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\begin{document}

\title{Extracellular expression and characterization of an \selectlanguage{greek}α-\selectlanguage{english}glucosidase from
Oryza sativa and its transglycosylation for synthesis of
2-O-\selectlanguage{greek}α-\selectlanguage{english}D-glucopyranosyl-L-ascorbic acid\selectlanguage{english}}



\author[1]{Xuelian Qi}%
\author[1]{Junlan Shao}%
\author[1]{Yinchu Cheng}%
\author[1]{Xiaoying He}%
\author[1]{Yan Li}%
\author[1]{Honghua Jia}%
\author[1]{Ming Yan}%
\affil[1]{Nanjing Tech University}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Abstract: 2-O-\selectlanguage{greek}α-\selectlanguage{english}D-Glucopyranosyl-L-ascorbic acid (AA-2G) is an important
industrial derivative of L-ascorbic acid (AA), which has the distinct
advantages of non-reducibility, antioxidation, and reproducible
decomposition into L-ascorbic acid and glucose. Enzymatic synthesis is a
preferred method for AA-2G production over alternative chemical
synthesis owing to the regioselective glycosylation reaction.
\selectlanguage{greek}α-\selectlanguage{english}Glucosidase, an enzyme classed into O- glycoside hydrolases, may be
used in glycosylation reactions to synthesize AA-2G. Here, one
\selectlanguage{greek}α-\selectlanguage{english}glucosidase from Oryza sativa (rAGL) was recombinantly produced in
Pichia pastoris GS115 and used for biosynthesis of AA-2G with few
intermediates and byproducts. The extracellular rAGL reached 9.11 U/mL
after fed-batch cultivation for 102 h in a 5-L fermenter. The specific
activity of purified rAGL is 49.83 U/mg at 37 \selectlanguage{ngerman}°C and pH 4.0. The optimal
temperature of rAGL was 65 °C, and it was stable below 55 °C. rAGL was
active over the range of pH 3.0--7.0, with the maximal activity at pH
4.0. Under the condition of 37 °C , pH 4.0, equimolar maltose and AA·Na,
8.7±0.4 g/L of AA-2G was synthesized by rAGL. These studies lay the
basis for the industrial application of recombinant \selectlanguage{greek}α-\selectlanguage{english}glucosidase.
Keywords: \selectlanguage{greek}α-\selectlanguage{english}Glucosidase; Oryza sativa; 2-O-\selectlanguage{greek}α-\selectlanguage{english}D-glucopyranosyl-L-ascorbic
acid; Transglycosylation; Pichia pastoris%
\end{abstract}%



\sloppy


\selectlanguage{greek}\textbf{Extracellular expression and characterization of an
\emph{α}-glucosidase from \emph{Oryza sativa}and its transglycosylation
for synthesis of 2-O-\emph{α}-D-glucopyranosyl-L-ascorbic acid}\selectlanguage{english}

Xuelian Qi, Junlan Shao, Yinchu Cheng, Xiaoying He, Yan Li*, Honghua
Jia*, Ming Yan

College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech
University, Nanjing 211816, China

* Corresponding author. Tel.: +86-25-58139368; Fax: +86-25-58139368

liyan@njtech.edu.cn (Y.Li); hhjia@njtech.edu.cn (H. Jia)



\textbf{Abstract:}

2-O-\selectlanguage{greek}α-\selectlanguage{english}D-Glucopyranosyl-L-ascorbic acid (AA-2G) is an important
industrial derivative of L-ascorbic acid (AA), which has the distinct
advantages of non-reducibility, antioxidation, and reproducible
decomposition into L-ascorbic acid and glucose. Enzymatic synthesis is a
preferred method for AA-2G production over alternative chemical
synthesis owing to the regioselective glycosylation reaction.\selectlanguage{greek}\emph{α}
-\selectlanguage{english}Glucosidase, an enzyme classed into \emph{O} - glycoside hydrolases,
may be used in glycosylation reactions to synthesize AA-2G. Here, one
\selectlanguage{greek}α-\selectlanguage{english}glucosidase from \emph{Oryza sativa} (rAGL) was recombinantly produced
in \emph{Pichia pastoris} GS115 and used for biosynthesis of AA-2G with
few intermediates and byproducts. The extracellular rAGL reached 9.11
U/mL after fed-batch cultivation for 102 h in a 5-L fermenter. The
specific activity of purified rAGL is 49.83 U/mg at 37 \selectlanguage{ngerman}°C and pH 4.0.
The optimal temperature of rAGL was 65 °C, and it was stable below 55
°C. rAGL was active over the range of pH 3.0--7.0, with the maximal
activity at pH 4.0. Under the condition of 37 °C , pH 4.0, equimolar
maltose and AA·Na, 8.7±0.4 g/L of AA-2G was synthesized by rAGL. These
studies lay the basis for the industrial application of recombinant
\selectlanguage{greek}\emph{α} -\selectlanguage{english}glucosidase.

\textbf{Keywords:} \selectlanguage{greek}\emph{α} -\selectlanguage{english}Glucosidase; \emph{Oryza sativa} ;
2-O-\selectlanguage{greek}\emph{α} -\selectlanguage{english}D-glucopyranosyl-L-ascorbic acid; Transglycosylation;

\emph{Pichia pastoris}

\textbf{1 Introduction}

L-Ascorbic acid (L-AA) is a water-soluble vitamin, commonly known as
vitamin C (VC), which participates in various physiological activities
and plays an important role in maintaining and promoting human
health\textsuperscript{{[}1-3{]}}. VC is an essential nutrient that
cannot be synthesized by the human body, and must be obtained from foods
and supplements \textsuperscript{{[}4-5{]}}. However, the extreme
instability in aqueous solutions, especially under specific oxidation
conditions, directly reduces its biological activity, resulting in its
applications being greatly restricted \textsuperscript{{[}6-9{]}}. In
order to maintain the same biological activity and improve its
stability, various VC derivatives including ascorbyl phosphate, ascorbyl
sulfate \textsuperscript{{[}10-11{]}}, ascorbyl
palmitate\textsuperscript{{[}12-13{]}} and ascorbyl
glucoside\textsuperscript{{[}14-15{]}} have been chemically or
biologically synthesized, among which 2-O-\selectlanguage{greek}α-\selectlanguage{english}D-glucopyranosyl-L-ascorbic
acid (AA-2G) is extremely stable and nonreducible, which has attracted
considerable attention with its wide range of applications in cosmetics,
food, and medicine \textsuperscript{{[}16-17{]}}.

