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\begin{document}

\title{Halo-tolerant and thermostable mechanisms of an endoglucanase from
marine \emph{Aspergillus niger}}



\author[1]{Li-Nian Cai}%
\author[2]{Tao Lu}%
\author[1]{Dong-Qiang Lin}%
\author[1]{Shanjing   Yao}%
\affil[1]{Zhejiang University}%
\affil[2]{Zhejiang University of Technology}%


\vspace{-1em}



  
  \date{June 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The cellulase cocktail of marine \emph{Aspergillus niger} exhibited
halo-tolerant and thermostable properties, which is of great potential
in industrial application. In order to excavate single tolerant
cellulase components from the complex cellulase cocktail, constitutive
homologous expression was employed for direct obtainment of the
endoglucanase (\emph{An}EGL). The constitutive expression system seems
as ``separation factor'' to be implanted into cell for conveniently
obtaining a single target enzyme. Enzymatic property study revealed that
\emph{An}EGL exhibited a property of halo-tolerance and an outstanding
thermostability in high salt environment. Significantly, its activity
increased by 29\% and the half-life at 65 \selectlanguage{ngerman}°C increased by 26.7-fold with
the presence of 4.5 M NaCl. Molecular dynamics (MD) simulation revealed
that Na\textsuperscript{+} and Cl\selectlanguage{english}\textsuperscript{-} could form salt
bridges with charged residues, and then influenced the activity of loops
and the stability of substrate binding pocket, which accounted for the
halo-tolerance and thermostability. Further, site-specific mutagenesis
study proved that the residues Asp95 and Asp99 in the pocket were of
great concern for the halo-tolerance and thermostability. The
halo-tolerant and thermostable \emph{An}EGL was of great value in
lignocellulosic utilization and the conjectural mechanisms were of
referential significance for other tolerant enzymes.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Halo-tolerant and thermostable mechanisms of an endoglucanase
from marine \emph{Aspergillus niger}}

\emph{Li-Nian Cai\textsuperscript{1}, Sheng-Nan Xu\textsuperscript{1},
Tao Lu\textsuperscript{2}, Dong-Qiang Lin\textsuperscript{1}, Shan-Jing
Yao\textsuperscript{1*}}

\textsuperscript{1} Key Laboratory of Biomass Chemical Engineering of
Ministry of Education, College of Chemical and Biological Engineering,
Zhejiang University, Hangzhou 310027, China

\textsuperscript{2} College of Environment, Zhejiang University of
Technology, Hangzhou 310014, China

\textsuperscript{*} \textbf{Corresponding author} :

Prof. Shan-Jing Yao

College of Chemical and Biological Engineering

Zhejiang University

Hangzhou 310027, China

E-mail: yaosj@zju.edu.cn

Tel/Fax: +86-571-87951982

ORCID: 0000-0003-3199-3044

\textbf{E-mail address of each author:}

Li-Nian Cai: 11428065@zju.edu.cn

Sheng-Nan Xu: 21728068@zju.edu.cn

Tao Lu: lutao@zjut.edu.cn

Dong-Qiang Lin: lindq@zju.edu.cn

Shan-Jing Yao: yaosj@zju.edu.cn

\textbf{Abstract}

The cellulase cocktail of marine \emph{Aspergillus niger} exhibited
halo-tolerant and thermostable properties, which is of great potential
in industrial application. In order to excavate single tolerant
cellulase components from the complex cellulase cocktail, constitutive
homologous expression was employed for direct obtainment of the
endoglucanase (\emph{An} EGL). The constitutive expression system seems
as ``separation factor'' to be implanted into cell for conveniently
obtaining a single target enzyme. Enzymatic property study revealed
that\emph{An} EGL exhibited a property of halo-tolerance and an
outstanding thermostability in high salt environment. Significantly, its
activity increased by 29\% and the half-life at 65 \selectlanguage{ngerman}°C increased by
26.7-fold with the presence of 4.5 M NaCl. Molecular dynamics (MD)
simulation revealed that Na\textsuperscript{+} and
Cl\selectlanguage{english}\textsuperscript{-} could form salt bridges with charged residues,
and then influenced the activity of loops and the stability of substrate
binding pocket, which accounted for the halo-tolerance and
thermostability. Further, site-specific mutagenesis study proved that
the residues Asp95 and Asp99 in the pocket were of great concern for the
halo-tolerance and thermostability. The halo-tolerant and thermostable
\emph{An} EGL was of great value in lignocellulosic utilization and the
conjectural mechanisms were of referential significance for other
tolerant enzymes.

\textbf{Keywords} : constitutive homologous expression; endoglucanase;
marine \emph{Aspergillus niger;} halo-tolerance; thermostability; salt
bridge

\textbf{Abbreviations:}

BGL: \selectlanguage{greek}β-\selectlanguage{english}glucosidase

EGL: endo-1,4-\selectlanguage{greek}β-\selectlanguage{english}glucanase

CBH: cellobiohydrolase or exo-1,4-\selectlanguage{greek}β-\selectlanguage{english}glucanase

LPMO: lytic polysaccharide monooxygenase

AMT: \emph{Agrobacterium tumefaciens} mediated transformation

GH: glycoside hydrolase

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

MD: molecular dynamics

CMC-Na: sodium carboxymethylcellulose

\emph{K\textsubscript{m}} : Michaelis constant

DNS: 3,5-dinitrosalicylic acid

RMSD: root mean square deviation

RMSF: root mean square fluctuation

\textbf{1. Introduction}

Cellulase is an enzyme cocktail that catalyzes the hydrolysis of
cellulose to glucose.\textsuperscript{{[}1{]}} It can be roughly divided
into cellobiohydrolase (CBH), endoglucanase (EGL), \selectlanguage{greek}β-\selectlanguage{english}glucosidase (BGL)
and lytic polysaccharide monooxygenase (LPMO).\textsuperscript{{[}1{]}}
Enzymatic hydrolysis of cellulose relies on the synergistic action of
various cellulases,\emph{i.e.} , CBH degrades cellulose from both ends
of the cellulose chain to form cello-oligosaccharide, EGL and LPMO act
respectively on the non-crystalline and crystalline regions of cellulose
and degrade the long cellulose chain into short ones, and BGL degrades
the cello-oligosaccharide into glucose.\textsuperscript{{[}1, 2{]}}

