AEM Accepted Manuscript Posted Online 2 September 2016 Appl. Environ. Microbiol. doi:10.1128/AEM.01768-16 Copyright © 2016 Kojima et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
1 Characterization of an LPMO from the brown-rot fungus Gloeophyllum
2 trabeum with broad xyloglucan specificity, and its action on cellulose-
3 xyloglucan complexes
5 Yuka Kojima3'*, Aniko Varnaib'*, Takuya Ishidac, Naoki Sunagawac, Dejan M. Petrovicb,
6 Kiyohiko Igarashic'd', Jody Jellisone, Barry Goodellf, Gry Alfredsen6, Bj0rge Westerengb,
7 Vincent G.H. Eijsinkb't,#' Makoto Yoshida^*
9 aDepartment of Environmental and Natural Resource Science, Tokyo University of
10 Agriculture and Technology, Fuchu, Tokyo, Japan
11 bDepartment of Chemistry, Biotechnology and Food Science, Norwegian University of Life
12 Sciences (NMBU), As, Norway
13 cDepartment of Biomaterial Sciences, Graduate School of Agriculture and Life Sciences, The
14 University of Tokyo, Bunkyo-ku, Tokyo, Japan
15 dVTT Technical Research Centre of Finland, Espoo, Finland
16 eCenter for Agriculture, Food and the Environment, University of Massachusetts, Amherst,
17 Massachusetts, USA
18 'Department of Sustainable Biomaterials, Virginia Polytechnic Institute and State University,
19 Blacksburg, Virginia, USA
20 gNorwegian Institute of Bioeconomy Research, As, Norway
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22 Running title: Enzymatic properties of a brown-rot LPMO
24 Section: enzymology and protein engineering
26 Keywords: brown-rot, LPMO, AA9, viscosity, xyloglucan, cellulose
28 #To whom correspondence should be addressed: V.G.H. Eijsink, vincent.eijsink@nmbu.no;
29 M. Yoshida, ymakoto@cc.tuat.ac.jp.
30 *,f: These authors contributed equally to the manuscript.
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33 Abstract
34 Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases
35 (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum,
36 tend to have few LPMOs and information on these enzymes is scarce. The genome of G.
37 trabeum encodes four AA9 LPMOs, whose coding sequences were amplified from cDNA. Due
38 to alternative splicing, two variants of GtLPMO9A seem to be produced, a single domain
39 variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal
40 domain comprising approximately 55 residues without a predicted function. We have
41 overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated
42 its properties. Standard analyses, using HPAEC-PAD and MS, showed that GtLPMO9A-2 is
43 active on cellulose, carboxymethylcellulose and xyloglucan. Importantly, compared to other
44 known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position
45 along the P-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic
46 viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a
47 well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa, revealed that
48 GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurments also
49 revealed minor activity on glucomannan that could not be detected by the analysis of
50 soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C.
51 Experiments with co-polymeric substrates showed an inhibitory effect of hemicellulose-
52 coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2.
53 These results provide insight into the LPMO-potential of G. trabeum and provide a novel
54 sensitive method, measurement of dynamic viscosity, for monitoring LPMO activity.
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55 Importance
56 Currently, there are only a few methods available to analyze end-products of lytic
57 polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid
58 chromatography and mass spectrometry. Here we present an alternative and sensitive
59 method based on measurement of dynamic viscosity, for real-time continuous monitoring of
60 LPMO activity in the presence of water-soluble hemicelluloses such as xyloglucan. We have
61 used both this novel and existing analytical methods to characterize a xyloglucan-active
62 LPMO from a brown rot fungus. This enzyme, GtLPMO9A-2, differs from previously
63 characterized LPMOs, in having broad substrate specificity, enabling almost random
64 cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan,
65 suggesting a preference for xyloglucan chains that tether cellulose fibres together. The
66 xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength
67 at the initial stage of brown-rot, through degradation of the primary cell wall.
69 Introduction
70 For decades, the enzymatic degradation of cellulose by filamentous fungi was considered
71 to proceed through the hydrolytic action of cellulases. As early as in 1950, the involvement
72 of additional factors in cellulose conversion was proposed (1), and in 1974, Eriksson et al.
73 showed that enzymatic hydrolysis of cellulose by a crude fungal enzyme mixture is promoted
74 by the presence of molecular oxygen, suggesting a role for redox reactions (2). The
75 explanation for the observations by Eriksson et al. came almost four decades later, when
76 copper-dependent lytic polysaccharide monooxygenases (LPMOs), which are currently
77 classified into auxiliary activities (AA) family 9, 10, 11, and 13 in the CAZy database (3, 4),
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78 were first described (5-7). Since their discovery, LPMOs received much attention and are
79 currently considered as one of the key enzymes in fungal cellulose degradation (8). LPMOs
80 employ molecular oxygen and an external electron donor (5, 9-12) to carry out oxidative
81 cleavage of the p-1,4-glucosidic bonds in cellulose. Some LPMOs exclusively oxidize C1,
82 others exclusively oxidize C4, whereas a third type of LPMOs yields a mixture of C1- and C4-
83 oxidized products. Recently, LPMOs have been found to show oxidative activity against
84 various other plant polysaccharides, including xyloglucan, glucomannan, xylan and starch
85 (13-19). Thus, the physiological roles of LPMOs in biomass decomposition are likely to be
86 more complex and varied than initially assumed.
87 Brown-rot fungi are a group of wood-rotting basidiomycetous fungi and represent the
88 dominant wood decay fungi in northern coniferous forest ecosystems. They are able to
89 remove plant cell wall polysaccharides, such as cellulose and hemicelluloses together with
90 extensive lignin depolymerization and modification, but without lignin metabolism (20-22).
91 Brown-rot fungi employ a unique system of wood degradation, in which they combine
92 enzymatic and chemical mechanisms. The chemical mechanism implies the formation of
93 hydroxyl radicals (*OH) through a chelator-mediated Fenton (CMF) reaction that randomly
94 attack the wood cell wall components (22-26). This process causes structural and chemical
95 changes in the wood cell wall, potentially providing carbohydrate-active enzymes with
96 greater access to the substrate by generating holes large enough for the enzymes to
97 infiltrate the cell wall (21). Compared to soft-rot and white-rot fungi, the majority of brown-
98 rot fungi seem to have an incomplete enzymatic system for cellulose degradation. In
99 particular, they do not produce processive cellobiohydrolases, which are key enzymes in the
100 degradation of crystalline cellulose (27-29). Brown-rot fungi also often lack cellobiose
101 dehydrogenase (CDH), which is considered to be a natural electron donor of fungal LPMOs
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102 (9, 10, 12), albeit not the only one (11, 30, 31). While several cellulases and hemicellulases
103 from brown-rot fungi have been characterized, little is known about brown-rot LPMOs (32).
104 In general, the physiological function of oxidative enzyme systems in wood degradation by
105 brown-rot fungi is still unclear.
106 Currently, detection of LPMO activity on polymeric substrates is based on measurment of
107 the production of soluble oligomeric products which are released as a result of LPMO
108 cleavage near polymer chain ends or cleavage of the same polymer chain twice at nearby
109 positions. Such products can be detected using various types of chromatography (33, 34) or
110 mass spectrometry (5). The activity of strictly C4-oxidizing LPMOs may also be quantified
111 directly using a standard method for detection of the newly generated reducing ends (13).
112 Recently, a semiquantitative, high-throughput method for screening activity towards water-
113 soluble substrates has been reported (35), which is suitable for a wide variety of
114 carbohydrate-acting enzymes and can be adapted for LPMOs (13). LPMO-action on solid
115 substrates may also be quantified using confocal laser scanning microscopy after labeling C1-
116 oxidized cellulose chain ends with a fluorescence dye that is specific for carboxylic acids (36).
117 Unlike for endo-acting glycoside hydrolases, the use of gel permeation chromatography or
118 viscosity measurements for detecting LPMO-generated decreases in the molecular weight of
119 carbohydrate polymers has not yet been reported. Such analytical tools could potentially
120 reveal activities that cannot be detected by the other available methods and are also of
121 industrial relevance because they relate to (reducing) biomass viscosity.
122 In the present study, we cloned five cDNAs encoding putative LPMO9s (GtLPMO9s) from
123 Gloeophyllum trabeum, which is one of the most studied brown-rot fungi. We
124 heterologously expressed the one LPMO that, by phylogenetic analysis, seemed distant from
125 most other known LPMOs, namely GtLPMO9A-2. This enzyme has the longer amino acid
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126 sequence of two naturally occurring variants of GtLPMO9A (see below) and was expressed in
127 Pichia pastoris. As part of the in-depth characterization of GtLPMO9A-2, we developed a
128 method for monitoring LPMO-activity on hemicelluloses by measuring reduction in viscosity.
129 We show that GtLPMO9A-2 is a promiscuous LPMO with a unique ability to cleave
130 xyloglucan regardless of the substitutions of the p-1,4-linked glucan backbone. Using
131 dynamic viscosity measurements we were able to detect activity on additional
132 hemicelluloses that remained undetected in the standard chromatographic and mass
133 spectrometry analyses.
135 Materials and Methods
136 Strains and enzymes
137 Gloeophyllum trabeum strain NBRC 6430 was used as a source of LPMO genes.
138 Escherichia coli strain JM109 (Takara Bio, Shiga, Japan) and Pichia pastoris strain KM71H
139 (Invitrogen, Carlsbad, CA) were used as hosts for subcloning experiment and heterologous
140 production of recombinant GtLPMO9A-2, respectively.