Enzymatic synthesis is a preferred method for AA-2G production over
alternative chemical synthesis owing to the regioselective glucosylation
reaction \textsuperscript{{[}16{]}}. Up to now, AA-2G has mainly been
synthesized by transglucosylation of L-AA catalyzed by several enzymes,
including \selectlanguage{greek}α-\selectlanguage{english}glucosidase \textsuperscript{{[}18-19{]}}, cyclodextrin
glycosyltransferase (CGTase) \textsuperscript{{[}20-21{]}},
amylase\textsuperscript{{[}22{]}}, sucrose
phosphorylase\textsuperscript{{[}23-24{]}}, and
\selectlanguage{greek}α-\selectlanguage{english}isomaltosylglucosaccharide synthase\textsuperscript{{[}25{]}}. Among
them, the synthesis of AA-2G by \selectlanguage{greek}α-\selectlanguage{english}glucosidase from higher eukaryote such
as rice seed and mammals has the advantages of less intermediates and
by-products\textsuperscript{{[}18, 26, 27{]}}. However, due to the low
efficiency and limited source of glycoside hydrolases, there are only a
few related reports in recent years \textsuperscript{{[}16, 28{]}}.

\selectlanguage{greek}\emph{α} -\selectlanguage{english}Glucosidase (AGL; EC 3.2.1.20), mainly classified into the
glycoside hydrolase families GH13 and GH31, represents a group of
exoglycosidases widely distributed in microorganisms, plants, and
animals, which can react with the \selectlanguage{greek}\emph{α} -\selectlanguage{english}glucosidic bond at the
nonreducing terminal of substrates and release \selectlanguage{greek}\emph{α}
-\selectlanguage{english}glucose\textsuperscript{{[}29{]}}. Under high substrate concentration,
GH31 AGL can also catalyze transglucosylation to synthesize
oligosaccharides or other \selectlanguage{greek}α-\selectlanguage{english}glucosylated compounds
\textsuperscript{{[}29-30{]}}. The glycosylated~hydroxyl group of
the~receptor of \selectlanguage{greek}α-\selectlanguage{english}glucosidase transglycosylation~activity~is
independent~on its~hydrolysis~specificity,~and~the substrate specificity
of GH 31 AGL~hydrolysis~activity~varies~with enzyme
source\textsuperscript{{[}31{]}}. It is possible to synthesize AA-2G by
glycoside hydrolases belonging to family GH31, because these are
retaining enzymes with the catalytic mechanism allowing them to act as
transferases. The pattern of transglycosylation products of glycoside
hydrolase mainly depends on the structure of the enzyme, especially its
catalytic center\textsuperscript{{[}32-34{]}}.

\emph{Pichia pastoris} is the excellent expression host with
well-established genetic tools and cultivation strategies for scale-up
producing heterologous proteins, particularly industrial enzymes and
biopharmaceuticals \textsuperscript{{[}35{]}}. Several AGL
from\emph{Aspergillus} species \textsuperscript{{[}28, 36, 37{]}}
and\emph{Apis cerana indica} \textsuperscript{{[}38{]}} have been
successfully expressed in \emph{P. pastoris} . The inducible alcohol
oxidase I promoter (P\textsubscript{AOX1}) and the constitutive
glyceraldehyde-3-phosphate dehydrogenase promoter (P\textsubscript{GAP})
are commonly used in genetic engineering of \emph{P. pastoris} . Unlike
P\textsubscript{AOX1}, P\textsubscript{GAP} simplifies the cultivation
by avoiding the use of toxic methanol as a carbon source and
inducer\textsuperscript{{[}39{]}}. In the present study, the AGL gene
from\emph{Oryza sativa} was codon-optimized and synthesized for
extracellular production of the rAGL in \emph{P. pastoris} under the
regulation of P\textsubscript{GAP}. The rAGL was characterized, and its
potential for AA2G production via transglycosylation of L-AA was
investigated.

\textbf{2 Experimental Section}

\textbf{2.1 Strains, media, and chemicals}

\emph{Escherichia coli} DH5\selectlanguage{greek}α \selectlanguage{english}(TransGen Biotech, Beijing, China)
and\emph{P. pastoris} GS115 (Invitrogen, Carlsbad, USA) were used as the
host strains for DNA cloning and protein expression, respectively. The
plasmid pPIC9K (Invitrogen, Carlsbad, USA) was selected as a backbone
vector to generate the recombinant expression vectors for \emph{P.
pastoris.}

\emph{E. coli} DH5\selectlanguage{greek}α \selectlanguage{english}was grown at 37\selectlanguage{ngerman}°C in Luria-Bertani (LB) medium (50
mg/mL Kanamycin supplemented when necessary). Yeast strains were
cultivated in the yeast extract peptone dextrose (YPD) medium, buffered
minimal glycerol (BMGY) medium, or minimal dextrose (MD) medium
following the pPIC9K manual. YPD medium containing different
concentrations of G418 sulfate (geneticin, Yuanye Bio-Technology,
Shanghai, China), which is applied to select geneticin resistant
strains.

FastPure Plasmid Mini Kit and Taq DNA polymerase were obtained from
Vazyme (Nanjing, China). The restriction enzymes were purchased from New
England Biolabs (Beverly, USA). The nucleotide sequences used in this
study were synthesized by Genscript (Nanjing, China). D-Glucose assay
kit based on the combined action of glucose oxidase (GOD) and peroxidase
(POD), was obtained from Yuanye Bio-Technology Co., Ltd (Shanghai,
China). AA-2G was purchased from Tokyo Chemical Industry Co. Ltd.
(Japan).

\textbf{2.2 Plasmid construction and transformation}

According to the \emph{P. pastoris} GS115~chromosome, the sequence of
P\textsubscript{GAP} (Supplementary Data 1) was synthesized and cloned
between \emph{Sac} I and \emph{Bam} HI restriction sites of pPIC9K,
generating the plasmid pGAP9K with P\textsubscript{GAP}. The AGL
(UniProtKB accession no. Q653V7) encoding sequence from the \emph{Oryza
sativa} subsp. japonica (Rice) was synthesized with optimized codons for
heterologous expression in \emph{P. pastoris} (Supplementary Data 1),
and inserted into the \emph{Eco} RI site of pGAP9K with a 6-histidine
tag at the \emph{N} -terminus to replace the original signal peptide of
AGL (1 - 33 amino acid residues), thus obtaining the plasmid pPIC9K-AGL.
(Fig. 1).

Figure 1

Plasmid transformations were performed according to a condensed
electroporation protocol previously described\textsuperscript{{[}40{]}}.
The plasmid pGAP9K-AGL was linearized with\emph{Psh} AI and introduced
into \emph{P. pastoris} GS115 by electroporation to facilitate
integration into the host genome. Multi-copy inserts of the
transformants were selected by MD plates and the grown clones were
transferred to YPD plates containing different concentrations of
geneticin (0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, and 4 mg/mL)
\textsuperscript{{[}41{]}}. The geneticin-resistant clones were
confirmed by Polymerase Chain Reaction (PCR) amplification with pairs of
primers listed in Supplementary Table 1, and the PCR-positive clones
were selected for the consequent expression tests. As a negative
control, transformation was also performed using only the \emph{Psh} AI
linearized pGAP9K vector.

