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
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
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
Bairoch A (2000) The enzyme database in 2000. Nucleic Acids Res 28:304–305
Bathgate G (1979) The institute of brewing analysis committee the determination of endo-β-glucanase activity in malt. J Inst Brew 85:92–94
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
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
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
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
Classen H (1996) Cereal grain starch and exogenous enzymes in poultry diets. Anim Feed Sci Technol 62:21–27
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
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
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
Hasan F, Shah A, Hameed A (2006) Industrial applications of microbial lipases. Enzyme Microb Technol 39(2):235–251
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
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
Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
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
Manchenko G (2003) Handbook of detection of enzymes on electrophoretic gels, 2nd edn. CRC Press, Boca Raton
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
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
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
Miller G (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428
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
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
Pitson S, Seviour R, McDougall B (1993) Noncellulolytic fungal β-glucanases: their physiology and regulation. Enzyme Microb Technol 15:178–192
Planas A (2000) Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochim Biophys Acta 1543:361–382
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
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
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
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
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
Walsh G (2002) Protein: biochemistry and biotechnology. Wiley, Chicester
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
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This work was funded by Enterprise Ireland.
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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