Two-step biosynthesis of D-allulose via a multienzyme cascade for the bioconversion of fruit juices

Article abstract of DOI:10.1016/j.foodchem.2021.129746

D-Allulose, a low-calorie rare sugar with potential as sucrose substitute for diabetics, can be produced using D-allulose 3-epimerase (DAE). Here, we characterized a putative thermostable DAE from Pirellula sp. SH-Sr6A (PsDAE), with a half-life of 6 h at 60 °C. Bioconversion of 500 g/L D-fructose using immobilized PsDAE on epoxy support yielded 152.7 g/L D-allulose, which maintained 80% of the initial activity after 11 reuse cycles. A multienzyme cascade system was developed to convert sucrose to D-allulose comprising sucrose invertase, D-glucose isomerase and PsDAE. Fruit juices were treated using this system to convert the high-calorie sugars, such as sucrose, D-glucose, and D-fructose, into D-allulose. The content of D-allulose among total monosaccharides in the treated fruit juice remained between 16 and 19% during 15 reaction cycles. This study provides an efficient strategy for the development of functional fruit juices containing D-allulose for diabetics and other special consumer categories.

Full text of DOI:10.1016/j.foodchem.2021.129746

Food Chemistry 357 (2021) 129746
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Two-step biosynthesis of D-allulose via a multienzyme cascade for the
bioconversion of fruit juices
,
,*
Chao Lia, Lei Lia, Zhiyuan Fenga, Lijun Guanb, Fuping Lua *, Hui-Min Qina
a Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology,
Tianjin University of Science and Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, PR China
b Institute of Food Processing, Heilongjiang Academy of Agricultural Sciences, Harbin, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
D-Allulose, a low-calorie rare sugar with potential as sucrose substitute for diabetics, can be produced using D-
allulose 3-epimerase (DAE). Here, we characterized a putative thermostable DAE from Pirellula sp. SH-Sr6A
(PsDAE), with a half-life of 6 h at 60 ◦C. Bioconversion of 500 g/L D-fructose using immobilized PsDAE on
epoxy support yielded 152.7 g/L D-allulose, which maintained 80% of the initial activity after 11 reuse cycles. A
multienzyme cascade system was developed to convert sucrose to D-allulose comprising sucrose invertase, D-
glucose isomerase and PsDAE. Fruit juices were treated using this system to convert the high-calorie sugars, such
as sucrose, D-glucose, and D-fructose, into D-allulose. The content of D-allulose among total monosaccharides in
the treated fruit juice remained between 16 and 19% during 15 reaction cycles. This study provides an efficient
strategy for the development of functional fruit juices containing D-allulose for diabetics and other special
consumer categories.
D-Allulose
D-Allulose 3-epimerase
Immobilized enzyme
Multienzyme cascade system
Fruit juices
1. Introduction
extraction from natural resources is not practical, and the chemical
synthesis of D-allulose is difficult due to the complex multi-step reactions
D-Allulose is a C-3 epimer of D-fructose that is rarely found in nature,
but it has aroused significant interest due to its suitability as an ultra-
low-calorie functional sweetener (Matsuo, Suzuki, Hashiguchi, & Izu-
mori, 2002). D-Allulose is an attractive sucrose substitute for food pro-
duction due to its significantly reduced caloric content of about 0.2 kcal/
g, while maintaining approximately 70% of the sweetness of sucrose
(Mooradian, 2019). Furthermore, D-allulose improves the gelling prop-
erties of food and reduces oxidation via the Maillard reaction in food
processing, better preserving the food flavor (Zeng, Zhang, Guan, & Sun,
2011). Notably, it has been granted the generally recognized as safe
(GRAS) status by the US Food and Drug Administration (FDA) (Moora-
dian, 2019). In addition to potential uses as a food additive, studies have
demonstrated that D-allulose also has notable physiological effects
associated with blood glucose suppression, neuroprotective effects,
reactive oxygen species (ROS) scavenging activity, and therapeutic ef-
fects against atherosclerosis (Zhang, Fang, Xing, Zhou, Jiang, & Mu,
2013; Zhang, Yu, Zhang, Jiang, & Mu, 2016). Accordingly, D-allulose is
an attractive natural functional ingredient due to its health-promoting
attributes such as mitigation of obesity, reducing lipidemia, and
fighting diabetes (Shintani et al., 2017). The production of D-allulose by
and functional group protection-deprotection steps. Moreover, chemical
synthesis can produce toxic byproducts that render the final product
unsuitable for food use (Emmadi & Kulkarni, 2014). Instead, a sustain-
able bioprocess based on Izumoring strategy has been established to
biosynthesize D-allulose from D-fructose via the enzymatic and micro-
biological synthesis (Mu, Yu, Zhang, Zhang, & Jiang, 2015).
