Despite a number of reports describing exo-1,3-b-glucanases from different sources [19,20,23 – 25], the subsite structure of the substrate binding site as well as the affinity and the nu
Trang 1Isolation, enzymatic properties, and mode of action of an
Anna A Kulminskaya1, Karl K Thomsen2,*, Konstantin A Shabalin1, Irina A Sidorenko1, Elena V Eneyskaya1, Andrew N Savel’ev3and Kirill N Neustroev1
1 Petersburg Nuclear Physics Institute, Russian Academy of Science, Russia;2Carlsberg Laboratory, Department of Physiology,
Copenhagen, Denmark;3St Petersburg Technical University, Biophysics Department, Russia
An exo-1,3-b-glucanase has been isolated from cultural
filtrate of Trichoderma viride AZ36 The N-terminal
sequence of the purified enzyme (m ¼ 61 ^ 1 kDa)
showed no significant homology to other known glucanases
The 1,3-b-glucanase displayed high activity against
laminarins, curdlan, and 1,3-b-oligoglucosides, but acted
slowly on 1,3-1,4-b-oligoglucosides No significant activity
was detected against high molecular mass
1,3-1,4-b-glucans The enzyme carried out hydrolysis with inversion
of the anomeric configuration Whereas only glucose was
released from the nonreducing terminus during hydrolysis of
1,3-b-oligoglucosides, transient accumulation of gentiobiose
was observed during hydrolysis of laminarins The gentiobiose was subsequently degraded to glucose The Michaelis constants Kmand Vmaxhave been determined for the hydrolysis of 1,3-b-oligoglucosides with degrees of polymerization ranging from 2 to 6 Based on these data, binding affinities for subsites were calculated Substrate binding site contained at least five binding sites for sugar residues
Keywords: exo-1; 3-b-glucanase; Trichoderma viride; anomerity of hydrolysis
Enzymes hydrolyzing 1,3-b-D-glucans occur in a variety of
organisms [1] 1,3-b-Glucanases hydrolyze the
O-glyco-sidic linkages of 1,3-b-linked glucans and are classified
according to their mode of action The exo-1,3-b-glucanases
(EC 3.2.1.58) sequentially release glucose residues from the
nonreducing terminus of a substrate while the
endo-1,3-b-glucanases (EC 3.2.1.39) are capable of cleaving internal
1,3-b-linkages at random sites along the polysaccharide
chain, releasing short oligosaccharides 1,3-b-Glucanases
have been isolated from bacteria [2], yeast and fungi [3 – 5],
plants [6,7], and marine organisms [8,9] It has been
suggested that plant 1,3-b-glucanases may protect the
germinating grain against pathogen attack [10] Microbial
1,3-b-glucanases play an essential role in development and differentiation of saprophyte and mycoparasite cultures [11 – 13] while 1,3-b-glucanases from the filamentous fungi Coprinas seem to be involved in the process of stipe elongation [14] In Saccharomyces cerevisiae the produc-tion of exo-1,3-b-glucanases is growth-associated and cell-cycle regulated, suggesting that their activities are required
at specific stages during morphogenesis [15,16] Most organisms synthesize multiple 1,3-b-glucanases rather than
a single enzyme [17] and complete degradation of 1,3-b-glucans by fungi is often accomplished by synergistic action
of endo- and exo-glucanases [18] These enzymes have received attention in many fields of science and biotech-nology because many cultures of microorganisms widely used in industry produce 1,3-b-glucanases, which are essen-tial for cell-cycle functions [19,20] and due to their increasing importance in modification of b-glucans for pharmaceutical purposes [21,22] Despite a number of reports describing exo-1,3-b-glucanases from different sources [19,20,23 – 25], the subsite structure of the substrate binding site as well
as the affinity and the number of subsites have not been analyzed for most of these enzymes The present study describes the isolation and characterization of an exo-1,3-b-glucanase from the filamentous fungus Trichoderma viride AZ36 The subsite structure was evaluated by steady-state kinetics using 1,3-b-oligoglucosides with a different degree
of polymerization The mode of action and specificity as well as stereoselectivity of hydrolysis catalyzed by the exo-1,3-b-glucanase were studied by NMR spectroscopy
M A T E R I A L S A N D M E T H O D S
Substrates Laminarin from Laminaria digitata, barley 1,3-1,4-b-glucan, lichenan from Cetraria islandica, gentiobiose, cellulose,
Correspondence to K N Neustroev, Petersburg Nuclear Physics
Institute, Gatchina, St Petersburg, 188350, Russia.
