Catalase-peroxidase StKatG2 from Salinicola tamaricis: a versatile Mn(II) oxidase that decolorizes malachite green

Abstract

Manganese (Mn) oxidation processes have garnered significant attention recently due to their potential for degrading organic pollutants. These processes are primarily catalyzed by Mn(II) oxidases. Salinicola tamaricis F01, an endophytic bacterium derived from wetland plants, has demonstrated Mn(II)-oxidizing capacity. In this study, a catalase-peroxidase, StKatG2, was cloned and overexpressed in Escherichia coli from the strain F01. The purified recombinant StKatG2 exhibited Mn(II)-oxidizing activity with Km and Kcat values of 2.529 mmol/L and 2.82 min⁻¹, respectively. Optimal catalytic conditions for StKatG2 were observed at pH 7.5 and 55°C, with 45.1% activity retention after an 8-h exposure to 80°C. The biogenic manganese oxides produced by StKatG2 exhibited mixed-valence states with Mn(II), including Mn(III), Mn(IV), and Mn(VII). Furthermore, StKatG2 demonstrated superior decolorization efficiency for malachite green (MG), achieving decolorization rates of 73.38% for 20 mg/L MG and 60.08% for 50 mg/L MG, while degrading MG into 4-(dimethylamino)benzophenone. Therefore, the catalase-peroxidase StKatG2 exhibits multifunctionality in Mn(II)-oxidizing activity and has the potential to serve as an environmentally friendly enzyme for MG removal.

Full text

Frontiers in Microbiology 01 frontiersin.org
Catalase-peroxidase StKatG2
from Salinicola tamaricis: a
versatile Mn(II) oxidase that
decolorizes malachite green
MengyaoDing
†, WenjingWang
†, ZhenkunLu , YuhuiSun ,
XinzhenQiao , MeixueDai and GuoyanZhao *
College of Life Science, Shandong Normal University, Jinan, China
Manganese (Mn) oxidation processes have garnered significant attention recently
due to their potential for degrading organic pollutants. These processes are
primarily catalyzed by Mn(II) oxidases. Salinicola tamaricis F01, an endophytic
bacterium derived from wetland plants, has demonstrated Mn(II)-oxidizing capacity.
In this study, a catalase-peroxidase, StKatG2, was cloned and overexpressed in
Escherichia coli from the strain F01. The purified recombinant StKatG2 exhibited
Mn(II)-oxidizing activity with K
m
and K
cat
values of 2.529 mmol/L and 2.82 min
−1
,
respectively. Optimal catalytic conditions for StKatG2 were observed at pH
7.5 and 55°C, with 45.1% activity retention after an 8-h exposure to 80°C. The
biogenic manganese oxides produced by StKatG2 exhibited mixed-valence
states with Mn(II), including Mn(III), Mn(IV), and Mn(VII). Furthermore, StKatG2
demonstrated superior decolorization eciency for malachite green (MG),
achieving decolorization rates of 73.38% for 20 mg/L MG and 60.08% for 50 mg/L
MG, while degrading MG into 4-(dimethylamino)benzophenone. Therefore,
the catalase-peroxidase StKatG2 exhibits multifunctionality in Mn(II)-oxidizing
activity and has the potential to serve as an environmentally friendly enzyme
for MG removal.
KEYWORDS
catalase-peroxidase, Mn(II) oxidases, malachite green, decolorization ability, enzymic
activity
1 Introduction
Malachite green (4-[(4-dimethylaminophenyl)-phenyl-methyl]-N, N-dimethyl aniline;
MG) is a synthetic triarylmethane dye used in the food industry, medical disinfection, and
for coloring various materials like leather and fabrics (Rashtbari and Dehghan, 2021; Sahu
and Poler, 2024). However, MG in wastewater poses serious health risks, including
teratogenic, carcinogenic, and mutagenic eects (Ng etal., 2013; Sartape etal., 2013; Shivaraju
etal., 2017; Rout and Jena, 2021). MG is highly stable and resistant to degradation due to its
three benzene rings (Xu etal., 2006). Only a few enzymes, such as triphenylmethane
reductase (TMR) (Wang etal., 2012; Shang etal., 2019), manganese peroxidase (Yang etal.,
2016), laccase (Casas etal., 2009), and tyrosinase (Shedbalkar etal., 2008), can degrade or
modify MG. However, incomplete enzymatic conversion can increase the toxicity of MG
by-products (Zhou etal., 2019). Moreover, the limited natural production and instability of
some enzymes under high temperatures and ionic conditions restrict their applicability
(Martins etal., 2015). erefore, it is crucial to develop new and viable enzymatic approaches
for the degradation of these dyes.
OPEN ACCESS
EDITED BY
Muhammad Ali,
Trinity College Dublin, Ireland
REVIEWED BY
Shamkant B. Badgujar,
Independent Researcher, Jalgaon, India
Xizi Long,
National Institute for Materials Science, Japan
*CORRESPONDENCE
Guoyan Zhao
zhaoguoyan@sdnu.edu.cn
†These authors have contributed equally to
this work
RECEIVED 09 August 2024
ACCEPTED 21 October 2024
PUBLISHED 05 November 2024
CITATION
Ding M, Wang W, Lu Z, Sun Y, Qiao X,
Dai M and Zhao G (2024) Catalase-peroxidase
StKatG2 from Salinicola tamaricis: a versatile
Mn(II) oxidase that decolorizes malachite
green.
