Abstract
A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45–70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.
Similar content being viewed by others
References
Khattak WA, Ul-Islam M, Park JK (2012) Prospects of reusable endogenous hydrolyzing enzymes in bioethanol production by simultaneous saccharification and fermentation. Korean J Chem Eng 29(11):1467–1482
Ha JH, Shah N, Ul-Islam M, Park JK (2011) Potential of the waste from beer fermentation broth for bio-ethanol production without any additional enzyme, microbial cells and carbohydrates. Enzyme Microb Tech 49:298–304
Shehzad O, Khan S, Khan T, Park JK (2009) Production of bacterial cellulose in static conditions by a simple fed-batch cultivation strategy. Korean J Chem Eng 26:1689–1992
Khan T, Hyun SH, Park JK (2007) Production of glucuronan oligosaccharides using the waste of beer fermentation broth as a basal medium. Enzyme Microb Technol 42:89–92
Ha JH, Gang MK, Khan T, Park JK (2012) Evaluation of sediments of the waste from beer fermentation broth for bioethanol production. Korean J Chem Eng 29:1224–1231
Park JK, Hyun SH, Ahn WS (2006) Production of bacterial cellulose using waste of beer fermentation broth. Korean Chem Eng Res 44:52–57
Wood TM (1991) Fungal cellulases. In: Haigler CH et al (eds) Biosynthesis and biodegradation of cellulose. Macel Dekker Inc., New York
Han M, Kim Y, Kim Y, Chung B, Choi GW (2011) Bioethanol production from optimized pretreatment of cassava stem. Korean J Chem Eng 28:119–125
Lipke PN, Ovalle R (1998) Cell wall architecture in yeast: new structure and new challenges. J Bacteriol 180:3735–3740
Kádár Z, Szengyel Z, Réczey K (2004) Simultaneous saccharification and fermentation (SSF) of industrial wastes for the production of ethanol. Ind Crop Prod 20:103–110
Pilkington PH, Margaritis A, Mensour NA, Russell I (1998) Fundamentals of immobilized yeast cells for continuous beer fermentation: a review. J Inst Brew 104:19–31
Bischof JC, Padanilam J, Holmes WH, Ezzell RM, Lee RC, Tomkins RG, Yarmush ML, Toner M (1995) Dynamics of cell membrane permeability changes at supraphysiological temperatures. Biophys J 68:2608–2614
Buchner E (1897) Alkoholische Gährung ohne Hefezellen. Ber Dtsch Chem Ges 30(117–124):1110–1113
Kim TW, Chokhawala HA, Hess M, Dana CM, Baer Z, Sczyrba A, Rubin EM, Blanch HW, Clark DS (2011) High-throughput in vitro glycoside hydrolase (HIGH) screening for enzyme discovery. Angew Chem Int Ed 50:11215–11218
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Hatfield R, Nevins DJ (1986) Purification and properties of an endoglucanase isolated from the cell walls of Zea mays seedlings. Carbohyd Res 148:265–278
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Mandels M, Hontz L, Nystrom J (1974) Enzymatic hydrolysis of waste cellulose. Biotechnol Bioeng 16:1471–1493
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 7:248–254
Bamforth CW (2009) Current perspectives on the role of enzymes in brewing. J Cereal Sci 50:353–357
Woodward JR, Fincher GB (1982) Purification and chemical properties of two β-glucan endohydrolases from germinating barley. Eur J Biochem 121:663–669
Aastrup S, Bautista N, Janser E, Dörreich K (2004) Choice of enzyme solution should determine choice of raw materials and process. In: Proceedings of the presentation given at world brewing conference, San Diego, USA
Chithra M, Muralikrishna G (2008) Characterization of purified xylanase from finger millet (Eleusine coracana-Indaf 15) malt. Eur Food Res Technol 227:587–597
Bamforth CW, Martin HL, Wainwright T (1979) A role for carboxypeptidase in the solubilization of barley β-glucan. J Inst Brew 85:334–338
Sørensen SB, Svendsen I, Breddam K (1989) Primary structure of carboxypeptidase III from malted barley. Carlsberg Res Commun 54:193–202
MacGregor AW (1987) α-Amylase, limit dextrinase and α-glucosidase enzymes in barley and malt. CRC CR Rev Biotechnol 5:117–128
Jamar C, du Jardin P, Fauconnier ML (2011) Cell wall polysaccharides hydrolysis of malting barley (Hordeum vulgare L.): a review. Biotechnol Agron Soc Environ 15:301–313
Trudel J, Grenier J, Asselin A (1998) Detection of enzymes active on various β-1,3-glucans after denaturing polyacrylamide gel electrophoresis. Electrophoresis 19:1788–1792
Kumar RS, Singh SA, Rao AG (2005) Thermal stability of alpha-amylase from malted jowar (Sorghum bicolor). J Agric Food Chem 53:6883–6888
Acquistucci R, Turfani V, Aureli G (2011) Amylase modiWcation induced by the germination process in organic barley. Eur Food Res Technol 232:583–590
Burton RA, Zhang XQ, Hrmova M, Fincher GB (1999) A single limit dextrinase gene is expressed both in the developing endosperm and in germinated grains of barley. Plant Physiol 119:859–871
Koehler SM, Ho THD (1990) A major gibberellic acid-induced barley aleurone cysteine proteinase which digests hordein. Plant Physiol 94:251–258
