Tài liệu Báo cáo khoa học: 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 doc

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:
(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
and 19) of glycoside hydrolases [3,4]. Recently, a com-
plete survey of Trichoderma chitinases suggested a fur-
ther classification of family 18 chitinases into
subgroups A (bacterial ⁄ fungal), B (plant ⁄ fungal) and
C (killer toxin-like chitinases) [5]. Family 18 chitinases
are represented in most living organisms, whereas fam-
ily 19 enzymes are mostly found in plants, where they
contribute to defence against chitinous pathogens.
As a result of the recalcitrance of chitinous matrices,
microorganisms have devised a variety of complemen-
tary strategies to gain access to and degrade individual
polymer chains. First, the chains are degraded by both
endochitinases, that attack the chitin chain randomly,
and exochitinases, that attack the chitin chains from
either the reducing or nonreducing end [6,7]. As endo-
acting enzymes increase substrate availability for exo-
acting enzymes, synergistic effects are observed [8–10].
Second, some chitinases act processively, that is, they
remain associated with one and the same polymer
chain whilst cleaving off consecutive dimers (also

called ‘multiple attack’ mechanism [11]). Processivity is
considered to be beneficial when degrading crystalline
substrates, because it prevents detached individual
polymer chains from re-associating with insoluble
material [12,13]. Furthermore, the majority of chitinases
targeting crystalline chitin are equipped with additional
chitin-binding domains [also called modules or carbo-
hydrate-binding modules (CBMs)] that are thought to
increase the affinity of the enzyme for the insoluble
substrate [14–16]. In addition to the enzyme machinery
that decomposes the polymers, chitin-degrading micro-
organisms produce an N-acetylhexosaminidase (chito-
biase) that converts chitobiose to N-acetylglucosamine.
Recently, an additional strategy for chitin degrada-
tion was identified, which involves the secretion of a
nonhydrolytic chitin-binding protein (CBP) that acts
synergistically with chitinases, presumably by increas-
ing substrate accessibility [10,17]. These nonhydrolytic
proteins are classified as family 33 CBMs [3,18], but,
with one exception [19], they occur as individual pro-
teins rather than as auxiliary domains in hydrolytic
enzymes. Genome analyses indicate that secreted
family 33 CBPs are produced by most chitin-degrading
microorganisms [17], but only a few have been charac-
terized biochemically. Binding studies of family 33
CBPs have been conducted for CBP21 from Serra-
tia marcescens [17,20], ChbB [21] and Chb3 [22] from
Streptomyces coelicolor, CHB1 from Streptomyces oli-
vaceoviridis [23], CHB2 from Streptomyces reticuli [24],
CbpD from Pseudomonas aeruginosa [25] and proteins

E7 and E8 from Thermobifdia fusca [26], showing a
large diversity of binding preferences. The function of
family 33 CBPs was first demonstrated for CBP21
from S. marcescens [10], and a second example has
been described recently in a study on carbohydrate-
binding proteins and domains from T. fusca [26].
Genes encoding family 33 CBPs occur even in bacte-
ria containing otherwise seemingly simple chitinolytic
machineries, such as in the lactic acid bacterium
(LAB) Lactococcus lactis ssp lactis IL1403. LABs are
Gram-positive, facultatively, anaerobic, fermentative
bacteria that are of major importance in the food
industry for the generation of fermented products. In
general, there is not much known about the ability of
LABs to degrade chitin, but one study has shown that
L. lactis is able to grow on a minimal medium contain-
ing N-acetylglucosamine oligomers as the sole carbon
source [27]. According to the CAZy database [3], only
a few of the sequenced LAB genomes contain genes
that together encode a complete chitinolytic machin-
ery. The genome sequence of L. lactis [28] shows three
genes potentially involved in chitin turnover, coding
for the following: a secreted family 18 chitinase (gene
name chiA; protein referred to as LlChi18A); a
secreted family 33 CBP (yucG; protein referred to as
LlCBP33A); and a family 20 N-acetylhexosaminidase
(LnbA). The chiA and yucG genes are separated by
19 bp in an operon starting with a putative transcrip-
tional regulator positioned 166 bp upstream from the
chitinase start codon. In this study, we have followed

a biochemical approach to the question of whether
L. lactis contains a functional chitinolytic machinery.
The genes encoding LlChi18A and LlCBP33A were
cloned and the gene products were characterized. In
addition to yielding insight into the chitinolytic poten-
tial of L. lactis, the present results provide only the
third example of the role of family 33 CBPs in the deg-
radation of recalcitrant polysaccharides. Furthermore,
the results provide the first example of a family 33
CBP that promotes the degradation of a-chitin, the
most abundant chitin form in nature.
Results and Discussion
Preliminary assessment of the production of
chitinases by L. lactis
Apart from one study showing that L. lactis can grow
on chito-oligosaccharides [27], nothing is known about
the ability of LABs to metabolize chitin. We attempted
to culture L. lactis IL1403 on minimal medium con-
taining various chitin forms as the sole carbon source.
The chitin-containing media (sterilized by autoclaving)
were inoculated with cells from an overnight culture
that had been washed in sterile 0.9% saline buffer in
order to remove traces of glucose. Under these condi-
G. Vaaje-Kolstad et al. L. lactis chitinase and chitin-binding protein
FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS 2403
tions, the bacterium did not grow, and we could not
detect chitinolytic activity in the culture supernatants
even after several days of incubation.
Most microorganisms secrete a variety of hydrolytic
enzymes when starved, in order to access new sources

of carbon. In order to further analyse whether L. lactis
would look for chitin as an alternative source of car-
bon, the bacterium was grown in a medium containing
only 0.1% (w ⁄ v) glucose. During the growth period
and starvation period, culture samples were taken and
assayed for chitinolytic activity. Chitinolytic activity
was detected, peaking 7 h after inoculation (Fig. 1).
After 7 h, chitinolytic activity declined, but still remained
significant. We could not detect chitinolytic activity in
uninoculated culture medium or in cultures grown with
normal glucose concentrations.
Cloning and purification of LlChi18A and
LlCBP33A
The gene fragments coding for the mature proteins of
LlChi18A and LlCBP33A were successfully cloned into
the pETM11 and pET30 Xa ⁄ LIC expression vectors,
respectively.
When expressed in Escherichia coli BL21 DE3, both
proteins were produced in large amounts, although
partly (LlChi18A) or almost exclusively (LlCBP33A)
in an insoluble form (inclusion bodies). The culture
conditions (temperature, isopropyl thio-b-d-galactoside
concentrations and duration of culture) were varied in
an attempt to obtain soluble protein. For LlChi18A,
this resulted in the production of sufficient amounts
of soluble protein. Soluble LlCBP33A was obtained
through refolding of protein obtained from the inclu-
sion bodies. After testing several denaturation and
refolding protocols, we adopted a protocol based on
denaturation in 8 m urea, pH 8.0 for 3 h and refolding