Fax: 1 781271 32303, Tel.: 1 781271 32014,
E-mail: neustk@omrb.pnpi.spb.ru
Enzymes: 1,3-b-glucanase, 1,3-b- D -glucan glucanohydrolase,
laminarinase (3.2.1.39); exo-b-1,3-glucanase, 1,3-b- D -glucan
glucohydrolase (EC 3.2.1.58); a-glucosidase (EC 3.2.1.20);
glucoamylase (EC 3.2.1.3); b- D -glucosidase, b- D -glucoside
glucohydrolase (EC 3.2.1.21).
Definition: G4G4G3G, b- D -Glcp-(1 !4)-b- D -Glcp-(1
!4)-b-D -Glcp-(1 !3)-b- D -Glcp; G4G3G, b- D -Glcp-(1 !4)-b- D
-Glcp-(1 !3)-b- D -Glcp; G3G3G3G3G, b- D -Glcp-(1 !3)-b- D -Glcp-(1
!3)-b- D -Glcp-(1 !3)-b- D -Glcp-b- D -Glcp-(1 !3)-b- D -Glcp; G3G3G3G,
b- D -Glcp-(1 !3)-b- D -Glcp-(1 !3)-b- D -Glcp-(1 !3)-b- D -Glcp;
G3G3G, b- D -Glcp-(1 !3)-b- D -Glcp-(1 !3)-b- D -Glcp; G3G,
b- D -Glcp-(1 !3)-b- D -Glcp; G6G, gentiobiose.
*Present address: Fussingsvej 8, I, DK-8700 Horsens, Denmark.
(Received 3 July 2001, revised 20 September 2001, accepted
27 September 2001)
Abbreviations: DP, degree of polymerization; PHMB,
p-hydroxymercuribenzoic acid sodium salt.
Trang 2cellobiose, p-nitrophenyl cellotrioside, p-nitrophenyl
b-D-glucopyranoside were from Sigma Chemical Co (St
Louis, MO, USA) Curdlan from Alcaligenes faecalis and
pustulan from Umbilicaria popullosa were kindly donated
by I J.Goldstein (Michigan University, USA) Laminarin
from Laminaria cichorioides was kindly donated by A V
Kir’yanov (Institute of Organic Chemistry, Moscow,
Russia) p-Hydroxymercuribenzoic acid sodium salt
(PHMB) was from Merck (Germany)
Mixed linkage oligosaccharides (for oligosaccharide
definitions, see footnotes): G4G4G3G and G4G3G were
produced by digestion of barley glucan with a
1,3-1,4-b-glucanase and purified [26]
The G3G3G3G3G3G, G3G3G3G3G, and G3G3G3G were
prepared by formic acid hydrolysis of curdlan followed by
purification of 1,3-b-oligoglucosides [26,27] G3G and
G3G3G were produced by digestion of laminarin with a
commercially available laminarinase from Trichoderma sp
purchased from Sigma Chemical Co Laminarin (80 mg)
was dissolved in 2 mL of 20 mM sodium acetate buffer,
pH 5.0, and digested using < 0.005 units of the enzyme
per mg of laminarin Incubation was at 37 8C for
60 min The reaction mixture was fractionated on a
Sephadex G-25 (Fine) column equilibrated in water,
separating oligosaccharides of degree of polymerization
(DP) 2 – 6 from the high molecular mass fraction Following
freeze drying the 1,3-b-oligoglucosides were fractionated
on a TSK NH2-60 column (5 mm, 4.6 250 mm) from
Pharmacia Biotech (Uppsala, Sweden) in 80% acetonitrile
in water (v/v)
The purity of b-oligoglucosides was analyzed by TLC
and 1H and 13C NMR spectroscopy as described below
Published values for1H and13C chemical shifts of
b-oligo-saccharides with different DP and linkage types [26,28 –30]
were used for structure determination of the obtained
compounds
General methods
SDS/PAGE was carried out according to Laemmli [31] and
isoelectric focusing was on Servalyt PRECOATES plates
3 – 10 (Serva Electrophoresis GmbH, Heidelberg, Germany)
Protein concentration was measured following the Lowry
procedure using BSA as a standard [32].1H-NMR spectra
and 13C-NMR spectra were recorded with an AMX-500
Bruker spectrometer Prior to NMR analysis laminarin,
buffer components, and the enzyme were freeze-dried twice
from D2O The measurements were made in 20 mMsodium
phosphate buffer (pD 6.0) at room temperature and 50 mM
4,4-dimethyl-4-silapentane sodium sulfonate was used as an
internal standard in 0.5 mL 99.8% D2O The solvent
reson-ance was presaturated for 0.5 s with a decoupler operating at
24 dB in the CW mode Data were acquired after a 758 pulse
into 16K points, with a spectral width of 10 kHz and 2053
scans, including first five dummy scans The spectra were
Lorentz-broadened by 1 Hz The
4,4-dimethyl-4-silapen-tane sodium sulfonate signal was used for adjustment of
phase and amplitude parameters in order to obtain correct
differential spectra Oligosaccharide substrates and products