The biosynthesis of D-allulose via the enzymatic Izumoring strategy is
more efficient, with higher substrate specificity and mild reaction con-
ditions offering increased sustainability (Izumori, 2006). Biosynthesis of
D-allulose can be realized via the epimerization of D-fructose by ketose 3-
epimerases (KEases), which can be classified as D-tagatose 3-epimerases
(DTEs, EC 5.1.3.-) and D-allulose 3-epimerase (DAEs, also named DPEs,
EC 5.3.1) based on substrate specificity (Zhang, Mu, Jiang, & Zhang,
2009; Zhang et al., 2013). To date, at least 20 KEases from various
bacterial species and strains have been characterized, such as the DTEs
from Sinorhizobium sp. (Zhu, et al., 2019), as well as the DAEs from Dorea
sp. CAG317 (Zhang et al., 2015) and Arthrobacter globiformis M30
(Yoshihara et al., 2017). Enzymatic catalysis offers many advantages
with few or no side reactions, facile control and optimization of reaction
conditions, and avoidance of rate-limiting diffusion across the cell
* Corresponding authors.
E-mail addresses: lfp@tust.edu.cn (F. Lu), huiminqin@tust.edu.cn (H.-M. Qin).
https://doi.org/10.1016/j.foodchem.2021.129746
Received 16 December 2020; Received in revised form 10 March 2021; Accepted 31 March 2021
Available online 5 April 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.

C. Li et al.
Food Chemistry 357 (2021) 129746
Fig. 1. Schematic representation of D-allulose synthesis from sucrose via the multienzyme cascade catalysis system developed in this study. The enzymes used are
invertase (INV), D-glucose isomerase (GI), and D-allulose 3-epimerase (DAE).
membrane (Bujara, Schuemperli, Billerbeek, Heinemann, & Panke,
2010; Finnigan, Cutlan, Snajdrova, Adams, Littlechild, & Harmer, 2019;
Hold & Panke, 2009; Xue & Woodley, 2012). Although enzymatic syn-
thesis is considered a green and sustainable approach, the large-scale
industrial application of enzymes to produce D-allulose is restricted by
expensive unrecyclable biocatalysts, poor operational stability, and high
production cost (Choi, Han, & Kim, 2015; DiCosimo, McAuliffe, Poulose,
& Bohlmann, 2013; Klein-Marcuschamer, Oleskowicz-Popiel, Simmons,
& Blanch, 2012). Notably, enzyme immobilization has been demon-
strated as an efficient approach for improving the physical properties,
recovery and reusability of enzymes to reduce process costs (Nawani,
Singh, & Kaur, 2006; Su, Zhang, Zhu, Xu, Yang, & Li, 2013).
2. Materials and methods
2.1. Strains, plasmids, and reagents
The strains and plasmids used in this study are listed in Table S1. The
pET22b vector was used for protein expression in Escherichia coli BL21
(DE3). The sucrose, D-glucose, D-fructose and D-allulose standard were
purchased from Sigma-Aldrich (Shanghai, China). The epoxy support
ES-103B was purchased from Tianjin Nankai HECHENG (Tianjin,
China). INV and immobilized GI were acquired from Novozymes
Biotechnology Inc. (Suzhou, China). Oligonucleotides were synthesized
by GENEWIZ (Suzhou, China). All other chemicals were of analytical
grade and were obtained from Sigma-Aldrich (Shanghai, China) or
Sangon Biotech (Shanghai, China).
