F, ZHANG X M, YIN Y R, et al.Cloning, expression and characterization of a novel GH5 exo/endoglucanase of Thermobifida halotolerans YIM 90462T by genome mining [J].Journal of Bioscience and Bioengineering, 2015, 120(6):644-649.
[18] SANANGELANTONI A M, MALATRASI M, TRIVELLONI E, et al.A novel β-propeller phytase from the dioxin-degrading bacterium Sphingomonas wittichii RW-1[J].Applied Microbiology and Biotechnology, 2018, 102(19):8351-8358.
[19] 周茂志, 唐存多, 许建和, 等.新型扁桃酸消旋酶的基因挖掘和性能表征 [J].生物工程学报, 2018, 34(6):897-905.
ZHOU M Z, TANG C D, XU J H.et al.Genome mining and characterization of a new mandelate racemase[J].Chinese Journal of Biotechnology, 2018, 34(6):897-905.
[20] LIN L, QIN N, GUAN L. A novel cold-adapted endoglucanase (M6A) from Microbacterium kitamiense S12 isolated from Qinghai-Tibetan Plateau. Biotechnology and Bioprocess Engineering, 2019, 24: 544-551.
[21] DHUME G M, MAHARANA A K, TSUJI M, et al.Cold-tolerant endoglucanase producing ability of Mrakia robertii A2-3 isolated from cryoconites, Hamtha glacier, Himalaya [J].Journal of Basic Microbiology, 2019, 59(7):667-679.
[22] HONG J, TAMAKI H, AKIBA S, et al.Cloning of a gene encoding a highly stable endo-β-1, 4-glucanase from Aspergillus niger and its expression in yeast [J].Journal of Bioscience and Bioengineering, 2001, 92(5):434-441.
[23] MILLER G L.Use of dinitrosalicylic acid reagent for determination of reducing sugar [J].Analytical Chemistry, 1959, 31(3):426-428.
[24] SUN J P, PHILLIPS C M, ANDERSON C T, et al.Expression and characterization of the Neurospora crassa endoglucanase GH5-1[J].Protein Expression and Purification, 2011, 75(2):147-154.
[25] SINGH J, BATRA N, SOBTI R C.Purification and characterisation of alkaline cellulase produced by a novel isolate, Bacillus sphaericus JS1 [J].Journal of Industrial Microbiology and Biotechnology, 2004, 31(2):51-56.
[26] SIDDIQUI K S, CAVICCHIOLI R.Cold-adapted enzymes [J].Annual Review of Biochemistry, 2006, 75:403-433.
[27] LIANG C N, XUE Y F, FIORONI M, et al.Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4[J].Applied Microbiology and Biotechnology, 2011, 89(2):315-326.