The utilization of cellulose resources is one of the potential ways to
alleviate or solve energy crisis, in which the pretreatment of
lignocellulosic biomass and the enzymatic hydrolysis of cellulose are
two key steps.\textsuperscript{{[}3, 4{]}} However, pretreatment may
also put some pressure on the enzymatic hydrolysis
step.\textsuperscript{{[}5{]}} The method of acid/alkali pretreatment
makes the cellulose substrate rich in salt, the method of ionic liquid
pretreatment also introduces considerable ions in the substrate. Without
desalination, the subsequent enzymatic hydrolysis step is bound to be
carried out in high salt environment, which is obviously unfavorable for
enzymatic efficiency. The introduction of halo-tolerant cellulases can
eliminate or alleviate the adverse effects caused by acid/alkali and
ionic liquid pretreatment.\textsuperscript{{[}6, 7{]}} The halo-tolerant
cellulases refer to the ones that maintain high activity even in high
salt concentration. Recently, different kinds of halo-tolerant
cellulases, including CBHs from glycoside hydrolase family 5 (GH5) and
GH7\textsuperscript{{[}8, 9{]}}, BGLs from GH1 and
GH3\textsuperscript{{[}10,11{]}} and EGLs from GH1, GH5, GH8, GH44 and
GH45\textsuperscript{{[}12-16{]}}, have been reported. Among them, the
EGLs from GH5 were reported the most. Most of the halo-tolerant
cellulases were found from halophiles or in high salt
environments.\textsuperscript{{[}8, 10, 12, 15{]}} In our previous
studies, the cellulases from marine \emph{A. niger} have been proved to
be more halo-tolerant than those from terrestrial \emph{A. niger}
.\textsuperscript{{[}17-21{]}} Fortunately, a halo-tolerant BGL from
marine \emph{A. niger} has been successfully
characterized.\textsuperscript{{[}10{]}} Some studies have provided
speculation on the mechanisms of halo-tolerance.\textsuperscript{{[}8,
22{]}} The generally accepted mechanism is that a large excess of acidic
residues distributed in protein surface contribute to bind large amounts
of water molecules and metal ions to maintain the activity of protein.
In addition, other factors such as ordered secondary structure, salt
bridge, etc., also have positive effects on
halo-tolerance.\textsuperscript{{[}23{]}}

During the enzymatic hydrolysis process, temperature has an important
influence on the enzymatic efficiency.\textsuperscript{{[}24{]}} As the
temperature increases, on the one hand, the viscosity of the reaction
system decreases, and the mass transfer effect increases. On the other
hand, the intensification of Brownian motion increases the contact
frequency between enzyme and substrate. Therefore, a higher reaction
temperature is preferred during enzymatic hydrolysis of cellulose and
the thermostability of cellulase is of crucial significance.
Thermostable cellulases are widely available from many organisms,
including fungi and bacteria.\textsubscript{{[}25, 26{]}} At present, a
large number of thermostable cellulases have been discovered, which are
almost exclusively derived from thermophiles.\textsuperscript{{[}25,
26{]}} The study on the mechanisms of thermostability is of great
significance for directed evolution to obtain more thermostable enzymes.
A number of breakthroughs have been made in the study on the
mechanisms.\textsuperscript{{[}27, 28{]}} The thermostability of protein
is related with many factors, including amino acid composition,
disulfide bond, hydrophobic interaction, aromatic interaction, hydrogen
bond, salt bridge, etc.

For understanding the mechanisms of thermostability and halo-tolerance,
Fourier transform infrared spectroscopy\textsuperscript{{[}29{]}},
circular dichroism spectrum\textsuperscript{{[}30{]}} and nuclear
magnetic resonance spectroscopy\textsuperscript{{[}31{]}} can be used to
measure the changes in protein secondary structure, and Michaelis
constant (\emph{K\textsubscript{m}} ) can reflect the substrate binding
capacity. However, the deeper mechanisms involving the interaction
between residues or atoms still cannot be explained clearly. The
tertiary structure or the crystal structure of protein is prerequisite
but not sufficient for the study on the mechanisms. The crystal
structure is always obtained under a certain static condition, while the
demonstration of thermostability and halo-tolerance needs a condition
with higher temperature or higher salt concentration. Molecular dynamics
(MD) simulation provides an effective way for dynamic studying the
mechanisms of halo-tolerance and thermostability.\textsuperscript{{[}8,
32{]}} By simulating the changes at different salt concentrations or
temperatures, it is possible to elucidate the tolerant mechanisms. The
changes include secondary structure, salt bridge, solvent accessible
surface, hydrogen bond, residue activity, etc.

In this study, we would prepare an EGL (\emph{An} EGL) of marine
\emph{A. niger} directly from the fermentation broth by homologous
constitutive expression. And then a pure \emph{An} EGL would be obtained
by simple separation and purification to determine its properties of
halo-tolerance and thermostability. As an important part, we would like
to analyze the corresponding molecular mechanisms of halo-tolerance and
thermostability by combining MD simulation with site directed
mutagenesis study.

\textbf{2. Material and methods}

\textbf{2.1 Main strains, plasmids, media and primers}

\emph{A. niger} ZJUBE-1 was stored at the China Center for Type Culture
Collection (Conservation No. CCTCC M2010132) and used for homologous
expression of \emph{An} EGL. \emph{Trichoderma reesei} RUT-C30 was used
as the donor of \emph{cbh1} terminator gene. \emph{Agrobacterium
tumefaciens} AGL-1 was used for transformation of marine \emph{A. niger}
. GPY medium (40 g/L glucose, 20 g/L peptone, 5 g/L yeast extract, 4 g/L
KH\textsubscript{2}PO\textsubscript{4}) was used for propagation of
marine \emph{A. niger} and constitutive expression of \emph{An} EGL. PDA
medium (20 g/L potato powder, 20 g/L glucose, 20 g/L agar) was used for
the generation and preservation of marine \emph{A. niger} . The induced
medium (IM) and minimal medium (MM) used for \emph{A. tumefaciens}
-mediated transformation (AMT) of marine \emph{A. niger}were identical
with the protocol.\textsuperscript{{[}33{]}} The plasmids and primers
used in this study were shown in \textbf{Figure S1} and\textbf{Table S1}
, respectively.

\textbf{2.2 Cloning of \emph{egl} gene}

About 10\textsuperscript{7} spores of \emph{A. niger} ZJUBE-1 were
inoculated in 100 ml GPY medium in 250 ml shake flask and cultivated for
3 d at 180 rpm, 30 \selectlanguage{ngerman}°C. Then the genomic DNA was extracted using Rapid
Fungi Genomic DNA Isolation Kit (Sangon, Shanghai, China). Subsequently,
the \emph{egl} gene was PCR-amplified from the genomic DNA using
primers, eglc-F and eglc-R. Finally, the cloned gene was ligated into
pUCm-T vector (Sangon, Shanghai, China) and the recombinant plasmid was
sequenced using the primers, M13F and M13R.

