One-pot biocatalytic conversion of lactose to gluconic acid and galacto-oligosaccharides using immobilized β-galactosidase and glucose oxidase

Article abstract of DOI:10.1016/j.cattod.2020.06.090

One-pot cascade reaction systems offer several process and technological benefits, completed by the opportunity of the second enzyme to act on a convenient concentration of its substrate. Most of the reported cascade reactions involve immobilization of both biocatalysts on a single support, but in this case the catalytic performance of the whole system is affected if the activity of either enzyme drops. In this work, tailor-made immobilization of two β-galactosidases (from Kluyveromyces lactis and Aspergillus oryzae) and a glucose oxidase (from Aspergillus niger) was investigated, to subsequent utilization of the best performing immobilized biocatalyst of each type for the one-pot conversion of lactose to gluconic acid and galacto-oligosaccharides. The immobilization was performed by covalent binding onto amino-terminated Ni_0.4Co_0.2Zn_0.4Fe₂O₄ and Ni_0,5Zn_0,5Fe₂O₄ magnetic particles and commercial amino- and epoxy-functionalized methacrylate supports. The highest activity for β-galactosidases was achieved on Ni_0.5Zn_0.5-Fe₂O₄ magnetic particles, meanwhile the glucose oxidase showed higher affinity for the supports with epoxy active groups. The immobilized biocatalysts have been characterized in detail, demonstrating enhanced storage, pH, and thermal stability as well as better operational stability compared to the soluble enzymes.

Full text of DOI:10.1016/j.cattod.2020.06.090

Journal Pre-proof
One-pot biocatalytic conversion of lactose to gluconic acid and
galacto-oligosaccharides using immobilized ␤-galactosidase and glucose
oxidase
Anamaria Todea (Conceptualization) (Methodology) (Investigation)
(
Writing - original draft) (Writing - review and editing), Ioana Cristina
Benea (Investigation) (Writing - original draft), Ioan B ˆı tcan
Investigation) (Methodology) (Validation) (Formal analysis),
Francisc P e´ ter (Conceptualization) (Supervision) (Resources)
Validation) (Writing - original draft) (Writing - review and editing),
Szilvia Kl e´ bert (Investigation) (Methodology), Tivadar Feczk o´
(
(
(
Resources) (Validation) (Formal analysis), Zolt a´ n K a´ roly
(
Investigation) (Methodology), Emese Bir o´ (Investigation)
(Validation) (Writing - original draft) (Writing - review and editing)
PII:
S0920-5861(20)30532-0
DOI:
https://doi.org/10.1016/j.cattod.2020.06.090
CATTOD 13064
Reference:
To appear in:
Catalysis Today
Received Date:
Revised Date:
Accepted Date:
15 November 2019
7 June 2020
29 June 2020
Please cite this article as: { doi: https://doi.org/

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One-pot biocatalytic conversion of lactose to gluconic acid and galacto-oligosaccharides
using immobilized β-galactosidase and glucose oxidase
1
1
1
1
2
Anamaria Todea* , Ioana Cristina Benea , Ioan Bîtcan , Francisc Péter , Szilvia Klébert ,
2
,3
2
2
Tivadar Feczkó , Zoltán Károly , Emese Biró*
1Politehnica University of Timişoara, Faculty of Industrial Chemistry and Environmental
Engineering, Carol Telbisz 6, 300001 Timisoara, Romania, Email: anamaria.todea@upt.ro
2Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences,
Hungarian Academy of Sciences, Magyar Tudósok körútja 2, H-1117 Budapest, Hungary, Email:
emese.biro@gmail.com
3Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Egyetem
u. 10, H-8200, Veszprém, Hungary
Graphical abstract
Highlights

Experimental design of lactose conversion to gluconic acid in one-pot system
1




Novel immobilized β-galactosidases on Ni0.4Co0.2Fe
2
O
4
and Ni0,5Zn0,5Fe
2
O particles
4
High operational stability of the immobilized β-galactosidase and glucose oxidase
One-pot cascade system for synthesis of gluconic acid galacto-oligosaccharides
Abstract
One-pot cascade reaction systems offer several process and technological benefits, completed by
the opportunity of the second enzyme to act on a convenient concentration of its substrate. Most
of the reported cascade reactions involve immobilization of both biocatalysts on a single support,
but in this case the catalytic performance of the whole system is affected if the activity of either
enzyme drops. In this work, tailor-made immobilization of two β-galactosidases (from
Kluyveromyces lactis and Aspergillus oryzae) and a glucose oxidase (from Aspergillus niger) was
investigated, to subsequent utilization of the best performing immobilized biocatalyst of each
type for the one-pot conversion of lactose to gluconic acid and galacto-oligosaccharides. The
immobilization was performed by covalent binding onto amino-terminated Ni0.4Co0.2Zn0.4Fe
magnetic particles and commercial amino- and epoxy-functionalized
methacrylate supports. The highest activity for β-galactosidases was achieved on Ni0.5Zn0.5-Fe
2
O
4
and Ni0,5Zn0,5Fe
2
O
4
2
O
4
magnetic particles, meanwhile the glucose oxidase showed higher affinity for the supports with
epoxy active groups. The immobilized biocatalysts have been characterized in detail,
demonstrating enhanced storage, pH, and thermal stability as well as better operational stability
compared to the soluble enzymes.
Keywords: one-pot biocatalysis, β-galactosidase, glucose oxidase, immobilization, gluconic acid,
oligosaccharides
1
. Introduction
The continuously growing importance of industrial biocatalysis led to the wide development of
techniques for stabilization of the biocatalysts. Thus, immobilization of enzymes and whole cells
is recognized nowadays as a key strategy for developing efficient and economically viable
processes [1]. Reusability of the biocatalyst and maintenance of the activity for long period of
time is an important feature in applied biocatalysis, in terms of productivity increase and
reduction of the production costs [2, 3]. However, the enzyme immobilization can be considered
still a rather empiric technology. Therefore, the rational design on the immobilization protocols
2