of enzymatic hydrolysis were analyzed by TLC on
Kieselgel 60 plates from Merck (Darmstadt, Germany)
with a mobile phase of ethyl acetate/acetic acid/water
(2 : 1 : 1) Plates were developed at room temperature, air
dried and sprayed with 5% H2SO4in 1-propanol followed by incubation at 120 8C for 8 min N-Terminal amino-acid sequencing was conducted using the Edman degradation and phenylisothiocyanate amino-acid analysis The Procise Protein Sequencing System (Applied Biosystems, Foster City, California 94404) was employed
Growth conditions The T viride AZ36 from Petersburg Nuclear Physics Insti-tute strain collection was grown at 30 ^ 1 8C for 72 h in a 10-L fermentor with constant stirring The growth medium contained (g per L) KH2PO4, 1; NaNO3, 1.5; (NH4)2SO4, 1.5; MgSO4 7H2), 0.5; wheat bran, 40
Purification of the exo-1,3-b-glucanase All steps were carried out at 4 8C Mycelium was removed
by centrifugation (3000 g, 30 min), and the supernatant was concentrated 20-fold by use of hollow fibers with a nominal molecular mass limit of 25 kDa (‘Kirishi’, Kirishi, Russia) During the process the buffer was changed to 20 mM
Tris/HCl, pH 7.5 (buffer A) The crude 1,3-b-glucanase preparation was loaded on a DEAE-Sephadex column (50 200 mm) equilibrated with buffer A and bound pro-tein was eluted with 1MNaCl in the same buffer Following concentration to 25 mL on an Amicon PM-30 membrane and dialysis against buffer A, the fraction was loaded onto a TSK column (21.5 150 mm) equilibrated with buffer
A Elution was performed by using a linear 0 – 0.5MNaCl gradient in buffer A Fractions containing exo-1,3-b-glucanase activity were concentrated to 4 mL on an Amicon PM-30 membrane, dialyzed against 20 mM Tris/HCl,
pH 7.2 (buffer B) and chromatographed on a Mono Q HR (5/5) (Pharmacia, Sweden) Bound protein was eluted by applying a linear 0 – 0.5M NaCl gradient in buffer B Finally, the exo-1,3-b-glucanase preparation was concen-trated to 3 mL using an Amicon PM-30 membrane, dialysed against 20 mM sodium acetate buffer, pH 5.0 (buffer C) Saturated (NH4)2SO4 in buffer C was added to a final concentration of 1.7Mbefore applying the enzyme prepar-ation onto a phenyl-Superose HR (5/5) column (Pharmacia, Sweden) equilibrated with 1.7M (NH4)2SO4 in buffer C Bound protein was eluted by using a linear gradient (0 – 100%) of 20 mM sodium acetate buffer, pH 5.0, and fractions with the exo-1,3-b-glucanase activity were dialyzed against buffer C, then against deionized water, and freeze dried
Enzyme assays Exo-1,3-b-glucanase activity was analyzed by measuring the amount of glucose released from laminarin Standard assays (0.25 mL) were in 20 mM sodium acetate buffer,
pH 4.5, and contained 0.5 mg of laminarin and crude or purified enzyme extract corresponding to at least 0.2 mg
of pure exo-1,3-b-glucanase Incubation was at 37 8C for
10 – 30 min and the reaction was stopped by boiling for
5 min Glucose formation was measured by the glucose oxidase method [33] Alternatively, enzyme activity was evaluated by measuring the formation of reducing sugars according to the Somogyi-Nelson method [34] One unit of the enzyme activity produced 1 mmol of glucose:min21at
Trang 3pH 4.5, 37 8C, with laminarin as substrate b-Glucosidase
activity was measured using p-nitrophenyl b-D
-glucopyr-anoside as substrate according to [35]
The influence of pH and temperature on activity of the
enzyme was studied by the glucose oxidase method The
effect of pH on activity was measured at 37 8C for 10 min in
the range pH 3 – 9 in 100 mM sodium phosphate/citrate
buffers Reaction mixture: 400 mL of buffer and 10 mL of
enzyme (0.01 U) in 2 mM sodium acetate buffer, pH 4.5,
was mixed with 50 mL of laminarin (20 mg:mL21in water)
or with 20 mL of G3G3G3G (40 mM in water) The
influence of pH on stability of the enzyme was studied by
incubating samples of the enzyme at 20 8C for 16 h in
100 mM sodium phosphate/citrate buffers ranging from
