Enzymatic glycosylation of indoxyglycosides catalyzed by a novel maltose phosphorylase from Emticicia oligotrophica

Article abstract of DOI:10.1080/07328303.2016.1238479

Maltose phosphorylases (EC 2.4.1.8) catalyze the reversible conversion of maltose to glucose and glucose-1-phosphate in the presence of inorganic phosphate. Herein, we describe for the first time the use of a maltose phosphorylase for the synthesis of various anomerically modified diglycosides. The maltose phosphorylase used was isolated from the bacterium Emticicia oligotrophica and showed a high selectivity towards the phosphorolysis of maltose, whereas no phosphorolysis was observed using other glucose-containing disaccharides such as cellobiose, melibiose, sucrose and trehalose. The addition of glucose to various 5-bromo-4-chloro-3-indolyl-glycosides (X-sugars) was used to evaluate the promiscuity of the maltose phosphorylase, and product formation was verified by LC-ESI-MS and MALDI-TOF-MS. The simple expression and purification protocol and the use of maltose as an inexpensive starting material make this maltose phosphorylase from Emticicia oligotrophica a valuable novel biocatalyst for the synthesis of glucose-containing glycosides.

Full text of DOI:10.1080/07328303.2016.1238479

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
Enzymatic glycosylation of indoxyglycosides
catalyzed by a novel maltose phosphorylase from
Emticicia oligotrophica
Faisal Nureldin Awad, Anna Kulinich, Ming Jun Yao, Xu Chu Duan, Zhi Peng
Cai, Bin Gu, Li Liu & Josef Voglmeir
To cite this article: Faisal Nureldin Awad, Anna Kulinich, Ming Jun Yao, Xu Chu Duan, Zhi
Peng Cai, Bin Gu, Li Liu & Josef Voglmeir (2016): Enzymatic glycosylation of indoxyglycosides
catalyzed by a novel maltose phosphorylase from Emticicia oligotrophica, Journal of
Carbohydrate Chemistry, DOI: 10.1080/07328303.2016.1238479
To link to this article: http://dx.doi.org/10.1080/07328303.2016.1238479
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Date: 11 November 2016, At: 11:03

JOURNAL OF CARBOHYDRATE CHEMISTRY
, VOL. , NO. , –
http://dx.doi.org/./..
Enzymatic glycosylation of indoxyglycosides catalyzed by a
novel maltose phosphorylase from Emticicia oligotrophica
Faisal Nureldin Awada,b, Anna Kulinicha, Ming Jun Yaoa, Xu Chu Duana,
a
Zhi Peng Caia, Bin Gua, Li Liua,c, and Josef Voglmeir
aGlycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and
Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China; bNational Food Research
Centre, Khartoum North, Sudan; cQlyco Ltd., Nanjing, People’s Republic of China
ABSTRACT
ARTICLE HISTORY
Received  March 
Accepted  September 
Maltose phosphorylases (EC 2.4.1.8) catalyze the reversible con-
version of maltose to glucose and glucose-1-phosphate in the
presence of inorganic phosphate. Herein, we describe for the
first time the use of a maltose phosphorylase for the synthe-
sis of various anomerically modified diglycosides. The maltose
phosphorylase used was isolated from the bacterium Emticicia
oligotrophica and showed a high selectivity towards the phos-
phorolysis of maltose, whereas no phosphorolysis was observed
using other glucose-containing disaccharides such as cellobiose,
melibiose, sucrose and trehalose. The addition of glucose to var-
ious 5-bromo-4-chloro-3-indolyl-glycosides (X-sugars) was used
to evaluate the promiscuity of the maltose phosphorylase,
and product formation was verified by LC-ESI-MS and MALDI-
TOF-MS. The simple expression and purification protocol and
the use of maltose as an inexpensive starting material make
this maltose phosphorylase from Emticicia oligotrophica a valu-
able novel biocatalyst for the synthesis of glucose-containing
glycosides.
KEYWORDS
X-glycosides; Emticicia
oligotrophica; maltose
phosphorylase; maltose;
phosphorolysis; beta glucose
 phosphate
GRAPHICAL ABSTRACT
CONTACT Josef Voglmeir
ple’s Republic of China
josef.voglmeir@njau.edu.cn and
liu.lichen@qlyco.com
Qlyco Ltd., Nanjing, Peo-
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lcar.
Supplemental data for this article can be accessed on the publisher’s website.
©  Taylor & Francis Group, LLC