141 Recombinant NcLPMO9C from Neurospora crassa (UniProt:Q7SHI8) was prepared
142 according to Kittl et al. (37). Endoglucanases AfCel12A from Aspergillus fumigatus
143 (UniProt:Q8TG26) and TaCel5A from Thermoascus aurantiacus (UniProt:Q8TG26) were
144 produced and purified as previously described (38).
145 Sequence analysis
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146 Multiple sequence alignments were generated using MAFFT, version 7.295 (39), as
147 available at the European Bioinformatics Institute website. For phylogenetic analysis of
148 LPMO9s, amino acid sequences were aligned using MAFFT and then manually edited using
149 Sea View (40). A phylogenetic tree was generated from this alignment by the neighbor-
150 joining method (41) in ClustalX software (42), with 1,000 bootstraps as previously described
151 (43).
152 Cloning of genes encoding GtLPMO9s
153 G. trabeum was cultivated in Highley's medium (44) containing 0.5% glucose (Wako,
154 Osaka, Japan) as the sole carbon source, and without thiamine hydrochloride. After 7 days of
155 cultivation at 23°C on 120 rpm, total RNA was extracted using the RNeasy Plant Mini Kit
156 (Qiagen, Venlo, The Netherlands). First-strand cDNA was then synthesized using reverse
157 transcriptase (SuperScript III, Invitrogen) with 3' rapid amplification of the cDNA ends
158 (3'RACE) using the GeneRacer Kit (Invitrogen) according to the manufacturer's instructions;
159 the 3'RACE primers used are listed in Table 1. The regions encoding the mature LPMOs were
160 sequenced after PCR amplification from first-strand cDNA using the ORF primers. The
161 nucleotide sequences of the genes (cDNA) encoding GtLPMO9A-1, GtLPMO9A-2, GtLPMO9C,
162 and GtLPMO9D have been deposited in the DDBJ database under accession numbers
163 LC157847, LC157848, LC157849, and LC157850, respectively. The cDNA sequence of
164 GtLPMO9B has already been described by Jung et al. (32) and has been deposited in the
165 GenBank database under accession number AEJ35168 as a endo-p-1,4-glucanase (named
166 Cel61G in the CAZy database). For expression, the insertion fragments of the GtLPMO9s,
167 except for GtLPMO9A-2, were amplified by expression primers appending the XhoI site and
168 the Kex2 cleavage site at the 5' end and the NotI site at the 3' end of the coding sequences
169 without signal peptide (Table 1) and cloned into pPICZa (Invitrogen) by restriction digestion
170 and ligation. After amplification with expression primers, the insertion fragment of
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171 GtLPMO9A-2 was inserted into pPICZa (Invitrogen) using the In-Fusion HD Cloning Kit
172 (Takara Bio); this strategy was chosen because the ORF encoding GtLPMO9A-2 contains a
173 NotI cleavage site.
174 Heterologous expression of the GtLPMO9s and purification of GtLPMO9A-2
175 Approximately 10 |g of pPICZa expression plasmid DNA was linearized with Bpu1102I
176 (Takara Bio) prior to transformation into P. pastoris. Electroporation, selection of
177 transformants, and production of recombinant protein were carried out according to the
178 instruction manual of the EasySelect Pichia expression kit (Invitrogen). After cultivating for 4
179 days in YP media using methanol as the carbon source, the cells were removed by
180 centrifugation (30 min at 10,000 g), and then the supernatants were applied for SDS-PAGE
181 analysis to evaluate the production of recombinant proteins.
182 For purification of GtLPMO9A-2, all steps were carried out at 4 °C, unless indicated
183 otherwise. The P. pastoris strain expressing GtLPMO9A-2 was grown in 4 liter YP medium
184 containing 1% glycerol for 1 day at 30 °C at 180 rpm. The cells were collected by
185 centrifugation at 1,500 g for 5 min and resuspended in 400 ml YP medium containing 1%
186 (v/v) methanol to induce expression and incubated further at 30 °C. Every 24 hours,
187 methanol was added to the culture, to a final concentration of 1% (v/v). After 4 days, the
188 cell-free supernatant was harvested by centrifugation at 10,000 g for 30 min. Ammonium
189 sulfate was added to the cell-free broth, to a final concentration of 50% (w/w). After
190 removing the precipitate by centrifugation (30 min at 10,000 g), the supernatant was diluted
191 approximately 2.2 times with 20 mM sodium acetate buffer (pH 4.5), to a final concentration
192 of approximately 1 M ammonium sulfate, and applied to a Toyopearl Phenyl-650S column
193 (020 mm by 160 mm; TOSOH, Tokyo, Japan) equilibrated with 20 mM sodium acetate buffer
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194 (pH 4.5) containing 1 M ammonium sulfate. The protein was eluted by applying a 100-mL
195 linear reverse-gradient to 20 mM sodium acetate buffer (pH 4.5). Fractions containing the
196 recombinant protein were collected and pooled, and the buffer was changed to 20 mM
197 sodium acetate buffer (pH 4.0) using Vivaspin 20 centrifugal concentrator tubes with a 10
198 kDa MWCO membrane (Sartorius AG, Gottingen, Germany). Then, the sample was applied to
199 a Toyopearl SP-650S column (020 mm by 250 mm; TOSOH) equilibrated with the same
200 buffer. The flow-through, containing the recombinant protein, was collected, buffer
201 exchanged to 20 mM sodium acetate buffer (pH 5.5) with Vivaspin 20 centrifugal
202 concentrator tubes, and ca. 13.1 mg proteins were treated with 1 ^L Endo H (500 Units; New
203 England Biolabs, Ipswich, MA) at 30 °C overnight. The protein solution was then buffer-
204 exchanged to 20 mM Tris-HCl buffer (pH 7.0) with Vivaspin 20 centrifugal concentrator tubes
205 and applied to a Toyopearl DEAE-650S column (020 mm by 250 mm; TOSOH) equilibrated
206 with the same buffer. The recombinant protein was eluted from the column with a linear
207 NaCl gradient, and the fractions eluting from 28 to 52 mM NaCl were collected. Protein
208 purity was analyzed by SDS-PAGE analysis with 12% polyacrylamide gels, and the N-terminal
209 amino acid sequence was determined with a protein sequencer (model 491 cLC; Applied
210 Biosystems, Foster City, CA).
211 Substrates
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212 The following substrates were used to characterize the substrate specificity of
213 GttPMO9A-2: phosphoric acid swollen cellulose (PASC), carboxymethylcellulose (CMC),
214 cellohexaose, cellopentaose, tamarind xyloglucan - partially arabinosylated (XG), xyloglucan
215 oligosaccharide (XG-oligomers), xyloglucan heptasaccharide (XG7), konjac glucomannan
216 (GM), ivory nut mannan, wheat arabinoxylan, oat flour mixed-linkage (ß-1,3-1,4) glucan, oat
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217 spelt xylan, beech wood xylan and birchwood xylan. PASC was prepared as described earlier
218 (45); birchwood xylan was purchased from Carl Roth GmbH (Karlsruhe, Germany). Konjac
219 glucomannan used in the dynamic viscosity experiments was purchased from Wako; konjac
220 glucomannan used in the coating experiments was purchased from Megazyme (Wicklow,
221 Ireland). Oat spelt xylan was purchased from Serva Electrophoresis GmbH (Heidelberg,
222 Germany); beech wood xylan was purchased from Sigma-Aldrich (St. Louis, MO). All other
223 substrates were obtained from Megazyme.
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224 LPMO-activity measurements
225 Dynamic viscosity experiments
226 Dynamic viscosity of the reaction solution was determined using an AMVn Automated
227 Micro Viscometer (Anton Paar, Graz, Austria), which is a falling ball-type viscometer
228 measuring the rolling time of a ball through a liquid in a capillary. Reaction mixtures of 1 mL
229 in total contained 1 ^M copper-saturated enzyme (see below), 0.5 mM dithiothreitol (DTT),
230 and substrate, in 50 mM sodium acetate buffer (pH 5.0). The concentration of each
231 substrate was adjusted, based on its intrinsic viscosity, so that the viscosity falls within the
232 optimal range of the viscometer. The concentrations of konjac glucomannan, tamarind
233 xyloglucan, arabinoxylan, and CMC were 0.05%, 0.15%, 0.2%, and 0.5% (w/v), respectively.
234 The reactions were carried out inside a glass capillary (01.6 mm) containing a steel ball (01.5
235 mm), thermostated at 30 °C. The capillary was positioned at a 50-degree angle relative to
236 horizontal (causing the ball to move through the reaction mixture along the capillary) and
237 was automatically inverted when the ball reached its lower end. The rolling time of the steel
238 ball was continuously recorded with the viscometer while the capillary was inverted 2000
239 times. End-point samples (after 16 hours of incubation, when the change in viscosity had
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240 levelled off) were analyzed by HPAEC-PAD and MALDI-ToF MS to check for the formation of
241 soluble oxidized oligosaccharides, as described below.
242 Dynamic viscosity was calculated as K-(p - Po)-T, where K is the calibration constant of the
243 system (mPa-cmVg), p is the density of the ball (g/cm3), p0 is the density of the reaction
244 mixture (g/cm3), and T is the rolling time of ball (sec). The calibration constant, K was
245 experimentally determined to be 0.00845 mPa-cm3/g using water as a standard.
