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Action of Trichoderma reesei mannanase on galactoglucomannan in pine kraft pulp
ELSEVIER
Journal
of Biotechnology
57 (1997) 191-204
Action of Trichoderma reesei mannanase on galactoglucomannan in pine kraft pulp Maija Tenkanen
+*, Mari Makkonen ‘TV,Marjukka Anita Teleman b
Perttula
‘, Liisa Viikari a,
a VTT Biotechnology
and Food Research, P.O. Box 1500, FIN-02044 VTT, Finland b VTT Chemical Technology, P.O.Box 1400, FIN-02044 VTT, Finland
Received
15 October
1996; received
in revised
form
10 March
1997; accepted
3 April
1997
Abstract The di-, tri- and tetrasaccharides formed during Trichoderma reesei endo-B-D-mannanase treatment of pine kraft pulp were studied. The oligosaccharides in the hydrolysate were fractionated using size-exclusion, anion exchange and activated carbon chromatography. The primary sequence of the purified oligomers was determined by two-dimensional NMR techniques. The T. reesei mannanase cleaves the /J-1,4-glycosidic linkage of D-mannOSy residues attached either to D-mannose or D-glucose. The D-mannosyl residue may also be substituted by a D-galaCtOSy1 group. The main disaccharide produced was mannobiose, but a significant amount of 4-O-jI-D-glucopyranosyl-D-mannopyranose (GlcMan) was also produced. After extensive hydrolysis the main trisaccharides produced were 4-0-b-Dmannopyranosyl-[6-O-a-galactopyranosyl]-D-mannopyranose (GallMan,) and 4-O-/I-D-glucopyranosyl-4-O-P-Dglucopyranosyl-D-mannopyranose (Glc,Man). Some mannotriose 4-0-/?-D-glucopyranosyl-4-O-/?-D-mannopyranosyl-D-mannopyranose (GlcMan,) and 4-0-8-D-glucopyranosyl-[6-O-cc-galactopyranosyl]-D-mannopyranose (Gal’GlcMan) were also detected in the hydrolysate. The structures of two tetrasaccharides were studied. They 4-O-~-D-gluCOpyranOsyl-4-0-~-D-gluCOpyranOsyl-4-O-~-D-glucopyranosy~-D-mannopyranose appeared to be (Glc,Man) and 4-0-~-D-glucopyranosyl-4-O-~-D-mannopyranosyl-4-0-~-D-glucopyranosyl-D-mannopyranose (GlcManGlcMan). According to the results obtained, the galactoglucomannan in pine contains regions in which two or three glucose units are linked together, which further means that it may contain regions with several successive mannose residues. The galactose side groups were found to be attached only to mannose. 0 1997 Elsevier Science B.V. Keywords: Endomannanase; troscopy
* Corresponding Ol68-1656/97/$17.00
Trichoderma reesei; Pine kraft pulp; Hydrolysis;
Manno-oligosaccharides;
author. Tel.: + 358 9 4565141; fax: + 358 9 4552028; e-mail: [email protected] 0 1997 Elsevier
PII SO168-1656(97)00099-O
Science
B.V. All rights
reserved.
NMR
spec-
192
M. Tenkanen et al. /Journal
1. Introduction The major softwood hemicellulose is an Oacetyl-galactoglucomannan (15-20% of wood) with various structural variations (Timell, 1967). In hardwood a low amount of glucomannan (25% of wood) is also present. During pulping the acetyl groups of hemicelluloses are totally removed. In kraft pulping about half of the galactoglucomannan is dissolved and further degraded, whereas the other half is present in softwood kraft pulps (SjiistrGm, 1981). The utilisation of endo-BD-mannanases (E.C. 3.2.1.78) has been shown to enhance delignification and to improve the bleachability of softwood pulps (Clark et al., 1990; Buchert et al., 1993a; SuurnCkki et al., 1994, 1996a,b; Saake et al., 1995). Hydrolysis of mannan-type polysaccharides by mannanases yields an array of oligomers, including the p-o-manno-oligosaccharides and a range of mixed oligosaccharides containing D-mannose, D-glucose and D-galactose. The nature of the oligosaccharides produced by different mannanases varies, indicating that there are differences in the action patterns of the enzymes (Kusakabe et al., 1988; McCIeary and Matheson, 1983; McCleary, 1991). Enzymes also differ in their action towards fibre-bound substrates. For example mannanase from Trichoderma reesei was able to hydrolyse galactoglucomannan in pine kraft pulp, whereas the mannanase from Bacillus subtilis was not (Ratta et al., 1993). Moreover, even if an enzyme is able to hydrolyse a fibrebound substrate, this does not necessarily correspond with delignification. In a comparative study of T. reesei, Aspergillus niger and Caldocellulosiruptor saccharolyticus mannanases, the T. reesei mannanase was found to be superior in bleach-boosting (Suurnlkki et al., 1996b). The mannanases from T. reesei and C. saccharolyticus belong to the same glycosyl hydrolase family 5, whereas the Bacillus subtilis mannanase has been assigned to the family 26 (Henrissat and Bairoch, 1996). However, the T. reesei and C. mannanases have only weak saccharolyticus amino acid similarities. The A. niger mannanase has not yet been classified but the mannanase from A. aculeatus, which has a high amino acid
0s Biotechnology
57 (1997)
191-204
similarity with the T. reesei mannanase (Stglbrand et al., 1995) and clear N-terminal similarity with the A. niger mannanase (StAlbrand, 1995), also belongs to the family 5 of glycosyl hydrolases. Mannanases in different families may have different catalytic properties, as in the case of xylanases (Biely et al., 1997). The differences in catalytic properties might explain the differences in bleachboosting efficiencies. Degradation of linear manno-oligosaccharides and polymeric mannan by T. reesei mannanase has been studied previously (Stilbrand et al., 1993; Harjunptig et al., 1995). Hydrolysis of both isolated and fibre-bound galactoglucomannans from pine wood and pine kraft pulp has also been studied in some detail (Rattii et al., 1993), but the structure of the oligosaccharides produced has not been verified and thus the action pattern and the substrate binding requirements of T. reesei mannanase are not known. In this work, the mode of action of T. reesei mannanase on galactoglucomannan in unbleached pine kraft pulp was studied and the substrate specificity was compared with other mannanases studied earlier.
2. Materials 2.1. Enzymes
and methods and substrates
The mannanase from T. reesei was purified as described by R&ii et al. (1993). P-Glucosidase from Aspergillus niger was purchased from Megazyme (Australia), p -mannosidase from A. niger was kindly provided by Folke Tjerneld and Pia Ademark from Lund University (Sweden) and cl-galactosidase I from Penicillium simplicissium was purified as described by Luonteri et al. (1997). The enzyme activities were determined according to the methods in Rgttii and Poutanen (1988). The pine kraft pulp with kappa number 25.8 was cooked in the laboratory. The production and properties of this pulp have been described previously (Teleman et al., 1995a). Locust bean gum (galactomannan, G-0753) was purchased from Sigma.
M. Tenkanen et al. /Journal of Biotechnology 57 (1997) 191-204
2.2. Hydrolysis experiments Pine kraft pulp (50 g l- ’ was hydrolysed with 200 or 2000 nkat g - ’ of T. reesei mannanase for 2 or 48 h, respectively, at pH 5 and 40°C. /?Mannosidase and P-glucosidase treatments of oligosaccharides were performed by hydrolysing with 500 nkat of enzyme per g of oligosaccharides for 24 h at pH 5 and 40°C. After secondary enzymatic hydrolysis of the released oligosaccharides to monosaccharides (Buchert et al., 1993b), the degree of hydrolysis was calculated by dividing the total amount of mannose in the hydrolysate by the amount in the pulp. 2.3. Isolation of oligosaccharides The pine kraft pulp (10 g) was suspended in water (20 g 1- ’ and the pH was adjusted to 5.0 with 0.1 M H,SO,, after which the pulp was hydrolysed for 48 h at 40°C with 2000 nkat gg ’ of T. reesei mannanase. The hydrolysate was concentrated to 30 ml by rotary evaporation and fractionated in three batches with Biogel P-2 (Bio-Rad, packed in 5 x 85 cm column) using water (2 ml min - ‘) as eluent. Fractions containing di-, tri- and tetrasaccharides were selected according to TLC. The fractions containing disaccharides were pooled and concentrated to 2 ml. According to TLC the disaccharide fraction contained mannobiose and an unknown disaccharide (M2_,). M,., was further purified with cation exchange resin, Dowex 5Ow*8 in Ba2+ form (200-400 mesh, Fluka, packed in 1.6 x 80 cm column) at 50°C using water (1 ml min - ‘) as eluent. Fractions were analyzed by TLC, after which the fractions containing M,_, were pooled, concentrated and lyophilized before further analysis by NMR spectroscopy. After pooling and concentration of the fractions containing trisaccharides the mixture (4 ml), which according to TLC contained at least three different compounds, was run through a column of Dowex 5Ow*8 as described above. According to TLC analysis of the fractions ob-
193
tained, only mannotriose was separated to some extent from the other unidentified trisaccharides, which were pooled, concentrated and separated further using activated carbon. A lo-ml sample was applied to a column (1.6 x 7 cm) of activated carbon (Oy Flinkenberg Ab, Finland). The column was washed with water, after which the bound oligosaccharides were eluted with a linear ethanol gradient from 0 to 25% v/v using a flow rate of 0.15 ml min ~ ‘. According to TLC analysis three different oligosaccharide fractions (M,_,, M,_,, M,.,) were pooled, which were concentrated and lyophilized. The tetrasaccharide fraction was further fractionated using the activated carbon column (1.6 x 7 cm) as described above, except that after washing with water the column was washed with 20% v/v ethanol. The bound oligosaccharides were separated with a linear ethanol gradient from 20 to 50% v/v. The fractions obtained were pooled in five pools and analyzed further by HPAEC-PAD, which showed that the first fractions contained trisaccharide (M3_J and the last fractions contained a new compound (M4_r). The fractionation of the hydrolysate is presented in Fig. 1. The Gal’Man, used as reference material was prepared from locust bean gum. First, 1 g of locust bean gum was mixed with 200 ml of 50 mM Na-acetate buffer, pH 5 and then heated to boiling point to dissolve the galactomannan. After cooling, the mixture was hydrolysed with 5000 nkat g _ ’ of T. reesei mannanase for 48 h at 40°C. The hydrolysate was concentrated lofold and then fractionated by gel filtration in two batches using Biogel P-2 gel as described above. GallMan, was further separated from mannotriose with Dowex 5Ow*8 resin as described above. 2.4. Analytical techniques Separation of manno-oligosaccharides on TLC was achieved using precoated aluminium sheets of silica gel (Kieselgel 60, Merck). The mobile phase contained ethylacetate: acetic acid: water = 9:6:6. Visualisation was performed by
194
M. Tenkanen et al. /Journal
of’ Biotechnology 57 (1997) 191-204
Hydrolysis with T. reesei mannanase
1Mixture of oligosaccharides 1 Biogel P-2
Dowex 50 W in Ba2+ form
l
Dowex 50 W
8
l
8 Activated carbon
Man,
Activated carbon
4,
Gal’ Man,
Fig. 1. Purification procedure of oligosaccharides in isolated preparations are presented.
