Skip to main content
SpringerLink
Log in

Purification and characterization of a novel thermophilic β-galactosidase from Picrophilus torridus of potential industrial application

  • Original Paper
  • Published:
Extremophiles Aims and scope Submit manuscript

Abstract

Intracellular β-galactosidase (E.C 3.2.1.23) produced by the thermoacidophilic archeon Picrophilus torridus DSM 9790 was purified to homogeneity using a combination of DEAE Sepharose, gel filtration, hydroxyapatite and chromatofocusing chromatographies. LC–MS/MS analysis was used to confirm the identity of the purified protein. The enzyme was found to be a homotrimer, with a molecular mass of 157.0 kDa and an isoelectric point of 5.7. To our knowledge, this enzyme has the lowest pH optimum of any intracellular β-galactosidase characterized to date. Maximal activity was exhibited at acidic pH values of 5.0–5.5 and at 70 °C. The enzyme retained > 95% activity after heating to 70 °C for 1 h, or after incubation at pH 5.5 for 1 h. The enzyme may be of interest for high-temperature bioprocessing, such as in the production of lactulose. This investigation suggests that the β-galactosidase activity produced by P. torridus is potentially more useful than several enzymes already characterized for such an application.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

ONP:

Ortho-nitrophenol

ONPG:

Ortho-nitrophenol-β-d-galactopyranoside

Ylactulose :

Yield of lactulose to initial concentration of lactose

References

  • Aburto C, Castillo C, Cornejo F, Arenas-Salinas M, Vásquez C, Guerrero C, Arenas F, Illanes A, Vera C (2019) β-Galactosidase from Exiguobacterium acetylicum: cloning, expression, purification and characterization. Bioresour Technol 277:211–215

    CAS  PubMed  Google Scholar 

  • Adamczak M, Charubin D, Bednarski W (2009) Influence of reaction medium composition on enzymatic synthesis of galacto-oligosaccharides and lactulose from lactose concentrates prepared from whey permeate. Chem Pap 63:111–116

    CAS  Google Scholar 

  • Akiyama K, Takase M, Horikoshi K, Okonogi S (2001) Production of galacto-oligosaccharides from lactose using a β-glucosidase from Thermus sp. Z-1. Biosci Biotechnol Biochem 65:438–441

    CAS  PubMed  Google Scholar 

  • Albers SV, Jonuscheit M, Dinkelaker S, Urich T, Kletzin A, Tampe R, Driessen AJM, Schleper C (2006) Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus. Appl Environ Microbiol 72:102–111

    CAS  PubMed  PubMed Central  Google Scholar 

  • Angelov AS (2004) Genome sequence analysis and characterization of recombinant enzymes from the thermoacidophilic archaeon Picrophilus torridus. PhD thesis

  • Angelov A, Futterer O, Valerius O, Braus GH, Liebl W (2005) Properties of the recombinant glucose/galactose dehydrogenase from the extreme thermoacidophile, Picrophilus torridus. FEBS J 272:1054–1062

    CAS  PubMed  Google Scholar 

  • Angelov A, Putyrski M, Liebl W (2006) Molecular and biochemical characterisation of α-glucosidase and α-mannosidase and their clustered genes from the thermoacidophilic archaeon Picrophilus torridus. J Bacteriol 188:7123–7131

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bertoldo C, Dock C, Antranikian G (2004) Thermoacidophilic microorganisms and their novel biocatalysts. Eng Life Sci 4:521–532

    CAS  Google Scholar 

  • BIO-RAD (2004) Model 111 mini IEF cell instruction manual

  • Buonocore V, Sgambati O, Rosa M, Esposito E, Gambacorta A (1980) A constitutive β-galactosidase from the extreme thermoacidophile archaebacterium Caldariella acidophila: properties of the enzyme in the free state and in immobilised whole cells. J Appl Biochem 2:390–397

    CAS  Google Scholar 

  • Carninci P, Nishiyama Y, Westover A, Itoh M, Nagaoka S, Sasaki N, Okazaki Y, Muramatsu M, Hayashizaki Y (1998) Thermostabilisation and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. PNAS 95:520–524

    CAS  PubMed  Google Scholar 

  • Chakraborti S, Sani RK, Banerjee UC, Sobti RC (2000) Purification and characterisation of a novel β-galactosidase from Bacillus sp. MTCC 3088. J Ind Microbiol Biotechnol 24:58–63