246 The dynamic viscosity data were fit to an exponential decay formula (y=a-e(b'x)+c) with
247 DeltaGraph (SPSS Inc., CA), where y is the dynamic viscosity, x is the time, and a, b, and c are
248 constants. The constant c is the final viscosity of the substrate. The initial decline rate of
249 dynamic viscosity was calculated by differentiating the formula with respect to x=0. In case
250 of arabinoxylan and CMC, the correlation between dynamic viscosity and time was linear.
251 Endoglucanase treatment
252 To convert oxidized products into short oligosaccharides, the end-point samples of the
253 dynamic viscosity experiments were subjected to endoglucanase treatment and the resulting
254 oligomer mixtures were analyzed by HPAEC-PAD and MALDI-ToF MS (see below). Purified
255 endoglucanases, AfCel12A and IaCel5A, were diluted to a concentration of 100 |M, and 1 |l
256 AfCel12A or IaCel5A solution was added to 50 |l of the reaction mixtures containing LPMO-
257 treated xyloglucan or glucomannan. Subsequently, the reactions were incubated for 20
258 hours at 30 °C.
259 Screening for substrate specificity
260 After Cu(II)-saturation (46), GtLPMO9A-2 (or NcLPMO9C) was incubated with various
261 substrates in a 100 |L total reaction volume containing 1 |M enzyme, 0.5 mM dithiothreitol
262 (DTT) or 1 mM ascorbic acid, and substrate in 50 mM sodium acetate buffer (pH 5.0). The
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263 concentrations of substrates were as follows: 0.2% (w/v) PASC or Avicel, 0.2% (w/v) CMC,
264 0.05% (w/v) GM, 0.15% (w/v) XG, 0.2 g/l cellopentaose, cellohexaose, XG7 or XG-oligomer,
265 0.5% (w/v) mannan, xylan, or mixed-linkage glucan. Reactions were incubated at 30 °C in an
266 Eppendorf Thermomixer C (Eppendorf AG, Hamburg, Germany) with shaking at 1,000 rpm
267 for 16 hours. The reactions were run in at least duplicates, the reaction mixtures were boiled
268 for 10 minutes after sampling, and the solids were separated by centrifugation at 10 000 g
269 for 5 min. The supernatants were analyzed by HPAEC-PAD and MALDI-ToF MS (see below) to
270 check for the formation of soluble oxidized oligosaccharides.
271 Activity on complex substrates
272 In order to evaluate LPMO activity on complex substrates, hemicellulose was coated on
273 cellulose by pre-mixing aqueous solution of 1% (w/v) tamarind XG or 1% (w/v) konjac GM
274 with 1% (w/v) PASC in a 1:1 (v/v) ratio for 15 minutes (at room temperature without shaking,
275 in 20 mM Na-acetate buffer, pH 5.0) prior to enzyme addition. After the pre-incubation
276 reactions, reaction mixtures were prepared in 20 mM Na-acetate buffer (pH 5.0) containing
277 0.2% (w/v) PASC and 0.2% (w/v) XG or GM. Reaction mixtures containing 0.2% (w/v) of XG,
278 GM, or PASC as single substrate were also prepared. Further, the reaction mixtures
279 contained 1 ^M GtLPMO9A-2 or WcLPMO9C (copper-saturated according to Loose et al. (46))
280 and 1 mM ascorbic acid, and they were incubated at 30 °C, for 24 hours. The reactions were
281 run in at least duplicates, the reaction mixtures were boiled for 10 minutes after sampling,
282 and the solids were separated by centrifugation at 10 000 g for 5 min. Products were
283 analyzed using HPAEC-PAD as described below. Control experiments were performed
284 without electron donor or enzyme.
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285 Detection of oxidized oligosaccharides
286 The oligosaccharides released by LPMO action and sequential LPMO-endoglucanase
287 treatment were analyzed by high-performance anion exchange chromatography (HPAEC) on
288 a Dionex ICS3000 system equipped with pulsed amperometric detection (PAD), using a 50289 minute gradient (33) for cellulosic and a 75-minute gradient (13) for hemicellulosic
290 substrates.
291 The oligosaccharides were further analyzed using MALDI-ToF mass spectrometry (MALDI-
292 ToF MS). The analysis was carried out on an Ultraflex MALDI-ToF/ToF instrument (Bruker
293 Daltonics, Bremen, Germany) equipped with nitrogen 337 nm laser beam as described
294 earlier (5). Samples (2 |l) were applied to an MTP 384 ground steel target plate TF (Bruker
295 Daltonics) together with 4.5 mg 2,5-dihydroxybenzoic acid (DHB) matrix dissolved in 0.5 ml
296 30% acetonitrile. Data were collected with the lowest laser energy necessary to obtain
297 sufficient quality spectra, using Bruker's flexControl software. Spectra were analyzed using
298 Bruker's flexAnalysis software. All samples analyzed in this study contained 50 mM sodium
299 acetate, which suppressed the formation of potassium adducts.
301 Results
302 Nucleotide and amino acid sequences
303 The publicly available genome database of G. trabeum shows four genes encoding AA9
304 enzymes (29), which we have named lmpo9A (location in the G. trabeum genome database
305 v1.0: scaffold_00011:177323-178510), lmpo9B (scaffold_00011:179858-181138), lmpo9C
306 (scaffold_00002:3232078-3233061), and lmpo9D (scaffold_00001:4158823-4160504). So far,
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307 only one of these genes, lmpo9B, has been cloned (called Cel61G in the CAZy database);
308 studies of the recombinant protein have suggested that it acts on cellulose but the enzyme
309 remains largely uncharacterized (32). In the present study, the cDNA species transcribed
310 from the lpmo genes were cloned by RT-PCR using total RNA extracted from mycelium of G.
311 trabeum. The lmpo9A gene was transcribed into two different mRNAs (named lmpo9A-1 and
312 -2), the first 706 nucleotides of which were identical. The first 706 nucleotides were followed
313 by another 53 nucleotides, corresponding to an exon for the shorter splicing variant
314 (GtLPMO9A-1) or an intron for the longer splicing variant (GtLPMO9A-2). The latter, longer
315 transcript continued with another 407 nucleotides (Fig. S1).
316 The coding transcripts of lmpo9A-1, A-2, B, C, and D consist of 756, 1110, 756, 768, and
317 1032 bp, respectively, excluding the stop codon. Excluding the N-terminal signal peptide
318 from the translated amino acid sequences, as predicted by the SignalP program (47), the
319 mature GtLPMO9A-2 protein consists of 351 amino acids with a molecular weight (MW) of
320 36.0 kDa and a predicted pI of 4.56. For the other LPMOs these values are: GtLPMO9A-1, 233
321 amino acids, MW 25.1 kDa, pI 4.94; GtLPMO9B, 232 amino acids, MW 24.5 kDa, pI 6.19;
322 GtLPMO9C, 239 amino acids, MW 25.8 kDa, pI 4.68; GtLPMO9D, 321 amino acids, MW 33.1
323 kDa, pI 5.84.
324 In a phylogenetic tree of LPMO9s, GtLPMO9A-1, A-2, and B cluster in the same group,
325 whereas GtLPMO9C and D ended up in a different cluster each (Fig. 1). GtLPMO9A-1, A-2,
326 and B are closely related to a C1/C4 oxidizing LPMO from Neurospora crassa (NcLPMO9M or
327 NCU-07898; (48, 49)). Among characterized LPMOs, the ones being closest to GtLPMO9C are
328 the strictly C4-oxidizing LPMOs NcLPMO9A (NCU-02240; (49)), NcLPMO9C (a well-
329 characterized LPMO from Neurospora crassa with broad P-1,4-glucan-degrading abilities;
330 NCU-02916; (13, 15, 50)), and NcLPMO9D (NCU-01050; (48, 49)). GtLPMO9D is part of a
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331 clade that seems quite distinct from all LPMO9 enzymes characterized so far. Fig. 2 shows a
332 multiple sequence alignment including the GtLPMO9s. The catalytic domains of all four
333 LPMOs contain the two characteristic copper-binding histidines (His1 and His86 in
334 GtLPMO9A-2) as well as a conserved tyrosine that occupies one of the axial positions in the
335 copper coordination sphere (Tyr175 in GtLPMO9A-2) (6).
336 GtLPMO9A-2 and D have an additional C-terminal domain that starts with a region of low
337 sequence complexity that could possibly be a linker (Fig. S2). We were not able to detect
338 sequence similarity with known carbohydrate-binding domains (CBMs) or with other
339 domains of known function. For more detailed sequence analysis of the C-terminal domains,
340 see the Supplementary material (including Figs. S2 - S4). Notably, the sequence alignments
341 of Fig. S3 & S4 show that the C-terminal domains of GtLPMO9A-2 and GtLPMO9D also occur
342 in other LPMOs. Furthermore, the alignment shows that the C-terminal domain of
343 GtLPMO9A-2 has features not unlike those of well-known cellulose-binding domains.
344 As a first step towards mapping the ability of G. trabeum to oxidatively cleave plant
345 polysaccharides, we have overexpressed and characterized GtLPMO9A-2. This enzyme was
346 selected as a representative for three (GtLPMO9A-1, A-2, and B) of the five G. trabeum
347 LPMOs that are closely related (Fig. 1) and that cluster in an area of the phylogenetic tree
348 with little available functional data. Also, the coding sequence of GtLPMO9A-2 has not been
349 reported before. GtLPMO9D was also considered interesting, but we were not able to
350 express this enzyme in sufficient amounts.