As a functional rare sugar, D-allulose has significant commercial
potential as a low-calorie sweetener in fruit juices. Some high-calorie
sugars in fruit juices, such as sucrose, D-glucose, and D-fructose, which
are considered to be equivalent to high-calorie sugars in sugar-
sweetened beverages can be utilized as substrates for multienzyme
cascade systems (Zolot, 2017). Studies have demonstrated that excessive
consumption of high-calorie sugars increases the risk of diabetes,
obesity, and metabolic disease (Imamura et al., 2015; Yang et al., 2019).
Notably, D-allulose can reduce food calorie intake and promote glucose
tolerance in healthy persons as well as obese diabetics, since it induces
glucagon-like peptide-1 (GLP-1) release and activates vagal afferent
signaling following oral administration (Iwasaki et al., 2018). The
conversion of high-calorie sugars into D-allulose can be achieved via
enzymatic biocatalysis or fermentation technology, which are increas-
ingly being viewed as attractive technologies for promoting better
health in recent years (Men, Zhu, Zeng, Izumori, Sun, & Ma, 2014). Yang
et al. reported that an engineered Corynebacterium glutamicum strain
expressing DAE and a thermostable invertase (INV) can produce D-
allulose using inexpensive sugarcane molasses as feedstock (Yang et al.,
2019). Enzymatic catalysis pathways using glucose isomerase (GI) and
DAE have enabled the conversion of D-glucose and D-fructose in high-
fructose corn syrup into D-allulose (Men et al., 2014).
2.2. Cloning and expression of the PsDAE gene
The coding sequence of PsDAE (GenBank: WP_146677337.1) was
codon-optimized for E. coli, ordered from Genewiz (Suzhou, China) as
synthetic DNA, and subcloned into the pET22b vector (Novagen, Mad-
ison, WI, USA) between the NdeI and EcoRI restriction sites. The re-
combinant pET22b plasmid containing the PsDAE gene and a His6 tag at
the C-terminus (pET-PsDAE) was introduced into E. coli BL21 (DE3),
which was cultured in lysogeny broth (LB) containing ampicillin (100
◦
μ
g/mL) at 37 C and 220 rpm until the OD600 reached 0.6–0.8. Then,
isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final con-
centration of 0.5 mM to induce the expression of PsDAE, which was
continued at 16 ◦C for 16–18 h.
2.3. Purification of PsDAE
The recombinant cells expressing the protein were collected by
centrifugation at 5000×g and 4 ◦C for 15 min, resuspended in lysis
buffer (20 mM Tris-HCl, 10 mM imidazole, 500 mM NaCl, 1 mM
dithiothreitol (DTT), 1 mg/mL lysozyme, and 1 mM phenylmethane
sulfonyl fluoride (PMSF), pH 8.0), disrupted by sonication, and the
resulting crude lysate cleared by centrifugation at 40,000×g and 4 ◦C for
30 min. The cleared supernatant containing His-tagged-PsDAE was
loaded onto a column containing Ni-NTA Superflow resin (Qiagen,
Hilden, Germany) equilibrated with lysis buffer. The recombinant His-
tagged PsDAE was eluted using 10 mL of elution buffer (20 mM Tris-
HCl, 300 mM imidazole, 100 mM NaCl, and 1 mM DTT, pH 8.0) and
dialyzed against 20 mM Tris-HCl pH 8.0 with 1 mM DTT to remove the
imidazole, and used directly for the activity assays and biosynthesis of D-
allulose. The protein concentration was determined using a BCA assay
kit (Solarbio, China) with bovine serum albumin as the standard. The
purity of the target protein was assessed by sodium dodecyl sulfate
In this study, a thermostable D-allulose 3-epimerase from Pirellula sp.
SH-Sr6A (PsDAE) has been characterized. Additionally, PsDAE was
immobilized and used as a recyclable biocatalyst to biosynthesize D-
allulose from D-fructose with high bioconversion efficiency. Further-
more, a multienzyme strategy was developed to convert high-calorie
sugars in fruit juice into D-allulose via two-step reaction strategies.
The reaction system consisted of free INV, immobilized GI, and immo-
bilized PsDAE (Fig. 1). INV was firstly used to catalyze the hydrolysis of
sucrose into D-glucose and D-fructose. D-Glucose was then converted into
D-fructose by GI, while D-fructose was transformed into D-allulose by
PsDAE. A variety of fruit juices were subjected to treatment using this
multienzyme cascade catalysis system to convert high-calorie sugars
into D-allulose with high efficiency.
