Endo ⁄exo mechanism and processivity of family 18chitinases produced by Serratia marcescensSvein J. Horn1, Audun Sørbotten2, Bjørnar Synstad1, Pawel Sikorski2,3, Morten Sørlie1,Kjell M. Varum2 and Vincent G. H. Eijsink1

1 Department of Chemistry, Biotechnology and Food Science, the Norwegian University of Life Sciences, As, Norway

2 NOBIPOL, Department of Biotechnology, the Norwegian University of Science and Technology, Trondheim, Norway

3 Biophysics and Medical Technology, Department of Physics, Trondheim, Norway

Chitin is a linear insoluble polymer of b-1,4 linked

N-acetylglucosamine (GlcNAc or A), which is synthes-

ized by crustaceans, molluscs, algae, insects, fungi and

yeasts. Second only to cellulose, chitin is an abundant

biopolymer with an annual production of 100 billon

tons [1]. In nature, two major types of chitin occur

which are characterized by an antiparallel (a-chitin) ora parallel (b-chitin) arrangement of the N-acetylgluco-

samine chains [2,3]. One industrial exploitation route

for chitin involves its conversion to chitosan, a water-

soluble copolymer of GlcNAc and d-glucosamine

(GlcN or D), which may be obtained by partial de-

N-acetylation of chitin, and for which several appli-

cations exist [4]. The term chitosan refers collectively

to water-soluble copolymers which are N-acetylated to

different extents. Chitosans prepared by homogeneous

de-N-acetylation of chitin have been found to have

a random distribution of A and D units [5–7]. The

Keywords

chitinase A; chitinase C; chitin degradation;

chitosan degradation; processivity

Correspondence

V.G.H. Eijsink, Department of Chemistry,

Biotechnology and Food Science, The

Norwegian University of Life Sciences,

PO Box 5040, 1432 As, Norway

Tel: +47 64965892

Fax: +47 64965901

E-mail: vincent.eijsink@umb.no

Website: http://www.umb.no

(Received 23 October 2005, accepted

29 November 2005)

doi:10.1111/j.1742-4658.2005.05079.x

We present a comparative study of ChiA, ChiB, and ChiC, the three family

18 chitinases produced by Serratia marcescens. All three enzymes eventu-

ally converted chitin to N-acetylglucosamine dimers (GlcNAc2) and a

minor fraction of monomers. ChiC differed from ChiA and ChiB in that it

initially produced longer oligosaccharides from chitin and had lower activ-

ity towards an oligomeric substrate, GlcNAc6. ChiA and ChiB could

convert GlcNAc6 directly to three dimers, whereas ChiC produced equal

amounts of tetramers and dimers, suggesting that the former two enzymes

can act processively. Further insight was obtained by studying degradation

of the soluble, partly deacetylated chitin-derivative chitosan. Because there

exist nonproductive binding modes for this substrate, it was possible to dis-

criminate between independent binding events and processive binding

events. In reactions with ChiA and ChiB the polymer disappeared very

slowly, while the initially produced oligomers almost exclusively had even-

numbered chain lengths in the 2–12 range. This demonstrates a processive

mode of action in which the substrate chain moves by two sugar units at a

time, regardless of whether complexes formed along the way are produc-

tive. In contrast, reactions with ChiC showed rapid disappearance of the

polymer and production of a continuum of odd- and even-numbered oligo-

mers. These results are discussed in the light of recent literature data on

directionality and synergistic effects of ChiA, ChiB and ChiC, leading to

the conclusion that ChiA and ChiB are processive chitinases that degrade

chitin chains in opposite directions, while ChiC is a nonprocessive endo-

chitinase.

Abbreviations

CBM, carbohydrate-binding module; DP, degree of polymerization; GlcNAc or A, N-acetylglucosamine; GlcN or D, D-glucosamine.

FEBS Journal 273 (2006) 491–503 ª 2006 The Authors Journal compilation ª 2006 FEBS 491

successive sugar units in the chitin ⁄ chitosan chain are

rotated 180� relative to each other. Thus, the func-

tional and structural unit in these polymers is a disac-

charide.

Despite its robust nature, its insolubility and its

abundant production, chitin does not accumulate in

most ecosystems, indicating that nature has developed

effective processes for chitin degradation. Bacteria cap-

able of degrading chitin usually produce a battery of

chitinases [8–10]. consisting of a catalytic domain and,

often, one or more smaller domains involved in sub-

strate binding [11–14]. While several individual chitin-

ases have been characterized in detail [15–18], little is

known about important issues such as exo- vs. endo-

action and processivity. This precludes full understand-

ing of nature’s chitinolytic machineries.

Serratia marcescens is one of the most intensively

studied chitinolytic bacteria. When grown on chitin,

S. marcescens produces three chitinases (ChiA, ChiB,

and ChiC), a chitin-binding protein (Cbp21) lacking

chitinase activity, and a hexosaminidase which further

degrades the major end product of the chitinases, Glc-

NAc2 [8,16,19–22]. The chitinase genes have been

cloned from several S. marcescens strains by several

research groups [17,20,23]. All three chitinases belong

to the family 18 of glycosyl hydrolases [24], which pos-

sess a (b ⁄a)8 barrel catalytic domain with approxi-

mately six sugar subsites [11,13,25–27]. Since chitin

hydrolysis by family 18 chitinases directly involves the

N-acetyl group of the sugar in the )1 subsite [28,29],

productive substrate binding requires an N-acetylgluco-

samine to be bound in this subsite. Other subsites

show less stringency in this respect, and may produc-

tively bind to, e.g. GlcN [30]. Indeed it has been shown

that ChiB can degrade chitosans, even those with low

degrees of acetylation [31].