Front. Microbiol. 15:1478305.
doi: 10.3389/fmicb.2024.1478305
COPYRIGHT
© 2024 Ding, Wang, Lu, Sun, Qiao, Dai and
Zhao. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 05 November 2024
DOI 10.3389/fmicb.2024.1478305

Ding et al. 10.3389/fmicb.2024.1478305
Frontiers in Microbiology 02 frontiersin.org
Biogenic manganese (Mn) oxidation, driven by Mn(II)-
oxidizing microorganisms, is an eective method for degrading
organic pollutants (Tran etal., 2018; Li etal., 2021; Liu etal., 2023).
ese microorganisms use enzymatic oxidases to produce biogenic
manganese oxides (BioMnOx), which have high redox potential
and strong adsorptive capacity. BioMnOx can oxidize various
organic pollutants, such as phenols, chlorinated phenols,
chlorinated anilines, atrazine, and various inorganic pollutants.
For example, a Mn(II)-oxidizing microbial community in rivulet
sediment, comprising core microbes such as Sphingobacterium and
Bacillus, eectively decolorizes dyes (Wan etal., 2020). Moreover,
the combination of the Mn(II)-oxidizing bacterium Pantoea
eucrina SS01 and its BioMnOx eciently and sustainably degrade
MG (Sun et al., 2021). Previous research shows that Mn(II)-
oxidizing bacteria use Mn(II) oxidases, which include Multi-
Copper Oxidases (MCOs), Mn peroxidases (MnPs), and Mn
catalases, for Mn(II) oxidation (Zhou and Fu, 2020). ese Mn
oxidases show minimal sequence similarities and are
predominantly large enzymes with challenges in heterologous gene
expression (Zhou and Fu, 2020). eir role in dye degradation is
not well-explored. Among these, only MnP has been identied as
a viable system for Mn(II)-dependent reactions, enabling the in
vitro degradation of MG and recalcitrant polymeric dyes (Moreira
et al., 2001; Champagne and Ramsay, 2005; Saravanakumar
etal., 2013).
Recently, a catalase-peroxidase designated as StKatG (referred
to as StKatG1in this study) from Salinicola tamaricis F01, an
endophytic Mn(II)-oxidizing bacterium isolated from the
halophyte Tamarix chinensis Lour, has been identified as a distinct
clade of bacterial Mn(II) oxidases (Zhao etal., 2023). This enzyme
is capable of converting Mn(II) into biogenic Mn oxides, including
MnO
2
, Mn
3
O
4
, and Mn oxalate (Zhao etal., 2023), which may hold
potential applications in the adsorption and oxidation of organic
pollutants. Beyond its Mn(II) oxidizing capabilities, catalase-
peroxidase is a versatile enzyme with Mn(II) oxidizing, catalase,
peroxidase, and peroxynitritase activities. It can catalyze multiple
reactions, such as hydroxylation, epoxidation, halogenation of
C-H bonds, and transformations of aromatic groups and
biophenols (Takio etal., 2021; Shen and Wang, 2024). Although
limited information exists regarding the application of catalase-
peroxidase in the degradation of organic pollutants, a thermostable
catalase-peroxidase from Thermobacillus xylanilyticus has shown
efficacy in oxidizing small aromatic compounds derived from
lignins (Fall et al., 2023). Further research into the potential
enhancement of catalase-peroxidase activity by Mn(II) could
bevaluable, as it may expand the enzyme’s applicability in organic
pollutant degradation.
e study aimed to investigate the decolorization capability of the
catalase-peroxidases toward MG. Salinicola tamaricis F01 contains two
catalase-peroxidases, both of which demonstrated the ability to
decolorize MG, with StKatG2 exhibiting superior ecacy. Previous
research has identied StKatG1 as a Mn(II) oxidase. is study further
characterized the Mn(II)-oxidizing activity and MG decolorization
capability of StKatG2. Additionally, the ecient heterologous
expression of StKatG2 enhanced its potential utility in
environmental remediation.
2 Materials and methods
2.1 Phylogenetic analysis and structural
alignment
e catalase-peroxidase sequences were obtained from
PeroxiBase1 (Passardi etal., 2007), where they have been veried and
annotated. Multiple sequence alignment was performed using Clustal
Omega2, while structural alignment was conducted with the ESPript
program suite
3
. e phylogenetic tree was generated using MEGA7,
employing the maximum likelihood method as described by Kumar
etal. (2016) and the Jones-Taylor-ornton (JTT) model, with 1,000
bootstrap replicates (Jones etal., 1992). e structure of StKatG1 and
StKatG2 was predicted using the machine learning algorithm
AlphaFold3 (v3.0)
4
. PyMOL soware
5
was utilized for the structural
alignment of the homology model with the AlphaFold3 structure, as
well as for calculating and visualizing root mean square deviations.
2.2 Cloning of stkatG2 gene from Salinicola
tamaricis F01
e Salinicola tamaricis F01 strain was cultured on LB5 medium
(peptone 10 g/L, yeast powder 5 g/L, NaCl 50 g/L, agar 15 g/L) at 30°C
for 48 h. Genomic DNA of strain F01 was isolated using the Qiagen
genomic DNA isolation kit. e stkatG2 gene was amplied from the
genomic DNA using the primers 5’-GGAATTCCATATGATGAGT
GAAGAGATCAAGATCGGTGG-3′ (with the NdeI restriction site
underlined) and 5’-CGCGGATCCGAGACGTCGAAGCGGTC
GTG-3′ (with the BamHI restriction site underlined) for cloning into
the pET-22b (+) expression vector, which includes a C-terminal
hexahistidine tag. e recombinant plasmid was constructed as
described and subsequently transformed into Escherichia coli BL21
competent cells (Vazyme, Nanjing, China). Transformants were
selected on LB24 medium (peptone 10 g/L, yeast powder 24 g/L, NaCl
50 g/L, agar 15 g/L) with 50 μg/mL ampicillin.