\textbf{2.3 Construction ofMini-Ti vector}

The construction of Mini-Ti vector pCAMBIA-hph-egl was shown
in\textbf{Figure S2} . Firstly, the hygromycin phosphotransferase gene
(\emph{hph} ) was PCR-amplified from pAN7-1 using primers, hph-F and
hph-R. The genes including pCAMBIA skeleton and \emph{trpC} promoter
were PCR-amplified from pCAMBIA0380TB using primers, PtrpC-F and
vector-R. The empty Mini-Ti vector pCAMBIA-hph was constructed by
ligating these 2 fragments using pEASY-Uni Seamless Cloning and Assembly
Kit (TransGen, Beijing, China; all of the ligation steps in this study
were using this kit). Secondly, the \emph{gpdA} promoter was
PCR-amplified from pAN7-1 using primers, PgpdA-F and PgpdA-R. The
\emph{egl} gene was PCR-amplified from the genomic DNA using primers,
egl-F and egl-R. The \emph{cbh1} terminator was PCR-amplified from the
genomic DNA of \emph{T. reesei} using primers, Tcbh1-F and Tcbh1-R. The
linearized pUC19 was obtained by PCR using primers, pUC19-F and pUC19-R.
The vector pUC-PgpdA-egl-Tcbh1 was constructed by ligating these 4
fragments. Thirdly, the linearized empty Mini-Ti vector was obtained by
PCR using primers, vector-F and vector-R. The \emph{egl} expression
cassettes were PCR-amplified from pUC-PgpdA-egl-Tcbh1 using primers,
cassette-F and cassette-R. The target Mini-Ti vector pCAMBIA-hph-egl was
constructed by ligating these 2 fragments. The sequences of
PgpdA-egl-Tcbh1-hph-PtrpC were checked by DNA sequencing.

\textbf{2.4 Transformation of marine \emph{A. niger}}

\emph{A. tumefaciens} AGL-1 was transformed with the Mini-Ti vector
pCAMBIA-hph-egl using freeze-thaw
method.\textsuperscript{{[}34{]}}Standard procedures of AMT were used as
described in the protocol.\textsuperscript{{[}33{]}} Briefly, the fresh
\emph{A. tumefaciens} recombinant was inoculated in IM broth, cultivated
until OD\textsubscript{600} reached 0.6-0.8. About 100 \selectlanguage{greek}μ\selectlanguage{english}l induced
\emph{A. tumefaciens} recombinant and 10\textsuperscript{6} spores of
marine\emph{A. niger} were mixed and spread on cellophane of IM agar,
cultivated for 2 d at 23 \selectlanguage{ngerman}°C. Then the cellophane as well as the strains
was transferred onto MM agar with 200 \selectlanguage{greek}μ\selectlanguage{english}g/ml hygromycin B and 200 \selectlanguage{greek}μ\selectlanguage{english}g/ml
cefotaxime sodium. Additional MM agar with equal concentration of
antibiotics was poured onto the cellophane to enhance the screening
effect. This interlayer medium was cultivated at 30 \selectlanguage{ngerman}°C until
recombinants grew out. Positive recombinants were verified by PCR
identification using primers, hph-F and hph-R. Single conidiospore
isolation was performed for the acquisition of homozygote. Spore
suspensions of recombinants (10\textsuperscript{7}/ml) were prepared
using blood cell counting plate, and then 10\textsuperscript{2} spores
obtained by progressive dilution were spread on PDA medium with 200
\selectlanguage{greek}μ\selectlanguage{english}g/ml hygromycin B, cultivated at 30 \selectlanguage{ngerman}°C. After the spores were mature,
repeat the step for 4 times. The resulting recombinants were stored at 4
°C.

\textbf{2.5 Enzyme assay}

EGL activity was assayed using sodium carboxymethylcellulose (CMC-Na) as
substrate. The enzymatic reaction mixtures (500 \selectlanguage{greek}µ\selectlanguage{english}l) containing 50 \selectlanguage{greek}µ\selectlanguage{english}l
enzyme solution and 450 \selectlanguage{greek}µ\selectlanguage{english}l CMC-Na solution (1\% (w/v) CMC-Na, 0.1 M
sodium citrate/citric acid, pH 4.0) were incubated for 5 min at 50 \selectlanguage{ngerman}°C.
The amount of reducing sugar released was determined by
3,5-dinitrosalicylic acid (DNS) assay.\textsuperscript{{[}35{]}} One
unit of EGL activity was defined as the amount of enzyme required for
release 1 \selectlanguage{greek}µ\selectlanguage{english}mol glucose equivalent per minute. The values in following
text were determined by this method unless otherwise stated.

\textbf{2.6 Protein expression}

For the constitutive expression of \emph{An} EGL,
10\textsuperscript{7}spores of recombinant were inoculated in 250 ml GPY
medium in 1000 ml shake flask, cultivated at 30 \selectlanguage{ngerman}°C, 200 rpm for 6 d.
After fermentation, the supernatant was obtained by filtration with 2
layers of gauze and concentrated about 5-fold by ultrafiltration with
molecular weight cut-off 10 kDa. Then the expression was analyzed by
12\% sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE).

\textbf{2.7 Purification}

After constitutive expression, the mycelia were removed using 2 layers
of gauze. Then the supernatant was concentrated and the saline ions as
well as the impurities with small molecule were replaced by water using
ultrafiltration (molecular weight cut off 10 kDa). The pH of
ultrafiltrate was adjusted to 7.0 with 0.1 M NaOH and then loaded on
DEAE-Sepharose column pre-equilibrated with 20 mM sodium phosphate
buffer pH 7.0. The fractions were eluted with the gradient of 0-0.5 M
NaCl at a flow rate of 1 ml/min. The purity of \emph{An} EGL in each
eluent was determined by 12\% SDS-PAGE. Then the purified \emph{An} EGL
was concentrated and the buffer was replaced by 20 mM sodium acetate
buffer pH 4.0 by ultrafiltration (molecular weight cut off 10 kDa). The
resulting enzyme solution was stored at 4 °C.

