Benzylamine as Hydrogen Transfer Agent: Cobalt-Catalyzed Chemoselective C=C Bond Reduction of β-Trifluoromethylated α,β-Unsaturated Ketones via 1,5-Hydrogen Transfer

Article abstract of DOI:10.1002/asia.201901210

An efficient cobalt-catalyzed chemoselective reduction of β-CF₃-α,β-unsaturated ketones using benzylamine as hydrogen transfer agent involving intramolecular 1,5-hydrogen transfer is reported. The reaction proceeded smoothly with a relatively w

Full text of DOI:10.1002/asia.201901210

Article
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Cite This: J. Agric. Food Chem. 2019, 67, 5486−5495
Mapping the Secretome and Its N‑Linked Glycosylation of Pleurotus
eryngii and Pleurotus ostreatus Grown on Hemp Stalks
Chunliang Xie,† Wenbing Gong,† Zuohua Zhu,† Yingjun Zhou,† Li Yan,† Zhenxiu Hu,†
Lianzhong Ai,*,†,‡ and Yuande Peng*,†
†Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China
‡Shanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food Engineering, University of
Shanghai for Science and Technology, Shanghai 200093, China
S
* Supporting Information
ABSTRACT: Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment
of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for
the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were
identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation
were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked
glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and
Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-
galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic
saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase
were reduced after deglycosylation.
KEYWORDS: hemp stalks, N-linked glycosylation, Pleurotus eryngii, Pleurotus ostreatus, secretome
might help to identify effective enzyme cocktails to degrade
hemp lignin during the pretreatment process.
INTRODUCTION
■
Industrial hemp (Cannabis sativa L.) is an important fiber
crop.1 Ninety percent of the biomass of hemp is stalk and
leaves. The hemp stalk is mainly composed of 37.3% cellulose,
19.79% hemicellulose, and 12.35% lignin. However, stalks used
for the production of industrial products require depolymeri-
zation to mono- and oligosaccharides.2,3 This conversion
process is achieved through a variety of extracellular
lignocellulolytic enzymes by microbes, especially white rot
fungi. In our previous research, the white-rot fungi Pleurotus
eryngii (P. eryngii) and Pleurotus ostreatus (P. ostreatus) were
found to be effective for pretreatment of industrial hemp stalks
to improve enzymatic saccharification.4 These two fungi can
produce various extracellular secreted enzymes to transform
cellulose, hemicellulose, and lignin of plant biomass and can
preferentially degrade lignin during the pretreatment process.
Laccase, lignin peroxidase (LiP), manganese peroxidase
(MnP), β-glucosidase, endo-1,4-β-glucanase, exo-1,4-β-gluca-
nase, acetyl xylan esterase, endo-1,4-β-xylanase, α-L-arabinofur-
anosidase, β-xylosidase, and α-glucuronidase, mainly involved
in cellulose, hemicellulose, and lignin degradation, were
identified.5−8 In addition, the secretome of P. eryngii cultured
on ramie stalk substrates was reported. Cellulases, hemi-
cellulases, and ligninase described as above were identified.9,10
The secretome of P. ostreatus was identified by other
laboratories, and similar results were obtained.11 Because the
lignocellulolytic enzymes from P. eryngii and P. ostreatus play
an important role in hemp stalk pretreatment, the secretomic
analysis of P. eryngii and P. ostreatus grown on hemp stalks
The efficiency of lignocellulose utilization depends on
localization, protein−protein interactions, activities, and post-
translational modifications (PTMs) of extracellular en-
zymes.12−15 Among all PTMs, N-linked glycosylation is the
most common and can strongly influence protein folding,
secretion, cellular localization, and biological activity.16 Asn-
X(aa)-Ser/Thr sequon is the dominant N-glycosylation region,
where X denotes any amino acid except proline.17,18 With the
recent advances in glycomic technologies, hydrazide chemistry,
hydrophilic interaction, and lectin affinity were developed as
specific N-glycopeptide enrichment methods.19 At the same
time, LC-MS/MS has been used to analyze and characterize
both N-glycoproteins and N-glycosylated sites.20 N-glycosyla-
tion has been well studied in several biotechnologically
important yeasts and filamentous fungi, including Saccharo-
myces cerevisiae, Ashbya gossypii, Aspergillus nidulans, and
Phanerochaete chrysosporium (P. chrysosporium).21−24 In
addition, the glycan structure of laccase from P. ostreatus has
been reported.25 However, the N-glycosylation information
available on the composition and structural characteristics of
other proteins from P. eryngii and P. ostreatus secretomes is
limited.