2.3 Expression and purification of
recombinant StKatG2
e correct transformants were cultured to an OD
600 nm
of 0.6in
LB24 medium containing 50 μg/mL ampicillin. To induce the expression
of recombinant StKatG2, 0.05 mmol/L isopropyl-β--
thiogalactopyranoside (IPTG) was added. e culture was incubated at
20°C for 10 h. Cells were lysed by suspending them in a 50 mmol/L
solution of Tris–HCl (pH 7.4) and sonicated on ice to facilitate further
disruption (20 kHz power for 12 min with 5 s burst sonication cycles at
5 s intervals). At 4°C, the crude enzyme solution was puried using a
1 http://peroxibase.toulouse.inra.fr
2 https://www.genome.jp/tools-bin/clustalw
3 http://espript.ibcp.fr/ESPript/ESPript
4 https://golgi.sandbox.google.com/
5 http://www.pymol.org

Ding et al. 10.3389/fmicb.2024.1478305
Frontiers in Microbiology 03 frontiersin.org
nickel column (Yeasen, Shanghai, China), with 4 mL of HisSep Ni-NTP
Agarose Resin loaded into the purication column. Lysis Buer
(50 mmol/L NaH2PO4, 300 mmol/L NaCl, 10 mmol/L imidazole, pH 8.0)
was employed for equilibration, followed by the introduction of 20 mL
of crude enzyme solution into the nickel column resin. Aer washing
away impurities with Wash Buer (50 mmol/L NaH
2
PO
4
, 300 mmol/L
NaCl, 20 mmol/L imidazole, pH 8.0), StKatG2 was eluted using Elution
Buer (50 mmol/L NaH
2
PO
4
, 300 mmol/L NaCl, 250 mmol/L imidazole,
pH 8.0) and 7.5 mL was collected, and subsequently dialyzed for 12 h in
a 50 mmol/L Tris–HCl solution (pH 7.4). e puried protein was
analyzed by SDS-PAGE, and the StKatG2 protein concentration was
determined using a BCA kit (Yeasen, Shanghai, China).
To evaluate the molecular weight of the proteins, puried protein
samples were subjected to chromatographic separation and molecular
weight determination using High-Performance Liquid
Chromatography-Mass spectrometry (HPLC-MS) technology (ermo
Fisher Scientic, UnitedStates, Ultimate 3,000 Ultra High Performance
Liquid Chromatography System, Q Exactiv™ Hybrid Quadrupole-
Orbitrap™ Mass Spectrometer High Resolution Mass Spectrometer).
2.4 Mn(II)-oxidizing activity analysis
Manganese chloride (MnCl
2
) was employed as the substrate to
evaluate the Mn(II) oxidation activity of the recombinant StKatG2
enzyme in HEPES buer at pH 8.0. e buer composition included
59 mmol/L NaCl, 10 mmol/L CaCl2, 0.4 mmol/L NADH·Na2 (Sigma),
1 mmol/L H
2
O
2
, 1 μmol/L heme (Sigma) in 50 mmol/L, and 10 μmol/L
pyrroloquinoline quinone (Sigma) (Kono and Fridovich, 1983). e
reaction mixture was incubated for 10 h at 30°C. A 300 μL aliquot of
each sample was reacted for 2 h at 30°C with 60 μL of LBB (Sigma,
0.04%, w/v) (Jones etal., 2019) and 900 μL of acetic acid (45 mmol/L).
e activity was assessed by monitoring the absorbance at 620 nm and
the equilibrated potassium permanganate concentration.
To determine the kinetic constant of recombinant StKatG2,
various concentrations of MnCl
2
(ranging from 0–80mmol/L) were
incubated with 0.4 mg/mL of StKatG2 for 10 h at 30°C in 50 mmol/L
Tris–HCl (pH 7.4) (Jang etal., 2005). e values for Km and Vmax were
derived using non-linear regression tting.
2.5 Impact of temperature, pH, and metal
ions
e eect of temperature on the enzymatic activity of StKatG2
was investigated by incubating the reaction mixture at temperatures
ranging from 25 to 80°C under standard assay conditions at pH 7.0.
e residual enzyme activity was evaluated at 50°C, with the
maximum enzyme activity dened as 100%. Additionally, the optimal
pH was determined by varying pH values from 4.0 to 8.0 while
maintaining a constant temperature of 50°C.
To assess thermostability, StKatG2 samples were incubated at
dierent temperatures (50, 60, 70, and 80°C) for various time
intervals. Subsequently, residual activity was measured at 50°C and
compared with protein samples stored on ice, which was considered
to represent 100% activity.
EDTA and several metal ions, including Ca
2+
, Co
2+
, Cu
2+
, Fe
3+
,
Mg
2+
, Zn
2+
, and Ni
2+
, were incubated with StKatG2 at nal
concentrations ranging from 0.1 mmol/L to 10 mmol/L at 50°C for a
duration of 10 h. e eects of these ions on the activity of recombinant
StKatG2 were then evaluated.
2.6 Characterization of BioMnOx
e MnCl
2
(50 mmol/L) and various auxiliary components were
reacted with recombinant StKatG2 (0.5 mg/mL) at 55°C for 10 h. e
fresh Mn oxides (BioMnOx) pellets were collected by centrifugation
(10,000 × g, 10 min), and washed three times with deionized water
before drying for 12 h at 55°C. Surface topography observations were
performed using a scanning electron microscope (ermo Fisher
Scientic, UnitedStates, Apreo 2C) at 5.00 kV, equipped with an
Oxford X-Max 80T EDX system (Oxford Instruments Ultim Max 65,
Oxford, UnitedKingdom) for energy dispersive analysis of BioMnOx.
e phases were determined by scanning the dry form of the
dioxide using an X-ray diractometer (Beijing Purkinje General
Instrument Co, China, X-D6) in the 2θ range of 5–85°. e valence
state and composition of Mn were identied using the X-ray
photoelectron spectroscopy (XPS; ESCALAB XI+, ermo Fischer,
UnitedStates).