through dialysis of concentrated denatured protein in
a large volume of 20 mm Tris ⁄ HCl, pH 8.0 (see Mate-
rials and methods for more details). At most, the puri-
fication scheme resulted in 10 mg of purified LlChi18A
and 7.1 mg of purified LlCBP33A per litre of culture.
After purification, His-tags were removed from
LlChi18A and LlCBP33A with tobacco etch virus
(TEV) protease and factor Xa, respectively, with no
significant loss of cleaved protein. The purity of the
recombinant proteins after His-tag removal was
assessed by SDS-PAGE to be better than 95%.
Sequence analysis and modelling of LlChi18A
and LlCBP33A
The closest homologue of LlChi18A (when performing
a standard blast search with the LlChi18A sequence)
is ChiC1 from S. marcescens (49% sequence identity
when aligning full-length sequences, 78.5% when align-
ing catalytic domains only). Like ChiC1 from S. mar-
cescens, LlChi18A is predicted to be a three-domain
protein consisting of a catalytic domain belonging to
glycoside hydrolase family 18 subgroup A, according
to the classification of family 18 chitinases suggested
by Seidl et al. [5], followed by a Fibronectin-III (FnIII)
module and a family 5 CBM [3,18], respectively.
ChiC1 has the same domain structure, but the FnIII
domain is followed by a family 12 CBM, which is dis-
tantly related to the family 5 CBM found in LlChi18A.
Sequence analysis also shows that the catalytic module
lacks an a
+ b-fold insertion between b-sheets 7 and 8

of the TIM-barrel fold (Fig. 2A), which is responsible
for deepening the substrate-binding groove in many
family 18 chitinases [29]. A deep substrate-binding
groove is considered to be characteristic of enzymes
that act in an exo-fashion and ⁄ or that tend to stick
tightly to the substrate whilst degrading it in a proces-
sive manner [30,31]. Enzymes lacking the a + b-fold
insertion have a shallow catalytic cleft, as illustrated
by the crystal structure of the plant family 18 sub-
group B chitinase hevamine [32]. Such shallow cata-
lytic clefts are typically seen amongst endo-acting,
nonprocessive carbohydrate-degrading enzymes.
Detailed studies using chitosan as substrate have
shown that ChiC1 from S. marcescens is indeed a non-
processive endo-acting enzyme [30,33]. A model of
LlChi18A automatically generated by 3d-jigsaw [34]
using the structure of hevamine (Protein Data
Bank code: 2HVM) as template suggested that the two
Fig. 1. Chitinolytic activity produced by cultured L. lactis. Bar chart
of chitinolytic activity measured in the culture supernatant of a
starved L. lactis culture at specific time points. The bar labelled as
‘LM17’ indicates the chitinolytic activity present in fresh culture
medium. Activity was recorded by measuring the hydrolysis of the
fluorogenic substrate 4MU-(GlcNAc)
3
. All experiments were run in
triplicate.
L. lactis chitinase and chitin-binding protein G. Vaaje-Kolstad et al.
2404 FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS
A

B
Fig. 2. Sequence alignments for LlChi18A and LlCBP33A. (A) Catalytic domains of LlChi18A (chitinase of L. lactis ssp. lactis), ChiC1 (chitin-
ase C from S. marcescens BJL200), Heva (hevamine from Hevea brasiliensis), ChiA (chitinase A from S. marcescens BJL200) and ChiB
(chitinase B from S. marcescens BJL200). The ChiC1 and LlChi18A sequences are aligned with a previously generated structural alignment
of ChiA, ChiB and hevamine (see [49]). Conserved residues are shaded black. The stretches of residues constituting the a + b domain pres-
ent in ChiA and ChiB, but lacking in LlChi18A, ChiC1 and hevamine, are shaded grey. Asterisks mark residues that are identical in LlChi18A
and ChiC1. Small insertions in the hevamine sequence have been replaced by the letter ‘X’. Diagnostic sequence motifs containing residues
that are crucial for catalysis (SXGG and DXXDXDXE) are shown below the alignment. Arrows indicate Ala126, replacing S in the SXGG motif,
as well as two other residues, Tyr48 and Asn230, that presumably play a major role in catalysis (see text). (B) Full-length sequences of
LlCBP33A (family 33 CBP of L. lactis ssp. lactis), ChbB (family 33 CBP from B. amyloliquefaciens) and CBP21 (family 33 CBP from S. mar-
cescens). Fully conserved residues are shaded in black. Asterisks indicate residues that are thought to be located in the binding surface for
chitin (as derived from the crystal structure of CBP21, as well as mutagenesis studies [10,17]). Residues involved in the chitin-binding and
functional properties of CBP21 [10,17], but not conserved in LlCBP33A or ChbB, are shaded grey. The arrow indicates the terminal amino
acid of the N-terminal signal sequence for all three proteins. The putatively surface-exposed aromatic amino acids in the first LlCBP33A
insert are indicated by (d; Trp51) and (s; Phe55).
G. Vaaje-Kolstad et al. L. lactis chitinase and chitin-binding protein
FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS 2405
proteins indeed have similar shallow and open sub-
strate-binding clefts (results not shown).
As shown in Fig. 2A, LlChi18A contains all residues
known to be important for catalysis in family 18
chitinases, except for the serine in the diagnostic
SXGG sequence motif, which is replaced by alanine
(residue 126 in LlChi18A). The role of this serine in
the catalytic mechanism of family 18 glycosyl hydrolas-
es is to help in the stabilization of a temporary surplus
of negative charge that develops on the first aspartate
of the catalytic sequence motif DXDXE during cataly-
sis [7,35]. For ChiB from S. marcescens, it was shown
that this charge stabilization is in fact achieved by two

