Advertisement

Applied Microbiology and Biotechnology

, Volume 72, Issue 4, pp 705–712 | Cite as

Cloning of β-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3)

  • Da Teng
  • Jian-hua Wang
  • Ying Fan
  • Ya-lin Yang
  • Zi-gang Tian
  • Jin Luo
  • Guan-pin Yang
  • Fan Zhang
Biotechnologically Relevant Enzymes and Proteins

Abstract

β-1,3-1,4-Glucanase has been applied in the brewing and animal feed additive industry. It can effectively improve digestibility of barley-based diets and reduce enteritis. It also reduces viscosity during mashing for high-quality brewers malt. The aim of this work is to clone β-1,3-1,4-glucanase-encoding gene and express it heterogeneously. The gene was amplified by polymerase chain reaction using Bacillus licheniformis genomic DNA as the template and ligated into the expression vector pET28a. The recombinant vector was transformed into Escherichia coli. The estimated molecular weight of the recombinant enzyme with a six-His tag at the N terminus was about 28 kDa, and its activities in cell lysate supernatant were 1,286 and 986 U ml−1 for 1% (w/v) barley β-glucan and 1% (w/v) lichenan, respectively. Accordingly, the specific activities were 2,479 and 1,906 U mg−1 for these two substrates. The expression level of recombinant β-1,3-1,4-glucanase was about 60.9% of the total protein and about 12.5% of the total soluble protein in crude cell lysate supernatant. Acidity and temperature optimal for this recombinant enzyme was pH 5.6 and 40°C, respectively.

Keywords

Glycosidic Bond Recombinant Enzyme Bacillus Licheniformis Glucanase Activity Glucanase Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgement

This project is funded by the Chinese National Hi-Tech R&D Program (Chinese 863 Program No. 2001 AA 246041 and 2004 AA 246040).