351 Physical properties of recombinant GtLPMO9A-2
352 GtLPMO9A-2 was expressed in Pichia pastoris and purified to homogeneity by 3 steps of
353 column chromatography (Fig. S5). Purified GtLPMO9A-2 appeared to have a significantly
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354 higher MW (ca. 60 kDa) than that estimated from the amino acid sequence (36.0 kDa).
355 Treatment with EndoH led to a small decrease in MW (to approx. 57 kDa), indicating N-
356 glycosylation of the protein. The remaining mass difference suggests that the recombinant
357 enzyme was also O-glycosylated, perhaps in the serine-rich linker region. Correct processing
358 of the purified protein was confirmed by determination of N-terminal amino acid sequence
359 of the purified enzyme, yielding HGYVDQVTIG, which is identical to the amino acid sequence
360 deduced from the nucleotide sequence. As observed previously for LPMO9s expressed in P.
361 pastoris, the N-terminal histidine was not methylated (14, 15, 51).
362 Cellulolytic activity of recombinant GtLPMO9A-2
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363 GtLPMO9A-2 was active on phosphoric acid-swollen cellulose (PASC) (Fig. 3), similar to all
364 LPMO9s characterized so far (52), and Avicel (data not shown). HPAEC analysis showed
365 diagnostic product patterns that reflect a mixed C1/C4 oxidation pattern, similar to the
366 phylogenetically closest characterized LPMO (Fig. 1), WcLPMO9M (NCU-07898; (49)). The
367 type of electron donor, ascorbic acid or DTT, had no effect on the product profile of the
368 LPMO (Fig. S6) but the reaction was slower when using DTT. GtLPMO9A-2 was inactive on
369 shorter cello-oligosaccharides, such as cellopentaose (Fig. S7A) and cellohexaose (not
370 shown), compared to WcLPMO9C. Neither GtLPMO9A-2 nor WcLPMO9C showed activity on
371 XG7 (not shown). Interestingly, GtLPMO9A-2 was active on carboxymethylcellulose (CMC)
372 (Fig. S8), suggesting that the enzyme can act on soluble polymeric ß-glucans and is not
373 restricted by substitutions on the D-glucose backbone.
374 Hemicellulolytic activity of recombinant GtLPMO9A-2
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The activity of GtLPMO9A-2 against hemicellulosic substrates was assayed using dynamic
376 viscosity analysis, using NcLPMO9C, with known activity towards several p-1,4-glucans (13),
377 as positive control. As expected, significant decreases in viscosity were observed when
378 xyloglucan (XG, Fig. 4b) or glucomannan (GM, Fig. 4d) was incubated with NcLPMO9C,
379 whereas no significant change of viscosity was detected in the reaction with arabinoxylan
380 (Fig. 4f). Similarly to NcLPMO9C, GtLPMO9A-2 also caused a decrease in viscosity in the
381 reactions with XG (Fig. 4a) and GM (Fig. 4c) but not with arabinoxylan (Fig. 4e). Both
382 enzymes also reduced slightly the viscosity in reactions with CMC (Fig. 4g,h). The dynamic
383 viscosity remained unchanged in reactions without the LPMO or an electron donor, showing
384 that the observed effects are due to oxidative cleavage by the LPMOs and not to e.g.
385 contaminating hydrolases. The fact that treatment with the LPMOs led to a drop in viscosity
386 in the presence (but not in the absence) of an electron donor clearly indicates that both
387 enzymes have an endo-type of action on XG and GM.
388 Comparative studies revealed that GtLPMO9A-2 reduced the viscosity of XG almost two-
389 fold faster than NcLPMO9C, whereas the difference in final viscosity was neglectable (Table
390 2). The viscosity data indicated that the Gloeophyllum enzyme was slightly less active than
391 the Neurospora enzyme on GM, both in terms of initial rate and final viscosity. Both
392 GtLPMO9A-2 and NcLPMO9C were 5-10-fold slower in reducing the viscosity of a CMC
393 solution (0.5%, w/v) as compared with XG (0.15%, w/v).
394 We used HPAEC-PAD and MALDI-ToF MS to analyse water-soluble oligosaccharides
395 potentially released by GtLPMO9A-2 from a selection of hemicellulosic substrates. No
396 products could be detected upon incubating the LPMO (+ electron donor) with ivory nut
397 mannan (a linear P-1,4-linked mannan), wheat arabinoxylan, oat spelt xylan, beech wood
398 xylan, and birchwood xylan (heteropolymers with P-1,4-linked xylan backbone) or mixed
399 linked p-glucan (with a P-1,3 linkage at every third linkage in the p-1,4-linked glucan
400 backbone) (data not shown). Data for XG and GM are discussed below.
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401 Identification of oligosaccharides released from xyloglucan and glucomannan
402 To confirm LPMO-action on hemicelluloses, which eventually would lead to the formation
403 of oligosaccharides, we subjected the end-point samples from the dynamic viscosity
404 experiments to HPAEC-PAD analysis. The HPAEC-PAD-profiles show that GtLPMO9A-2
405 released a broad range of xyloglucan oligosaccharides eluting from 25 to 60 minutes,
406 whereas the major products generated by NcLPMO9C eluted between 45 and 60 minutes
407 (Fig. 5A). No products could be detected when either of the LPMOs or the reducing agent
408 was incubated with the substrate alone. To simplify the product profiles, we treated the
409 samples with AfCel12A, a XG-active endoglucanase, which depolymerizes XG into its
410 repeating oligomeric units by cleaving next to non-substitued glucose units. The known
411 dominant products of AfCel12A are XXXG, XXLG/XLXG, XLLG (G, glucose; X, glucose carrying a
412 xylose substitution at C6; L, X with a further galactose appended to the C2 of the xylose).
413 Note that the basic repeating unit in tamarind xyloglucan is XXXG carrying galactosylations at
414 various positions (13, 53). In addition, the tamarind xyloglucan was partially arabinosylated,
415 carrying 0.27 arabinosyl unit per repeating (XXXG) unit (compositional data provided by the
416 manufacturer, Megazyme).
417 The resulting chromatograms (Fig. 5B) are dominated by standard (non-oxidized) XG
418 breakdown products that were released by the endoglucanase from the still mainly
419 polymeric XG remaining after the LPMO treatment. The dominating products with a four
420 glucan backbone (XXXG fragments with varying degrees of galactosylation) elute between 30
421 and 40 minutes, whereas less abundant longer products elute between 45 and 55 minutes in
422 discrete peaks (Fig. 5B, black line). The latter products are likely to be a variety of oligomers
423 of DP 12-18, some of which contain arabinosyl units (54). Interestingly, some of these
424 oligomeric products (marked with grey dashed line in Fig. 5B) were absent in samples that
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425 had been treated with LPMO + reductant (compare reactions without, light blue and red,
426 and with, dark blue and red lines, reducing agent in Fig. 5B). Next to these standard
427 products, the product profiles from reactions that had been pretreated with an active LPMO
428 showed additional peaks, and the patterns of these additional peaks reveal differences
429 between the two enzymes. Endoglucanase treatment of the product mixture generated by
430 GtLPMO9A-2 yielded a wide variety of additional products, with elution times ranging from
431 20 to 60 minutes and including products eluting earlier than XXXG (XG7; Fig. 5B, green line).
432 In contrast, endoglucanase treatment of NcLPMO9C-generated material yielded a less varied
433 collection of additional products, which almost exclusively eluted at 45 minutes or later and
434 which included a cluster of products eluting at 61-66 minutes that is lacking from the
435 reactions with GtLPMO9A-2. The product profile obtained here with NcLPMO9C is similar to
436 what has been observed before for this C4-oxidizing enzyme (13), whereas the product
437 profile for GtLPMO9A-2 is clearly different. Based on data presented by Bennati-Granier et
438 al. (14), the peak eluting at 43 min that is unique for GtLPMO9A-2 could be C1-oxidized XG7.
439 HPAEC-PAD chromatograms for the reactions with konjac GM showed clear effects of
440 NcLPMO9C whereas the effects of GtLPMO9A-2 on GM were less pronounced (Fig. S9A).
441 Treatment of GM with NcLPMO9C led to a clear shift in the polymer peak, and small
442 amounts of oligmeric products were also observed. For the reaction with GtLPMO9A-2, only
443 a minor shift in the polymeric peak was observed. Subsequent endoglucanase treatment
444 with TaCel5A led to the accummulation of chromatographically distinct products in case of
445 NcLPMO9C, while similar products were not observed after the combined action of
446 GtLPMO9C and TaCel5A (Fig. S9B).
447 MALDI-ToF MS analysis of the reaction mixtures obtained after the dynamic viscosity
448 experiments confirmed major differences in the product profiles generated by GtLPMO9A-2
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449 and NcLPMO9C (Fig. 6). While NcLPMO9C released clusters of oxidized xyloglucan fragments
450 with the number of xylose residues being a multiple of three, GtLPMO9A released a much
451 broader range of oxidized oligosaccharides. The results for NcLPMO9C are in accordance
452 with previous work (13) showing that the enzyme cleaves XG at the non-reducing end of the
453 non-substituted glucosyl residue in the (XXXG)n backbone of xyloglucan, forming C4-oxidized
454 XG fragments. On the other hand, the data for GtLPMO9A-2 suggest that this LPMO can
455 cleave almost anywhere in the XG chain, since reactions with this enzyme yield oxidized XG
456 fragments with a number of xylose residues not being only a multiple of three. Addition of
457 LPMO in the absence of reducing agent did not have an impact on the product profiles
458 obtained from XG after endoglucanase treatment. This indicates that the GtLPMO9A-2 and
459 NcLPMO9C enzyme preparations were not contaminated with background activity of an
460 enzyme that would cleave off xylosyl units of XG, and that all XG fragments in Fig. 6 were
461 products of LPMO-treatment. Upon treating GM with GtLPMO9A-2, we could not detect
462 oxidized oligosaccharides with MALDI-ToF MS.