\textbf{2.8 Enzymatic properties}

The purified \emph{An} EGL was used for the study on enzymatic
properties. In terms of pH, the enzymatic reaction mixtures (500 \selectlanguage{greek}μ\selectlanguage{english}l)
including 50 \selectlanguage{greek}μ\selectlanguage{english}l properly diluted enzyme solution and 450 \selectlanguage{greek}μ\selectlanguage{english}l CMC-Na
solution (1\% (w/v) CMC-Na, 0.1 M sodium citrate/citric acid, pH 2.5,
3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5) were incubated in water bath
at 50 \selectlanguage{ngerman}°C for 5 min. In terms of temperature, the enzymatic reaction
mixtures (500 \selectlanguage{greek}μ\selectlanguage{english}l) including 50 \selectlanguage{greek}μ\selectlanguage{english}l properly diluted enzyme solution and
450 \selectlanguage{greek}μ\selectlanguage{english}l CMC-Na solution (1\% (w/v) CMC-Na, 0.1 M sodium citrate/citric
acid, pH 4.0) were incubated in water bath at different temperature (20,
30, 40, 50, 60, 70 and 80 \selectlanguage{ngerman}°C) for 5 min. In terms of metal ions (5 mM
and 10 mM), the enzymatic reaction mixtures (500 \selectlanguage{greek}μ\selectlanguage{english}l) including 50 \selectlanguage{greek}μ\selectlanguage{english}l
properly diluted enzyme solution, 5 \selectlanguage{greek}μ\selectlanguage{english}l or 10 \selectlanguage{greek}μ\selectlanguage{english}l metal ions solution
(NaCl, LiCl, AgNO\textsubscript{3}, MgCl\textsubscript{2},
CaCl\textsubscript{2}, NiCl\textsubscript{2}, CuSO\textsubscript{4},
ZnSO\textsubscript{4}, CrCl\textsubscript{2}, HgSO\textsubscript{4},
FeCl\textsubscript{3}, 0.5 M) and 445 \selectlanguage{greek}μ\selectlanguage{english}l or 440 \selectlanguage{greek}μ\selectlanguage{english}l CMC-Na solution (1\%
(w/v) CMC-Na, 0.1 M sodium citrate/citric acid, pH 4.0) were incubated
in water bath at 50 \selectlanguage{ngerman}°C for 5 min. In terms of salt concentration, the
enzymatic reaction mixtures (500 \selectlanguage{greek}μ\selectlanguage{english}l) including 50 \selectlanguage{greek}μ\selectlanguage{english}l properly diluted
enzyme solution, 450 \selectlanguage{greek}μ\selectlanguage{english}l CMC-Na solution (1\% (w/v) CMC-Na, 0, 1.0, 2.0,
3.0, 4.0, 5.0 M NaCl, 0.1 M sodium citrate/citric acid, pH 4.0) were
incubated in water bath at 50 \selectlanguage{ngerman}°C for 5 min. In terms of thermostability,
50 \selectlanguage{greek}μ\selectlanguage{english}l properly diluted enzyme solution was added into 450 \selectlanguage{greek}μ\selectlanguage{english}l CMC-Na
solution (1\% (w/v) CMC-Na, 0.1 M sodium citrate/citric acid, pH 4.0)
after incubated at 50, 60, 65, 70 \selectlanguage{ngerman}°C for 5, 10, 15, 20, 30, 40, 50, 60,
90, 120 min. Then the enzymatic reaction mixtures were incubated in
water bath at 50 °C for 5 min. In terms of the influence of salt
concentration on thermostability, enzyme solution was diluted properly
with 0.1 M sodium citrate/citric acid buffer pH 4.0 with different NaCl
concentration (0, 1.0, 2.0, 3.0, 4.0, 5.0 M), incubated at 65 °C for 4,
8, 12, 16, 20, 30, 60 min. Subsequently, 50 \selectlanguage{greek}μ\selectlanguage{english}l of the diluted enzyme
solution was added into 450 \selectlanguage{greek}μ\selectlanguage{english}l CMC-Na solution (1\% (w/v) CMC-Na, 0.1 M
sodium citrate/citric acid, pH 4.0). Then the enzymatic reaction
mixtures were incubated in water bath at 50 \selectlanguage{ngerman}°C for 5 min. The highest
EGL activity was defined as 100\%.

\textbf{2.9 MD simulation}

The amino acid sequence of \emph{An} EGL was translated from its
nucleotide sequence, and then its signal peptide was predicted by
SignalP (http://www.cbs.dtu.dk/services/SignalP-4.0/). The alignment of
amino acid sequences with other solved EGLs was accomplished with DNAMAN
8. The homology modeling of \emph{An} EGL was accomplished with
Discovery Studio 3.0 using the crystal structure of \selectlanguage{greek}β-\selectlanguage{english}1,4-endoglucanase
from \emph{A. niger} (EglA, PDB ID: 1KS4) as
template.\textsuperscript{{[}36{]}} The conserved substrate binding
sites and catalytic residues were determined according to the solved
structure of EglA. The MD simulation was carried out with GROMACS at two
temperatures (300 and 350 K) and three salt concentrations (0, 2 and 4 M
NaCl). After simulation, the root mean square deviation (RMSD), root
mean square fluctuation (RMSF) and salt bridges were calculated by
GROMACS. The visualization of trajectories was accomplished by VMD.

\textbf{2.10 Obtainment of mutant \emph{An}EGLs}

In order to simplify protein expression and enzyme assay, the
mutant\emph{An} EGLs as well as original \emph{An} EGL were expressed
in\emph{E. coli} Rosetta(DE3). Firstly, the plasmid pET-egl was
constructed. In detail, the \emph{egl} gene was PCR-amplified from the
genomic DNA using primers, eglec-F and eglec-R; The linearized pET28a
was obtained by PCR using primers, pET-F and pET-R; The plasmid pET-egl
was constructed by ligating these 2 fragments. Secondly, the plasmid
pET-mutant1 was constructed by site-saturation mutagenesis using
primers, mutant1-F and mutant1-R. Thirdly, the plasmid pET-mutant1 was
constructed. In detail, the fragments 1-4 of \emph{egl} gene were
PCR-amplified from the genomic DNA using primers, mutant2-1-F and
mutant2-1-R, mutant2-2-F and mutant2-2-R, mutant2-3-F and mutant2-3-R,
mutant2-4-F and mutant2-4-R, respectively; The linearized pET28a was
obtained by PCR using primers, pET2-F and pET2-R; The plasmid
pET-mutant2 was constructed by ligating these 5 fragments. The
recombinant plasmids mentioned above were transformed into \emph{E.
coli} Rosetta(DE3). As for protein expression, the recombinants were
inoculated into 5 ml LB broth, cultivated at 37 \selectlanguage{ngerman}°C 200 rpm overnight;
The strains were inoculated into 50 ml LB broth, cultivated at 37 °C 200
rpm until OD\textsubscript{600} reached 0.4-0.6;
Isopropyl-\selectlanguage{greek}β-\selectlanguage{english}d-thiogalactoside (IPTG) was added into the culture to 0.1
mM of final concentration, cultivated at 16 \selectlanguage{ngerman}°C 200 rpm overnight. After
expression, the crude enzyme solutions were obtained by cell disruption
with ultraphonic. The influences of salt and temperature on activity
were determined as mentioned in section 2.8.