Received: January 3, 2019
Revised: April 21, 2019
Accepted: April 23, 2019
Published: April 23, 2019
© 2019 American Chemical Society
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ion mode. A complete MS scan was followed by five MS/MS scans of
those highest peaks. The data files were generated by Mascot Distiller
v.2.3.2.0 software (Matrix Science Ltd.) and submitted to the P.
eryngii and P. ostreatus genome from JGI. The search parameters were
set as follows: fixed modification (carbamidomethylation), optional
modification (methionine oxidation), and enzyme (trypsin with one
missed cleavage); tolerance (10 ppm and 1 Da for precursor ions and
fragment ions, respectively). The following criteria were required to
consider a protein successfully identified: two or more unique
peptides identified; p-value < 0.1 or a confidence level of 95%.
P. eryngii and P. ostreatus Secretome Deglycosylation by
PNGaseF. Secretomes were extracted from 10 g (wet weight) of
substrate incubated at 28 °C for 21 days as described above. The
secretomes were deglycosylated using N-glycosidase F (PNGaseF) as
follows. First, 500 U of PNGase F was added to 10 mL of secretome
supernatants, and the mixture was digested at 37 °C at 900 rpm for 10
h. When only partial deglycosylation was achieved, an additional 500
U PNGase F was added and allowed to react overnight. The reactions
were monitored by SDS-PAGE. The completed deglycosylated
secretomes were used directly in the following enzyme assay and
hemp stalk pretreatment experiment.
In this study, secreted proteins and CAZymes in the P.
eryngii and P. ostreatus genome were predicted, and the
secretomes of P. eryngii and P. ostreatus were analyzed by
proteomic methods. In addition, we comprehensively identi-
fied protein N-glycosylation of P. eryngii and P. ostreatus by
combining a hydrazide enrichment method with MS-based
proteomics techniques. Furthermore, the comparisons of the
secretomes and glycoproteins in these two fungi were
presented. The function of protein glycosylation of P. eryngii
and P. ostreatus in the activities and stabilities of enzymes and
hemp stalk enzymatic saccharification was also discussed. The
objectives of this research were to reveal the composition and
N-glycosylation of secretomes of P. eryngii and P. ostreatus
cultivated on hemp stalks for 21 days and to explore the effects
of N-glycosylation on enzyme activity, stability, and enzymatic
saccharification of hemp stalks, so as to provide a theoretical
basis for the construction of a compound enzyme for rapid
lignin degradation of stalks for pretreatment in the future.
Enzyme Assay and Thermal Stability Test. In order to
investigate the effect of glycosylation on enzyme activity and stability,
secretomes with or without deglycosylation were prepared as
described above for enzyme activity determination. The enzyme
activity results of experiments were the average results of 20 replicates.
The substrate used to measure laccase activity was ABTS.28 α-
Galactosidase activity was determined by the pNPG method.29
Phenol oxidase activity was determined by the methods according to
Benjamin and Montgomery.30 The optimum temperature for the
enzymes was detected by incubating the secretome at different
temperatures ranging from 30 to 80 °C for 10 min. Then, the enzymes
were maintained at 70 °C for 10−70 min, and activity was measured
every 10 min.
MATERIALS AND METHODS
■
Strains and Substrates. P. eryngii (CICC50126) and P. ostreatus
(bio-67015) were obtained from the inquiry network for microbial
strains of China (http://www.biobw.org/). The two white rot fungal
strains were precultured on PDA plates at 28 °C for 7 days. The hemp
stalks were cut into small chips (400−800 μm). Hemp stalks (3 g)
and 7 mL of distilled water were placed in 300 mL Erlenmeyer flasks
and sterilized at 121 °C for 20 min. Four pieces of P. eryngii preculture
(approximately 40 mm2 piece-1) were inoculated into the substrates.
P. ostreatus was inoculated using the same method as that for P.
eryngii. These cultures were statically incubated at 28 °C for 21 days.
All of the experiments were carried out in triplicate.
Effect of P. eryngii and P. ostreatus Secretome Glycosyla-
tion on Enzymatic Hydrolysis. A normal or deglycosylated
secretome was used to pretreat hemp stalks for 24, 48, and 72 h at
37 °C, pH 5.0. The ratio of hemp stalks to secretome was 1:20 (solid
to liquid ratio). After this reaction, the pretreated hemp stalks were
centrifuged at 3000g for 10 min. The supernatant was discarded.