463 Closer inspection of Fig. 6 (see Fig. S10) showed that the labeled signals correspond to
464 clusters containing signals for the native, the non-hydrated oxidized, and the hydrated
465 oxidized oligosaccharide, with the former two signals dominating. Signals for the sodium salt
466 of the aldonic acid and double-oxidized species, which would be indicative of C1 oxidation,
467 were not observed. Although it is not certain that oxidized xyloglucan oligosaccharides
468 behave like oxidized cello-oligomers under these analytical conditions (see legend to Fig. 3),
469 the data suggest that C4-oxidation dominated during the degradation of XG.
470 In addition to the expected masses originating from galactosylated XG fragments, the
471 MALDI-ToF spectra for the reactions with GtLPMO9A-2 contained a few species with more
472 than three pentose units per repeating unit (4 hexoses), and hence are predicted to carry an
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473 arabinosylated X-unit (Fig. 6; (54)). Since the arabinosyl and xylosyl groups (both pentoses)
474 cannot be distinguished by MS, we cannot exclude that some of the other fragments
475 generated by GtLPMO9A-2 also contain an arabinosyl group replacing a xylosyl unit (see
476 proposed structures by Niemann et al. (54)).
477 The chromatogram of Fig. 5B shows that incubation of the LPMO-treated xyloglucan
478 sample with AfCel12A resulted in production of shorter xyloglucan oligosaccharides,
479 producing mostly native oligosaccharides with DP 7-9 and a wide variety of putatively
480 oxidized products, particularly in the case of GtLPMO9A-2 (Fig. 5B). MALDI-ToF MS (results
481 not shown) showed that many of the higher DP species visible in Figs. 5B and 6A were no
482 longer present after combined endoglucanase-LPMO action. The combined action of
483 GtLPMO9A-2 and AfCel12A on XG resulted in the formation of shorter fragments (in
484 particular, m/z 494.1 (oxidized XG/GX or L) and 791.4 (native XXG/XGX/GXX or XL/LX)). These
485 shorter native species may correspond to the compounds eluting before the XG7 peak, at
486 20-30 min, in Fig. 5B. It is noteworthy that the native species observed with HPAEC may also
487 originate from C4-oxidized products as it has recently been shown that C4-oxidized products
488 are unstable and tend to loose the oxidized monosugar under the chromatographic
489 conditions used here (34).
490 LPMO activity on complex substrates
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491 In order to evaluate possible additional differences between GtLPMO9A-2 and WcLPMO9C
492 and to study LPMO action on more natural substrates, we then carried out experiments with
493 co-polymeric substrates generated by mixing xyloglucan or glucomannan with PASC, using
494 ascorbic acid as reducing agent (Fig. 7; note that the reaction is faster when using ascorbic
495 acid, explaining the apparent differences between Fig. 7 and Fig. 5A). These experiments
496 were inspired by the findings by Frommhagen et al. (17), who demonstrated LPMO activity
497 on xylan, but only if the xylan was grafted onto cellulose. In general, hemicellulose coating
498 hindered LPMO-activity on cellulose, reducing the amount of oxidized cello-oligosaccharides
499 released (compare blue and purple lines in Figs. 7 and S10). Interestingly, coating XG on
500 cellulose also seemed to have a negative effect on the activity of both LPMOs towards XG
501 itself (compare red and purple lines in Fig. 7). Experiments with GM-coated PASC and
502 GtLPMO9A-2 showed that coating with GM completely abolished GtLPMO9A-2 activity on
503 PASC, whereas soluble GM products were not detected by the HPAEC-PAD analysis (Fig.
504 S11). Interestingly, control reactions with WcLPMO9C, with much higher activity on GM,
505 showed that this LPMO degraded the GM and that, consequently, degradation of PASC was
506 still observed after coating with GM. We carried out several experiments with birchwood
507 xylan, alone or mixed with PASC, without detecting any activity on xylan.
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509 Discussion
510 The present data show that GtLPMO9A-2 is a C1/C4-oxidizing LPMO with high activity on
511 cellulose and xyloglucan, negligible activity on cellodextrins and a minor activity on
512 glucomannan. The enzyme has the exceptional capability of cleaving any Glc-Glc bond in
513 xyloglucan, regardless of substitutions, thus functionally expanding the arsenal of
514 hemicellulolytic LPMOs so far described (13, 14, 16, 17). Xylosyl substitutions at the C6
515 position in XG seemed to be less inhibitory than carboxymethyl substitutions in CMC, which
516 may be located at the C2, C3, or C6 positions on the glucan backbone. The fact that
517 GtLPMO9A-2 was not active on cello-oligosaccharides, nor on mixed-linked glucan, suggests
518 that GtLPMO9A-2, in contrast to WcLPMO9C, requires longer stretches of p-1,4-glucosidic
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519 linkages in the polysaccharide backbone for substrate recognition. Perhaps, the minor
520 depolymerizing activity that we observed with konjac glucomannan could reflect the
521 occasional presence of longer stretches of p-1,4-linked glucose units. The fact that
522 GtLPMO9A-2 was inactive towards XG7 (consisting of a cellotetraose backbone),
523 cellopentaose, and cellohexaose may be taken to imply that the enzyme requires at least
524 seven glucosyl units for productive substrate binding.
525 So far, only three xyloglucan-active LPMOs have been studied in detail, NcLPMO9C from
526 Neurospora crassa (13), PaLPMO9H from Podospora anserina (14), and An3046 from
527 Aspergillus nidulans (16). All these LPMOs cleave xyloglucan next to the non-substituted
528 glucosyl residue (i.e. G-unit), yielding a clustered product profile on MALDI-ToF MS (as in Fig.
529 6B). In contrast, the oxidized products released by GtLPMO9A-2 had a wide variety of
530 masses (Fig. 6A), showing that GtLPMO9A-2 activity is unaffected by substitution of the
531 glycosyl units at the C6 position and that GtLPMO9A-2 can cleave everywhere in the p-
532 glucan main chain. These observations are further supported by the wide range of products
533 observed upon the combined action of GtLPMO9A-2 and AfCel12A (which only cleaves at
534 non-substituted G's), including substituted products with dimeric or trimeric backbones that
535 only can emerge upon cleavage in between two substituted main chain sugars.
536 The oxidative regioselectivities of xyloglucan-active LPMOs on cellulose show that all
537 variants occur, C1 (An3046; literature data are not conclusive; C1/C4 activity is also possible),
538 C4 (NcLPMO9C) and C1/C4 (PaLPMO9H and GtLPMO9A-2). However, there is little data on
539 the oxidative regioselectivity of these enzymes on xyloglucan. Our own previous data for
540 NcLPMO9C and the present data for GtLPMO9A-2 indicate that these enzymes almost
541 exclusively oxidize C4 when acting on XG.
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542 The sequence alignment of Fig. 2 shows that GtLPMO9A-2 has an extended L2 loop
543 (compared to NcLPMO9C and PoLPMO9H), while it lacks the L3 loop and also has a deletion
544 (relative to NcLPMO9C and PoLPMO9H) in the so-called LC-loop, very close to a conserved
545 surface-exposed aromatic residue (Tyr204 in NcLPMO9C and Tyr215 in GtLPMO9A-2). The
546 same applies to An3046, although this was not noted by the authors (16). The L3 loop has
547 been proposed to be a structural determinant of xyloglucan activity (14), and a recent NMR
548 study of enzyme-substrate interactions in NcLPMO9C showed that the L3 loop indeed
549 interacts with xyloglucan (55). The present data, and data for An3046, show that xyloglucan
550 cleavage can also be achieved by LPMOs lacking the L3 loop. The extension of the L2 loop
551 may compensate for the deletion of the neighboring L3 loop and it is interesting to note that
552 the extension and deletion are of the same length, 14 amino acids. Lack of the L3 loop and
553 the seemingly correlated deletion near Tyr204/215 may have effects on the specificity of
554 xyloglucan cleavage and/or the ability to cleave shorter substrates. For example, only the
555 two xyloglucan-active LPMOs with the L3 loop and with no deletion in the LC loop, i.e.
556 NcLPMO9C and PoLPMO9H, have been shown to cleave soluble cellodextrins (14, 50).
557 Viscosity measurements have occasionally been used to assess activity of endoglucanases
558 on water-soluble polysaccharides (56-58). Here we show that dynamic viscosity
559 measurements provide an alternative and sensitive method for assessing and comparing
560 LPMO-activity on water-soluble polysaccharides. This simple method proved to be a good
561 choice for both characterizing substrate specificity and comparing depolymerization rates. In
562 fact, by relying solely on the standard methods (HPAEC-PAD and MALDI-ToF MS) that are
563 based on monitoring released oligomeric products, we would have overlooked the low
564 activity of GtLPMO9A-2 on glucomannan. These findings show the need for using
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565 complementary methods, including methods capable of monitoring the polymeric fraction,
566 when characterizing LPMOs.
567 Furthermore, while quantification of LPMO activity is complex, due to product
568 heterogeneity and lack of product solubility, the dynamic viscosity measurements provide a
569 direct measurement of the number of cuts introduced in the polysaccharide substrate. This
570 method is independent of oxidative regioselectivity and the cleavage pattern. In the present
571 study, the dynamic viscosity experiments showed that WcLPMO9C is more active on
572 glucomannan compared to GtLPMO9A-2, whereas the latter depolymerized xyloglucan at a
573 higher rate. The higher activity of GtLPMO9A-2 on xyloglucan could be due to its broader
574 cleavage specifity, which implies that there are more productive binding sites on the
575 substrate.