2.7 Decolorization of MG
e MG decolorization experiments were conducted in 2 mL
tubes using a reciprocating shaker set at 30°C and 120 rpm. e
reaction mixture comprised a 50 mmol/L Tris–HCl buer containing
20 mg/L, 50 mg/L, and 80 mg/L of MG, along with 500 μg/mL of
puried recombinant StKatG1 or StKatG2. Control groups were
established that excluded the StKatG1 or StKatG2 enzymes. e
absorbance of MG at 620 nm was measured using a UV
spectrophotometer. e formula for calculating the decolorization rate
is as follows:
( )
Decolorization %
initial MG absorbance final MG absorbance 100%
initial MG absorbance
=
−∗
All experiments were performed in triplicate.
2.8 HPLC-MS analyses of MG metabolites
High-Performance Liquid Chromatography-Mass Spectrometry
(HPLC-MS) was employed to detect malachite green (MG) and its
degradation products (Daneshvar etal., 2007). e malachite green or
its metabolites were dried, dissolved in 1 mL of methanol, and
subsequently injected into an Agilent 1,290 Innity II HPLC system
(Agilent, Santa Clara, UnitedStates) at a volume of 3 μL. is system
was equipped with a reversed-phase C-18 analytical column and
coupled to a quadrupole mass spectrometer. Methanol served as the
mobile phase, with a ow rate of 0.6 mL min
−1
. e initial methanol
proportion was set at 5%, which was gradually increased to 95% over
10 min, maintained at 95% for an additional 2 min, and then reduced
back to 5% over 2 min. e entire analytical process lasted for a total

Ding et al. 10.3389/fmicb.2024.1478305
Frontiers in Microbiology 04 frontiersin.org
of 20 min (Han et al., 2020). e analysis utilized electrospray
ionization (ESI) in positive ion mode, and the data were processed
using Qualitative Analysis B.05.00 soware (Agilent, UnitedStates).
2.9 Molecular docking studies
e protonation state of all compounds was set at pH 7.4, and the
compounds were converted to three-dimensional structures using
Open Babel (O'Boyle et al., 2011). AutoDock Tools (ADT3) were
utilized to prepare and parametrize the receptor protein and ligands.
Docking grid les were generated using the AutoGrid feature of
Sitemap, and docking simulations were performed with AutoDock
Vina (1.2.0) (Trott and Olson, 2010; Eberhardt etal., 2021). e
optimal pose was selected for interaction analysis. Finally, the protein-
ligand interaction gure was generated using PyMOL.
3 Results
3.1 Sequence and structural dierences
between StKatG1 and StKatG2 proteins
Two catalase-peroxidases, StKatG1 (~80 kDa) and StKatG2
(~85 kDa), were identied from S. tamaricis F01. While StKatG1 has
been conrmed to exhibit Mn(II) oxidation activities in previous
research (Zhao etal., 2017; Zhao etal., 2023), the function of StKatG2
remains uncharacterized. StKatG2 shares 53.6% amino acid sequence
identity with StKatG1 (Figure1A and Supplementary Figure S1).
Phylogenetic analysis revealed that StKatG1 clusters with catalase-
peroxidases from Burkholderia pseudomallei, Salmonella enterica, and
Escherichia coli, among others, whereas StKatG2 is distantly related
(Figure 1B). Alphafold3 (v3.0) analysis indicated that StKatG2
possesses a typical catalase-peroxidase structure, comprising 23
α-helices, 22 short 3 10 helices, and 13 β-strands, with the critical
histidine-arginine pair (His-112 and Arg-108) located in α-helix 4,
essential for H
2
O
2
breakdown (Poulos and Kraut, 1980). Additionally,
StKatG2 also contains the triad His295-Asp406-Trp347 as the
proximal heme iron ligand, along with Asp-130, Arg-444, and
Tyr-248, which act as a molecular switch regulating catalase activity
(Regelsberger etal., 2000; Jakopitsch etal., 2003; Yu etal., 2003; Singh
etal., 2004; Carpena etal., 2005). A comparison of the structures of
StKatG1 and StKatG2 revealed signicant similarities, with a root
mean square deviation (RMSD) of 0.98 Å. Both StKatG1 and StKatG2
exhibit three types of secondary structures: α-helices, short 3_10
helices, and β-strands. StKatG1 contains 24 α-helices, whereas
StKatG2 has 23. In contrast, StKatG1 features 17 3_10 helices, while
StKatG2 possesses 22. Regarding β-strands, StKatG1 has 10, compared
to 13in StKatG2 (Figure1C). In conclusion, despite being derived
from the same organism, StKatG1 and StKatG2 demonstrate notable
distinctions in both sequence and structure.
3.2 Expression and purification of
recombinant StKatG2
To investigate the function of the StKatG2 protein, the full-length
gene (2,297 bp) was cloned and inserted into the pET-22b (+)
expression vector with a His6-tag at the C-terminal
(Supplementary Figures S2, S3). e resulting recombinant plasmid
was conrmed through double digestion with NdeI and BamHI
restriction enzymes, yielding a correct 2.3 kb DNA fragment.