residues: serine in the SXGG motif and a tyrosine resi-
due. Although LlChi18A lacks serine, it does contain
this tyrosine residue (Tyr48, corresponding to Tyr10 in
S. marcescens ChiB). A multiple sequence alignment of
the 50 family 18 catalytic modules that are most simi-
lar to the LlChi18A catalytic module (not shown)
showed that about one-half of the proteins had a sub-
stitution at either the conserved serine or tyrosine,
whereas none had substitutions at both positions.
Thus, it appears that family 18 glycosyl hydrolases are
tolerant to substitutions of either of the discussed
amino acids, as long as both are not substituted.
Another conspicuous sequence characteristic of
LlChi18A is the presence of an asparagine residue at
position 230. The presence of an asparagine at this
position is characteristic for family 18 chitinases with
acidic pH optima for activity, whereas enzymes with
more neutral pH optima have an aspartic acid at this
position. For the latter type of enzyme, it has been
shown that mutation of aspartic acid to asparagine
leads to a drastic acidic shift of the pH optimum [35].
Indeed, LlChi18A was found to have an acidic pH
optimum for activity (see below).
LlCBP33A is a family 33 CBP. The only available
three-dimensional structure of a family 33 CBP is that
of CBP21 from S. marcescens, which binds exclusively
to b-chitin [17,20]. The combination of sequence and
structural information with the results of site-directed
mutagenesis studies showed that the surface of fam-
ily 33 CBPs contains a patch of highly conserved,

mostly polar residues that are important for binding to
chitin and for the positive effect on chitinase efficiency
[10,17] (Figs 2 and 3). Comparison of the LlCBP33A
and CBP21 sequences shows two substitutions in the
conserved surface patch, both concerning residues that
are known to be important for CBP21 functionality
[10]: (a) Ser63 occurs at a position at which CBP21
has a tyrosine (Tyr54) and where several other fam-
ily 33 CBPs have another aromatic residue, tryptophan
(e.g. Trp57 in CHB1 from St. olivaceoviridis, which
has been shown to be important for the ability of
CHB1 to bind a-chitin [36]); (b) Asn64 occurs instead
of a glutamate residue (Glu55 in CBP21). Interestingly,
the closest homologue of LlCBP33A from species
other than L. lactis is ChbB from Bacillus amylolique-
faciens (66% sequence identity), which binds both
a- and b-chitin [21]. As shown in Fig. 2B, ChbB differs
from CBP21 in the same two positions as LlCBP33A:
Tyr54 is replaced by Asp62 and Glu55 is replaced by
Asn63. In addition to these sequence differences,
LlCBP33A and ChbB differ from CBP21 in that they
have two short inserts (Figs 2B and 3). Although it is
not possible to model the structural position of these
inserts accurately, it is clear that they are located close
to the binding surface and may thus affect functional-
ity (Fig. 3B). The possible implications of the observed
differences within family 33 CBPs are discussed further
in the context of the experimental results (see below).
Enzyme pH optimum, stability and kinetics
Activity measurements with the artificial substrate

4-methylumbelliferyl-b-d-N, N¢,N¢-diacetylchitobioside
[4MU-(GlcNAc)
3
] showed that LlChi18A has a narrow
pH activity profile with an optimum at pH 3.8
(Fig. 4A). Studies on pH stability showed that the
AB
Fig. 3. Structural comparison of CBP21 and LlCBP33A. Illustrations
of the CBP21 structure (A) and a structural model of LlCBP33A (B)
shown in a surface representation. The surface thought to be
involved in chitin binding is coloured blue. The side-chains of resi-
dues marked with an asterisk in the sequence alignment of Fig. 2B
are shown as blue sticks. Residues important for chitin binding and
the function of CBP21 [10,17], but not conserved in LlCBP33A, are
shown as blue sticks and labelled. For illustration purposes only,
the figure also shows the small inserts in LlCBP33A (orange) as
they were rendered by the structure prediction program. Note that,
as no template structure residues are available for modelling the
inserts, the structural prediction of these inserts is highly inaccu-
rate. Phe55 is coloured magenta and its side-chain is shown. Trp
(Trp51) in the LlCBP33A insert is hidden from view. The model of
LlCBP33A was generated by SwissModel (http://swissmodel.
expasy.org//SWISS-MODEL.html; [50]), using CBP21 (Protein Data
Bank code: 2BEM) as structural template. The model of LlCBP33A
is deposited in the PMDB database (PMDB code: PM0075054).
L. lactis chitinase and chitin-binding protein G. Vaaje-Kolstad et al.
2406 FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS
enzyme was unstable at pH 3.8 and below, whereas
enzyme activity remained stable for more than a week
at bench temperature when dissolved in buffers with a

pH higher or equal to pH 5 (results not shown). At
shorter incubation times (e.g. up to the 20 min used in
the enzyme assays), LlChi18A was stable at pH values
as low as pH 3.4. Thus, kinetic parameters could be
determined with confidence at the pH optimum.
Both artificial substrates [4-methylumbelliferyl N-di-
acetyl-b-d-glucosaminide (4MU-(GlcNAc)
2
) and 4MU-
(GlcNAc)
3
] were used to determine the enzyme kinetics
of LlChi18A. Degradation of 4MU-(GlcNAc)
2
gave
sigmoidal kinetics that proved difficult to interpret
(results not shown). 4MU-(GlcNAc)
3
, however, gave a
regular hyperbolic curve that could be fitted to the
Michaelis–Menten equation using nonlinear regression
(Fig. 4B). The curve fitting showed LlChi18A to have
a turnover rate ( k
cat
) of 2.8 ± 0.2 s
)1
and a K
m
value
of 94 ± 10 lm. These are typical values for family 18

chitinases with shallow substrate-binding clefts [37–39].
Processive chitinases with their characteristic deep sub-
strate-binding grooves usually have about 10-fold
higher k
cat
and 10-fold lower K
m
values for oligomeric
substrates [39].
Initial rate measurements with (GlcNAc)
3
and (Glc-
NAc)
4
as substrates yielded specific activities of 0.64
and 11.6 s
)1
, respectively (Fig. 4C), within the range of
other results reported in the literature (e.g. ChiC1 from
S. marcescens [30]). The products observed for (Glc-
NAc)
3
degradation were GlcNAc and (GlcNAc)
2
.
(GlcNAc)
4
degradation resulted in the exclusive forma-
tion of (GlcNAc)
2