pH 3 to pH 9, followed by measuring the residual activity of
the enzyme using standard conditions
The effect of temperature on activity was measured at
pH 4.5 in 100 mMsodium citrate buffer over a temperature
range of 25 – 80 8C The reaction mixture (0.4 mL)
con-taining 1 mg of laminarin was preincubated at various
temperatures in the range mentioned above Then 50 mL
(0.005 U) of enzyme solution in the same buffer was added
and incubated for 10 min The reaction was stopped by
addition of 1 mL of copper reagent (Somogyi reagent) and
boiling for 5 min Liberated glucose was measured by the
Somogyi-Nelson method [34] To study stability in relation
to temperature, the purified exo-1,3-b-glucanase was
incubated at various temperatures for 10 min, in 100 mM
sodium citrate buffer, pH 4.5 After cooling, the residual
activity was determined by the glucose-oxidase method as
described above
Effect of metal ions on activity was determined in 50 mM
sodium acetate buffer, pH 4.5, at 37 8C after 15 min of
incubation with different ions
Kinetic parameters
The Michaelis –Menten constants Kmand Vmaxwere
deter-mined from the Lineweaver– Burk representation of data
obtained by measuring the initial rate of substrate hydrolysis
The purified exo-1,3-b-glucanase (0.01 –1 U) was incubated
with different substrates in 0.5 mL of 20 mMsodium acetate
buffer, pH 4.5 After the reaction was stopped by boiling,
liberated glucose was measured as described above
Concentrations of laminarins from 1 to 0.05 mg:mL21,
curdlan concentrations from 15 to 1 mg:mL21, and
b-oligoglucoside concentrations from 12 to 0.1 mM were
used
Enzyme specificity The specificity of the exo-1,3-b-glucanase was studied by analyzing the products of hydrolysis of different substrates Laminarins with different degrees of ramification as well as curdlan, pustulan, and b-oligoglucosides differing in DP and linkage type were used Hydrolysis was stopped after appropriate time intervals by boiling for 5 min The resulting products were analysed qualitatively by TLC and quantitatively by HPLC using a TSK-NH2-60 column (5 mm, 4.6 250 mm) from Pharmacia Biotech (Uppsala, Sweden) in 80% acetonitrile in water (v/v) with refracto-metric detection Glucose and 1,3-b-oligoglucosides of DP
2 – 6 were used as standards To investigate activity of the exo-1,3-b-glucanase towards different soluble and insoluble b-glucans, substrates at a concentration of 5 mg:mL21were incubated with 2 – 3 U of enzyme at 37 8C for various time intervals
For studies of the mode of action in the hydroysis of laminarins, intermediate products were fractionated by HPLC on a Dextro-Pak cartrige column (8 10 mm) WATO85650 from Millipore-Waters (Bedford, MA, USA) using isocratic elution with water The purified products of hydrolysis were analyzed by1H and13C NMR spectroscopy
as described previously [30]
Determination of the stereochemical course of hydrolysis The enzyme was dissolved in 20 mM sodium phosphate buffer, pH 6.0, made up in D2O The reaction mixture of 0.6 mL contained 20 mg:mL21 laminarin in sodium phosphate buffer All NMR measurements were performed
at ambient temperature (<17 8C) After accumulation of the initial spectrum, 80 U of the exo-1,3-b-glucanase was added and the reaction mixture was brought to 37 8C within 5 min The stereochemical course of hydrolysis was monitored by collecting spectra at 6, 20, and 40 min after addition of the enzyme Direct1H NMR analysis of the exo-1,3-b-glucanase action on 1,3-b-oligoglucosides was carried out at a substrate concentration of <10 mMin 0.6 mL of the same buffer
R E S U L T S A N D D I S C U S S I O N
Enzyme purification The T viride AZ36 used in this study is a producer of mainly exo-1,3-b-glucanase An exo-1,3-b-glucanase was purified 25-fold from concentrated culture supernatants of
T viride in four chromatographic steps About 40% of the initial activity was recovered and the purified enzyme
Table 1 Purification of the exo-1,3-b-glucanase.