Subsequently, the recombinant plasmid was transformed into E. coli
BL21, and transformants on LB medium supplemented with
ampicillin (50 μg/mL) were selected via colony PCR. e puried
recombinant StKatG2 protein was obtained using an immobilized
Ni2+-anity column. A total of 20 mL of crude proteins (1792.71 μg/
mL) was subjected to an immobilized Ni2+-anity column, resulting
in 7.5 mL of eluted proteins with a concentration of 229 μg/
mL. Analysis by SDS-PAGE (10%) revealed a band at approximately
about 85 kDa (Supplementary Figures S4, S5), consistent with the
expected mass of StKatG2. e HPLC-MS result demonstrated that
the actual molecular weight of StKatG2 was 86.97 kDa
(Supplementary Figure S6B and Supplementary Tables S1, S2).
3.3 Characterization of the
manganese-oxidizing activity of StKatG2
e manganese-oxidizing activity of recombinant StKatG2 was
assessed using the LBB method (Jones etal., 2019). Upon reaction of
StKatG2 with MnCl2 in 50 mmol/L Tris–HCl (pH 7.4) buer, insoluble
brown particles were observed. Subsequent mixing of the reaction
solution with the LBB solution resulted in a blue color change
(Figure2A). ese reactions conrmed the formation of Mn oxides,
demonstrating the ability of recombinant StKatG2 to oxidize Mn(II).
e oxidation ability of StKatG2 toward Mn(II) was further
analyzed by calculating kinetic values at dierent concentrations of
MnCl
2
. A Michaelis–Menten plot was generated through non-linear
regression curve tting (Figure2B). Table1 provides a comparison of
the kinetic characteristics of various manganese-catalases. It is evident
that the K
m
value of StKatG2 is 2.529 mmol/L, higher than that of
StKatG1 (Zhao etal., 2023). Moreover, the Km value of StKatG2 was
found to belower than those of most reported multicopper oxidases,
suggesting that StKatG2 has a higher anity for the substrate and
greater manganese oxidation capacity. e V
max
value for StKatG2 was
determined to be10.07 μmol/L·min
−1
, slightly lower than StKatG1
(Zhao etal., 2023), which exhibited a Vmax of 10.3 μmol/L·min−1. is
implies that StKatG1 has a slightly higher maximum velocity in the
catalytic reaction compared to StKatG2. e K
cat
of StKatG2 is
2.82 min
−1
, slightly higher than that of StKatG1 with a K
cat
value of
2.78 min
−1
, indicating a slightly superior catalytic performance in
terms of substrate turnover for StKatG2. ese dierences in kinetic
values between StKatG1 and StKatG2 may beattributed to variances
in enzyme structure, active site residues, or the microenvironment
surrounding the active site, which could impact the eciency of the
catalytic reaction.
3.4 Characterization of BioMnOx
In order to further characterize the biogenic manganese oxides
(BioMnOx) catalyzed by StKatG2, scanning electron microscopy
(SEM) combined with energy dispersive spectroscopy (EDS) was
conducted. Unlike layered sheet-like structure of BioMnOx catalyzed
by StKatG1 (Zhao etal., 2023), the BioMnOx nanoparticles produced

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Frontiers in Microbiology 05 frontiersin.org
by StKatG2 appeared somewhat spherical or irregular, arranged
closely with void spaces between them (Figures3A,B). EDS analysis
revealed the elemental composition on the surface of the BioMnOx
particles, with mass fractions of 21.97% C, 13.64% O, and 64.39% Mn,
and atomic fractions of 47.46% C, 22.12% O, and 30.41% Mn
(Figure3C).
To analyze the phase of the product, X-ray diraction (XRD) was
utilized for qualitative analysis of the phase composition of
bio-manganese oxide (Figure 3D). e results revealed that the
primary phase composition of bio-manganese oxide consisted of
MnO
2
(JCPDS 39–0735), Mn
2
O
3
(JCPDS 24–0734), and C
2
MnO
4
(JCPDS 32–0646). ese ndings suggest that the Mn compounds
catalyzed by StKatG2 exhibit multiple oxidation states. Furthermore,
notable peaks of NaCl (JCPDS 99–0059) were observed, likely
attributed to the incorporation of sodium chloride during the
preparation of BioMnOx.
Valence states compositions in the BioMnOx were identied
through photoelectron spectroscopy (XPS). e XPS analysis showed
two peaks in Mn2p
1/2
and ve in Mn2p
3/2
(Figure3E), consistently
indicating that the BioMnOx produced by StKatG2 is a mixed-valence
manganese compound. Specically, XPS measurements revealed the
presence of four valence states: Mn(II), Mn(III), Mn(IV), and Mn(VII)
in the BioMnOx (Figure3F). e multiple oxidation states in the
BioMnOx product catalyzed by StKatG2 are similar to those catalyzed
by StKatG1, which also exhibits mixed valences (Zhao etal., 2023).
3.5 The eect of pH and temperature on
the Mn(II)-oxidizing activity of StKatG2
e impact of pH conditions on the Mn(II)-oxidizing activity of
StKatG2 was investigated. e optimal pH for StKatG2 to exhibit
FIGURE1
Comparison of amino acid sequences of StKatG homologs. (A) Sequence alignment of StKatG1 and StKatG2. The sequences were aligned using the
Clustal Omega and treated with ESPript 3 (https://espript.ibcp.fr/). The secondary structure elements presented on top were obtained from the
predicted StKatG2 structure using Alphafold3 (α, α-helices; η, 3 10 helices; β, β-strands. TT, turns). Identical and similar residues are displayed in red and
blue boxes, respectively. (B) Phylogenetic tree of KatG proteins from Salinicola tamaricis and other species. StKatG1 and StKatG2, Salinicola tamaricis
KatGs; DkKatG, Drosophila kikkawai KatG; BpKatG, Burkholderia pseudomallei KatG; SeKatG, Salmonella enterica KatG; EcKatG, Escherichia coli KatG;
SdKatG, Shigella dysenteriae KatG; RpKatG, Ralstonia pickettii KatG; BtKatG, Burkholderia thailandensis KatG; MpKatG, Methylibium petroleiphilum
KatG; AvCP01, Azotobacter vinelandii KatG. The evolutionary analyses were conducted based on amino acid homology using the MEGA7. Bootstrap
values are shown at branch points. (C) Predicted structures of StKatG1 and StKatG2 using AlphaFold3, with the manganese binding site in StKatG2
represented as a purple sphere.