, indicating preference for binding
D
C
A
B
Fig. 4. Enzymatic properties of LlChi18A. (A) Relative specific activities of LlChi18A measured at pH values of 3.4, 3.8, 4.0, 4.2, 4.6, 5.0,
6.0, 7.0 and 8.0 using 4MU-(GlcNAc)
3
as substrate at 37 °C. (B) Kinetics of LlChi18A towards 4MU-(GlcNAc)
3
at pH 3.8 and 37 °C. The data
were fitted to the Michaelis–Menten equation by nonlinear regression (represented by the curve drawn). The kinetic parameters k
cat
and K
m
derived from the data are shown in the figure. (C) Time course of the degradation of (GlcNAc)
3
(r) and (GlcNAc)
4
( )byLlChi18A, illustrated
by the production of (GlcNAc)
2
during the initial linear phase of the degradation reaction. Note that the enzyme concentrations used in the
two reactions differed by a factor of 10 (see Materials and methods). (D) Chromatogram of (GlcNAc)
6
degradation products generated by
LlChi18 after 2 min of incubation with 1 n
M of enzyme. The double peaks represent the a- and b-anomers of the oligomers. Using standard
curves, the total concentrations of dimer, trimer and tetramer were calculated to be 25, 10 and 24 l
M, respectively. The peak marked ‘X’

represents a nonhydrolysable background oligosaccharide that is also seen (with equal peak area) in control samples without enzyme. Glc-
NAc was not observed before all (GlcNAc)
6
was degraded. Although the experiments in (D) were not conducted to preserve anomeric ratios
generated by the enzyme, one important trend is still visible: the combination of a relative predominance of b-anomers for the (GlcNAc)
2
product and the approximately equilibrium anomeric ratio for the tetrameric product suggests that the conversion of (GlcNAc)
6
to (GlcNAc)
2
and (GlcNAc)
4
primarily results from binding of the nonreducing end of the substrate in subsite )2.
G. Vaaje-Kolstad et al. L. lactis chitinase and chitin-binding protein
FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS 2407
subsites )2 to +2. Analysis of the initial degradation
products formed from (GlcNAc)
6
showed a 1 : 1 ratio
of (GlcNAc)
2
to (GlcNAc)
4
, which indicates a nonpro-
cessive mode of action (Fig. 4D). Processive chitinases
tend to convert (GlcNAc)
6
processively into three (Glc-
NAc)
2

moieties, leading to a characteristic initial (Glc-
NAc)
2
⁄ (GlcNAc)
4
product ratio that is considerably
larger than unity (see, for example [40]). The product
profile obtained with (GlcNAc)
6
further shows that
approximately 30% of (GlcNAc)
6
is converted into
two (GlcNAc)
3
molecules. The data suggest that con-
version of (GlcNAc)
6
to (GlcNAc)
2
and (GlcNAc)
4
predominantly results from binding of the substrate
with its nonreducing end in subsite )2 (see legend to
Fig. 4), meaning that the longer part of the substrate
interacts with + subsites. In a detailed analysis of
product profiles [39], a similar conclusion was drawn
for ChiC1 from S. marcescens. The fact that the longer
part of the substrate extends towards the + side of the
catalytic centre is compatible with the notion that the

C-terminal substrate-binding domains are likely to be
located on this side, which again suggests that this side
of the enzyme is optimized for interacting with the
longer (polymeric) part of the substrate. In conclusion,
these experimental data and the inferences made from
the sequence and structural comparisons above indi-
cate that LlChi18A is a nonprocessive endo-acting
chitinase, with overall properties that are quite simi-
lar to those of, for example, the nonprocessive endo-
chitinase ChiC1 from S. marcescens.
Binding preferences for LlCBP33A
Some family 33 CBPs bind to a broad selection of
insoluble carbohydrates (e.g. ChbB, which binds both
a- and b-chitin [21], and Chb3 from St. coelicolor,
which binds a-chitin, b-chitin, colloidal chitin and
chitosan [22]), whereas others bind only to a specific
substrate variant (e.g. CBP21 from S. marcescens
which strictly binds to b-chitin [20] and CHB1 from
St. olivaceoviridis [23] and CHB2 from St. reticuli [24]
which strictly bind to a-chitin). A common property is
that binding is influenced by pH (e.g. CBP21 from
S. marcescens does not bind at pH < 4.5 [20]).
The binding preferences of LlCBP33A were investi-
gated by incubating the protein with various types of
chitin and other insoluble polymeric substrates. As
noncrystalline ⁄ amorphous chitin variants, chitin beads
(re-acetylated chitosan beads) and colloidal chitin (chi-
tin processed with strong acid to disrupt the ordered
crystalline properties of native chitin to render it amor-
phous) were used. Preliminary experiments showed

that binding of LlCBP33A to chitin was relatively slow
and that approximately 24 h of incubation at room
temperature were needed to reach binding equilibrium.
The extent and specificity of LlCBP33A binding was
analysed by SDS-PAGE (Fig. 5A,B). The amount of
LlCBP33A bound was also analysed by determining
the protein concentrations in the supernatants of the
reaction mixtures after 24 h of incubation. The results
(Fig. 5C) show that LlCBP33A binds equally well to
a- and b-chitin ( 40% of the protein in solution was
bound at equilibrium), whereas binding to chitin beads
(noncrystalline chitin, chitin beads; no binding
detected) and colloidal chitin (amorphous chitin;
 10% bound) was lower. As no or low binding was
observed for the amorphous ⁄ noncrystalline chitin vari-
ants, it seems that LlCBP33A has a preference for
A
B
C
Fig. 5. Substrate preferences for LlCBP33A at pH 6.0. (A, B) Bind-
ing of LlCBP33A visualized by SDS-PAGE. (A) LlCBP33A present in
the supernatant after 24 h of incubation with a-chitin (lane 2), b-chi-
tin (lane 3), Avicel (lane 4), chitin beads (lane 5) and colloidal chitin
(lane 6). Lane 1 shows the control incubation (0.4 mgÆmL
)1
LlCBP33A incubated for 24 h in 50 mM citrate–phosphate buffer,
pH 6.0). (B) LlCBP33A bound to a-chitin (lane 2), b-chitin (lane 3),
Avicel (lane 4), chitin beads (lane 5) and colloidal chitin (lane 6).
Lane 1 shows controls (LlCBP33A bound to the sample tube wall).
The proteins were removed from the solid substrates by boiling in

SDS-PAGE sample buffer after the substrates had been washed to
remove nonspecifically bound protein. Note that the samples in (B)
are approximately sixfold concentrated compared with the corre-
sponding samples in (A) (A shows 20 lL of a 300 lL supernatant;
B shows 20 lL samples of bound protein resolubilized in 50 lLof
SDS-PAGE sample buffer). (C) Bar chart quantifying the binding of
LlCBP33A to a variety of insoluble substrates. Bound protein was
determined indirectly by measuring the concentration of free
protein in the supernatants after 24 h of incubation.
L. lactis chitinase and chitin-binding protein G. Vaaje-Kolstad et al.
2408 FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS
binding the ordered, crystalline chitin forms rather
than individual chitin chains. Interestingly, LlCBP33A
also showed some binding to Avicel (microcrystalline
cellulose,  20% bound), as has also been observed
for other family 33 CBMs [21,41].
In terms of binding to the various chitin forms, the
characteristics of LlCBP33A are similar to those of
ChbB from B. amyloliquefaciens, in that both proteins
bind well to both a- and b-chitin. As noted above,
ChbB is the closest homologue of Ll CBP33A and the
two proteins share sequence characteristics that sepa-
rate them from the ‘one-substrate binders’ such as
CBP21 [17,20] and CHB1 [23]. It is conceivable that
the above-mentioned two mutations in the binding sur-
face and the two insertions that are putatively close to
this surface (Fig. 3) endorse LlCBP33A and ChbB
with the ability to bind a wider variety of substrates
than do CBP21 and CHB1.
Degradation of a- and b-chitin