Purification step
Volume (mL)
Total protein (mg)
Total activity (U)
Specific activity (U:mg 21 )
Purification (fold)
Yield (%)
Trang 4had a specific activity of 63 U:mg21 (Table 1) In SDS/
PAGE analysis the protein appeared as a single polypeptide
with an apparent molecular mass of 61 ^ 1 kDa (Fig 1)
Small amounts of endo-1,3-b-glucanase were separated
from the main fraction during the first steps of purification
During the purification process, the exo-1,3-b-glucanase
and b-glucosidase activities were efficiently separated, and
the purified exo-1,3-b-glucanase preparation possessed
less than 0.1% b-glucosidase activity Analytical gel
filtration on a Superose 12 column demonstrated that the
exo-1,3-b-glucanase had an apparent molecular mass of
60 ^ 5 kDa suggesting that the enzyme was a monomer
(data not shown)
Multiple forms of Trichoderma harzianum
exo-1,3-b-glucanases with a wide range of molecular masses up to
35 kDa have been reported [13] The multiplicity of
secreted forms of the Trichoderma enzymes might be
caused by postsecretory proteolysis by acid proteases also
secreted by the fungus [36,37] The strain T viride AZ36
employed in the present study has a lower level of secreted
proteases than the other Trichoderma species (data not
shown), which is likely to be one of the reasons that a
homogenous exo-1,3-b-glucanase preparation could be
obtained
General properties The purified exo-1,3-b-glucanase displayed optimal activity
in hydrolysis of laminarin from L digitata and 1,3-b-oligoglucosides at pH 4.5 The optimal temperature was
55 8C Activity decreased rapidly at temperatures above
60 8C At room temperature the enzyme was stable for 24 h
in the pH range 3.5 to 7.5 The isolelectric point was 4.2 ^ 0.05 and the N-terminal amino-acid sequence was: AVDDFAPNTKQTPIALNNVLL There are a number of reports providing amino-acid sequence data for fungal exo-1,3-b-glucanases such as Cochliobolus carbonum [38], yeast C albicans [39], Saccharomyces cerevisiae [40], Yarrowia lipolitica [41] but none of these display significant homology in their N-terminal amino-acid sequence to the exo-1,3-b-glucanase described in here
Whereas some divalent metal ions as well as EDTA had a slight inhibitory effect on the exo-1,3-b-glucanase, MnSO4 and MnCl2 increased the activity towards laminarin (Table 2) Similar dependence of the enzymic activity on metal ions was reported for endo-1,6-a-mannanase from a soil bacterium [42] Based on the observation that neither
Hg21nor PHMB caused any inhibition of enzyme activity,
it is assumed that no essential SH-groups are involved as functional groups Most exo-1,3-b-glucanases of fungal origin typically display optimal enzymatic activity in the pH range between 4 and 6 [19,23,43] Similarly the enzyme from T viride AZ36 displayed optimal activity at pH 4.5 The stability in relation to pH and temperature was also similar to that of other fungal exo- and endo-glucanases [11], and like most exo-1,3-b-glucanases that have been studied, the enzyme described here was not inhibited by heavy metal ions and PHMB
The mode of action of the exo-1,3-b-glucanase was analyzed employing 1,3-b-oligoglucosides with DP 3 – 6 and curdlan from A faecalis [26] as substrates Using TLC and HPLC analyses it was demonstrated that glucose was the only product formed during the enzyme-catalysed reaction 1H NMR techniques was also used to study the mode of action of purified exo-1,3-b-glucanase on curdlan and reduced G3G3G3G3G A signal at d ¼ 5.248 p.p.m corresponding to reducing ends [26] did not increase during hydrolysis This observation unequivocally demonstrates the absence of endo-type hydrolysis Consequently, this glucanase should be classified as an exo-glucohydrolase (Fig 2) The enzyme displayed only very low activity against 1,3-1,4-b-glucans, lichenan, and barley glucan, and
Fig 1 SDS/PAGE of the exo-1,3-b-glucanase from T viride Lane
1, purified exo-1,3-b-glucanase from T viride; lane 2, protein
standards – phosphorylase a (98 kDa), BSA (67 kDa), ovalbumin
(45 kDa), a-chymotrypsin (25 kDa).