Tetrasaccharides
Trisaccharides
Disaccharides
M3-2 Glc Man, Gall Glc Man
M3-3
h-1
Glc, Man
Glc, Man Glc Man Glc Man
from the pine kraft pulp hydrolysate.
spraying with a solution containing 80% ethanol, 10% H,SO,, 10% water and 0.2% orcin (Merck). manno-oligosaccharides (Man,-Man,, Linear Megazyme, Australia) were used as standards. The high-performance anion-exchange chromatography (HPAEC-PAD) of oligosaccharides was performed using a CarboPac PA-l column (Dionex Corp, USA) in a Dionex DX 500 series chromatograph equipped with pulse amperometric detection (Dionex ED 40). The system was equilibrated with 2.5 mM NaOH. After sample injection 2.5 mM NaOH was run through the column for 23 min and a linear gradient from 2.5 to 100 mM NaOH was created during the next 17 min. The second linear gradient from 100 mM NaOH to 100 mM NaOH + 150 mM Na-acetate was run in the column during the next 20 min, after which the column was washed with 100 mM NaOH + 300 mM Na-acetate and then with 300 mM NaOH. The flow rate used was 1 ml min- ‘. The standards used were commercially available linear manno-oligosaccharides and Gal’Man, (Megazyme).
Only the structures
of main oligosaccharides
For NMR analysis the lyophilized samples were dissolved in D,O (99.8 atom%, Fluka) and the pH was adjusted to 7 with NaOD and/or DCl. The ‘H NMR and 13C NMR spectra were obtained at 599.86 and 150.85 MHz, respectively, on a Varian UNITY 600 MHz spectrometer. Typical acquisition parameters were for 1D ‘H NMR (1D 13C NMR) a 70” pulse of 14.8 ps (8 ps), a spectral width of 8000 Hz (40000 Hz) and a repetition time of 22 s (3 s). Spectra were obtained at 70°C. The chemical shifts are reported relative to an internal acetone standard at 2.225 ppm for ‘H spectra. Standard pulse sequences and phase cycling were utilised to obtain phase-sensitive COSY, relay COSY, TOCSY, ROESY and NOESY 2D spectra. A spectral width of 1500 Hz (2100 Hz) for GlcMan (Gal’Man,) was employed in both dimensions and the relaxation delay was 2.2 s (2.5 s). ‘H-detected HMQC and HMBC spectra were acquired over a t, spectral window of 7500 Hz and a t2 spectral window of 1500 Hz (2100 Hz). Full details of the 2D experiments have been reported earlier (Teleman et al., 1996).
M. Tenkanen et al. /Journal
of Biotechnology 57 (1997) 191-204
3. Results 3.1. Extensive
hydrolysis
Table 1 Relative amounts of sugar residues in the mannanase hydrolysate of pine kraft pulp after extensive hydrolysis (2000 nkat g-‘, 48 h)
of pine kraft pulp
The action of Trichoderma reesei mannanase on pine pulp galactoglucomannan was first studied by analysing the whole hydrolysate obtained after extensive enzyme treatment (2000 nkat g- ‘, 48 h) by ‘H NMR spectroscopy and HPAEC-PAD (Figs. 2 and 3). After the enzyme treatment 20.4 wt% of pulp galactoglucomannan was hydrolysed to soluble oligosaccharides. The galactoglucomannan solubilised from pine kraft pulp was extensively hydrolysed to a mean degree of polymerization of 2.1. Almost all of the oligosaccharides produced had a mannose residue at the reducing end and only about 2 mol% had a reducing end glucose (Fig. 2). The observed reducing end glucose most probably originated from reducing ends of the polymers. The galactose groups in the oligosaccharides formed were attached by a-(1 +6)-glycosidic linkages only to mannose. The ratio of Man:Glc:Gal in the hydrolysate was 3.6:1.0:0.3 as analysed by ‘H NMR and 3.5:1.0:0.3 as analysed by HPAEC-PAD (Table 1). According to HPAEC-PAD analysis the main product formed was mannobiose (350 mg l- ‘, 40 wt% of solubilised oligosaccharides). A rather high amount of mannose (95 mg l- ‘, 10%) was also produced, whereas only a small amount of mannotriose (8 mg l- ‘, 1 %) was detected in the hydrolysate. In addition to these quantified MZUI reducing end a
M.Ul reducing end $
I
internal +
Glc B(1-w internal +
terminal
I”“~““,““,““l~“~,~“.,““,““,““,~,~~,..’~r
5.5
5.4
5.3
5.2
5.1
5.0
4.9
4.8
4.1
4.6
195
4.5 ppm
Fig. 2. Anomeric proton region of the ‘H NMR spectrum of the pine kraft pulp hydrolysate after extensive mannanase treatment (2000 nkat g-‘, 48 h).
Method
Carbohydrate Man
‘H NMR HPAEC-PAD
mol (‘%J) 73.4 mol (“XI) 71.8 mg 1-l 663
content
Glc
Gal
Others
20.4 20.3 188
5.8 6.9 64
0.4 1.0 9
manno-oligosaccharides many other oligosaccharides, including three main and several minor unknown compounds, were formed (Fig. 3A). The other oligosaccharides represented 49% of the soluble sugars formed and they contained 29% of hydrolysed mannose and all the hydrolysed glucose and galactose. In order to obtain information about the structure of the unidentified oligosaccharides the hydrolysate was treated with P-mannosidase and /3-glucosidase. During P-mannosidase treatment mannobiose and mannotriose were degraded to mannose (Fig. 3B). The peak with a retention time of 43.77 min (peak 5) was slightly decreased and the peak at 34.43 min was slightly increased. No detectable differences were observed in the other main peaks. After /?-glucosidase treatment the main peaks with retention times 45.43 (peak 6) and 51.28 (peak 9) min and the minor peaks with retention times 48.07 (peak 7) and 53.50 (peak 10) min decreased (Fig. 3C). These peaks obviously contained oligosaccharides with terminal glucose units. 3.2. Identljication
of disaccharides
The main unidentified components were isolated from the hydrolysate for further structural studies (Fig. 1). The isolation of the oligosaccharides was started by running the hydrolysate through a gel filtration column (Biogel P-2) which separated the sugars according to their size. The fractions were analysed by TLC and those containing di- tri- and tetrasaccharides were pooled for further purification.
196
M. Tenkanen
et al. /Journal
oJ’ Biotechnology
57 (1997) 191-204
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Fig. 3. HPAEC-PAD chromatogram of the pine kraft pulp hydrolysate after extensive mannanase treatment (2000 nkat g ‘, 48 h); (A) after further hydrolysis of the hydrolysate in A with p-mannosidase (B) and b-glucosidase (C) and after limited mannanase treatment (200 nkat g ‘, 2 h) (D). See text for the peak identification. IS, internal standard; 1, Glc; 2, Man; 3, Man,; 4, Man,; 5, Gal’Man,; 6, GlcMan; 7, GlcMan,; 8, Gal’GlcMan; 9, GlcGlcMan; 10, tetrasdccharides; 11, Gal’ Man,.
The disaccharide fraction was further fractionated using Dowex 5Ow*8 resin in Ba* + form. The unidentified disaccharide (M2_,) was separated from mannobiose and its structure was solved with NMR spectroscopy analysis. The structure of M,., was 4-0-p-D-glucopyranosyl-D-mannopyranose (GlcMan). The assignment of ‘H and 13C NMR resonances was based on phase-sensitive COSY, relay COSY and TOCSY experiments starting from the anomeric protons (Table 2). The signal ratio for Man H-la and H-l/? was 67:33, which is consistent with the literature data (McCleary et al., 1982; Harjunpaa et al., 1995). In the rotatingframe NOE spectroscopy (ROESY) spectrum the Glc H-l signals at 6 4.5 15 and 4.506 have crosspeaks with H-4 of Man at 6 3.80 and 3.87, thereby
establishing the sequence of GlcMan. The structure was also verified by p -mannosidase and B -glucosidase treatments. ,6’-Mannosidase did not whereas p -glucosidase hydegrade GlcMan, drolysed it to glucose and mannose in a ratio of 1.2:1 (results not shown). No disaccharides with reducing end glucose (ManGlc or cellobiose) were found in the disaccharide fraction. In the HPAECPAD analysis the isolated GlcMan had a retention time (45.40 min) close to that of the main unidentified peak in Fig. 3A (45.43 min, peak 6), which indicated that the peak was GlcMan. 3.3. IdentiJication The trisaccharide
of trisacchurides fraction,
which
according
to
M. Tenkanen et al. /Journal
,
“,
.
.
.
.
.
.
of Biotechnology 57 (1997) 191-204
.
.
.
.
,
.
.
.
.
10
20
30
40
t,“‘,““I,,,,,,,,‘,‘, 10 0
20
30
40
0
.
.
.
.
197
,
.
.
,
.
(
50
60
50
60
,‘,“‘,I
Retention time (minutes) Fig. 3 (contd.)
TLC contained at least three different compounds, was further fractionated using Dowex 5Ow*8 resin and activated carbon (Fig. 1). The different trisaccharides obtained were not completely separated from each other, but the primary structures of the major components could be determined by ‘H NMR spectroscopy and treatments with /?-mannosidase, /?-glucosidase and e-galactosidase. The amount of isolated oligosaccharides was too low to obtain r3C NMR data. For all of the trisaccharides the reducing residue was readily identified as mannose because of the chemical shifts, coupling constants and relatively smaller intensities in the ‘H NMR spectra of the anomeric proton of the cx and /? forms. The ‘H NMR resonances were assigned by 2D NMR techniques starting from the anomeric protons (Table 2).