    CAS  Google Scholar 

  • Chen YS, Lee GC, Shaw JF (2006) Gene cloning, expression, and biochemical characterisation of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli. J Agric Food Chem 54:7098–7104

    CAS  PubMed  Google Scholar 

  • Fütterer O, Angelov A, Liesegang H, Gottschalk G, Schleper C, Schepers B, Dock C, Antranikian G, Liebl W (2004) Genome sequence of Picrophilus torridus and its implications for life around pH 0. PNAS 101:9091–9096

    PubMed  Google Scholar 

  • Grogan DW (1991) Evidence that β-galactosidase of Sulfolobus solfataricus is only one of several activities of a thermostable β-d-glycosidase. Appl Environ Microbiol 57:1644–1649

    CAS  PubMed  PubMed Central  Google Scholar 

  • Guerrero C, Vera C, Plou F, Illanes A (2011) Influence of reaction conditions on the selectivity of the synthesis of lactulose with microbial β-galactosidases. J Mol Catal B Enzym 72:206–212

    CAS  Google Scholar 

  • Guerrero C, Vera C, Araya E, Conejeros R, Illanes A (2015) Repeated-batch operation for the synthesis of lactulose with β-galactosidase immobilized by aggregation and crosslinking. Bioresour Technol 190:122–131

    CAS  PubMed  Google Scholar 

  • Hess M, Katzer M, Antranikian G (2008) Extremely thermostable esterases from the thermoacidophilic euryarchaeon Picrophilus torridus. Extremophiles 12:351–364

    CAS  PubMed  Google Scholar 

  • Holmes ML, Scopes RK, Moritz RL, Simpson RJ, Englert C, Pfeifer F, Dyall-Smith ML (1997) Purification and analysis of an extremely halophilic β-galactosidase from Haloferax alicantei. Biochim Biophys Acta 1337:276–286

    CAS  PubMed  Google Scholar 

  • Horikoshi K (2011) Extremophiles handbook. Springer, Berlin

    Google Scholar 

  • Hoyoux A, Jennes I, Dubois P, Genicot S, Dubail F, Francois JM, Baise E, Feller G, Gerday C (2001) Cold-adapted β-galactosidase from the Antarctic psychrophile Pseudoalteromonas haloplanktis. Appl Environ Microbiol 67:1529–1535

    CAS  PubMed  PubMed Central  Google Scholar 

  • Juajun O, Nguyen TH, Maischberger T, Iqbal S, Haltrich D, Yamabhai M (2011) Cloning, purification, and characterisation of β-galactosidase from Bacillus licheniformis DSM 13. Appl Microbiol Biotechnol 89:645–654

    CAS  PubMed  Google Scholar 

  • Karan R, Capes MD, DasSarma P, DasSarma S (2013) Cloning, overexpression, purification, and characterisation of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi. BMC Biotechnol 13:11

    Google Scholar 

  • Kim CS, Ji ES, Oh DK (2003) Expression and characterisation of Kluyveromyces lactis β-galactosidase in Escherichia coli. Biotechnol Lett 25:1769–1774

    CAS  PubMed  Google Scholar 

  • Kim YS, Park CS, Oh DK (2006) Lactulose production from lactose and fructose by a thermostable β-galactosidase from Sulfolobus solfataricus. Enzyme Microb Technol 39:903–908

    CAS  Google Scholar 

  • Kosugi A, Murashima K, Doi RH (2002) Characterisation of two noncellulosomal subunits, ArfA and BgaA, from Clostridium cellulovorans that cooperate with the cellulosome in plant cell wall degradation. J Bacteriol 184:6859–6865

    CAS  PubMed  PubMed Central  Google Scholar 

  • Krüger A, Schäfers C, Schröder C, Antranikian G (2018) Towards a sustainable biobased industry—highlighting the impact of extremophiles. New Biotechnol 40:144–153

    Google Scholar 

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

    CAS  Google Scholar 

  • Lee MS (2012) Mass spectrometry handbook. Pharmaceutical science and biotechnology: practices, applications and methods. Wiley

  • Lee YJ, Kim CS, Oh DK (2004) Lactulose production by β-galactosidase in permeabilised cells of Kluyveromyces lactis. Appl Microbiol Biotechnol 64:787–793