\textbf{3. Results}

\textbf{3.1 Cloning of \emph{egl} gene}

The \emph{egl} gene was cloned into pUCm-T vector and sequenced. The
sequence of \emph{egl} gene has been submitted to GenBank (GenBank
accession No. \textbf{MK587440} ). The distribution of intron was
confirmed according to the genomic gene with 100\% identity (GenBank
accession No. \textbf{ABL\_05547} ). Considering that the introns as
well as peptide can be well recognized in marine \emph{A. niger} , the
cloned gene was directly inserted into expression vector.

\textbf{3.2 Constitutive expression and purification}

The constitutive expression of \emph{An} EGL in GPY medium was checked
by 12\% SDS-PAGE. Obvious bands were observed in the supernatant of
recombinant at molecular weight of about 26 kDa (\textbf{Figure 1A} ).
EGL activity was detected in the fermentation broth when cultivated the
original strain in GPY medium. Therefore, it is necessary to purify the
crude \emph{An} EGL before studying on its enzymatic properties. For the
few contaminants in fermentation broth, the purification of \emph{An}
EGL was accomplished by one step of anion exchange chromatography.
The\emph{An} EGL was eluted with 20 mM sodium phosphate buffer pH 7.0
and the contaminants were eluted with 0.1-0.5 M NaCl (\textbf{Figure 1B}
). Although the volume hadn't been pre-eluted, the purity of \emph{An}
EGL was still very high. In addition, gradient elution with NaCl was
performed on the supernatant of the original strain, and it was found
that the impurity with EGL activity was concentrated in the eluent of
0.2-0.3 M NaCl (\textbf{Figure S3} ). Therefore, the purified\emph{An}
EGL could be used for the subsequent study.

\textbf{3.3 Enzymatic properties of \emph{An}EGL}

The influence of pH on the activity of \emph{An} EGL was shown
in\textbf{Figure 2A} . The purified \emph{An} EGL showed maximal
activity at pH 3.0-4.0 and retained 75\% of activity at pH 2.5. However,
the activity decreased sharply when pH was over 4.5 and was hardly
detected when pH was up to 6.0. The influence of temperature was shown
in\textbf{Figure 2B} . It showed the optimal activity was exhibited at
40 °C. The influence of salt concentration was shown in \textbf{Figure
2C} . The activity increased as the NaCl concentration increased and
reached the maximum (increased by 29\%) in the presence of 4.5 M NaCl,
suggesting it had a property of halo-tolerance. The thermostability
of\emph{An} EGL was shown in \textbf{Figure 2D} . The activity kept
constant when \emph{An} EGL was incubated at 50 °C for 4 h. The
half-lives of\emph{An} EGL were about 210, 6.5 and 2.5 min at 60, 65 and
70 °C, respectively. And the activity was completely abolished after
incubation at 65 °C for 20 min or at 70 °C for 15 min. It seemed that
the thermostability was not very different from that of mesophilic EGL.
The influence of salt concentration on the thermostability of \emph{An}
EGL was shown in \textbf{Figure 2E} . As the NaCl concentration
increased,\emph{An} EGL retained higher activity after incubation. The
half-lives of \emph{An} EGL were about 6.5, 14, 80, 120 and 180 min at
65 °C in the presence of 0.9, 1.8, 2.7, 3.6 and 4.5 M NaCl,
respectively. The thermostability of \emph{An} EGL was enhanced with the
increase of NaCl concentration, especially when NaCl concentration was
higher than 3.6 M, which was comparable to the EGL from
thermophiles.\textsuperscript{{[}25{]}} Among various metal ions (5 mM)
tested for their effect on the activity of \emph{An} EGL (\textbf{Table
1} ), a better enhancement (119\%) of activity was observed with 5 mM
Mg\textsuperscript{2+}, but the activity was strongly inhibited by
Ag\textsuperscript{+}, Cu\textsuperscript{2+} and
Hg\textsuperscript{2+}. When the concentration was up to 10 mM,\emph{An}
EGL was sensitive to most metal ions except with Na\textsuperscript{+},
Mg\textsuperscript{2+} and Fe\textsuperscript{3+}. The activity was
almost undetectable in the presence of 10 mM Ag\textsuperscript{+},
Cu\textsuperscript{2+} and Hg\textsuperscript{2+}.

\textbf{3.4 Sequence alignment and homology modeling}

Sequence alignment and homology modeling of \emph{An} EGL were shown
in\textbf{Figure 3A} and \textbf{Figure 3B} and the modeled structure
was provided in the supplementary materials (\textbf{AnEGL.pdb}
).\emph{An} EGL shows higher homology to EglA (89.7\% identity), which
belongs to GH12.\textsuperscript{{[}36{]}} The overall fold of\emph{An}
EGL strongly resembles that of EglA, which has a ``jelly-roll'' fold
with two antiparallel-sheets.\textsuperscript{{[}36{]}} As is shown in
\textbf{Figure 3B} , the substrate binding sites and their spatial
position from subsite -3 to +3 (Tyr7, Trp22, Tyr61, Phe101, Phe206,
Trp120, Pro129 and Trp147) are consistent with those of
EglA.\textsuperscript{{[}36{]}} Besides, the catalytic residues, Glu116
and Glu204, are highly conserved throughout the EGLs from GH12
(\textbf{Figure 3A} ). Here, we noticed that most of the substrate
binding residues were located in the loops. For the sake of analysis,
the loops were numbered from loop 1 to loop 5 (\textbf{Figure 3C} ).
Although the homology between \emph{An} EGL and the reported EglA was
very high, the study on EglA did not involve the properties of
halo-tolerance and thermostability.

