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Production, purification and application-relevant characterisation of an endo-1,3(4)-β-glucanase from Rhizomucor miehei

Applied Microbiology and Biotechnology volume 76, pages 835–841 (2007)Cite this article

Abstract

Growth on a wheat bran media induced production of an extracellular β-glucanase by Rhizomucor miehei (DSM 1330). The enzyme was purified to homogeneity. Substrate specificity studies coupled with protein database similarity searching using mass spectrometry-derived sequence data indicate it to be an endo-1,3(4)-β-glucanase (EC 3.2.1.6). The enzyme was characterised in terms of potential suitability for use in animal (poultry) feed. Significant activity was observed over the entire pH range typical of the avian upper digestive tract (pH 2.6–6.5). The enzyme was also found to be more thermostable than current commercialized β-glucanases, particularly when heated at a high enzyme concentration, and retained twice as much residual activity as the latter upon exposure to simulated avian digestive tract conditions. There are no previous reports of the production, purification or characterization of a β-glucanase from a Rhizomucor, and the enzyme’s application-relevant physicochemical characteristics render it potentially suited for use in animal feed.

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References

  • Akiba S, Kimura Y, Yamamoto K, Kumagai H (1995) Purification and characterization of a protease-resistant cellulase from Aspergillus niger. J Ferment Bioeng 79(2):125–130

    CAS  Article  Google Scholar 

  • Akiyama T, Pillai M, Sentoku N (2004) Cloning, characterisation and expression of OsGLN2, a rice endo-1,3-β-glucanase gene regulated developmentally in flowers and hormonally in germinating seeds. Planta 220(1):129–139

    CAS  Article  Google Scholar 

  • Almirall M, Esteve-Garcia E (1995) In vitro stability of a β-glucanase preparation from Trichoderma longibrachiatum and its effect in a barley based diet fed to broiler chicks. Anim Feed Sci Technol 54:149–158

    CAS  Article  Google Scholar 

  • Bairoch A (2000) The enzyme database in 2000. Nucleic Acids Res 28:304–305

    CAS  Article  Google Scholar 

  • Bathgate G (1979) The institute of brewing analysis committee the determination of endo-β-glucanase activity in malt. J Inst Brew 85:92–94

    CAS  Article  Google Scholar 

  • 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 72:248–254

    CAS  Article  Google Scholar 

  • Choct M (2001) Enzyme supplementation of poultry diets based on viscous cereals. In: Bedford M, Partridge G (eds) Enzymes in farm animal nutrition. CABI Publishing, Oxon UK, pp 145–160

    Google Scholar 

  • Choct M (2002) Non-starch polysaccharides: effect on nutritive value. In: McNab J, Boorman K (eds) Poultry feedstuffs, supply, composition and nutritive value, Poultry Science Symposium Series, vol 26. CABI Publishing, Oxon UK, pp 221–235

    Chapter  Google Scholar 

  • Christakopoulos P, Hatzinikolaou D, Fountoukidis G, Kekos D, Claeyssens M, Macris B (1999) Purification and mode of action of an alkali-resistant endo-1,4-β-glucanase from Bacillus pumilus. Arch Biochem Biophys 364(1):61–66

    CAS  Article  Google Scholar 

  • Classen H (1996) Cereal grain starch and exogenous enzymes in poultry diets. Anim Feed Sci Technol 62:21–27

    CAS  Article  Google Scholar 

  • Coutinho P, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach. In: Gilbert H, Davies G, Henrissat B, Svensson B (eds) Recent advances in carbohydrate bioengineering. The Royal Society of Chemistry, Cambridge UK, pp 3–12

    Google Scholar 

  • European Commission (2007) Community register of feed additives pursuant to Regulation (EC) No 1831/2003, Appendixes 3 & 4, Annex: List of additives (released Feb 2007 Rev 9), available http://ec.europa.eu/food

  • Gerard C (1990) Purification of glycoproteins. In: Deutscher M (ed) Methods in enzymology: guide to protein purification, vol 182. Academic, London, pp 529–539

    Chapter  Google Scholar 

  • Grishutin S, Gusakov A, Dzedzyulya E, Sinitsyn A (2006) A lichenase-like family 12 endo-(1→4)-beta-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

    CAS  Article  Google Scholar 

  • Hasan F, Shah A, Hameed A (2006) Industrial applications of microbial lipases. Enzyme Microb Technol 39(2):235–251

    CAS  Article  Google Scholar 

  • Kawai R, Igarashi K, Yoshida M, Kitaoka M, Samejima M (2006) Hydrolysis of β-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-beta-glucanase from the basidiomycete Phanerochaete chrysosporium. Appl Microbiol Biotechnol 71(6):898–906

    CAS  Article  Google Scholar 

  • Kim Y, Kim H, Bae K, Yu J, Oh T (1998) Purification and properties of a thermostable phytase from Bacillus sp. DS11. Enzyme Microb Technol 22(1):2–7