The crystal structures of ChiA [11] and ChiB [13]

revealed deep substrate-binding clefts, in part due to

the presence of a 70–90-residue insertion in the cata-

lytic domain (‘a + b domain’ [11]), which is character-

istic for these chitinases but which is absent in other

chitinases (such as in hevamine and ChiC [25]; see

below). Both ChiA and ChiB contain an additional

substrate-binding domain which extents the substrate-

binding cleft on the side where the nonreducing end of

the substrate binds in ChiA [11] and on the side where

the reducing end of the substrate binds in ChiB [13].

In ChiA the deep substrate-binding cleft seems rather

accessible, whereas the substrate-binding cleft of ChiB

is more ‘tunnel-like’ [29,32]. On the basis of structural

characteristics [11,13] and enzymological work

[14,16,17,33] it has been suggested that ChiA and ChiB

are exochitinases which degrade chitin chains from

opposite ends, ChiA from the reducing end and ChiB

from the nonreducing end (see Fig. 1).

The structure of ChiC is not known, but its amino

acid sequence shows that it consists of a catalytic

domain and two putative chitin-binding domains,

which are located C-terminally in the sequence [20].

The catalytic domain of ChiC lacks the a + b domain

which makes up a wall in the substrate binding

grooves of ChiA and ChiB. This suggests that ChiC

has a much more open substrate-binding groove, as

observed in the crystal structure of the endochitinase

hevamine [25]. ChiC often occurs in two forms in

cultures of S. marcescens: the complete protein, some-

times called ChiC1, and a proteolytically truncated

variant, called ChiC2, which lacks the two putative

chitin-binding domains [20,34].

Some enzymatic properties of ChiA, ChiB and ChiC

have previously been studied and compared [16,17,33],

showing, among other things, that these chitinases may

act synergistically when present in certain combinations.

Nevertheless, there still is limited insight into important

properties of these enzymes (as concluded in recent

studies by Suzuki et al. [16] and Hult et al. [33]).

Remaining important issues concern the endo- or exo-

character of the enzymes [17,33] and the possible occur-

rence of multiple attack mechanisms (processivity)

[35,36]. Processive enzyme action has been studied

extensively for cellulases [37–39] and has been suggested

to occur in ChiA on the basis of the observation (by

microscopy) that ChiA sharpened one end of b-chitinmicrofibrils [14]. In the present study, we have

addressed these remaining issues, primarily by studies

of the degradation of the water-soluble GlcNAc ⁄GlcN

heteropolymer chitosan, which provide insight into the

exo- or endo- character of the enzymes as well as into

the occurrence of processivity. In addition, we have

compared the action of ChiA, ChiB and ChiC towards

two types of chitin and GlcNAc6.

ChiA

ChiB CBM5

Fn3-2 -1 +1 +2

-2-3 -1 +1 +2 +3

Fig. 1. Schematic drawing of subsites, chitin binding domains and

proposed orientation of polymer substrates in ChiA and ChiB. Fn3,

Fibronectin type 3 domain; CBM5, chitin binding module. Dotted

lines indicate that the polymer substrates are much longer than

shown in the figure. Reducing sugars are shown in grey.

The chitinolytic machinery of S. marcescens S. J. Horn et al.

492 FEBS Journal 273 (2006) 491–503 ª 2006 The Authors Journal compilation ª 2006 FEBS

Results

Degradation of hexamer (A6)

Figure 2 shows the initial time course of the degrada-

tion of A6 by ChiA, B and C. ChiA and ChiB degra-

ded the hexamer at approximately the same speed,

whereas ChiC reacted more slowly (approximately

threefold; Table 1). For all enzymes the end products

obtained upon prolonged incubation were A and A2

(results not shown; transglycosylation was never

observed). Since the monomer is not expected nor has

been observed to be produced directly from a tetramer,

pentamer or hexamer ([15–17]; Horn and Eijsink,

unpublished observations; Fig. 2) it must be derived

from trimers produced during the reaction. Thus, pro-

ductive binding of the hexamer to ChiA, ChiB or ChiC

has three possible outcomes: A2+A4, A3+A3, or, if

the enzymes operate processively, A2+A2+A2. The

degradation patterns of ChiA and ChiB were very sim-

ilar with A2 as the dominant product, whereas ChiC

produced similar amounts of A2 and A4. The fact that

A2 dominates very early in the reaction (i.e. when the

concentration of A6, is much higher than that of the

intermediate product A4), suggests that part of the

A6 substrate is degraded processively, producing

A2 + A2 + A2 as initial products.

The percentages of A6 that were converted to

A3 + A3 were calculated by dividing half the molar

amount of A3 by the decrease in the molar amount

of A6 concentration multiplied by 100. For ChiA and

ChiB, after 5 min, these percentages were 21.2% and

21.7%, respectively. For ChiC the percentage was

lower, amounting to 16.3% after 10 minutes. Thus,

all three enzymes have a preference for producing

dimers.