\textbf{3.5 MD simulation}

MD simulation was carried out at different temperatures and salt
concentrations in order to study the halo-tolerant and thermostable
mechanisms of \emph{An} EGL. The RMSD results were shown
in\textbf{Figure 4A} (300 K) and \textbf{Figure 4B} (350 K). At 300 K,
the RMSD achieved a balance quickly after about 5 ns MD simulation at
different NaCl concentrations. At 350 K, its structure kept deviating
from the initial position in the absence of NaCl while this deviation
tendency was relieved with the increase of NaCl concentration. The
structure tended to be stable after 30 ns and 10 ns in the presence of 2
M and 4 M NaCl, respectively. The RMSF results were shown
in\textbf{Figure 4C} (300 K) and \textbf{Figure 4D} (350 K). At 300 K,
with the increase of NaCl concentration, the regions of residue 22-29,
52-57, 107-113 and 123-144 became active while the region of residue
152-157 became inactive. At 350 K, things are different. The regions of
residue 22-29 and 107-113 were most active in the absence of NaCl. In
order to better demonstrate the activity of different regions under
different conditions, the trajectories of \emph{An} EGL during 20 to 50
ns were overlapped and colored by RMSF of atom (\textbf{Figure 4E}
and\textbf{Figure 4F} ). The regions of residue 22-29, 52-57, 107-113,
123-144 and 152-157 corresponded to loop 2, 1, 4, 5 and 3. At 300 K,
except for loop 3, the other loops showed a more active trend with the
increase of NaCl concentration, of which loop 5 was the most obvious
one. At 350 K, the activity of loop 1 to 4 decreased with the increase
of NaCl concentration. In addition, it was found that in the absence of
NaCl, the substrate binding pocket tended to be exposed and the
structure deviated significantly from the initial one at 350 K
(\textbf{Figure 5F} ).

\textbf{3.6 Halo-tolerance and thermostability of mutant \emph{An}EGLs}

According to the MD results, two mutant \emph{An} EGLs (mutant1: N95D,
N99D; mutant2: Q55E, N114D, Q157E, T159K) were designed. The expression
of \emph{An} EGL, mutant1 and mutant2 in \emph{E. coli} were shown
in\textbf{Figure S4} . The apparent molecular weights of \emph{An} EGL,
mutant1 and mutant2 were accordance with the \emph{An} EGL homologously
expressed, which suggested that no glycosylation was occurred
in\emph{An} EGL. Considering that no EGL activity was detected in
original\emph{E. coli} , the crude enzyme solutions prepared with
ultrasonication were directly used for subsequent determination of
halo-tolerance and thermostability.

The influence of salt concentration on EGL activity was shown
in\textbf{Figure 6A} . It was found that the activity of mutant 1
significantly decreased with the increase of NaCl concentration, while
the activity of mutant 1 increased at the range of 0-1.8 M NaCl and
slightly decreased when NaCl concentration was above 1.8 M.
Comparatively, mutant 1 was more halo-sensitive than mutant 2. The
influence of salt concentration on the thermostability of mutants was
shown in \textbf{Figure 6B} . In the presence of 4.5 M NaCl, compared
with the activity of \emph{An} EGL, when incubated at 65 °C the activity
of mutant 1 sharply decreased; After incubated at 65 °C for 60 min, the
activity of mutant 1 was almost deactivated. In comparison, the activity
of mutant 2 decreased more slowly. After incubated at 65 °C for 60 min,
the activity of mutant 2 remained 45.9\%.

\textbf{4. Discussion}

Cellulase is an enzyme cocktail and produced in the presence of inducer.
As a result, there are always various components of cellulases in
fermentation broth. Direct separation to the cellulase cocktail is
hardly competent to obtain a single cellulase with high purity. Here, we
adopted constitutive expression to prevent the expression of other
inducible cellulase, which was named as ``directed
expression''.\textsuperscript{{[}10{]}} The expression cassettes
contained the \emph{gpdA} promoter, the target genes and the
\emph{cbh1}terminator, which realized constitutive expression in marine
\emph{A. niger} . In other word, the constitutive expression use in this
work seems as ``separation factor'' to be implanted into cell for
conveniently obtaining a single target enzyme from a complex inducible
enzyme system\textbf{.}

Acid treatment is widely used in the pretreatment of lignocellulosic
biomass.\textsuperscript{{[}5{]}} However, the treated biomass will be
acidic without neutralization. \emph{An} EGL exhibited an acidophilic
property, which conferred it with the potential to hydrolyze cellulose
under acidic condition, and then reduce the cost of neutralization. In
addition, \emph{An} EGL exhibited the property of halo-tolerance and
outstanding thermostability under high salt environment, which would
greatly expand its application, especially in the cellulose hydrolysis
at high salt concentration and high temperature. Moreover, \emph{An} EGL
was the first reported GH12 endoglucanase with halo-tolerant property.
Some halo-tolerant EGLs were summarized in \textbf{Table 2} , most of
which belonged to GH5.

RMSD and RMSF results revealed that high salt environment could active
some loops at normal temperature (300 K) and stabilize the structure at
high temperature (350 K). Many substrate binding sites were located on
the loops, and the loops with proper activity were of important
guarantee for substrate binding.\textsuperscript{{[}36{]}} The relative
active loops in high NaCl concentration might be the reason why the
activity of \emph{An} EGL increased slightly with the increase of NaCl
concentration. Overactive loops might result in some irreversible
changes and permanent loss of enzyme
activity.\textsuperscript{{[}43{]}}At 350 K, with the increase of NaCl
concentration, some loops as well as the substrate binding pocket became
more stable. Therefore, it could be concluded that high NaCl
concentration played important roles in stabilizing the structure of
\emph{An} EGL. How did NaCl make the loops stable or active and make the
pocket tend to be closed?