    CAS  Article  Google Scholar 

  • Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    CAS  Article  Google Scholar 

  • Mackey A, Haystead T, Pearson W (2002) Getting more from less-algorithms for rapid protein identification with multiple short peptide sequences. Mol Cell Proteomics 1:39–147

    Article  Google Scholar 

  • Manchenko G (2003) Handbook of detection of enzymes on electrophoretic gels, 2nd edn. CRC Press, Boca Raton

    Google Scholar 

  • Martin J, Murphy R, Power R (2006) Purification and physico-chemical characterisation of genetically modified phytases expressed in Aspergillus awamori. Bioresour Technol 97:1703–1708

    CAS  Article  Google Scholar 

  • McCarthy T, Hanniffy O, Savage A, Tuohy M (2003) Catalytic properties and mode of action of three endo-β-glucanases from Talaromyces emersonii on soluble β-1,4-and β-1,3;1,4-linked glucans. Int J Biol Macromol 33:141–148

    CAS  Article  Google Scholar 

  • McCleary B (2001) Analysis of feed enzymes. In: Bedford M, Partridge G (eds) Enzymes in farm animal nutrition. CABI Publishing, Oxon UK, pp 85–107

    Google Scholar 

  • Miller G (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428

    CAS  Article  Google Scholar 

  • Moran A, Walsh G (2004) A comparison of the physicochemical properties of β-glucanases relevant to their application as supplemental digestive aids in monogastric animal feeds. Proceedings of the 6th international conference on protein stabilization. Bratislava, Slovakia, September 26–29, 2004. Slovak Society of Chemical Engineering, Bratislava, p 86

  • Nevalainen K (1981) Induction, isolation and characterisation of Aspergillus niger mutant strains producing elevated levels of β-galactosidase. Appl Environ Microbiol 41(3):593–596

    CAS  Article  Google Scholar 

  • Perkins D, Pappin D, Creasy D, Cottrell J (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567

    CAS  Article  Google Scholar 

  • Pitson S, Seviour R, McDougall B (1993) Noncellulolytic fungal β-glucanases: their physiology and regulation. Enzyme Microb Technol 15:178–192

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  • Sarath G, De La Motte R, Wagner F (1989) Protease assay methods. In: Beynon R, Bond J (eds) Proteolytic enzymes, a practical approach. IRL, Oxford UK, pp 25–55

    Google Scholar 

  • Shevchenko A, Sunyaev S, Loboda A, Shevchenko A, Bork P, Ens W, Standing K (2001) Charting the proteomes of organisms with unsequenced genomes by MALDI-Quadrupole time-of-flight mass spectrometry and BLAST homology searching. Anal Chem 73(9):1917–1926

    CAS  Article  Google Scholar 

  • Teng D, Wang J, Fan Y, Yang Y, Tian Z, Luo J, Yang G and 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(4):705–712

    CAS  Article  Google Scholar 

  • Tervila-Wilo A, Parkkonen T, Morgan A, Hopeakoski-Nurminen M, Poutanen K, Heikkinen P, Autio K (1996) In vitro digestion of wheat microstructure with xylanase and cellulase from Trichoderma reesei. J Cereal Sci 24:215–225

    CAS  Article  Google Scholar 

  • Vahjen W, Simon O (1999) Biochemical characteristics of non starch polysaccharide hydrolyzing enzyme preparations designed as feed additives for poultry and piglet nutrition. Arch Anim Nutr 52:1–14

    CAS  Google Scholar 

  • Walsh G (2002) Protein: biochemistry and biotechnology. Wiley, Chicester

    Google Scholar 

  • Zyla K, Gogol D, Koreleski J, Swiatkiewicz S, Ledoux D (1999) Simultaneous application of phytase and xylanase to broiler feeds based on wheat: in vitro measurements of phosphorus and pentose release from wheats and wheat-based feeds. J Sci Food Agric 79:1832–1840

    CAS  Article  Google Scholar 

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Acknowledgement

This work was funded by Enterprise Ireland.

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  1. Department of Chemical and Environmental Sciences and Materials and Surface Science Institute, University of Limerick, Limerick City, Ireland

    A. Boyce & G. Walsh

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  1. A. Boyce
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  2. G. Walsh
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Correspondence to G. Walsh.

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Boyce, A., Walsh, G. Production, purification and application-relevant characterisation of an endo-1,3(4)-β-glucanase from Rhizomucor miehei . Appl Microbiol Biotechnol 76, 835–841 (2007). https://doi.org/10.1007/s00253-007-1058-x

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  • DOI: https://doi.org/10.1007/s00253-007-1058-x

Keywords

  • β-glucanase
  • β-glucan
  • Rhizomucor miehei
  • Animal feed