2
F. N. AWAD ET AL.
ABBREVIATIONS: α-PGM: α-phosphoglucomutase; β-Glc-1-P:
β-Glucose-1-phosphate;
β-PGM:
β-phosphoglucomutase;
DHB: 2,5-dihydroxybenzoic acid; E. coli: Escherichia coli; EDTA:
ethylenediaminetetraacetic acid; ESI-MS: electrospray ion-
ization – mass spectrometry; HPLC: high-performance liquid
chromatography; IPTG: isopropyl-β-D-thiogalactopyranoside;
MALDI-TOF: matrix-assisted laser desorption/ionization;
PAGE: polyacrylamide gel electrophoresis; PMSF: phenyl-
methylsulfonyl fluoride; SDS: sodium dodecyl sulphate;
SIM: selected ion monitoring; TFA: trifluoroacetic acid; Tris:
tris(hydroxymethyl)aminomethane; X-β-GlcNAc: 5-bromo-4-
chloro-3-indolyl-acetyl-beta-D-glucosaminide; X-β-GlcNAc-Glc:
5-bromo-4-chloro-3-indolyl-acetyl-beta-D-glucosaminide-
α1,4-Glc
Introduction
Enzymes involved in bacterial starch and maltose metabolism have been intensively
studied for more than 60 years.[1] Maltose degradation can be either catalyzed by the
hydrolytic action of α-glucosidases, or by maltose phosphorylases in the presence
of inorganic phosphate.[2,3] The product of the phosphorolysis reaction, glucose-
1-phosphate, can be utilized after subsequent isomerization by phosphoglucomu-
tase for the glycolytic energy pathway in bacteria.[4] Maltose phosphorylase (EC
2.4.1.8) belongs to glycosyl hydrolase family GH 65 and catalyzes the phosphorol-
ysis of maltose with the inversion of anomeric configuration to form β-glucose-1-
phosphate (β-Glc-1-P) and glucose. Many probiotic as well as pathogenic bacteria
have been shown to bear maltose phosphorylase or closely related enzymes in their
genome.[5–7] Several maltose phosphorylase homologues have already been cloned
and characterized,[8,9] and the crystal structure of the maltose phosphorylase from
Lactobacillus brevis has been solved.[10] The high specificity of this enzyme towards
maltose has permitted applications such as the determination of α-amylase activ-
ity,[11] or the detection of maltose in oligosaccharide mixtures.[12] Furthermore,
maltose phosphorylases have been used in conductometric sensors for detecting
phosphate in foodstuffs.[13]
Maltose phosphorylases have also been used for the synthesis of unnatural dis-
accharides such as glucosylfucose,[14] or for the synthesis of monoglucosides such
as salicylglucose.[15] Although a number of novel maltose phosporylases have been
reported over the last years, the search for enzymes with broader substrate promis-
cuity remains challenging.
Here we describe the identification of a previously uncharacterized maltose phos-
phorylase from Emticicia oligotrophica, a bacterium initially isolated from warm
spring water in Assam, India.[16] Although the bacterium was discovered almost a
decade ago, the biochemical potential of this organism remains unexplored. Using E.
oligotrophica maltose phosphorylase for the enzymatic synthesis of indoxyldiglyco-
sides is the first step in broadening our knowledge of the biotechnological potential
of this microorganism.