Ding et al. 10.3389/fmicb.2024.1478305
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Mn(II)-oxidizing activity was found to be7.5 (Figure4A). e relative
enzyme activity of StKatG2 is only 8.3% at pH 4.0, and the highest
enzyme activity was observed around 7.5, which indicated that neutral
or slightly alkaline environments were benecial to the Mn(II)-
oxidizing activity of StKatG2. is trend is consistent with other studies
on manganese oxidases, such as CopA with an optimal pH of 8.0.
CueO, another manganese oxidase, shows peak activity between pH
7.6 and 8.2, with minimal oxidation activity below pH 7.0 (Su etal.,
2014). ese ndings suggest that most manganese oxidases involved
in biomanganese oxidation exhibit maximal enzyme activity in neutral
to alkaline pH ranges, while chemical oxidation of manganese becomes
predominant at pH levels above 8.0, as demonstrated by the control
sample without StKatG2 (Supplementary Figure S7).
To determine the optimal temperature for Mn(II) oxidizing
activity, StKatG2 was evaluated across a temperature range of
25–80°C. e study revealed that the recombinant StKatG2 exhibited
its peak enzymatic activity at 55°C (Figure4B). Notably, the enzyme
retained 40% activity at lower temperatures (25°C) and maintained
over 80% activity within the 45–55°C range. However, enzyme activity
signicantly decreased beyond 55°C, with a sharp drop to 38.3% at
80°C. ese ndings indicate that StKatG2 is a mesophilic enzyme.
Moreover, the optimal temperature for manganese oxidase activity in
recombinant StKatG2 was higher than that observed for StKatG1
(50°C) (Zhao etal., 2023).
ermostability of StKatG2 was evaluated by subjecting the
protein to dierent temperatures (50, 60, 70, and 80°C) for a specic
duration, followed by measuring residual activity at 55°C (Figure4C).
Aer an 8-h exposure to 50°C, the enzyme retained 82.3% of its
activity, surpassing StKatG1, which only maintained 73.6% activity
under similar conditions (Zhao et al., 2023). Notably, StKatG2
demonstrated superior thermal stability, retaining 45.1% activity aer
8-h exposure to 80°C. In contrast, StKatG1 lost 92% of its activity
when exposed to 80°C for 8 h (Zhao et al., 2023). ese results
collectively indicate that StKatG2 exhibits robust thermal stability in
high-temperature environments and shows promise for broader
applications in such environments.
3.6 The eect of metal ions on the
enzymatic activity of StKatG2
e impact of metal ions and the chelating agent EDTA on the
enzymatic activity of StKatG2 was assessed (Figure 5). EDTA
signicantly suppressed the Mn(II)-oxidizing activity of StKatG2,
suggesting a reliance on divalent metal ions. At a concentration of
0.1 mmol/L, Fe
3+
inhibited the enzyme activity (Figure5A), likely
because of its structural and chemical similarity to Mn, leading to
potential competition for binding sites on the enzyme and consequent
disruption of its structure. Moreover, the addition of 0.1 mmol/L Cu2+,
Mg
2+
, and Zn
2+
enhanced the enzyme activity, possibly by forming a
metalloenzyme complex that boosts enzymatic function. However, a
higher concentration (10 mmol/L) of Cu2+ and Zn2+ almost completely
inhibited the enzymatic activity, while Fe3+ at the same concentration
aected the nal detection outcome due to its inherent color, which
holds no analytical value (Figure5C).
3.7 Decolorization ability of recombinant
StKatG2 on MG
To evaluate the performance of recombinant StKatG2, toxic
malachite green was utilized as the research material. Upon mixing
with the enzyme solution, the decolorization ability was monitored.
StKatG2 exhibited the fastest decolorization eciency within the
initial 15 min for both 20 mg/L and 50 mg/L of malachite green,
peaking around 30 min, with a decolorization rate of up to 30% aer
FIGURE2
Characterization of StKatG2. (A) The LBB assay for the formation of
Mn oxides by incubating purified recombinant StKatG2 (0.3 mg
protein/mL) with MnCl2 (50 mmol/L). Tube 1–3: sample after 24 h of
incubation in the presence of StKatG2; tube 4: Negative control after
24 h in the absence of StKatG2. (B) The reaction kinetics of StKatG2
(0.3 mg protein/mL) were analyzed using various concentrations of
MnCl2. The data presented are the averages of three independent
experiments, accompanied by standard error bars. The curve was
fitted using the Michaelis–Menten equation.
TABLE1 Comparison of the kinetic parameters for StKatG2 and other
characterized manganese oxidases.
Name KmVmax Kcat Reference
CueO 17.33 mM 9.33 μM min−12.09 s−1Su etal. (2014)
CotA 14.85 mM 3.01 μM min−10.32 s−1Su etal. (2013)
MopA 154 μM 1.08 μM min−11.60 min−1Medina etal. (2018)
StKatG1 1.29 mM 10.3 μM min−12.78 min−1Zhao etal. (2023)
StKatG2 2.529 mM 10.07 μM min−12.82 min−1is study

Ding et al. 10.3389/fmicb.2024.1478305
Frontiers in Microbiology 07 frontiersin.org
4 h of action (Figure6). Although the BioMnOx structures of StKatG1
and StKatG2 are similar and the kinetic values of Mn(II) are also
comparable (Supplementary Table S3), StKatG2 exhibits excellent
decolorization eciency for malachite green. Aer 4 h of treatments,
the maximum decolorization rates of StKatG2 reached 73.38% for
20 mg/L MG and 60.08% for 50 mg/L MG (Figure6A), while StKatG1
for 20 mg/L mg and 50 mg/L mg were 63.91 and 51.78%, respectively
(Figure6B). When the concentration of MG reaches 80 mg/L, the
decolorization rates of StKatG1 and StKatG2 were 34.63 and 18.37%,
respectively (Supplementary Figure S8). ese ndings highlight
StKatG2 as a promising biological approach for the ecient
degradation of malachite green within a short period.