Degradation of chitin

As shown in Fig. 3A, degradation of b-chitin with

ChiA and ChiB initially yielded A2 and small amounts

of A and A3. ChiC also yielded A2 as the dominant

product, but the relative amount of A3 was larger than

for the other two chitinases. In addition, ChiC initially

also yielded small amounts of A and A4 and very

minor amounts of A5 (hardly detectable in Fig. 3A).

Fig. 2. Product formation during degradation

of GlcNAc6 with ChiA, ChiB and ChiC. The re-

action mixtures contained identical amounts

of substrate (100 lM) and enzyme (3.2 nM).

The concentrations of the various products in

the reaction mixtures were calculated from

HPLC chromatograms, as described in Exper-

imental procedures.s, dimers; h, tetramers;

d, hexamers;m, trimers; n, monomers.

Table 1. Degradation of chitin with different chitinases.

Enzyme

Final A2 ⁄A3

ratio(b-chitin)1Final A2 ⁄A3 ratio

(a-chitin)aInitial rate

(b-chitin)b (s)1)

Initial rate

(a-chitin)b (s)1)

Initial rate

[(GlcNAc)6] (s)1)

ChiA 7.3 5.9 0.52 0.24 46

ChiB 12.6 11.4 0.24 0.16 35

ChiC 4.1 3.0 0.38 0.16 14

aMolar A2 ⁄A3 ratio calculated from observed A and A2 concentrations as (A2-A) ⁄A; see text. bFormation of chitobiose in the initial linear

phase of degradation.

S. J. Horn et al. The chitinolytic machinery of S. marcescens

FEBS Journal 273 (2006) 491–503 ª 2006 The Authors Journal compilation ª 2006 FEBS 493

As the degradation proceeded some of the oligosaccha-

rides initially produced were degraded along with chi-

tin, and the end products of all three enzymes were A

and A2 (Fig. 3B).

Considering what is known about chitinases in

general and previous experimental observations for

Serratia chitinases [15–17], it is very unlikely that mo-

nomers are produced directly from chitin. Instead, they

are produced when A3 is degraded to A2 and A. Thus,

the molar amount of monomer seen after complete

degradation of the substrate (Fig. 3B) may be taken to

represent the cumulative molar amount of trimer

formed during the degradation reaction. The molar

amount of A2 directly produced from chitin (i.e. not

from degradation of A3) equals the observed molar

amount of A2 minus the observed molar amount of A.

The (A2 ) A) ⁄A ratio (¼ A2 ⁄A3 ratio) is interesting

because it can give information about substrate-bind-

ing modes and ⁄or processivity [37,40], as discussed fur-

ther below. Interestingly, the three enzymes show clear

differences in their A2 ⁄A3 ratios, ChiB producing the

highest ratio (Table 1).

Product formation curves for b-chitin degradation

(Fig. 4A) had a short linear part, which permitted

determination of initial degradation rates (Table 1).

After prolonged incubation, no remaining b-chitincould be observed in the reaction mixtures of ChiA

and ChiC. However, in the case of ChiB, which is only

about twofold less effective than the other two

enzymes in terms of initial rate (Table 1), complete

conversion of the substrate was never obtained, not

even after several weeks of incubation with repeated

addition of excessive amounts of enzyme.

Degradation of a-chitin yielded similar mixtures of

chito-oligosaccharides, both initially and at the end of

the reaction, and yielded similar A2 ⁄A3 ratios as degra-

dation of b-chitin (Table 1). Degradation of a-chitinoccurred with slightly lower initial rates than degrada-

tion of b-chitin and none of the enzymes was capable

of completely degrading the substrate. The fraction of

the substrate degraded by ChiA was approximately

three times higher than the fraction converted by ChiB

or ChiC (Fig. 4B).

Fig. 3. HPLC analysis of the degradation of b-chitin by ChiA, ChiB

and ChiC. The reaction mixtures contained 360 nM of enzyme and

1 mgÆmL)1 b-chitin. (A) Initial products after 10 min. (B) Final prod-

ucts after 48 h; only monomers and dimers are observed. Note that

each oligosaccharide produces two peaks, representing the a ano-

mer and the b anomer; the a anomer elutes first.

Fig. 4. Production of chitobiose during de-

gradation of b-chitin (A) or a-chitin (B) by

ChiA, B and C. The reaction mixtures con-

tained 50 nM (A) or 360 nM (B) of ChiA

(squares), ChiB (circles) or ChiC (triangles)

and 0.1 mgÆmL)1 b-chitin (A) or 1 mgÆmL)1

a-chitin (B). The level of chitobiose obtained

with ChiB on b-chitin after 215 h (A) is about

10% lower than the maximum level

obtained after several weeks of incubation.

The chitinolytic machinery of S. marcescens S. J. Horn et al.

494 FEBS Journal 273 (2006) 491–503 ª 2006 The Authors Journal compilation ª 2006 FEBS

Degradation of chitosan

Reactions with chitosan were run for periods between

15 min and 1 week, as described in Experimental pro-

cedures. To verify the occurrence of enzyme depletion

and ⁄or product inhibition, reaction mixtures that had

been incubated for several days and in which the reac-

tion had proceeded to a supposedly final stage, were

supplied with either new enzyme or new substrate,

incubated, and analysed for product formation. These

control reactions showed, for all three enzymes, that

neither enzyme depletion nor product inhibition

occurred under the conditions used in this study (data

not shown).

