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β-1,3-1,4-glucanase gene from Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi

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Abstract

Bacillus velezensis is a known antifungal bacteria. To understand the role of β-1, 3-1, 4-glucanase played on B. velezensis about the mechanism which exerts effect on fungi, we isolated and cloned the β-1, 3-1, 4-glucanase gene (Bglu1) from B. velezensis ZJ20. The Bglu1 open reading frame was 732 bp that encoded a protein with 243 amino acids and a calculated molecular weight of 27.3 kDa. The same gene without the signal peptide, termed Bglu2, was also cloned and expressed in E. coli BL21. Among the two variants, only Bglu2 protein was expressed. Purified Bglu2 could be eluted with imidazole solution at concentrations ranging from 100 to 500 mM although the highest expression was observed at 150 and 200 mM and the purest was at 500 mM. In addition, activity of the crude enzyme was 1527 U ml−1 and the highest activity of the purified enzyme was 1706 U ml−1. The purified β-1, 3-1, 4-glucanase had activity on a wide range of pH and temperatures and displayed optimal activity at pH 5.0 and 35 °C. More importantly, the mycelial morphology of three pathogenic fungi was destroyed by the purified β-1, 3-1, 4-glucanase. In conclusion, β-1, 3-1, 4-glucanase from B. velezensis ZJ20 can be highly expressed in E. coli BL21 and the recombinant protein is pathogenic to fungi.

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References

  • Adams DJ (2004) Fungal cell wall chitinases and glucanases. Microbiology 150:2029–2035

    Article  CAS  Google Scholar 

  • Akita M, Kaytama K, Hatada Y, Ito S, Horikoshi K (2005) A nove β-glucanase gene from Bacillus halodurans C-125. FEMS Microbiol Lett 248:9–15

    Article  CAS  Google Scholar 

  • Ana IFM, Mario JVF, Juan ACR, Jorge ML, José ANM, Isidro BP (2008) A pure culture of strain AH2 of the Bacillus velezensis species and a product for the biological control of phytopathogenic fungi. Europäisches Pantentamt 08736711:6

    Google Scholar 

  • Anderson MA, Stone BA (1975) A new substrate for investigating the specifity of beta-glucan hydrolases. FEBS Lett 52(2):202–207

    Article  CAS  Google Scholar 

  • Barbeyron T, Gerard A, Potin P, Henrissat B, Kloareg B (1998) The kappa-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases. Mol Biol Evol 15(5):528–537

    Article  CAS  Google Scholar 

  • Borriss R, Buettner K, Maentsaelae P (1990) Structure of the β -1, 3-1, 4-glucanase of Bacillus macerans: homologies to other β-glucanase. Mol Gen Genet 222:278–283

    Article  CAS  Google Scholar 

  • Brennan CS, Cleary LJ (2005) The potential use of cereal (1 → 3, 1 → 4)-β-d-glucans as functional food ingredients. J Cereal Sci 42:1–13

    Article  CAS  Google Scholar 

  • Burton RA, Fincher GB (2009) (1, 3; 1, 4)-β-d-Glucans in cell walls of the Poaceae, lower plants, and fungi: a tale of two linkages. Mol Plant 2(5):873–882

    Article  CAS  Google Scholar 

  • Celestino KR, Cunha RB, Felix CR (2006) Characterization of a β-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry. BMC Biochem 7:23

    Article  CAS  Google Scholar 

  • Chen H, Li XL, Ljungdahl LG (1997) Sequencing of a 1, 3-1, 4-β-d-glucanase (lichenase) from the anaerobic fungus Orpinomyces strain PC-2: properties of the enzyme expressed in Escherichia coli and evidence that the gene has a bacterial origin. J Bacteriol 179:6028–6034

    CAS  Google Scholar 

  • Chen JL, Tsai LC, Wen TN, Tang JB, Yuan HS, Shyur LF (2001) Directed mutagenesis of specific active site residues on Fibrobacter succinogenes 1, 3-1, 4-β-glucanase significantly affects catalysis and enzyme structural stability. J Biol Chem 276:17895–17901

    Article  CAS  Google Scholar 

  • Ekinci MS, Flint HJ (2001) Expression of bifunctional genes encoding xylanase and β (1, 3-1, 4)-glucanase in Gram-positive bacteria. Turk J Vet Anim Sci 25:771–775

    Google Scholar 

  • Ekinci MS, McCrae SI, Flint HJ (1997) Isolation and overexpression of a gene encoding an extracellular β-(1, 3-1, 4)-glucanase from Streptococcus bovis JB1. Appl Environ Microbiol 63(10):3752–3756

    CAS  Google Scholar 

  • Farkas V (1979) Biosynthesis of cell walls of fungi. Microbiol Mol Biol R 43(4):519