In fact, before performing the MD simulation at 300 K without NaCl, 8
additional sodium ions were added into the system to balance the
negative charge. It was these traces of
Na\textsuperscript{+}(approximately 50 mM) that entered the substrate
binding pocket after 5 ns simulation (\textbf{Figure 5A} and
\textbf{Figure 5B} ). Similar results were obtained under other
simulation conditions. Analysis of the surface electrostatic potential
of \emph{An} EGL revealed that the substrate binding pocket exhibited a
strong electronegativity (\textbf{Figure 5C} ), which explained the
reason why traces of Na\textsuperscript{+} could enter the pocket in
such a short time. Subsequently, the charged amino acid residues of
\emph{An} EGL were analyzed (\textbf{Figure 5D} ) and it was found that
more electropositive residues were located in the pocket. Among them,
Asp95, Asp99, Glu116 and Glu204 had a catching effect on
Na\textsuperscript{+}, which could also be reflected by the change of
salt bridges between residues and Na\textsuperscript{+}. Similar
phenomenon that the formation of
R-COO\selectlanguage{english}\textsuperscript{-}***Na\textsuperscript{+}***\selectlanguage{english}\textsuperscript{-}OOC-R
salt bridges was crucial for enhanced thermostability in high salt
concentration has been reported.\textsuperscript{{[}44{]}}

The instability of the loops mainly comes from two aspects, one is the
interaction of the internal residues, and the other is the interaction
between the residues of loops and the molecules in
solution.\textsuperscript{{[}28{]}} At lower temperature, the
interaction between Na\textsuperscript{+} and residues of loops was
weaker than that of the internal residues. Thus, at 300 K, protein
structure as well as loop activity did not change much in different NaCl
concentration. The decrease activity of loop 3 may be due to the
elimination of the interaction between loop 3 and loop 4 by
Na\textsuperscript{+} and Cl\selectlanguage{english}\textsuperscript{-}, which made the force
acting on loop 3 tend to be simple and ultimately reduced its activity.
There was only one Lys residue (Lys132) in loop 5 and as the
Cl\selectlanguage{english}\textsuperscript{-} concentration increased, the frequency of
interaction between Cl\selectlanguage{english}\textsuperscript{-} and loop 5 increased, thereby
increasing its activity. At 350 K, the activity of the whole protein
increased. At this time, the frequency of interaction between
Na\textsuperscript{+} and Cl\selectlanguage{english}\textsuperscript{-} and loops increased
accordingly. As the concentration of NaCl increased, the force between
the loops was weakened. Similar to that at 300 K, the activity of loop 3
was greatly reduced. In detail, salt bridges could be formed between
Lys159 in loop 3 and Asp157 in loop 3 or between Lys159 in loop 3 and
Asp114 in loop 4. Due to the limitation of steric hindrance and the
interference of Na\textsuperscript{+} and Cl\selectlanguage{english}\textsuperscript{-}, the
salt bridge between Lys159 and Asp114 became unstable with the increase
of NaCl concentration (\textbf{Table S2} ), which weakened the
interaction between loop 3 and loop 4.

Because the substrate binding pocket was rich in electronegative
residues, the pocket exhibits strong electronegativity. In the pocket,
in addition to Asp95, Asp99, Glu116, Glu204, there were other
electronegative residues, such as Glu55 in loop 1, Asp157 in loop 3 and
Asp114 in loop 4. If there was not enough Na\textsuperscript{+} to
balance the negative electricity, the electronegative residues would
produce a strong repulsive force to expand the pocket. At 300 K, due to
the lower activity of Na\textsuperscript{+} and residues, two
Na\textsuperscript{+} were basically located in the pocket during the
simulation, and thereby the expansion of the pocket was not obvious.
When the temperature was up to 350 K, the activity of
Na\textsuperscript{+} increased, and the captured
Na\textsuperscript{+}might escape from the pocket. When the
Na\textsuperscript{+}concentration was low, the negative charge of the
pocket was not well balanced, so the pocket tended to expand. With the
increase of Na\textsuperscript{+} concentration, despite the escape of
Na\textsuperscript{+}, the Na\textsuperscript{+} outside the pocket
would enter the pocket due to the differential concentration. Thus the
protein pocket did not expand to a large extent in high NaCl
concentration. Further, site directed mutagenesis was employed to
verified the conjectural mechanisms by MD simulation. It was found that
the charged residues Asp95 and Asp99 in the pocket were more significant
for the halo-tolerance and thermostability than those in the loops.

In summary, the strategy of directed expression was employed for
efficient obtainment of the target enzyme \emph{An} EGL.\emph{An} EGL
exhibited a halo-tolerant activity and an outstanding thermostability in
high salt concentration, which was potential and competitive in
industrial application. MD simulation revealed that the salt bridges
formed between charged residues and Na\textsuperscript{+} and
Cl\selectlanguage{english}\textsuperscript{-}influenced the activity of loops and the stability
of pocket, and then conferred on \emph{An} EGL the halo-tolerance and
outstanding thermostability in high salt environment, which were
verified by site directed mutagenesis. These conjectural mechanisms were
of reference value for the study on halo-tolerance and thermostability.

\textbf{Acknowledgments}

This study was funded by National Natural Science Foundation of China
{[}21576233 and 21878263{]} and Fundamental Research Funds for the
Central Universities.

\textbf{Conflicts of interest}

The authors declare no financial or commercial conflict of interest.

\textbf{Author contributions}

Shan-Jing Yao and Li-Nian Cai designed the research; Li-Nian Cai and Tao
Lu performed the research; Li-Nian Cai, Tao Lu, and Dong-Qiang Lin
analyzed the data; Li-Nian Cai wrote the manuscript. All the authors
have read and approved the submitted manuscript.

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7.

44. Liang, C., Xue, Y., Fioroni, M., Rodr\selectlanguage{ngerman}íguez-Ropero, F., Zhou, C.,
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Biotechnol. 89, 315-326.

\textbf{Figure captions and legends}

\textbf{FIGURE 1} Constitutive expression and purification

(A) Constitutive expression in GPY medium. Lane M: protein marker; lane
1: concentrated supernatant of original strain; lane 2 to 6:
concentrated supernatant of recombinant 1 to 5. (B) Purification
of\emph{An} EGL by anion exchange chromatography. Lane M: protein
marker; lane 7: ultrafiltrate; lane 8: wash with 20 mM pH 7.0 sodium
phosphate buffer; lane 9 to 12: elution with 0.1, 0.2, 0.3, 0.5 M NaCl.

\textbf{FIGURE 2} Enzymatic properties of \emph{An} EGL

(A) Influence of pH on the activity. (B) Influence of temperature on the
activity. (C) Influence of salt concentration on the activity. (D)
Thermostability. (E) Influence of salt concentration on the
thermostability.

\textbf{FIGURE 3} Analysis on sequence and structure of \emph{An} EGL

(A) Amino acid sequence alignment of EGLs. 1KS4, 5GM3, 5M2D, 4H7M, 1OA2,
1H8V and 1OLQ are the PDB IDs of EGLs. The residues with 100\% homology
level were highlighted with yellow. The substrate binding residues and
catalytic residues of \emph{An} EGL were marked with red solid circles
and pentagrams, respectively. (B) Structure and key residues of\emph{An}
EGL. The structures of EglA and \emph{An} EGL were displayed with light
green and light orange, respectively; Key residues of EglA and \emph{An}
EGL were displayed with green and orange, respectively. (C) Distribution
of key loops involved in substrate binding.

