Application of a Novel Alkali-Tolerant Thermostable DyP-Type Peroxidase from Saccharomonospora viridis DSM 43017 in Biobleaching of Eucalyptus Kraft Pulp
et al. (2014) Application of a Novel Alkali-Tolerant Thermostable DyP-Type Peroxidase from
Saccharomonospora viridis DSM 43017 in Biobleaching of Eucalyptus Kraft Pulp. PLoS ONE 9(10): e110319. doi:10.1371/journal.pone.0110319
Application of a Novel Alkali-Tolerant Thermostable DyP- Type Peroxidase from Saccharomonospora viridis DSM 43017 in Biobleaching of Eucalyptus Kraft Pulp
Wangning Yu 0
Weina Liu 0
Huoqing Huang 0
Fei Zheng 0
Xiaoyu Wang 0
Yuying Wu 0
Kangjia Li 0
Xiangming Xie 0
Yi Jin 0
Daniele Daffonchio, University of Milan, Italy
0 1 College of Biological Sciences and Technology, Beijing Forestry University , Beijing , PR China , 2 Feed Research Institute Chinese Academy of Agricultural Sciences , Beijing , PR China , 3 College of Materials Science and Technology, Beijing Forestry University , Beijing , PR China
Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70uC, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining.63% of its maximum activity at 50-80uC. It also showed broad pH adaptability (.35% activity at pH 4.0-9.0) and alkali-tolerance (.80% activity after incubation at pH 5-10 for 1 h at 37uC), and was highly thermostable (.60% activity after incubation at 70uC for 2 h at pH 7.0). SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO) in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The svidyp gene sequence are available from
GenBank (accession number = KF444221).
Funding: This work was supported by the Fundamental Research Funds for the Central University (No. TD2012-03) and the National Natural Science Foundation
of China (No. 31070635, No. 30870028, and No. J1103516). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Heme peroxidases, containing prosthetic heme groups, have
been divided into two large groups, animal and plant peroxidases
. The plant peroxidases are divided into classes I, II, and III,
which include fungal (class II) and bacterial peroxidases (class I),
according to primary structural homology . Class I peroxidases
contain mitochondrial yeast cytochrome c peroxidase, chloroplast
and cytosol ascorbate peroxidases, and gene duplicated bacterial
peroxidase . Class II enzymes include secretory fungal
peroxidases, such as lignin peroxidase , manganese peroxidase
, and versatile peroxidase . Secretory plant peroxidases such
as horseradish peroxidase and barley grain peroxidase belong to
class III peroxidases. The recently described DyP-type peroxidases
(DyPs) (EC 188.8.131.52) superfamily comprises a novel class of heme
peroxidases, and are found in both fungi and bacteria .
DyPs have been classified into four phylogenetically distinct
subfamilies, with fungal enzymes belonging to subfamily D and
bacterial enzymes constituting subfamilies AC [15,16]. These
enzymes are very promising for use in biotechnological
applications. Despite the large number of putative DyP sequences
registered in PeroxiBase (http://peroxibase.toulouse.inra.fr/), only
a few studies have described the isolation and characterization of
peroxidases, including four proteins from actinomycetes [17-21].
However, no DyP-type peroxidase genes have been identified in
Pseudonocardiaceae to date.
Biobleaching is the bleaching of pulps using ligninolytic
microorganisms or enzymes that reduce the amount of chemical
bleach required to obtain a desirable brightness of pulps. Some
enzymes from fungi and bacteria, such as extracellular xylanase
and peroxidase from Streptomyces sp [22,23], laccase and
manganese peroxidase produced by Trametes versicolor [24,25],
have been studied for biobleaching of paper pulp and other
industrial applications, as they degraded lignin or xylan and
decolorized the pulp [26,27]. However, no DyP-type peroxidase
enzymes has been characterized and applied in the pulp bleaching
In the current work, a novel peroxidase, SviDyP, related to a
putative DyP-type peroxidase enzyme, was isolated, purified, and
characterized from Saccharomonospora viridis, a
pentachlorophenol-degrading thermophilic actinomycete . In addition, the
catalytic properties of the enzyme and its application as an
enzymatic pretreatment for kraft pulp bleaching were investigated.
SviDyP was an alkali-tolerant thermostable dye-decolorizing
peroxidase reported from the Pseudonocardiaceae family, which
belongs to the genus of Saccharomonospora, and it had an
accelerated action during the biobleaching of eucalyptus kraft
pulp. These favorable properties made SviDyP a good candidate
for application in the pulp and paper industries.
