Biologia, Bratislava, 57/Suppl. 11: 155-162, 2002.

ISSN 0006-3088 (Biologia).

 

Full Paper

Expression and characterisation of the catalytic domain of an archaeal family 57 pullulanase type II.

 

Florent Chang-Pi-Hin1, Marta Erra-Pujada1, Manuel Dauchez2, Philippe Debeire1, Francis Duchiron1 & Michael J. O’Donohue1* J. O'Donohue1

 

1 Technologie Enzymatique et Physicochimie des Agroressources, Institut National de la Recherche Agronomique, UMR 614 FARE, 8, rue Gabriel Viosin, BP 316, 51686 Reims cedex 02, France; tel.: ++ 33 326 355 365, fax: ++ 33 326 355 369, e-mail: michael.odonohue@univ-reims.fr

2 Laboratoire de Spectroscopies et Structures Biomoleculaires, Bat 18, Moulin de la House, BP 1039, 51687 Reims cedex 02, France

* corresponding author

Received: October 4, 2001 / Accepted: February 12, 2002

 

Abstract

The catalytic domain (ApuD2) of the thermostable family 57 pullulanase type II from the archaeabacterium Thermococcus hydrothermalis has been cloned. The activity of ApuD2 in the presence of various reagents has been characterised and the effects of pH and temperature have been determined. Our results show that ApuD2 possesses an extreme thermostability, identical to that of the wild type parent enzyme, which may be mainly due to electrostatic interactions at the protein level. Furthermore, data indicate that this thermostability is increased in the presence of calcium and in the presence of substrates, and particularly non-reducing malto-oligosaccharides. Finally, although N-bromosuccinimide treatment leads to the indiscriminate inactivation of both activities, pH dependence and acarbose inhibition data have revealed differences in the behaviour of ApuD2 towards a-1,6 and a-1,4 glucosidic linkages. Therefore, overall these results suggest that ApuD2 may possess two active sites. Using a combination of spectroscopic methods and secondary structure predictions, the ApuD2 secondary structure has been examined. Good agreement between the experimentally-adjusted predictions has allowed the generation of a high quality consensus prediction which indicates that ApuD2 may be composed of several distinct structural elements. Notably, this analysis has allowed the identification of a major region (amino acids 127 to 578) which exhibits an alternating pattern of b-strands and a-helices, which is highly reminiscent of the (a/b)8 fold which is commonly associated with family 13 amylolytic enzymes.

 

Key words:

pPullulanase, F-57, glycosyl hydrolase, thermostable, Thermococcales, acarbose.