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C. Li et al.
Food Chemistry 357 (2021) 129746
polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant
Blue staining.
2.6. Enzyme activity assay
The catalytic activity of free and immobilized PsDAE was determined
by measuring the formation of D-allulose from D-fructose. The reaction
mixtures in PBS (pH 7.4) contained 10 g/L D-fructose, 1 mM Mg2+, and
0.5 g/L free PsDAE or 20 g/L immobilized PsDAE. The reaction was
conducted at 60 ◦C for 10 min in a final volume of 1 mL. The reactions
were stopped by boiling for 5 min, and the amount of the D-allulose
produced by PsDAE was assayed using HPLC as described above. All
measurements were conducted in triplicate, and the results were
expressed as the means ± standard deviation (SD).
2.4. Preparation of immobilized PsDAE enzyme
To immobilize PsDAE, the epoxy support ES-103B was first washed
with 0.1 M potassium phosphate buffer pH 8.0, followed by distilled
water. Then, 1 g of epoxy resin ES-103B and 30 mg PsDAE were mixed in
15 mL 1.5 M sodium phosphate buffer pH 6.5 and gently shaken for 24 h
at 25 ◦C. The immobilized PsDAE was washed non-covalently bound
enzyme with phosphate buffer saline (PBS) buffer. The amount of
enzyme covalently immobilized onto the epoxy support was determined
by calculating the difference between the initial PsDAE concentration in
the solution and the amount of protein in the supernatant and washing
fractions.
To determine the optimal pH of PsDAE for D-allulose production, the
reactions were conducted at 60 ◦C for 10 min across a pH range of 5.5 to
11.0 in different buffers (NaAc-HAc: 4.5–5.5, 4-morpholineethanesul-
fonic acid (MES): pH 5.5–6.5, PBS: 7.0–8.0, Tris-HCl: 8.5–9.0, 3-
(cyclohexylamino)-1-propanesulfonic acid (CAPS): pH 9.5–11.0). The
optimal temperature of PsDAE was determined by conducting the
2.5. Identification of sugars by HPLC
◦
enzyme activity assay at temperatures from 30 to 90 C. The effect of
temperature on the stability of free and immobilized enzyme was tested
The qualitative and quantitative analysis of D-allulose, D-fructose, D-
glucose, and sucrose was conducted using high-performance liquid
chromatography (HPLC) on an Agilent 1260 instrument (USA) equipped
◦
by incubating the enzyme at different temperatures (40–70 C) in 20
mM PBS buffer (pH 7.4) for 12 h, and analyzing the residual activity
every 2 h. To investigate the effects of various metal ions on the activity
of PsDAE, the activity assay was supplemented with 1 mM Ca2+, Mg2+
,
with a prevail Carbohydrate ES column-W (5 μm, 4.6 × 250 mm, Agela
Zn2+, Cu2+, Mn2+, Co2+, or Fe2+. In addition, 1 mM EDTA was added to
test the metal-dependence of the enzyme. The activity without adding
metal ions was defined as 100%.
Technologies, China), an evaporative light-scattering detector (ELSD)
(Agilent 1260 Infinity, USA), an Agilent (USA) multichannel interface,
and a XWK-III pump. Acetonitrile (75%) was used as the mobile phase at
a flow rate of 0.8 mL/min and the column was kept at 40 ◦C. All mea-
surements were repeated three times.
The substrate specificity of PsDAE was measured under standard
Fig. 2. Biochemical prosperities of free and immobilized PsDAE. pH dependence (a), temperature dependence (b), thermostability analysis of free PsDAE (c),
thermostability analysis of immobilized PsDAE (d), the effect of mental ions on activity of PsDAE (e), substrate specificity of PsDAE (f).