Materials and Methods
Microorganism, culture conditions, and chemicals
Saccharomonospora viridis DSM 43017 was obtained from the
China General Microbiological Culture Collection Center, Beijing
(reference number CGMCC 4.1324). The strain was cultivated in
a shaker flask at 45uC in STS medium (1.0% (w/v) soy peptone,
1.0% (w/v) glucose, 0.2% (w/v) yeast extract, 0.2% (w/v) NaCl,
and 0.2% casein enzymatic hydrolysate, all from Biodee, Beijing,
China), adjusted pH to 8.0 with NaOH prior to autoclaving.
Escherichia coli DH5a and E. coli BL21 (Tiangen, Beijing,
China) (were cultivated in Luria Bertani (LB) medium at 37uC for
gene cloning, sequencing, and expression. The pEASY-T3 vector
(TransGen, Beijing, China) was used for plasmid gene cloning and
sequencing. The plasmid pET-28a (+) (Takara Bio, Otsu, Japan)
was used as an expression vector. Manganese peroxidase (MnP),
azure B, brilliant green, reactive blue 19, reactive green 19,
reactive yellow 2, reactive black 5, reactive red 120, malachite
green and crystal violet were purchased from Sigma (St. Louis,
MO, USA). All other reagents used were of analytical grade unless
Decolorization assay for S. viridis on solid medium
Azure B, which was membrane-filtered with a 0.45-mm cellulose
nitrate filter, was mixed with STS medium to give a final
concentration of 0.1 g l21 and used to make agar plates. S. viridis
was inoculated onto the plates with a sterile spreader. The plates
were then incubated at 45uC and their surface appearance was
observed daily. A ring of decoloration around the colonies on the
blue medium is a qualitative signal of presence or absence of
dyedecolorizing enzyme DyP-type peroxidase. The presence of
bleaching around colonies on the blue medium indicates the
production of the dye-decolorizing enzyme.
Bioinformatic and phylogenetic analysis and homology
The whole genome of S. viridis DSM 43017 is available from
the GenBank database under the accession number NC_013159
. By performing a sequence homology comparison between
four well characterized DyP-type peroxidases from Shewanella
oneidensis TyrA , Bacteroides thetaiotaomicron BtDyP ,
Rhodococcus jostii DyPB , and E. coli YcdB , one
unannotated DyP-type peroxidase gene (Gene YP_003135694)
was identified in the genome of S. viridis DSM 43017. The open
reading frame, which we tentatively named svidyp, showed
sequence similarity to peroxidase genes in other dye-decolorizing
microbes. The DNA sequence of svidyp was analyzed using the
software package DNAMAN 6.0 (Lynnon Corporation, Quebec,
Canada). Nucleotide and the corresponding deduced amino acid
sequence homology searches were carried out using the BLASTn
and BLASTp programs, respectively, at the NCBI website (http://
www.ncbi.nlm.nih.gov/BLAST/). Multiple sequence alignment
was performed by ClustalW . A phylogenetic tree containing
the closest homologs of the DyP protein was constructed using
MEGA 5.2 with the neighbor-joining algorithm . The
deduced tertiary structure of SviDyP was derived from the amino
acid sequence using SWISS-MODEL .
Gene cloning, heterologous expression and enzyme
Genomic DNA from S. viridis was isolated following overnight
growth in STS liquid medium. Cells were harvested by
centrifugation (10,0006g, 25uC for 2 min). DNA extraction was
then performed using the Bacterial Genomic DNA Extraction Kit
V.3.0 (Takara) according to the manufacturers instructions. Two
primers were designed at the 59 and 39 ends of the svidyp gene
with the following sequences: Svidyp-F:
59-GGCGAATTCATGAAGGGCCGGCGGTTC-39 and Svidyp-R:
59-GCCAAGCTTTCAGGCTTCCAACAGCGG-39, which contained the
restriction sites EcoRI and HindIII (underlined), respectively, to
enable directional cloning into the pET-28a (+) expression vector.
Using these primers, the complete svidyp gene was PCR-amplified
from S. viridis genomic DNA. PCR amplification was performed
using Taq DNA polymerase, 106 PCR buffer (Mg2+ Plus), and
dNTP mixture (Takara) under the following conditions: 5 cycles of
94uC for 30 s, 67.3uC for 30 s, and 72uC for 1 min, followed by 30
cycles of 94uC for 30 s, 63.7uC for 30 s, and 72uC for 1 min.