576 The experiments with co-polymeric substrates yielded somewhat surprising results. We
577 show that hemicellulose coating of PASC reduces LPMO activity on PASC dramatically. This
578 provides a rationale for hemicellulolytic activity among LMPOs, as exemplified by the studies
579 with glucomannan. WcLPMO9C, with its glucomannan activity was able to degrade GM-
580 coated PASC (degrading both GM and PASC), wheras GtLPMO9A-2 with only very weak GM
581 activity, could not degrade the mixture of these two polymers. On the other hand, in
582 mixtures of cellulose and XG, the activity on both polymers was strongly reduced, for both
583 tested LPMOs (see below for further discussion).
584 To date, there is limited information on LPMOs from brown-rot fungi. Vanden
585 Wymelenberg et al. showed low expression levels of LPMO genes in the brown-rot fungus
586 Postia placenta even when cultivated on aspen wood (59). Expression of LPMOs was
587 undetectable in proteome analyses for the brown-rot fungi Fomitopsis pinicola and
588 Wolfiporia cocos growing on aspen wood (60). These results indicate that LPMOs may not
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589 constitute a major enzyme component in the wood degradation system of brown-rot fungi.
590 However, recently, Jung et al. reported that saccharification of oak and kenaf pretreated
591 with popping was promoted by a homologue of GtLPMO9B, suggesting the importance of
592 this enzyme in the enzymatic degradation of plant biomass by brown-rot fungi (32).
593 Moreover, we were able to amplify all G. trabeum LPMOs from mRNA, meaning that the
594 genes are expressed, and we show that GtLPMO9A-2 is active on biomass.
595 Brown-rot fungi, such as G. trabeum, preferentially invade softwood. In softwood, the
596 main hemicelluloses are glucomannan and glucuronoxylan, which occur in the secondary cell
597 wall. The secondary cell wall is surrounded by a primary cell wall, where the most abundant
598 hemicelluloses are xyloglucan and pectin (61-63). The genome of G. trabeum encodes sets of
599 CAZymes that target xyloglucan (GH12s and GH74) and pectin (10 GH28s, GH43s, 2 CE8s, and
600 CE15), besides cellulose (e.g. GH3s, GH5s and GH12s), glucomannan (GH3s and GH5s), and
601 xylan (3 GH10s, GH43s, CE1, and 6 CE16s) (29). Due to its substrate promiscuity, GtLPMO9A-
602 2 could play a role in the depolymerization of not only cellulose but also xyloglucan during
603 biomass-degradation. Perhaps the enzyme has additional capabilities that remain
604 undiscovered, perhaps activities on natural co-polymeric plant cell walls. It could be of
605 interest to test other electron donors, since there are indications that the electron donor
606 affects LPMO performance, including substrate specificity (11, 31). By degrading xyloglucan,
607 GtLPMO9A-2 may loosen the adhesion between cellulose bundles, thus creating access for
608 various CAZymes. The fact that GtLPMO9A-2 (and also WcLPMO9C) acted preferentially on
609 free XG as compared to cellulose-bound XG suggests that GtLPMO9A-2 may preferentially
610 cut XG chains that tether cellulose fibres together as opposed to XG chains that adhere to
611 cellulose fiber surfaces.
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612 It is well known that wood strength rapidly decreases at the initial stage of brown-rot
613 even before the loss of weight in wood (64). As the fungus invades the wood cells from the
614 lumen, depolymerization of cell wall components using a low molecular weight decay system
615 facilitates enzyme penetration into the cell wall and enables enzymatic degradation of the
616 outer primary cell wall concurrent with the inner secondary cell wall layers (22). The rapid
617 decrease in cellulose DP throughout the wood cell wall and the degradation of the primary
618 cell wall are likely to be primary causes of strength reduction at this decay stage (22). It is
619 tempting to speculate that GtLPMO9A-2 plays a role in this process, i.e. primary cell wall
620 degradation at the initial stage of brown-rot.
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622 Funding information
623 The work was carried out as part of the Biomim project funded by the Norwegian
624 Research Council through grant 243663, and partly co-funded by the Bilateral Joint Research
625 Projects programme of JSPS in Japan (to M.Y.), and JSPS KAKENHI (grant number 16J03946
626 to Y.K., 15H04526 to M.Y., and 16K14952 to M.Y.).
628 References
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03 Q. CD Q.
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846 Figure legends
847 Figure 1. Position of G. trabeum LPMOs in the phylogenetic tree of LPMO9s. The
848 phylogenetic tree was created from catalytic domains of LPMO9s by using the neighbor-
849 joining method in ClustalX (v. 2.1). Several LPMOs are labeled and their cleavage specificities
850 are indicated (C1, C4, or both, C1/C4; N. D. means not determined). For all labeled non-
851 GtLPMOs, activity on cellulose has been demonstrated. An * indicates that additional activity
852 on xyloglucan has been detected. The functional data for GtLPMO9A-2 stem from the
853 present report; other functional data come from references (13, 14, 16, 49, 65).
855 Figure 2. Multiple sequence alignment of the catalytic domains of selected LPMO9s. Fully
856 conserved residues are printed in white on a black background. Active site histidines (black-
857 filled triangles) and a tyrosine (gray-filled triangle) involved in copper coordination are
858 indicated. The labeled bars over the sequences indicate known variable regions in LPMO9s
859 (15, 51). Note that GtLPMO9A-2 and GtLPMO9D have C-terminal extensions; for more
860 information see Figs. S2-S4. This alignment was generated using MAFFT, version 7.295 (39),
861 available at the European Bioinformatics Institute website
862 (https://www.ebi.ac.uk/Tools/msa/mafft/).
864 Figure 3. Products generated by GtLPMO9A-2 or NcLPMO9C on PASC. (A) HPAEC-PAD
865 chromatograms showing cello-oligosaccharides released by GtLPMO9A-2 (blue) and
866 WcLPMO9C (red) from PASC. Peaks were assigned based on previous assignments by Isaksen
867 et al. (50); native cello-oligosaccharides are labeled as Glcn where n is the degree of
868 polymerization (DP). Note that it has recently been shown that C4-oxidized products are
Ш Q. CD Q.
Ш СП
r—t-
СГ CD
CD i О Ш
869 unstable under these chromatographic conditions and that the peaks labeled C4 are, in fact,
870 diagnostic degradation products (34). The fractions of native products are high because C4-
871 oxidized products tend to loose the oxidized monosugar under these chromatographic
872 conditions. (B) MALDI-ToF spectrum of cello-oligosaccharides released by GtLPMO9A-2 from
873 PASC, where the inset shows details for the heptamer ion cluster (sodium adducts only).
874 Possible products in these clusters are the native Glc7 (m/z 1175.8), the Cl-oxidized lactone
875 or C4-oxidized ketoaldose (anhydrated species, m/z 1173.8), the Cl-oxidized aldonic acid or
876 C4-oxidized gemdiol (hydrated species, m/z 1191.8), and the sodium adduct of the aldonic
877 acid sodium salt (1213.8). The double-oxidized species corresponds to m/z 1171.8
878 (anhydrated form, lactone-ketoaldose species), 1189.8 (hydrated form), and 1211.8 (the
879 sodium salt of the sodium adduct). The presence of sodium salts is diagnostic for C1-
880 oxidation, since only this oxidation yields an aldonic acid. The strong signals for dehydrated
881 oxidized species in MALDI-ToF are diagnostic for C4-oxidation. The 4-keto to gemdiol
882 equilibrium is less skewed towards the hydrated form compared to the lactone - aldonic
883 acid equilibrium, and, besides, C4-oxidized products are more efficiently dehydrated during
884 spotting on MALDI sample plates (see Forsberg et al. (66) for further discussion).
885 Experiments with Avicel yielded similar product patterns (data not shown).
887 Figure 4. Assessing LPMO-activity on hemicelluloses with dynamic viscosity experiments.
888 (a, c, e, g) GtLPMO9A-2 or (b, d, f, h) WcLPMO9C were incubated with (a, b) 0.15% (w/v)
889 xyloglucan, (c, d) 0.05% (w/v) glucomannan, (e, f) 0.2% (w/v) arabinoxylan, and (g, h) 0.5%
890 (w/v) carboxymethylcellulose in the presence (red lines) or absence (green lines) of DTT as a
891 reducing agent. Reactions with only DTT and no LPMO are shown by blue lines.
03 Q. 0 Q.
03 C/J
o »o cr 0
—5 0
0 i o'
893 Figure 5. HPAEC-PAD analysis of reaction products generated from xyloglucan (XG) by
894 GtLPMO9A-2 and WcLPMO9C in the dynamic viscosity experiments. Samples were taken
895 after 16 hours of incubation and the chromatograms show the product profiles before (A)
896 and after (B) a subsequent treatment with AfCel12A. Green line: XG7 standard (XXXG); grey
897 line: XG-oligo standard (this is a mixture of shorter XG oligomers with DP in the range of 14898 27). Black line: XG incubated with DTT only. Red lines: products generated from XG with
899 WcLPMO9C in the presence (dark red) or absence (light red) of DTT. Blue lines: products
900 generated from XG with GtLPMO9A-2 in the presence (dark blue) or absence (light blue) of
901 DTT. Grey dashed lines indicate oligosaccharides the concentration of which was lower as a
902 result of LPMO activity. The reaction conditions are specified in the Materials and Methods
903 section. Nb. Fig. 7 provides an even clearer example of the difference between WcLPMO9C
904 and GtLPMO9A-2 with respect to the activity on xyloglucan.
