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Purification and characterization of recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II with high hydrolytic activity on sophorose

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Abstract

β-Glucosidase II (Bgl II), encoded by the βglu2 gene of the thermo-tolerant yeast Pichia etchellsii, was purified from recombinant Escherichia coli pBG22:JM109. The enzyme had a molecular mass of 176 kDa and was a dimer with an apparent subunit mass of 83 kDa. It exhibited broad substrate specificity and hydrolyzed β-linked gluco-disaccharides and oligosaccharides, salicin, and cyanogenic glucoside amygladin. The unusually high hydrolytic activity of 7,680 units min−1 g−1 protein was obtained on sophorose. Competition experiments performed using differently linked β-disaccharides indicated these to be hydrolyzed at the same active site. Transglycosylation activity leading to the biosynthesis of several disaccharides and oligosaccharides was observed. The enzyme was placed in glycosyl hydrolase family 3, based on a statistical approach using amino acid composition data. The involvement of His as a catalytically important residue was confirmed by diethylpyrocarbonate modification. Pre-incubation of the purified enzyme with 5 mM p-nitrophenyl-β-d-glucoside offered 2.5-fold higher residual activity compared with unbound enzyme, indicating protection at the active site. The feasibility of this enzyme as a biocatalyst of choice for the synthesis of glyco-conjugates is discussed.

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References

  • Ajisaka K, Nishida H, Fujimoto H (1987) The synthesis of oligosaccharides by the reversed hydrolysis reaction of β-glucosidase at high temperature. Biotechnol Lett 9:243–248

    CAS  Google Scholar 

  • Bacchawat P, Mishra S, Bhatia Y, Bisaria VS (2004) Enzymatic synthesis of oligosaccharides, alkyl- and terpene glucosides by recombinant Escherichia coli expressed Pichia etchellsii β-glucosidase II. Appl Biochem Biotechnol 118:269–282

    Article  PubMed  Google Scholar 

  • Bhatia Y, Mishra S, Bisaria VS (2002a) Microbial β-glucosidases: cloning, properties and applications. Crit Rev Biotechnol 22:375–407

    CAS  PubMed  Google Scholar 

  • Bhatia Y, Mishra S, Bisaria VS (2002b) Biosynthetic activity of recombinant Escherichia coli expressed Pichia etchellsii β-glucosidase II. Appl Biochem Biotechnol 102/103:367–379

    Article  Google Scholar 

  • Burnstein Y, Walsh KA, Neurath H (1974) Evidence of an essential histidine residue in thermolysin. Biochemistry 13:205–210

    PubMed  Google Scholar 

  • Chen Q-X, Zhang Z, Zhou X-W, Zhuang Z-L (2000) Kinetics of inhibition of β-glucosidase from Ampullarium crossean by bromoacetic acid. Int J Biochem Cell Biol 32:717–723

    Article  CAS  PubMed  Google Scholar 

  • Chou PY (1989) Prediction of protein structural classes from amino acid compositions. In: Fasman GD (ed) Prediction of protein structure and the principles of protein conformation. Plenum, New York, pp 549–586

    Google Scholar 

  • Clarke AJ (1990) Chemical modification of a β-glucosidase from Schizophyllum commune: evidence for essential carboxyl groups. Biochim Biophys Acta 1040:145–152

    Article  CAS  PubMed  Google Scholar 

  • Coutinho PM, Henrisaat B (2004) Carbohydrate active enzymes server. http://afmb.cnrs-mrs.fr/∼pedro/CAZY

  • Fersht A (1985) Enzyme structure and mechanism, 2nd edn. Freeman, New York

    Google Scholar 

  • Gallagher S, Winston SE, Fuller SA, Huriell JGR (1993) Unit 10.8. Analysis of proteins. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology. Wiley Interscience, New York

    Google Scholar 

  • Geurtsen R, Cote F, Hahn MG, Boons GJ (1999) Chemoselective glycosylation strategy for the convergent assembly of phytoalexin-elicitor active oligosaccharides and their photoreactive derivatives. J Org Chem 64:7828–7835

    Article  CAS  Google Scholar 

  • Gunata Z, Vallier MJ, Sapis JC, Baumes R, Bayonove C (1994) Enzymic synthesis of monoterpenyl β-d-glucosides by various β-glucosidases. Enzyme Microb Technol 16:1055–1058

    Article  CAS  Google Scholar 

  • Hiromi K, Hamauzu Z, Takahashi K, Ono S (1966) Kinetic studies on glucoamylase II. Competition between two types of substrates having α1→4 and α1→6 glucosidic linkages. J Biochem 59:411–418

    CAS  PubMed  Google Scholar 

  • Iwashita K, Nagahara T, Kimura H, Takano M, Shimoi H, Ito K (1999) The bglA gene of Aspergillus kawachii encodes both extracellular and cell wall bound β-glucosidases. Appl Environ Microbiol 65:5546–5553

    CAS  PubMed  Google Scholar 

  • Kannan T, Loganathan D, Bhatia Y, Mishra S, Bisaria VS (2004) Transglycosylation catalyzed by almonds β-glucosidase and cloned Pichia etchellsii β-glucosidase II using glycosylasparagine mimetics as novel acceptors. Biocatal Biotransform 22:1–7

    Article  CAS  Google Scholar 

  • Kempton JB, Withers SG (1992) Mechanism of Agrobacterium β-glucosidase: kinetic studies. Biochemistry 31:9961–9969

    CAS  PubMed  Google Scholar 

  • Kumble KD, Kumble S, Jaffar MB (1992) Inactivation of β-glucosidase from Arthrobotrys conoides by diethylpyrocarbonate: evidence of histidine at the active site. Indian J Exp Biol 30:99–102