The resulting PCR product was gel-purified, digested with
EcoRI and HindIII, and cloned into the corresponding site of the
pET28a (+) vector to construct the recombinant plasmid
pET28asvidyp. The plasmid was then transformed into chemically
competent E. coli BL21 cells. Plasmid DNA from recombinant
E. coli was isolated using a Plasmid Purification Kit (Takara).
DNA concentration was estimated by the absorbance at 260 nm,
and DNA integrity was verified by agarose gel electrophoresis.
Transformants were identified by PCR analysis and enzymatic
digestion, both as described above, and were then confirmed by
DNA sequencing using the primers T7 and T7-terminor.
Sequencing was carried out by Beijing AuGCT DNA-SYN
Biotechnology Co., Beijing, China.
A positive transformant harboring pET28a-svidyp was isolated
as a single colony for gene expression. The transformant was
cultured overnight at 37uC in 10 ml LB medium containing
kanamycin at 50 mg ml21. The culture was then inoculated into
fresh LB medium (1:100 dilutions) containing kanamycin and was
grown at 37uC to an OD600 of approximately 0.6.
Isopropyl-b-D1-thiogalactopyranoside (IPTG) was then added to a final
concentration of 1.0 mM for induction, and the culture was
further cultivated at different temperature and time. Following
induction with 1.0 mM IPTG at 25uC for 3 h, little activity was
detected from the cell lysate, while incubation at 16uC for 12 h the
activity in the lysate increased to 9.4 U ml21. Thus, to effectively
induce enzyme expression, induction with 1.0 mM IPTG at 16uC
overnight was selected for further production of the recombinant
DyP peroxidise. No DyP peroxidase activity was detected from the
uninduced transformant or from the transformant harboring the
empty pET-28a (+) vector.
Cultures of induced recombinant E. coli (200 ml) were
centrifuged (10,0006g, 4uC for 10 min) and the cell pellets were
resuspended in 10 ml sodium phosphate buffer (50 mM, pH 8.0).
The cell suspension was then lysed by sonication (pause on: 10 s;
pause off: 10 s; power: 130 W; time: 30 min) using a cell ultra
sonicator (Sonics* VCX130; Sonics & Materials, CT, USA).
Following sonication, the unbroken cells and large debris were
removed by centrifugation (12,0006g, 4uC for 20 min) and the
Specific activity (U mg21)
supernatant was collected. Because the enzyme was expressed as a
His-tagged protein, the supernatant was loaded onto a Ni-affinity
column (NiNTA Agarose; Qiagen, Valencia, CA, USA) and
eluted with a linear gradient of imidazole (20250 mM, 25 ml) in
sodium phosphate buffer (20 mM phosphate, 300 mM NaCl,
pH 8.0), at a flow rate of 1 ml min21. Fractions displaying
DyPperoxidase activity were combined and applied to a
SephadexG75 (106200 mm) column pre-equilibrated with 0.1 M
phosphate buffer (pH 7.0) and eluted with the same buffer at a flow
rate of 0.5 ml min21. The purified enzyme with purification yields
of 40.3% (Table 1) was stored at 220uC until further
characterization. The protein concentration was determined using a
Bradford assay  using bovine serum albumin as a standard.
The purity of SviDyP was verified by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) analysis on a 12%
gel as described previously  and by Western blotting analysis
using an anti-His6 tag mouse monoclonal antibody .
Brilliant green, a representative triarylmethane dye, was used as
the substrate. The substrate solution consisted of 125 mM brilliant
green in 50 mM phosphate buffer (pH 7.0). One hundred
microliters of the purified SviDyP enzyme solution (0.01 mg
ml21) was mixed with the substrate solution. All reactions were
started by addition of H2O2 (0.1 mM for all tests). The total
volume of the enzyme reaction mixture was then adjusted to 3 ml.
The enzyme activity was calculated by measuring the variation in
absorbance at 623 nm using the molar extinction coefficient
of brilliant green (max. wavelength: 623 nm; e623 = 18,000
M21cm21). One unit of enzyme activity was defined as the
amount of enzyme that decolorized 1 mmol of brilliant green at
60uC in 1 min . The same method was used to study the
substrate range of the enzyme using reactive blue 19 (max.