JOURNAL OF CARBOHYDRATE CHEMISTRY
3
Figure . SDS-PAGE analysis and phylogenic relationship of EoMP. (A) SDS-PAGE at different
expression and purification stages: M – protein marker; 1 – cell pellet before induction; 2 – cell pellet
after induction with IPTG; 3 – supernatant after cell lysis; 4 – purified protein. (B) Phylogenetic rela-
tionship of maltose and kojibiose phosphorylases from selected bacterial species to E. oligotrophica
isoform. UniProt identifiers are shown in parenthesis. Asterisks indicate putative, non-characterized
enzymes.
Results and discussion
Cloning, expression, and purification of maltose phosphorylase from
E. oligotrophica
The putative maltose phosphorylase gene was chosen from the annotated genome
sequence of E. oligotrophica (eo.ggbrc.com) provided by the Pathosystems Resource
Integration Center (PATRIC).[17] The open reading frame consisted of 2325 base
pairs and was annotated based on its consecutive gene number (EoMP4038) in the
genome sequence. The screening of the genome sequence revealed a second open
reading frame belonging to glycoside hydrolase family 65, which maltose phospho-
rylases amongst trehalases and other phosphorylases belong to. However, the low
homology of only 18% to any other characterized family member suggests a dif-
ferent functionality of this homologue. The amplified gene product of EoMP4038
was successfully cloned and ligated into a pET30a expression vector construct con-
taining a hexa-histidine fusion tag, to facilitate protein purification using Ni+2-
nitrilotriacetate agarose affinity chromatography. The concentration of the purified
protein was determined to be 1.8 mg/mL. A single protein band with an apparent
molecular weight between 70 and 100 kDa was observed using SDS-PAGE (Fig. 1A),
which is in good agreement with the theoretical mass of the expression product
(89 kDa).
The amino acid sequence of EoMP4038 was compared to the amino acid
sequences of various homologues of bacterial origin using a translated nucleotide
database search (TBLASTN). The best homology scores were found for kojibiose
phosphorylases from Runella limosa (UniProt: C6W3J4) and Dyadobacter fermen-
tas (UniProt: F8EPQ9), both sharing 68% homology with EoMP4038. Furthermore,
EoMP4038 shares close homologies with maltose phosphorylases from Paenibacil-
lus sp. (58%, UniProt: Q50LH0), Bacillus selenitireducens (56%, UniProt: D6XUS4),
Bacillus sp. strain RK-1 (47%, UniProt: Q84IX5), Lactobacillus acidophilus (47%,

4
F. N. AWAD ET AL.
Figure . Analysis of disaccharide phosphorolysis catalysed by EoMP: (A) TLC-based assay;
positive (+ve) and negative (−ve) are reaction mixtures with EoMP and without EoMP,
respectively. Arrows indicate the phosphorolysis products, glucose and glucose--phosphate. (B)
enzyme-coupled microplate-based assay.
UniProt: Q5FI04), Lactobacillus brevis (46%, UniProt: Q7SIE1), and Lactobacillus
sanfranciscensis (45%, UniProt: O87772) (Fig. 1B, Supplementary Fig. S1).
Phosphorolytic specificity of EoMP4038
The specificity of EoMP4038 phosphorolysis was initially tested by thin layer chro-
matography (TLC) using a range of disaccharides as substrates. Only maltose was
converted to glucose and β-Glc-1-P, indicating that the enzyme had high selectiv-
ity to α-1,4-glycosydic linkage. No phosphorolysis was observed for sucrose, cel-
lobiose, trehalose, and melibiose (Fig. 2A). Activity of EoMP4038 towards maltose
was further confirmed using a photometric assay by monitoring the formation of
NADPH resulting from the coupled enzymatic reactions catalyzed by EoMP4038,
β-phosphoglucomutase and glucose-6-phosphate dehydrogenase (Fig. 2B). The
exchange of β-phosphoglucomutase with α-phosphoglucomutase led to no forma-
tion of NADPH, which indicates that β-Glc-1-P, but not α-Glc-1-P, was produced
from maltose in the phosphorolylis reaction.
The strict specificity of EoMP4038 toward maltose is in agreement with the speci-
ficity described for maltose phosphorylases from several other bacteria such as B.
selenitireducens,[18] L. acidophilus[19] and Paenibacillus sp.[8] One exception is the
maltose phosphorylase from Bacillus sp. RK-1, which also showed activity towards
the α-1,6- and β-1,6-linkages present in isomaltose and gentiobiose, respectively.[20]
Enzymatic properties of EoMP4038
The pH optimum was determined at pH 7.0, and showed relatively good activities
over a pH range between pH 5.0 and 9.0 (Fig. 3A). This pH optimum is comparable
with the ones reported for maltose phosphorylases from Paenibacillus sp. (pH 6.0
to 7.0)[8] and for the Bacillus sp. (pH 7.0 to 7.5).[20] The temperature optimum was
45°C, and although EoMP4038 still performed well up to 65°C, the enzyme’s activity