The main galactose-containing oligosaccharide formed from galactomannan by T. reesei mannanase, 4-U-@-D-mannopyranosyl-[6-O-ol-galactopyranosyll-D-mannopyranose (GallMan,), was isolated from the locust bean gum hydrolysate and used as a reference compound in the ‘H NMR and HPAEC-PAD analyses of the puipderived trisaccharides. The assignments of ‘H (Table 2) and 13C (results not shown) NMR data for GallMan, were based on phase-sensitive COSY, relay COSY, TOCSY and HMQC experiments starting from the anomeric protons. Having assigned all of the ‘H and 13C resonances, the primary sequence was directIy obtainable from the HMBC spectrum, in which three-bond interresidue ‘H/13C correlations were detectable. The observed cross-peaks bH- 1/a&-4, bH- 1/afi C-4, cH-l/a&-6, cH-l/a/3C-6, aaH-4/bC-1, a,BH-4/ bC- 1, aaH-6/cC- 1, ap H-6/cC- 1 and a/? H-6’/cC- 1
M. Tenkanen et al. /Journal
198 Table 2 ‘H NMR
data
Compound”
for isolated
oligosaccharides
Residue
Chemical
shiftb (coupling
constant”)
H-2
H-3
H-4
H-5
H-6
H-6’
H-l Glc-Man b a
of Biotechnology 57 (1997) 191-204
a-Manp-a p-Manp-a p-GIcp-b, /i’-Glcp-b,j
5.187 4.904 4.515 4.506
(1.8) (1.1) (7.9) (7.9)
3.99 4.01 3.33 3.32
3.96 3.77 3.53 3.52
3.87 3.80 3.43 3.43
3.92 3.52 3.50 3.52
3.92 3.96 3.93 3.93
3.85 3.82 3.74 3.74
x-Manp-a /j’-Manp-a /J-Manp-b, p-Manp-b, r-Galp-c, r -Galp-c,j
5.184 4.910 4.741 4.736 5.021 5.030
(1.7) (1.1)
4.00 4.01 4.08
4.01 3.82 3.64
3.95 3.89 3.62
4.08 3.67 3.42
3.99 3.95 3.94
3.76 3.80 3.76
3.84
3.88
4.02
3.91
3.77
3.77
a-Manp-a p-Manp-a p-Manp-b, p-Manp-b,, p-Glcp-c
5.184 4.904 4.758 4.751 4.503
(-1.7) (1 .O) (-0.8) (-0.8) (7.9)
3.99 4.01 4.12
3.98 3.80 3.77
3.91 3.82 3.82
3.7-3.9 3.6-4.0 3.58
3.773.9 3.664.0 4.01
3.7-3.9 3.6-4.0 3.84
3.32
3.52
3.42
3.51
3.93
3.74
a-Mary-a /J-Manp-a p-Glcp-b, p-Glcp-b, 8-Glcp-c
5.186 4.904 4.542 4.532 4.516
(1.7) (1 .O) (7.9) (7.9) (7.9)
3.99 4.00 3.39
3.97 3.77 3.66
3.90 3.83 3.61
3.773.9 3.664.0 3.65
3.7-3.9 3.6-4.0 3.99
3.773.9 3.664.0 3.82
3.33
3.52
3.43
3.49
3.92
3.74
c
I Gal Man-Man b a
Clc-Man-Man c b a
Glc-Glc-Man c b a
(1.O) (1 .O) (3.8) (3.8)
‘- = /J-1,4-linkage; 1= r-l ,6-linkage. The reducing end is denoted as a. bin ppm relative to internal acetone at 2.225 ppm (D,O, 70°C and pH 7) acquired “Observed first order coupling in Hz.
established the sequence of Gal’Man,. The chemical shifts for the carbons are in good agreement with published values (McCleary et al., 1982). The trisaccharides were fractionated in three fractions (M,.,, M,., and M,_,). The main component in M,_, according to ‘H NMR and HPAECPAD analysis was Gal’Man, (peak 5, - 80%). The fraction also contained some mannotriose (peak 4, - 20%) (Fig. 4A). The main component in M,_, (Fig. 4C) was 4-O-j?-D-glucopyranosyl-40-p-D-glucopyranosyl-D-mannopyranose (Glc,M,_, also conMan, peak 9, - 70%). Fraction tained some of the tetrasaccharides found in M,_, (see below). According to HPAEC-PAD analysis M,., contained three different oligosaccharides (Fig. 4B). According to ‘H NMR analysis the main component in M,., was
at 600 MHz.
4-0-p-D-glucopyranosyl-P-D-mannobiose (Glc60%). The primary sequences of Man,, Glc,Man and GlcMan, were obtainable from the ROESY and NOESY spectra, in which interresidue cross-peaks were observed at the H-l frequencies. M,_, also contained some Glc,Man ( - 10%) and an oligosaccharide that had galactose linked to a reducing end mannose ( - 30%). However, the amount of Gal’Man, was very low. In the anomeric proton region of the ‘H NMR spectrum of fraction M,., there were two additional non-reducing end (terminal) glucose resonances (6 4.501 J,,, = 7.9, 6 4.497 J,,, = 7.9). Thus it seemed that the third trisaccharide in fraction MS-2 could be 4-O-p-glucopyranosyl-[6-O-a-Dgalactosyll-D-mannopyranose (Gal’GlcMan). M,_ 2 was treated with a-galactosidase which hydrolysed the peak with retention time 49.08 min
M. Tenkanen el al. /Journal
of Biolechnology 57 (1997) 191-204
199
A 200
0 0
10
20
30
40
60
50
B 78
0
10
20
30
40
50
60
0
10
20
30
40
50
60
0
Retention time (minutes) Fig. 4. HPAEC-PAD identification.
chromatograms
of the isolated
trisaccharide
(peak 8) to produce galactose and GlcMan (re sults not shown). Thus this peak contained
fractions;
M,.,
(A); M,.,
(B) and M,.,
(C). See Fig. 3 for peak
Gal’GlcMan and the peak with 48.00 min (peak 7) was GlcMan,.
retention
time
200
M. Tenkanen
3.4. IdentiJication
et al. /Journal
of tetrasaccharides
The amount of sugars in the tetrasaccharide fraction was very low and after further purification on the activated carbon column only one sample M,_, was obtained for the ‘H NMR analysis. According to HPAEC-PAD, M,_, contained one main peak. /J’-Glucosidase treatment reduced the size of the main peak, producing glucose and GlcMan but no mannobiose (results not shown). The amount of isolated tetrasaccharides was too low to obtain 2D NMR data. However, the structural characterisation can be based on the chemical shifts and coupling constants of the so-called structural reporter signals, i.e. well separated resonances mainly originating from the anomeric proton of the monosaccharide residues. This method relies on a good spectral library of related compounds. The data in Table 2, as well as mannooligosaccharide and cello-oligosaccharide data reported in the literature (Teleman et al., 1995b; Harjunpaa et al., 1995), was used for the structural characterisation. Almost all of the tetrasaccharides produced have a mannose residue at the reducing end. From the relative proportions of the different structural elements (Fig. 5), it can be internal Glc * linked
toGlc 0raMan
,
terminal Glc
I
,
1linked internal Man
I
’
linked to Glc
I terminal Man ,bl
linked to Man
qf Biotechnolog)~ 57 (1997) 1916204
concluded that the most abundent structure of the tetrasaccharides is 4-O-/?-D-glucopyranosyl-4-Op-D-glucopyranosyl-4-O-P_D-glucopyranosyl-Dmannopyranose (Glc,Man) and that the second most abundant is most probably 4-o-p-D-glucopyranosyl-4-0-P-D-mannopyranosyl-4-O-PD-glucopyranosyl-D-mannopyranose (GlcManGlcMan). 3.5. Limited
hydrolysis
The pine kraft pulp was also hydrolysed for 2 h by 200 nkat g ~ ’ of mannanase in order to study the oligosaccharides produced in the limited hydrolysis used in mannanase aided bleaching of kraft pulps. The degree of hydrolysis of pulp galactoglucomannan was 3.5 wt%. The main product was mannobiose (38 mg l- ‘, 23 % of solubilised oligosaccharides) (Fig. 3D). The amount of mannose produced was below the detection limit but a significant amount of mannotriose (28 mg 1~ ‘, 17 %) was formed. Mannobiose and mannotriose represented 61% of the solubilised mannose. The mixed oligosaccharides produced represented 60 and 39% of solubilised sugars and mannose, respectively. According to HPAEC-PAD the main mixed oligosaccharides were GlcMan (peak 6) and GlcMan, (peak 7) (Fig. 3D). GalMan, (peak 5) and GlcGlcMan (peak 9) were also detected in the hydrolysate. The main unidentified peak, which was not detected after extensive hydrolysis, had a retention time 46.25 min (peak 11). This peak disappeared after /5-mannosidase treatment but p-glucosidase treatment did not affect it (results not shown). Thus it contained a non-reducing end mannose. The peak was identified to be Gal’Man, by comparing the retention time to commercially available Gal’Man,. The hydrolysate also clearly contained oligosaccharides with a degree of polymerisation higher than 4 and retention times longer than 54 min.
4. Discussion 4.76
4.14
4.72
4.55
4.53
4.51
4.49
ppm
Fig. 5. Analysis of tetrasaccharide fraction M,, by and proton NMR. The different structural elements are according to Table 2, (Teleman et al. (1995b) and HarjunpZi et al. (1995)).
According to the present study the mannanase from T. reesei is able to cleave not only p-1,4linkages between two mannosyl residues, but also
M. Tenkanen
Isolated di-andtrisaccharides
,
et al. /Journal
of Biotechnology
57 (1997) 191-204
l
--
-
,
I
201
4
I
Gal
Gal
$4 &J 14 J J 4 A 4 I$ -Man-Man-Glc-Man-Man-Man-Man-Man-Man-M~-~-M~-Glc-Glc-M~-Man-Gic-M~-Man$
$
5T
5
Fig. 6. Hypothetical presentation of the action of T. reesei mannanase on galactoglucomannan from pine kraft pulp. linkage; 1= r-1,6-linkage; 1 = linkage that can be hydrolysed; X with arrow = linkage that cannot be hydrolysed.
after mannosyl residue attached to glucose. It is not able to hydrolyse the linkage after glucosyl residue. The mannosyl residue can also be substituted by a galactosyl group, as the smallest galactose-containing oligosaccharide obtained was Gal’Man,. The action of T. reesei mannanase on galactoglucomannan from pine kraft pulp is summarized in Fig. 6. After extensive hydrolysis (2000 nkat g - ‘, 48 h) the galactoglucomannan was mainly hydrolysed to mannobiose and GlcMan (Fig. 3A). The main trisaccharides produced were Gal’Man, and Glc,Man. After limited hydrolysis (200 nkat, 2 h) mannobiose and GlcMan were produced in same ratio as after extensive hydrolysis, but clear differences were observed in the trisaccharides formed (Fig. 3A, D). After limited hydrolysis the amounts of mannotriose and GlcMan, were rather high as compared to those of Gal’Man, and Glc,Man, and thus more mannose-containing trisaccharides were formed at the early stage of hydrolysis. The amount of mixed oligosaccharides was clearly higher after limited hydrolysis (60% of solubilized oligosaccharides) than after extensive hydrolysis (49%). The enzyme thus seems to act first on regions with successive unsubstituted mannose units, producing mixed oligosaccharides. After limited hydrolysis the hydrolysate also contained long oligosaccharides having a DP higher than 4 (retention time longer than 54 min), which disappeared during longer incubation. T. reesei mannanase could further degrade mannotriose and GlcMan,, as their amounts were very low after extensive hydrolysis. The hydrolysis might proceed through transglycosylation of trisaccharides first to hexasaccharides which are then degraded further (Harjunpaa et al., 1995).