Pretreated hemp samples were washed with ddH2O three times, dried
at 50 °C for 3 days, and used for determination of weight loss,
cellulose, and lignin contents and enzymatic hydrolysis detection
according to the Laboratory Analytical Procedure of the National
Renewable Energy Laboratory.31
Enzymatic hydrolysis was performed as follows. First, 0.16 g of
pretreated or untreated hemp stalks in citric acid buffer (pH 4.8) were
hydrolyzed with 30 FPU/g cellulase at 50 °C. After 48 h, the yield of
reducing sugar in the reaction supernatants was detected with the
DNS method. The amounts of reducing sugar (mg/g) were calculated
as follows:
Secretome Protein Sample Preparation. For secretome
analysis, extracellular proteins were extracted as follows. Sodium
acetate buffer (200 mL, 50 mM, pH 5.0) was added to 10 g (wet
weight) of substrate cultivated for 21 days and stirred at 180 rpm
overnight in an ice bath. The supernatants were collected by
centrifugation at 8000g for 30 min at 4 °C. Then, the supernatants
were mixed with an equal volume of cold 40% trichloroacetic acid
(TCA) on ice overnight. The precipitate was collected, washed with
20% TCA, followed by washing with acetone twice. Subsequently, the
protein precipitate was dried and lyophilized. Lysis buffer (10 mL),
including 8 M urea, 2 M thiourea, 2% CHAPS, 20 mM Tris−HCl,
and 30 mM DTT, was added to a 1 mg lyophilized pellet, incubated
on ice for 4 h, and centrifuged at 12000g for 30 min. The supernatants
were collected, and the protein concentrations in the supernatant
were determined by the BCA method according to the manufacturer’s
instructions.
Peptide Extraction and Glycopeptide Enrichment. The
peptide-level enrichment method was used as described.26 Briefly, 1
mg of protein was reduced and alkylated with 10 mM DTT and 55
mM IAA. Then, the alkylated proteins were digested at 37 °C
overnight with trypsin (Promega, Madison, WI, USA). The tryptic
peptides were purified with a C18 desalted column. The glycopeptide
enriched by solid phase extraction using hydrazide chemistry followed
by enzymatic release of glycans by PNGase F was done according to
previous research.27 NaIO4 (15 mM) was used to oxidize the digested
peptides. Then, the excess NaIO4 was removed by SPEC-18. Oxidized
peptides were eluted by 80% ACN and 0.1% TFA and incubated
overnight with 10 mg of hydrazide magnetic beads. Glycopeptides on
the beads were released and collected as described.27
LC−MS/MS Analysis. The glycopeptide mixture was analyzed by
an LTQ Velos Orbitrap mass spectrometer (Thermo Fisher
Scientific) combined with an EASY-nLC system (Proxeon Bio-
systems). The peptides were separated along a linear gradient using
buffer A (0.1% formic acid) and 0−60% of buffer B (acetonitrile/
water, 98:2, v/v, 0.1% formic acid) over a period of 150 min, with a
flow rate of 200 nL/min. The LC-MS/MS was operated in a positive
reducing sugar amounts
amount of reducing sugar after enzymatic hydrolysis
=
amount of dry hemp woody core
Secreted Protein and CAZyme Annotation. There are four
steps to predict secreted proteins. First, SignalP v4.1 was used to
predict whether proteins contained N-terminal signal peptides;32
then, TMHMM v2.0 was used to predict whether the protein had a
transmembrane region.33 Lipid-anchored proteins were analyzed by a
big-PI Fungal predictor, and TargetP v1 was used to predict the
subcellular localization of proteins.34 CAZyme annotation was
predicted by dbCAN (version V5.0), which consists of 680974
sequences (http://csbl.bmb.uga.edu/dbCAN/download/CAZyDB.
07152016.fa).35 The Blast p e-value threshold was 1 × 10−5.
Statistical Analysis. All of the experimental values presented in
graphs and tables are the mean SD calculated using Excel 2007.
The enzyme activity results were the average results of 20 replicates
from four independent repetition, and the enzymatic hydrolysis and
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Article
component analysis of treated and untreated hemp chips were
conducted in triplicate. Multiple comparison tests were performed
with a t test (significance level = 0.05).
RESULTS
■
Prediction of Secreted Proteins and CAZymes in the
P. eryngii and P. ostreatus Genome. Previous research
showed that secreted proteins and CAZymes play important
roles in lignocellulose degradation.36 To identify all of the
secreted proteins and CAZymes in P. eryngii and P. ostreatus,
ORFs (10678 entries) were downloaded from the JGI Genome
Database. Secreted proteins should have the following
characteristics: (1) N-terminal signal peptide; (2) no trans-
membrane domain; (3) no GPI anchor point; and (4) no
predictive location signal for transferring protein to mitochon-
dria or other intracellular organelles. On the basis of the
principles mentioned above, 301 and 359 secreted proteins
were identified in P. eryngii and P. ostreatus genomes,
respectively (Table 1), with respect to the 501 and 521
Table 1. Number of Predicted Secretory Proteins and
CAZymes in the Pleurotus eryngii and Pleurotus ostreatus
Genomes
Pleurotus eryngii
Pleurotus ostreatus
secreted proteins
CAZymes
secreted CAZymes
301
501
249
359
521
242
CAZymes, which were annotated by dbCAN (automated
CAZymes annotation) in P. eryngii and P. ostreatus genomes,
respectively. In total, 249 and 242 proteins belonging to both
secreted proteins and CAZymes in P. eryngii and P. ostreatus
genomes were identified, respectively.