JOURNAL OF CARBOHYDRATE CHEMISTRY
5
Figure . Biochemical properties of EoMP. (A) Effects of temperature and (B) pH-dependency of
on the activity of the enzyme.
declined drastrically at temperatures above 70°C (Fig. 3B). This temperature opti-
mum is lower compared to Paenibacillus sp. and Bacillus sp. maltose phosphorylases
(50°C and 65°C, respectively).[8,20]
The kinetic parameters of EoMP4038 phosphorolytic activity are listed in Table 1.
The KM value of 0.59 mM is lower than the data reported for characterized maltose
phosphorylases from Paenibacillus sp. (4.0 mM)[8] and Bacillus sp. (12.3 mM),[20]
which might be advantageous when using EoMP4038 in reactions with low maltose
concentrations.
Synthesis of indoxyldiglycosides by reverse phosphorolysis
The enzymatic addition of α-1,4-linked glucose to various indoxysugars was exam-
ined in a coupled reaction. An excess of maltose and inorganic phosphate was used
to generate β-glucose-1-phosphate by phosphorolysis, which was consequently uti-
lized as a glucosyl donor for the glycosylation of the indoxysugars (Fig. 4).
The advantage of this coupled reaction is that inexpensive maltose and inorganic
phosphate can be used to generate the glucose donor β-glucose-1-phosphate. The
use of indoxyglycosides as acceptors allowed facile separation by reversed phase
HPLC and monitoring of the donor substrates and reaction product due to the pres-
ence of the 5-bromo-4-chloro-3-indolyl chromophore.
The conjugation of α-1,4-linked glucose was observed when X-β-GlcNAc, X-α-
Glc, X-β-Glc were used as acceptor substrates in a reaction catalyzed by EoMP4038,
whereas no product formation was observed for the other acceptor substrates tested
(Table 2).
Table . Kinetic parameters of EoMP.
Enzyme
KM (mM)
Vmax (mM/min)
Kcat (/s)
EoMP
. .
. .
. 

6
F. N. AWAD ET AL.
Figure . Scheme of indoxyldiglycoside synthesis (here exemplified with X-GlcNAc) using maltose
phosphorylase in a coupled enzymatic reaction. The aromatic indoxyl groups are highlighted.
The analysis of the enzymatic glucosylation reaction using HPLC-ESI-MS and
MALDI-TOF-MS for the acceptor substrate X-β-acetylglucosamine (X-β-GlcNAc)
is exemplified in Figs. 5 and 6. Although a product yield of only 3.1% could be
achieved after 16 h of reaction, the resulting X-β-acetylglucosamine-α1,4-glucose
could be cleanly separated using reversed phase HPLC with a retention time of
7.6 min (Fig. 5A). Treatment of the reaction mixture with β-hexosaminidase led
to complete hydrolysis of the starting material X-β-GlcNAc, resulting in the gen-
eration of free GlcNAc and an insoluble, dark blue indigo dye, which can easily be
removed by centrifugation. Further sample analysis was performed using HPLC-
ESI-MS: signals at m/z values corresponding to both substrate and product (459.00
[M + H]+ and 611.00 [M + H]+, respectively) were detected in reaction mixtures
containing EoMP4038, whereas a signal corresponding to the product could not be
detected in mixtures which did not contain EoMP4038 (Fig. 5B). In addition, the
identity of the synthesized disaccharide was also confirmed by MALDI–TOF mass
spectrometry (Fig. 6).
Several maltose phosphorylases have been reported to accept a broader variety
of free monosaccharide substrates, such as the maltose phosphorylases from Lac-
tobacillus acidophilus and Propionibacterium freudenreichi which can also utilize
mannose-, fucose-, and xylose[14,19] and achieve product yields of more than 50%.
Interestingly, maltose phosphorylase from Enterococcus hirae has been used to α-
glucosylate various alcohols, such as butanol and benzyl alcohol.[15] Furthermore, a
maltose phosphorylase from Bacillus celenitireducens has been applied to the synthe-
sis of branched glucose containing trisaccharides.[18] However, despite the relatively
Table . Transglycosylation activity of EoMP.
Acceptor
Expected product
Product yield, %
X-β-acetylglucosamine
X-α-glucose
X-β-glucose
X-α-galactose
X-α-fucose
X-α-mannose
X-β-mannose
X-β-xylose
X-β-acetylglucosamine-α,-glucose
X-α-glucose-α,-glucose
X-β-glucose-α,-glucose
X-α-galactose-α,-glucose
X-α-fucose-α,-glucose
X-α-mannose-α,-glucose
X-β-mannose-α,-glucose
X-β-xylose-α,-glucose
.
.
.
ND
ND
ND
ND
ND
ND – product formation not detected.