-
= p-1,4-
The ratio of Man:Glc:Gal in the hydrolysate was 3.5:1.0:0.3. This is in good agreement with earlier reported value of 3.5:1.0:0.4 analysed from pine pulp hydrolysate produced by T. reesei mannanase (Buchert et al., 1993a). It seems that T. reesei mannanase is able to hydrolyse both types of galactoglucomannans found in softwoods, having ratios of Man:Glc:Gal 3:l:l and 4:l:O.l (Timell, 1967). The action of several mannanases from legume seeds and micro-organisms on various galactomannans and glucomannans has been studied in detail (Emi et al., 1972; McCleary, 1979; Shimazu and Ishihara, 1983; McCleary et al., 1983; McCleary and Matheson, 1983; McCleary, 1991; Takahashi et al., 1984; Kusakabe et al., 1988; Park and Chang, 1992). However, only a few studies have been performed with galactoglucomannans and there is only one study in which the products formed after the hydrolysis of galactoglucomannan from softwood kraft pulp have been analysed (Ratto et al., 1993). The extent of hydrolysis of galactomannans has been shown to be dependent on the extent of substitution and the pattern of distribution of substituents. The action of mannanases on glucomannans is dependent on the glucose/mannose ratio. The microbial mannanases studied include those from Bacillus subtilis, Irpex lacteus, Aspergillus niger, Tyromyces palustris, Streptomyces sp, Penicillium purpurogenum and Penicillium kloeckeri. GallMan, was the smallest substituted oligosaccharide produced in the hydrolysis of galactomannans by A. niger, I. lacteus and P. purpurogenum mannanases and P. kloeckeri mannanase 2 (McCleary and Matheson, 1983; Park and Chang, 1992; Biely et al., 1996). B. subtilis
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mannanase and P. kloeckeri mannanase 1 have a more limited ability to hydrolyse galactomannans. GalMan, was the shortest substituted product formed by B. subtilis mannanase and the main monosubstituted oligosaccharide was GalMan, (Emi et al., 1972; McCleary and Matheson, 1983). P. kloeckeri mannanase 1 produced GalMan, as the shortest substitued oligosaccharide (Biely et al., 1996). The structure of oligosaccharides found after the hydrolysis of glucomannans has also varied. All mannanases studied have produced GlcMan and GlcMan,. P. purpurogenum and Streptomyces sp. mannanases produced Glc,Man, which was not detected in the hydrolysates produced by A. niger and T. palustris mannanases. There also appear to be differences in the structures of tetrasaccharides containing glucose and mannose produced by these mannanases (McCleary and Matheson, 1983; Shimazu and Ishihara, 1983; Takahashi et al., 1984; Kusakabe et al., 1988). The action patterns of T. reesei and P. purpurogenum mannanases appeared to be similar. A. niger mannanase showed similar action on galactomannans to the T. reesei mannanase but differed in its action on glucomannans. A. niger mannanase is not reported to produce any tri- or two adjacent glucose tetrasaccharides with residues, whereas Glc,Man was the main linear trisaccharide produced by T. reesei mannanase. Thus it is seems that the binding requirements of the subsites of the T. reesei mannanase are different from those of A. niger mannanase. However, the substrate used for A. niger mannanase was salep glucomannan, which might have a different distribution of glucose and mannose units from that of the konjac glucomannan used as substrate for P. purpurogenum and Streptomyces sp. mannanases and pine pulp galactoglucomannan studied in this work. The ratio of mannose to glucose is 4:l in salep glucomannan, and thus the possibility of successive glucose units is equal to that in pine pulp galactoglucomannan, whereas konjac glucomannan contains much more glucose (Man:Glc = 1.5:l.O). Of the above-mentioned mannanases only the actions of mannanases from A. niger, B. subtilis and P. kloeckeri have been studied on galactoglu-
of Biotechnology
57 (1997) 191-204
comannans. It was reported that a mixed Gal’GlcMan was produced by A. niger mannanase, but the structures of other oligosaccharides formed during hydrolysis were not reported (McCleary, 1991). The structures of oligosaccharides formed by B. subtilis and P. kloeckeri mannanases were also not studied in detail (Ratto et al., 1993; Biely et al., 1996). Mannanases in different families might have different catalytic properties towards substituted mannans, similarly to the case of xylanases (Biely et al., 1997). T. reesei and A. niger mannanases, which appear to belong to the same glycosyl hydrolase family (family 5) (Henrissat and Bairoch, 1996; Stalbrand, 1995), produced Gal’Man, from galactomannan whereas mannanase from B. subtilis, which produced Gal’Man, as the shortest substitued oligosaccharide, has been assigned to a different family (family 26) (Henrissat and Bairoch, 1996). The differences in the action patterns of these enzymes do not, however, explain the differences observed in the mannanase-aided bleaching of pine kraft pulps (Suurnakki et al., 1996b). The action of A. niger and B. subtilis mannanases on pine pulp galactoglucomannan should also be studied in detail before any conclusions can be drawn about the action mechanisms of these enzymes and their effects on pulp bleachability. Enzymes are powerful tools, not only in the modification of polymers but also in their structural analysis. According to the present study it appeares that galactoglucomannan in pine contains regions with several glucose units in secan form strong quence. These structures intermolecular hydrogen bonds with cellulose, which might be important for cellulose-galactoglucomannan interactions in wood and pulp. If the backbone contains 2-3 successive glucose units it might correspondingly contain 7710 successive mannose units. This type of structure in wood galactoglucomannan have also earlier been suggested by Holmbom (1996) (personal communication). The mannanase from T. reesei is known to contain a cellulose-binding domain (CBD), the role of which is unclear (Tenkanen et al., 1995; Stalbrand et al., 1995). Because galactoglucoman-
M. Tenkanen et al. /Journal
of Biotechnology 57 (1997) 191-204
nan and cellulose are closely associated in plant tissues, Stalbrand et al. (1995) suggested that the CBD, by binding on cellulose, might target the mannanase close to the substrate. The T. reesei mannanase does not specifically bind on mannan (Tenkanen et al., 1995) but several sequential glucose units in galactoglucomannan may bind the CBD. However, the binding of CBD on cellulose requires six successive glucose units (Linder et al., 1995) which were not found in the oligosaccharides isolated in this study but may be present in native softwood galactoglucomannas.
Acknowledgements Matti
Siika-aho
is thanked for purification of Folke Tjerneld and Pia Ademark for p-mannosidase, Elina Luonteri for c1galactosidase, Maria Sandelin, Tapani Vuorinen and Timo Paakkiinen for help in the oligosaccharide purification, Ulla Lahtinen and Paivi Matikainen for the HPAEC-PAD analysis and Riitta Isoniemi for technical assistance. The work was mainly financed by TEKES (Technology Development Centre, Finland). T. reesei mannanase,
References Biely, P., McCay, D.L., Farrell, R.L., 1996. Catalytic properties of two major endo+l,4-mannanases of Penicillium kloeckeri. In: Messner, K., Srebotnik, E. (Eds.), Biotechnology in the Pulps and Paper Industry: Advances and Fundamental Research. WUA-Universitatslag, Vienna, Austria. pp. 479-484. Biely, P., Vrsanska, M., Tenkanen, M., Kluepfel, D., 1997. Endo-/3-l-4-xylanase families: Difference in catalytic properties. J. Biotechnol. this issue Buchert, J., Salminen, J., Siika-aho, M., Viikari, L., 1993a. The role of T. reesei xylanase and mannanase in treatment of softwood kraft pulp prior to bleaching. Holzforschung 47, 413-478. Buchert, J., Siika-aho, M., Bailey, M., Pulps, J., Valkeajarvi, A., Pere, J., Viikari, L., 1993b. Quantitative determination of wood-derived soluble oligosaccharides by HPLC. Biotechnol. Tech. 7, 7855790. Clark, T.A., McDonald, A.G., Senior, D.J., Mayers, P.R., 1990. Mannanase and xylanase treatment of softwood chemical pulps: effects on pulp properties and bleachability. In: Kirk, K.T., Chang, H.-M. (Eds.), Biotechnology in
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Pulp and Paper Manufacture. Butterworth-Heinemann, Boston, pp. 153-167. Emi, S., Fukumoto, J., Yamamoto, T., 1972. Crystallization and some properties of mannanase. Agric. Biol. Chem. 36, 991-1001. Henrissat, B., Bairoch, A., 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 3 16, 695 696. Harjunpaa, V., Teleman, A., Siika-aho, M., Drakenberg, T., 1995. Kinetic and stereochemical studies of mannooligosaccharide hydrolysis catalyzed by /?-mannanases from Trichoderma reesei. Eur. J. Biochem. 234, 278-283. Kusakabe, I., Park, G.G., Kumita, N., Yasui, T., Murakami, K., 1988. Specificity of p-mannanase from Penicillium purpurogenum for konjac glucomannan. Agric. Biol. Chem. 52, 519-524. Linder, M., Mattinen, M.-L., Kontteli, M., Linderberg, G., Stalberg, J., Drakenberg, T., Reinikainen, T., Pettersson, G., Annila, A., 1995. Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I. Protein Sci. 4, 1056-1064. Luonteri, E., Alatalo, E., Siika-aho, M., Penttill, M., Tenkanen, M., 1997. n-Galactosidases of Penicillium simplicissium; production, purification and characterization of gene encoding AGL I (submitted). McCleary, B.V., 1979. Modes of action of /I-mannanase enzymes of diverse origin on legume seed galactomannans. Phytochemistry 18, 757-763. McCleary, B.V., Taravel, F.R., Cheetham, N.W.H., 1982. Preparative-scale isolation and characterisation of 6'-X-Dgalactosyl-(1,4)-p-D-mannobiose and 6’-r-D-galactosyl(1,4)-b-D-mannobiose. Carbohydr. Res. 104, 2855297. McCleary, B.V., Matheson, N.K., 1983. Action patterns and substrate-binding requirements of /?-D-mannanase with mannosaccharides and mannan-type polysaccharides. Carbohydr. Res. 119, 191-219. McCleary, B.V., Nurthen, E., Taravel, F.R., Joseleau, J.-P., 1983. Characterisation of the oligosaccharides produced on hydrolysis of galactomannan with /?-D-mannanase. Carbohydr. Res. 118, 91-109. McCleary, B.V., 1991. Comparison of endolytic hydrolases that depolymerize 1,4-B-D-mannan, 1,5-cc-L.-arabinan, and 1,4-/I’-D-galactan. In: Leatham, G.F., Himmel, M.E. (Eds.), Enzymes in Biomass Conversion. ACS Symp., American Chemical Society, Washington, DC, Ser 460, pp. 437-449. Park, G.G., Chang, H.G., 1992. Separation and preparation of galactosylmanno-oligosaccharides from copra galactomannan by mannanase from Penicillium purpurogenum. J. Microbiol. Biotechnol. 2, 204-208. R&to, M., Poutanen, K., 1988. Production of mannan-degrading enzymes. Biotechnol. Lett. 10, 661-664. Ratto, M., Siika-aho, M., Buchert, J., Valkeajarvi, A., Viikari, L., 1993. Enzymatic hydrolysis of isolatated and fibrebound galactoglucomannans from pine-wood and pine kraft pulp. Appl. Microbial. Biotechnol. 40, 4499454.