Figure 1. Functions classification of the identified proteins of
Pleurotus eryngii and Pleurotus ostreatus on hemp stalk medium at
21 days. (A) The function of the identified secretome of Pleurotus
eryngii (B) The function of the identified secretome of Pleurotus
ostreatus.
Secretomes of P. eryngii and P. ostreatus Cultivated
in Hemp Stalk Medium for 21 Days Identified by LC-
MS/MS. The secretomes cultivated in hemp stalk medium
were analyzed. From two measurements, 185 proteins were
identified, and 169 and 155 proteins were identified in P.
eryngii and P. ostreatus, respectively; 139 proteins were
identified in both fungi (Tables S1−S5). According to the
annotation reported in the UniProt database, Figure 1A shows
that 52 proteins (31.7%) from P. eryngii were identified as
CAZymes. Another 117 identified proteins were involved in (i)
glucose, amino-acid, and lipid metabolism (46), (ii) cell-wall
biogenesis and biodegradation (6), (iii) transporters (14), (iv)
protease and peptidase (24), and (VI) cell signaling (27). As
shown in Figure 1B, protein function classification and
distribution of P. ostreatus were similar to those of P. eryngii.
Interestingly, CAZymes account for more than 50% of the total
lignocellulose degradation proteins. In total, 52 and 50
CAZymes were identified in the P. eryngii and P. ostreatus
secretomes, respectively. Among these CAZymes, 16 GHs, 3
GTs, 3 CEs, 1 PL, and 25 AAs were identified in the P. eryngii
secretome. At the same time, 18 GHs, 2 GTs, 2 CEs, 1 PL, and
22 AAs were identified in the P. eryngii secretome (Figure 2).
The number and distribution of CAZymes isolated from P.
eryngii and P. ostreatus did not differ.
Figure 2. Differentially expressed CAZymes in Pleurotus eryngii and
Pleurotus ostreatus cultivated in hemp stalk medium at 21 days.
present research, 27 and 24 N-glycopeptides were detected in
P. eryngii and P. ostreatus secretomes, respectively, and 23, 1,
and 1 proteins had single, double, and triple glycosylated
peptides, respectively, in P. eryngii (Table 2). In total, 20 and 2
proteins had single and double glycosylation peptides,
respectively, in P. ostreatus (Table 3). The majority of the
glycoproteins are involved in lignocellulose degradation. Most
importantly, 6 and 5 glycosylated peptides of laccase were
Global N-Glycosylated Protein Profiling of P. eryngii
and P. ostreatus Cultivated in Hemp Stalk Medium. To
understand the glycosylation status of the enzymes, the
hydrazide enrichment method was combined with MS-based
proteomics techniques to identify the N-glycopeptides. In the
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Table 2. Global N-Glycosylated Peptides Profiling of Pleurotus eryngii Cultivated in Hemp Stalk Medium
protein
accession
matched motif
(N-X-S/T)
peptide
unique
mass
description
P13860
A0A0N9GYX4
B5MAF5
G4XU44
J0DB01
LYVQNGKVIQNSSVK
LTNETMK
DVVNTGVSTDDNVTIR
PIFRDVVSTGTPAAGDNVTIRFR
SSPANITQGTR
NSS
NET
NVT
NVT
NIT
Y
Y
Y
Y
Y
1681.9022 exoglucanase 1
838.3992 laccase
1709.8091 phenol oxidase
2491.3069 laccase 2
1133.5563 FAD-binding domain-
containing protein
K5UJU4
K5ULW4
K5W0L8
LPLAGGTNNTNPALR
QNGSLTANASESTEPWGIWLNGGPGSSSLLGLLFENGPLHIR
NFSGNPANGTGGNVFVK
NNT
NAS
NFS
Y
Y
Y
1516.7754 trehalase
4379.1709 carboxypeptidase
1681.7946 glycoside hydrolase family 92
protein
K5W209
K5W5A5
M2PGV5
M2QK38
M2QVI5
AMLNNSHIYHQTLHDLK
IPSHAGENGSATSGILK
NLTDQIHAMGFK
DVVSIGGATDNVTIR
GNSTNVLARNVTCR
NNS
NGS
NLT
NVT
Y
Y
Y
Y
Y
2039.9871 dihydrolipoyl dehydrogenase
1640.8256 cation-transporting ATPase
1376.6644 α-galactosidase
1518.7775 laccase
1569.7439 glycoside hydrolase family 28
protein
NST;NVT
M2RUP8
M2RUP8
O60199
Q1W6B1
Q50JG6
R7S0G4
LTSGTFRGLSLPNGTDR
NNITVIHDDFNLNK
RDVVSIGDDPTDNVTIR