JOURNAL OF CARBOHYDRATE CHEMISTRY
7
Figure . HPLC and MS analysis of the enzymatic α-glucosylation transfer: (A) HPLC chromatogram
of the reaction mixture, purified product (X-β-GlcNAc-α,-Glc) and the substrate standard (X-β-
GlcNAc). The arrows indicate the product peak; (B) HPLC-ESI-MS signal response of the corresponding
substrate and product masses and substrate masses for reaction mixtures with EoMP (top panel)
and without EoMP (bottom panel).
low yield of glucose-modified indoxyglycosides by EoMP4038, this first example of
the use of sugar phosphorylases for the synthesis of anomerically modified glyco-
sides shows the potential and promiscuity of these class of enzymes.
Conclusion
In this report we describe the identification of maltose phosphorylase from E. olig-
otrophica, a maltose-specific enzyme capable of reverse phosphorolysis using var-
ious indoxyglycosides as acceptor substrates. The synthesis of several α-glucose-
terminated indoxyldisaccharides could be demonstrated by analyzing reaction mix-
tures using LC-MS and MALDI-TOF-MS. The screening of other maltose phospho-
rylases and the optimization of the reaction conditions to obtain higher product
yields are part of our current research effort.
Experimental
Bacterial strains and chemicals
Emticicia oligotrophica strain DSM 17448 was obtained from the German Collection
of Microorganisms and Cell Cultures (DSMZ). DNA polymerases were obtained

8
F. N. AWAD ET AL.
Figure . MALDI-TOF-MS spectra of the acceptor substrate (X-β-GlcNAc, top panel), the enzymatic
reaction reaction mixture (middle panel), and the isolated product after HPLC purification (X-β-
GlcNAc-α,-Glc,lowerpanel).Thenaturalisotoperatioforbromine(Br:Br=.:.)andforchlo-
rine (Cl:Cl = .:.) substituents of the indoxyl group are the reason for the additional masses
(signals with the + and + Da) observed in the spectra.
from Takara (Japan); restriction endonucleases and T4 ligase were from Thermo
Fisher Scientific (Shanghai); DNA Gel Purification Kit and Plasmid Extraction
Kit were purchased from Axygen (Beijing); maltose was obtained from Aladdin
(Shanghai, China); 5-bromo-4-chloro-3-indolyl-glycoside derivatives of α-glucose,
β-glucose, β-acetylglucosamine, α-galactose, α-L-fucose, α-mannose, β-mannose
and β-xylose were obtained from Carbosynth (Berkshire, UK). All chemicals used
in the work were of the highest grade available.
Gene cloning and construction of the expression plasmid
Genomic DNA from E. oligotrophica was isolated as previously described.[21]
The putative maltose phosphorylase gene was designated EoMP4038 accord-
ing to its sequential gene number in the E. oligotrophica genome provided by
the Pathosystems Resource Integration Center (PATRIC)[17] (eo.ggbrc.com).
The gene encoding EoMP4038 was amplified via polymerase chain reac-
tion (PCR) with Primestar HS DNA Polymerase, using the genomic DNA
of E. oligotrophica as a template with the following primer pair: forward,
5ꢀ-CGGGATCCATGAAAAATTATATAACACACGATG-3ꢀ and reverse, 5ꢀ-
CCGCTCGAGTTAGTATTCTACACTGGCCGAAG-3ꢀ containing BamHI and

JOURNAL OF CARBOHYDRATE CHEMISTRY
9
Figure . Microplate-based spectrophotometric detection of maltose phophorylase activity using a
coupled enzymatic assay with β-phosphoglucomutase and glucose--phospate dehydrogenase (left
branch). A more commonly applied application of the microplate based assay for determining the
activities of starch phosphorylase or sucrose phosphorylase is shown in the right branch of the figure.
XhoI restriction sites (underlined), respectively. In brief, 35 PCR cycles consisting
of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at
72°C for 3 min were performed. The resulting PCR product was digested using
the corresponding restriction enzymes, purified and ligated into the predigested
expression vector pET30a (Novagen). The EoMP4038-containing vector construct
was transformed into E. coli Mach1 T1 cells (Invitrogen) and plated onto Luria–
Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 1% NaCl and 1.5% agarose)
supplemented with kanamycin (50 μg/mL). The gene sequence of candidate clones
was further verified by Sanger sequencing (Genscript, Nanjing).
Expression and purification of recombinant protein
E. coli BL21 (DE3) cells were used as a host for recombinant protein expression
which was carried out as described previously.[22] Briefly, cells were cultured in