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Saake, B., Clark, T., Puls, J., 1995. Investigations on the reaction mechanism of xylanases and mannanases on sprucewood chemical pulps. Holzforschung 49, 60-68. Shimazu, K., Ishihara, M., 1983. Isolation and characterization of oligosaccharides from the hydrolysate of larch wood glucomannan with endo-p-D-mannanase. Agric. Biol. Chem. 47, 949. Sjostrom, E., 1981. Wood Chemistry, Fundamentals and Applications, Academic Press, New York. Stalbrand, H., Siika-aho, M., Tenkanen, M., Viikari, L., 1993. Purification and characterization of two /!-mannanases from Trichoderma reesei. J. Biotechnol. 29, 2299242. Stalbrand, H., 1995. Hemicellulose-degrading enzymes from fungi, PhD Thesis, Lund University. Stalbrand, H., Saloheimo, A., Vehmaanpera, J., Henrissat, B., Penttila, M., 1995. Cloning and expression in Succharomyces cereuisiae of a Trichoderma reesei /?-mannanase gene containing a cellulose binding domain. Appl. Environ. Microbial. 61, 1090- 1097. Suurnakki, A., Kantelinen, A., Buchert, J., Viikari, L., 1994. Enzyme aided bleaching of industrial softwood kraft pulp. Tappi J. 77, 111-116. Suurnakki, A., Clark, T., Allison, R., Viikari, L., Buchert, J., 1996a. Xylanaseand mannanase-aided ECF and TCF bleaching. Tappi J. 79, 1 l- 117. Suurnakki, A., Clark, T., Allison, R., Buchert, J., Viikari. L., 1996b. Mannanase-aided bleaching of softwood kraft pulps. In: Messner, K., Srebotnik, E. (Eds.), Biotechnology in Pulp and Paper Industry: Advances in Applied and
of Biotechnology 57 (1997) 191-204 Fundamental Research, WUV-Universitltsverlag, Vienna, Austria, pp. 69-74. Takahashi, R., Kusakabe, l., Kusama, S., Sakurai, Y., Murakami, K., Maekawa, A., Suzuki, T., 1984. Structures of glucomanno-oligosaccharides from the hydrolytic products of konjac glucomannan produced by a b-mannanase from Streptomyces sp.. Agric. Biol. Chem. 48, 2943-2950. Tenkanen, M., Buchert, J., Viikari, L., 1995. Binding of hemicellulases on isolated polysaccharide substrates. Enzyme Microb. Biol. 17, 4999505. Teleman, A., HarjunpG, V., Tenkanen, M., Buchert, J., Hausalo, T., Drakenberg, T., Vuorinen, T., 1995a. Characterization of 4-deoxy-b-L-threo-hex-4-enopyranosyluronic acid attached to xylan in pine kraft pulp and pulping liquor by ‘H and “C NMR spectroscopy. Carbohydr. Res. 272, 55571. Teleman, A., Koivula, A., Reinikainen, T., Valkeajarvi, A., Teere, T.T., Drakenberg, T., 1995b. Progress-curve analysis shows that glucose inhibits the cellotriose hydrolysis catalysed by cellobiohydrolase II from Trichodermu reesei.. Eur. J. Biochem. 231, 250-258. Teleman, A., Hausalo, T., Tenkanen, M., Vuorinen, T., 1996. Identification of the acidic degradation products of hexenuronic acid and characterisation of hexenuronic acidsubstituted xylooligosaccharides by NMR spectroscopy. Carbohydr. Res. 280, 1977208. Timell, T.E., 1967. Recent progress in the chemistry of wood hemicelluloses. Wood Sci. Technol. 1, 45570.
Journal
of Biotechnology
57 (1997) 191-204
Action of Trichoderma reesei mannanase on galactoglucomannan in pine kraft pulp Maija Tenkanen
+*, Mari Makkonen ‘TV,Marjukka Anita Teleman b
Perttula
‘, Liisa Viikari a,
a VTT Biotechnology
and Food Research, P.O. Box 1500, FIN-02044 VTT, Finland b VTT Chemical Technology, P.O.Box 1400, FIN-02044 VTT, Finland
Received
15 October
1996; received
in revised
form
10 March
1997; accepted
3 April
1997
Abstract The di-, tri- and tetrasaccharides formed during Trichoderma reesei endo-B-D-mannanase treatment of pine kraft pulp were studied. The oligosaccharides in the hydrolysate were fractionated using size-exclusion, anion exchange and activated carbon chromatography. The primary sequence of the purified oligomers was determined by two-dimensional NMR techniques. The T. reesei mannanase cleaves the /J-1,4-glycosidic linkage of D-mannOSy residues attached either to D-mannose or D-glucose. The D-mannosyl residue may also be substituted by a D-galaCtOSy1 group. The main disaccharide produced was mannobiose, but a significant amount of 4-O-jI-D-glucopyranosyl-D-mannopyranose (GlcMan) was also produced. After extensive hydrolysis the main trisaccharides produced were 4-0-b-Dmannopyranosyl-[6-O-a-galactopyranosyl]-D-mannopyranose (GallMan,) and 4-O-/I-D-glucopyranosyl-4-O-P-Dglucopyranosyl-D-mannopyranose (Glc,Man). Some mannotriose 4-0-/?-D-glucopyranosyl-4-O-/?-D-mannopyranosyl-D-mannopyranose (GlcMan,) and 4-0-8-D-glucopyranosyl-[6-O-cc-galactopyranosyl]-D-mannopyranose (Gal’GlcMan) were also detected in the hydrolysate. The structures of two tetrasaccharides were studied. They 4-O-~-D-gluCOpyranOsyl-4-0-~-D-gluCOpyranOsyl-4-O-~-D-glucopyranosy~-D-mannopyranose appeared to be (Glc,Man) and 4-0-~-D-glucopyranosyl-4-O-~-D-mannopyranosyl-4-0-~-D-glucopyranosyl-D-mannopyranose (GlcManGlcMan). According to the results obtained, the galactoglucomannan in pine contains regions in which two or three glucose units are linked together, which further means that it may contain regions with several successive mannose residues. The galactose side groups were found to be attached only to mannose. 0 1997 Elsevier Science B.V. Keywords: Endomannanase; troscopy
* Corresponding Ol68-1656/97/$17.00
Trichoderma reesei; Pine kraft pulp; Hydrolysis;
Manno-oligosaccharides;
author. Tel.: + 358 9 4565141; fax: + 358 9 4552028; e-mail: [email protected] 0 1997 Elsevier
PII SO168-1656(97)00099-O
Science
B.V. All rights
reserved.
NMR
spec-
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M. Tenkanen et al. /Journal
1. Introduction The major softwood hemicellulose is an Oacetyl-galactoglucomannan (15-20% of wood) with various structural variations (Timell, 1967). In hardwood a low amount of glucomannan (25% of wood) is also present. During pulping the acetyl groups of hemicelluloses are totally removed. In kraft pulping about half of the galactoglucomannan is dissolved and further degraded, whereas the other half is present in softwood kraft pulps (SjiistrGm, 1981). The utilisation of endo-BD-mannanases (E.C. 3.2.1.78) has been shown to enhance delignification and to improve the bleachability of softwood pulps (Clark et al., 1990; Buchert et al., 1993a; SuurnCkki et al., 1994, 1996a,b; Saake et al., 1995). Hydrolysis of mannan-type polysaccharides by mannanases yields an array of oligomers, including the p-o-manno-oligosaccharides and a range of mixed oligosaccharides containing D-mannose, D-glucose and D-galactose. The nature of the oligosaccharides produced by different mannanases varies, indicating that there are differences in the action patterns of the enzymes (Kusakabe et al., 1988; McCIeary and Matheson, 1983; McCleary, 1991). Enzymes also differ in their action towards fibre-bound substrates. For example mannanase from Trichoderma reesei was able to hydrolyse galactoglucomannan in pine kraft pulp, whereas the mannanase from Bacillus subtilis was not (Ratta et al., 1993). Moreover, even if an enzyme is able to hydrolyse a fibrebound substrate, this does not necessarily correspond with delignification. In a comparative study of T. reesei, Aspergillus niger and Caldocellulosiruptor saccharolyticus mannanases, the T. reesei mannanase was found to be superior in bleach-boosting (Suurnlkki et al., 1996b). The mannanases from T. reesei and C. saccharolyticus belong to the same glycosyl hydrolase family 5, whereas the Bacillus subtilis mannanase has been assigned to the family 26 (Henrissat and Bairoch, 1996). However, the T. reesei and C. mannanases have only weak saccharolyticus amino acid similarities. The A. niger mannanase has not yet been classified but the mannanase from A. aculeatus, which has a high amino acid
0s Biotechnology
57 (1997)
191-204
similarity with the T. reesei mannanase (Stglbrand et al., 1995) and clear N-terminal similarity with the A. niger mannanase (StAlbrand, 1995), also belongs to the family 5 of glycosyl hydrolases. Mannanases in different families may have different catalytic properties, as in the case of xylanases (Biely et al., 1997). The differences in catalytic properties might explain the differences in bleachboosting efficiencies. Degradation of linear manno-oligosaccharides and polymeric mannan by T. reesei mannanase has been studied previously (Stilbrand et al., 1993; Harjunptig et al., 1995). Hydrolysis of both isolated and fibre-bound galactoglucomannans from pine wood and pine kraft pulp has also been studied in some detail (Rattii et al., 1993), but the structure of the oligosaccharides produced has not been verified and thus the action pattern and the substrate binding requirements of T. reesei mannanase are not known. In this work, the mode of action of T. reesei mannanase on galactoglucomannan in unbleached pine kraft pulp was studied and the substrate specificity was compared with other mannanases studied earlier.
2. Materials 2.1. Enzymes
and methods and substrates
The mannanase from T. reesei was purified as described by R&ii et al. (1993). P-Glucosidase from Aspergillus niger was purchased from Megazyme (Australia), p -mannosidase from A. niger was kindly provided by Folke Tjerneld and Pia Ademark from Lund University (Sweden) and cl-galactosidase I from Penicillium simplicissium was purified as described by Luonteri et al. (1997). The enzyme activities were determined according to the methods in Rgttii and Poutanen (1988). The pine kraft pulp with kappa number 25.8 was cooked in the laboratory. The production and properties of this pulp have been described previously (Teleman et al., 1995a). Locust bean gum (galactomannan, G-0753) was purchased from Sigma.