\textbf{2.3 Expression of AGL in \emph{P. pastoris}}

Well-grown colonies were incubated in 5 mL of YPD medium (10 g/L yeast
extract, 20 g /L peptone, and 20 g/L glucose) and grew overnight at 30°C
and 200 rpm. Then, 0.5 mL of the culture was inoculated into 50 ml of
BMGY medium (2\% glycerol, 1\% yeast extract, 2\% peptone, 1.34\% yeast
nitrogen base w/o amino acids, 4 x 10\textsuperscript{-5} \% biotin and
100 mM sodium phosphate buffer, pH 6.0) in a 250-mL flask and incubated
at 30°C and 200 rpm. When the culture turbidity (OD\textsubscript{600})
reached approximately 18--20, it was centrifuged (2000 x \emph{g} ) for
10 min at 4 °C and the cell pellet was resuspended in 200 mL of
BMGY\textsuperscript{{[}42{]}}. The cells were cultured at 30 °C and 200
rpm for another 72 h. Glucose was added every 12 h to a final
concentration of 1\%. The cell culture (1 mL) was separated by
centrifugation at 6,000 x \emph{g} for 10 min and the supernatant was
kept at 4 °C for AGL activity assay.

The recombinant \emph{P. pastoris} with the highest yield of AGL was
used for scale-up fermentation in a 5-L fermenter. The fermentation
began in 2 L Basal Salt Medium (4\% glycerol, 26.7 mL 85\%
H\textsubscript{3}PO\textsubscript{4}, 0.93 g CaSO\textsubscript{4},
18.2 g K\textsubscript{2}SO\textsubscript{4}, 14.9 g
MgSO\textsubscript{4}·7H\textsubscript{2}O, 4.13 g KOH and 2 mL 5\%
antifoam) containing 4.35 mL/L PTM1 salt solution (2 g/L
CuSO\textsubscript{4}·5H\textsubscript{2}O, 7 g/L ZnCl\textsubscript{2},
0.08 g/L NaI, 22.0 g/L FeSO\textsubscript{4}.7H\textsubscript{2}O, 3.0
g/L MnSO\textsubscript{4}·7H\textsubscript{2}O, 0.2 g/L biotin, 0.2 g/L
Na\textsubscript{2}MoO\textsubscript{4}.2H\textsubscript{2}O, 0.02 g/L
boric acid, 0.5 g/L CoCl\textsubscript{2}, 2 mL
H\textsubscript{2}SO\textsubscript{4}) and lasted for about 16 h. Using
50\% glycerol (w/v) containing 12 mL/L of PTM1 as carbon sources, the
fed-batch fermentation of glycerol was initiated at 16 mL/L/h for 3 h.
In the subsequent fermentation, the feeding rate was kept at 10 mL/L/h
for 90 h. The temperature and pH during fermentation were maintained at
30 °C and 5.0, respectively. The dissolved oxygen was kept at around
20\% and was automatically adjusted by cascading agitation speed
(600--1000 rpm) \textsuperscript{{[}42{]}}. The cell cultures (10 mL)
were collected at the same time intervals, and the supernatants were
obtained by centrifugation at 6,000 x \emph{g} and 4 °C for 20 min for
the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
analysis \textsuperscript{{[}43{]}} and AGL activity determinations.

\textbf{2.4 Enzyme purification}

The culture supernatant was filtered with 0.45 \selectlanguage{greek}μ\selectlanguage{english}m filters before being
loaded on the column. Purification of the rAGL was performed with the
AKTA Prime Plus (GEHealthcare Bio-science, Uppsala, Sweden) using a 5-mL
high affinity Ni-Charged resin FF prepacked column (Genscript, Nanjing,
China) \textsuperscript{{[}44{]}}. After exchanging with sodium acetate
buffer (100 mM, pH 4.0) and concentrated using an
Amicon\selectlanguage{ngerman}\textsuperscript{®}~Ultra-15 Centrifugal~Filter Unit with an
Ultracel-30 membrane (Merck Millipore Ltd., Ireland), the elute solution
containing AGL was collected and stored at 4 °C for further experiments.
The molecular weight of rAGL and protein purity were determined by 8\%
(w/v) SDS-PAGE. The protein concentration was determined using the
Bradford method \textsuperscript{{[}45{]}}.

\textbf{2.5 Enzyme assay}

AGL activity was measured by the release of glucose from
maltose\textsuperscript{{[}46{]}}. Each reaction mixture (100 \selectlanguage{greek}μ\selectlanguage{english}L)
containing 25 mM maltose, 100 mM sodium acetate buffer (pH 4.0), and 10
\selectlanguage{greek}μ\selectlanguage{english}g purified enzyme (or 20 \selectlanguage{greek}μ\selectlanguage{english}L culture supernatant) was incubated for 10
min at 37 \selectlanguage{ngerman}°C. After heating at 100 °C for 7 min, the glucose of 10 \selectlanguage{greek}μ\selectlanguage{english}L
reaction mixture was assayed using the D-glucose GOD-POD colorimetric
assay kit\textsuperscript{{[}47-48{]}}. One activity unit was defined as
the amount of enzyme that liberate 2 \selectlanguage{greek}μ\selectlanguage{english}mol of glucose from maltose per
minute under the above assay conditions.

The pH optimum of rAGL was measured in the range of pH 3.0--7.0 using
100 mM sodium citrate buffer (pH 3.0--3.5), sodium acetate buffer (pH
4.0--5.5) and sodium phosphate buffer (pH 6.0--7.0), respectively. To
determine the pH stability, rAGL (2.12 mg/mL) was preincubated in the
various buffers described above at 4 \selectlanguage{ngerman}°C for 24 h and 48 h, and then the
residual activity was assayed under the standard assay
conditions\textsuperscript{{[}28{]}}.

The optimal temperature of rAGL was measured at pH 4.0 and temperatures
range from 37 °C to 70 °C, and maltose was used as substrate in 100 mM
acetate buffer. The thermal stability was determined by incubating rAGL
(2.12 mg/mL) in 100 mM acetate buffer (pH 4.0) at different temperatures
(30--70 °C). At each temperature, the buffer and rAGL were preincubated
for 15 min, 30 min, 45 min and 1 h, respectively. The reaction was
initiated by adding maltose, and allowed to proceed for another 10 min.
At different time intervals, samples were collected and the residual
activity was assayed under the standard assay
conditions\textsuperscript{{[}28, 37{]}}.

