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Degradation of Chitosans with Chitinase G from Streptomyces coelicolor A3(2): Production of Chito-oligosaccharides and Insight into Subsite Specificities

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Ellinor B. Heggset^†, Ingunn A. Hoell^‡, Marius Kristoffersen^†, Vincent G. H. Eijsink^‡ and Kjell M. Vårum*^†

Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway, and Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1432 Ås, Norway

Biomacromolecules, 2009, 10 (4), pp 892–899

DOI: 10.1021/bm801418p

Publication Date (Web): February 17, 2009

* To whom correspondence should be addressed. E-mail: kjell.morten.vaarum@biotech.ntnu.no., †

Norwegian University of Science and Technology.

, ‡

Norwegian University of Life Sciences.

Section:

Enzymes

Abstract

We have studied the degradation of soluble heteropolymeric chitosans with a bacterial family 19 chitinase, ChiG from Streptomyces coelicolor A3(2), to obtain insight into the mode of action of ChiG, to determine subsite preferences for acetylated and deacetylated sugar units, and to evaluate the potential of ChiG for production of chito-oligosaccharides. Degradation of chitosans with varying degrees of acetylation was followed using NMR for the identity (acetylated/deacetylated) of new reducing and nonreducing ends as well as their nearest neighbors and using gel filtration to analyze the size distribution of the oligomeric products. Degradation of a 64% acetylated chitosan yielded a continuum of oligomers, showing that ChiG operates according to a nonprocessive, endo mode of action. The kinetics of the degradation showed an initial rapid phase dominated by cleavage of three consecutive acetylated units (A; occupying subsites −2, −1, and +1), and a slower kinetic phase reflecting the cleavage of the glycosidic linkage between a deacetylated unit (D, occupying subsite −1) and an A (occupying subsite +1). Characterization of isolated oligomer fractions obtained at the end of the initial rapid phase and at the end of the slower kinetic phase confirmed the preference for A binding in subsites −2, −1, and +1 and showed that oligomers with a deacetylated reducing end appeared only during the second kinetic phase. After maximum conversion of the chitosan, the dimers AD/AA and the trimer AAD were the dominating products. Degradation of chitosans with varying degrees of acetylation to maximum degree of scission produced a wide variety of oligomer mixtures, differing in chain length and composition of acetylated/deacetylated units. These results provide insight into the properties of bacterial family 19 chitinases and show how these enzymes may be used to convert chitosans to several types of chito-oligosaccharide mixtures.

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This article has been cited by 2 ACS Journal articles (2 most recent appear below).

Human Chitotriosidase-Catalyzed Hydrolysis of Chitosan
Kristine Bistrup Eide, Anne Line Norberg, Ellinor Bævre Heggset, Anne Rita Lindbom, Kjell Morten Vårum, Vincent G. H. Eijsink, and Morten Sørlie
Biochemistry2012 51 (1), 487-495
- Human Chitotriosidase-Catalyzed Hydrolysis of Chitosan
  Kristine Bistrup Eide, Anne Line Norberg, Ellinor Bævre Heggset, Anne Rita Lindbom, Kjell Morten Vårum, Vincent G. H. Eijsink, and Morten Sørlie
  Biochemistry2012 51 (1), 487-495
  Chitotriosidase (HCHT) is one of two family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the human defense mechanism against fungal parasites, but its precise role and the ...
  Abstract | Full Text HTML | Hi-Res PDF | PDF w/ Links
Degradation of Chitosans with a Family 46 Chitosanase from Streptomyces coelicolor A3(2)
Ellinor B. Heggset, Anette I. Dybvik, Ingunn A. Hoell, Anne Line Norberg, Morten Sørlie, Vincent G. H. Eijsink, and Kjell M. Vårum
Biomacromolecules2010 11 (9), 2487-2497
- Degradation of Chitosans with a Family 46 Chitosanase from Streptomyces coelicolor A3(2)
  Ellinor B. Heggset, Anette I. Dybvik, Ingunn A. Hoell, Anne Line Norberg, Morten Sørlie, Vincent G. H. Eijsink, and Kjell M. Vårum
  Biomacromolecules2010 11 (9), 2487-2497
  We have studied the degradation of well-characterized soluble heteropolymeric chitosans by a novel family 46 chitosanase, ScCsn46A from Streptomyces coelicolor A3(2), to obtain insight into the enzyme’s mode of action and to determine its potential for ...
  Abstract | Full Text HTML | Hi-Res PDF | PDF w/ Links