wavelength: 590 nm; e590 = 10,000 M21 cm21), reactive green 19
(max. wavelength: 630 nm; e630 = 22,000 M21 cm21), reactive
yellow 2 (max. wavelength: 390 nm; e390 = 8,000 M21 cm21),
reactive black 5 (max. wavelength: 598 nm; e598 = 30,000 M21
cm21), reactive red 120 (max. wavelength: 535 nm;
e535 = 28,000 M21 cm21), malachite green (max. wavelength:
617 nm; e617 = 8,400 M21 cm21), crystal violet (max. wavelength:
583 nm; e583 = 21,900 M21 cm21), azure B (max. wavelength:
651 nm; e630 = 22,000 M21 cm21), 2, 6 - Dimethoxyphenol (max.
wavelength: 469 nm; e469 = 2,750 M21 cm21) and veratryl alcohol
(max. wavelength: 310 nm; e310 = 9,300 M21 cm21) as substrates
under their corresponding maximum absorption wavelength.
Control treatments without H2O2 and/or without enzyme were
performed. MnP activity was measured at 25uC by the formation
of Mn+3-tartrate complex (e238 = 6,500 M21 cm21) during the
oxidation of 0.5 mM MnSO4 in 100 mM tartrate bufferat pH 4.5
with 0.1 mM H2O2 . One unit of MnP activity corresponds to
the amount of enzyme which oxidizes 1 mmol Mn2+ to Mn3+ per
minute at pH 4.5 and 25uC (in the presence of 0.1 mM H2O2).
Aliquots of the recombinant SviDyP sample (1 mg ml21) were
put under various conditions to test its stability. The optimal pH
for enzyme activity was determined by measuring the reaction rate
in buffers covering a pH range of 3.012.0. The buffers used were
50 mM citric acid-Na2HPO4 (pH 3.08.0), 50 mM Tris-HCl
(pH 8.09.0), and 50 mM glycine-NaOH (pH 9.012.0). The pH
stability was measured by adding enzyme to buffers ranging from
pH 2.010.0 at 37uC for 1 h without the substrate solutions, and
then, its residual activity was analyzed as described above. The
highest activity of the enzyme, treated as above, was defined as
100%. The optimal temperature for enzyme activity was
examined over the temperature range of 3090uC. To estimate
the thermal stability, the enzymes were incubated at 50uC, 60uC,
70uC, or 80uC for 0.5, 1, 2, 3, 4, and 5 h without substrate, and
the residual enzyme activity was measured with the method as
described above. To determine the effect of different metal ions
and chemical reagents on the enzyme activity, the enzyme sample
(1 mg ml21) was incubated for 30 min at 37uC and pH 7.0 in
0.05 M BrittonRobinson buffer, supplemented with the following
additives respectively (final concentration, 5 mM): SDS,
bmercaptoethanol, ethylenediaminetetraacetic acid (EDTA),
ZnCl2, BaCl2, MnCl2, MgCl2, AlCl3, CoCl2, CaCl2, LiCl, FeCl2,
and FeCl3. The residual activity was then assayed using the
method as described above. And a system without any additives
was used as a control.
The Km, and Vmax values for the purified SviDyP protein were
determined by several substrates including reactive blue 19,
reactive yellow 2 and brilliant green. Lineweaver-Burk plots were
generated from the initial rates obtained at varying substrate
concentrations with a constant concentration of the second
substrate . Each experiment was repeated three times and
each experiment included three replicates.
Physico-chemical characterization of kraft pulps and
biobleaching with SviDyP
An unbleached eucalyptus kraft pulp was prepared by cooking
the dried wood material (provided by JinHai Pulp & Paper
Limited Company, Hainan, China) with sulfate process under the
following conditions: total alkali, 16.0% (w/v), sulfureted degree
25% (w/v), liquid ratio 1:6, and a cooking temperature of 160uC
for 2 h. The optimization of enzyme dose and reaction time for
biobleaching was carried out by treating pulp with varying doses of
DyP-type peroxidase, ranging from 06.0 U g21 dry pulp, for
various time intervals up to 6 h. Pulp properties were investigated
at regular intervals. Enzyme-treated and untreated pulp samples at
10% pulp consistency were incubated at 65uC with SviDyP (5
milligram protein per gram dried pulp, equivalently enzyme
dosage as 3 U g21 which was in terms of enzyme activity towards
brilliant green) in 50 mM citric acid-Na2HPO4 buffer (pH 7.0) for
2 h, whereas the biobleaching by MnP with 0.5 mM MnSO4 was
performed at 25uC in pH 4.5 for 2 h as the positive control.
