Báo cáo khoa học: Degradation of chitosans with chitinase B from Serratia marcescens Production of chito-oligosaccharides and insight into enzyme processivity docx

Degradation of chitosans with chitinase B from
Serratia marcescens
Production of chito-oligosaccharides and insight into enzyme
processivity
Audun Sørbotten
1
, Svein J. Horn
2
, Vincent G. H. Eijsink
2
and Kjell M. Va
˚
rum
1
1 Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology,
Trondheim, Norway
2 Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, A
˚
s, Norway
Chitosans are linear, cationic polysaccharides composed
of (1 fi 4)-linked units of 2-amino-2-deoxy-b-d-gluco-
pyranose (GlcN, D-unit) which may be N-acetylated to
varying extents. Chitosans are soluble in acidic solution,
whereas chitin, a structural polysaccharide occurring
mainly in the exo-skeleton of arthropods, is insoluble
[1]. Chitin is composed of (1 fi 4)-linked units of 2-ace-
tamido-2-deoxy-b-d-glucopyranose (GlcNAc, A-unit).
Chitin shares many similarities with cellulose, e.g. the
conformation of the monomers, di-equatorial glycosidic
Keywords
chitinase B; chitosan degradation;

oligosaccharides; processive enzymes;
subsite analysis
Correspondence
K. M. Va
˚
rum, Norwegian Biopolymer
Laboratory (NOBIPOL), Department of
Biotechnology, Norwegian University of
Science and Technology, 7491 Trondheim,
Norway
Fax: +47 73591283
Tel: +47 73593324
E-mail:
(Received 12 October 2004, accepted 18
November 2004)
doi:10.1111/j.1742-4658.2004.04495.x
Family 18 chitinases such as chitinase B (ChiB) from Serratia marcescens
catalyze glycoside hydrolysis via a mechanism involving the N-acetyl group
of the sugar bound to the )1 subsite. We have studied the degradation of
the soluble heteropolymer chitosan, to obtain further insight into catalysis
in ChiB and to experimentally assess the proposed processive action of this
enzyme. Degradation of chitosans with varying degrees of acetylation was
monitored by following the size-distribution of oligomers, and oligomers
were isolated and partly sequenced using
1
H-NMR spectroscopy. Degrada-
tion of a chitosan with 65% acetylated units showed that ChiB is an exo-
enzyme which degrades the polymer chains from their nonreducing ends.
The degradation showed biphasic kinetics: the faster phase is dominated by
cleavage on the reducing side of two acetylated units (occupying subsites

)2 and )1), while the slower kinetic phase reflects cleavage on the reducing
side of a deacetylated and an acetylated unit (bound to subsites )2 and )1,
respectively). The enzyme did not show preferences with respect to acetyla-
tion of the sugar bound in the +1 subsite. Thus, the preference for an
acetylated unit is absolute in the )1 subsite, whereas substrate specificity is
less stringent in the )2 and +1 subsites. Consequently, even chitosans with
low degrees of acetylation could be degraded by ChiB, permitting the pro-
duction of mixtures of oligosaccharides with different size distributions and
chemical composition. Initially, the degradation of the 65% acetylated
chitosan almost exclusively yielded oligomers with even-numbered chain
lengths. This provides experimental evidence for a processive mode of
action, moving the sugar chain two residues at a time. The results show
that nonproductive binding events are not necessarily followed by substrate
release but rather by consecutive relocations of the sugar chain.
Abbreviations
ChiB, chitinase B; DP, degree of polymerization; D-units, GlcN-units; A-units, GlcNAc-units; a, degree of scission; F
A
, fraction of acetylated
units.
538 FEBS Journal 272 (2005) 538–549 ª 2005 FEBS
linkages, crystalline structure, and lack of solubility in
aqueous solvents.
The detailed chemical composition of chitosans will
vary, depending on the fraction of acetylated units
(F
A
) and their synthesis or preparation. This variation
affects many properties of chitosan, such as solubility
as a function of pH [2], binding to lysozyme [3], sus-
ceptibility to degradation by lysozyme [4,5], as well as

functional properties in drug delivery [6] and gene
delivery systems [7]. Previous studies have shown that
chitosans obtained by homogeneous de-N-acetylation
of chitin, such as those used in the present study, have
a random distribution of N-acetyl groups [8–10].
Chitinases and chitosanases are capable of convert-
ing chitin and chitosans to low molecular mass prod-
ucts (oligosaccharides) by hydrolyzing the b(1 fi 4)
glycosidic linkages between the sugar units. There are
several families of chitinases (glycoside hydrolase fam-
ilies 18 and 19) and chitosanases (glycoside hydrolase
families 46, 75 and 80) [11]. These enzymes differ with
respect to their preference for A- and D-units on each
side of the scissile glycosidic bond and, consequently,
differ with respect to their activities towards different
types of chitosan. Like many other chitinolytic bac-
teria, the Gram-negative soil bacterium Serratia mar-
cescens produces multiple chitinases (ChiA, ChiB,
ChiC1 and ChiC2) [12–14]. These enzymes all belong
to family 18 chitinases, which have a characteristic cat-
alytic mechanism which depends on participation of
the N-acetyl group of the sugar unit bound to the )1
subsite [15–19]. This substrate-assisted catalytic mech-
anism implies that family 18 chitinases are expected to
have an absolute preference for A-units in the )1 sub-
site and that the presentation of a deacetylated sugar
(D-unit) to the )1 subsite would represent nonproduc-
tive binding.
Chitinase B (ChiB; EC 3.2.1.14) from S. marcescens
is a two-domain family 18 chitinase [20]. The active site

of ChiB has defined subsites from )3 to +3 [16,20], and
the substrate-binding cleft is tunnel-like, permitting tight
interaction with the polymeric substrate. The aglycon-
side of the substrate-binding groove is extended by the
aromatic surface of a putative chitin-binding domain.
The crystal structure of ChiB further suggests that the
active site cleft is partially blocked at the )3 subsite [20].
Together with studies showing that ChiB converts chitin
primarily to dimers [14,21], the structure of ChiB sug-
gests that the enzyme exerts exo-activity and degrades
chitin chains from their nonreducing ends [16,20].
It is generally accepted that enzymes that degrade
carbohydrate polymers can do so by three principally
different mechanisms, as first described by Robyt and
French [22,23]: (a) A multiple-chain mechanism, where
the enzyme-substrate complex dissociates after each
reaction. (b) A single-chain mechanism, where the
enzyme remains associated with the substrate until
every cleavable linkage in the chain has been hydro-
lyzed. (c) A multiple attack mechanism, where a given
average number of attacks are performed after the
enzyme-substrate complex has been formed. The latter
two mechanisms are often referred to as ‘processive’.
Carbohydrate degrading enzymes with tunnel-like act-
ive site clefts are often suggested to act processively
[24]. For example, cellobiohydrolases are thought to
degrade cellulose according to a multiple attack mech-
anism, where dimers are cleaved off from the end of
the polysaccharide chain [24–27]. These cellulose-
degrading enzymes convert cellulose to cellobiose,

along with small amounts of monomers and trimers,
which are thought to be the result of the initial bind-
ing ⁄ cleaving event. Whereas plausible models exist for
cellulase action, mainly based on the results of site-
directed mutagenesis and structural studies, less is
known about processivity in chitinases. The only avail-
able information comes from Uchiyama et al. [28],
who used electron microscopy to study the degradation
of b-chitin fibers by ChiA from S. marcescens and who
concluded that their results were compatible with a
processive mode of action.
One goal of the present study was to exploit the
unique heteropolymeric nature of chitosan to study the
mechanism of action of ChiB. A second goal of this
study was to explore possibilities of producing chito-
oligosaccharide mixtures by degrading chitosans with
ChiB. This latter goal is of interest because chito-
oligosaccharides have a number of potential applica-
tions [29–32]. To obtain these goals, we have analyzed
the degradation of well-characterized, fully water-
soluble, high molecular mass chitosans with ChiB,
using a newly developed chromatographic method for
oligomer separation and previously established NMR-
based methods [5,9,10,33] for analysis of oligomer com-
position. For example, we studied the time course of
depolymerization of a highly acetylated but still water-
soluble chitosan of high molecular mass, and the result-
ing oligosaccharides were characterized with respect to
their chain length and chemical composition. In addi-
tion, high molecular mass chitosans of varying F

A
were
degraded, and the resulting oligosaccharides were char-
acterized in the same way. The results show that ChiB
acts as an exo-chitinase ⁄ -chitosanase with specific
requirements towards acetylated ⁄ deacetylated sugar
units in subsites )2 and )1, which is reflected in the
formation of oligomers with widely varying size distri-
butions and chemical compositions, depending on the
type of chitosan used. The results also provide unequi-
A. Sørbotten et al. Degradation of chitosans with ChiB
FEBS Journal 272 (2005) 538–549 ª 2005 FEBS 539
vocal experimental evidence for a processive mode of
action in ChiB.
Results
Size-exclusion chromatography of chito-
oligosaccharides
The size-exclusion chromatographic system was exten-
sively calibrated using fully acetylated and fully
de-N-acetylated oligomer standards. Figure 1 shows
the degree of polymerization as a function of the dis-
tribution coefficient
½K
av
¼ðV
e
À V
0
Þ=ðV
t

À V
0
Þ
where V
e
is the elution volume of the actual oligo-
mer, and V
0
and V
t
is the void volume and the total
volume of the column, respectively. Although the two
homologous oligomer-series do not elute at exactly
the same K
av
values, the calibration lines are close
and nearly parallel. Extrapolation of the calibration
lines to K
av
¼ 0 revealed that the corresponding
degree of polymerization (DP)-value was 40. The cal-
ibration lines of the acetylated- and the de-N-acetylat-
ed oligomers did not intercept (an interception of the
calibration lines would mean that a fully deacetylated
oligomer would co-elute with an acetylated oligomer
of different chain length). Thus, the chromatographic
system allows the separation of mixtures of partially
N-acetylated oligomers according to size (DP) and
not according to chemical composition, at least in the
separation range between DP 4 (see below for details)

and a DP of approximately 20 (Fig. 4; these are the
longest oligomers that can be observed separately). It
should be noted that the fully acetylated and fully
de-N-acetylated standards represent the extreme limits
of the possible variation in chemical composition of
an oligomer with a given DP, while the enzymatic
degradation of chitosans results in oligomer fractions
with less variation in chemical composition. As shown
in Fig. 1 and by the results presented below, the fully
acetylated dimer (AA) and trimer (AAA) are resolved
from the corresponding deacetylated dimer (DD) and
trimer (DDD).
ChiB degradation of a high molecular mass
chitosan with F
A
¼ 0.65
Time course of the reaction
Progress in chitosan degradation can be determined as
the degree of scission (a), i.e. the fraction of glycosidic
linkages in the chitosan that has been cleaved by the
enzyme, by monitoring the increase in reducing end
resonances relative to resonances from internal anomer
protons in a
1
H-NMR spectrum of the reaction mix-
ture. Figure 2 shows the anomer region of the
1
H-NMR-spectrum of a high molecular mass chitosan
with F
A

of 0.65 which had been incubated with ChiB
for periods from 15 min (a ¼ 0.03) to 168 h (a ¼
0.37). These spectra were assigned from previously
published assignments of
1
H-NMR spectra of hydro-
lysed chitosans [33,38]. The b-anomer reducing end
resonance appears as a doublet due to the relatively
large coupling constant of 8.1 Hz to H-2. Depending
on whether the preceding unit is an A-unit or a D-unit,
the b-anomer reducing end resonance appears as two
doublets at 4.705 p.p.m. (–AA) and 4.742 p.p.m. (–
DA). This assignment was made from
1
H-NMR data
on acid hydrolysis of two chitosans with F
A
of 0.65
and 0.30. The relative intensities of the a ⁄ b-anomers
correspond to the equilibrium ratio between the two
anomers, which is 60 : 40 at pH 4–5 [39]. The a- and
b-anomer reducing end resonances from a D-unit,
which would be expected to appear at 5.43 p.p.m. [–
D(a)] and 4.92 p.p.m. [–D(b)] [33], are completely
absent in the spectrum. The internal D- and A-units
resonate at around 4.9 p.p.m. (–D-) and 4.6 p.p.m.
(–A-) [9]. Using these assignments, the ratio between
internal protons and reducing end protons can easily
be calculated.
The time course of hydrolysis of the F

A
¼ 0.65
chitosan (Fig. 3) shows that the product formation
rate is constant until the degree of scission reaches
approximately 0.19 (i.e. 19% of the glycosidic linkages
has been cleaved). After this initial phase, a second,
much slower kinetic phase becomes apparent in which
Fig. 1. Calibration of SEC-columns. Degree of polymerization (DP)
plotted as a function of the distribution coefficient (K
av
) for
fully acetylated and fully de-N-acetylated oligomers (monomer
to hexamer). The lines correspond to the following equations:
log DP ¼ )1.826 K
av
+1.597 (fully acetylated); log DP ¼ )1.714 K
av
+1.604 (fully de-N-acetylated).
Degradation of chitosans with ChiB A. Sørbotten et al.
540 FEBS Journal 272 (2005) 538–549 ª 2005 FEBS
the rate of the enzymatic reaction is decreased by a
factor of 4.1. This slow phase continues until the
degree of scission reaches 0.37, that is, a situation
where on average more than one out of three glycosi-
dic linkages have been cleaved (note that for an
enzyme producing dimers only, the maximum value of
a is 0.5 and that the presence of D-units lowers the
maximum value of a).
Size distribution of oligomers as a function of a
(F

A
¼ 0.65)
The size distributions of oligomers resulting from
hydrolysis of F
A
¼ 0.65 chitosan at different a-values
are shown in Fig. 4. Undegraded chitosan, with a
number average relative molecular mass of 160 000,
elutes in the void volume of the column, as do all
chitosan chains with DP > 40. Dimer fractions were
found to elute in two peaks, which were identified (by
1
H-NMR spectroscopy) as being AA (the dimer with
the lowest K
av
value) and DA. Similarly, trimer and
tetramer fractions were found to elute in two peaks.
During the initial phase of the degradation process,
i.e. at a-values lower than 0.2 (Fig. 3), the peak eluting
in the void volume disappeared slowly, while short,
mainly even-numbered oligomers (DP 2–12) were pro-
duced (Fig. 4). Around a ¼ 0.20, a transition took
place: as the void peak disappeared, the reaction
slowed down (Fig. 3), and the relative amounts of olig-
omers with an odd number of sugar units began to
increase. Thus, it seems that the slow phase of the reac-
tion starts as the polymer eluting in the void volume is
used up. In the final product mixture (a ¼ 0.37), oligo-
mer frequencies appear to be determined by oligomer
length only, and do not seem to depend on the pres-

ence of an odd- or even number of sugar residues. The
increased appearance of odd numbered oligomers coin-
cides with the appearance of the second, slower kinetic
phase visible in Fig. 3.
Chemical composition of oligomers
To obtain further insight into the chemical composi-
tion of the oligomers, we isolated several fractions
Fig. 2. Anomer region of the
1
H-NMR spectrum of chitosan (F
A
¼
0.65) after incubation with ChiB for periods ranging from 15 min
(a ¼ 0.03) to 10 080 min (a ¼ 0.37) at 37 °C. The reducing end of
the a-anomer of an acetylated unit resonates at 5.19 p.p.m., while
the b-anomer from the same unit resonates at 4.7–4.8 p.p.m. [33].
D-andA-units within the chain and at nonreducing ends resonate
near 4.9 and 4.6 p.p.m., respectively [9]. -AA
b
and -DA
b
are the redu-
cing end b-anomer where the preceding unit is an A-unit or a D-unit,
respectively.
Fig. 3. Time course of ChiB degradation of a chitosan with F
A
¼
0.65. The graph shows the degree of scission (a) as a function of
time. The slope of the line at low a is 1.8 · 10
)3

min
)1
,andat
higher a-values 4.3 · 10
)4
min
)1
.Thea -values continue to increase
to a ¼ 0.37.
A. Sørbotten et al. Degradation of chitosans with ChiB
FEBS Journal 272 (2005) 538–549 ª 2005 FEBS 541
obtained at a ¼ 0.11 and a ¼ 0.37, and analyzed these
by
1
H-NMR spectroscopy. The yield of odd-num-
bered oligomers (a ¼ 0.11) was too low to obtain
well-resolved NMR spectra. The results of these analy-
ses are summarized in Table 1.
Because of an absolute preference for A-units at
newly formed reducing ends (i.e. at subsite )1 of the
enzyme), only two dimers are formed, AA and DA. In
accordance with the NMR analyses shown in Fig. 2,
the chromatographic data (Fig. 4) show that, at low a,
the dimer mainly consists of AA (86% at a ¼ 0.11).
By the end of the reaction, the relative amount of DA
starts to increase, finally reaching 34% of the total
amount of dimers.
NMR analyses of the tetramer fraction at a ¼ 0.11
(Fig. 5A) revealed a F
A

of 0.73 and a DP of 4.1. Elec-
trospray ionization mass spectroscopy (ESI-MS) ana-
lysis showed that the dominating tetramer was
composed of three A-units and 1 D-unit (results not
shown). The minor resonances between 4.8 and
4.9 p.p.m. suggest that minor amounts of DDAA are
present. The absence of a doublet at 4.742 p.p.m. in
Fig. 5A (–DA; Fig. 2) shows that all tetramers has an
A-unit next to the reducing end (–AA). The presence
of only one major D-unit resonance at 4.865 p.p.m.,
and the F
A
of 0.73 show that there is one dominating
tetramer which can be ADAA or DAAA.
1
H-NMR analyses of the tetramer fraction at a ¼
0.37 revealed a DP of 4.2 and an F
A
of 0.51 (Fig. 5B).
ESI-MS data showed that the majority of the tetramers
were composed of two A-units, whereas minor amounts
of tetramers with one and three A-units could be detec-
ted (results not shown). There are three possibilities for
tetramers containing two A-units with an acetylated
reducing end, which are DDAA, DADA and ADDA.
The fact that at least three different doublet resonances
from internal D-units are observed (at 4.855, 4.895 and
4.905 p.p.m.; Fig. 5B) indicates that indeed several
oligomers occur. The relative intensities of the doublets
at 4.705 p.p.m. (–AA) and 4.742 p.p.m. (–DA)

(Fig. 5B) show that the nearest-neighbour to the redu-
cing ends are 70% acetylated and 30% deacetylated.
Thus, one dominating tetramer must be DDAA. The
other possible tetramers with an acetylated reducing
end are DDDA, ADDA, DADA, DAAA, ADAA and
AADA.
Fig. 4. Size-distribution of oligomers after ChiB degradation (F
A
¼
0.65). Chromatograms showing the size-distribution of oligomers
obtained after hydrolysis of chitosan (F
A
¼ 0.65) with ChiB to
degrees of scission (a) varying from 0.03 to 0.37. Peaks are
labelled with DP-values or with the sequence of the oligomer.
Degradation of chitosans with ChiB A. Sørbotten et al.
542 FEBS Journal 272 (2005) 538–549 ª 2005 FEBS
NMR analysis of the trimer fraction (a ¼ 0.37;
Fig. 5C) showed a DP of 2.90 and an F
A
of 0.65.
The resonance at 4.705 p.p.m. and the absence of a
resonance at 4.742 p.p.m. show that the nearest
neighbour to the reducing end unit is an A-unit
(–AA). This is confirmed by the two doublet reso-
nances at 4.648 and 4.660 p.p.m. with a coupling con-
stant of 8.1 Hz, which are assigned to an A-unit next
to the reducing end and where the ratio between the
two doublets reflects the a ⁄ b-anomer equilibrium at
the neighbouring reducing end unit. The resonance at

4.880 p.p.m. is assigned to a deacetylated unit which
must be located at the nonreducing end. Thus, the
trimer is identified as DAA. Two very small doublet
resonances at 4.742 p.p.m. reflect the presence of
minor amounts of the trimers with a D-unit next to
the acetylated reducing end (i.e. DDA and ⁄ or ADA).
Based on the F
A
-value of 0.65 found for the trimer
fraction, relative amounts of the trimers with two
acetylated units (DAA and ADA) and the trimer with
one acetylated unit (DDA) were calculated to be 95
and 5%, respectively (Table 1).
Table 1 also shows F
A
values for longer oligomers,
as determined by
1
H-NMR spectroscopy. The results
show that the relative amount of D-units in the longer
oligomers increases at high a. This indicates that some
D-containing sequences are only hydrolyzed as other,
more favourable substrates become scarce.
Degradation of chitosans with varying F
A
Three high molecular mass chitosans with F
A
¼ 0.13,
0.32 and 0.50 (Table 2) were incubated with ChiB, and
the chitosans were extensively depolymerized to maxi-

mum a. The results (Fig. 6) show that the size distribu-
tion of the product mixtures shifts towards higher
oligomer lengths for substrates with lower F
A
values
and, thus, longer and noncleavable stretches of con-
secutive D-units. In addition to a shift to longer
oligomers, Fig. 6 shows a reduction in the AA ⁄ DA
ratio, as would be expected when lowering the F
A
of the chitosan. A highly deacetylated chitosan
Table 1. Composition of isolated oligomers. Chemical composition and sequence of isolated oligomer fractions obtained after degradation of
chitosan (FA ¼ 0.65) with ChiB to degrees of scission (a) of 0.11 and 0.37. ND, not determined.
Dimer Trimer Tetramer Pentamer Hexamer Octamer
a ¼ 0.11 F
A
¼ 0.93 ND F
A
¼ 0.73 ND F
A
¼ 0.66 F
A
¼ 0.61
14% DA ADAA ⁄ DAAA
86% AA
a ¼ 0.37 F
A
¼ 0.83 F
A
¼ 0.65 F

A
¼ 0.51 F
A
¼ 0.57 F
A
¼ 0.53 ND
34% DA DAA DDAA
66% AA DDA ⁄ ADA (ADDA ⁄ DADA ⁄
DAAA ⁄ ADAA ⁄
AADA ⁄ DDDA)
Fig. 5.
1
H-NMR spectra (anomer region) of isolated oligomers
obtained after hydrolysis of a chitosan with F
A
¼ 0.65 with ChiB.
(A) Tetramer fraction (a ¼ 0.11). (B) Tetramer fraction (a ¼ 0.37).
(C) Trimer fraction (a ¼ 0.37).
A. Sørbotten et al. Degradation of chitosans with ChiB
FEBS Journal 272 (2005) 538–549 ª 2005 FEBS 543
(F
A
< 0.001) was not degraded when incubated with
an excess of ChiB (result not shown).
The chemical compositions of selected oligo-
mers (trimer to hexamer) from the experiments with
chitosan (F
A
¼ 0.32) were investigated by
1

H-NMR
spectroscopy, using the methods described above
(results not shown). The trimer fraction had a DP of
3.04 and a F
A
of 0.44, and was concluded to consist
mainly of DDA and DAA, in addition to minor
amounts of ADA. The tetramer fraction showed a DP
of 4.1 and a F
A
of 0.33 and the dominant tetramers
were found to be DDDA and DDAA.
1
H-NMR
analyses of the longer oligomers (pentamers, hexamers
and heptamers) revealed two dominating oligomers
which both contained A-units at the reducing end but
which varied with respect to the neighbouring unit (A
or D). All other units were deacetylated.
Discussion
The NMR analysis of reaction products obtained upon
degradation of chitosan by ChiB (Figs 2 and 5)
showed that all oligomers had acetylated units at their
reducing ends. This is in full agreement with the pro-
Table 2. Characterization of chitosans. Fraction of acetylated units (F
A
), diad frequencies (F
AA
, F
AD

, F
DA
and F
DD
), number-average block
lengths (N
D
and N
A
) as determined from 600 MHz
1
H-NMR spectroscopy [9]. The intrinsic viscosities ([g]) were determined as described
previously [31] and the number-average molecular masses (M
n
) were calculated from the intrinsic viscosities [32]. The experimentally deter-
mined diad frequencies and block lengths of each chitosan are compared with the calculated diad frequencies and block lengths of a chito-
san with a random (Bernoullian) distribution of A- and D-units (numbers given in brackets). ND, not determined.
F
A
[g](mlÆg
)1
) M
n
F
AA
F
AD
¼ F
DA
F

DD
N
D
N
A
Chitosan 65% 0.65 740 160 000 0.42(0.42) 0.23(0.23) 0.12(0.12) 1.5(1.5) 2.8(2.9)
Chitosan 50% 0.50 850 210 000 0.27(0.25) 0.24(0.25) 0.26(0.25) 2.1(2.0) 2.1(2.0)
Chitosan 32% 0.32 820 250 000 0.12(0.10) 0.20(0.22) 0.48(0.46) 3.4(3.1) 1.6(1.5)
Chitosan 13% 0.13 910 230 000 ND ND ND ND ND
Fig. 6. Size-distribution of oligomers after extended hydrolysis of various chitosans with ChiB. Chromatograms showing the size-distribution
of oligomers obtained upon extended ChiB-hydrolysis of chitosans with F
A
of 0.65, 0.50, 0.32 and 0.13 to a -values (corresponding DP
n
-val-
ues in brackets) of 0.37 (2.7), 0.34 (2.9), 0.22 (4.5) and 0.11 (9.5), respectively.
Degradation of chitosans with ChiB A. Sørbotten et al.
544 FEBS Journal 272 (2005) 538–549 ª 2005 FEBS
posed substrate-assisted catalytic mechanism of ChiB
[15,16]. Whereas many details are known about the
catalytic mechanism of ChiB and other family 18
chitinases, much less is known about processivity.
Some work has been carried out on ChiA, another
family 18 chitinases from S. marcescens which, like
ChiB, has a deep active site cleft. It was shown that
ChiA is an exoenzyme and degrades the polymer from
its reducing end [14,21,28]. On the basis of electron
microscopy studies, Uchiyama et al. [28] proposed that
ChiA acts processively.
In the present study, we have used a new approach

to study the properties of ChiB, including possible
processivity. When ChiB acts on chitin, it only produ-
ces dimers and trimers, indicating that ChiB is an
exoenzyme, which can bind either two (subsites )1 and
)2) or three (subsites )3, )2 and )1) sugar units in
the product binding site [14,21]. The degradation of
chitosans yielded products with more than three sugar
units very early during the reaction (Fig. 4). This
shows that, in the case of a chitosan substrate, the
putative physical barrier at the )3 subsite in ChiB [20]
does not prevent the enzyme from productive binding
events that position more than three sugars on the gly-
con side of the catalytic centre. The observation of
longer oligomers early during the reaction may seem
to contrast with the putative exo-activity of the
enzyme, but can be explained by processivity, as dis-
cussed below. The slow disappearance of the void vol-
ume peak and the early appearance of only shorter
oligomers (Fig. 4) indicate that ChiB does indeed
degrade the chitosan chains from their ends (as
opposed to an endo-action), and thus is an exoenzyme.
If ChiB were to be a processive enzyme, one would
expect chain movements by an even number of sugar
units, as successive glycoside units in chitin ⁄ chitosan are
rotated by 180° along the chain, meaning that the cata-
lytically important N-acetyl group is positioned cor-
rectly in every second sugar only, as proposed for
processive cellulases [40–42]. The clear dominance of
even-numbered oligomers during the initial phase of the
degradation of chitosan with F

A
0.65 (Fig. 4) can only
be explained by a processive mode of action. If each
binding and, for productive binding, cleavage event
would be followed by separation and rebinding, the lon-
ger initial products would be equally divided between
odd-numbered and even-numbered oligomers. The
enzyme does not have more than three subsites ()3, )2
and )1) on the glycon side of the catalytic centre, mean-
ing that preferential production of, e.g. hexamers and
octamers (as opposed to pentamers and heptamers) can-
not be explained by specific binding interactions with
the enzyme in a nonprocessive mode of action.
Taking together all information and observations,
ChiB is likely to initially bind the chitosan chain with
similar chances of having an odd or even number of
sugar units positioned on the glycon side (most likely
three, occupying subsites )3to)1, or two, occupying
subsites )2 and )1). Depending on the sequence of the
bound chain, the first cleavage may then occur directly
or after one or more consecutive movements by two
sugar units at the time. This would lead to the initial
production of an odd- or even-numbered oligomer,
respectively. If the enzyme would act processively, all
subsequent products coming out of the same binding
event will be even-numbered regardless of the initial
binding mode, leading to the observed initial domin-
ance of even-numbered oligomers. Interestingly, the
initial formation of even-numbered oligomers all the
way to at least decamers not only reveals the proces-

sive mechanism, but also shows that processivity is not
interrupted when a nonproductive or less-preferred
enzyme–substrate complex emerges (for example a
complex that positions a D-unit in the )1 subsite). In
the later stages of enzymatic degradation odd-num-
bered oligomers appear in larger amounts. This is a
consequence of the increased depolymerization of the
substrate, which leads to more ‘initial attacks’.
It is generally accepted that cellobiohydrolases,
which convert cellulose (which is closely related to and
equally insoluble as chitin) mainly to dimers, act pro-
cessively [40–42]. It is not straightforward to give evi-
dence of a processive mechanism, as the parameter
that is often used [the dimer ⁄ (trimer + monomer)
ratio] strictly speaking does not discriminate between
processivity and different initial binding events that
occur with particular frequencies, leading to particular
dimer ⁄ (trimer + monomer) ratios. The availability of
the water-soluble, heteropolymeric chitosan, acting as
a ‘pseudo-substrate’ for family 18 chitinases and
resembling both chitin and cellulose, allowed us to
obtain clear experimental data which unequivocally
show that the enzyme acts processively.
The current data also provide some insight into the
preference for A-orD-units in subsites )2 and +1.
Because the nonreducing end sugar of an oligomer
must have been bound productively in the +1 subsite
in a previous cleavage, the occurrence of both D and
A nonreducing ends shows that there is no clear pref-
erence for A or D in the +1 subsite. In the initial, fas-

ter stage of the depolymerization reaction (a < 0.2),
most oligomers formed had an A-unit as nearest neigh-
bour to the reducing end (–AA), indicating that most
of the productive enzyme–substrate complexes had an
A-unit bound in subsite )2. Upon more extensive
hydrolysis, presumably leading to depletion of binding
A. Sørbotten et al. Degradation of chitosans with ChiB
FEBS Journal 272 (2005) 538–549 ª 2005 FEBS 545
sites with the optimal AA sequence in )2 and )1, the
reaction becomes slower, and an increase in oligomers
with a D-unit as nearest neighbour to the reducing end
(–DA) is observed. Thus, ChiB has a clear preference
for A in subsite )2.
Catalysis in family 18 chitinases requires distortion
of the sugar in the )1 subsite [16]. In analogy with,
e.g. lysozyme, a certain minimum amount of binding
energy is likely to be required to achieve this distortion
and formation of a productive enzyme–substrate com-
plex. This is reflected by some of the observations
made in this study. For example, the trimer AAA is
degraded, whereas the trimer DAA (which in principle
could bind productively to the )2, )1 and +1 sub-
sites) is not. Likewise, several of the tetramers with an
–AA reducing end are not degraded while these two
A-units could bind productively to the )1 and +1 sub-
sites. These observations suggest that the ability to
cleave a substrate that positions a D in the )2 subsite
depends on how many of the subsites on the aglycone
side are occupied by sugars.
The distribution of oligomers formed upon extensive

ChiB depolymerization of chitosans with varying F
A
shifted towards higher oligomers with decreasing F
A
.
This was expected because the average chain length of
D-blocks that will not be attacked by the chitinase
increases with decreasing F
A
(Table 2). By extensive
ChiB-degradation of chitosans with F
A
from 0.1 to
0.5, we were able to produce longer oligomers
(DP > 4) which were composed of only D-units,
except for the reducing end unit and in some cases its
nearest neighbour. By selecting the chitosan with the
appropriate F
A
and the extent of degradation
(a-value), oligomers with predetermined composition
and chain length can be prepared.
In conclusion, we have found that chitosans with
full water-solubility and known random distribution of
A- and D-units are useful substrates for obtaining dee-
per insight into the mode of action of ChiB, including
its processive character. The catalytic mechanism of
ChiB requires an acetylated unit in the )1 subsite, but
this does not prevent the enzyme from degrading a
wide range of chitosans with varying chemical compo-

sitions. Thus, ChiB may be used to convert these
chitosans to mixtures of oligosaccharides with predict-
able chain lengths and chemical compositions.
Experimental procedures
Chitosans
Chitin was isolated from shrimp shells by the method of
Hackman [34] and milled in a hammer mill to pass through
a 1.0-mm sieve. Chitosans, with degrees of N-acetylation of
65, 50, 32 and 13% (F
A
¼ 0.65, 0.50, 0.32 and 0.13), were
prepared by homogeneous de-N-acetylation of chitin [35].
The chemical properties of the chitosans (F
A
, diad frequen-
cies, DP
n
) were characterized by
1
H-NMR spectroscopy
[5,9], and by intrinsic viscosity measurements [36]. Number-
average molecular masses were calculated from the Mark–
Houwink–Sakurada equation, as reported by Anthonsen
et al. [37]. Characteristic features of the chitosans are given
in Table 2, including the fraction of acetylated units (F
A
),
the diad frequencies, the intrinsic viscosities ([g]), the num-
ber-average molecular mass (M
n

) and the average length of
the D-blocks (N
D
). These characteristics show that the
chitosans have a random distribution of acetylated units,
i.e. according to Bernoullian distribution.
Chitinase B
ChiB was overexpressed in Escherichia coli and purified by a
protocol consisting of a previously described hydrophobic
interaction chromatography step [20,21], preceded by ion-
exchange chromatography on Q-Sepharose Fast Flow
(Amersham Pharmacia Biotech AB, Uppsala, Sweden). The
final protein material was collected in an ammonium carbon-
ate buffer [21], and freeze-dried. Before use, the enzyme was
dissolved to 1 mgÆmL
)1
in 20 mm trizma base, pH 7.5. Pro-
tein concentrations were determined with the Bio-Rad Pro-
tein Assay (Bio-Rad Laboratories Inc., Hercules, CA, USA).
The purified protein used in this work displayed the same
specific activity and kinetic parameters as ChiB used in previ-
ous studies, including the protein used for successful X-ray
diffraction studies [20].
Kinetics of chitosan degradation
Samples of 10 mg chitosan (F
A
¼ 0.65) were dissolved in
1.0 mL H
2
O and added to 1.0 mL 0.08 m NaAc buffer,

pH 5.5, containing 0.2 m NaCl and 0.2 mg BSA. The sam-
ples were placed in a shaking waterbath at 37 °C. The
depolymerization reaction was started by adding 5 lgof
ChiB and was stopped after between 15 and 10 080 min by
adjusting the pH to 2.5 with 1.0 m HCl, and immersing the
samples in boiling water for 2 min. The samples were
stored at )18 °C until further analysis.
Extended depolymerization of chitosans with
varying degree of acetylation (F
A
)
Four samples of 10 mg chitosan (F
A
¼ 0.13, 0.32, 0.50 and
0.65) were each dissolved in 1.0 mL H
2
O and added to
1.0 mL 0.08 m NaAc buffer, pH 5.5, containing 0.2 m NaCl
and 0.2 mg BSA. The samples were immersed in a shaking
waterbath at 37 °C. The depolymerization reactions were
started by adding 10 lg of ChiB and the reactions were
stopped after 1 week as described above.
Degradation of chitosans with ChiB A. Sørbotten et al.
546 FEBS Journal 272 (2005) 538–549 ª 2005 FEBS
1
H-NMR-spectroscopy
The samples were dissolved in D
2
O and the pD was adjus-
ted to 4 with DCl. The deuterium resonance was used as a

field-frequency lock, and the chemical shifts were refer-
enced to internal sodium 3-(trimethylsilyl)propionate-d
4
(0.00 p.p.m.). The
1
H-NMR spectra were obtained at 90 °C
at 300.13, 400.13 or 600.13 MHz as previously described
[5,9], and F
A
values were calculated as described by Va
˚
rum
et al. [9]. The number average degree of polymerization,
DP
n
, of enzyme-degraded chitosans was determined from
the anomer (H-1) resonances as follows: DP
n
¼ [area of all
H-1-resonances (internal and reducing end)] ⁄ (area of redu-
cing end resonances). The degree of scission, a, was calcula-
ted as a ¼ 1 ⁄ DP
n
.
Size-exclusion chromatography (SEC)
Oligomers from enzymatic depolymerization reactions were
separated on three columns in series, packed with Superdexä
30, from Amersham Pharmacia Biotech (overall dimensions
2.60 · 180 cm). The column was eluted with 0.15 m
ammonium acetate, pH 4.5 at a flow rate of 0.8 mLÆmin

)1
.
The effluent was monitored with an online refractive index
(RI) detector (Shimadzu RID 6 A, Shimadzu Schweiz
GmbH, Reinach, Switzerland), coupled to a datalogger.
Fractions of 3.2 mL were collected for analysis. Standard
samples contained 10 mg of partially depolymerized
chitosan. In experiments where oligomers were collected for
further analysis by NMR spectroscopy, samples containing
up to 200 mg of partially depolymerized chitosan were
injected without loss of resolution. Fully acetylated and fully
de-N-acetylated oligomer were used as standards (monomer
to hexamer; Seikagaku Corporation, Tokyo, Japan).
To verify the possibility to quantify amounts of oligosac-
charides with varying DP and F
A
from the refractive index
detector response, we checked the detector response in the
following way: exact amounts (1 mg) of the fully acetylated
and fully de-N-acetylated oligomers (dimer, tetramer and
hexamer) were dissolved in the mobile phase and injected
on the columns, and the refractive index detector signal
was used to determine the area of the peaks after elution
from the column. These analyses showed that, within the
accuracy of the area determination, there was a linear rela-
tionship between peak areas and the amount (mass) of
injected oligomer, irrespective of DP and degree of acetyla-
tion.
Acknowledgements
This work was supported by grants from the Norwegian

Research Council (140497 ⁄ 420 and 134674 ⁄ I10). We
thank Hege Grindheim for carrying out some of the
experiments.
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