10
F. N. AWAD ET AL.
400 mL of LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) with shak-
ing at 37°C, 250 rpm until the optical density (OD600) reached 0.5. Target protein
expression was induced by addition of 1 mM isopropyl-β-D-thiogalactopyranoside
(IPTG) with 24 h of further culture shaking at 18°C. Cells were harvested by cen-
trifugation (5000 g, 20 min, 4°C), resuspended in 10 mL of lysis buffer (50 mM
Tris/HCl, 1% Triton X-100, 100 mM NaCl and 1 mM phenylmethylsulfonyl fluoride
(PMSF), pH 8.0) and disintegrated by ultrasonication (40 on/off cycles with 20 μm
amplitude for 15 s at 4°C) for 20 min in ice water. Cell debris were removed by cen-
trifugation (20000 g, 20 min, 4°C). The resulting supernatant was loaded onto Ni2+-
nitrilotriacetate agarose affinity column (2 mL bed volume, Qiagen), washed with
15 column volumes of protein binding buffer (50 mM NaCl, and 50 mM Tris/HCl,
pH 8.0) and 5 column volumes of washing buffer (50 mM NaCl, 50 mM Tris/HCl,
and 10 mM imidazole, pH 8.0) to avoid unspecific protein adsorption. The target
protein with a N-terminally fused His6-tag was eluted from the affinity column
using elution buffer (50 mM NaCl, 50 mM Tris/HCl, and 500 mM imidazole, pH
8.0). The purity of the recombinant protein was evaluated by sodium dodecyl sul-
fate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions as
described by Laemmli,[23] and visualized by staining with Coomassie Brilliant Blue
G-250. The purest fractions were pooled and protein concentration was performed
using a Bradford-based protein quantification assay kit (Sangon Biotech).
Chromatographic analysis of phosphorolysis products
Thin-layer chromatography was employed to detect the products of the phospho-
rolysis reactions. A 20 µL reaction mixture containing 20 mM NaH2PO4, 50 mM
Tris/HCl buffer (pH 7.5), 0.5 mM MgCl2, 95 mU of EoMP4038, and 50 mM of
a disaccharide substrate (maltose, sucrose, cellobiose, trehalose, or melibiose) was
incubated overnight at 37°C and spotted on silica-gel TLC plate Merck KG (Type 60
F
254, Darmstadt, Germany). The plates were developed using a mobile phase mix-
ture and staining solutions as described previously[24] (acetonitrile-acetic acid-H2O,
7:1.5:1.5, v/v/v). Substrate and product spots from the reaction were visualized by
soaking the plate in a 5% solution of methanolic H2SO4 followed by heating at 95°C
for 10 min. Samples lacking the enzyme, and sugar standards such as glucose-1-
phosphate (Glc-α-1P) and glucose were used as controls.
Enzymatic assay
The activity of EoMP4038 was determined using a coupled assay which allows the
generation of NADPH to be monitored at a wavelength of 340 nm. Typically, the
reaction mixture (25 µL) contained 50 mM maltose, 10 mM NaH2PO4, 0.2 mM
MgCl2, 50 mM Tris/HCl buffer (pH 7.5), 1 mM NADP+, and 0.5 mU, 0.5 mU
and 50 mU of EoMP4038, β-phosphoglucomutase (EC 5.4.2.6 Figs. S2 and 3), and
glucose-6-phosphate dehydrogenase (EC 1.1.1.49, Sigma aldrich), respectively. In