    Google Scholar 

  • Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59(2):257–268

    Article  CAS  Google Scholar 

  • Görlach JM, van der Knaap E, Walton JD (1998) Cloning and targeted disruption of MLG1, a gene encoding two of three extracellular mixed-linked glucanases of Cochliobolus carbonum. Appl Environ Microbiol 64(2):385–391

    Google Scholar 

  • Grishutin SG, Gusakov AV, Dzedzyulya EI, Sinitsyn AP (2006) A lichenase-like family 12 endo-(1, 4)-β-glucanase from Aspergillus japonicus: study of the substrate specificity and mode of action on β-glucans in comparison with other glycoside hydrolases. Carbohydr Res 341:218–229

    Article  CAS  Google Scholar 

  • Hinchliff E, Wendy GB (1984) Expression of the cloned endo-β-1, 3-1, 4-glucanase gene of Bacillus subtilis in Saccharomyces cerevisiae. Curr Genet 8:471–475

    Article  Google Scholar 

  • Høj PB, Hartman DJ, Morrice NA, Doan DNP, Fincher GB (1989) Purification of (1 → 3)-β-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone. Plant Mol Biol 13:31–42

    Article  Google Scholar 

  • Hua CW, Yan QJ, Jiang ZQ, Li YN, Katrolia P (2010) High-level expression of a specific β-1, 3-1, 4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris. Appl Microbiol Biotechnol 88:509–518

    Article  CAS  Google Scholar 

  • Kim P, Chung KC (2004) Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908. FEMS Microbiol Lett 234(1):177–183

    Article  CAS  Google Scholar 

  • Kitamura E, Myouga H, Kamei Y (2002) Polysaccharolytic activities of bacterial enzymes that degrade the cell walls of Pythium porphyrae, a causative fungus of red rot disease in Porphyra yezoensis. Fish Sci 68(2):436–445

    Article  CAS  Google Scholar 

  • Kour J, Maheshwari DK, Mehta P (1993) Microbial degradation of aquatic biomass by Trichoderma viride 992 and Aspergillus wentii 669 with reference to the physical structure. J Basic Microbiol 33:19–25

    Article  CAS  Google Scholar 

  • Li S, Sauer WC, Huang SX, Gabert VM (1996) Effect of beta-glucanase supplementation to hulless barley-or wheat-soybean meal diets on the digestibilities of energy, protein, beta-glucans, and amino acids in young pigs. J Anim Sci 74(7):1649–1656

    CAS  Google Scholar 

  • Liu X, Ren B, Chen M, Wang H, Kokare CR, Zhou X, Wang JD, Dai H, Song F, Liu M, Wang J, Wang S, Zhang L (2010) Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3. Appl Microbiol Biotechnol 87(5):1881–1893

    Article  CAS  Google Scholar 

  • Luo H, Yang J, Yang P, Li J, Huang H, Shi P, Bai Y, Wang Y, Fan Y, Yao B (2010) Gene cloning and expression of a new acidic family 7 endo-β-1, 3-1, 4-glucanase from the acidophilic fungus Bispora sp. MEY-1. Appl Microbiol Biotechnol 85(4):1015–1023

    Article  CAS  Google Scholar 

  • Mathlouthi N, Serge MS, Luc S, Bernard Q, Michel L (2002) Effects of xylanase and β-glucanase addition on performance, nutrient digestibility, and physico-chemical conditions in the small intestine contents and fecal microflora of broiler chickens fed a wheat and barley-based diet. Anim Res 51:395–406

    Article  CAS  Google Scholar 

  • McCleary BV (1988) Purification of (1 → 3), (1 → 4)-β-d-glucan from barley flour. Method Enzymol 160:511–514

    Article  CAS  Google Scholar 

  • Murray PG, Grassick A, Laffey CD, Cuffe MM, Higgins T, Savage AV, Planas A, Tuohy MG (2001) Isolation and characterization of a thermostable endo-β-glucanase active on 1, 3-1, 4-β-d-glucans from the aerobic fungus Talaromyces emersonii CBS814. 70. Enzyme Microb Technol 29(1):90–98

    Article  CAS  Google Scholar 

  • Olsen O, Borriss R, Simon O, Thomsen KK (1991) Hybrid Bacillus (1-3, 1-4)-β-glucanase: engineering thermostable enzymes by construction of hybrid genes. Mol Gen Genet 225:177–185

    Article  CAS  Google Scholar 

  • Parrish FW, Perlin AS, Reese ET (1960) Selective enzymolysis of poly-β-d-glucans, and the structure of the polymers. Can J Chem 38(11):2094–2104

    Article  CAS  Google Scholar 

  • Planas A (2000) Bacterial 1, 3-1, 4-β-glucanase: structure, function and protein engineering. Biochim Biophys Acta 1543(2):361–382