3

C. Li et al.
Food Chemistry 357 (2021) 129746
reaction conditions using 100 mM D-allulose, D-fructose, D-tagatose, or D-
sorbose as the substrate, respectively. One unit of enzyme activity was
Table 1
Kinetic parameters of free and immobilized PsDAE for D-fructose and D-allulose.
defined as the amount of the enzyme required to catalyze 1 μmol sub-
Substrate
Kinetic parameters
Free enzyme
Immobilized enzyme
strate per minute under standard assay conditions. The kinetic param-
eters of free and immobilized PsDAE were determined under standard
conditions using a range of D-fructose concentrations (10–500 mM). The
Michaelis-Menten constant (Km), turnover number (kcat), and catalytic
efficiency (kcat/Km), were calculated using a Lineweaver Burk plot.
D-Fructose
Km (mM)
74.7 ± 2.5
47.2 ± 1.3
0.63
122.4 ± 3.2
42.2 ± 1.5
0.34
kcat (sꢀ 1
)
k
cat/Km (s-1mMꢀ 1
)
)
D-Allulose
Km (mM)
41.7 ± 1.2
63.6 ± 1.7
1.53
71.1 ± 1.8
58.0 ± 1.4
0.82
kcat (sꢀ 1
)
k
cat/Km (s-1mMꢀ 1
2.7. Production of D-allulose using free and immobilized PsDAE
3.2. Characterization of PsDAE
For the biocatalytic production of D-allulose, solutions containing 50
g/L, 100 g/L, and 500 g/L of D-fructose were treated with 205U isolated
PsDAE or 0.2 g epoxy resin ES-103 with immobilized PsDAE (containing
205U PsDAE) in a 10.0 mL reaction mixture comprising 1.0 mM Mg2+ in
20 mM PBS buffer (pH 7.4). The reaction mixtures were incubated at
60 ◦C and samples were drawn at regular time intervals to measure the D-
allulose production by HPLC. At the end of the reaction, immobilized
PsDAE was washed three times with 20 mM PBS buffer (pH 7.4) and
reused for the next cycle of reaction. The reaction procedure was carried
out in 15 sequential batches. The reusability of immobilized PsDAE was
analyzed based on the residual activity after every cycle, whereby the
activity during the initial cycle was defined as 100%.
The coding sequence of PsDAE was codon-optimized, overexpressed
in E. coli BL21 (DE3) with a His6 tag, and purified using Ni-affinity
chromatography. The molecular weight of PsDAE was estimated based
on SDS-PAGE, indicating a size of approximately 32 kDa (Fig. S2).
The biochemical properties of free and immobilized PsDAE were
characterized using D-fructose as the substrate (Fig. 2). The HPLC
retention times of D-allulose and D-fructose were 8.4 and 10.5 min,
respectively (Fig. S3a). PsDAE was optimally active in PBS buffer pH 7.5,
and was active in a pH range from 5.0 to 11.0. Notably, immobilized
PsDAE showed high activity within a wider pH range (pH 6.5–11.0) than
the free enzyme (pH 7.0–9.0) (Fig. 2a). The temperature-activity profile
for both the free and immobilized PsDAE revealed an optimal temper-
◦
ature of 60 C, with more than 80% relative activity between 55 and
2.8. Biocatalytic production of D-allulose via a multienzyme cascade
65 ◦C. Moreover, the immobilized PsDAE exhibited higher activities at
60–80 ◦C than the free enzyme (Fig. 2b). The free enzyme was stable for
12 h at temperatures ranging from 40 to 50 ◦C. Moreover, PsDAE
retained over 50% residual activity after 6 h at 60 ◦C. However, the
enzyme was completely inactivated after 3 h at 70 ◦C (Fig. 2c). By
contrast, other DAEs from the same family, including those from
A. tumefaciens (Kim, Hyun, Kim, Lee, & Oh, 2006), C. cellulolyticum (Mu,
Chu, Xing, Yu, Zhou, & Jiang, 2011a), and C. scindens (Zhang et al.,
2013) were relatively stable below 45–50 ◦C, and their relative activity
decreased significantly at 55 ◦C. Furthermore, the DAE from Desmospora
sp. was inactive at temperatures over 60 ◦C (Xing, 2013). Therefore,
PsDAE exhibited higher thermostability than other characterized DAEs.