M. Tenkanen et al. /Journal of Biotechnology 57 (1997) 191-204
2.2. Hydrolysis experiments Pine kraft pulp (50 g l- ’ was hydrolysed with 200 or 2000 nkat g - ’ of T. reesei mannanase for 2 or 48 h, respectively, at pH 5 and 40°C. /?Mannosidase and P-glucosidase treatments of oligosaccharides were performed by hydrolysing with 500 nkat of enzyme per g of oligosaccharides for 24 h at pH 5 and 40°C. After secondary enzymatic hydrolysis of the released oligosaccharides to monosaccharides (Buchert et al., 1993b), the degree of hydrolysis was calculated by dividing the total amount of mannose in the hydrolysate by the amount in the pulp. 2.3. Isolation of oligosaccharides The pine kraft pulp (10 g) was suspended in water (20 g 1- ’ and the pH was adjusted to 5.0 with 0.1 M H,SO,, after which the pulp was hydrolysed for 48 h at 40°C with 2000 nkat gg ’ of T. reesei mannanase. The hydrolysate was concentrated to 30 ml by rotary evaporation and fractionated in three batches with Biogel P-2 (Bio-Rad, packed in 5 x 85 cm column) using water (2 ml min - ‘) as eluent. Fractions containing di-, tri- and tetrasaccharides were selected according to TLC. The fractions containing disaccharides were pooled and concentrated to 2 ml. According to TLC the disaccharide fraction contained mannobiose and an unknown disaccharide (M2_,). M,., was further purified with cation exchange resin, Dowex 5Ow*8 in Ba2+ form (200-400 mesh, Fluka, packed in 1.6 x 80 cm column) at 50°C using water (1 ml min - ‘) as eluent. Fractions were analyzed by TLC, after which the fractions containing M,_, were pooled, concentrated and lyophilized before further analysis by NMR spectroscopy. After pooling and concentration of the fractions containing trisaccharides the mixture (4 ml), which according to TLC contained at least three different compounds, was run through a column of Dowex 5Ow*8 as described above. According to TLC analysis of the fractions ob-
193
tained, only mannotriose was separated to some extent from the other unidentified trisaccharides, which were pooled, concentrated and separated further using activated carbon. A lo-ml sample was applied to a column (1.6 x 7 cm) of activated carbon (Oy Flinkenberg Ab, Finland). The column was washed with water, after which the bound oligosaccharides were eluted with a linear ethanol gradient from 0 to 25% v/v using a flow rate of 0.15 ml min ~ ‘. According to TLC analysis three different oligosaccharide fractions (M,_,, M,_,, M,.,) were pooled, which were concentrated and lyophilized. The tetrasaccharide fraction was further fractionated using the activated carbon column (1.6 x 7 cm) as described above, except that after washing with water the column was washed with 20% v/v ethanol. The bound oligosaccharides were separated with a linear ethanol gradient from 20 to 50% v/v. The fractions obtained were pooled in five pools and analyzed further by HPAEC-PAD, which showed that the first fractions contained trisaccharide (M3_J and the last fractions contained a new compound (M4_r). The fractionation of the hydrolysate is presented in Fig. 1. The Gal’Man, used as reference material was prepared from locust bean gum. First, 1 g of locust bean gum was mixed with 200 ml of 50 mM Na-acetate buffer, pH 5 and then heated to boiling point to dissolve the galactomannan. After cooling, the mixture was hydrolysed with 5000 nkat g _ ’ of T. reesei mannanase for 48 h at 40°C. The hydrolysate was concentrated lofold and then fractionated by gel filtration in two batches using Biogel P-2 gel as described above. GallMan, was further separated from mannotriose with Dowex 5Ow*8 resin as described above. 2.4. Analytical techniques Separation of manno-oligosaccharides on TLC was achieved using precoated aluminium sheets of silica gel (Kieselgel 60, Merck). The mobile phase contained ethylacetate: acetic acid: water = 9:6:6. Visualisation was performed by
194
M. Tenkanen et al. /Journal
of’ Biotechnology 57 (1997) 191-204
Hydrolysis with T. reesei mannanase
1Mixture of oligosaccharides 1 Biogel P-2
Dowex 50 W in Ba2+ form
l
Dowex 50 W
8
l
8 Activated carbon
Man,
Activated carbon
4,
Gal’ Man,
Fig. 1. Purification procedure of oligosaccharides in isolated preparations are presented.
Tetrasaccharides
Trisaccharides
Disaccharides
M3-2 Glc Man, Gall Glc Man
M3-3
h-1
Glc, Man
Glc, Man Glc Man Glc Man
from the pine kraft pulp hydrolysate.
spraying with a solution containing 80% ethanol, 10% H,SO,, 10% water and 0.2% orcin (Merck). manno-oligosaccharides (Man,-Man,, Linear Megazyme, Australia) were used as standards. The high-performance anion-exchange chromatography (HPAEC-PAD) of oligosaccharides was performed using a CarboPac PA-l column (Dionex Corp, USA) in a Dionex DX 500 series chromatograph equipped with pulse amperometric detection (Dionex ED 40). The system was equilibrated with 2.5 mM NaOH. After sample injection 2.5 mM NaOH was run through the column for 23 min and a linear gradient from 2.5 to 100 mM NaOH was created during the next 17 min. The second linear gradient from 100 mM NaOH to 100 mM NaOH + 150 mM Na-acetate was run in the column during the next 20 min, after which the column was washed with 100 mM NaOH + 300 mM Na-acetate and then with 300 mM NaOH. The flow rate used was 1 ml min- ‘. The standards used were commercially available linear manno-oligosaccharides and Gal’Man, (Megazyme).
Only the structures
of main oligosaccharides
For NMR analysis the lyophilized samples were dissolved in D,O (99.8 atom%, Fluka) and the pH was adjusted to 7 with NaOD and/or DCl. The ‘H NMR and 13C NMR spectra were obtained at 599.86 and 150.85 MHz, respectively, on a Varian UNITY 600 MHz spectrometer. Typical acquisition parameters were for 1D ‘H NMR (1D 13C NMR) a 70” pulse of 14.8 ps (8 ps), a spectral width of 8000 Hz (40000 Hz) and a repetition time of 22 s (3 s). Spectra were obtained at 70°C. The chemical shifts are reported relative to an internal acetone standard at 2.225 ppm for ‘H spectra. Standard pulse sequences and phase cycling were utilised to obtain phase-sensitive COSY, relay COSY, TOCSY, ROESY and NOESY 2D spectra. A spectral width of 1500 Hz (2100 Hz) for GlcMan (Gal’Man,) was employed in both dimensions and the relaxation delay was 2.2 s (2.5 s). ‘H-detected HMQC and HMBC spectra were acquired over a t, spectral window of 7500 Hz and a t2 spectral window of 1500 Hz (2100 Hz). Full details of the 2D experiments have been reported earlier (Teleman et al., 1996).
M. Tenkanen et al. /Journal
of Biotechnology 57 (1997) 191-204
3. Results 3.1. Extensive
hydrolysis
Table 1 Relative amounts of sugar residues in the mannanase hydrolysate of pine kraft pulp after extensive hydrolysis (2000 nkat g-‘, 48 h)
of pine kraft pulp
The action of Trichoderma reesei mannanase on pine pulp galactoglucomannan was first studied by analysing the whole hydrolysate obtained after extensive enzyme treatment (2000 nkat g- ‘, 48 h) by ‘H NMR spectroscopy and HPAEC-PAD (Figs. 2 and 3). After the enzyme treatment 20.4 wt% of pulp galactoglucomannan was hydrolysed to soluble oligosaccharides. The galactoglucomannan solubilised from pine kraft pulp was extensively hydrolysed to a mean degree of polymerization of 2.1. Almost all of the oligosaccharides produced had a mannose residue at the reducing end and only about 2 mol% had a reducing end glucose (Fig. 2). The observed reducing end glucose most probably originated from reducing ends of the polymers. The galactose groups in the oligosaccharides formed were attached by a-(1 +6)-glycosidic linkages only to mannose. The ratio of Man:Glc:Gal in the hydrolysate was 3.6:1.0:0.3 as analysed by ‘H NMR and 3.5:1.0:0.3 as analysed by HPAEC-PAD (Table 1). According to HPAEC-PAD analysis the main product formed was mannobiose (350 mg l- ‘, 40 wt% of solubilised oligosaccharides). A rather high amount of mannose (95 mg l- ‘, 10%) was also produced, whereas only a small amount of mannotriose (8 mg l- ‘, 1 %) was detected in the hydrolysate. In addition to these quantified MZUI reducing end a
M.Ul reducing end $
I
internal +
Glc B(1-w internal +
terminal
I”“~““,““,““l~“~,~“.,““,““,““,~,~~,..’~r
5.5
5.4
5.3
5.2
5.1
5.0
4.9
4.8
4.1
4.6
195
4.5 ppm
Fig. 2. Anomeric proton region of the ‘H NMR spectrum of the pine kraft pulp hydrolysate after extensive mannanase treatment (2000 nkat g-‘, 48 h).
Method
Carbohydrate Man
‘H NMR HPAEC-PAD
mol (‘%J) 73.4 mol (“XI) 71.8 mg 1-l 663
content
Glc
Gal
Others
20.4 20.3 188
5.8 6.9 64
0.4 1.0 9
manno-oligosaccharides many other oligosaccharides, including three main and several minor unknown compounds, were formed (Fig. 3A). The other oligosaccharides represented 49% of the soluble sugars formed and they contained 29% of hydrolysed mannose and all the hydrolysed glucose and galactose. In order to obtain information about the structure of the unidentified oligosaccharides the hydrolysate was treated with P-mannosidase and /3-glucosidase. During P-mannosidase treatment mannobiose and mannotriose were degraded to mannose (Fig. 3B). The peak with a retention time of 43.77 min (peak 5) was slightly decreased and the peak at 34.43 min was slightly increased. No detectable differences were observed in the other main peaks. After /?-glucosidase treatment the main peaks with retention times 45.43 (peak 6) and 51.28 (peak 9) min and the minor peaks with retention times 48.07 (peak 7) and 53.50 (peak 10) min decreased (Fig. 3C). These peaks obviously contained oligosaccharides with terminal glucose units. 3.2. Identljication
of disaccharides
The main unidentified components were isolated from the hydrolysate for further structural studies (Fig. 1). The isolation of the oligosaccharides was started by running the hydrolysate through a gel filtration column (Biogel P-2) which separated the sugars according to their size. The fractions were analysed by TLC and those containing di- tri- and tetrasaccharides were pooled for further purification.