Table 2 Effect of metal ions, PHMB, and EDTA on the exo-1,3-b-glucanase activity towards laminarin.
Effector (1 m M ) Relative activity (%)
Trang 5no activity was detected against 1,4-b-glucan,
1,4-b-oligo-glucosides, pustullan (1,6-b-glucan), cellulose, and
p-nitro-phenyl b-glucoside Whereas the Km values for mixed
linked 1,3-1,4-b-oligoglucosides and the corresponding
1,3-b-oligoglucosides were similar (Tables 3 and 4), the
Vmaxvalue for hydrolysis of G4G3G was <260-fold lower
than for G3G3G For the corresponding tetraoses the
difference was < 4000-fold
The mode of action of the T viride exo-1,3-b-glucanase
in hydrolysis of 1,3-b-oligoglucosides and curdlan is typical
of fungal exo-1,3-b-glucanases [17,19,23] The enzyme also displayed some activity towards 1,3-1,4-b-oligoglucosides
In contrast to barley 1,3-b-glucanase [44] and exo-1,3-b-glucanase from C albicans [45], the enzyme was inactive against cellooligosaccharides and p-nitrophenyl b-glucopyranoside
Subsite stucture of the active center For determination of the active center subsite structure the kinetic parameters Kmand Vmax of the hydrolytic reaction were measured using 1,3-b-oligoglucosides of DP 2 – 6 as substrates The initial rate of hydrolysis was measured as a function of substrate concentration For all 1,3-b-oligo-glucosides the reaction follows Michaelis – Menten kinetics Values for Km, Vmax and Vmax/Km for different substrates shown in Table 4 indicate that oligoglucosides with lower
DP have lower Vmax Because this enzyme exclusively displays exo-activity in the hydrolysis of 1,3-b-oligogluco-sides, the subsite theory of Hiromi [46,47] was employed for construction of a subsite map for the estimation of affinities and number of subsites in the active center of the enzyme According to this theory the kinetic parameters can be expressed in a unified way in terms of subsite affinities Aiof
m subsites and the intrinsic rate constant Kint for glucosyl bond cleavage [46] As shown in [47], the subsite affinities for sites A225 of the exo-1,3-b-glucanase according to Davies et al numbering system for exo-glycanases [48]can
be calculated from the Eqn (1):
lnðVmax/ KmÞn 112 lnðVmax/ KmÞn¼ 2ðAn 11/ RTÞ ð1Þ where Kmand Vmaxare the kinetic parameters of the exo-1,3-b-glucanase-catalyzed hydrolysis of 1,3-b-oligoglucosides of
DP 3 – 6 Assuming that there is only one nonproductive complex A21can be obtained from the Hiromi dependence
of 1/Vmaxon exp[ – An11/(RT )] [47] The value for A1can be estimated from the Eqn (2) based on preliminary calculated affinities A2 – A5:
exp ÿXnÿ1 i¼ÿ1
Ai
RT1 exp ÿ
Xn i¼1
Ai
RT1 …
The calculated affinities A21and A1– A5are represented in Fig 3A The diagram shows that negative values for binding energy were at subsites 21 to 1 4, so that the exo-1,3-b-glucanase from T viride strain AZ336 possesses at least five binding subsites of monomeric units at its binding site A
Fig 2 HPLC analysis of the hydrolytic products resulting from the
exo-1,3-b-glucanase action on curdlan Separation was performed on
a Lichrosorb NH 2 -60 column in 80% acetonitrile in water Elution rate
was 1 mL:min21: 1, glucose Inset: elution profile for a Lichrosorb
NH 2 -60 column separation of a mixture of 1,3-b-oligoglucosides:
1, glucose; 2, laminariobiose; 3, laminariotriose; 4, laminariotetraose;
5, laminariopentaose.
Table 3 Relative rate of hydrolysis of b -glucans, and oligosaccharides by the exo-1,3-b-glucanase.