    CAS  PubMed  Google Scholar 

  • Lee JH, Kim YS, Yeom SJ, Oh DK (2011) Characterisation of a glycoside hydrolase family 42 β-galactosidase from Deinococcus geothermalis. Biotechnol Lett 33:577–583

    CAS  PubMed  Google Scholar 

  • Li Y, Wang H, Lu L, Li Z, Xu X, Xiao M (2009) Purification and characterisation of a novel β-galactosidase with transglycosylation activity from Bacillus megaterium 2037-4-1. Appl Biochem Biotechnol 158:192–199

    CAS  PubMed  Google Scholar 

  • Liao XY, Zheng QW, Zhou QL, Lin JF, Guo LQ, Yun F (2016) Characterization of recombinant β-galactosidase and its use in enzymatic synthesis of lactulose from lactose and fructose. J Mol Catal B Enzym 134:253–260

    CAS  Google Scholar 

  • Linares-Pasten JA, Andersson M, Karlsson EN (2014) Thermostable glycoside hydrolases in biorefinery technologies. Curr Biotechnol 3:26–44

    CAS  Google Scholar 

  • Mayer J, Conrad J, Klaiber I, Lutz-Wahl S, Beifuss U, Fischer L (2004) Enzymatic production and complete nuclear magnetic resonance assignment of the sugar lactulose. J Agric Food Chem 52:6983–6990

    CAS  PubMed  Google Scholar 

  • Naessens M, Vandamme EJ (2003) Multiple forms of microbial enzymes. Biotechnol Lett 25:1119–1124

    CAS  PubMed  Google Scholar 

  • Nagy Z, Kiss T, Szentirmai A, Biro S (2001) β-Galactosidase of Penicillium chrysogenum: production, purification, and characterisation of the enzyme. Protein Expr Purif 21:24–29

    CAS  PubMed  Google Scholar 

  • Nooshkam M, Babazedeh A, Jooyandeh H (2018) Lactulose: Properties, techno-functional food applications, and food grade delivery system. Trends Food Sci Technol 80:23–34

    CAS  Google Scholar 

  • O’Connell S, Walsh G (2007) Purification and properties of a β-galactosidase with potential application as a digestive supplement. Appl Biochem Biotechnol 141:1–14

    PubMed  Google Scholar 

  • O’Connell S, Walsh G (2010) A novel acid-stable, acid-active β-galactosidase potentially suited to the alleviation of lactose intolerance. Appl Microbiol Biotechnol 86:517–524

    PubMed  Google Scholar 

  • Onishi N, Tanaka T (1995) Purification and characterisation of a novel thermostable galacto-oligosaccharide-producing β-galactosidase from Sterigmatomyces elviae CBS8119. Appl Environ Microbiol 61:4026–4030

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Pisani FM, Rella R, Raia CA, Rozzo C, Nucci R, Gambacorta A, Derosa M, Rossi M (1990) Thermostable β-galactosidase from the archaebacterium Sulfolobus solfataricus—purification and properties. Eur J Biochem 187:321–328

    CAS  PubMed  Google Scholar 

  • Rajput R, Verma VV, Chaudhary V, Gupta R (2013) A hydrolytic γ-glutamyl transpeptidase from thermoacidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation. Extremophiles 17:29–41

    CAS  PubMed  Google Scholar 

  • Rasouli I, Kulkarni PR (1994) Enhancement of β-galactosidase productivity of Aspergillus niger NCIM-616. J Appl Bacteriol 77:359–361

    CAS  PubMed  Google Scholar 

  • Reher M, Bott M, Schonheit P (2006) Characterisation of glycerate kinase (2-phosphoglycerate forming), a key enzyme of the nonphosphorylative Entner–Doudoroff pathway, from the thermoacidophilic euryarchaeon Picrophilus torridus. FEMS Microbiol Lett 259:113–119

    CAS  PubMed  Google Scholar 

  • Saishin N, Ueta M, Wada A, Yamamoto I (2010) Properties of a β-galactosidase purified from Bifidobacterium longum subsp Longum JCM 7052 grown on gum Arabic. Int J Biol Macromol 10:23–31