196
M. Tenkanen
et al. /Journal
oJ’ Biotechnology
57 (1997) 191-204
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Fig. 3. HPAEC-PAD chromatogram of the pine kraft pulp hydrolysate after extensive mannanase treatment (2000 nkat g ‘, 48 h); (A) after further hydrolysis of the hydrolysate in A with p-mannosidase (B) and b-glucosidase (C) and after limited mannanase treatment (200 nkat g ‘, 2 h) (D). See text for the peak identification. IS, internal standard; 1, Glc; 2, Man; 3, Man,; 4, Man,; 5, Gal’Man,; 6, GlcMan; 7, GlcMan,; 8, Gal’GlcMan; 9, GlcGlcMan; 10, tetrasdccharides; 11, Gal’ Man,.
The disaccharide fraction was further fractionated using Dowex 5Ow*8 resin in Ba* + form. The unidentified disaccharide (M2_,) was separated from mannobiose and its structure was solved with NMR spectroscopy analysis. The structure of M,., was 4-0-p-D-glucopyranosyl-D-mannopyranose (GlcMan). The assignment of ‘H and 13C NMR resonances was based on phase-sensitive COSY, relay COSY and TOCSY experiments starting from the anomeric protons (Table 2). The signal ratio for Man H-la and H-l/? was 67:33, which is consistent with the literature data (McCleary et al., 1982; Harjunpaa et al., 1995). In the rotatingframe NOE spectroscopy (ROESY) spectrum the Glc H-l signals at 6 4.5 15 and 4.506 have crosspeaks with H-4 of Man at 6 3.80 and 3.87, thereby
establishing the sequence of GlcMan. The structure was also verified by p -mannosidase and B -glucosidase treatments. ,6’-Mannosidase did not whereas p -glucosidase hydegrade GlcMan, drolysed it to glucose and mannose in a ratio of 1.2:1 (results not shown). No disaccharides with reducing end glucose (ManGlc or cellobiose) were found in the disaccharide fraction. In the HPAECPAD analysis the isolated GlcMan had a retention time (45.40 min) close to that of the main unidentified peak in Fig. 3A (45.43 min, peak 6), which indicated that the peak was GlcMan. 3.3. IdentiJication The trisaccharide
of trisacchurides fraction,
which
according
to
M. Tenkanen et al. /Journal
,
“,
.
.
.
.
.
.
of Biotechnology 57 (1997) 191-204
.
.
.
.
,
.
.
.
.
10
20
30
40
t,“‘,““I,,,,,,,,‘,‘, 10 0
20
30
40
0
.
.
.
.
197
,
.
.
,
.
(
50
60
50
60
,‘,“‘,I
Retention time (minutes) Fig. 3 (contd.)
TLC contained at least three different compounds, was further fractionated using Dowex 5Ow*8 resin and activated carbon (Fig. 1). The different trisaccharides obtained were not completely separated from each other, but the primary structures of the major components could be determined by ‘H NMR spectroscopy and treatments with /?-mannosidase, /?-glucosidase and e-galactosidase. The amount of isolated oligosaccharides was too low to obtain r3C NMR data. For all of the trisaccharides the reducing residue was readily identified as mannose because of the chemical shifts, coupling constants and relatively smaller intensities in the ‘H NMR spectra of the anomeric proton of the cx and /? forms. The ‘H NMR resonances were assigned by 2D NMR techniques starting from the anomeric protons (Table 2).
The main galactose-containing oligosaccharide formed from galactomannan by T. reesei mannanase, 4-U-@-D-mannopyranosyl-[6-O-ol-galactopyranosyll-D-mannopyranose (GallMan,), was isolated from the locust bean gum hydrolysate and used as a reference compound in the ‘H NMR and HPAEC-PAD analyses of the puipderived trisaccharides. The assignments of ‘H (Table 2) and 13C (results not shown) NMR data for GallMan, were based on phase-sensitive COSY, relay COSY, TOCSY and HMQC experiments starting from the anomeric protons. Having assigned all of the ‘H and 13C resonances, the primary sequence was directIy obtainable from the HMBC spectrum, in which three-bond interresidue ‘H/13C correlations were detectable. The observed cross-peaks bH- 1/a&-4, bH- 1/afi C-4, cH-l/a&-6, cH-l/a/3C-6, aaH-4/bC-1, a,BH-4/ bC- 1, aaH-6/cC- 1, ap H-6/cC- 1 and a/? H-6’/cC- 1
M. Tenkanen et al. /Journal
198 Table 2 ‘H NMR
data
Compound”
for isolated
oligosaccharides
Residue
Chemical
shiftb (coupling
constant”)
H-2
H-3
H-4
H-5
H-6
H-6’
H-l Glc-Man b a
of Biotechnology 57 (1997) 191-204
a-Manp-a p-Manp-a p-GIcp-b, /i’-Glcp-b,j
5.187 4.904 4.515 4.506
(1.8) (1.1) (7.9) (7.9)
3.99 4.01 3.33 3.32
3.96 3.77 3.53 3.52
3.87 3.80 3.43 3.43
3.92 3.52 3.50 3.52
3.92 3.96 3.93 3.93
3.85 3.82 3.74 3.74
x-Manp-a /j’-Manp-a /J-Manp-b, p-Manp-b, r-Galp-c, r -Galp-c,j
5.184 4.910 4.741 4.736 5.021 5.030
(1.7) (1.1)
4.00 4.01 4.08
4.01 3.82 3.64
3.95 3.89 3.62
4.08 3.67 3.42
3.99 3.95 3.94
3.76 3.80 3.76
3.84
3.88
4.02
3.91
3.77
3.77
a-Manp-a p-Manp-a p-Manp-b, p-Manp-b,, p-Glcp-c
5.184 4.904 4.758 4.751 4.503
(-1.7) (1 .O) (-0.8) (-0.8) (7.9)
3.99 4.01 4.12
3.98 3.80 3.77
3.91 3.82 3.82
3.7-3.9 3.6-4.0 3.58
3.773.9 3.664.0 4.01
3.7-3.9 3.6-4.0 3.84
3.32
3.52
3.42
3.51
3.93
3.74
a-Mary-a /J-Manp-a p-Glcp-b, p-Glcp-b, 8-Glcp-c
5.186 4.904 4.542 4.532 4.516
(1.7) (1 .O) (7.9) (7.9) (7.9)
3.99 4.00 3.39
3.97 3.77 3.66
3.90 3.83 3.61
3.773.9 3.664.0 3.65
3.7-3.9 3.6-4.0 3.99
3.773.9 3.664.0 3.82
3.33
3.52
3.43
3.49
3.92
3.74
c
I Gal Man-Man b a
Clc-Man-Man c b a
Glc-Glc-Man c b a
(1.O) (1 .O) (3.8) (3.8)
‘- = /J-1,4-linkage; 1= r-l ,6-linkage. The reducing end is denoted as a. bin ppm relative to internal acetone at 2.225 ppm (D,O, 70°C and pH 7) acquired “Observed first order coupling in Hz.
established the sequence of Gal’Man,. The chemical shifts for the carbons are in good agreement with published values (McCleary et al., 1982). The trisaccharides were fractionated in three fractions (M,.,, M,., and M,_,). The main component in M,_, according to ‘H NMR and HPAECPAD analysis was Gal’Man, (peak 5, - 80%). The fraction also contained some mannotriose (peak 4, - 20%) (Fig. 4A). The main component in M,_, (Fig. 4C) was 4-O-j?-D-glucopyranosyl-40-p-D-glucopyranosyl-D-mannopyranose (Glc,M,_, also conMan, peak 9, - 70%). Fraction tained some of the tetrasaccharides found in M,_, (see below). According to HPAEC-PAD analysis M,., contained three different oligosaccharides (Fig. 4B). According to ‘H NMR analysis the main component in M,., was
at 600 MHz.
4-0-p-D-glucopyranosyl-P-D-mannobiose (Glc60%). The primary sequences of Man,, Glc,Man and GlcMan, were obtainable from the ROESY and NOESY spectra, in which interresidue cross-peaks were observed at the H-l frequencies. M,_, also contained some Glc,Man ( - 10%) and an oligosaccharide that had galactose linked to a reducing end mannose ( - 30%). However, the amount of Gal’Man, was very low. In the anomeric proton region of the ‘H NMR spectrum of fraction M,., there were two additional non-reducing end (terminal) glucose resonances (6 4.501 J,,, = 7.9, 6 4.497 J,,, = 7.9). Thus it seemed that the third trisaccharide in fraction MS-2 could be 4-O-p-glucopyranosyl-[6-O-a-Dgalactosyll-D-mannopyranose (Gal’GlcMan). M,_ 2 was treated with a-galactosidase which hydrolysed the peak with retention time 49.08 min
M. Tenkanen el al. /Journal
of Biolechnology 57 (1997) 191-204
199
A 200
0 0
10
20
30
40
60
50
B 78
0
10
20
30
40
50
60
0
10
20
30
40
50
60
0
Retention time (minutes) Fig. 4. HPAEC-PAD identification.
chromatograms
of the isolated
trisaccharide
(peak 8) to produce galactose and GlcMan (re sults not shown). Thus this peak contained
fractions;
M,.,
(A); M,.,
(B) and M,.,
(C). See Fig. 3 for peak
Gal’GlcMan and the peak with 48.00 min (peak 7) was GlcMan,.
retention
time
200
M. Tenkanen
3.4. IdentiJication
et al. /Journal
of tetrasaccharides
The amount of sugars in the tetrasaccharide fraction was very low and after further purification on the activated carbon column only one sample M,_, was obtained for the ‘H NMR analysis. According to HPAEC-PAD, M,_, contained one main peak. /J’-Glucosidase treatment reduced the size of the main peak, producing glucose and GlcMan but no mannobiose (results not shown). The amount of isolated tetrasaccharides was too low to obtain 2D NMR data. However, the structural characterisation can be based on the chemical shifts and coupling constants of the so-called structural reporter signals, i.e. well separated resonances mainly originating from the anomeric proton of the monosaccharide residues. This method relies on a good spectral library of related compounds. The data in Table 2, as well as mannooligosaccharide and cello-oligosaccharide data reported in the literature (Teleman et al., 1995b; Harjunpaa et al., 1995), was used for the structural characterisation. Almost all of the tetrasaccharides produced have a mannose residue at the reducing end. From the relative proportions of the different structural elements (Fig. 5), it can be internal Glc * linked
toGlc 0raMan
,
terminal Glc
I
,
1linked internal Man
I
’
linked to Glc
I terminal Man ,bl
linked to Man
qf Biotechnolog)~ 57 (1997) 1916204
concluded that the most abundent structure of the tetrasaccharides is 4-O-/?-D-glucopyranosyl-4-Op-D-glucopyranosyl-4-O-P_D-glucopyranosyl-Dmannopyranose (Glc,Man) and that the second most abundant is most probably 4-o-p-D-glucopyranosyl-4-0-P-D-mannopyranosyl-4-O-PD-glucopyranosyl-D-mannopyranose (GlcManGlcMan). 3.5. Limited
hydrolysis
The pine kraft pulp was also hydrolysed for 2 h by 200 nkat g ~ ’ of mannanase in order to study the oligosaccharides produced in the limited hydrolysis used in mannanase aided bleaching of kraft pulps. The degree of hydrolysis of pulp galactoglucomannan was 3.5 wt%. The main product was mannobiose (38 mg l- ‘, 23 % of solubilised oligosaccharides) (Fig. 3D). The amount of mannose produced was below the detection limit but a significant amount of mannotriose (28 mg 1~ ‘, 17 %) was formed. Mannobiose and mannotriose represented 61% of the solubilised mannose. The mixed oligosaccharides produced represented 60 and 39% of solubilised sugars and mannose, respectively. According to HPAEC-PAD the main mixed oligosaccharides were GlcMan (peak 6) and GlcMan, (peak 7) (Fig. 3D). GalMan, (peak 5) and GlcGlcMan (peak 9) were also detected in the hydrolysate. The main unidentified peak, which was not detected after extensive hydrolysis, had a retention time 46.25 min (peak 11). This peak disappeared after /5-mannosidase treatment but p-glucosidase treatment did not affect it (results not shown). Thus it contained a non-reducing end mannose. The peak was identified to be Gal’Man, by comparing the retention time to commercially available Gal’Man,. The hydrolysate also clearly contained oligosaccharides with a degree of polymerisation higher than 4 and retention times longer than 54 min.