0.1 mM H2O2 was added in all the assays. During the bleaching
treatment, kneading the pulp mixture every 15 min was needed in
order to make sure all ingredients can mix well. After the enzyme
bleaching, pulps were thoroughly washed with deionized water to
remove soluble reducing sugars and any free soluble residual
lignin, and then made into pieces of paper by the sheet forming
machine. Pulp sheet preparation was conducted as described in
the paper . Degree of brightness of pulp according to ISO
(International Standards Organization) was measured on an
YQZ-48A (Qingtong, Hangzhou, China) brightness color tester. Pulp
kappa number was determined by using TAPPI useful method
T236 (Technical Association of the Pulp and Paper Industry,
Atlanta, Ga.). All reported data are averages of experiments
performed in at least six replicates.
Scanning electron microscopy
Samples of pulp fibers were processed for scanning electron
microscopy (SEM). Paper pulp samples were formed into hand
sheets using a Buchner funnel. Small pieces of fiber were vacuum
dried in the presence of P2O5, placed on the stubs and mounted
with silver tape, and then sputter coated with gold using fine coat.
The samples were examined at 5.0 kV under a scanning electron
microscope (Hitachi S-4300, Ibaraki, Japan) at various
Figure 3. Multiple sequence alignment of the SviDyP amino acid sequence with other members of DyP-type peroxidases family. The
sequence alignments were performed using ClustalW (Thompson et al. 1994). Perfect match residues are displayed in white on black shade. The
unique GXXDG motif is boxed. The conserved residues of the active site are indicated by asterisks. Residues indicated by triangles are residuals
important for coordinating the heme at the active site.
Decolorization assay for S. viridis on solid medium
Some bleaching laps were observed on STS medium mixed
with 0.1 g l21 azure B around colonies of S. viridis (Fig. 1), which
were produced after incubating at 45uC for 3 days, indicating the
presence of a dye-decolorizing enzyme. This indicated one or
more extracellular dye-decolorizing enzymes produced in the
cultivating process of S. viridis.
SviDyP is a member of the DyP-type family enzymes
To isolate the gene encoding the DyP-type peroxidase from S.
viridis DSM 43017, two specific primers, Svidyp-F and Svidyp-R,
were designed. PCR amplification of the DyP-peroxidase gene
(GenBank under accession number KF444221) of S. viridis
generated a DNA fragment of 1215 bp, which encoded a
polypeptide of 404 amino acids with a predicted molecular mass
of 44.5 kDa and a deduced isoelectric point of 5.17. Sequence
analysis showed that overall G + C content of the svidyp gene was
The deduced amino acid sequence of SviDyP showed 36%
amino acids identity with Bacillus subtilis BsDyP  and R. jostii
DyPB , 33% E. coli YcdB , 27% to S. oneidensis TyrA
 and 21% to B. thetaiotaomicron BtDyP . Twenty typical
and hotspot DyPs proteins, such as Thermobifida fusca TfuDyP
, S. oneidensis TyrA , B. subtilis BsDyP , and R. jostii
DyPB , were used to phylogenetic analysis. Phylogenetic trees
and multiple sequence alignments showed that SviDyP was most
closely related to Mycobacterium sp. MyspDyP, Thermobifida fusca
TfuDyP, Rhodococcus jostii DyPA, Escherichia coli EfeB/YcdB,
Bacillus subtilis BsDyP  and the putative uncharacterized
protein Sco3963 from Streptomyces coelicolor A3 (2), all of which
belong to subfamily A (Fig 2). Therefore the enzyme SviDyP was
grouped into subfamily A, and typical motifs, such as conserved
GXXDG motif and some conserved amino acid residues (D223,
N227, R328, H313, E342, D371), were found in DyP-type
Reactive Green 19f
Reactive Yellow 2f
Reactive Red 120f
Figure 5. Characterization of SviDyP. a. pH optimization. The enzyme was assayed at different pH, as described in the Materials and
Methodssection. b. Temperature optimization. The enzyme samples were incubated in pH 7.0 at 30, 40, 50, 60, 70, 80, and 90uC for 30 min and their
residual activities were assayed, as described in the Materials and Methodssection. c. pH stability. Enzyme samples (1 mg ml21) were incubated for
30 min at different pH levels at 37uC, and then their residual activities were assayed at the optimum pH,as described in the Materials and Methods
section. d. Temperature stability. The enzyme samples were incubated in pH 7.0 at 50, 60, 70, or 80uC, and then their residual activities were assayed
at different times.
peroxidases (Fig 3). The model structure of SviDyP showed two
domains consisting of a four-stranded antiparallel b-sheet and
peripheral a-helices which is typical in the DyP-type family (Figure
Heterologous expression and purification of SviDyP
The svidyp gene was successfully cloned into the pET28a vector
and expressed in E. coli BL21, forming the recombinant clone E.
coli BL21/pET28a-svidyp, with the expression of His-tagged
protein. The purified SviDyP protein, as verified by SDS-PAGE
analysis and Western Blot analysis (Fig. 4a) by anti-6xHis
antibody, was about 45 kDa, consistent with the theoretical value
of 44.5 kDa.