    CAS  Google Scholar 

  • Schepers B, Thiemann V, Antranikian G (2006) Characterisation of a novel glucoamylase from the thermoacidophilic Archaeon Picrophilus torridus heterologously expressed in E. coli. Eng Life Sci 6:311–317

    CAS  Google Scholar 

  • Schleper C, Puehler G, Holz I, Gambacorta A, Janekovic D, Santarius U, Klenk HP, Zillig W (1995) Picrophilus gen. nov., fam. nov.—a novel aerobic, heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth around pH 0. J Bacteriol 177:7050–7059

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sen S, Ray L, Chattopadhyay P (2012) Production, purification, immobilisation, and characterisation of a thermostable β-galactosidase from Aspergillus alliaceus. Appl Biochem Biotechnol 167:1938–1953

    CAS  PubMed  Google Scholar 

  • Serour E, Antranikian G (2002) Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum, Picrophilus torridus and Picrophilus oshimae. Anton Leeuw Int J G 81:73–83

    CAS  Google Scholar 

  • Shaikh SA, Khire JM, Khan MI (1999) Characterisation of a thermostable extracellular β-galactosidase from a thermophilic fungus Rhizomucor sp. Biochim Biophys Acta 1472:14–322

    Google Scholar 

  • Sharma S, Vaid S, Bhat B, Singh S, Bajaj BK (2019) Thermostable enzymes for industrial biotechnology. Adv Enzyme Biotechnol 17:469–495

    Google Scholar 

  • Shipkowski S, Brenchley JE (2006) Bioinformatic, genetic, and biochemical evidence that some glycoside hydrolase family 42 β-galactosidases are arabinogalactan type I oligomer hydrolases. Appl Environ Microbiol 72:7730–7738

    CAS  PubMed  PubMed Central  Google Scholar 

  • Silvério S, Macedo EA, Teixeira JA, Rodrigues LR (2017) New β-galactosidase producers with potential for prebiotic synthesis. Bioresour Technol 250:131–139

    PubMed  Google Scholar 

  • Song YS, Lee HU, Park C, Kim SW (2012a) Batch and continuous synthesis of lactulose from whey lactose by immobilised β-galactosidase. Food Chem 136:689–694

    PubMed  Google Scholar 

  • Song YS, Shin HY, Lee JY, Park C, Kim S (2012b) β-Galactosidase-immobilised microreactor fabricated using a novel technique for enzyme immobilisation and its application for continuous synthesis of lactulose. Food Chem 133:611–617

    CAS  Google Scholar 

  • Takagi M, Tamaki H, Miyamoto Y, Leonardi R, Hanada S, Jackowski S, Chohnan S (2010) Pantothenate kinase from the thermoacidophilic Archaeon Picrophilus torridus. J Bacteriol 192:233–241

    CAS  PubMed  Google Scholar 

  • Thurmer A, Voigt B, Angelov A, Albrecht D, Hecker M, Liebl W (2011) Proteomic analysis of the extremely thermoacidophilic archaeon Picrophilus torridus at pH and temperature values close to its growth limit. Proteomics 11:4559–4568

    PubMed  Google Scholar 

  • van Laere KM, Abee T, Schols HA, Beldman G, Voragen AG (2000) Characterisation of a novel β-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalacto-oligosaccharides. Appl Environ Microbiol 66:1379–1384

    PubMed  PubMed Central  Google Scholar 

  • Wierzbicka-Wos A, Cieslinski H, Wanarska M, Kozlowska-Tylingo K, Hildebrandt P, Kur J (2011) A novel cold-active β-d-galactosidase from the Paracoccus sp. 32d-gene cloning, purification and characterisation. Microb Cell Fact 10:108

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work has been funded by the Irish Research Council under the Embark Initiative.

Author information

Authors and Affiliations

  1. Department of Chemical and Environmental Sciences, University of Limerick, Limerick City, Ireland

    Jayne Murphy & Gary Walsh

Authors
  1. Jayne Murphy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gary Walsh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jayne Murphy.

Additional information

Communicated by M. Moracci.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 26 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Murphy, J., Walsh, G. Purification and characterization of a novel thermophilic β-galactosidase from Picrophilus torridus of potential industrial application. Extremophiles 23, 783–792 (2019). https://doi.org/10.1007/s00792-019-01133-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00792-019-01133-4

Keywords

Search

Navigation