906 Figure 6. MALDI-ToF MS analysis of the reaction products generated by (A) GtLPMO9A-2 and
907 (B) WcLPMO9C during the dynamic viscosity experiments with tamarind xyloglucan (XG) in
908 the presence of reducing agent (DTT). The samples analyzed were the end-point samples (16
909 hours). The reaction conditions are specified in the Materials and Methods and were similar
910 to the conditions used for generating Fig. 5. GtLPMO9A-2 generated a wide range of
911 oligosaccharides with all possible combination of hexose (Hex) and pentose (Pen) units,
912 whereas WcLPMO9C generated clusters of oligosaccharides with the number of pentose
913 residues being a multiple of three. In both spectra, oxidized species (-2, as compared with
914 the mass of native species) are marked with #; species with masses indicating
915 arabinosylation are labeled green (see text). Note that most labeled peaks in fact are a
Q. CD Q.
Ш СП
r—t-
СГ CD
К) О
cluster of signals; see Fig. S10 and text. All labeled species are Na+-adducts. No species were detected below m/z 1000.
Figure 7. HPAEC-PAD analysis of the reaction products generated by (A) GtLPMO9A-2 or (B) WcLPMO9C on xyloglucan-coated PASC. Green line: native cello-oligosaccharides with DP 26; yellow line: XG7 standard (XXXG); grey line: XG-oligo standard with DP 14-27. Brown line: mixture of PASC and XG incubated with LPMO only. Black line: mixture of PASC and XG incubated with ascorbic acid (ASC) only. Blue line: products generated from PASC with LPMO in the presence of ASC. Red line: products generated from XG with LPMO in the presence of ASC. Purple line: products generated from xyloglucan-coated PASC with LPMO in the presence of ASC. The reaction conditions are specified in the Materials and Methods section. Note that the chromatographic conditions were similar to those used in Fig. 5 but different from those used in Fig. 3. This explains why the retention times of cello-oligomers differ between Fig. 3 and Fig. 5.
CD Q. 0 Q.
03 C/J
o »o cr 0
—5 0
0 i o'
931 Tables
933 Table 1. Oligonucleotide primers.
Primers
Sequences (5'-sequence-3'
GtLPMO9A_3'RACE_forward
GtLPMO9B_3'RACE_forward
Gt LPMO9C_3'RACE_forward
GtLPMO9D_3'RACE_forward
3'RACE_reverse
Gt LPMO9A_O RF_forward
GtLPMO9B_ORF_forward
GtLPMO9C_ORF_forward
GtLPMO9D_ORF_forward
GtLPMO9A-1_ORF_reverse
GtLPMO9A-2_ORF_reverse
GtLPMO9B_ORF_reverse
GtLPMO9C_ORF_reverse
GtLPMO9D_ORF_reverse
GtLPMO9A-1_expression_forward
GtLPMO9A-2_expression_forward
GtLPMO9B_expression_forwa rd
GtLPMO9C_expression_forward
GtLPMO9D_expression_forward
GtLPMO9A-1_expression_reverse
GtLPMO9A-2_expression_reverse
GtLPMO9B_expression_reverse
GtLPMO9C_expression_reverse
GtLPMO9D_expression_reverse
ATCTCACACTCCTTGACATCCAAT
GTTCAATGCAACGGGGAGAAT
CCCACCATAGACGACGTCAA
TGCAGCAGGAAATCATGAAC
G CTGTCAACGATACGCTACGTAACG
TCCTTGCTTTGGTTCTGC
CCAAGACATTCTTCGCCATCG
CTTCATTCAGCCGCTCTAG
CCACGACTAGGACGATGA
TCATTGCTCGTCCAGACA
CACAGCCAATTGCTTCAAG
GAGTCCTGAGTACACGGGTGAGC
TACGCGACATCAGCTGAT
AGCGCGTGAGTGAGCTATA
AACTCGAGAAAAGACATGGATATGTTGATCAAGTCAC
AGGGGTATCTCTCGAGAAAAGACATGGATATGTTGATCAAGTCAC
AACTCGAGAAAAGACACGGGTACGTTGATACCC
AACTCGAGAAAAGACACACAATATTTCAGAGGGTATATG
AACTCGAGAAAAGACACGGCTTCGTGTCCAAG
AAGCGGCCGCCTATGAATTTATTTGTGTTAGGATTTG
AGAAAGCTGGCGGCCTTAGAAAGAGAGACGTCCCAG
AAGCGGCCGCTTAATTGCCGGTCCAGAC
AAGCGGCCGCTTAACCCTCTGCCGGCATAC
AAGCGGCCGCCTACCACTGGTGCGCCGTCGTCT
The restriction sites Xhol (CTCGAG; forward expression primers) and NotI (GCGGCCGC; reverse expression primers; only partial in the GtLPMO9A-2 primer) and the sequence encoding the cleavage site of Kex2 (AAAAGA, encoding Lys-Arg; forward primers) are printed in italics. ORF sequences are underlined. Expression primers were designed with a
CD Q. 0 Q.
CD C/J
r—t-
o cr 0
—5 0
protecting AA sequence at the 5' end. GtLPMO9A-2 expression primers, for In-Fusion cloning, were designed to contain 15 extra bases at the 5' end, overlapping with the Kex2 (forward) or NotI restriction cleavage site (reverse) of pPICZa.
CD Q. CD Q.
QJ C/J
O f—t-O cr CD
—5 0
Table 2. Parameters obtained after fitting the dynamic viscosity curves. The correlation between dynamic viscosity and time (data from Fig. 4) was fitted to an exponential (Exp.) curve (y=a-e(b'x)+c) or to a linear (Lin.) equation (y=a\x+b), where y is the dynamic viscosity (in mP), x is the time (in ks), and a, b, and c are constants. The constant c is the final viscosity of substrate after completion of LPMO9 action. The initial depolymerization rate (IDR) was calculated as the first derivative for the formula at x=0. The high R-square values (>0.97) reflect a good fit of the model.
CD Q. CD Q.
Substrate Enzyme Fit Parameters a (mP) b (ks-1) c (mP) R-square IDR (mP^ks-1)
XG GtLPMO9A-2 Exp. 0.5748 -0.1514 0.9812 0.9927 0.0870
NcLPMO9C Exp. 0.4633 -0.1067 0.9947 0.9950 0.0495
GM GtLPMO9A-2 Exp. 0.7115 -0.07416 1.0496 0.9966 0.0528
NcLPMO9C Exp. 0.3907 -0.1537 0.9367 0.9832 0.0600
WAX GtLPMO9A-2 Lin. -0.001454 1.7106 - 0.9936 0.001454
NcLPMO9C Lin. -0.001654 1.663 - 0.9731 0.001653
CMC GtLPMO9A-2 Exp. 0.3713 -0.01999 1.7082 0.9998 0.0074
NcLPMO9C Exp. 0.4455 -0.02732 1.6661 0.9991 0.0122
03 C/J
o »o cr CD
CD i O' 03
Л/с1_РМ09Р, С4 A/CLPM09A, С4
GfLPM09C, N.D.
A/CLPM09E, Cl
PaLPM09H, С1/С4*
NcLPM09C, C4*
AN3046, N.D.*
GÍLPM09D, N.D.
PcLPM09D, Cl
A/CLPM09M, C1/C4
NCU07760, C1/C4
Gfl_PM09A-l, N.D. GfLPM09A-2, C1/C4* GtLPM09B, N.D.
CL CD CL
Ш СЯ
r—t-
О СГ CD
GtLPM09A-l
GtLPM09A-2
GtLPM09B
GtLPM09C
GtLPM09D
NcLPM09M
PaLPM09H
NcLPM09C
AN3046
PcLPM09D
GtLPM09A-l
GtLPM09A-2
GtLPM09B
GtLPM09C
GtLPM09D
NCLPM09M
PaLPM09H
NcLPM09C
AN3046
PCLPM09D
GtLPM09A-l
GtLPM09A-2
GtLPM09B
GtLPM09C
GtLPM09D
NcLPM09M
PaLPM09H
NcLPM09C
AN3046
PcLPM09D
GtLPM09A-l
GtLPM09A-2
GtLPM09B
GtLPM09C
GtLPM09D
NCLPM09M
PaLPM09H
NcLPM09C
AN3046
PCLPM09D
GYV-DQVTIGe GYV-DQVTIr GYV-DTLNVGG TIF-QRVYVDp GFV-SKW GFV-DNATI 5IF-QKVS' riF-QKVS' VFF-DTLVIDI YTFPDFIEP!
í JVYTGYQPYQDPYESPVPQRIE Ge JVYTGYQPYQDPYESPVPQRIE GS rQYTGYLPYNDPYTTPAPQRIE i i/G-EGHLSG-----------1-
£ 3SYAGNTPGGD-
-TSPSPI-
ï3F---YQPYQDPYMGSPPDRIS
t i/D-QGQLKG-----------V! \D-QGQLKG-----------1-
J 3E-TTPNQY-----------V-
S0TV-TGDWVY-----------V-
ЭА---IPGNG
¡A---IPGNG
Эр---IPGNG
HI---PESNV\
HQ---ISTIS
Эк---IPGNG
HA---PYSNF
HA---PANNN
HSNTRPEKYN ¡¡ETQNHYSNG
l/EDLTL-l/EDLTL-й I/TALTT-¡IMDLSS-* l/KGAAN-
j l/EDVTS ¡IENVNH
-KDMF -LAK PDFAl
¡l/TDVMS--------SDI] !