The size distributions of oligomers obtained at var-

ious stages of degradation of a highly acetylated, high

molecular weight and water-soluble chitosan (FA ¼0.65; Mn ¼ 160 000) are shown in Fig. 5 (ChiA),

Fig. 6 (ChiB) and Fig. 7 (ChiC). The results for ChiB

have been described and discussed previously [31]. The

stage of the reaction is characterized by the a value,

which is the fraction of hydrolysed glycosidic bonds

(a would be 0.5 if a long polymer were to be converted

to dimers only).

Figures 5–7 reveal clear differences between ChiC

and the two other enzymes. The degradation by

ChiC resulted in the disappearance of the void peak

already at an a-value as low as 0.05, while the pro-

duced oligomers had lengths spanning the complete

detectable range (2 to approximately 40; Fig. 7). This

clearly indicates endo-activity. In contrast, in the

reactions with ChiA and ChiB, the void peak did

not disappear completely until the a-value was

higher than 0.20 (Figs 5 and 6). This would be

expected for enzymes that act in an exo-fashion

and ⁄or have a highly processive mode of action (see

below). Most importantly, the initial degradation

products obtained with ChiA and ChiB consisted

almost exclusively of an even number of sugar units.

This prooves that ChiA and ChiB act processively,

as discussed below. Later in the degradation process,

concomitantly with the depletion of chitosan, the

longer oligomers initially released during processive

action (see below) were degraded further, resulting in

the production of odd-numbered oligomers. The final

product mixtures obtained with ChiA and ChiB con-

tained a continuum of odd- and even-numbered

oligomers (see a¼ 0.35 and 0.38 in Figs 5 and 6,

respectively), with the AA dimer as dominant prod-

uct.

Comparison of Figs 5 and 6 reveals several differ-

ences between ChiA and ChiB. For example, in the

initial phase of the degradation reaction (up to a¼

0.20) the oligomer fractions obtained with ChiB

became larger with decreasing oligomer length

(Fig. 6). Degradation with ChiA yielded a slightly

different pattern: initially, the hexamer and octamer

peaks had similar sizes, while the tetramer peak was

Fig. 5. Size-distribution of oligomers during the degradation of

chitosan (FA ¼ 0.65) by ChiA. The peaks are marked by numbers

which indicate the lengths (DP) of the oligomers they contain, or, in

the case of peaks containing only one known compound, by the

sequence of the oligomer. The annotation of the peaks is based on

the use of standard samples, as well as NMR analyses (see Experi-

mental procedures and [31]). The a-values denote the degree of

scission; the lower panel represents the maximum obtainable

a-value. Undegraded chitosan elutes in the void volume of the

column, as do all chitosan fragments with a DP > 40. See Experi-

mental procedures section for more details.

S. J. Horn et al. The chitinolytic machinery of S. marcescens

FEBS Journal 273 (2006) 491–503 ª 2006 The Authors Journal compilation ª 2006 FEBS 495

much smaller. Also, ChiA produced less odd-num-

bered oligomers than ChiB in the initial phase of the

reaction.

Using NMR, the composition and (partial)

sequences of the oligomers present in the dimer to

tetramer fractions obtained with the various enzymes

were determined (Table 2; see Experimental proce-

dures and [31] for a description of the methodology).

The results show that in all cases the reducing ends

of the oligomers consisted exclusively of A units, as

expected for family 18 chitinases. In the case of

ChiC, the sugar preceding the reducing end was

almost exclusively an A, but a small amount DA

dimers was formed towards the end of the reaction.

In the case of ChiA and ChiB, D was observed more

frequently, approaching 35% (i.e. as in the substrate)

in the dimer fraction towards the end of the reaction.

Thus, ChiA and ChiB have a preference for A in

their )2 subsites, but this preference is less strong

than in the case of ChiC. Principally, all three

enzymes are capable of cleaving after –DA– if –AA–

containing substrates become depleted. In the initial

phases of the reactions, newly formed nonreducing

ends had an A ⁄D ratio of 65 ⁄ 35 (determined by car-

bon NMR, data not shown), which is the same ratio

as in the substrate. Thus, none of the enzymes have

preferences for A or D in the +1 subsite that are

strong enough to be noticeable in these experiments.

Fig. 6. Size distribution of oligomers during the degradation of

chitosan (FA ¼ 0.65) by ChiB. See legend to Fig. 4 for details.

Fig. 7. Size distribution of oligomers during the degradation of

chitosan (FA ¼ 0.65) by ChiC. See legend to Fig. 4 for details.

The chitinolytic machinery of S. marcescens S. J. Horn et al.

496 FEBS Journal 273 (2006) 491–503 ª 2006 The Authors Journal compilation ª 2006 FEBS

Discussion

In the present study, we used natural substrates and

chitosan to characterize the family 18 chitinases pro-

duced by S. marcescens. Studies with insoluble, resili-

ent polymer substrates such as chitin (or cellulose) are

intrinsically difficult because it is hard to analyse the

substrate fraction and because intermediately formed

soluble oligomers are much better substrates than the

insoluble polymer. In the case of chitinases, these

problems can be partly avoided by studying the degra-

dation of chitosan.