Biochemical and spectroscopic characterization of SviDyP
The UV-visible spectrum of SviDyP at resting state showed the
main Soret band at 406 nm and two charge transfer bands at
503 nm and 636 nm (Fig. 4b), as well as a Reinheitszahl ratio
(A406/A280) of 1.7. The molar absorption coefficient of SviDyP,
e406, was 97,000 M21 cm21, similar to values found for other
peroxidases and DyPs [8,44].
The recombinant enzyme expressed and purified from E. coli
BL21 was then biochemically characterized. The controls of
without H2O2 and/or without enzyme were without significant
statistical difference. Substrate specificity of the enzyme was
determined by using a subset of well-known peroxidase substrates
and anthraquinone and azo dyes (Table 2). The enzyme displayed
maximal activity towards the anthraquinone dye reactive blue 19
(1.29 U mg21), the azo dyes reactive green 19, reactive yellow 2,
reactive black 5, and reactive red 120 (1.32, 4.86, 0.96 and 0.69 U
mg21, respectively), and the triarylmethane dyes brilliant green,
malachite green, and crystal violet (12.24, 8.4 and 4.11 U mg21,
respectively). SviDyP was able to act on veratryl alcohol (0.03 U
mg21), but not very efficiently (Table 2), and also showed activity
towards azure B (1.62 U mg21), a methylated thiazine dye that is a
good substrate for lignin peroxidase . Additionally, SviDyP
showed a modest activity towards 2, 6 - dimethoxyphenol (0.06 U
mg21) which is a typical methoxylated phenol substrate for plant
peroxidases. From the above, it is true that SviDyP displays
(DyPtype) peroxidase activity.
To study the behavior of the enzyme under various pH and
temperature conditions, brilliant green was used as the substrate.
Residual activity (%) 5 mM
UD, undetectable activity.
EDTA, ethylenediaminetetraacetic acid.
SDS, sodium dodecyl sulfate.
The optimal pH of SviDyP at 60uC was 7.0. It had broad pH
adaptability (.35% activity at pH 4.09.0) and retained over 62%
of the peak activity at pH 6.08.0 (Fig. 5a). SviDyP was highly
alkali-tolerant, retaining at least 80% of its initial activity after
incubation at 37uC for 1 h in buffers ranging from pH 5.010.0
(Fig. 5b). The optimal temperature for SviDyP activity was 70uC,
at pH 7.0 (Fig. 5c). SviDyP was highly thermophilic, retaining at
least 63% of its maximum activity at 5080uC, and approximately
95% at 80uC. The thermostability of the enzyme was investigated
for a period of 5 h at temperatures ranging from 5080uC
(Fig. 5d). It was observed that SviDyP maintained approximately
60% of maximal activity at 60uC and 70uC for at least 3 h and
2 h, respectively.
The effect of different metal ions and metal chelating agents on
the enzymatic activity was also assayed, using brilliant green as the
substrate (Table 3). The activity of control without any additives
was defined as 100%. The activity of the enzyme was not affected
(residual activity,.80%) by the presence of 5 mM final
concentration of Ba2+, Mn2+, Mg2+, Fe2+, or EDTA. An obvious
reduction in catalytic activity was found in solutions containing
5 mM Fe3+ or SDS. Al3+ and Co2+ caused 27.6% and 16.9%
activation of SviDyP, respectively. The addition of 5 mM Cu2+,
Zn2+, Ca2+, or Li+ had similar but less activating effects on the
activity of SviDyP, probably through promoting an oxidation
reaction. b-mercaptoethanol highly boosted the enzymatic activity
by almost a factor of 8.5.
The kinetic analysis of purified SviDyP corroborated the
38.4660.98 mmol l21 min21 and a Km of 58.8764.7 mVmmoalx l2o1f
Michaelis-Menten behavior of the enzyme, with a
for brilliant green, a Vmax of 3.760.41 mmol l21 min21 and a Km
of 187.2616.6 mmol l21 for reactive yellow 2, and a Vmax of
2.260.26 mmol l21 min21 and a Km of 210.2616.5 mmol l21 for
reactive blue 19. Such kinetic behavior strongly suggested that the
cloned enzyme might be a DyP-type peroxidase.