Notably, immobilization of PsDAE onto the epoxy support ES-103B
greatly improved its thermal stability, reaching a half-life of 12 h at
60 ◦C (Fig. 2d). The effects of different divalent metal ions on the activity
of free PsDAE are shown in Fig. 2e. PsDAE displayed activity in the
absence of a metal cofactor, but its activity was increased in the presence
of Co2+, Mg2+, and Mn2+ by 1.75-, 1.28-, and 1.17-fold, respectively.
These results indicated that PsDAE is a metalloenzyme, similar to other
homologous members of the same family (Kim et al., 2006; Mu, Chu,
Xing, Yu, Zhou, & Jiang, 2011).
Biosynthetic production of D-allulose from sucrose was performed
using a multienzyme cascade system with 500 g/L sucrose in a 10.0 mL
reaction mixture. The reaction was conducted at 55 ◦C for 2 h with 500
g/L sucrose and 0.5 g/L INV (200000 U/g) in NaAc-HAc buffer (pH 4.5)
in the first step. Subsequently, the pH of the reaction mixture was
adjusted to 7.5 using 1 M NaOH and then 50–200 g/L immobilized GI
(50000 U/g) and 20 g/L immobilized PsDAE were added into the re-
action system. The reaction was implemented for another 1.5 h,
whereby the substrate consumption and product formation were
analyzed by HPLC every 30 min.
2.9. Conversion of native high-calorie sugars in fruit juices into D-allulose
in situ
Pure mango juice, orange juice, and sugar cane juice were individ-
ually treated with INV, GI, and PsDAE via the two-step reaction strategy.
In the initial step, 0.5 g/L INV was directly added into 10 mL of the
100% fruit juice. The reaction was carried out at 55 ◦C for 1 h. In the
next step, the pH of the fruit juices was adjusted to 7.5 using 1 M NaOH
and then 150 g/L immobilized GI and 20 g/L immobilized PsDAE were
added to the fruit juices. The reaction was performed for another 1 h,
and D-allulose formation was followed using HPLC every 10 min.
The substrate specificity of PsDAE was investigated using D-allulose,
D-fructose D-tagatose, and D-sorbose as substrates. The maximal activity
of PsDAE was observed with D-allulose, while the activity decreased for
other substrates in the order D-fructose, D-tagatose, D-sorbose (Fig. 2f).
This result was similar to the DAEs from C. cellulolyticum and Rumino-
coccus sp. (Mu et al., 2011; Zhu et al., 2012). Due to the high specificity
of PsDAE for D-allulose, it can be classified as a D-allulose 3-epimerase.
The kinetic parameters of the free and immobilized PsDAE for D-allu-
lose and D-fructose were also investigated. For D-allulose, the free and
immobilized PsDAE showed Km 41.7 vs 71.1 mM and kcat/Km 1.53 vs
0.82 s-1mMꢀ 1. As for D-fructose, they showed Km 74.7 vs 122.4 mM and
3. Results and discussion
3.1. Multiple sequence alignment
A search in the NCBI database for DAE sequences yielded an unusual
epimerase from Pirellula sp. SH-Sr6A (PsDAE; GenBank Accession No.
AMV33274.1). A phylogenetic tree was constructed based on the protein
sequence of PsDAE, together with characterized DAEs and DTEs from
other organisms. The phylogenetic tree revealed that PsDAE was closely
related to homologs from Rhodobacter sphaeroides (GenBank:
ACO59490.1) and Sinorhizobium sp. (GenBank: WP_069063284.1).
Moreover, PsDAE exhibited the highest amino acid sequence identity
with DAE from Sinorhizobium sp. (26.95%), and DTE from Rhodobacter
sphaeroides (26.88%) (Fig. S1). This percentage was still low and indi-
cated the uniqueness of PsDAE.
k
cat/Km 0.63 vs 0.34 s-1mMꢀ 1. These indicated that both free and
immobilized PsDAE exhibited higher affinity and catalytic efficiency
with D-allulose than D-fructose (Table 1). Notably, the immobilized
PsDAE enzyme exhibited a kcat that was nearly the same as that of its free
form, revealed that immobilization did not significantly alter the enzy-
matic properties of PsDAE (Table 1 and Fig. 2).