Sopanen T, Mikola J (1975) Purification and partial characterization of barley leucine aminopeptidase. Plant Physiol 55:809–814
Jones BL (2005) The endogenous endoproteinase inhibitors of barley and malt and their roles in malting and brewing. J Cereal Sci 42:271–280
Skriver K, Leah R, Mulleruri F, Olsen FL, Mundy J (1992) Structure and expression of the barley lipid transfer protein gene Ltp 1. Plant Mol Biol 18:585–589
Baxter ED (1984) Recognition of two lipases from barley and green malt. J Inst Brew 90:277–281
Doderer A, Kokkelink I, Van der Veen S, Valk BE, Douma AC (1991) Purification and characterization of lipoxygenase from germinating barley. In: Proceedings of the 23rd European Brewery convention congress, Lisbon, pp 109–116
Aehle W (2007) Enzymes in industry production and applications. Wiley-VCH, Weinhheim
Antrobus CJ, Large PJ, Bamforth CW (1997) Changes in the cationic isoenzymesof peroxidase during the malting of barley. I. Tissue location studies. J Inst Brewing 103:227–231
Chun CZ, Hur SB, Kim YT (1997) Purification and characterization of an endoglucanase from the marine rotifer, Brachionus plicatilis. Biochem Mol Biol Int 43:241–249
Farinas CS, Scarpelini LM, Miranda EA, Neto VB (2011) Evaluation of operational parameters on the precipitation of endoglucanase and xylanase produced by solid state fermentation of Aspergillus niger. Braz J Chem Eng 28:17–26
Kavitha S, Nagarajan P (2011) Fermentative production of endoglucanse—kinetics and modeling. Int J Eng Sci Tech 3:1894–1898
Tao YM, Zhu XZ, Huang JZ, Ma SJ, Wu XB, Long MN, Chen QX (2010) Purification and properties of endoglucanase from a sugar cane bagasse hydrolyzing strain, Aspergillus glaucus XC9. J Agric Food Chem 58:6126–6130
Kim DW, Kim TS (1994) Purification of cellulase from Trichoderma viride and properties of its component enzymes. Bull Korean Chem Soc 15:719–724
Gajendra SN, Kaul P, Prakash V (2007) Purification and characterization of a new endoglucanase from Aspergillus aculeatus. J Agric Food Chem 55:7566–7572
Onsori H, Zamani MR, Motallebi M, Zarghami N (2005) Identification of over producer strain of endo-β-1,4-glucanase in Aspergillus species: characterization of crude carboxymethyl cellulose. African J Biotechnol 4:26–30
Karnchanatat A, Petsom A, Sangvanich P, Piapukiewb J, Whalley AJS, Reynolds CD, Gadd GM, Sihanonth P (2008) A novel thermostable endoglucanase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm. Enzyme Microb Technol 42:404–413
Immanuel G, Bhagavath CMA, Raj PI, Esakkiraj P, Palavesam A (2007) Production and partial purification of cellulase by Aspergillus niger and A. fumigatus fermented in coir waste and sawdust. Int J Microbiol 3:1–20
Dicko MH, Leeuwen MJFSV, Traore AS, Hilhorst R, Beldman G (2001) Polysaccharide hydrolases from Boscia senegalensis: purification and characterization of endo-1,3-beta-glucanase. Appl Biochem Biotech 94:225–241
Gasch AP, Werner-Washburne M (2002) The genomics of yeast responses to environmental stress and starvation. Funct Integr Genomics 2:181–192
Gasch A, Spellman P, Kao C, Carmel-Harel O, Eisen M, Storz G, Botstein D, Brown P (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11:4241–4257
Swan TM, Watson K (1997) Membrane fatty acid composition and membrane fluidity as parameters of stress tolerance in yeast. Can J Microbiol 43:70–77
Lu C, Brauer MJ, Botstein D (2009) Slow growth induces heat-shock resistance in normal and respiratory-deficient yeast. Mol Biol Cell 20:891–903
Banat IM, Nigam P, Singh D, Marchant R, McHale AP (1998) Review: ethanol production at elevated temperatures and alcohol concentrations: part I-yeasts in general. World J Microbiol Biotechnol 14:809–821
Halanych KM (1995) The phylogenetic position of the pterobranch Hemichordates based on 18S rDNA sequence data. Mol Phylogenet Evol 4:72–76
Lin Y, Tanaka S (2006) Ethanol fermentation from biomass resources: current state and prospects. Appl Microbiol Biotechnol 69:627–642
Han B, Kiers JL, Nout RM (1999) Solid-substrate fermentation of soybeans with Rhizopus spp.: comparison of discontinuous rotation with stationery bed fermentation. J Biosci Bioeng 88:205–209
Salvadó Z, Arroyo-López FN, Guillamón JM, Salazar G, Querol A, Barrio E (2011) Temperature adaptation markedly determines evolution within the genus Saccharomyces. Appl Environ Microbiol 77:2292–2302
Allain EJ (2007) Perspective Cell-free ethanol production: the future of fuel ethanol? Chem Technol Biotechnol 82:117–120
Acknowledgments
This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education, Science and Technology (No. 2011-0016965).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Khattak, W.A., Kang, M., Ul-Islam, M. et al. Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth. Bioprocess Biosyst Eng 36, 737–747 (2013). https://doi.org/10.1007/s00449-013-0899-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00449-013-0899-1