\textbf{FIGURE 4} RMSD and RMSF analysis at different temperatures and
NaCl concentrations

(A) RMSD of \emph{An} EGL at 300 K within 50 ns MD simulation. (B) RMSD
of \emph{An} EGL at 350 K within 50 ns MD simulation. (C) RMSF of C-\selectlanguage{greek}α \selectlanguage{english}at
300 K within 20 to 50 ns MD simulation. (D) RMSD of C-\selectlanguage{greek}α \selectlanguage{english}at 350 K within
20 to 50 ns MD simulation. (E) Activity analysis on \emph{An} EGL at 300
K within 20 to 50 ns MD simulation. (F) Activity analysis on\emph{An}
EGL at 350 K within 20 to 50 ns MD simulation. The proteins were colored
according to the RMSF of atom within 20 to 50 ns MD simulation. The
activity from strong to weak is shown as colored gradient from red to
blue.

\textbf{FIGURE 5} The function of NaCl on halo-tolerance and
thermostability

(A) The spatial position of Na\textsuperscript{+} before MD simulation
at 300 K in the absence of NaCl. (B) The spatial position of
Na\textsuperscript{+} after 5 ns MD simulation at 300 K in the absence
of NaCl. The red sphere represented Na\textsuperscript{+}. (C) Surface
electrostatic potential of \emph{An} EGL. The region circled with the
ellipse was the substrate binding pocket. Electrostatic potential
between -5 kT/e and 5 kT/e was shown as colored gradient from red to
white to blue. (D) Charged residues of \emph{An} EGL. The
electropositive and electronegative residues were colored with blue and
red, respectively.

\textbf{FIGURE} \textbf{6} The halo-tolerance and thermostability
of\emph{An} EGL and its mutants

(A) Influence of NaCl concentration on activity; (B) The thermostability
in presence of 5 M NaCl.

\textbf{Table 1} The influence of metal ions on the activity of\emph{An}
EGL\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
Metal ions & Relative activity (\%) & Relative activity (\%) & Metal
ions & Relative activity (\%) & Relative activity (\%) & Relative
activity (\%) & Relative activity (\%)\tabularnewline
\midrule
\endhead
& 5 mM & 10 mM & & 5 mM & 5 mM & 10 mM & 10 mM\tabularnewline
CK\textsuperscript{a} & 100 & 100 & Ni\textsuperscript{2+}
(NiCl\textsubscript{2}) & Ni\textsuperscript{2+} (NiCl\textsubscript{2})
& 98 & 98 & 58\tabularnewline
Na\textsuperscript{+} (NaCl) & 100 & 101 & Cu\textsuperscript{2+}
(CuSO\textsubscript{4}) & Cu\textsuperscript{2+} (CuSO\textsubscript{4})
& 76 & 76 & 8\tabularnewline
Li\textsuperscript{+} (LiCl) & 105 & 82 & Zn\textsuperscript{2+}
(ZnSO\textsubscript{4}) & Zn\textsuperscript{2+} (ZnSO\textsubscript{4})
& 105 & 105 & 85\tabularnewline
Ag\textsuperscript{+} (AgNO\textsubscript{3}) & 54 & 5 &
Hg\textsuperscript{2+} (HgSO\textsubscript{4}) & Hg\textsuperscript{2+}
(HgSO\textsubscript{4}) & 29 & 29 & 4\tabularnewline
Mg\textsuperscript{2+} (MgCl\textsubscript{2}) & 119 & 101 &
Fe\textsuperscript{3+} (FeCl\textsubscript{3}) & Fe\textsuperscript{3+}
(FeCl\textsubscript{3}) & 108 & 108 & 97\tabularnewline
Ca\textsuperscript{2+} (CaCl\textsubscript{2}) & 107 & 67 &
Cr\textsuperscript{3+} (CrCl\textsubscript{3}) & Cr\textsuperscript{3+}
(CrCl\textsubscript{3}) & 98 & 98 & 83\tabularnewline
\bottomrule
\end{longtable}

a. The control check, which was not added with metal ions.

\textbf{Table 2} Halo-tolerant and halophilic EGLs\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
Cellulase name & Source & GH family & pH optimum & Temperature optimum
(\selectlanguage{ngerman}°C) & Salt concentration optimum & Relative activity (\%) &
Reference\tabularnewline
\midrule
\endhead
EG1 & \emph{Stachybotrys microspora} & NM\textsuperscript{a} & 7.0 & 50
& 5 M NaCl & 152 & {[}37{]}\tabularnewline
endoglucanase & \emph{Haloarcula} sp. G10 & NM & 9.0 & 60 & 3 M NaCl &
NC\textsuperscript{b} & {[}38{]}\tabularnewline
egl01 & \emph{Bacillus licheniformis} & 5 & 5 & 50 & 2-3 M NaCl/KCl & >
120 & {[}39{]}\tabularnewline
EG-PY2 & \emph{Paenibacillus} sp. Y2 & NM & 4.5 & 30 & 0.5 M NaCl &
211.5 & {[}40{]}\tabularnewline
Cel5A & \emph{Vibrio} sp. G21 & 5 & 6.5-7.5 & 50 & 0.5 M NaCl & 160 &
{[}14{]}\tabularnewline
En5H & \emph{Alkalilimnicola} sp. NM-DCM1 & 5 & 8.8 & 55 & 2.5 M NaCl &
500 & {[}41{]}\tabularnewline
MgCel44 & mangrove soil metagenomic library & 44 & 6.0 & 45 & 0.5 M NaCl
& 160 & {[}16{]}\tabularnewline
AgCMCase & \emph{Aspergillus glaucus} CCHA & 5 & 5.0 & 55 & 1.0-4.0 M
NaCl & 230 & {[}42{]}\tabularnewline
\emph{An}EGL & \emph{A.} \emph{niger} ZJUBE-1 & 12 & 3.5-4.0 & 40 & 5.0
M NaCl & 129 & This study\tabularnewline
\bottomrule
\end{longtable}

a. Not mentioned

b. The relative activity cannot be calculated because there was no
activity in the absence of NaCl.

\textbf{FIGURE 1}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{FIGURE 2}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{FIGURE 3}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{FIGURE 4}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{FIGURE 5}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{FIGURE} \textbf{6}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\selectlanguage{english}
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