Recombinant expression and characterization of β-glucosidase from Bacillus thermoamylovorans

LIU Yang1,2 PENG Hui2 ZHANG Cuan1 DONG Yi-Ning1 SUN Xing1 LUO Xia1 CAI Hua1 ZHAO Wei-Ping1

(1.College of Biological and Food Engineering, Chuzhou University, Chuzhou, Anhui, China 239000)
(2.Collaborative Innovation Center of Modern Bio-Manufacture, Anhui University, Hefei, Anhui, China 230601)
【Knowledge Link】β-glucosidase

【Abstract】 [Background] β-glucosidase (EC 3.2.1.21) is an important component of cellulase system. At present, most β-glucosidases used in industry come from plants and fungi, but few come from bacteria, and there are some problems such as low enzyme activity, poor thermal stability, narrow reaction conditions, products inhibition, which increase the economic cost. Thermophilic microorganisms have special genetic information resources, so it is possible to excavate novel β-glucosidases with good enzymatic characterization from the genome to solve the industrial problems. [Objective] A novel β-glucosidase gene was extracted from the genome of Bacillus thermoamylovorans, and purified protein was obtained by gene recombination, heterologous expression and protein purification. The enzymatic characterization was studied systematically. It can lay the foundation for the application of β-glucosidase in the fields of cellulose hydrolysis. [Methods] The recombinant plasmid pET22b-bgl52 was constructed and transformed into Escherichia coli BL21 (DE3) by electric pulse method. The recombinant protein was expressed in soluble form and purified by Ni-NTA affinity chromatography. [Results] The recombinant plasmid pET22b-bgl52 was expressed in E. coli BL21 (DE3) and purified β-glucosidase Bgl52 protein was obtained. The molecular weight of the Bgl52 was 52 kD and it showed the best activity at 70°C and pH 6.5. With p-nitrophenyl-β-D-glucopyranoside (pNPG) as substrate, the specific enzyme activity was (223.7 ± 5.3) U/mg, Km was (9.3 ± 1.2) mmol/L, and Vmax was (270.3 ± 4.3) μmol/ (min·mg). Bgl52 preferred substrate for hydrolysis of β-1,4 glycosidic bond. Fe2+and Mg2+activated the enzyme activity obviously. Co2+, Cu2+and SDS inhibited the activity of enzyme. Bgl52 is one of the few glucose and xylose-activated glucosidases. A maximal 2.84-fold stimulation by glucose was observed at 0.2 mol/L, and a maximal 3.24-fold stimulation by xylose was found at 0.4 mol/L. At the same time, under physiological conditions, Bgl52 was not substantially inhibited by the feedback of the product glucose. [Conclusion] Using the genetic information resources contained in the genomes of thermophilic microorganisms, and through modern biotechnological methods such as gene synthesis, we can excavate the β-glucosidase with excellent enzymatic characterization. It lays a foundation for its application in cellulose degradation and other industrial fields.

【Keywords】 Bacillus thermoamylovorans; β-glucosidase; Recombinant expression; Enzymatic characterization;

【DOI】

【Funds】 Key Projects of Natural Science Research in Universities of Anhui Province (KJ2018A0426, KJ2019A0639) Open Laboratory Project of Collaborative Innovation Center of Modern Bio-Manufacture of Anhui University (BM2017003) Open Fund of Key Laboratory of Ministry of Agriculture Crop Gene Resources and Germplasm Creation in East China (ECG2018001)

Download this article

    References

    [1] Counts JA, Zeldes BM, Lee LL, et al. Physiological, metabolic and biotechnological features of extremely thermophilic microorganisms [J]. Wiley Interdisciplinary Reviews: Systems Biology and Medicine, 2017, 9 (3): e1377

    [2] Donati ER, Castro C, Urbieta MS. Thermophilic microorganisms in biomining [J]. World Journal of Microbiology and Biotechnology, 2016, 32 (11): 179

    [3] del Cueto J, Møller BL, Dicenta F, et al. β-Glucosidase activity in almond seeds [J]. Plant Physiology and Biochemistry, 2018, 126: 163–172

    [4] Mc Donald JE, Houghton JNI, Rooks DJ, et al. The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters [J]. Environmental Microbiology, 2012, 14 (4): 1077–1087

    [5] Wang HT, Yang JT, Chen KI, et al. Hydrolyzation of mogrosides: Immobilized β-glucosidase for mogrosides deglycosylation from Lo Han Kuo [J]. Food Science&Nutrition, 2019, 7 (2): 834–843

    [6] Asati V, Sharma PK. Purification and characterization of an isoflavones conjugate hydrolyzing β-glucosidase (ICHG) from Cyamopsis tetragonoloba (guar) [J]. Biochemistry and Biophysics Reports, 2019, 20: 100669

    [7] Nguyen TTH, Seo C, Kwak SH, et al. Enzymatic production of steviol glucosides using β-glucosidase and their applications [A] //Kuddus M. Enzymes in Food Biotechnology [M]. London: Academic Press, 2019: 405–418