The degradation rates of a- and b-chitin were assayed
with LlChi18A in the presence or absence of
LlCBP33A. As both chitin variants, and especially
a-chitin, are highly resistant to enzymatic hydrolysis,
the time span of the assay was 2 weeks using a rela-
tively high concentration of LlChi18A and LlCBP33A
(1.0 and 3.0 lm, respectively). The production of
(GlcNAc)
2
(the major end-product of enzymatic
hydrolysis by family 18 glycosyl hydrolases) was
recorded at regular time intervals.
The degradation of a-chitin by LlChi18A started
with a rapid phase, regardless of the presence of
LlCBP33A. In the presence of LlCBP33A, the fast
initial phase was maintained longer than in the absence
of LlCBP33A, indicating that LlCBP33A acts synergis-
tically with LlChi18A. However, the effect of
LlCBP33A was small and ceased after approximately
48 h (Fig. 6A). This indicates that LlCBP33A only acts
on a specific minor subfraction of a-chitin. Thus,
LlCBP33A has an effect on the degradation of a-chi-
tin, but the effect is smaller than the effects of CBP21
[10] or LlCBP33A (below) on b-chitin.
The degradation of b-chitin by LlChi18A was much
more rapid than the degradation of a-chitin. More-
over, although about 85% of a-chitin was left after
2 weeks of incubation, all of the b-chitin was com-
pletely solubilized by LlChi18A, in both the absence
and presence of LlCBP33A. In the absence of

LlCBP33A, the end-point of the reaction (i.e. solubili-
zation of all chitin) was reached after approximately
2 weeks. When LlCBP33A was present in the reaction,
the degradation rate was substantially higher, the
end-point being reached after approximately 48 h
(Fig. 6B). Thus, LlCBP33A clearly acts synergistically
with LlChi18A in the degradation of b-chitin. The
increase in LlChi18A efficiency on addition of
LlCBP33A is comparable with the increase observed
when adding CBP21 during the degradation of b-chitin
with ChiC1 from S. marcescens [10].
Although the occurrence of family 33 CBPs has been
known for some time [23], the present results provide
only the third demonstration of the accessory function
of these proteins. The effect of LlCBP33A on b-chitin
degradation is of the same order of magnitude as the
effect of CBP21. The effect on a-chitin degradation is
unique for LlCBP33A, but is rather modest (Fig. 6A).
It should be noted that, in nature, chitin is often found
as a composite where layers ⁄ sheets of chitin are inter-
woven with proteins and ⁄ or minerals in a recalcitrant
A
B
180
160
140
120
100
80
GlcNAc

2
(µM)
GlcNAc
2
(µM)
GlcNAc (µ
M)
60
40
20
0
0
50
100
150
200
250
300
350
400
Time (h)
0
50
100
150
200
250
300
350
400

Time (h)
200
180
160
140
120
100
80
60
40
20
0
200
Fig. 6. Chitin degradation by LlChi18A in the absence and presence
of LlCBP33A at pH 6.0, 37 °C. (A) Full lines show the degradation
of 0.5 mgÆmL
)1
a-chitin by LlChi18A ( ) and LlChi18A in the pres-
ence of LlCBP33A (d) with nonstatic incubation. (B) Full lines show
the degradation of 0.1 mgÆmL
)1
b-chitin by Ll Chi18A ( ) and
LlChi18A in the presence of LlCBP33A (d) with static incubation.
For comparison, the production of the minor end-product GlcNAc is
also shown (dotted lines through squares for LlChi18A; dotted lines
through circles for LlChi18A in the presence of LlCBP33A). The
production of GlcNAc in the reaction with a-chitin could not be
quantified accurately, but was of the same order of magnitude.
G. Vaaje-Kolstad et al. L. lactis chitinase and chitin-binding protein
FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS 2409

heteropolymer. The crystalline chitin used in most
experiments in the chitin ⁄ chitinase field has been trea-
ted by strong acids and bases in order to remove the
protein and ⁄ or the mineral fraction. It is conceivable
that the real natural substrates of the CBP proteins
differ from the substrates used here and in other stud-
ies. There may exist composite natural chitinous sub-
strates that are more susceptible to the action of
CBPs.
Structure–function studies of CBP21 have shown
that this protein may act by disrupting the crystalline
substrate through interactions that involve polar resi-
dues in a conserved surface patch [10,17]. The lack of
aromatic residues in the binding surface of CBP21
(there is only one, Tyr54) was somewhat surprising,
because aromatic residues are generally considered to
play important roles in enzyme–carbohydrate interac-
tions [18]. As CBP21 and LlCBP33A have different
binding properties, a structural comparison of the two
proteins could provide more insight into the mecha-
nism and specificity of CBP action. Unfortunately,
despite extensive attempts, we have so far been unable
to obtain crystals of LlCBP33A. The most obvious
structural difference between the two proteins is
formed by the two inserts in LlCBP33A that seem
positioned close to the conserved surface patch and
that could extend the binding surface (Fig. 3B). Inter-
estingly, the largest insert contains two aromatic amino
acids (Trp51 and Phe55), which could interact with the
surface of a chitin crystal. Another interesting observa-

tion is that a disulfide bridge on the surface, close to
the important Tyr54 in CBP21 (Cys41–Cys49 in
CBP21), is missing in LlCBP33A, which has the 50–57
insert in this area (Fig. 2B). This could affect the bind-
ing properties of the protein, as it may introduce flexi-
bility and ⁄ or structural changes in this crucial region.
Conclusions
The present data show that the putative chitinase
and CBP genes in L. lactis code for a functional
chitinolytic machinery capable of converting chitin to
GlcNAc and (GlcNAc)
2
. The primary product of this
machinery is (GlcNAc)
2
, which can be converted to
mono-sugars by the putative N-acetylglucosaminidase
encoded by LnbA. We were able to show that L. lactis
indeed produces chitinolytic activity under certain
conditions. However, further work is needed to analyse
the role and regulation of the chitinolytic system of
this bacterium. LlChi18A was shown to be active and
relatively stable at low pH, which agrees with the
ability of L. lactis to grow and thrive in mildly acidic
environments.
The finding of nonhydrolytic accessory proteins for
chitinases has reinforced interest in the question as to
whether such proteins may also exist for cellulose. The
existence of substrate-disrupting accessory proteins and
domains that act synergistically with cellulases has

