The chitinolytic system of Lactococcus lactis ssp. lactis comprises a nonprocessive chitinase and a...

The chitinolytic system of Lactococcus lactis ssp. lactiscomprises a nonprocessive chitinase and a chitin-bindingprotein that promotes the degradation of a- and b-chitinGustav Vaaje-Kolstad, Anne C. Bunæs, Geir Mathiesen and Vincent G. H. Eijsink

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, As, 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 ⁄orminerals 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 As, 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. LlCBP33Aincreased 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-galactosideconcentrations 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-foldinsertion 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.


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).



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

A B

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).



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 (kcat) of 2.8 ± 0.2 s)1 and a Km 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 kcat and 10-fold lower Km 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

DC

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 kcat and Km

derived from the data are shown in the figure. (C) Time course of the degradation of (GlcNAc)3 (r) and (GlcNAc)4 ( ) by LlChi18A, 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 nM 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 lM, 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.



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)4predominantly 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 lL of

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.



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 LlCBP33A 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-chitinwith 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-chitindegradation 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

Glc

NA

c 2 (µ

M)

Glc

NA

c 2 (µ

M)

Glc

NA

c (µ

M)

60

40

20

00 50 100 150 200 250 300 350 400

Time (h)

0 50 100 150 200 250 300 350 400Time (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 LlChi18A ( ) 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.



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 �Cwithout 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-chitinisolated 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. A3392 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



https://www.researchgate.net/publication/8279670_Biological_Degradation_of_Soluble_Cellulose_Derivatives_and_its_Relationship_to_the_Mechanism_of_Cellulose_Hydrolysis?el=1_x_8&enrichId=rgreq-fd4e54d3-1f1e-453c-b4c6-4ecea13c1bb6&enrichSource=Y292ZXJQYWdlOzI0NzY2NjA4OTtBUzoxNzM1Mzc4MDEyODU2MzJAMTQxODM4NjA0NDYwNw==

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 (http://www.cbs.dtu.dk/services/

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

pCR�4Blunt-TOPO�Zero Blunt TOPO (Invitrogen),

excised from the TOPO vector using XhoI and BsaI, and

ligated to NdeI–XhoI-digested pETM11 vector (Gunter

Stier, EMBL Heidelberg, Germany). The pETM11 vector

contains a T7 promoter sequence for expression and an

N-terminal His6 tag for immobilized metal affinity chroma-

tography purification.

The putative CBP was cloned using the pET30 Xa ⁄LICkit (Merck Chemicals Ltd, Nottingham, UK), which

provides a ligation-independent method for cloning a gene

of interest. The expression vector (pET30 Xa ⁄LIC) providesan N-terminal His6 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 BigDye� Terminator v3.1 Cycle Sequencing Kit (Perkin-

Elmer ⁄Applied Biosystems, Foster City, CA, USA) and an

ABI PRISM� 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 (D600), 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 NaH2PO4, 10 mm 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 NaH2PO4 and

10 mm Tris ⁄HCl, pH 8.0 as running buffer, and 8 m urea,

0.1 m NaH2PO4, 10 mm 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 His6 tags was performed

by the addition of recombinant TEV protease (1 U per



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

CaCl2, 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 His6 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-chitinfrom 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 ddH2O to

yield a 20 mgÆmL)1 stock suspension. Binding was assayed

in 1 mL reactions in Eppendorf tubes containing

5 mgÆmL)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 Km 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 Na2CO3. 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



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)6degradation 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 lL of

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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