4. Discussion 4.76
4.14
4.72
4.55
4.53
4.51
4.49
ppm
Fig. 5. Analysis of tetrasaccharide fraction M,, by and proton NMR. The different structural elements are according to Table 2, (Teleman et al. (1995b) and HarjunpZi et al. (1995)).
According to the present study the mannanase from T. reesei is able to cleave not only p-1,4linkages between two mannosyl residues, but also
M. Tenkanen
Isolated di-andtrisaccharides
,
et al. /Journal
of Biotechnology
57 (1997) 191-204
l
--
-
,
I
201
4
I
Gal
Gal
$4 &J 14 J J 4 A 4 I$ -Man-Man-Glc-Man-Man-Man-Man-Man-Man-M~-~-M~-Glc-Glc-M~-Man-Gic-M~-Man$
$
5T
5
Fig. 6. Hypothetical presentation of the action of T. reesei mannanase on galactoglucomannan from pine kraft pulp. linkage; 1= r-1,6-linkage; 1 = linkage that can be hydrolysed; X with arrow = linkage that cannot be hydrolysed.
after mannosyl residue attached to glucose. It is not able to hydrolyse the linkage after glucosyl residue. The mannosyl residue can also be substituted by a galactosyl group, as the smallest galactose-containing oligosaccharide obtained was Gal’Man,. The action of T. reesei mannanase on galactoglucomannan from pine kraft pulp is summarized in Fig. 6. After extensive hydrolysis (2000 nkat g - ‘, 48 h) the galactoglucomannan was mainly hydrolysed to mannobiose and GlcMan (Fig. 3A). The main trisaccharides produced were Gal’Man, and Glc,Man. After limited hydrolysis (200 nkat, 2 h) mannobiose and GlcMan were produced in same ratio as after extensive hydrolysis, but clear differences were observed in the trisaccharides formed (Fig. 3A, D). After limited hydrolysis the amounts of mannotriose and GlcMan, were rather high as compared to those of Gal’Man, and Glc,Man, and thus more mannose-containing trisaccharides were formed at the early stage of hydrolysis. The amount of mixed oligosaccharides was clearly higher after limited hydrolysis (60% of solubilized oligosaccharides) than after extensive hydrolysis (49%). The enzyme thus seems to act first on regions with successive unsubstituted mannose units, producing mixed oligosaccharides. After limited hydrolysis the hydrolysate also contained long oligosaccharides having a DP higher than 4 (retention time longer than 54 min), which disappeared during longer incubation. T. reesei mannanase could further degrade mannotriose and GlcMan,, as their amounts were very low after extensive hydrolysis. The hydrolysis might proceed through transglycosylation of trisaccharides first to hexasaccharides which are then degraded further (Harjunpaa et al., 1995).
-
= p-1,4-
The ratio of Man:Glc:Gal in the hydrolysate was 3.5:1.0:0.3. This is in good agreement with earlier reported value of 3.5:1.0:0.4 analysed from pine pulp hydrolysate produced by T. reesei mannanase (Buchert et al., 1993a). It seems that T. reesei mannanase is able to hydrolyse both types of galactoglucomannans found in softwoods, having ratios of Man:Glc:Gal 3:l:l and 4:l:O.l (Timell, 1967). The action of several mannanases from legume seeds and micro-organisms on various galactomannans and glucomannans has been studied in detail (Emi et al., 1972; McCleary, 1979; Shimazu and Ishihara, 1983; McCleary et al., 1983; McCleary and Matheson, 1983; McCleary, 1991; Takahashi et al., 1984; Kusakabe et al., 1988; Park and Chang, 1992). However, only a few studies have been performed with galactoglucomannans and there is only one study in which the products formed after the hydrolysis of galactoglucomannan from softwood kraft pulp have been analysed (Ratto et al., 1993). The extent of hydrolysis of galactomannans has been shown to be dependent on the extent of substitution and the pattern of distribution of substituents. The action of mannanases on glucomannans is dependent on the glucose/mannose ratio. The microbial mannanases studied include those from Bacillus subtilis, Irpex lacteus, Aspergillus niger, Tyromyces palustris, Streptomyces sp, Penicillium purpurogenum and Penicillium kloeckeri. GallMan, was the smallest substituted oligosaccharide produced in the hydrolysis of galactomannans by A. niger, I. lacteus and P. purpurogenum mannanases and P. kloeckeri mannanase 2 (McCleary and Matheson, 1983; Park and Chang, 1992; Biely et al., 1996). B. subtilis
202
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mannanase and P. kloeckeri mannanase 1 have a more limited ability to hydrolyse galactomannans. GalMan, was the shortest substituted product formed by B. subtilis mannanase and the main monosubstituted oligosaccharide was GalMan, (Emi et al., 1972; McCleary and Matheson, 1983). P. kloeckeri mannanase 1 produced GalMan, as the shortest substitued oligosaccharide (Biely et al., 1996). The structure of oligosaccharides found after the hydrolysis of glucomannans has also varied. All mannanases studied have produced GlcMan and GlcMan,. P. purpurogenum and Streptomyces sp. mannanases produced Glc,Man, which was not detected in the hydrolysates produced by A. niger and T. palustris mannanases. There also appear to be differences in the structures of tetrasaccharides containing glucose and mannose produced by these mannanases (McCleary and Matheson, 1983; Shimazu and Ishihara, 1983; Takahashi et al., 1984; Kusakabe et al., 1988). The action patterns of T. reesei and P. purpurogenum mannanases appeared to be similar. A. niger mannanase showed similar action on galactomannans to the T. reesei mannanase but differed in its action on glucomannans. A. niger mannanase is not reported to produce any tri- or two adjacent glucose tetrasaccharides with residues, whereas Glc,Man was the main linear trisaccharide produced by T. reesei mannanase. Thus it is seems that the binding requirements of the subsites of the T. reesei mannanase are different from those of A. niger mannanase. However, the substrate used for A. niger mannanase was salep glucomannan, which might have a different distribution of glucose and mannose units from that of the konjac glucomannan used as substrate for P. purpurogenum and Streptomyces sp. mannanases and pine pulp galactoglucomannan studied in this work. The ratio of mannose to glucose is 4:l in salep glucomannan, and thus the possibility of successive glucose units is equal to that in pine pulp galactoglucomannan, whereas konjac glucomannan contains much more glucose (Man:Glc = 1.5:l.O). Of the above-mentioned mannanases only the actions of mannanases from A. niger, B. subtilis and P. kloeckeri have been studied on galactoglu-
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comannans. It was reported that a mixed Gal’GlcMan was produced by A. niger mannanase, but the structures of other oligosaccharides formed during hydrolysis were not reported (McCleary, 1991). The structures of oligosaccharides formed by B. subtilis and P. kloeckeri mannanases were also not studied in detail (Ratto et al., 1993; Biely et al., 1996). Mannanases in different families might have different catalytic properties towards substituted mannans, similarly to the case of xylanases (Biely et al., 1997). T. reesei and A. niger mannanases, which appear to belong to the same glycosyl hydrolase family (family 5) (Henrissat and Bairoch, 1996; Stalbrand, 1995), produced Gal’Man, from galactomannan whereas mannanase from B. subtilis, which produced Gal’Man, as the shortest substitued oligosaccharide, has been assigned to a different family (family 26) (Henrissat and Bairoch, 1996). The differences in the action patterns of these enzymes do not, however, explain the differences observed in the mannanase-aided bleaching of pine kraft pulps (Suurnakki et al., 1996b). The action of A. niger and B. subtilis mannanases on pine pulp galactoglucomannan should also be studied in detail before any conclusions can be drawn about the action mechanisms of these enzymes and their effects on pulp bleachability. Enzymes are powerful tools, not only in the modification of polymers but also in their structural analysis. According to the present study it appeares that galactoglucomannan in pine contains regions with several glucose units in secan form strong quence. These structures intermolecular hydrogen bonds with cellulose, which might be important for cellulose-galactoglucomannan interactions in wood and pulp. If the backbone contains 2-3 successive glucose units it might correspondingly contain 7710 successive mannose units. This type of structure in wood galactoglucomannan have also earlier been suggested by Holmbom (1996) (personal communication). The mannanase from T. reesei is known to contain a cellulose-binding domain (CBD), the role of which is unclear (Tenkanen et al., 1995; Stalbrand et al., 1995). Because galactoglucoman-
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nan and cellulose are closely associated in plant tissues, Stalbrand et al. (1995) suggested that the CBD, by binding on cellulose, might target the mannanase close to the substrate. The T. reesei mannanase does not specifically bind on mannan (Tenkanen et al., 1995) but several sequential glucose units in galactoglucomannan may bind the CBD. However, the binding of CBD on cellulose requires six successive glucose units (Linder et al., 1995) which were not found in the oligosaccharides isolated in this study but may be present in native softwood galactoglucomannas.
Acknowledgements Matti
Siika-aho
is thanked for purification of Folke Tjerneld and Pia Ademark for p-mannosidase, Elina Luonteri for c1galactosidase, Maria Sandelin, Tapani Vuorinen and Timo Paakkiinen for help in the oligosaccharide purification, Ulla Lahtinen and Paivi Matikainen for the HPAEC-PAD analysis and Riitta Isoniemi for technical assistance. The work was mainly financed by TEKES (Technology Development Centre, Finland). T. reesei mannanase,
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