Applied Microbiology and Biotechnology

, Volume 26, Issue 5, pp 427–433 | Cite as

Purification and preliminary characterization of an extracellular pullulanase from Thermoanaerobium Tok6-B1

  • Adrian R. Plant
  • Robyn M. Clemens
  • Roy M. Daniel
  • Hugh W. Morgan
Biotechnology

Summary

Extracellular pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) was purified from cell free culture supernatants of Thermoanaerobium Tok6-B1 by ammonium sulphate precipitation, affinity precipitation, gel exclusion and ion exchange chromatography. A final purification factor of over 1600 was achieved. A molecular weight of 120 kD was determined by steric exclusion HPLC. Enzyme activity was specifically directed towards the α 1–6 glucosidic linkages of pullulan resulting in 100% conversion to maltotriose and also possessed activity towards α 1–4 linkages of starch, amylopectin and amylose producing maltooligosaccharides (DP2-DP4) as products. Maltotetraose was slowly hydrolysed to maltose. Values of Km (% w/v) were 7.3×10-3 for pullulan, 2.7×10-3 for amylopectin and 4.7×10-3 for Lintner's starch. Pullulanase activity was resistant to 6 M urea and was thermostable at temperatures up to 80°C (t1/2 in the order of hours). Above 80°C thermal denaturation was significant (t1/2=17 min at 85°C; 5 min at 90°C) but became less so in the presence of substrate (pullulan or starch). Thermostability was greatest at the pH activity optimum (pH 5.5) and was promoted by Ca2+ ions.

Abbreviations

BSA

bovine serum albumin

EDTA

ethylenediamine tetracetic acid

HPLC

high performance liquid chromatography

MES

2-[N-Morpholino] ethanesulphonic acid

MOPS

3-[N-Morpholino] propanesulphonic acid

Tris

tris-(hydroxymethyl)methylamine

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Copyright information

© Springer-Verlag 1987

Authors and Affiliations

  • Adrian R. Plant
    • 1
  • Robyn M. Clemens
    • 1
  • Roy M. Daniel
    • 1
  • Hugh W. Morgan
    • 1
  1. 1.Microbial Biochemistry and Biotechnology Unit, School of ScienceUniversity of WaikatoHamiltonNew Zealand

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