Enzyme effects on pulp bleaching
Enzyme effects on pulp bleaching were reflected by measuring
the physicochemical properties of paper pulp biobleached by
enzymic preparations. The unbleached/raw eucalyptus kraft pulp
had a kappa number of 11.0 and a brightness of 33.6% (ISO) .
With the increase of enzyme dosage and reaction time, the pulp
brightness was increased and kappa number was reduced
gradually, and then they keep invariant (Figure S2). When the
enzyme dosage was 3 U g21 (Figure S2a, b) and the reaction time
was 2 h (Figure S2c, d), the pulp brightness reached the
maximum, and pulp kappa number reached the lowest value.
Therefore, the choice of 3 U g21 as an optimal enzyme dosage
and 2 h as an optimal reaction time were the best condition of
SviDyP biobleaching kraft pulp (Figure S2). The experiments of
pulp bleaching by recombinant protein SviDyP showed that
enzyme treatment to improve the brightness of the pulp 2.98%,
pulp kappa number reduced 21.8% compared with control
(Table 4), indicating that the enzyme treatment can significantly
improve the brightness of the pulp, but reduce kappa value will
help subsequent bleaching. Application of MnP in the pulp
bleaching experiment showed that MnP reduced the specific pulp
kappa number by 31.8%, and increased the whiteness of pulp by
5.90% at pH 4.5, 25uC (Table 4), whereas it reduced the specific
pulp kappa number by 24.8% and increased the whiteness of pulp
by 3.26% at pH 7, 65uC.
To study the effect of the enzyme on fiber surface morphology
in SviDyP-bleached pulp, eucalyptus kraft pulp fiber morphology
and surface changes were observed by SEM (Fig. 6). Without any
enzymatic bleaching, eucalyptus kraft pulp fibers were stiff, and
the surface structure was relatively smooth with no damage or
cracks (Fig. 6a). The surface morphology of fibers treated with
MnP is shown in Fig. 6b and the fibers treated with DyP-type
peroxidase from S. viridis are shown in Fig. 6c and 6d. The pulp
fiber surface treated with DyP-type peroxidase from S. viridis
appeared rough and wrinkled and showed signs of slight fissures as
well as that treated with MnP. There were also many holes in the
aThe control samples and the test samples were incubated in the selected buffer at the set temperature with and without corresponding enzyme.
Brightness increase (%)
Figure 6. Morphology of bleached pulp fiber surface. Scanning electron micrograph of unbleached (control) eucalyptus kraft pulp (a).
Scanning electron micrograph of MnP treated eucalyptus kraft pulp (b). Scanning electron micrograph of SviDyP treated eucalyptus kraft pulp (c, d).
surface fibers, and fibers began to increase in flexibility (Fig. 6b, 6c
and 6d). These representative images show the obvious cracks,
grooves and spalling in the surface of the fibers, with some fibers
having holes with an aperture diameter of 7 mm, which increases
The pentachlorophenol-degrading thermophilic actinomycete
S. viridis and its extracellular enzymes offer great potential for
different biotechnological applications. Both a crude extracellular
xylanase  and a purified xylanase , apparently in the
absence of cellulase or endoglucanase activity, have been described
in S. viridis, and may represent a potential new enzyme source for
use in pulp bleaching preparations. In the current study, we
reported the cloning, expression, purification, characterization,
and pulp biobleaching application of an alkali-tolerant
thermostable peroxidase SviDyP from S. viridis DSM 43017.
Bioinformatic analysis revealed one unannotated DyP-type
peroxidase gene in the genome of S. viridis DSM 43017 that
had sequence similarity with other DyPs at the primary structure
level showing the characteristic conserved residues in the
hemebinding site and GXXDG motif . Based on the phylogenetic
analysis, SviDyP belongs to subfamily A of DyPs. Compared with
other DyPs from subfamily A, the purified recombinant SviDyP,
appeared remarkably broad range substrate specificity of which
could degrade various dyes, such as triarylmethane dyes,
anthraquinonic and azo dyes, at different levels, much better than
some enzymes grouped into subfamily A, for example, YcdB ,
TfuDyP . The enzyme could efficiently decolorize
triarylmethane dyes to other triarylmethane dye-degrading
microorganisms [49,50], and was more active towards crystal violet [51,52]
and malachite green [51,53,54]. SviDyP also showed the ability to
degrade anthraquinonic and azo dyes under neutral to alkaline
conditions. Hence, such a multifunctional peroxidase with wide
substrate specificity was likely capable of decolorizing other,
currently unidentified, xenobiotics, Which suggested that SviDyP
might be applied directly to solve various environmental and
industrial problems, such as the disposal of dye wastewater and
pulp biobleaching rather than the DyPs [8,9] which usually
worked under acid condition.