DVVSTGSPGDNVTIR
DVVSTGNRAAGDNVTIR
MFNVSYDVLRYR
NGT
NIT
NVT
NVT
NVT
NVS
Y
Y
Y
Y
Y
Y
1793.9158 carboxylic ester hydrolase
1664.7915 carboxylic ester hydrolase
1873.9266 laccase
1518.7411 laccase
1746.8746 laccase1
1622.7649 DDE-domain-containing
protein (fragment)
R7S8W6
R7S8W6
R7S8W6
FANGVFNFTD
NFT
NGT
NTT
Y
Y
Y
1133.4915 glycoside hydrolase family 92
protein
1331.6720 glycoside hydrolase family 92
protein
1838.9397 glycoside hydrolase family 92
protein
NGTGGHVFVQSVK
EISFQNPVLNTTTTIR
R7SIS8
R7SJ57
NITMLNAQNGAR
MVVTNYSSK
NIT
NYS
Y
Y
1310.6158 pectin lyase-like protein
1030.4891 ARM repeat-containing
protein
R7SJI7
R7SR93
TVNVTSPFTNK
ASNQTPTAFVPD
NVT
NQT
Y
Y
1209.6127 trehalase
1249.5712 FMN-linked oxidoreductase
(fragment)
R7T134
NETRSERSVDNK
NET
Y
1439.6624 general substrate transporter
identified in P. eryngii and P. ostreatus secretomes, respectively.
Three and two glycosylated peptides of glycoside hydrolase
family 92 protein were identified in P. eryngii and P. ostreatus
secretomes, respectively. Both glycosylated peptides of
carboxylic ester hydrolase were identified in P. eryngii and P.
ostreatus. In addition, exoglucanase, phenol oxidase, α-
galactosidase, carboxylic ester hydrolase, and pectin lyase
participating in lignocellulose degradation were also identified
as important glycoproteins. Among all of the identified N-
glycopeptides, the N-X-T and N-X-S sequences accounted for
80% and 20%, respectively.
Effect of Deglycosylation on Laccase, α-Galactosi-
dase, and Phenol Oxidase Activities and Thermal
Stability. Before the enzyme activity test, the secretomes
were deglycosylated using PNGase F. The reactions were
monitored by SDS-PAGE. Figure 3 shows that the
deglycosylated protein strips migrated toward the smaller
molecular weight direction, suggesting that deglycosylation
occurred within approximately 20 h. The effects of
deglycosylation on laccase, α-galactosidase, and phenol oxidase
activities and thermal stability were detected. The activities of
the enzymes were detected by incubating the secretome of P.
eryngii and P. ostreatus at different temperatures ranging from
30 to 80 °C for 10 min. Figure 4 shows that the optimum
temperatures for the P. eryngii laccase, α-galactosidase, and
phenol oxidase activities were 55, 50, and 45 °C, respectively,
similar to P. ostreatus. The activity increased gradually from 30
°C to the optimum temperature. Beyond the optimum
temperature, the residual activity started to decline quickly.
Table 4 shows that, at the optimum temperature, laccase and
phenol oxidase activities of P. eryngii were approximately 2-
and 1.3-fold higher, respectively, than those of P. ostreatus,
while the α-galactosidase activity of P. ostreatus was 1.12-fold
higher than that of P. eryngii. The results also showed that after
deglycosylation, laccase, α-galactosidase, and phenol oxidase
activities were precipitously decreased about 2.4-, 3-, and 3.2-
fold, respectively. These results suggest that enzyme glyco-
sylation can affect enzymatic activities.
Figure 5 shows that normal laccase had higher half-life values
than deglycosylated enzyme at 70 °C. In contrast, the
thermostabilities of normal α-galactosidase and phenol oxidase
did not differ significantly from those of deglycosylated
enzymes. The thermostability of laccase was significantly
enhanced by glycosylation modification.
Effect of Deglycosylation on Hemp Stalk Pretreat-
ment and Enzymatic Hydrolysis. In order to reveal the
effect of glycosylation on hemp stalk pretreatment and
enzymatic hydrolysis, hemp stalks were first pretreated with
the secretomes. The results in Table 5 show that at 72 h, the
efficiency of enzymatic saccharification was equivalent to P.