been a topic in cellulose research ever since the studies
by Reese et al. around 1950 [42]. Cases as clear cut
as the two cases from the chitinase field ([10], this
paper) do not yet exist. However, more recent studies
indicate that nonhydrolytic proteins that are either
dedicated to cellulose degradation [43,44] or that can
be exploited for this purpose (expansins [45]; see also
[46]) do exist.
Materials and methods
Bacterial strains and plasmids and cultivation
Lactococcus lactis ssp. lactis IL1403 is a derivative of the
strain IL594 that was isolated from a cheese starter culture
[47]. For the isolation of genomic DNA and the creation of
stock cultures, the bacterium was grown overnight at 30 °C
without aeration in M17 medium (Oxoid, Basingstoke,
Hampshire, UK) supplemented with 0.2% (w ⁄ v) glucose
(GM17). The bacterium was maintained as frozen stocks at
)80 °C in liquid medium containing 17% (v ⁄ v) glycerol.
To investigate whether L. lactis was able to produce
chitinolytic activity, overnight cultures of L. lactis grown in
GM17 were diluted to an attenuance D at 600 nm of
approximately 0.1 in modified M17 medium (LM17),
composed of Maritex Fish peptone (5.0 gÆL
)1
) [48], bacto
yeast (Difco Laboratories, Sparks, MD, USA) (5.0 gÆL
)1
),
ascorbic acid (Sigma, St Louis, MO, USA) (0.5 gÆL
)1

),
magnesium sulfate (0.25 gÆL
)1
) (Sigma), disodium glycerol-
ophosphate (19 gÆL
)1
) (Sigma) and manganese sulfate
(0.05 gÆL
)1
) (Sigma). As a carbon source, b-chitin isolated
from squid pen (France Chitin, Marseille, France), a-chitin
isolated from shrimp (Hov-Bio, Tromsø, Norway), colloidal
chitin and glucose were used (all chitin variants at a final
concentration of 1% w ⁄ v and glucose at final concentra-
tions of 0.1% or 0.4% w ⁄ v). The cultures were incubated
at 30 °C and samples were taken at various time points (4,
7, 8 and 10.5 h) in order to assay for chitinolytic activity in
the culture supernatants (see below for assay details).
Cloning of L. lactis chitinases and CBP
Genomic DNA from L. lactis was isolated from an over-
night culture using a midi-prep genomic DNA isolation kit
(Qiagen, Venlo, The Netherlands) and stored at ) 20 °C. A
3392 bp long region of the genome containing a putative
transcription regulator (GenBank ID: AAK06047.1), chitin-
ase gene (GenBank ID: AAK06048.1) and gene encoding a
family 33 CBP (GenBank ID: AAK06049.1) was amplified
L. lactis chitinase and chitin-binding protein G. Vaaje-Kolstad et al.
2410 FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS
by PCR using primers flanking 100 bp upstream of the first
ORF and 100 bp downstream of the third ORF (forward

primer, 5¢-GGATGAGCTCTATACTCACATCTTGAGC-
3¢; reverse primer, 5¢-TTGTGGGCCCAACCAATCTATG
AAGAATT-3¢). The PCR product was cloned using the
zero blunt TOPO-cloning kit (Invitrogen, Carlsbad, CA,
USA). The resulting plasmid was transformed into E. coli
TOP10 competent cells and the insert was sequenced using
a series of sequencing primers evenly distributed along the
cloned DNA sequence. Different strategies were used for
subsequent separate cloning of the chitinase and CBP,
as the N-terminus of the latter protein should be free of
non-native amino acids after removal of the affinity
tag (because amino acid one of the mature protein is
conserved and seems important; see [17]). Both primary
gene products were predicted to contain N-terminal leader
peptides directing sec-dependent secretion. The genes
were cloned without these leader peptide-encoding
parts. The start of the mature proteins was assigned
using the SignalP server ( />SignalP/).
Primers for cloning of the putative chitinase were
designed to amplify a fragment encoding amino acids
32–492 of the predicted gene product. The forward primer
(5¢-GGTCTCCCATGGATGCAGCTAGTGAAATGGTCA-3¢)
was designed with an NdeI-compatible BsaI restriction
site at the 5¢ end, leading to a one-residue (methionine)
N-terminal extension of the gene product. The reverse pri-
mer (5¢-CTCGAGTTATAGCTTTTTCCATGGACCAAA
ATCTC-3¢) contained a XhoI restriction site starting imme-
diately downstream of the stop codon of the chitinase gene.
The amplified chitinase fragment was ligated into vector
pCRÒ4Blunt-TOPOÒZero Blunt TOPO (Invitrogen),

excised from the TOPO vector using XhoI and BsaI, and
ligated to NdeI–XhoI-digested pETM11 vector (Gu
¨
nter
Stier, EMBL Heidelberg, Germany). The pETM11 vector
contains a T7 promoter sequence for expression and an
N-terminal His
6
tag for immobilized metal affinity chroma-
tography purification.
The putative CBP was cloned using the pET30 Xa ⁄ LIC
kit (Merck Chemicals Ltd, Nottingham, UK), which
provides a ligation-independent method for cloning a gene
of interest. The expression vector (pET30 Xa ⁄ LIC) provides
an N-terminal His
6
tag that can be removed from the
N-terminus of the purified protein using activated factor X
leaving no non-native amino acids. Cloning primers were
designed according to the suppliers’ instructions, containing
ends compatible with the expression vector (forward
primer, 5¢-GGTATTGAGGGTCGCCATGGTTATGTTC
AATCACCA-3¢; reverse primer, 5¢-AGAGGAGAGTTAG
AGCCTTACAAGAAGGGTCCAAAGA-3¢). The PCR
product was purified, treated with T4 exonuclease to
create vector-compatible overhangs and annealed to a
prepared expression vector (pET30 Xa ⁄ LIC) provided by
the supplier.
The final constructs (pETM11-LlChi18A and pET30Xa-
LIC-LlCBP33A) were transformed into E. coli BL21Star