3.8 Mechanism of MG removal
To further investigate the decolorization mechanism of MG by
treatment with StKatG2, the MG metabolites were analyzed using
FIGURE3
Morphology and composition of BioMnOx catalyzed by StKatG2. The BioMnOx were obtained through incubation of MnCl2 (50 mmol/L) and StKatG2
(0.5 mg/mL) at 50°C for 10 h. (A) The SEM photograph of the Mn oxide aggregates produced by StKatG2. Scale bar, 10 μm. (B) The SEM photograph of
the Mn oxide aggregates at a dierent magnification with (A). Scale bar, 1 μm. (C) The EDS spectrum showing the Mn composition of the aggregates.
The rectangle shown in figure A indicates the selected position of the aggregates. The unlabeled peaks are Au formed by the spray gold treatment.
(D) The XRD pattern of the BioMnOx produced by StKatG2. (E) XPS wide scan patterns. (F) The deconvoluted profile of the specific Mn 2p1/2 and Mn
2p2/3 spectrum for the BioMnOx.

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FIGURE4
Eects of temperature and pH on Mn(II)-oxidizing activities of StKatG2. (A) Mn(II)-oxidizing activities of StKatG2 at dierent pH values. (B) Mn(II)-
oxidizing activities of StKatG2 at dierent temperatures. (C) Thermostability of StKatG2 at various temperatures. StKatG2 was incubated at 50°C (red
circles), 60°C (green circles), 70°C (orange triangles), and 80°C (blue triangles) for dierent time intervals, with residual activities measured at 50°C. The
error bars indicate standard deviation.
FIGURE5
Eects of dierent metal ions and EDTA on the Mn(II)-oxidizing activity of StKatG2. Eects of dierent concentrations of metal ions and EDTA at
0.1 mM (A), 1 mM (B), and 10 mM (C) on enzymatic activity the Mn(II)-the oxidizing activity of StKatG2. **, ***, and **** represent significant dierences
from the control group (**p < 0.01, ***p < 0.001, ****p < 0.0001).

Ding et al. 10.3389/fmicb.2024.1478305
Frontiers in Microbiology 09 frontiersin.org
HPLC-MS. Following treatment with StKatG2, the MG peak, which
exhibited a retention time of 6.845 min, was found to bereduced
(Figure7A). In contrast, several peaks with retention times ranging
from 5 to 8 min increased (Figure 7B) when compared to the
control setup. ree distinct peaks were identied as MG
metabolites in the StKatG2 treatment setup (Figures 7C–E):
malachite green (7.153 min, m/z = 329), leucomalachite green
(6.845 min, m/z = 315), and 4-(dimethylamino) benzophenone
(7.791 min, m/z = 226). e latter is recognized as a degradation
metabolite of MG (Yang etal., 2015; Abu-Hussien etal., 2022). e
process of MG degradation catalyzed by StKatG2 is illustrated in
Figure7F.
Furthermore, We conducted an analysis of the interactions
between the proteins and MG (Supplementary Figure S9). Utilizing
these interaction forces, the binding energy of the StKatG1-MG and
StKatG2-MG complexes were determined to be−7.5 kcal/mol and
−8.0 kcal/mol, respectively (Supplementary Figure S9). Molecular
docking results indicate that MG interacts with both StKatG1 and
StKatG2, with a position away from the heme binding sites, Mn
2+
binding sites
,
and the H
2
O
2
catalytic pocket. Moreover, the docking
positions of StKatG1 and StKatG2 with MG are inconsistent, which
may contribute to the diering decolorization eciencies observed for
MG. When heme and Mn
2+
combine with StKatG2 for manganese
oxidation, this interaction may inuence the molecular conformation
of the proteins and release redox forces, ultimately leading to
MG decolorization.
4 Discussion
e issue of textile industry dyeing wastewater is a signicant
concern in terms of wastewater pollution. Traditional dye restoration
methods are oen costly and can lead to the production of harmful
byproducts. Enzymatic degradation technology oers a promising
solution to these challenges, paving the way for a more sustainable
environment. MG is a banned triphenylmethane dye that is still being
used illegally in certain regions (Bañ Uelos etal., 2016; Zhou etal.,
2019). Enzymes involved in the decolorization of triphenylmethane
dyes can becategorized into two groups based on their specicity. e
rst group includes manganese peroxidases (Yang etal., 2016) and
laccases (Casas etal., 2009), which act non-specically through radical
chain reactions to decolorize dyes (Azmi etal., 1998). e second
group consists of enzymes specically targeted at decolorizing
triphenylmethane dyes, with triphenylmethane reductases being the
sole members. However, these enzymes can only convert MG to LMG,
which remains toxic to humans and other organisms (Persson etal.,
2003; Zhou et al., 2019). In this study, we investigated the
decolorization capabilities of MG using the catalase-peroxidases
StKatG1 and StKatG2. With heme and methionine-tyrosine-
tryptophan cofactors, catalase-peroxidase displays versatile
functionality, exhibiting both peroxidase and catalase activities
(Zámocký etal., 2009). Previous research has shown its eectiveness
in treating wastewater containing Reactive Black 5, Allura Red,
Fuchsine, and Acid Red 37 (Taslimi etal., 2013). Our ndings further
FIGURE6
Decolorization of MG mediated by StKatGs. (A) The decolorization rate of MG by StKatG2. (B) The decolorization rate of MG by StKatG1.