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Table 3. Global N-Glycosylated Peptides Profiling of Pleurotus ostreatus Cultivated in Hemp Stalk Medium
protein
matched motif
(N-X-S/T)
accession
peptide
LYVQNGKVIQNSSVK
LTNETMK
DVVNTGVSTDDNVTIR
SSPANITQGTR
LPLAGGTNNTNPALR
NFSGNPANGTGGNVFVK
IPSHAGENGSATSGILK
NLTDQIHAMGFK
DVVSIGGATDNVTIR
GNSTNVLARNVTCR
LTSGTFRGLSLPNGTDR
NNITVIHDDFNLNK
RDVVSIGDDPTDNVTIR
DVVSTGSPGDNVTIR
DVVSTGNRAAGDNVTIR
GRIVIDSQFNTTVAGIKCIGDVTFGPMLAHK
MFNVSYDVLRYR
unique
mass
description
P13860
NSS
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
1681.9022 exoglucanase 1
838.3992 laccase
1709.8091 phenol oxidase
1133.5563 FAD-binding domain-containing protein
1516.7754 trehalase
1681.7946 glycoside hydrolase family 92 protein
1640.8256 cation-transporting ATPase
1376.6644 α-galactosidase
A0A0N9GYX4
B5MAF5
J0DB01
NET
NVT
NIT
NNT
NFS
NGS
NLT
NVT
NST;NVT
NGT
NIT
NVT
NVT
NVT
NTT
NVS
K5UJU4
K5W0L8
K5W5A5
M2PGV5
M2QK38
M2QVI5
M2RUP8
M2RUP8
O60199
1518.7775 laccase
1569.7439 glycoside hydrolase family 28 protein
1793.9158 carboxylic ester hydrolase
1664.7915 carboxylic ester hydrolase
1873.9266 laccase
1518.7411 laccase
1746.8746 laccase1
3363.7205 dihydrolipoyl dehydrogenase
1622.7649 DDE-domain-containing protein
(fragment)
Q1W6B1
Q50JG6
R7RZ26
R7S0G4
R7S8W6
R7S8W6
R7SIS8
R7SJ57
R7SJI7
FANGVFNFTD
NFT
NTT
NIT
NYS
NVT
NQT
NET
Y
Y
Y
Y
Y
Y
Y
1133.4915 glycoside hydrolase family 92 protein
1838.9397 glycoside hydrolase family 92 protein
1310.6158 pectin lyase-like protein
1030.4891 ARM repeat-containing protein
1209.6127 trehalase
EISFQNPVLNTTTTIR
NITMLNAQNGAR
MVVTNYSSK
TVNVTSPFTNK
ASNQTPTAFVPD
NETRSERSVDNK
R7SR93
R7T134
1249.5712 FMN-linked oxidoreductase (fragment)
1439.6624 general substrate transporter
when enzymatic saccharification was carried out with 30 FPU/
g cellulase loadings, the yields of reducing sugars also
decreased approximately 70% after deglycosylation (Table 6).
These results suggest that enzyme glycosylation can affect
hemp stalk pretreatment and enzymatic saccharification.
DISCUSSION
■
Large amounts of agricultural residues are produced
globally.37,38 In China, more than 400000 tons of hemp stalks
are produced as textile industry residue yearly.39,40 How to
degrade these stalks quickly, economically, and in an
environmentally friendly manner has become a popular
research topic.41−43 Physical, chemical, and biological methods
have been investigated to improve lignocellulose degrada-
tion.44 Biological methods, including the use of white rot fungi
and their enzyme extracts to degrade stalks, are considered to
be environmentally friendly and effective methods.45 Our
previous results showed that P. eryngii was the most effective
fungus to improve enzymatic saccharification of industrial
hemp. The yield of reducing sugars was 329 mg/g with 30
FPU/g cellulase loading after a 21-day treatment, which did
not differ significantly from that of P. ostreatus (315 mg/g) but
was significantly higher than that of Irpex lacteus (I. lacteus)
(288 mg/g) and P. chrysosporium (232 mg/g).4 However, the
molecular mechanism explaining why P. eryngii and P. ostreatus
are suitable for pretreatment remains unknown. Based on this,
the extracellular enzymes of P. eryngii and P. ostreatus cultivated
on hemp stalks for 21 days were analyzed to reveal the effective
enzyme cocktails for pretreatment of hemp stalks.
Figure 3. Deglycosylation reactions of Pleurotus eryngii and Pleurotus
ostreatus at 10 and 20 h monitored by SDS-PAGE.
eryngii treated for 21 days reported in our previous research.4
Therefore, the pretreatment time by the normal or
deglycosylation secretome was selected as 72 h in the following
experiments. Compared with the normal secretome pretreat-
ment group, degradation percentages of cellulose, hemi-
cellulose, and lignin decreased approximately 60% after
deglycosylation secretome pretreatment (Table 6). Moreover,
In the present research, 169 and 155 proteins were identified
in P. eryngii and P. ostreatus secretomes, respectively. In total,
28 proteins including carboxylic ester hydrolase, glucanase,
glycoside hydrolase family 92 protein, glycosyltransferase
family 2 protein, glycoside hydrolase family 35 protein, and
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Figure 4. Laccase, α-galactosidase, and phenol oxidase activities detected in secretome or deglycosylation secretome collected at 21 days of the
solid state fermentation.