    Article  CAS  Google Scholar 

  • Rooney AP, Price NPJ, Ehrhardt C, Swezey JL, Bannan JD (2009) Phylogeny and molecular taxonomy of the Bacillus subtilis specied complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov. Int J Syst Evol Micr 59:2429–2436

    Article  CAS  Google Scholar 

  • Ruiz-García C, Béjar V, Martínez-Checa F, Llamas I, Quesada E (2005) Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Vélez in Málaga, southern Spain. Int J Syst Evol Micr 55:191–195

    Article  CAS  Google Scholar 

  • Schimming S, Schwarz WH (1991) Properties of a thermoactive β-1, 3-1, 4-glucanase (Lichenase) from Clostridium thermocellum expressed in Escherichia coli. Biochem Biophys Res Commun 177(1):447–452

    Article  CAS  Google Scholar 

  • Teather RM, Erfle JD (1990) DNA sequence of a Fibrobacter succinogenes mixed-linkage 3-glucanase (1, 3-1, 4-β-d-glucan 4-glucanohydrolase) gene. J Bacteriol 172(7):3837–3841

    CAS  Google Scholar 

  • Teng D, Wang J, Fan Y, Yang Y, Tian Z, Luo J, Yang G, Zhang F (2006) Cloning of β-1, 3-1, 4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3). Appl Microbiol Biotechnol 72:705–712

    Article  CAS  Google Scholar 

  • Tjalsma H, Bolhuis A, Jongbloed JDH, Bron S, van Dijl JM (2000) Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol R 64(3):515–547

    Article  CAS  Google Scholar 

  • van Rensburg P, van Zyl WH, Pretorius IS (1991) Over-expression of the Saccharomyces cerevisiaeexo-β-1, 3-glucanase gene together with the Bacillus subtilis endo-β-1, 3-1, 4-glucanase gene and the Butyrivibrio fibrisolvensendo-β-1, 4-glucanase gene in yeast. J Biotechnol 55:43–53

    Article  Google Scholar 

  • Wang JL, Ruan H, Zhang HF, Zhang Q, Zhang HB, He GQ, Shen SR (2007) Characterization of a thermostable and acidic-tolerable β-glucanase from aerobic fungi Trichoderma koningii ZJU-T. J Food Sci 72(9):C452–C456

    Article  CAS  Google Scholar 

  • Wang LT, Lee FL, Tai CJ, Kuo HP (2008) Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens. Int J Syst Evol Micr 58(3):671–675

    Article  CAS  Google Scholar 

  • Wen TN, Chen JL, Lee SH, Yang NS, Shyur LF (2005) A truncated Fibrobacter succinogenes 1, 3-1, 4-β-glucanase with improved enzymatic activity and thermotolerance. Biochemistry 44:9197–9205

    Article  CAS  Google Scholar 

  • Xu T, Zhu TH, Li SJ, Qiao TM (2014) Fungus-inhibitory activity and gene Cloning of β-glucanase from Bacillus velezensis YB15. Chin J Biol Control 30(2):276–281

    Google Scholar 

  • Yaish M, Doxey A, McConkey B, Moffatt B, Griffith M (2006) Coldactive winter rye glucanases with ice-binding capacity. Plant Physiol 141(4):1459–1472

    Article  CAS  Google Scholar 

  • Yang P, Shi P, Wang Y, Bai Y, Meng K, Luo H, Yuan T, Yao B (2007) Cloning and overexpression of a Paenibacillus β-glucanase in Pichia pastoris: purification and characterization of the recombinant enzyme. J Microbiol Biotechnol 17(1):58–66

    Google Scholar 

  • Yang SQ, Yan QJ, Jiang ZQ, Fan GS, Wang L (2008) Biochemical characterization of a thermostable novel β-1, 3-1, 4-glucanase (lichenase) from Paecilomyces thermophila. J Agric Food Chem 56(13):5345–5351

    Article  CAS  Google Scholar 

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Acknowledgments

We acknowledge the College of Life Science of Sichuan Agriculture University for providing technical and equipment support.

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Authors and Affiliations

  1. College of Forestry, Sichuan Agricultural University, Ya’an, 625014, China

    Ting Xu & Tianhui Zhu

  2. Key Laboratory of Forest Protection of Sichuan Province, Ya’an, 625014, China

    Shujiang Li

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  1. Ting Xu
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  2. Tianhui Zhu
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Correspondence to Tianhui Zhu.

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Xu, T., Zhu, T. & Li, S. β-1,3-1,4-glucanase gene from Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi. World J Microbiol Biotechnol 32, 26 (2016). https://doi.org/10.1007/s11274-015-1985-0

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