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Food Chemistry 357 (2021) 129746
Fig. 3. (a) Bioconversion of D-allulose via isolated PsDTE enzyme with 50, 100, and 500 g/L D-fructose as substrate. Mean and error bars were calculated based on
biological triplicate experiments; (b) Bioconversion of D-allulose via immobilized PsDTE enzyme with 500 g/L as substrate; (c) Residual activity of immobilized
PsDAE during 15 reuses cycles.
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Food Chemistry 357 (2021) 129746
Fig. 4. (a) Effect of the ammount of immobilized glucose isomerase on D-allulose yield. (b) The conversion of sucrose to D-allulose catalyzed by 0.5 g/L INV, 150 g/L
immobilized GI, and 20 g/L immobilized PsDAE with two-step strategy.
3.3. D-Allulose production using free PsDAE enzyme
generated 150 g/L D-allulose from 500 g/L D-fructose, corresponding to a
conversion ratio of about 30% after 3 h. This result indicated that PsDAE
immobilized onto epoxy resin ES-103B had a similar bioconversion rate
to free enzyme (Fig. 3b). Next, the operational stability and reusability
of immobilized PsDAE were investigated via consecutive cycles of
repeated reactions. The relative activity of immobilized DAE barely
declined during 5 consecutive cycles, and it retained 80% of its initial
activity after 11 cycles of repeated reactions (Fig. 3c). Jung et al. re-
ported that a DAE enzyme immobilized onto graphene oxide retained
more than 50% of the initial activity after 5 continuous cycles, but lost
more than 80% of the initial activity after 10 cycles (Samir R. Dedania,
Patel, Patel, Akhani, & Patel, 2017). Dedania et al. reported that DAE
immobilized onto titanium dioxide lost 80% of its initial activity after 9
consecutive cycles of epimerization reactions (Dedania, Patel, Soni, &
Patel, 2020). Yang et al. reported that the catalytic rate of immobilized
cells of DAE barely decreased in 25 consecutive cycles and still retained
80% after 36 cycles of repeated reactions (Yang et al., 2019). Compared
to these earlier reports, the PsDAE enzyme immobilized onto the epoxy
support ES-103B showed excellent reusability, indicating its great
application potential for industrial D-allulose production.
Biocatalytic production of D-allulose was assessed with 50, 100, and
500 g/L D-fructose as the substrate, along with 0.5 g/L PsDAE in a 10.0
mL reaction volume. Under the optimal reaction conditions, PsDAE
produced 15.3, 30.4, and 152.7 g/L D-allulose from 50, 100, and 500 g/L
D-fructose, respectively. This corresponds to an equilibrium ratio of
30:70 between D-allulose and D-fructose (Fig. 3a), which was higher than
what was reported for other enzymes from the same family, i.e.
P. cichorii DTE (20:80) (Itoh, Okaya, Khan, Tajima, & Izumori, 1994)
and R. sphaeroides DTE (23:77) (Zhang et al., 2009), but lower than that
of A. tumefaciens DAE (32.9%) (Kim et al., 2006) and Staphylococcus
aureus (38.9%) (Zhu et al., 2019).
3.4. D-Allulose production using immobilized PsDAE
Enzyme immobilization on covalent supports with epoxy groups
provides a good strategy to prevent the shedding and leakage of enzymes
in biocatalytic processes (Ai, Yang, Li, Shi, Wang, & Jiang, 2014), in
which the epoxy groups of polymer resins can covalently bind with the
amino groups of the enzymes. In this study, we immobilized PsDAE
using epoxy resin ES-103B and determined its catalytic activity in the
conversion of D-fructose into D-allulose. The immobilized PsDAE
6

C. Li et al.
Food Chemistry 357 (2021) 129746
Fig. 5. D-Allulose synthesis in fruit juices catalyzed by INV and immobilized GI and PsDAE with a two-step strategy. (a) The content of sucrose, D-frucose and D-
allulose in untreated and treated fruit juices, respectively; (b) The effect of consecutive cycle reaction on the conversion of high-calorie sugar to D-allulose via
immobilized GI and PsDAE within 15 reuses cycles.