Table 4. Enzymatic Activities of Secretome at Optimum
Temperature
in the secretome of P. chrysosporium. These results suggested
that lower expression of cellulase and hemicellulase enzymes
and higher expression of lignin-degrading enzymes resulted in
selective degradation of lignin and retention of the cellulose of
P. eryngii and P. ostreatus during the pretreatment process. In
addition to these lignocellulose-degrading enzymes, proteins
that participated in sugar transport and intracellular metabo-
lism, peptidases, proteinases, phosphatases, and kinases were
also found. However, the roles of these proteins in the
degradation of lignocellulose are not very clear. Our research
provides a reference for the further design of an enzyme system
for stalk pretreatment.
glycosylation
deglycosylation
Pleurotus eryngii
laccase (IU/mL)
302 10.65
53.4 1.34
62.7 4.89
124 11.32
17.7 3.53
20.1 2.54
α-galactosidase (IU/mL)
phenol oxidase (IU/mL)
Pleurotus ostreatus
laccase (IU/mL)
α-galactosidase (IU/mL)
phenol oxidase (IU/mL)
145 7.98
57.8 2.54
59.3 1.77
62 1.89
20.1 2.44
15.4 0.39
Through comparison with our previous studies, we found
that culture substrate and culture time could influence the
number and variety of P. eryngii extracellular proteins. The
secretome of P. eryngii cultivated on cottonseed hulls, kenaf
stalks, ramie stalks, and bulrush stalks for 70 days was
analyzed.10 In total, 241 proteins were identified in the
secretome of the different cultures, with 179, 158, 158, and 144
proteins being specific to the ramie stalks, kenaf stalks,
cottonseed hulls, and bulrush stalks medium, respectively.
Compared with these results, there is a significant difference in
the number and type of proteins secreted in the culture
medium of hemp stalks over a 21-day period. As we described
in the Results Section, 301 proteins were predicted as secreted
proteins in P. eryngii, and this species was able to efficiently
pectin esterase involved in cellulose, hemicellulose, and pectin
degradation were identified. Compared with P. chrysosporium
and I. lacteus, there are fewer types of cellulases, hemicellulases,
and pectinases in the secretomes of P. eryngii and P.
ostreatus.46,47 Lignin-degrading enzymes including alcohol
dehydrogenase, aldehyde oxidase, glyoxal oxidase, GMC
oxidoreductase, MnP, laccase, and diphenol oxidase were
identified in the two species. At present, no single enzyme can
completely degrade the lignin of stalks, suggesting that the
degradation of lignin is achieved by the combined action of
various enzymes. Interestingly, ligninolytic enzymes secreted
by P. eryngii and P. ostreatus were different from those secreted
by P. chrysosporium and I. lacteus.46,47 For example, laccase was
detected in the secretomes of P. eryngii and P. ostreatus but not
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Figure 5. Laccase, α-galactosidase, and phenol oxidase thermal stability at 70 °C detected in secretome or deglycosylation secretome collected at 21
days of the solid state fermentation.
Table 5. Effect of Secretome on Hemp Stalks Pretreatment
and Enzymatic Hydrolysis at 24, 48, and 72 h
Table 6. Effect of Secretome Deglycosylation on Hemp
Stalks Pretreatment and Enzymatic Hydrolysis at 72 h
24 h
48 h
72 h
glycosylation deglycosylation
Pleurotus eryngii
Pleurotus eryngii
cellulose
degradation ratio
(%)
5.34 0.31
10.10 0.53
12.25 0.17
cellulose degradation rate (%)
hemicellulose degradation rate
(%)
lignin degradation rate (%)
reducing sugar yield (mg/g)
12.25 0.17
11.42 0.82
7.82 0.23
7.17 0.39
hemicellulose
degradation ratio
(%)
4.44 0.82
9.30 0.24
11.42 0.82
40.12 0.98
328 3.98
15.32 0.18
109.6 7.83
Pleurotus ostreatus
lignin degradation
ratio (%)
23.55 0.89
135 3.65
33.72 1.43
260 4.13
40.12 0.98
328 3.98
cellulose degradation rate (%)
hemicellulose degradation rate
(%)
lignin degradation rate (%)
reducing sugar yield (mg/g)
13.72 0.37
10.68 0.43
8.12 0.73
6.32 0.39
reducing sugar yield
(mg/g)
Pleurotus ostreatus
38.38 0.41
319 10.4
13.75 0.24
91.7 6.98
cellulose
degradation ratio
(%)
5.52 0.54
4.17 0.28
9.67 0.57
8.70 0.87
13.72 0.37
10.68 0.43
hemicellulose
degradation ratio
(%)
Most of the extracellular proteins secreted by fungi are
glycosylated proteins.48,49 Glycosylation can modulate a variety
of biochemical and cellular processes and aid in protein folding
and secretion.50 It is important to understand the N-
glycosylation of wild-type enzymes, as changes in glycosylation
status can affect the activities of these enzymes.51 It is reported
that protein glycosylation is closely related to fungal growth
conditions, the expression of the host, and glycan-modifying
enzymes, all of which can affect enzyme activity.52,53 For
example, most fungal laccases are monomeric glycoproteins.