JOURNAL OF CARBOHYDRATE CHEMISTRY
11
addition, an activity assay where β-phosphoglucomutase was replaced with 0.5 mU
of α-phosphoglucomutase (EC 5.4.2.2 Figs. S4 and 5) was carried out as a nega-
tive control (Fig. 7). Further negative controls included reaction mixtures lacking
EoMP4038, inorganic phosphate, or β-phosphoglucomutase. Absorbance was mon-
itored for 90 min continuously on a 384-well-microplate reader (Thermo Multi-
skan).
Determination of the substrate promiscuity of EoMP4038
The synthetic potential of EoMP4038 was evaluated using maltose as a β-Glc-
1-P donor and different 5-bromo-4-chloro-3-indolyl-glycoside derivatives (X-
glycosides), including X-α-Glc, X-β-Glc, X-β-GlcNAc, X-α-Gal, X-α-Fuc, X-β-Xyl,
X-α-Man and X-β-Man as acceptor substrates. Reaction mixtures (20 µL) contain
50 mM maltose, 50 mM Tris/HCl, pH 7.5, 2.5 mM NaH2PO4, 0.5 mM MgCl2, 1 mM
X-glycoside, and 86 mU of EoMP4038, were incubated at 37°C for 16 h. Reactions
were quenched by adding 20 µL of chloroform to the reaction mixture, followed
by centrifugation, and the upper phase containing the enzyme-free analyte was col-
lected and subjected to HPLC analysis as previously described,[25] with slight mod-
ifications. The analytes were separated using a reversed-phase HPLC column (Phe-
˚
nomenex Hyperclone, 5 µm, C18, 120 A, 250 × 4.6 mm) at a constant flow rate of
0.8 mL/min and continuous UV-detection at 300 nm. The mobile phases used were
aqueous ammonium formate (50 mM, pH 4.5) and acetonitrile (solvents A and B,
respectively). After sample injection (10 µL), a linear gradient of 30–60% solvent B
was applied from 0 to 5 min, followed by an increase of solvent B to 90% over 1 min,
and held at 90% for another 2 min. B was then decreased to 30% over 1 min, and the
column was equilibrated under the initial conditions for 6 min. Product and sub-
strate masses were detected by ESI-MS (Nexera MS 2020, Shimadzu Corporation,
Kyoto, Japan).
MALDI –TOF MS analysis of reverse phosphorolysis reaction compounds
The acceptor substrate, reaction mixtures and the β-hexosaminidase-treated reac-
tion product were lyophilized and resuspended in 10 µL H2O, and then mixed in
1:1 ratio with a solution of 2,5-dihydroxybenzoic acid (DHB) matrix containing
4 mg/mL DHB in 30% acetonitrile, 0.1% TFA, and 1 mM sodium chloride. Mass
spectrometry was performed using a Autoflex Speed MALDI-TOF mass spectrom-
eter (Bruker Daltonics, Germany).
Biochemical analysis
The optimum pH, optimum temperature and kinetic parameters of EoMP4038
were determined by measuring the amount of liberated glucose from maltose
during enzymatic phosphorylsis using a glucose oxidase-peroxidase assay kit which

12
F. N. AWAD ET AL.
was determined photometrically at 505 nm using 4-aminoantipyrine (Shanghai
Ronsheng Biotech Co. Ltd). The incubation time for all assays was 15 min and
the reaction was terminated by heating at 90°C for 10 min. For measuring the pH
optima, the reaction mixture contained maltose (20 mM), NaH2PO4 (10 mM),
MgCl2 (0.5 mM), and either sodium citrate buffer in the lower pH range (pH
3.5–7.5, 50 mM) or Tris/HCl in the higher pH range (pH 7.5–9.0, 50 mM); Sodium
citrate buffer (pH 7.0, 50 mM) was chosen for determining the temperature opti-
mum, by testing the enzyme’s activity at various temperatures ranging from 4–80°C.
The kinetic parameters of EoMP4038 were also determined photometrically, using
maltose concentrations between 0.05 and 5 mM, and by applying the non-linear
regression model provided by the Labplot data analysis software (version 2.0.1).
Funding
This work was supported in part by the Natural Science Foundation of China (grant number
31471703 and A0201300537 to J.V. and L.L.) and the 100 Foreign Talents Plan (grant number
JSB2014012 to J.V.).
Acknowledgments
The authors would like to thank Dr. Louis Conway (GGBRC, Nanjing) for language editing of
this manuscript.
ORCID
Josef Voglmeir http://orcid.org/0000-0002-4096-4926
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