To determine the influence of metal ions (Cu\textsuperscript{2+},
Ca\textsuperscript{2+}, Mg\textsuperscript{2+}, Mn\textsuperscript{2+},
Zn\textsuperscript{2+}, Fe\textsuperscript{3+}, Fe\textsuperscript{2+})
on enzyme activity, rAGL (2.12 mg/mL) was preincubated with each metal
ion (10 mM) in 100 mM acetate buffer (pH 4.0) at 37 °C for 1 h. The
residual enzyme activity was detected at 37 °C.

For determination of the Michaelis-Menten constants of rAGL, reactions
were carried out under the standard assay conditions with di\selectlanguage{english}fferent
concentrations of maltose (5--100 mM). After incubation for 10 min, the
reaction mixture was heated to 100 degC for 7 min and analyzed by the
GOD-POD method. On the basis of Michaelis-Menten kinetic model,
the\emph{K\textsubscript{m}} value of rAGL for maltose was determined by
non-linear curve-fitting.

All reactions were conducted in triplicate. The relative activity (\%)
was calculated in terms of that of the maximum activity (100\%).

\textbf{2.6 Transglycosylation catalyzed by rAGL}

The transglycosylation reaction was performed as previously described
with modification \textsuperscript{{[}18{]}}. Briefly, the reaction
mixture (0.3 mL) consisted of 267 mM maltose, 178 mM ascorbic acid
sodium salt (AA*Na), 13 mM thiourea, 1.12 U of rAGL (in 150 \selectlanguage{greek}μ\selectlanguage{english}L culture
supernatant) , and sodium acetate buffer (100 mM, pH 4.0), and was
incubated in the dark at 37 \selectlanguage{ngerman}°C for 1--6 h, unless otherwise specified.
The samples taken every hour were mixed with 600 \selectlanguage{greek}μ\selectlanguage{english}L of 1.06\% (w/v)
metaphosphoric acid to stop the reaction, and the solutions were
centrifuged at 12000 \selectlanguage{ngerman}× \emph{g} for 10 min. The supernatant was
collected and stored at -20 °C waiting for high performance liquid
chromatography (HPLC) analysis. On the basis of the initial reaction
condition, that is, the reaction last for 6 h at 37 °C, pH 4.0, with the
molar ratio of maltose to AA·Na set to 3:2, the in\selectlanguage{english}fluence of reaction
temperature (30, 37, 40, 45, and 50 degC), pH (100 mM sodium citrate
buffer at pH 3.0--3.5 and 100 mM sodium acetate buffer at pH 4.0--5.5),
and maltose/AA*Na molar ratio (1:1, 3:1 and 3:2) on the synthesis of
AA-2G catalyzed by rAGL was investigated. Besides, the influence of
enzyme concentrations on AA-2G synthesis was also investigated. The
reaction mixture was scaled up to 10 mL, containing 267 mM maltose, 267
mM AA*Na, 13 mM thiourea, various concentrations of rAGL (1, 2, 3 and 4
U/mL) , and sodium acetate buffer (100 mM, pH 4.0), and was incubated in
the dark with shaking at 37 degC for 1--8 h. All samples (200 \selectlanguage{greek}μ\selectlanguage{english}L)
collected each hour were mixed with 600 \selectlanguage{greek}μ\selectlanguage{english}L of 1.06\% (w/v)
metaphosphoric acid to stop the reaction. Other treatments to the
samples were the same as the above.

\textbf{2.7 HPLC analysis}

The above-mentioned supernatants were thawed at room temperature. After
proper dilution, the filtered samples were analyzed on an Agilent 1260
HPLC system (Agilent Technologies, The Netherlands), using an Agilent
TC-C18 column (150 mm x 4.6 mm; Agilent Technologies), maintaining at 25
\selectlanguage{ngerman}°C with UV detection at 238 nm. The \selectlanguage{english}flow rate was 0.8 mL/min using 1\%
HPLC grade methanol (pH adjusted to 2 with phosphoric acid) as the
mobile phase. The injection volume was 10 \selectlanguage{greek}μ\selectlanguage{english}L. The AA-2G concentration
was calculated on the basis of peak area of the standard samples.

\textbf{3 Results}

\textbf{3.1 Cloning and expression of rAGL in \emph{P. pastoris}}

The coding region derived from \emph{O. sativa} AGL excluding the
fragment of its original signal peptide but with an \emph{N} -terminal
6-histidine tag, was synthesized and cloned into the plasmid pGAP9K,
which was fused with \selectlanguage{greek}α-\selectlanguage{english}Factor signal sequence of the plasmid for
secretion in \emph{Pichia} under the control of P\textsubscript{GAP}.
Subsequently, the genomic integration was carried out by transformation
of the linearized plasmid DNA that was digested using \emph{Psh} AI,
into\emph{P. pastoris} GS115. The transformed cells were spread on MD
agar plates. Positive transformants containing high copy number of AGL
gene were obtained through screening on YPD plates with different
concentrations of geneticin. As a result, 12 clones were selected from
the plates containing 4 mg/mL geneticin, among which four clones were
confirmed by colony PCR with four sets of primers (Supplementary Figure
1) and selected for the subsequent expression experiments.

Expression levels of four selected clones were tested in the shake flask
culture. Samples were collected every 12 h, and protein concentration
and enzyme activity in the culture supernatant were detected. The growth
profiles of cell mass (OD\textsubscript{600}) for the four recombinant
clones were similar and could reach the highest value of 58.4\selectlanguage{ngerman}±3.7 at 72
h, when the one having the maximum protein concentration (0.114 mg/mL)
and enzyme activity (1.48 U/mL) was used for scale-up fermentation and
rAGL production.

\textbf{3.2 High-density fermentation in a 5-L fermenter}

A scale-up fermentation was carried out in a 5-L fermenter, and samples
were collected every 6 h to analyze OD\textsubscript{600}, total protein
concentration, and enzyme activity. The whole process of high-density
fermentation lasted for 108 h. As is showed in Fig. 2, in the fed-batch
with glycerol as carbon source, OD\textsubscript{600} increased
continuously, reaching the highest value of 716±15 at 102 h, and then
decreased, which may be due to the fact that the dissolved oxygen can
not maintain the growth of such high density cells under the
experimental conditions. In the 102-h sample, the total protein
concentration and AGL activity in the culture supernatant were as high
as 0.732 mg/mL and 9.11 U/mL, respectively, which exhibited the same
growth trend as cell mass.

Figure 2

\textbf{3.3 Purification of rAGL and kinetic analysis}

\emph{N} -terminal his-tagged rAGL in culture supernatant was purified
by Ni-NTA affinity chromatography. The details of purification are shown
in Table 1. At 37 °C and pH 4.0, the specific activity of rAGL is 49.83
U/ mg. In SDS-PAGE, rAGL with an \emph{N} -terminal 6-histidine showed a
band of roughly 100 kDa (Fig. 3).