    [8] Yang SQ, Wang LJ, Yan QJ, et al. Hydrolysis of soybean isoflavone glycosides by a thermostable β-glucosidase from Paecilomyces thermophila [J]. Food Chemistry, 2009, 115 (4): 1247–1252

    [9] Pentzold S, Jensen MK, Matthes A, et al. Spatial separation of the cyanogenic β-glucosidase ZfBGD2 and cyanogenic glucosides in the haemolymph of Zygaena larvae facilitates cyanide release [J]. Royal Society Open Science, 2017, 4 (6): 170262

    [10] Zhou X, Huang Z, Yang HW, et al. β-Glucosidase inhibition sensitizes breast cancer to chemotherapy [J]. Biomedicine & Pharmacotherapy, 2017, 91: 504–509

    [11] Rahikainen JL, Martin-Sampedro R, Heikkinen H, et al. Inhibitory effect of lignin during cellulose bioconversion: the effect of lignin chemistry on non-productive enzyme adsorption [J]. Bioresource Technology, 2013, 133: 270–278

    [12] Rahikainen JL, Moilanen U, Nurmi-Rantala S, et al. Effect of temperature on lignin-derived inhibition studied with three structurally different cellobiohydrolases [J]. Bioresource Technology, 2013, 146: 118–125

    [13] Cai L, Zheng SW, Shen YJ, et al. Complete genome sequence provides insights into the biodrying-related microbial function of Bacillus thermoamylovorans isolated from sewage sludge biodrying material [J]. Bioresource Technology, 2018, 260: 141–149

    [14] Arthornthurasuk S, Jenkhetkan W, Suwan E, et al. Molecular characterization and potential synthetic applications of GH1 β-glucosidase from higher termite Microcerotermes annandalei [J]. Applied Biochemistry and Biotechnology, 2018, 186 (4): 877–894

    [15] Cao LC, Wang ZJ, Ren GH, et al. Engineering a novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of sugarcane bagasse at high glucose concentration [J]. Biotechnology for Biofuels, 2015, 8: 202

    [16] Fang Z, Fang WM, Liu JJ, et al. Cloning and characterization of a β-glucosidase from marine microbial metagenome with excellent glucose tolerance [J]. Journal of Microbiology and Biotechnology, 2010, 20 (9): 1351–1358

    [17] Liu Y, Li R, Wang J, et al. Increased enzymatic hydrolysis of sugarcane bagasse by a novel glucose-and xylose-stimulated β-glucosidase from Anoxybacillus flavithermus subsp. yunnanensis E13T [J]. BMC Biochemistry, 2017, 18 (1): 4

    [18] Cao HF, Zhang YQ, Shi PJ, et al. A highly glucose-tolerant GH1 β-glucosidase with greater conversion rate of soybean isoflavones in monogastric animals [J]. Journal of Industrial Microbiology & Biotechnology, 2018, 45 (6): 369–378

    [19] Uchiyama T, Yaoi K, Miyazaki K. Glucose-tolerant β-glucosidase retrieved from a Kusaya gravy metagenome [J]. Frontiers in Microbiology, 2015, 6: 548

    [20] Yang F, Yang XF, Li Z, et al. Overexpression and characterization of a glucose-tolerant β-glucosidase from T. aotearoense with high specific activity for cellobiose [J]. Applied Microbiology and Biotechnology, 2015, 99 (21): 8903–8915

    [21] Crespim E, Zanphorlin LM, de Souza FHM, et al. A novel cold-adapted and glucose-tolerant GH1 β-glucosidase from Exiguobacterium antarcticum B7 [J]. International Journal of Biological Macromolecules, 2016, 82: 375–380

    [22] Mallek-Fakhfakh H, Belghith H. Physicochemical properties of thermotolerant extracellular β-glucosidase from Talaromyces thermophilus and enzymatic synthesis of cello-oligosaccharides [J]. Carbohydrate Research, 2016, 419: 41–50

    [23] Chamoli S, Kumar P, Navani NK, et al. Secretory expression, characterization and docking study of glucose-tolerant β-glucosidase from B. subtilis [J]. International Journal of Biological Macromolecules, 2016, 85: 425–433

    [24] Mai ZM, Yang J, Tian XP, et al. Gene cloning and characterization of a novel salt-tolerant and glucose-enhanced β-glucosidase from a marine Streptomycete [J]. Applied Biochemistry and Biotechnology, 2013, 169 (5): 1512–1522

    [25] Berlin A. No barriers to cellulose breakdown [J]. Science, 2013, 342 (6165): 1454–1456

    [26] Zanoelo FF, Polizeli MLTM, Terenzi HF, et al. β-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose [J]. FEMS Microbiology Letters, 2004, 240 (2): 137–143

    [27] Souza FHM, Meleiro LP, Machado CB, et al. Gene cloning, expression and biochemical characterization of a glucose-and xylose-stimulated β-glucosidase from Humicola insolens RP86 [J]. Journal of Molecular Catalysis B: Enzymatic, 2014, 106: 1–10

This Article

ISSN:0253-2654

CN: 11-1996/Q

Vol 47, No. 07, Pages 2050-2059

July 2020

Downloads:2

Share
Article Outline

Knowledge

Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion and conclusion
  • References