Generally, enzymatic degradation of polysaccharides

occurs from one of the chain ends (exo-mechanism) or

from a random point along the polymer chain (endo-

mechanism). Each of these two mechanisms can occur

in combination with a processive mode of action,

meaning that the substrate is not released after success-

ful cleavage but slides through the active site for the

next cleavage event to occur. Processivity reduces the

search space for enzymes from 3D to 1D and is

thought to be especially important when degrading

insoluble substrates [32,41,42]. For example, processive

cellobiohydrolases have deep, ‘tunnel-like’ substrate-

binding clefts that are thought to embrace and proces-

sively hydrolyse a single polymer chain detached from

the insoluble substrate [32,41,42]. It has been suggested

that aromatic residues lining these substrate-binding

clefts are important for the ‘sliding’ of the substrate

through the cleft [41,43]. Like cellobiohydrolases, ChiA

and ChiB have deep substrate-binding clefts lined with

aromatic residues [11,13], suggesting that the two

enzymes act processively. The experimental analysis of

processivity is not straightforward, as discussed below

and, e.g. in [44]. One approach is to study the shape of

the substrate during enzymatic degradation by micros-

copy [14,33,38,45]; here, sharpening of the fibril tips is

considered a sign of processive exo-action.

Because of the 180� rotation between consecutive

sugar units, processive action on chitin will yield

dimers, while trimers can only be produced by an

exo-chitinase in the first hydrolytic step. Therefore,

processivity may be assessed by studying the A2 ⁄A3

ratios in product mixtures [37,40]. One enzyme–sub-

strate association event followed by processive degra-

dation has two potential outcomes, depending on

whether the initial product is a dimer or trimer: (1)

production of X A2 or (2) production of 1 A3 and

[X)1] A2, respectively. If these events have equal fre-

quencies, the A2 ⁄A3 ratio will be X + [X)1] ¼ 2X)1,giving X-values of 4.2, 6.8 and 2.6 for ChiA, ChiB and

ChiC, respectively (derived from data in Table 1).

However, it must be emphasized that A2 ⁄A3 ratios

may also simply reflect different preferences for two

initial binding modes, which release dimers or trimers,

respectively. The experiments with A6 indicated that

ChiA and ChiB have a preference for initial cleavage

of a dimer. If this same preference would apply to the

situation in which a polymer is degraded, the actual

degree of processivity would be lower than what is

suggested by the A2 ⁄A3 ratios in Table 1. In other

words: a nonprocessive exo-enzyme with a strong pref-

erence for cleaving off dimers would also yield high

A2 ⁄A3 ratios. Even a nonprocessive endo-enzyme

could in principle yield high A2 ⁄A3 ratios if it would

have a strong preference for releasing dimers from

oligosaccharide intermediates. The latter seems to

apply to ChiC: this enzyme produces a majority of di-

mers in chitin degradation experiments, while the

chitosan experiments clearly show that this enzyme is

not processive (see below); the experiments with A6

show that, indeed, ChiC preferably cleaves off dimers

from short substrates. In conclusion, while the results

obtained with natural substrates do indicate processivi-

ty, they do not lead to unequivocal conclusions.

Chitosan is an interesting substrate for gaining more

insight into chitinase action, because the soluble sub-

strate is easier to analyse than chitin. In addition, and

most importantly, chitinases can bind nonproductively

to chitosan (e.g. complexes that place a deacetylated

sugar in the )1 subsite), which allows discrimination

between independent binding events and processive

binding events, as explained below.

Product mixtures obtained very early in reactions of

the putative exoenzymes ChiA and ChiB with chitosan

contained significant amounts of longer oligosaccha-

rides with predominantly even-numbered chain lengths

Table 2. Composition of dimer, trimer and tetramer fractions at

different a during degradation of chitosan (FA ¼ 0.65) by ChiA, B

and C.

Enzyme a Dimer Trimer Tetramer

ChiA 0.15 81% AA 81% DAA 100% -AA

19% DA 19% ADA

0.35 64% AA 51% DAA 56% -AA

36% DA 28% ADA

21% DDA

44% -DA

ChiB 0.11 86% AA 71% DDA 100% -AA

14% DA 29% AAA

0.38 66% AA 95% DAA 75% -AA

34% DA 3% DDA

2% ADA

25% -DA

ChiC 0.20 100% AA 66% DAA

34% AAA

100% -AA

0.38 81% AA 100% DAA 100% -AA

19% DA

S. J. Horn et al. The chitinolytic machinery of S. marcescens

FEBS Journal 273 (2006) 491–503 ª 2006 The Authors Journal compilation ª 2006 FEBS 497

(note that only dimers and trimers are observed during

degradation of chitin). Thus, both ChiA and ChiB are

capable of productive binding events in which parts of

the substrate extend from both sides of the active site

cleft. For ChiB this is somewhat unexpected because

association with substrate was originally thought to be

sterically blocked in front of the )3 subsite [13]. Struc-

tural inspections and modelling studies (data not

shown) using the available structure of a complex of

ChiA with A8 [46] superimposed on the ChiB structure

indicate though that the sterical barrier in ChiB is not

very strong and it is well conceivable that the chitosan

chain can bend to the extent that it’s association with

the enzyme is not hampered by this putative barrier.