The kinetic parameters of rAGL were analyzed using maltose as the
substrate at 37 °C and pH 4.0 (Supplementary Figure 2). As a result, the
kinetic constants \emph{K\textsubscript{m}} ,
\emph{V\textsubscript{max}}and \emph{K\textsubscript{cat}} of the rAGL
were found to be 61 mM, 184.64 U/mg, and 146.70 1/s, respectively.

Table 1

Figure 3

\textbf{3.4 Characterisation of biochemical properties}

The pH effect on rAGL activity was shown in Fig. 4a. rAGL activity
reached its maximum at pH 4.0. Under the standard condition, it showed
high enzymatic activity (\textgreater{}90\% of maximum value) between pH
3.0 and pH 5.0. Furthermore. rAGL was stable at pH 3.0--4.5, and
retained 99\% and 97\% activity after incubation at pH 4.0 for 48 h and
72 h, respectively (Fig. 4b).

The optimum temperature for rAGL was 65 °C, and it has high activity
between 60 °C and 65 °C (Fig.4c). rAGL was stable between 37 °C and 55
°C, and remained more than 80\% activity after 60 min of incubation
(Fig.4d). However, when the temperature was kept at 65 °C for 15 min,
rAGL lost 50\% of its activity.

In this study, the metal requirement of rAGL was investigated using 10
mM metal ions (Cu\textsuperscript{2+}, Ca\textsuperscript{2+},
Mg\textsuperscript{2+}, Mn\textsuperscript{2+}, Zn\textsuperscript{2+},
Fe\textsuperscript{3+} and Fe\textsuperscript{2+}). rAGL was
pre-incubated with each metal ion, and its activity was gauged. As is
displayed in Fig.5, none of these metals significantly affected rAGL
activity, which suggested that this enzyme is insensitive to the metal
ions under the experimental conditions.

Figure 4

Figure 5

\textbf{3.5 Formation of AA-2G catalyzed by rAGL}

In the present, rAGL from \emph{O. sativa} catalyzed the glycosylation
of AA·Na to produce AA-2G, a stable L-ascorbic acid derivative. The
proceeding of AA-2G synthesis was monitored by HPLC. As is shown in Fig.
6, using maltose as the glycosyl donor, the influences of pH,
temperature, substrate ratio and enzyme concentration on AA-2G
production catalyzed by rAGL were studied.

Among the pH range of 3.0 to 5.5 (Figure 6a), the product concentration
in the reaction mixture increased obviously in the first 3 h, and then
declined possibly because of the hydrolytic activity of rAGL. At pH 4.0,
the highest yield of AA-2G (6.73±0.02 g/L) was obtained within 3 h, and
after 3 h, the concentration of AA-2G decreased faster at pH 3.0 than at
other pH values.

As is indicated in Fig. 6b, the optimum temperature of glycosylation
reaction appears to be 37 °C, because the concentration of AA-2G remains
higher than other conditions within 6 h. Among the different reaction
temperatures, decrease of the AA-2G concentration was observed from the
beginning (reaction of 1 h) at 50 °C, probably degradation of the
substate AA·Na at high temperature is a factor that can not be ignored.
At 30 °C, AA-2G increased in 2 h, and then remained basically unchanged
within the next 4 h.

As for the influence of the molar ratio of maltose/AA·Na on the
synthesis of AA-2G (Fig. 6c), when maltose is kept at 267 mM in the
initial reaction system, the highest product concentration can increase
with the rise of AA·Na concentration (from 89 mM to 267 mM). Excessive
maltose increased the reaction rate and affected the yield from AA·Na.
In the case that the maltose/AA·Na molar ratio was 1:1, the AA-2G
concentration in the reaction mixture topped 8.7±0.4 g/L after reaction
for 3 h, and the yield from AA·Na was 9.7\%. The higher yield (10.6\%)
was achieved in 2 h at the maltose/AA·Na ratio of 3:2, although the
AA-2G concentration was only 6.4±0.1 g/L.

The enzyme concentration has an effect on the reaction rate as well, as
was presented in Fig. 6d. The formation patterns of AA-2G are similar
that the product increased firstly until it approaches the high summit,
and then the reaction decreased, but with the increase of enzyme amount,
the time to reach the highest concentration was shortened. Under the
condition of 4 U/mL rAGL, 7.5±0.3 g/L of AA-2G can be produced within 2
h, but its hydrolysis rate was also higher than other reactions with
lower enzyme concentration. The highest yield of AA-2G (8.3±0.7 g/L) was
reached in 5 h of the reaction with 2 U/mL of rAGL.

Figure 6

\textbf{4 Discussion}

A large number of AGLs were found in mammals, plants, and
microorganisms. Regarding the production of rAGL, up to now, most
bacterial enzymes from \emph{Sulfolobus
tokodaii}\textsuperscript{{[}49{]}}, \emph{Thermoplasma
acidophilum}\textsuperscript{{[}50{]}}, \emph{Pyrobaculum
aerophilum}\textsuperscript{{[}51{]}}, and \emph{Bifidobacterium
longum}\textsuperscript{{[}52{]}}, were overexpressed in \emph{E. coli}
, and several AGLs from plants and fungi like
barley\textsuperscript{{[}48{]}}, Rice
\textsuperscript{{[}53{]}},\emph{Aspergillus niger}
\textsuperscript{{[}54{]}}, were successfully produced in \emph{P.
pastoris} . In the previous work, efforts have been made to produce the
rAGL of glycoside hydrolase family GH31 from higher plants in \emph{E.
coli} , but resulted in no or rather low enzyme activity
\textsuperscript{{[}55-56{]}}. It was not until 2006 that the AGL from
barley (\emph{Hordeum vulgare} ) with fully activity was produced in
\emph{P. pastoris} for the first time\textsuperscript{{[}47-48{]}}.
Since then, \emph{P. pastoris} system that is widely used to produce
heterologous proteins from various organisms involving viruses,
bacteria, fungi, plants, animals and humans\textsuperscript{{[}57{]}},
also greatly promotes the heterologous production of AGL (Table 2).

Table 2

Previously, when two isoforms of ONG2 were purified from dry rice seeds,
native AGL from \emph{O. sativa} has been
characterized\textsuperscript{{[}53, 58{]}}\emph{.} In this study, rAGL
corresponding to the ONG2 was found only one bandlarger than 97.2 KDa
(Fig. 3), probably due to the lack of post-translational proteolysis
occurring in\emph{Pichia} . The \emph{K\textsubscript{m}} value of rAGL
for maltose was 61 mM, which indicates that the affinity to maltose is
lower than that of native ONG2-I and ONG2-II, which are 2.1 mM and 2.3
mM respectively. As well, rAGL appeared to possess lower specific
activity (49.83 U/mg) than that of the ONG2-I (122U/mg) and ONG2-II (124
U/mg), although the assay conditions were not completely the same.
Comparison of biochemical characteristics of native AGLs from \emph{O.
sativa} and recombinant enzyme from \emph{P. pastoris} indicated that
they had similar optimal pH and remained active in a wide pH range.
Native AGLs in plants prefer to be close to the temperature of the
growing environment. However, rAGL was stable at up to 55 °C and the
highest optimal temperature (65 °C) of enzyme activity, which
demonstrates better thermal stability than native informs, which was
stable as high as 40 °C, and completely lost its activity after
incubation at 60 °C for 10 min. It should be noted that thermal
stability of rAGL is an important factor related to its potential
applications in biotechnology.

Thanks to the transglycosylation activity, AGLs are potentially to be
effectively useful in the preparation of AA-2G (Fig. 7). The production
of AA-2G was facilitated under optimized conditions (40°C and pH 6.0) by
a truncated AGL from \emph{A. niger} and the highest yield of AA-2G
reached 0.528±0.04 g/L \textsuperscript{{[}19{]}}. As far as we know,
the yield of AA-2G in the present study represents the highest by AGLs
reported so far, reaching 8.7±0.4 g/L at 37 °C and pH 4.0. Among the
results of reactions, a typical `n'-shaped kinetic profile of the AA-2G
formation was observed. By understanding the glucosidase-catalyzed
glycosylation process in water-rich
environment\textsuperscript{{[}59{]}}, it can be concluded that
glycosylation is dominant at the beginning of reaction, which leads to
rapid synthesis of AA-2G. At the same time, the synthesized AA-2G
started to degrade with time. In the batch system, the AA conversion is
thermodynamically restricted, and the catalytic efficiency is mainly
impacted by the selectivity of AGL's glycosylation/hydrolysis. In the
case of the different maltose/AA·Na ratios (Fig. 6c), it can be
concluded that high glycosyl donor (maltose) content helped to slow down
the rate of hydrolysis and therefore have an influence on the catalytic
efficiency. However, the amount of rAGL only has the effect on the
reaction rate but does not affect the equilibrium of glycosylation and
hydrolysis (Fig. 6d). In order to maximize the product accumulation, the
decomposition of AA at inappropriate pH and temperatures should never be
neglected (Fig. 6a and 6b).

Figure 7

In summary, AGL from \emph{O. sativa} was expressed in \emph{P.
pastoris} , which proved to be beneficial in fermentation process,
allowing the production of enough enzyme and promoting enzymatic
synthesis of AA-2G by AGLs. To date, the full potential of rAGL has not
been fully realized in this study and further work is needed to improve
enzyme activity for transglycosylation, such as controlling the
conditions of glycosylation reactions \textsuperscript{{[}60{]}} and
enzyme technology to create a glycosynthase \textsuperscript{{[}61{]}}.
These studies will provide the basis for the application of rAGL in
AA-2G industrial production.

\textbf{Acknowledgments}

This study was funded by the NSFC (21878155), Qing Lan Project of
Jiangsu Universities, and Six Talent Peaks Project in Jiangsu Province,
the Jiangsu Synergetic Innovation Center for Advanced Bio-manufacture,
and PAPD.

\textbf{Author statement}

\textbf{Yan Li:} Conceptualization, Methodology, Supervision, Funding
acquisition

\textbf{Xuelian Qi:} Investigation, Writing - Original Draft,
Writing-review and editing

\textbf{Honghua Jia:} Resources, Project administration, Writing -
Review \& Editing Preparation

\textbf{Junlan Shao} : Investigation, Data curation

\textbf{Yinchu Chen:} Investigation, Visualization

\textbf{Xiaoying He:} Investigation\selectlanguage{english},Writing - Review

\textbf{Ming Yan:} Supervision, Resources

\textbf{Declaration of competing interest}

None

\textbf{References}

{[}1{]} S. Chambial, S. Dwivedi, K.K. Shukla, P.J. John, P. Sharma,
Indian J Clin Biochem 2013, 28, 314.

{[}2{]} S. Mousavi, S. Bereswill, M.M. Heimesaat, Eur J Microbiol
Immunol (Bp) 2019, 9, 73.

{[}3{]} J. Teng, A. Pourmand, M.M. Amirshahi, J Crit Care 2018, 43, 230.

{[}4{]} S.C. Rumsey, M. Levine, J Nutr Biochem 1998, 9, 116.

{[}5{]} M.C.D. Tullio, Nature Education 2010, 3, 48.

{[}6{]} S.H. LEE,T.P. LABUZA, J Food Sci 1975, 40, 370.

{[}7{]} M.H.Eison-Perchonok., T.W.Downes, J Food Sci 1982, 47, 765.

{[}8{]} A.M Rojas, L.N Gerschenson, J Sci Food Agr 1997, 74, 369.

{[}9{]} A.L. Herbig, C.M.G.C, Renard, Food Chem 2017, 220, 444.

{[}10{]} C.G.Mead, F.J. Finamore, Biochemistry 1969, 8, 2652.

{[}11{]} Ascorbic acid sulfate (AAS): A metabolite of ascorbic acid with
antiscorbutic activity. Nutrition Reviews 1973, 31, 251.

{[}12{]} H.J. Hsieh, G.R. Nair, W.T. WU, J Agric Food Chem 2006, 54,
5777.

{[}13{]} M.G. Yadav, M.R. Kavadia, R.N. Vadgama, A.A. Odaneth, A.M.
Lali, Appl Biochem Biotechnol 2018, 184, 1168.

{[}14{]} N. Muto, T.Nakamura, I. Yamamoto, J Biochem 1990, 107, 222.

{[}15{]} H.K. Jun, K.M. Bae, S.K. Kim, Biotechnol Lett 2001, 23, 1793.

{[}16{]} R. Han, L. Liu, J. Li, G. Du, J. Chen, Appl Microbiol
Biotechnol 2012, 95, 313.

{[}17{]} Y. Hanada, A. Iomori, R. Ishii, E. Gohda, A. Tai, Biosci
Biotechnol Biochem 2014, 78, 301.

{[}18{]} N. Muto, S. Suga, K. Fujii, K. Goto, I. Yamamoto, Agricultural
and Biological Chemistry 1990, 54, 1697.

{[}19{]} Z. Li, H. Han, B. Wang, J. Gao, B. Zhu, R. Peng, Q. Yao, J Food
Biochem 2017, 41.

{[}20{]} Y. Jiang, J. Zhou, R. Wu, F. Xin, W. Zhang, Y. Fang, J. Ma, W.
Dong, M. Jiang, BMC Biotechnol 2018, 18, 53.

{[}21{]} X. Tao, T. Wang, L. Su, J. Wu, J Agric Food Chem 2018, 66,
9052.

{[}22{]} S.B. Lee, K.C. Nam, S.J. Lee, J.H. Lee, K. Inouye, K.H. Park,
Biosci Biotechnol Biochem 2004, 68, 36.

{[}23{]} T. Kwon, C.T. Kim, J.H. Lee, Biotechnol Lett 2007, 29, 611.

{[}24{]} Y. Li, Z. Li, X. He, L. Chen, Y. Cheng, H. Jia, M. Yan, K.
Chen, J Biotechnol 2019, 305, 27.

{[}25{]} M. Kubota, K. Tsusaki, T. Higashiyama, S. Fukuda, T. Miyake,
Patent application, 2007.

{[}26{]} I. Yamamoto, N. Muto, K. Murakami, S. Suga, H. Yamaguchi, Chem
Pharm Bull (Tokyo) 1990, 38, 3020.

{[}27{]} I. Yamamoto, N. Muto, E. Nagata, T. Nakamura, Y. Suzuki,
Biochim Biophys Acta 1990, 1035, 44.

{[}28{]} D.L. Chen, X. Tong, S.W. Chen, S. Chen, D. Wu, S.G. Fang, J.
Wu, J. Chen, J Agric Food Chem 2010, 58, 4819.

{[}29{]} M. Okuyama, W. Saburi, H. Mori, A. Kimura, Cell Mol Life Sci
2016, 73, 2727.

{[}30{]} M. Okuyama, Biosci Biotechnol Biochem 2011, 75, 2269.

{[}31{]} M. Sugimoto, S. Furui, K. Sasaki, Y. Suzuki, Biosci Biotechnol
Biochem 2003, 67, 1160.

{[}32{]} S. Mala, H. Dvorakova, R. Hrabal, B. Kralova, Carbohyd Res
1999, 322, 209.

{[}33{]} K.M. Song, M. Okuyama, K. Kobayashi, H. Mori, A. Kimura, Biosci
Biotechnol Biochem 2013, 77, 2117.

{[}34{]} M. Casa-Villegas, J. Marin-Navarro, J. Polaina, ACS Omega 2017,
2, 8062.

{[}35{]} V. Looser, B. Bruhlmann, F. Bumbak, C. Stenger, M. Costa, A.
Camattari, D. Fotiadis, K. Kovar, Biotechnol Adv 2015, 33, 1177.

{[}36{]} S. Kumar, S. Mutturi, Enzyme Microb Technol 2020, 141, 109653.

{[}37{]} M. Ma, M. Okuyama, T. Tagami, A. Kikuchi, P. Klahan, A. Kimura,
J Agric Food Chem 2019, 67, 3380.

{[}38{]} J. Kaewmuangmoon, M. Kilaso, U. Leartsakulpanich, K. Kimura, A.
Kimura, C. Chanchao, BMC Biotechnol 2013, 13, 16.

{[}39{]} A. Zhang, J. Luo, T. Zhang,Y. Pan, Y. Tan, C. Fu, F. Tu, Mol
Biol Rep 2009, 36, 1611.

{[}40{]} J. Lin-Cereghino, W.W. Wong, S. Xiong, W. Giang, L.T. Luong, J.
Vu, S.D. Johnson, G.P. Lin-Cereghino, Biotechniques 2005, 38, 44.

{[}41{]} C.A. Scorer, J.J. Clare, W.R. McCombie, M.A. Romanos, K.
Sreekrishna, Biotechnology (N Y) 1994, 12, 181.

{[}42{]} M. Karaoglan, F.E. Karaoglan, M. Inan, Protein Expr Purif 2016,
121, 112.

{[}43{]} H.Schagger, G. von Jagow, Anal Biochem 1987, 166, 368.

{[}44{]} L. Chen, R. Cai, J. Weng, Y. Li, H. Jia, K. Chen, M. Yan, P.
Ouyang, Microb Biotechnol 2020, 13, 974.

{[}45{]} M.M. Bradford, Anal Biochem 1976, 72, 248.

{[}46{]} E.H. Muslin, A.M. Kanikula, S.E. Clark, C.A. Henson, Protein
Expr Purif 2000, 18, 20.

{[}47{]} T.P. Frandsen, F. Lok, E. Mirgorodskaya, P. Roepstorff, B.
Svensson, Plant Physiol 2000, 123, 275.

{[}48{]} H. Naested, B. Kramhoft, F. Lok, K. Bojsen, S. Yu, B. Svensson,
Protein Expr Purif 2006, 46, 56.

{[}49{]} J.E. Park, S.H. Park, J.Y. Woo, H.S. Hwang, J. Cha, H. Lee, J
Microbiol Biotechnol 2013, 23, 56.

{[}50{]} I. Park, H. Lee, J. Cha, Biotechnol Lett 2014, 36, 789.

{[}51{]} H. Jeon, H. Lee, D. Byun, H. Choi, J.-H. Shim, Food Sci
Biotechnol 2015, 24, 175.

{[}52{]} N.R. Kim, D.W. Jeong, D.S. Ko, J.H. Shim, Int J Biol Macromol
2017, 99, 594.

{[}53{]} H. Nakai, T. Ito, M. Hayashi, K. Kamiya, T. Yamamoto, K.
Matsubara, Y.M. Kim, W. Jintanart, M. Okuyama, H. Mori, S. Chiba, Y.
Sano, A. Kimura, Biochimie 2007, 89, 49.

{[}54{]} X. Liu, D. Wu, J. Wu, J. Chen, World J Microbiol Biotechnol
2013, 29, 533.

{[}55{]} B.K. Tibbot, C.A. Henson, R.W. Skadsen, Plant Mol Biol 1998,
38, 379.

{[}56{]} M.A. Taylor, H.A. Ross, D. McRae, F. Wright, R. Viola, H.V.
Davies, Planta 2001, 213, 258.

{[}57{]} J.L. Cereghino, J.M. Cregg, FEMS Microbiol Rev 2000, 24, 45.

{[}58{]} H. Nakai, S. Tanizawa, T. Ito, K. Kamiya, Y.M. Kim, T.
Yamamoto, K. Matsubara, M. Sakai, H. Sato, T. Imbe, M. Okuyama, H. Mori,
Y. Sano, S. Chiba, A. Kimura, J Biochem 2007, 142, 491.

{[}59{]} H. Chen, S. Yang, A. Xu, R. Jiang, Z. Tang, J. Wu, L. Zhu, S.
Liu, X. Chen, Y. Lu, Appl Microbiol Biotechnol 2019, 103, 9423.

{[}60{]} H. Chen, X. Jin, L. Zhu, Y. Lu, Z. Ma, S. Liu, X. Chen, Appl
Microbiol Biotechnol 2020, 104, 9523.

{[}61{]} M. Yang, G.J. Davies, B.G. Davis, Angew Chem Int Ed Engl 2007,
46, 3885.

\textbf{Tables}

Table 1 Summary of puri\selectlanguage{english}fication of rAGL from the recombinant \emph{P.
pastoris} .\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Step\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Total protein (mg)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Total activity (U)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Specific activity (U/mg)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Yield (\%)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Culture supernatant\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
208.8\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2653.85\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12.71\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
100\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Ni-NTA column purification\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12.69\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
632.34\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
49.83\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
23.8\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Table 2 rAGLs of glycoside hydrolase family GH31 produced in \emph{P.
pastoris} .\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Origin & Promoter & Enzyme activity \textsuperscript{a} & Enzyme
activity \textsuperscript{a} & References\tabularnewline
\midrule
\endhead
& & Culture supernatant & Purified rAGL &\tabularnewline
\emph{A. niger} & P\textsubscript{AOX1} & 2.07 U/mL; 2.21 U/mg & 2.52
U/mg & {[}28{]}\tabularnewline
\emph{H. vulgare} & P\textsubscript{AOX1} & 1 U/mg & 25.6 U/mg &
{[}48{]}\tabularnewline
\emph{Podospora anserina} & P\textsubscript{GAP} & 1.03 U/mL & 41.1 U/mg
& {[}33{]}\tabularnewline
\emph{A. niger} & P\textsubscript{GAP} & n.a. & 33.1 U/mg &
{[}37{]}\tabularnewline
\emph{O. sativa} & P\textsubscript{GAP} & 9.11 U/ mL; 12.71 U/mg & 49.83
U/mg & This study\tabularnewline
\bottomrule
\end{longtable}

Note: a) \textsuperscript{a} Maltose is used as the substrate for
activity assay; b) M.W., molecular weight; c) n.a., not available.

\textbf{Figure legends}

\textbf{Figure 1 The plasmid construct used for extracellular expression
of rAGL.}

\textbf{Figure 2} \textbf{Fermentation curves of the recombinant\emph{P.
pastoris} in a 5-L fermenter, including optical density}
(^),\textbf{protein concentration} (#), \textbf{and enzyme activity}
(*).

\textbf{Figure 3 SDS-PAGE analysis of the extracellular rAGL expressed
in the engineered \emph{P. pastoris}.} Lane M, molecular weight markers;
Lane 1, culture supernatant; Lane 2, purified rAGL. The band
corresponding to rAGL is indicated with an arrow.

\textbf{Figure 4 Effects of pH and temperature on the activity and
stability of rAGL} .

\textbf{Figure 5} \textbf{Effect of metal ions on the activity of rAGL.}

\textbf{Figure 6 Influence of the factors including the reaction
pH}(\textbf{A} ), \textbf{temperature} \textbf{(B)} ,
\selectlanguage{ngerman}\textbf{maltose/AA·Na molar ratio (C) and enzyme concentration (D) on
AA-2G production.}

\textbf{Figure 7 Synthesis of AA-2G by rAGL with maltose and AA as the
substrates.}

\textbf{Figures}

\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}

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/413834/articles/522021-extracellular-expression-and-characterization-of-an-%CE%B1-glucosidase-from-oryza-sativa-and-its-transglycosylation-for-synthesis-of-2-o-%CE%B1-d-glucopyranosyl-l-ascorbic-acid}

\textbf{Figure 3}

\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 4}

\textbf{A}

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/413834/articles/522021-extracellular-expression-and-characterization-of-an-%CE%B1-glucosidase-from-oryza-sativa-and-its-transglycosylation-for-synthesis-of-2-o-%CE%B1-d-glucopyranosyl-l-ascorbic-acid}

\textbf{B}

\textbf{Hosted file}

\verb`image5.emf`  available at \url{https://authorea.com/users/413834/articles/522021-extracellular-expression-and-characterization-of-an-%CE%B1-glucosidase-from-oryza-sativa-and-its-transglycosylation-for-synthesis-of-2-o-%CE%B1-d-glucopyranosyl-l-ascorbic-acid}



\textbf{C}

\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/413834/articles/522021-extracellular-expression-and-characterization-of-an-%CE%B1-glucosidase-from-oryza-sativa-and-its-transglycosylation-for-synthesis-of-2-o-%CE%B1-d-glucopyranosyl-l-ascorbic-acid}

\textbf{D}

\textbf{Hosted file}

\verb`image7.emf`  available at \url{https://authorea.com/users/413834/articles/522021-extracellular-expression-and-characterization-of-an-%CE%B1-glucosidase-from-oryza-sativa-and-its-transglycosylation-for-synthesis-of-2-o-%CE%B1-d-glucopyranosyl-l-ascorbic-acid}

\textbf{Figure 5}

\textbf{Hosted file}

\verb`image8.emf`  available at \url{https://authorea.com/users/413834/articles/522021-extracellular-expression-and-characterization-of-an-%CE%B1-glucosidase-from-oryza-sativa-and-its-transglycosylation-for-synthesis-of-2-o-%CE%B1-d-glucopyranosyl-l-ascorbic-acid}

\textbf{Figure 6}

\textbf{A}

\textbf{Hosted file}

\verb`image9.emf`  available at \url{https://authorea.com/users/413834/articles/522021-extracellular-expression-and-characterization-of-an-%CE%B1-glucosidase-from-oryza-sativa-and-its-transglycosylation-for-synthesis-of-2-o-%CE%B1-d-glucopyranosyl-l-ascorbic-acid}

\textbf{B}

\textbf{Hosted file}

\verb`image10.emf`  available at \url{https://authorea.com/users/413834/articles/522021-extracellular-expression-and-characterization-of-an-%CE%B1-glucosidase-from-oryza-sativa-and-its-transglycosylation-for-synthesis-of-2-o-%CE%B1-d-glucopyranosyl-l-ascorbic-acid}

\textbf{C}

\textbf{Hosted file}

\verb`image11.emf`  available at \url{https://authorea.com/users/413834/articles/522021-extracellular-expression-and-characterization-of-an-%CE%B1-glucosidase-from-oryza-sativa-and-its-transglycosylation-for-synthesis-of-2-o-%CE%B1-d-glucopyranosyl-l-ascorbic-acid}

\textbf{D}

\textbf{Hosted file}

\verb`image12.emf`  available at \url{https://authorea.com/users/413834/articles/522021-extracellular-expression-and-characterization-of-an-%CE%B1-glucosidase-from-oryza-sativa-and-its-transglycosylation-for-synthesis-of-2-o-%CE%B1-d-glucopyranosyl-l-ascorbic-acid}

\textbf{Figure 7}

\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

\end{center}
\end{figure}

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