(C) Decolorization of MG at an initial concentration of 20 mg/L by StKatG2 and StKatG1. (D) Decolorization of MG at an initial concentration of 50 mg/L
by StKatG2 and StKatG1.

Ding et al. 10.3389/fmicb.2024.1478305
Frontiers in Microbiology 10 frontiersin.org
highlight the potential of catalase-peroxidase in decolorizing MG in
textile bleaching euent.
e catalase-peroxidase degradation pathway, while not
completely understood, may play an indirect role in the degradation
of dyes. Textile dyes can induce oxidative stress and generate reactive
oxygen species (ROS). ese ROS are then converted into hydrogen
peroxide (H
2
O
2
) by superoxide dismutase (SOD), which is
subsequently neutralized by catalase-peroxidase, leading to ecient
dye removal under high-stress conditions (Takio etal., 2021). is
study indicates that catalase-peroxidase StKatG2 also exhibits Mn(II)-
oxidizing activities, which may synergistically enhance the removal of
MG. is process is similar to that of another Mn(II) oxidase, MnP
(Xiu, 2013). e heme in the enzyme reacts with H
2
O
2
to generate
highly reactive intermediates, causing the Mn(II) oxidase to transition
FIGURE7
HPLC-MS analysis of MG metabolites catalyzed by StKatG2. (A) HPLC-MS spectrum of the MG degradation products by StKatG2. (B) Higher
magnification view of the HPLC-MS spectrum of (A). (C) Malachite green (m/z = 329, retention time 7.153 min). (D) Leucomalachite Green (m/z = 315,
retention time 6.845 min). (E) 4-(dimethylamino) benzophenone (m/z = 226, retention time 7.791 min). (F) Proposed removal pathways of MG by
StKatG2, based on the results obtained from HPLC-MS.

Ding et al. 10.3389/fmicb.2024.1478305
Frontiers in Microbiology 11 frontiersin.org
into an oxidized state and facilitate the oxidation of Mn(II) to Mn(III).
Subsequently, through a mechanism involving two successive electron
transfer reactions, substrates like MG dyes can reduce the enzyme
back to its original form. Mn(III) can befurther oxidized to Mn(IV)
via disproportionation (Soldatova etal., 2017; Tao etal., 2017), acting
as an active oxidant and strong adsorbent, thereby enhancing the
adsorption and oxidation of textile dyes (Zhou etal., 2018; Sun etal.,
2021). Additionally, the BioMnOx produced by StKatG2 contains
multiple valences, including Mn(II), Mn(III), Mn(IV), and Mn(VII).
Mixed-valence Mn oxide generally exhibits higher electrical
conductivity compared to single-valence states like Mn(III) or
Mn(IV), making it more eective in adsorbing and oxidizing dyes
(Zener, 1951). e synergistic eects of StKatG2’s multifunctionality
may facilitate rapid start-up and stable operation in the removal of
MG contaminants.
Comparatively, the catalase-peroxidase StKatG2 demonstrates a
greater capacity to decolorize MG than StKatG1, despite their similar
BioMnOx structures and comparable kinetic values for Mn(II).
Although StKatG1 and StKatG2 share 53.6% amino acid identity,
phylogenetic analysis indicates that they are distantly related. Notably,
several dierences exist between the two proteins, primarily located
within the α-helix regions. Specically, StKatG1 lacks the α7 helix
found in StKatG2, while StKatG2 possesses a greater number of turns
compared to StKatG1. is variation in the number of secondary
structures may lead to deformation of the active pocket, resulting in
diering binding eciencies and varying decolorization eects.
Taken together, this study demonstrated that the catalase-
peroxidase StKatG2 not only exhibited Mn(II)-oxidizing activity but
also had the ability to decolorize MG. e successful cloning and
overexpression of the StKatG2 gene in E. coli suggest a potential large-
scale application for MG remediation in aquaculture. Furthermore,
StKatG2 showed high stability at high temperatures and a wide range
of pH and temperature tolerances, expanding its potential use in harsh
environments. e ndings of this research oer an environmentally-
friendly solution that could beapplied in aquaculture to eectively
remove MG and reduce its accumulation in aquatic organisms,
thereby ensuring the production of safe aquaculture products.
Data availability statement
e original contributions presented in the study are included in
the article/Supplementary material, further inquiries can bedirected
to the corresponding author.
Author contributions
MDi: Data curation, Investigation, Methodology, Writing –
original dra. WW: Data curation, Investigation, Methodology,
Writing – original dra. ZL: Data curation, Investigation, Writing –
original dra. YS: Data curation, Investigation, Writing – original
dra. XQ: Data curation, Investigation, Writing – original dra. MDa:
Data curation, Investigation, Writing – original dra. GZ:
Conceptualization, Data curation, Funding acquisition, Supervision,
Writing – review & editing.
Funding
e author(s) declare that nancial support was received for the
research, authorship, and/or publication of this article. is study was
funded by the Qingchuang Talents Induction Program of Shandong
Higher Education Institution in 2021, the Natural Science Foundation
of Shandong Province (Grant no. ZR2024MC202), and the National
Natural Science Foundation of China (Grant no. 31640002).
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their aliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may beevaluated in this article, or claim
that may bemade by its manufacturer, is not guaranteed or endorsed
by the publisher.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2024.1478305/
full#supplementary-material
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