Tài liệu Báo cáo khoa học: The chitinolytic system of Lactococcus lactis ssp. lactis comprises a

The chitinolytic system of Lactococcus lactis ssp. lactis

comprises a nonprocessive chitinase and a chitin-binding

protein that promotes the degradation of a- and b-chitin

Gustav Vaaje-Kolstad, Anne C. Bunæs, Geir Mathiesen and Vincent G. H. Eijsink

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, A˚ s, Norway

Chitin is a widespread biopolymer composed of b(1,4)-

linked N-acetylglucosamine that provides structural

and chemical resistance in the exoskeleton of crusta￾ceans and arthropods, as well as in the cell wall of

fungi. Chitin exists almost exclusively in an insoluble

crystalline form that complexes with proteins and ⁄ or

minerals to form a robust composite material. Three

naturally occurring crystalline polymorphs have been

described in the literature: the dominant polymorph

a-chitin (antiparallel packing of the chitin chains);

b-chitin (parallel packing of the chitin chains); and the

minor polymorph c-chitin (mixture of parallel and

antiparallel chain packing) [1,2]. In nature, chitin is

only exceeded in abundance by the structural biopoly￾mers of plants (cellulose and hemicellulose) and is an

important source of energy for a variety of organisms.

The primary degraders of chitin are microorganisms

that secrete one or several chitin-degrading enzymes

(chitinases). On the basis of sequence and structure,

chitinases are classified into two distinct families (18

Keywords

chitin; chitin binding; chitinase; lactic acid

bacterium; Lactococcus lactis

Correspondence

G. Vaaje-Kolstad, Department of Chemistry,

Biotechnology and Food Science,

Norwegian University of Life Sciences,

PO Box 5003, 1432 A˚ s, Norway

Fax: +47 64965901

Tel: +47 64965905

E-mail: [email protected]

(Received 23 December 2008, revised 10

February 2009, accepted 18 February 2009)

doi:10.1111/j.1742-4658.2009.06972.x

It has recently been shown that the Gram-negative bacterium Serratia

marcescens produces an accessory nonhydrolytic chitin-binding protein that

acts in synergy with chitinases. This provided the first example of the pro￾duction of dedicated helper proteins for the turnover of recalcitrant

polysaccharides. Chitin-binding proteins belong to family 33 of the carbo￾hydrate-binding modules, and genes putatively encoding these proteins

occur in many microorganisms. To obtain an impression of the functional

conservation of these proteins, we studied the chitinolytic system of the

Gram-positive Lactococcus lactis ssp. lactis IL1403. The genome of this lac￾tic acid bacterium harbours a simple chitinolytic machinery, consisting of

one family 18 chitinase (named LlChi18A), one family 33 chitin-binding

protein (named LlCBP33A) and one family 20 N-acetylhexosaminidase. We

cloned, overexpressed and characterized LlChi18A and LlCBP33A.

Sequence alignments and structural modelling indicated that LlChi18A has

a shallow substrate-binding groove characteristic of nonprocessive endoch￾itinases. Enzymology showed that LlChi18A was able to hydrolyse both

chitin oligomers and artificial substrates, with no sign of processivity.

Although the chitin-binding protein from S. marcescens only bound to

b-chitin, LlCBP33A was found to bind to both a- and b-chitin. LlCBP33A

increased the hydrolytic efficiency of LlChi18A to both a- and b-chitin.

These results show the general importance of chitin-binding proteins in

chitin turnover, and provide the first example of a family 33 chitin-binding

protein that increases chitinase efficiency towards a-chitin.

Abbreviations

CBM, carbohydrate-binding module; CBP, chitin-binding protein; FnIII, Fibronectin-III; LAB, lactic acid bacterium; 4MU-(GlcNAc)3,

4-methylumbelliferyl-b-D-N,N¢,N¢¢-diacetylchitobioside; TEV, tobacco etch virus.

2402 FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS