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

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 phas

Serratia marcescens

Production of chito-oligosaccharides and insight into enzyme

processivity

Audun Sørbotten1, Svein J Horn2, Vincent G H Eijsink2and Kjell M Va˚rum1

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 (1fi 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 (1fi 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: kjell.morten.vaarum@biotech.ntnu.no

(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 using1H-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; FA, fraction of acetylated units.

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

(FA) 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(1fi 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 processact-ively [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 FAwere 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-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

½Kav ¼ ðVe
V0Þ=ðVt
V0Þ

where Ve is the elution volume of the actual

oligo-mer, and V0 and Vt 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 Kav values, the calibration lines are close

and nearly parallel Extrapolation of the calibration

lines to Kav¼ 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 FA¼ 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 1H-NMR spectrum of the reaction mix-ture Figure 2 shows the anomer region of the

1H-NMR-spectrum of a high molecular mass chitosan with FA 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 1H-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 1H-NMR data

on acid hydrolysis of two chitosans with FA 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 FA¼ 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 (Kav) 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).

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 (FA¼ 0.65)

The size distributions of oligomers resulting from hydrolysis of FA¼ 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

1H-NMR spectroscopy) as being AA (the dimer with the lowest Kav 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 a¼ 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 (a¼ 0.37), oligo-mer frequencies appear to be determined by oligooligo-mer 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 fraccomposi-tions

Fig 2 Anomer region of the 1 H-NMR spectrum of chitosan (FA¼

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- and A-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)3min)1, and at higher a-values 4.3 · 10)4min)1 The a -values continue to increase

to a ¼ 0.37.

obtained at a¼ 0.11 and a ¼ 0.37, and analyzed these

by 1H-NMR spectroscopy The yield of odd-num-bered oligomers (a¼ 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 a¼ 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 a¼ 0.11 (Fig 5A) revealed a FA 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 FA of 0.73 show that there is one dominating tetramer which can be ADAA or DAAA

1H-NMR analyses of the tetramer fraction at a¼ 0.37 revealed a DP of 4.2 and an FAof 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 (FA¼ 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.

NMR analysis of the trimer fraction (a¼ 0.37;

Fig 5C) showed a DP of 2.90 and an FA 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 FA-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 FA values for longer oligomers,

as determined by 1H-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 FA Three high molecular mass chitosans with FA¼ 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 FA 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 FA

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 FA¼ 0.83 FA¼ 0.65 FA¼ 0.51 FA¼ 0.57 FA¼ 0.53 ND

34% DA DAA DDAA 66% AA DDA ⁄ ADA (ADDA ⁄ DADA ⁄

DAAA ⁄ ADAA ⁄ AADA ⁄ DDDA)

Fig 5. 1H-NMR spectra (anomer region) of isolated oligomers

obtained after hydrolysis of a chitosan with FA¼ 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).

(FA< 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 (FA¼ 0.32) were investigated by 1H-NMR

spectroscopy, using the methods described above

(results not shown) The trimer fraction had a DP of

3.04 and a FA 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 FA of 0.33 and the dominant tetramers

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 (FA), diad frequencies (FAA, FAD, FDAand FDD), number-average block lengths (NDand NA) 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.

FA [g] (mlÆg)1) Mn FAA FAD¼ F DA FDD ND NA 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 FAof 0.65, 0.50, 0.32 and 0.13 to a-values (corresponding DPn -val-ues in brackets) of 0.37 (2.7), 0.34 (2.9), 0.22 (4.5) and 0.11 (9.5), respectively.

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 FA 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 )3 to )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- or D-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

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 FA

shifted towards higher oligomers with decreasing FA

This was expected because the average chain length of

D-blocks that will not be attacked by the chitinase

increases with decreasing FA (Table 2) By extensive

ChiB-degradation of chitosans with FA 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 FA 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% (FA¼ 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 (FA, diad frequen-cies, DPn) were characterized by 1H-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 (FA), the diad frequencies, the intrinsic viscosities ([g]), the num-ber-average molecular mass (Mn) and the average length of the D-blocks (ND) 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)1in 20 mm trizma base, pH 7.5 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 (FA¼ 0.65) were dissolved in 1.0 mL H2O 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 lg of 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 (FA)

Four samples of 10 mg chitosan (FA¼ 0.13, 0.32, 0.50 and 0.65) were each dissolved in 1.0 mL H2O 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

The samples were dissolved in D2O 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-d4

(0.00 p.p.m.) The1H-NMR spectra were obtained at 90
C

at 300.13, 400.13 or 600.13 MHz as previously described

[5,9], and FAvalues were calculated as described by Va˚rum

et al [9] The number average degree of polymerization,

DPn, of enzyme-degraded chitosans was determined from

the anomer (H-1) resonances as follows: DPn¼ [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 a¼ 1 ⁄ DPn

Size-exclusion chromatography (SEC)

Oligomers from enzymatic depolymerization reactions were

separated on three columns in series, packed with Superdex

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 FAfrom 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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