Substrate
K m
(m M )
103:Vmax (mmol:min 21 :mg 21 )
Trang 6schematic model of the enzyme active centre is represented
in Fig 3B
The Hiromi subsite theory [46,47] is based on two
cardinal assumptions: firstly, each subsite has its own proper
affinity for a glucose residue of the oligoglucoside substrate
and there is no interaction between subsites; secondly, the
subsite affinities are additive The important thing is
regularity of a value for the intrinsic rate constant Kint
Accordingly, the substrate binding affinity becomes the sum
of the affinities for glucose residues of the substrate
accessible for binding; the distinction of Vmax(kcat) values is
conditioned by different quota of productive complexes for
various substrates Employing this model kinetic analysis of
the hydrolysis of oligosaccharides with different DP is an
effective tool for characterization of subsite structure of the
active center of exo-glucanases and glucosidases The Hiromi
subsite theory has initially been applied for glycoamylase
[47] Further it was employed in the determination of binding
affinities for various exo-glycosidases: a-glucosidase [49],
b-glucosidases [50,51] and glucoamylases [47,52] The
results obtained for a-glucosidase [49] as well as for
b-glucosidases from Aspergillus niger [50] and Candida
wickerhamii [51] demonstrated that for these enzymes the
active center affinity for substrates decreases as the DP of
the substrate increases: consequently, A21to A3have
posi-tive values A b-glucosidase from germinated barley has
been reported to have negative affinity at site 12 and
positive at the rest [53] This is in contrast to the results
obtained for the exo-1,3-b-glucanase described here where
the substrate affinity increases with increasing DP of the
substrate Thus, for this enzyme the affinities, A21to A4,
have negative values (Fig 3A) Negative values for Ai of
enzymes where Vmax increases with increasing DP of
oligosaccharide substrates is typical for glucoamylases from
several sources [47,52] and has also been described for an
exo-1,3-b-glucanase from C albicans [45] The latter enzyme
has similar structure of subsites at its active center where A21
has a negative value for affinity energy in contrast to values
for all other sites that are positive
Stereochemical course of action
1H NMR provides a direct method for determining the
stereochemical course of hydrolysis catalyzed by
glyca-nases Chemical shifts and coupling constants of the
anomeric protons in a- and b-glycosides as well as in the
product hemiacetals are distinct and readily observed When
sufficient exo-1,3-b-glucanase was used to complete
hydrolysis within 5 min the initially formed anomer
accu-mulated in sufficient amounts to permit detection before
mutarotation had occured to a significant extent NMR spectra reflecting the time course of laminarin hydrolysis catalyzed by exo-1,3-b-glucanase from T viride AZ36 are presented in Fig 4A Spectrum 1 shows the anomeric proton region of laminarin in buffer The large resonance peak at d 4.7 corresponds to the H2O protons Spectra 2 and 3 were recorded at different time intervals after addition of the
Fig 3 Subsite structure structure of the exo-1,3-b-glucanase (A) and schematic model of the exo-1,3-b-glucanase-binding site (B) (A) The subsite affinities A i are represented by the histogram (B) Schematic model of the exo-1,3-b-glucanase-binding site:
X, nonreducing terminus glucosyl residue; W, glucosyl residues; veritcal arrow, catalytic amino acids.
Table 4 Kinetic parameters of the hydrolysis of laminarioligosaccarides effected by the exo-1,3-b-glucanase.
Substrate (G n )
K m
(m M )
103:V max
(mmol:min 21 :mg 21 )
103:(V max /K m ) (min 21 : mg 21 )
ln (V max /K m )
Trang 7enzyme The resonance at d 5.23 (d, J ¼ 3.6 Hz) comes
from the equatorial anomeric proton of a-D-glucose In the
latter spectra (Figs 4A, 2 and 3), a resonance peak appears at
d 4.65 (d, J ¼ 7.9 Hz) from the axial anomeric proton of
b-D-glucose which is the product of mutarotation of the
initially formed a-glucose Mutarotation was considered
complete when the anomeric ratio (< 42% a, < 58% b) had
been established (about 40 min) Similar results were
obtained by1H NMR analysis of the hydrolysis of reduced
laminariotetraose (Fig 4B) The data unequivocally
demon-strate that enzymatic hydrolysis proceeds with inversion of
the anomeric configuration, presumably as a result of a
single displacement reaction [54] Moreover, as seen from
Fig 4B, the enzyme released only glucose from