lignin degradation
ratio (%)
21.98 0.33
129 1.9
30.56 0.89
237.8 12.3
38.38 0.41
319 10.4
reducing sugar yield
(mg/g)
express partial lignocellulosic enzymes induced by hemp stalks
in the medium, while expressions of other secreted proteins
were regulated by diverse factors.
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The molecular mass of laccase is 55−85 kDa with 3−10
potential glycosylation sites.54 Deglycosylation of laccase in
Pycnoporuss anguineus caused reduced activity and stability,
mostly at lower temperatures (20−30 °C).55 In addition,
deglycosylation of α-galactosidase can reduce the activity and
thermal stability of the enzyme. The activity of the
deglycosylated enzymes was significantly lower than that of
the native ones, i.e., 0.33 and 2.6 U/mg for Aspergillus niger α-
galactosidase, 3.3 and 32.5 U/mg for Cladosporium cladospor-
ioides (C. cladosporioides) α-galactosidase, and 11.66 and 31.1
U/mg for P. canescens α-galactosidase. A decrease of thermal
stability at 55 °C, by 20%, was shown for C. cladosporioides and
Prunus canescens α-galactosidases.56 In the present research, the
activities of laccase, α-galactosidase, and phenol oxidase and
the thermal stability of laccase were precipitously decreased
after deglycosylation. Based on these results, we reached a
similar conclusion as previous research works. In addition to
the three enzymes mentioned above, N-glycosylated peptides
of GH92, carboxypeptidase, pectin lyase, and carboxylic ester
hydrolase were also identified by LC−MS/MS after enrich-
ment by hydrazide-modified magnetic beads in P. eryngii and P.
ostreatus secretomes. Although the glycosylation sites of these
proteins have been identified in this study, the point at which
enzyme activity and stability will be affected requires further
demonstration through site-directed mutagenesis, which will
provide insight into the specific effects that glycosylation may
have on the performance of these lignocellulolytic enzymes.
Besides solid-state fermentation in the presence of white-rot
basidiomycetes to pretreat lignocellulosic bioenergy feedstock,
another commonly used method is to directly remove the
lignin by using the enzymes produced by these fungi.57 Our
results showed that after secretome treatment of hemp stalks at
72 h, the efficiency of enzymatic saccharification was
equivalent to P. eryngii treatment for 21 days. The results
also suggested that enzymatic saccharification with 30 FPU/g
cellulase loading and the yields of reducing sugars decreased
after enzyme deglycosylation. Based on the above information,
we speculated that deglycosylation reduces the activity and
stability of the enzymes in the reaction system and then affects
the pretreatment effect and enzymatic saccharification
efficiency of hemp stalks.
(Tables S1−S5 of the spreadsheet) All of the identified
proteins from Pleurotus eryngii and Pleurotus ostreatus
cultivated on hemp stalks at 21 days (XLSX)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: ailianzhong@hotmail.com (L.A.).
*E-mail: ibfcpyd313@126.com (Y.P.).
ORCID
Yuande Peng: 0000-0001-9281-7517
Funding
This work was financially supported by the Natural Science
Foundation of China (31600668), the China Agriculture
Research System for Bast and Leaf Fiber Crops (CARS-19-
E26), and the Project of Scientific Elitists in National
Agricultural Research and Agricultural Science and the
Technology Innovation Program of China (CAAS-ASTIP-
2019-IBFC).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
AA, auxiliary activity; ANOVA, analysis of variance; BE,
biological efficiency; BS, bulrush stalks; CAZy, carbohydrate-
active enzymes; CDH, cellobiose dehydrogenase; CH, cotton-
seed hull; DyP, decolorizing peroxidase; GH, glycoside
hydrolase; GMC, glucose-methanol-choline oxidoreductases;
HCD, higher energy collision-induced dissociation; KS, keanf
stalk; LiP, lignin peroxidase; MnP, manganese peroxidase; RS,
ramie stalk; VP, versatile peroxidase
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.9b00061.
Laccase activity assay; α-galactosidases assay; phenol
oxidase assay; determination of structural carbohydrates
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DOI: 10.1021/acs.jafc.9b00061
J. Agric. Food Chem. 2019, 67, 5486−5495