The observation that ChiA and ChiB almost exclu-

sively produce even-numbered oligomers in the initial

phase of the reaction with chitosan (a substrate having

a random distribution of A and D units [5–7]), is of cru-

cial importance since it provides unequivocal evidence

for processivity. If upon substrate binding the sugar in

subsite )1 is GlcNAc (A), ChiA and ChiB are in princi-

ple capable of hydrolysis and will cleave off an odd- or

en even-numbered oligomer (for an exo-enzyme this

would be a dimer or a trimer). However, if the )1 sub-

site contains a deacetylated unit (D), the enzyme cannot

hydrolyse the substrate. If the enzyme would release its

substrate after each productive or nonproductive bind-

ing event, the ratio between odd- and even-numbered

longer oligomers would be close to 1 : 1, since the puta-

tive product sites consist of only two (ChiA, +1 and

+2) or three (ChiB, )3 to )1) subsites (Fig. 1). Thus,

the enzymes cannot discriminate between, e.g. a hep-

tamer and an octamer in their product sites. If, how-

ever, the enzymes act processively, productive or

nonproductive initial binding events would be followed

by sliding of the substrate through the active site cleft

by two sugar units at the time, until a new productive

complex emerges and hydrolysis occurs. In such a mech-

anism the first product will be odd- or even-numbered,

whereas all other products resulting from the same

enzyme–substrate association event would be even-

numbered. In the case of chitin, all these even-numbered

products are dimers, whereas in the case of chitosan,

these products are longer because part of the complexes

formed during the processive movement are nonproduc-

tive. This also explains the somewhat counterintuitive

observation that the putative exoenzymes ChiA and

ChiB produce longer oligomers such as octa- and deca-

mers in the very beginning of the reaction. In these olig-

omers, the sequence of A and D units apparently is

such that productive complexes were only formed after,

e.g. four (octamer) or five (decamer) processive ‘moves’

through the active site cleft. Most of these longer oligo-

mers will still be cleavable (after rebinding) but only

through binding modes that have not previously been

explored during processive movement, i.e. binding

modes that cleave the even-numbered oligomers into

two odd-numbered products. This is exactly what is

observed in the later phases of the reactions. Interest-

ingly, the observation of longer even-numbered oligo-

mers in the beginning of the reaction implies that the

chitinases traverse stretches of unreactive polymer while

moving from one productive complex to the other, a

process sometimes referred to as ‘sliding’ or facilitated

diffusion [36,47].

The A2 ⁄A3 ratios of Table 1 indicate that ChiB has

the highest processivity when degrading the most nat-

ural substrate tested, chitin. In contrast, ChiA shows

the strongest predominance of even-numbered oligo-

mers in the reaction with chitosan (compare Figs 5

and 6), suggesting that this enzyme is the most proces-

sive one. It is possible that the suggested sterical hin-

drance beyond the )3 subsite in ChiB [13] limits

processivity in the case of a chitosan substrate, since

processive degradation of this substrate requires that

complexes are formed in which more than two or three

sugars are bound on the glycon side of the catalytic

centre. Since the substrate-binding cleft in ChiB is

more ‘tunnel’-like than in ChiA [13] one would a priori

expect that ChiB is the more processive enzyme

[32,39,41,48].

The degradation of b-chitin with ChiC revealed the

presence of longer oligomer products (A4 and A5) in

the initial phase of the reaction (such products have

not been detected previously; see [16]). ChiC showed

low activity towards A6 and no direct conversion of

A6 to three A2 molecules. Degradation of b-chitinresulted in a low A2 ⁄A3 ratios (Table 1). Taken

together, these observations suggest that ChiC is a

nonprocessive endo-acting enzyme. The most compel-

ling evidence for the ChiC reaction mechanism comes

from the studies with chitosan. Figure 6 shows that

ChiC converts chitosan to a continuum of oligomers

of different sizes and that the polymer peak disappears

early in the degradation reaction. Also, there is initially

no accumulation of dimers or other even numbered

oligomers.

The slow disappearance of the void peak in the reac-

tions of ChiA and ChiB with chitosan seems to con-

firm previous suggestions that these two chitinases act

in an exo-fashion. It should be noted though, that the

chromatographic analyses of product patterns shown

in Figs 5 and 6 cannot discriminate between processive

exoenzymes and highly processive enzymes that ini-

tially attack the substrate in an endo-fashion. The lat-

ter type of enzyme would only produce even numbered

The chitinolytic machinery of S. marcescens S. J. Horn et al.

498 FEBS Journal 273 (2006) 491–503 ª 2006 The Authors Journal compilation ª 2006 FEBS

oligomers directly from chitosan, but could produce

odd-numbered oligomers (which are observed, see

Figs 5 and 6) by reprocessing even numbered inter-

mediate oligomeric products. It is known that some

processive cellobiohydrolases occasionally bind the

substrate in an endo-fashion, showing that the loops

that form the ‘roof’ of the substrate-binding cleft may

open occasionally [49–51]. Since the ‘roofs’ of ChiA

and ChiB are rather open [11,13], it is conceivable that

these enzymes also show occasional endo-binding [48].

Hult et al. [33] have recently used microscopy to

study the degradation of b-chitin fibrils by ChiA and

ChiB, and their results support the idea that these two

chitinases are exo-acting enzymes, at least when hydro-

lysing insoluble chitin. Interestingly, by using micros-

copy to study the degradation of an end-labelled

substrate, these authors were capable of showing that

ChiA and ChiB degrade the chitin chains from the

reducing and nonreducing ends, respectively, as previ-

ously suggested on the basis of the enzyme crystal

structures [13].