¡TKWVNTRDDMTPDMPDFF J
l/TDVTS
Ï MGSGG-5 MGSGG-]MGENG-!MGGVNP
5 gy----
! MA----
5 M TN---
i MA----
. . J YKGSF-PEFRiVELDL-
/32 ß3
58 SGTKPAALIASAAA 58 SGTKPAALIASAAA 58 GGSSPAPLVATIAA 46 YHEPVSLAIIQVPA а 50 -DAQVASQVAAADF£ 52 -DSAPAKLHASAAA 43 -IQLRDNTVIKVPA 42 -VTMKDSNVLTVPA 56 -TFAGQTDTAEVKA 49 QNTAGQTQTATVS,
EIAFHWT-EIAFHWT-3GKIAFHWT-
j AR ¡AK J ¡SI' A0D
ÏSTITAEWHPTIDDV---
S 5KVTFTWS----GGGG-
!STVTLRWT---------
-----TWPSS
-----TWPSS
-----TWPSS
-NTTESIRPD ----QNWP—
----------IWPDS
RVGAWWGHEIGGAAGPNDPDHPIAAS KVGHFWGHEIGGAAGPNDADNPIAAS 3SKLAMKLG-------------VGATMQ !
-----------SAIY"
1TVGFKAN-
PVITYMGKVPSNTDITSYSPTGSD PVITYMGKVPSNTDITSYSPTGSD PVITYLGKVPSSTDVTKYSPTGSD
PVIAYLAKVPDAL-QT----DVAG
P LMTYMGAC EGTT-C DKY—TATD PVITYMARCPDTG-CQDWTPSASD
PIQVYLAKVNNAA-NA----GTSG
PIMVYLAKVDNAA-TT----GTSG
PGLVYMSKAPGAA-NQ----YEGD
3YLDVMMSPASPAA-NSPE—AGTG
VnßgJlD V]?¡SID
V] i IÍ
LS 9 j SI
AK ?j SID KV ? ; Ян Le ! i 3Vi LK ? j aVi 106 GD ?j Я1Н 100 QlIiSlY
113 113 113 106 105
106 105
:\GYE—NGKWAATDIMSAQNSTWTVT] : \GYE—NGKWAATDIMSAQNSTWTVT] : 3GYS—NGKWAATDVLSAQNSTWTVT] )GLS-DDGTWATDRLIANA-GKVNFT] : /GREANGGDWVQQEIMNG—GTYTVTL :3GREGTSNVWAATPLMTAP-ANYEYA] :}G LN—NGVWAVDNMISNG-GWHYFDf :\GLS—NGKWAVDDLIANN-GWSYFDf :EGICDTSKDIKTDAWCTWDKDRIEFT] :EKPQFENGQLVFD---TTQ-QEVTFT]
kalapE
KALAPe SSLAPe SCIQPß SNIA SCLKI ijSCVAl STCIA ¡ADLPDfj "KSLPSß
ь \L 3AETYPC Иоиа
¡I М. 3AETYPC 3qf
¡I \L JAQTYPC 3qs
¡I W <AETYP( 3qf
¡I M. -GMTEGC Je f
¡I M 5AYSYPC 3qf
L rL 5ASVRG/ 3qf
L rL YAGSQAC 3qf
fcv jAHD-GC iEF
И JM /ASSYGC QqfQ
176 176 176
174 TIFVFEVETAATPQMEiî
16 8--------------p^eJ
16 9--------------Pi
168--------------M
167--------------Ii
17 0--------------Yl
161--------------Ii
WVqvtgpHtetptsqalvsf
j F•VQVTGPgTETPTSQALVSF
IRVTGSj YKTPSGSYLVSF ~ LNITGP{5VVPSP—TATF Iß VRITGN { 5GTPNQ—TVSF LQVTGS J FKTPSS-GLVSF IEITGS j FNTGSN—FVSF INVTGG { 5ASPSN—TVSF ! VKVTGG e gGNPQD—TIKF LNVENGHGTPGP—LVS]
¡GI-TFNVYSGKHRGDVIRQILTQ
¡GI-TFNVYSGS------------
¡GI-AFNVYTNF------------
¡GI-TVDIERLI------------
¡GIWDKNVYDPS------------
j GV-TYDAYQAA------------
j GI-LVSIYDLQGRPT------NG
: GI-LINIYGGSGKTD------NG
SF-NFSVWGGM------------
¡GI-LINIYNLP----------KN
GtLPM09A-l 231 INS---
GtLPM09A-2 219 ITSYPI j i PPVWTSN
GtLPM09B 219 -TSYPI i PAVWTGN
GtLPM09C 229 —NYTV l "1PAEG—
GtLPM09D 210 -APYTF j i PPLSNL-
NcLPM09M 211 —TYTI j i PAVFTC-
PaLPM09H 215 GRPYTI î l PAPLTC-
NCLPM09C 214 GKPYQI i i PALFTC-
AN3046 211 -KDYPM j i PAVYTC-
PCLPM09D 204 FTGYPA i l PAVWQG-
CL CD CL
Ш СП
г—t-
О сг CD
Applied and Environmental à
Microbiology
Applied and Environmental Microbiology
ccepted Mârïïjscrrpt HostecTOnimë
Relative intensity
00 o o
H -Glcfi
- Glc7
Relative intensity
- GICq
00 o o
1171.8
/ 1173.8 X 1175.8
1189.8 X 1191.8 1211.8 1213.8
Signal (nC)
I—4 I—4 H* NJ
Ni>®(»0loj>01[»0 ooooooooooo
■t* o
■li U1
- Glc2
- Glc3 -Glc4
- Glee
AjBjqn |B0|p9|/\| ||9UJ0Q |||9AA Äq 9|,02 'l Jeqopo uo /ßJ0WSBW9B//:dHi| luoj^ p9pB0|UM0Q
со CL Е
со CL
сл ■>
Е со с
со CL
со о
о ■>
Е со с
1.6 1.4 1.2 1
2.4 2.2 2 1.8 1.6 1.4 1.2 1
10 20 30 40 50 Incubation time (ksec)
10 20 30 40 Incubation time (ksec)
0 10 20 30 40 50 Incubation time (ksec)
10 20 30 40 50 Incubation time (ksec)
ю о о
ел ■>
Е со с
со Q_
со о о
(п ■>
Е со с
1.8 г» 1.6^ 1.4 1.2 1
2.4 2.2 2 1.8 1.6 1.4 1.2 1
10 20 30 40 Incubation time (ksec)
10 20 30 40 Incubation time (ksec)
0 10 20 30 40 Incubation time (ksec)
10 20 30 40 Incubation time (ksec)
CL CD CL
Ш СП
О f—t-О er CD
Longer Polymer peak
Time (min)
Longer XG-oligosaccharides
Time (min)
XG + StLPM09A-2 + DTT
XG + A/CLPM09C+DTT
XG + GtLPM09A-2
XG + «CLPM09C
XG + DTT
XG-oligo
XG + GtLPM09A-2 + DTT + d/Cell2A ■ XG + NCLPM09C + DTT + AfCe I12A XG + GtLPM09A-2 + AfCe\12A XG + NcLPM09C + AJCe I12A XG + DTT + /\/Cell2A XG-oligo XG7
CD Q. CD Q.
03 C/J
O f—t-o cr CD
—5 0
150 -,
!" 100
** ^ LT) иэ
m с с С с 1Л с
с ai ф Ф CL) с LT) ш ф
ф о. CL Û_ Q_ CD с с Q_
Û_ г^ 00 00 CD û_ at cu tH
из X X X X ai û_ о ü_ о тЧ
X ai ai a) ai X X
Ф X X X X Ф T-i fH Ф
X X X X X
о о ^H ГМ ф Ф
CT) it rvj ^ Ю (X) ГМ X X LO
r^ О Ю СП (N 00
о w с Г-s 00 СП о LO т-i
LT) CD т—1 tH тН ГМ TH о (N KD
i-H С o_ (N гм LH..... fN
XG + GfLPM09A-2 + DTT
> 50 -
lO ID CT1 *H
rr> m 00 oo ел гм о *н
гм IN
XG + /VcLPM09C + DTT
CL CD CL
Ш СП
r—t-
150 П
r-. ni 00 о
^ШнМ^Мш.
Cl гм r\i
<N 1Л ■tf
»^.■..«И.ЛиЬитцЬ.Ц
U5 ID rv r->
мммА -1
u c 250
"5 200
OD 150
u c 250
"5 200
00 150
Longer XG-oligosaccharides
Cl-oxidized cello-
oligosaccharides
C4-oxidized cello-oligosaccharides
Time (min)
Time (min)
PASC + XG + GtLPM09A-2 + ASC
XG + GtLPM09A-2 + ASC
PASC + GiLPM09A-2 + ASC
PASC+ XG +ASC
PASC + XG + GtLPM09A-2
XG-oligo
Cello-oligos DP 2-6
PASC + XG + WcLPM09C + ASC
XG + A/cLPM09C + ASC
PASC+ A/CLPM09C +ASC
PASC+ XG +ASC
PASC + XG + A/cLPM09C
XG-oligo
Cello-oligos DP 2-6
CD Q. CD Q.
0) C/J
O o o er CD
CD i O' 0)