(DE3) (Invitrogen). DNA sequencing was performed using
a BigDyeÒ Terminator v3.1 Cycle Sequencing Kit (Perkin-
Elmer ⁄ Applied Biosystems, Foster City, CA, USA) and an
ABI PRISMÒ 3100 Genetic Analyser (Perkin-Elmer ⁄
Applied Biosystems).
Protein expression and purification
Overnight cultures grown from )80 °C stocks of BL21Star
(DE3) cells containing pETM11-LlA or pET30XaLIC-LlA
were used to inoculate 150 mL of Luria–Bertani medium
containing 50 lgÆmL
)1
of kanamycin. The cultures were
incubated at 37 °C and 250 r.p.m. When the cell density
reached 0.6 (D
600
), isopropyl thio-b-d-galactoside was
added to a final concentration of 0.05 mm, and the culture
was further incubated for 4 h at 37 °C, followed by harvest-
ing by centrifugation (11 325 g 10 min at 4 °C). The cell
pellet was resuspended in citrate–phosphate buffer pH 6.0,
and the cells were lysed by sonication at 20% amplitude
with 30 · 5 s pulses (with 5 s delay between pulses) on ice,
with a Vibra cell Ultrasonic Processor, converter model
CV33, equipped with a 3 mm probe (Sonics, Newtown, CT,
USA). The sonicated material was centrifuged at 11 325 g
for 10 min at 4 °C in order to pellet the insoluble cell
remains. At this stage, LlChi18A was found in the soluble
fraction and LlCBP33A was found as inclusion bodies in
the insoluble fraction. Thus, two separate protocols were
followed for subsequent purification. For LlChi18A, the

cleared lysate was applied to a 3 cm · 5 cm Ni-NTA col-
umn (Qiagen, Venlo, The Netherlands) equilibrated with
running buffer (100 mm Tris ⁄ HCl, pH 8.0). LlChi18A was
eluted by running four column volumes of elution buffer
(100 mm Tris ⁄ HCl, pH 8.0 and 100 mm imidazole) through
the column. The peak containing chitinase was collected
and concentrated using a Centricon P-20 unit (Millipore,
Billerica, MA, USA) and dialysed overnight in 20 mm
Tris ⁄ HCl, pH 8.0.
For LlCBP33A, the pellet resulting from centrifugation
of the sonicated cells was resuspended in denaturing buffer
containing 8 m urea, 0.1 m NaH
2
PO
4
,10mm Tris ⁄ HCl,
pH 8.0 and 25 mm dithiothreitol, and incubated at room
temperature for 3 h with gentle shaking. Subsequently, the
unfolded protein was purified on an Ni-NTA column under
denaturing conditions, using 8 m urea, 0.1 m NaH
2
PO
4
and
10 mm Tris ⁄ HCl, pH 8.0 as running buffer, and 8 m urea,
0.1 m NaH
2
PO
4
,10mm Tris ⁄ HCl, pH 8.0 and 100 mm

imidazole as elution buffer. The peak containing the pure
protein was concentrated using a Centricon P-20 unit (Mil-
lipore) and the protein was refolded by extensive dialysis in
20 mm Tris ⁄ HCl, pH 8.0 at 4 °C (two buffer changes in
24 h).
The removal of the N-terminal His
6
tags was performed
by the addition of recombinant TEV protease (1 U per
G. Vaaje-Kolstad et al. L. lactis chitinase and chitin-binding protein
FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS 2411
3 lg of target protein) or activated factor X (Merck
Chemicals Ltd.; 1 U per 70 lg of target protein) to purified
His-tagged LlChi18A and LlCBP33A, respectively. TEV
protease cleavage reactions were conducted in 20 mm
Tris ⁄ HCl, pH 8.0, 0.25 mm EDTA and 1 mm dithiothreitol,
incubated at 37 °C for 4 h. Factor Xa cleavage reactions
were conducted in 100 mm NaCl, 50 mm Tris ⁄ HCl, 5 mm
CaCl
2
, pH 8.0, incubated at room temperature for 16 h.
Cleavage reactions were followed by immobilized metal
affinity chromatography purification (as above), in which
the nonbound protein (cleaved LlChi18A or LlCBP33A)
was collected and the bound protein (free His
6
tag and
His-tagged TEV protease if present) was discarded.
Factor X was removed from the LlCBP33A cleavage reac-
tion by running the sample through a mini spin column

containing 50 lL of Xarrest Agarose (Merck Chemicals
Ltd). Both proteins were dialysed overnight in 20 mm
Tris ⁄ HCl, concentrated using Centricon P-20 units
(Millipore) and stored at 4 °C.
Protein purity was analysed by SDS-PAGE. Protein con-
centrations were determined using the Bradford micro-assay
(Bio-Rad, Hercules, CA, USA) according to the instruc-
tions provided by the supplier, employing purified bovine
serum albumin (New England Biolabs, Beverly, MA, USA)
as standard.
Chitin-binding assays
Binding studies were conducted using powdered a-chitin
from shrimp shells (Hov-Bio), powdered b -chitin from
squid pen (France Chitin), chitin beads (New England Biol-
abs), colloidal chitin and Avicel (microcrystalline cellulose;
Sigma). All chitin variants were suspended in ddH
2
Oto
yield a 20 mgÆmL
)1
stock suspension. Binding was assayed
in 1 mL reactions in Eppendorf tubes containing
5mgÆmL
)1
chitin and 400 lgÆmL
)1
LlCBP33A in 50 mm
citrate–phosphate buffer, pH 6.0. Reactions were mixed by
vertical rotation (60 r.p.m.) at room temperature for 24 h.
Subsequently, the chitin (with the bound protein fraction)

was pelleted by spinning the sample tubes for 5 min at
15 700 g in a microcentrifuge. The relative amount of
free protein in the supernatant was determined by measur-
ing the absorption at 280 nm (Eppendorf Biophotometer,
Eppendorf, Hamburg, Germany). All assays were per-
formed in triplicate and with blanks (buffer + 5 mgÆmL
)1
of the appropriate substrate) and controls to correct for
aspecific binding of the protein to the reaction vessels (buf-
fer + 400 lgÆmL
)1
LlCBP33A; the values derived from this
control sample were considered to represent 0% binding).
For further verification of binding, protein bound to the
substrate was analysed by SDS-PAGE after removal of
nonspecifically bound protein by washing with 1.5 mL of
50 mm Tris ⁄ HCl, pH 8.0. The pellets were resuspended in
50 lL SDS-PAGE sample buffer and boiled for 5 min in
order to strip bound protein of the substrate. Finally,
20 lL of sample was run on a pre-cast SDS-PAGE gel
(Novex 12%; Invitrogen). Gels were run for 30 min at
200 V, stained in a solution containing 0.5% (w ⁄ v) Coo-
massie brilliant blue, 50% (v ⁄ v) methanol and 10% (v ⁄ v)
acetic acid, and destained in a solution containing 10%
(v ⁄ v) methanol and acetic acid.
Enzymology
The kinetic properties of LlChi18A were determined using
the artificial substrate 4MU-(GlcNAc)
3
(Sigma). The maxi-