    CAS  PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  • Leah R, Kiegel J, Suendson IB, Mundy J (1995) Biochemical and molecular characterization of a barley seed β-glucosidase. J Biol Chem 22:15789–15796

    Google Scholar 

  • Levy MM, Leber PD, Ryan EM (1963) Inactivation of myosin by 2,4-dinitrophenol and protection by adenosine triphosphate and other phosphate compounds. J Biol Chem 238:3654–3659

    CAS  PubMed  Google Scholar 

  • Machida M, Ohtsuki I, Fukui S, Yamashita I (1988) Nucleotide sequences of Saccharomycopsis fibuligera gene for extracellular β-glucosidases as expressed in Saccharomyces cerevisiae. Appl Environ Microbiol 54:3147–3155

    CAS  PubMed  Google Scholar 

  • Marana SR, Jacobs-Lorena M, Terra WR, Ferreira C (2001) Amino acid residues involved in substrate binding and catalysis in an insect digestive β-glycosidase. Biochim Biophys Acta 1545:41–52

    Article  CAS  PubMed  Google Scholar 

  • Mata IH, Castillon MP, Dominguez JM, Macarron R, Acebal C (1993) Chemical modification of β-glucosidase from Trichoderma reesei QM9414. J Biochem 114:754–759

    PubMed  Google Scholar 

  • Muhlrad A, Hegyl G, Toth G (1967) Effect of diethylpyrocarbonate on proteins. I. Reaction of diethylpyrocarbonate with amino acids. Acta Biochim Biophys Acad Sci Hung 2:19–29

    CAS  Google Scholar 

  • Namchuk MN, Withers SG (1995) Mechanism of Agrobacterium β-glucosidase: kinetic analysis of the role of non covalent enzyme/substrate interactions. Biochemistry 34:16194–16202

    CAS  PubMed  Google Scholar 

  • Nossal NG, Heppel LA (1966) The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 241:3055–3062

    CAS  PubMed  Google Scholar 

  • Ovadi J, Libor S, Elodi P (1967) Spectrophotometric determination of histidine in proteins with diethyl pyrocarbonate. Acta Biochim Biophys Acad Sci Hung 2:455–458

    CAS  Google Scholar 

  • Pandey M, Mishra S (1995) Cloning and expression of β-glucosidase gene from the yeast Pichia etchellsii. J Ferment Bioeng 80:446–453

    Article  CAS  Google Scholar 

  • Pandey M, Mishra S (1997) Expression and characterization of Pichia etchellsii β-glucosidase in Escherichia coli. Gene 190 45–51

    Article  CAS  PubMed  Google Scholar 

  • Segal IH (1975) Enzyme kinetics. Wiley, New York

    Google Scholar 

  • Sethi B, Jain M, Chowdhary M, Soni Y, Bhatia Y, Sahai V, Mishra S (2002) Cloning, characterization of Pichia etchellsii β-glucosidase II and effect of media composition and feeding strategy on its production in a bioreactor. Biotechnol Bioprocess Eng 7:43–51

    CAS  Google Scholar 

  • Tomme P, Chauvaux S, Beguin P, Millet J, Aubert J-P, Claeyssens M (1991) Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum. J Biol Chem 266:10313–10318

    CAS  PubMed  Google Scholar 

  • Umezurike GM (1991) Octameric structure of β-glucosidase from Botryodiplodia theobromae Pat. Biochem J 275:721–725

    CAS  PubMed  Google Scholar 

  • Varghese JN, Hrmova M, Fincher GB (1999) Three-dimensional structure of a barley β-d-glucan exohydrolase, a family 3 glycosyl hydrolase. Struct 7:179–190

    Article  CAS  Google Scholar 

  • Vic G, Thomas D, Crout DHG (1997) Solvent effect on enzyme catalyzed synthesis of β-d-glucosides using the reverse hydrolysis method: application to the preparative scale synthesis of 2-hydroxybenzyl and octyl-β-d-glucopyranosides. Enzyme Microb Technol 20:597–603

    Article  CAS  Google Scholar 

  • Voet D, Voet JG (1995) Biochemistry, 2nd edn. Wiley, New York

    Google Scholar 

  • Wallecha A, Mishra S (2003) Purification and characterization of two β-glucosidases from a thermo-tolerant yeast Pichia etchellsii. Biochim Biophys Acta 1649:74–84

    Article  CAS  PubMed  Google Scholar 

  • Wang PG, Bertozzi CR (2001) Glycochemistry: principles, synthesis, and applications. Dekker, New York

    Google Scholar 

  • Wulff-Strobel CR, Wilson DB (1995) Cloning, sequencing, and characterization of a membrane associated Prevotella ruminicola B14 β-glucosidase with cellodextrinase and cyanoglycosidase activities. J Bacteriol 177:5884–5890

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors wish to thank the Department of Biotechnology, Ministry of Science and Technology, Government of India, for providing financial assistance. A scholarship awarded to Y.B. by the Council of Scientific and Industrial research (New Delhi) is duly acknowledged. The experiments carried out in this study comply with the current laws of the country.

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  1. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India

    Yukti Bhatia, Saroj Mishra & Virendra S. Bisaria

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  1. Yukti Bhatia
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  2. Saroj Mishra
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  3. Virendra S. Bisaria
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Correspondence to Saroj Mishra.

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Bhatia, Y., Mishra, S. & Bisaria, V.S. Purification and characterization of recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II with high hydrolytic activity on sophorose. Appl Microbiol Biotechnol 66, 527–535 (2005). https://doi.org/10.1007/s00253-004-1754-8

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  • DOI: https://doi.org/10.1007/s00253-004-1754-8

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