While an A bound to subsite )1 is an absolute

requirement for hydrolysis to occur, all three family 18

chitinases also showed a preference for an A in the )2subsite (seen as a preference for producing oligomers

with an AA at the reducing end; Table 2). This prefer-

ence was strongest for ChiC where DA only appeared

in the form of a dimer at very high a. We could not

detect any A ⁄D preference in the +1 subsites of the

three enzymes.

Synergetic effects of the three S. marcescens chitinas-

es have been shown with colloidal chitin [17], a-chitin[16] and b-chitin [33,52]. Taken together, the present

and previous studies [16,33] show that the three

chitinases have different and complementary activities

(endo- vs. exo-) and directionalities, which can explain

synergism. However, several observations remain unex-

plained, for example the observation that the exo-

enzyme ChiB and the endoenzyme ChiC show little,

if any, synergy [16,52]. The recent finding that the chi-

tin-binding protein (Cbp21) produced by S. marcescens

potentiates chitinase action by disrupting the structure

of the b-chitin substrate [52] points to another possible

explanation for synergistic effects. The three S. marces-

cens chitinases have different chitin-binding domains

(also called carbohydrate-binding modules, CBMs):

ChiA contains a fibronectin type III (FnIII)-like CBM;

ChiB contains a family 5 CBM and ChiC contains a

family 12 and an FnIII-like CBM (see http://afmb.cnrs-

mrs.fr/CAZY/for family classification). It is conceivable

that the primary role of these CBMs is to potentiate

catalytic activity by disrupting the substrate, rather

than simply to promote enzyme–substrate binding.

Synergistic effects could thus be due to one enzyme

increasing substrate accessibility for other enzymes by

hitherto unknown disruptive mechanisms involving the

CBMs. A similar role has occasionally been proposed

for cellulase-binding domains in cellulases [53,54].

Interestingly, Watanabe et al. have shown that deletion

of the two Fn III domains of ChiA1 from Bacillus cir-

culans did not affect chitin-binding, but strongly

reduced chitin hydrolysing activity [12]. Differences in

the ability to disrupt the substrate could also explain

why the three chitinases have different abilities to fully

convert a- and b-chitin (Fig. 4, see above), while dis-

playing rather similar initial degradation rates (Table 1;

these rates are likely to reflect degradation of amor-

phous, easily accessible regions in the substrate).

In conclusion, the chitinolytic machinery of S. mar-

cescens consists of two processive exo-enzymes with

different directionalities on chitin, ChiA and ChiB,

and a nonprocessive endo enzyme, ChiC. Thus, when

Serratia applies its battery of chitinases to degrade

chitin, ChiC is likely to supply the exo-enzymes with

new reducing and nonreducing ends, which are sub-

strates for ChiA and ChiB, respectively. The disrup-

tive effect of Cbp21 [52], and presumably of the

noncatalytic domains of the chitinases, makes the

crystalline regions of chitin more accessible for hydro-

lysis.

Experimental procedures

Chemicals

Squid pen b-chitin (3 lm in size) was from Seikagaku

(Tokyo, Japan; product number 400627; average molecular

weight 2 · 105 Da). Chitosan, with a degree of N-acetyla-

tion of 65% (FA ¼ 0.65), was prepared by homogeneous

N-deacetylation of milled (1.0 mm sieve) shrimp shell chitin

[55]. This procedure results in a chitosan with a random

sequence of acetylated and de-acetylated units [31]. Chito-

oligosaccharides, a-chitin (product number C-3641) and all

other chemicals were purchased from Sigma (St Louis,

MO, USA).

Enzymes

The chitinase genes chia, chib from S. marcescens strain

BJL200 were expressed in Escherichia coli DH5a (Life

Technologies, Rockville, MD, USA) under control of their

own promoters [17]. ChiA and ChiB were purified from

periplasmatic extracts of early stationary phase cultures,

essentially as described previously [13,17]. The extracts, in

0.65 mm MgCl2, 0.1 mm phenlymethylsulphonyl fluoride,

1 mm EDTA, were diluted 1.4-fold and adjusted to 20 mm

S. J. Horn et al. The chitinolytic machinery of S. marcescens

FEBS Journal 273 (2006) 491–503 ª 2006 The Authors Journal compilation ª 2006 FEBS 499

Tris ⁄HCl pH 8.0 and 0.4 m ammonium sulphate. Two mil-

lilitres of this dilution was loaded onto a phenyl-sepharose

HR 5 ⁄ 5 column (5 · 50 mm) in an FPLC system, equili-

brated in a buffer (20 mm Tris ⁄HCl pH 8.0, 1 mm EDTA,

0.1 mm phenlymethylsulphonyl fluoride) containing 0.4 m

ammonium sulphate. After loading the sample, the column

was washed with the starting buffer followed by a 5 mL lin-

ear gradient of 0.4–0 m ammonium sulphate. Subsequently,

a linear gradient of 0–6% (v ⁄ v) 2-propanol was applied to

elute the enzyme.

The chic gene (EMBL database, accession no. AJ630582)

was amplified from S. marcescens strain BL200 chromoso-

mal DNA and cloned into the T7 promoter expression vec-

tor pRSETB (Invitrogen), using primer 5¢-CGGGAA

TTCCATATGAGCACACAAATAACAC-3¢ (NdeIChiC)

to create an NdeI site for translational fusion, and a primer

located downstream of the putative terminator in the chic

gene [20]. The resulting plasmid (pRSETB-chic) was trans-

formed into the E. coli strain BL21 (DE3) star (Invitrogen).