mum substrate concentration used had to be limited to
about twice K
m
because of the occurrence of substrate
inhibition (which is usual in this type of assay; for
example, see [8]). Standard reaction mixtures contained
2.0 nm of LlChi18A, 0.1 mgÆmL
)1
bovine serum albumin
and 0–200 lm of the substrate in 50 mm citrate–phosphate
buffer, pH 3.8. The reaction mixtures were incubated at
37 °C and product formation was monitored by taking out
50 lL samples at different time points (0–20 min), in which
the reaction was stopped by the addition of 1.95 mL of
0.2 m Na
2
CO
3
. The amount of 4MU released was deter-
mined by measuring the fluorescence emitted at 460 nm on
excitation at 380 nm, using a DyNA 200 fluorimeter
(Hoefer Pharmacia Biotech, San Francisco, CA, USA). The
release of 4MU proved to be linear with time for all sub-
strate concentrations, allowing the straightforward calcula-
tion of enzyme velocities by linear regression (all curves
had correlation coefficients above 0.99). Kinetic para-
meters were calculated by directly fitting the data to the
Michaelis–Menten equation by nonlinear regression using
graphpad prism (GraphPad Software Inc., San Diego, CA,
USA).

The specific activity of LlChi18A towards a natural sub-
strate was determined by monitoring initial product release
during the degradation of (GlcNAc)
3
and (GlcNAc)
4
(Sei-
kagaku Co., Tokyo, Japan). Reactions were performed in
Eppendorf tubes containing 200 lm of oligosaccharide and
0.1 mgÆmL
)1
of bovine serum albumin in 50 mm citrate–
phosphate buffer, pH 3.8. The reaction was initiated by the
addition of LlChi18A, giving an end concentration of 15 or
1.5 nm of enzyme [for the degradation of (GlcNAc)
3
and
(GlcNAc)
4
, respectively]. Samples were taken at 0, 2, 4, 6, 8
and 10 min and mixed immediately 1 : 1 with 70% (v ⁄ v)
acetonitrile to stop hydrolysis [35% (v ⁄ v) acetonitrile abol-
ishes enzyme activity]. Samples were then analysed by iso-
cratic HPLC employing a 0.46 · 25 cm Amide-80 column
(Tosoh Bioscience, Montgomeryville, PA, USA), coupled to
a Gilson Unipoint HPLC system (Gilson, Middleton, WI,
USA). The liquid phase was 70% (v ⁄ v) acetonitrile and the
flow rate was 0.7 mLÆmin
)1
. Twenty microlitre samples

were injected using a Gilson 123 autoinjector. Eluted oligo-
saccharides were monitored by recording the absorption at
210 nm. Chromatograms were collected and analysed using
Gilson unipoint software (Gilson). A standard solution
L. lactis chitinase and chitin-binding protein G. Vaaje-Kolstad et al.
2412 FEBS Journal 276 (2009) 2402–2415 ª 2009 The Authors Journal compilation ª 2009 FEBS
containing 100 lm of (GlcNAc)
1–4
was analysed at the
start, in the middle and at the end of each series of sam-
ples. The resulting average values of the standards (display-
ing standard deviations of < 5%) were used for
calibration. All measurements were performed in triplicate.
Background was corrected for by subtracting the value of
samples taken at t = 0 min.
The determination of the initial products from (GlcNAc)
6
degradation was performed by incubating 1 nm of
LlChi18A with 200 lm of (GlcNAc)
6
in 50 mm citrate–
phosphate buffer, pH 6.0. Products formed after 2 min of
incubation at 37 °C were analysed using isocratic HPLC as
described above.
The presence of chitinolytic activity in culture super-
natants of L. lactis was assayed using 4MU-(GlcNAc)
2
as
substrate; 50 lL of supernatant was mixed with 50 lLof
50 mm citrate–phosphate buffer, pH 3.8, containing 50 lm

of substrate and 0.1 mgÆmL
)1
of bovine serum albumin,
giving a final volume of 100 lL. The reaction mixture was
incubated at room temperature overnight as the chitinase
concentration in the supernatant was low. The release of
4MU was determined as described above.
For the determination of the pH optimum, solutions of
4MU-(GlcNAc)
3
(50 lm) were prepared using 50 mm cit-
rate–phosphate buffer (pH range 3.4–7.0) and Tris ⁄ HCl at
pH 8.0, containing 0.1 mgÆmL
)1
of bovine serum albumin.
LlChi18A was added to a final concentration of 20 nm, and
samples were taken at 3, 6 and 9 min to record the release
of 4MU, as described above. All measurements were per-
formed in triplicate. Product release was linear over time in
all cases.
Degradation of a- and b-chitin
Determination of the enzyme activity towards insoluble chi-
tin was performed using b-chitin from a squid pen (France
Chitin) and a-chitin isolated from shrimp shells (Hov-Bio)
as substrates. Reaction mixtures contained 1 lm of
LlChi18A and ⁄ or 3 lm of LlCBP33A, 0.1 mgÆmL
)1
of puri-
fied bovine serum albumin, 0.1 mgÆmL
)1

of b-chitin powder
or 0.5 mgÆmL
)1
of a-chitin powder, in 50 mm citrate–phos-
phate buffer, pH 6.0. The reaction was buffered at a higher
pH than in the kinetic experiments as the long-term stabil-
ity (incubations exceeding 1 h) of LlChi18A (in the pres-
ence of bovine serum albumin) was better at a near-neutral
pH (at pH 6.0, there was no detectable loss of activity
under the conditions described below; A. C. Bunæs and
G. Vaaje-Kolstad, unpublished observations). Reaction
mixtures were incubated at 37 °C for up to 2 weeks with
vigorous shaking (a-chitin; 1300 r.p.m. in an Eppendorf
Thermomixer comfort; Eppendorf) or without shaking
(b-chitin). Initial experiments showed that the degradation
rate of a-chitin was slow when using static incubation
(results not shown); thus, to increase the amount of pro-
duct formed, and thereby the reliability of the assay, vig-
orous shaking was applied to promote the chitin–LlChi18A
and ⁄ or chitin–LlCBP33A contact. At time points ranging
from 2 to 340 h, 60 lL of the reaction was taken and
mixed with an equivalent amount of 70% acetonitrile in
an Eppendorf tube (the presence of acetonitrile arrests all
enzyme activity). All reactions were run in triplicate and
all samples were stored at )20 °C until product analysis by
HPLC as described above.
Acknowledgements
We thank Svein J. Horn for helpful discussions. This
work was funded by the Norwegian Research Council,
grants 171991 (GVK), 164653 (GVK), 159058 (GM)

and 140497 (ACB, VE).
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