References

  1. Akita M, Kaytama K, Hatada Y, Ito S, Horikoshi K (2005) A novel β-glucanase gene from Bacillus halodurans C-125. FEMS Microbiol Lett 248:9–15CrossRefPubMedGoogle Scholar
  2. Anderson MA, Stone BA (1975) A new substrate for investigating the specifity of beta-glucan hydrolases. FEBS Lett 52:202–207CrossRefPubMedGoogle Scholar
  3. Ausubel FM, Brent R, Kingston RE, Moore DD (1999) Short protocols in molecular biology, 4th edn. Wiley, New YorkGoogle Scholar
  4. Bielecki S, Galas E (1991) Microbial β-glucanase different from cellulases. Crit Rev Biotechnol 10:275–305PubMedGoogle Scholar
  5. 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 222:278–283CrossRefPubMedGoogle Scholar
  6. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  7. Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66PubMedCrossRefGoogle Scholar
  8. 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 276:17895–17901CrossRefPubMedGoogle Scholar
  9. Cheng HL, Tsai LC, Lin SS, Yuan HS, Yang NS, Lee SH, Shyur LF (2002) Mutagenesis of Trp(54) and Trp(203) residues on Fibrobacter succinogenes 1,3-1,4-beta-d- glucanase significantly affects catalytic activities of the enzyme. Biochemistry 41:8759–8766CrossRefPubMedGoogle Scholar
  10. Dubendorff JW, Studier FW (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol 219:45–59CrossRefPubMedGoogle Scholar
  11. 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 25:771–775Google Scholar
  12. Ekinci MS, Mccrae SI, Flint HJ (1997) Isolation and overexpression of a gene encoding an extracellular 1,3-1,4-glucanase from Streptococcus bovis. Appl Environ Microbiol 63:3752–3756PubMedGoogle Scholar
  13. Gosalbes MJ, Perez-Gonzalez JA, Gonzalez R, Navarro A (1991) Two β-glucanase genes are clustered in Bacillus polymyxa molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-β-(1,3)-(1,4)- glucanase. J Bacteriol 9:7705–7710Google Scholar
  14. Hahn M, Olsen O, Politz O, Borriss R, Heinemann U (1995) Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-glucanase. J Biol Chem 270:3081–3088CrossRefPubMedGoogle Scholar
  15. 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 8:471–475CrossRefGoogle Scholar
  16. Hirt B (1967) Selective extraction of polyoma DNA from infected mouse cell culture. J Mol Biol 26:365–369CrossRefPubMedGoogle Scholar
  17. Juncosa M, Pons J, Dot T, Querol E, Planas A (1994) Identification of active site carboxylic residues in Bacillus licheniformis 1,3-1,4-beta-d-glucan 4- glucanohydrolase by site-directed mutagenesis. J Biol Chem 269:14530–14535PubMedGoogle Scholar
  18. Kim JY (2003) Overproduction and secretion of Bacillus circulans endo-β-1,3-1,4- glucanase gene (bglBC1) in B. subtilis and B. megaterium. Biotechnol Lett 25:1445–1449CrossRefPubMedGoogle Scholar
  19. Li S, Sauer WC, Huang SX, Gabert VI (1996) Effect of beta-glucanase supplementation to hulless barley- or wheat-soybean meal diets on the digestibilities of energy, protein, beta-glucans and amino acid in young pigs. J Anim Sci 74:1649–1656PubMedGoogle Scholar
  20. Lloberas J, Perez-Pons JA, Querol E (1991) Molecular cloning, expression and nucleotide sequence of the endo-β-1,3-1,4-d-glucanase gene from Bacillus licheniformis. Eur J Biochem 197:337–343CrossRefPubMedGoogle Scholar
  21. 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 faecal microflora of broiler chickens fed a wheat and barley-based diet. Anim Res 51:395–406CrossRefGoogle Scholar
  22. Meldgaard M, Harthill J (1994) Different effect of N-glycosylation on the thermostability of highly homologous bacterial (1,3-1,4)-beta-glucanase secreted from yeast. Microbiology 140:153–157PubMedGoogle Scholar
  23. Michel G, Chantalat L, Duee E, Barbeyron T, Henrissat B, Kloareg B, Dideberg O (2001) The kappa-carrageenase of P. carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of Clan-B glycoside hydrolases. Structure (Camb) 9:513–525CrossRefGoogle Scholar
  24. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  25. 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 225:177–185CrossRefPubMedGoogle Scholar
  26. Parrish FW, Perlin AS, Reese ET (1960) Selective enzymolysis of poly-β-d glucan, and the structure of the polymers. Can J Chem 38:2094–2104CrossRefGoogle Scholar
  27. Planas A (2000) Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochim Biophys Acta 1543:361–382PubMedGoogle Scholar
  28. 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 177:447–452CrossRefPubMedGoogle Scholar
  29. Stemberger RS (1981) A general approach to the culture of planktonic rotifers. Can J Fish Aquat Sci 38:721–724CrossRefGoogle Scholar
  30. Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130CrossRefPubMedGoogle Scholar
  31. Supek F, Vlahovicek K (2005) Comparison of codon usage measures and their applicability in prediction of microbial gene expressivity. BMC Bioinformatics DOI 10.1186/1471-2105-6-182Google Scholar
  32. Teng D, Wang JH, Yao Y, Yang YL, Liu LH (2005) Cloning and expression of Bacillus licheniformis EG039 beta-1,3-1,4-glucanase in Pichia methanolica. In: Wang Jianhua (eds) Research and development of the new safe feed additives. China Science, Beijing, pp 102–109Google Scholar
  33. van Rensburg P, van Zyl WH, Pretorius IS (1997) Over-expression of the Saccharomyces cerevisiae exo-β-1,3-glucanase gene together with the Bacillus subtilis endo-β-1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-β-1,4-glucanase gene in yeast. J Biotechnol 55:43–53CrossRefPubMedGoogle Scholar
  34. 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 44:9197–9205CrossRefPubMedGoogle Scholar
  35. Wolf M, Geczi A, Simon O, Borriss R (1995) Genes encoding xylan and β-glucan hydrolyzing enzyme in Bacillus subtilis: characterization, mapping and construction of strain deficient in lichenase, cellulose and xylanase. Microbiology 4:281–290CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Da Teng
    • 1
  • Jian-hua Wang
    • 1
  • Ying Fan
    • 1
  • Ya-lin Yang
    • 1
  • Zi-gang Tian
    • 1
  • Jin Luo
    • 1
  • Guan-pin Yang
    • 2
  • Fan Zhang
    • 1
  1. 1.Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural SciencesBeijingPeople’s Republic of China
  2. 2.College of Marine Life ScienceOcean University of ChinaShandongPeople’s Republic of China

Personalised recommendations