For production of ChiC in E. coli BL21 (DE3) expression

of chic was induced by 0.4 mm isopropyl-b-D-thiogalacto-

side when the OD600 was between 0.5 and 0.7. After growth

in 150 min the cultures were harvested. ChiC was purified

using the same protocol as described above for ChiA and

ChiB[17; B. Synstad and V.G.H. Eijsink, unpublished

results].

Enzyme purity was verified by SDS ⁄PAGE and estimated

to be >95% in all cases (see [17] for an example). Protein

concentrations were determined using the Bio-Rad Protein

Assay (Bio-Rad Laboratories, Hercules, CA, USA), with

BSA as standard.

Enzymatic degradation of chitin, GlcNAc6 and

chitosan

For qualitative analysis of product formation, 1 mgÆmL)1

a- or b-chitin was hydrolysed at 37 �C in 50 mm sodium

acetate buffer (pH 6.1) with 360 nm of ChiA, B or C. Reac-

tions were stopped by adding 3 lL m HCl to 100 lL sam-

ples. The same conditions were used to estimate intitial

rates for a-chitin degradation. Quantitative analysis of

b-chitin degradation was conducted as described in [53].

Initial rates were estimated using the first linear part of the

product formation curve.

Hydrolysis of GlcNAc6 was carried out in 50 mm sodium

acetate with 50 lgÆmL)1 BSA at pH 6.1 and 37 �C. The

enzyme concentration was 3.2 nm for all three enzymes. All

reactions were stopped by adding 3 lL 2 m HCl to 100 lLsamples.

To analyse degradation of chitosan, 10 mg of the poly-

mer (FA ¼ 0.65), were dissolved in 1.0 mL H2O. After add-

ing 1.0 mL buffer (0.08 m NaAc, 0.2 m NaCl, pH 5.5) and

0.2 mg BSA, the samples were immersed in a shaking water

bath at 37.0 �C. The reactions were started by adding the

enzyme (5 lg ChiA or ChiB, or 3 lg ChiC) and the reac-

tions were allowed to proceed for 15 min to 1 week. Sam-

ples were taken at regular time intervals and reactions were

stopped by lowering the pH to 2.5 by addition of 1.0 m

HCl, and immersing the samples in boiling water for 2 min.

Chromatography of oligosaccharides

Mixtures of chito-oligosaccharides were analysed by HPLC

using a Tosoh TSK Amide 80 column (0.46 · 25 cm)

with an Amide 80 guard-column (Tosoh, Tokyo, Japan).

A 10-lL sample was injected on the column and the chitin

fragments were eluted isocratically at 0.7 mLÆmin)1 with

70% acetonitrile at room temperature. The chito-oligosac-

charides were monitored by measuring absorbance at

210 nm and the amounts were quantified by measuring

peak areas. Peak areas were compared to peak areas

obtained with standard samples with known concentrations

of chito-oligosaccharides. Using these standard samples, it

was established that there was a linear correlation between

peak area and oligosaccharide concentration within the

concentration range used in this study, for each of the

oligomers that were analysed.

Oligomers produced by enzymatic depolymerization of

chitosan were separated on three XK 26 columns, packed

with SuperdexTM 30, from Pharmacia Biotech (Uppsala,

Sweden), with an overall dimension of 2.60 · 180 cm. The

mobile phase was 0.15 m ammonium acetate, pH 4.50 and

the flow rate was 0.80 mLÆmin)1. The relative amounts of

oligomers were monitored with an online refractive index

detector (Shimadzu RID 6 A), and the data were logged with

a CR 510 Basic Data logger, from Campbell Scientific Inc

(Logan, UH, USA). Fractions of 3.2 mL were collected and,

where appropriate, pooled for analysis of the oligomers. This

method and its performance have been described in detail

previously [31]. It has been shown that this method allows the

separation of mixtures of partially N-acetylated oligomers

according to size (degree of polymerization, DPn), regardless

of chemical composition, in the separation range between

DP ¼ 4 and a DP of approximately 20. Within the mono-

mer–trimer range, some sequence specific separation was

observed, as indicated in the figures presented. Studies with

standard samples have shown that there is a linear relation-

ship between peak areas and the amount (mass) of injected

oligomer, irrespective of DP and degree of acetylation [31].

1H-NMR Spectroscopy

Proton NMR was used to partially sequence shorter oligo-

mers and to calculate the DPn in the reaction mixtures as

described previously [31]. The chitosan degradation is given

as the degree of scission, a (¼ 1 ⁄DPn, where DPn is the

number-average of the degree of polymerization), which

represents the fraction of glycosidic linkages that has been

cleaved. Complete conversion of the polymer to dimers

would yield an a of 0.50.

The chitinolytic machinery of S. marcescens S. J. Horn et al.

500 FEBS Journal 273 (2006) 491–503 ª 2006 The Authors Journal compilation ª 2006 FEBS

Acknowledgements

This work was supported by grants from the Nor-

wegian Research Council (140497 ⁄ 420 and 134674 ⁄110). We thank Xiaohong Jia for technical assistance.

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