To further explore the feasibility of using SviDyP in
biotechnological applications, enzyme stability was evaluated at different
pH levels and temperatures. More luckily, SviDyP was very stable
over a pH range of 510, and retained more than 80% of the
maximum residual activity. It also displayed high thermal stability,
maintaining 63% of residual activity after 4 h of incubation at
50uC, and more than 60% of residual activity after 2 h of
incubation at 60uC. SviDyP showed greater stability than DyP
from Anabaena sp. , which had an activity loss of 90%
following 3 h incubation at 50uC. Stability studies showed that
SviDyP exhibited a high degree of stability under alkaline pH and
high temperatures. This ability to work efficiently under such high
pH environments was a characteristic that distinguishes DyPs from
most peroxidases . These findings enriched DyP-type
peroxidases familys information databases and laid the foundation for
the study of structure, function and reaction mechanisms of
Concerning the influence factors of using SviDyP in the further
bleaching industrial application, the activation/inactivation effect
of the different metal ions and chemical reagents on the enzyme
activity was studied. Interestingly, addition of EDTA did not
inhibit the enzyme, which suggested that there are no essential
ions in the reaction mixture for the enzyme activity [7,8]. Al3+ had
similar activating effect between SviDyP and Pseudomonas
aeruginosa DyPPa , probably through promoting the oxidation.
SDS resulted in the complete activity loss of SviDyP and DyPPa
, which demonstrated that SDS could be an important DyP
inhibitor. b-mercaptoethanol highly boosted the enzymatic activity
presumably by counteracting the oxidation effects of the SS
linkage between cysteine residues [56,57].
The applicability of SviDyP in biobleaching of kraft pulp, a
process of high biotechnological interest, was examined by
prebleaching eucalyptus kraft pulp with the purified SviDyP
enzyme. Under 65uC, pH 7.0, SviDyP enzyme had positive effects
on the pulp bleaching process as well as MnP, while the
biobleaching effects of MnP at 25uC, pH 4.5, were better than
SviDyP at 65uC, pH 7.0. These data suggest a slightly
temperature and pH adjustment were needed in the usage of SviDyP in
bleaching pulp. Furthermore, the SEM studies revealed that the
existence of local fracture phenomena and the large area of the
trench structure containing holes would result in the fiber structure
becoming loose, and the internal structure of lignin being fully
exposed. This may help the bleaching agent permeate better, and
perfects delignification, which will happen later. Thereby, this
breakdown in structure means that much less bleaching agent was
needed, and less poisoned organochlorine was produced (data not
In addition, the prepared SviDyP peroxidase had hardly some
cellulase activity (data not shown), thereby it prevented reduction
of fiber strength and viscosity of thick liquid. The enzyme did show
significant heat and alkali resistance, and could directly and
effectively attack lignin. Therefore, SviDyP is applicable to the
process of pre-bleaching paper pulp for industrial production, and
shows excellent bleaching effects. With its wide substrate
specificities and thermophilic capabilities, SviDyP peroxidase is a
promising enzyme with considerable biotechnological and
commercial potential, especially in the pulp and paper industry.
Figure S1 Model structure of SviDyP. Model structure of
SviDyP generated using template sequences from putative
uncharacterized protein Sco3963 from Streptomyces coelicolor A3
(2) (PDB 4gt2A, GI: 541881521), whose amino acid sequence was
45.56% identical to SviDyP. The a-helices are shown in red and
the b-sheets in yellow.
Figure S2 Paper properties of SviDyP and
chemicallytreated eucalyptus kraft pulp. a. paper brightness on various
enzyme dosages. b. paper kappa number on various enzyme
dosages. c. paper brightness on various time intervals. d. paper
kappa number on various time intervals.
Conceived and designed the experiments: XX WY YJ. Performed the
experiments: WY WL. Analyzed the data: WY FZ. Contributed reagents/
materials/analysis tools: KL XW YW HH. Wrote the paper: WY WL.
Revised the first draft of the manuscript: HH. Read, corrected, and
approved the final manuscript: XX YJ.
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