1,3-b-oligoglucosides attacking the substrates in an exo-pattern
action
The anomer specificity of hydrolysis has been thoroughly
investigated for products of exo-1,3-b-glucanases from
Basidiomycete aphyllophorales [19] and barley malt
1,3-and 1,3-,1,4-b-glucanases [20] The first enzyme showed
inversion of the anomer configuration in contrast to the
barley enzymes Transglycosylating activity has been
demonstrated for 1,3-b-glucanases performing hydrolysis
with retention of anomeric configuration [20,26,45], but as
expected no such activity was observed for the inverting
exo-1,3-b-glucanase from T viride AZ36
Mode of action in the hydrolysis of laminarins
The mode of action of the exo-1,3-b-glucanase was studied
using laminarin from L digitata with a ratio of 1,3-b- and
1,6-b-linkages of 1 : 7 [55] and laminarin from L cichor-ioides, which has fewer side 1,6-b-Glc residues (<1 : 17) [56] The exo-1,3-b-glucanase exhibited a higher rate of hydrolysis of the glucan with the lower degree of ramification (Table 3) Kinetic parameters of the exo-1,3-b-glucanase in hydrolysis of curdlan from A faecalis, an essentially unbranched 1,3-b-glucan, are presented in Table 3 For this substrate, Kmis 10-fold higher and the rate of hydrolysis
is < twofold lower than for laminarin from L digitata The mode of action of the exo-1,3-b-glucanase on laminarin from L digitata was studied qualitatively by TLC and quantitatively by HPLC HPLC analysis of the reaction mixture revealed that glucose and gentiobiose
(6-O-b-D-glucopyranosyl-b-D-glucose) were liberated during hydrolysis (Fig 5) The latter was isolated from the reaction mixture and characterized by1H and13C NMR spectroscopy [30] Thus, the enzyme, although typically exo- in its mode of attack, can initiate endo-type cleavage of 1,3-b-bonds adjacent to 1,6-b-linkages At subsequent stages of hydrolysis, gentiobiose was degraded to glucose (Fig 5) Gentiobiose appeared to be a real substrate for the exo-1,3-b-glucanase The kinetic parameters are presented in Table 3 The values for Kmand Vmaxfor hydrolysis of G6G are similar to those of G3G (Tables 3 and 4) Therefore, it may be assumed that the enzyme has a mixed mode of action towards laminarins and that it is capable of degrading such branched b-glucans completely to glucose without syner-gistic action with a b-glucosidase Intermediate accumu-lation of gentiobiose was also observed during hydrolysis of scleroglucan by the exo-1,3-b-glucanase from Basidio-mycetes aphyllophorales [19] Similar action on laminarin from Eisenia bicyclis was reported for the exo-1,3-b-glucanase from Basidiomycetes sp QM 806 which released glucose, gentiobiose and gentiotriose [30], and the intra-cellular b-1,3-glucan hydrolase from Euglena gracilis [57]
In conclusion, it should be noted that Trichoderma species secrete a complex mixture of carbohydrases that are able
Fig 5 Kinetics of product formation during hydrolysis of laminarin from L digitata X, glucose; B, gentiobiose Laminarin from L digitata (4 mg) was incubated with exo-1,3-b-glucanase (5 U)
at 37 8C in 20 m M sodium acetate buffer, pH 4.5 After 25 min (left) and after 10 h (right) of the incubation the reaction was stopped by boiling The products were separated on a TSK-NH 2 -60 column (5 mm, 4.6 250 mm) with elution rate 0.8 mL:min21.
Fig 4 Time-course of a- and b-glucose formation (A) 1 H NMR
spectra of anomeric proton region of glucose showing the stereochemical
course of the hydrolysis of laminarin from L digitata by the
exo-1,3-b-glucanase 1, 1 H NMR spectrum of laminarin; 2, the spectrum of the
reaction mixture at an intermediate stage of hydrolysis (2 min); 3, after
40 min when mutarotation was close to completion (B) Kinetics of
a/b-glucose formation during the hydrolysis of reduced
laminariote-traose X, a-glucose; B, b-glucose; O, sum of a- and b-glucose.
Trang 8to affect components of the fungal cell wall such as chitin
and 1,3-b-glucan [58,59] Therefore, lytic enzymes
including endo- and exo-1,3-b-glucanases, exo-b-N
acetyl-glucosaminidases and chitinases may be important in
protecting plants against pathogenes Future detailed
investigation of different enzyme systems,
exo-1,3-b-glucanases, in particular, is essential for evaluating the
effectiveness of such multienzyme complex
A C K N O W L E G M E N T S
We thank Professor Irwin J Goldstein, Michigan University,
for his helpful assistance in sequencing the protein The
research was made possible by a grant 00-04-48878 from the
Russian Foundation for Basic Research
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