3.5. Production of D-allulose from sucrose via a multienzyme cascade
150 g/L immobilized GI (Fig. 4a). This reaction system yielded 101 g/L
D-allulose, and the D-allulose content among total monosaccharides
reached 20.2% after 1.5 h (Fig. 4b). This was a significantly higher yield
than that reported in a previous study using immobilized cells express-
ing DAE and INV, which produced 75 g/L D-allulose from 500 g/L su-
crose (Yang et al., 2019). Thus, the highest yield of D-allulose was
obtained via a two-step strategy catalyzed by INV followed by immo-
bilized GI and PsDAE.
We designed a multienzyme cascade combining invertase, D-glucose
isomerase, and PsDAE to produce D-allulose from sucrose. The reaction
was analyzed by HPLC, and the retention times of D-glucose and sucrose
were 13.9 and 19.7 min, respectively (Fig. S3b). Notably, INV showed
high catalytic activity under acidic conditions at pH 4.2–4.5, while GI
and PsDAE were inactivated under these conditions, having maximal
catalytic activity at slightly alkaline pH of 7.5. Initially, sucrose hydro-
lysis was performed simultaneously with epimerization of D-fructose
using INV, GI, and PsDAE. However, only a low yield (32.5 g/L) of D-
allulose was obtained due to discordant reaction conditions (Fig. S4).
Therefore, the one-pot strategy was not a viable approach to produce D-
allulose.
3.6. Application of the two-step strategy for D-allulose production in fruit
juices in situ
Fruit juices often contain high concentrations of sucrose, D-glucose,
and D-fructose, which are considered high-calorie sugars that promote
obesity and associated negative health consequences (Wojcicki & Hey-
man, 2012). In the present study, to convert high-calorie sugar of fruit
juices into the functional alternative D-allulose, a variety of fruit juice
containing sucrose, D-glucose, and D-fructose, such as mango juice, or-
ange juice, and sugar cane juice were subjected to treatment using the
two-step approach utilizing INV, GI, and DAE.
Consequently, we developed a two-step strategy in which sucrose
was initially hydrolyzed by INV to produce D-fructose and D-glucose.
This reaction was completed within 2 h, yielding 250 g/L of D-fructose
and D-glucose. Subsequently, the pH of the reaction mixture was
adjusted to 7.5, and 50–200 g/L of immobilized GI and 20 g/L immo-
bilized PsDAE were added to convert D-glucose and D-fructose to D-
allulose. The highest yield of D-allulose from sucrose was obtained using
The sugar content in the initial fruit juices (untreated sample) and
7

C. Li et al.
Food Chemistry 357 (2021) 129746
fruit juices subjected to treatment using the multienzyme cascade sys-
tem (treated sample) was determined by HPLC. Under the optimal re-
action conditions, the INV combined with immobilized GI and PsDAE
enzymes was able to effectively transform the high-calorie sugars in the
different juices into D-allulose. The percentage of D-allulose among total
monosaccharides in mango juice, orange juice, and sugar cane juice
reached 16.4% (11.9 g/L), 17.7% (6.9 g/L), and 19.3% (23.2 g/L),
respectively (Fig. 5a). The concentrations of D-fructose and D-glucose
were increased due to the hydrolysis of sucrose in juice. D-Fructose was
further converted to D-allulose (low-calorie) and thus the total kcal value
of fruit juices was thought to be decreased. Furthermore, after hydrolysis
of sucrose in the first step, the immobilized GI and PsDAE enzymes were
reused for up to 15 cycles without exhibiting an unacceptable loss of
activity. As shown in Fig. 5b, after fifteen successive use cycles, the
content of D-allulose among total monosaccharides in mango juice, or-
ange juice, and sugar cane juice remained at approximately 16, 17, and
19%, respectively. Considering the food safety, no metals have been
added in reaction system for bioconversion of D-allulose in furit juice,
although both GI and PsDAE are metal dependent enzymes. These re-
sults confirmed the high application potential of the immobilized PsDAE
for converting high-calorie sugars in fruit juices into D-allulose, offering
functional products for diabetics and other special consumers. Future
work will be focused on the directed evolution of PsDAE to improve its
thermostability and bioconversion efficiency for the production of D-
allulose.
Ethics approval and consent to participate
No animals or human subjects were used in the above research.
Consent for publication
Our manuscript does not contain any individual data in any form.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.129746.
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