will turn into a key issue to achieve stable, active, selective, and reusable enzymes [4]. This target
becomes even more important when two immobilized enzymes are exploited in the same reaction
system, as it happens in the case of one-pot cascade processes.
Enzyme-catalyzed cascade reactions have been increasingly studied in recent years. For reasons
such as selectivity, specificity, reduction of energy costs, lack of secondary reaction products,
mild reaction conditions and high yields, these systems can be effective solutions for the
replacement of chemical processes. Despite the existing achievements in the field of cascade
reactions, it is still necessary to develop new types of reaction systems for particular applications
[
5].
Whey is the principal by-product of dairy industry resulted from the manufacture of cheese and
casein from milk during the coagulation process. Although liquid whey is commonly considered
a waste product, it can be utilized as a cheap source of lactose and protein for food, dairy and
pharmaceutical industries. Lactose content in whey is in the range of 4.5-5% and it can be
directly fermented or hydrolyzed to glucose and galactose [6].

-Galactosidase (-GAL), mostly known as lactase, hydrolyzes lactose into glucose and
galactose in living organisms. The enzyme was already isolated from a variety of sources, but the
most important sources are fungi and yeast. β-GAL has potential applications in a wide array of
fields, such as food industry, bioremediation, biosensors, diagnostics and treatment of various
disorders. One of many industrial applications is the conversion of lactose to monosaccharides,
resulting lactose-free foods for lactose intolerant individuals [7]. β-GAL can also catalyze the
reverse synthetic reaction of transgalactosylation, leading to formation of galacto-
oligosaccharides (GOS) which contain a galactose unit chain with terminal glucose and are
considered prebiotic compounds [8, 9]. In aqueous systems transgalactosylation has to compete
with hydrolysis, therefore GOS mixtures always contain considerable amounts of remaining
lactose and monosaccharides [10] The synthesis of short-chain GOS by β-GALs has been
accomplished by different reaction systems, including whole cell systems, crude cell extracts,
purified enzymes, recombinant enzymes, and immobilized enzymes [11]. The structural analysis
of GOS produced by β-GAL from Aspergillus oryzae and Kluyveromyces lactis revealed a degree
of polymerization (DP) ranging from 1 to 7, with DP2 and DP3 as the dominant fractions [12].
Increasing the stability and allowing reuse of β-GAL, immobilization could be an important tool
to enhance the efficiency of both lactose hydrolysis and GOS synthesis processes. Several
3

immobilization methods were reported, including covalent immobilization on various inorganic
materials [13], or entrapment in sol-gels [14]. β-GALs produced by three microorganisms,
Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans, were most intensively studied
for immobilization. Several structural characteristics, like number of subunits (if multimeric),
location of the active center(s), molecular size, distribution of the amino acid residues on the
surface, should be considered for an appropriate immobilization strategy [15].
Gluconic acid (GA) is a multifunctional organic acid, belonging to the aldonic acid family and
used as a bulk chemical in the food, feed, pharmaceutical, textile, metallurgy, detergent, paper,
and construction industries [16]. Production methods of gluconic acid include chemical,
electrochemical, bioelectrochemical, and biotechnological methods. Since 1976 there are several
patents related to GA production and most include enzymatic routes [17]. Glucose oxidase
(GOX) is the enzyme that catalyzes the oxidation of β-D-glucose to gluconic acid, by utilizing
molecular oxygen as an electron acceptor, with simultaneous production of hydrogen peroxide as
side product [18]. GOX was immobilized on various supports, including porous glass silanized
with 3-aminopropyl-trimethoxysilane. Co-immobilization of GOX and catalase was also
investigated, catalase cleaving the formed hydrogen peroxide to oxygen and water [19].
Encouraged by the cascade metabolic processes existing in the living cells, we investigated in this
work an artificial biomimetic microsystem for lactose conversion. Two biocatalysts, β-GAL and
GOX, were tailor-made immobilized, characterized in detail, and their efficiency was evaluated
in a one-pot reaction system, to yield two important products, GA and GOS. The study is focused
on the demonstration of a novel concept for lactose conversion into GA and GOS using a one-pot
system with native and immobilized enzymes. Mixed spinel ferrites like Ni-Zn- and Ni-Zn-Co-
ferrites were for the first time used as magnetic cores for the covalent immobilization of these
enzymes. Another important goal was to demonstrate that the formation of GA or GOS products
can be directed as function of β-galactosidase source, immobilization method, and substrate
concentration.
2
. Experimental
2
.1. Materials
β-Galactosidases (β-GAL) from Kluyveromyces lactis and Aspergillus oryzae, gluconic acid,
stachyose, 4-aminoantipyrine, o-nitrophenyl-β-galactoside, glutaraldehyde, hydrogen peroxide
3
5%, horseradish peroxidase (POX) were supplied from Sigma-Aldrich (St. Louis, USA).
4

Glucose oxidase (GOX) from Aspergillus niger, glucose, lactose, phenol, peroxidase, bovine
serum albumin (BSA) were obtained from Merck (Darmstadt, Germany). Galactose, raffinose,
trehalose, glutaraldehyde solution 50% in water, K
2
HPO
4
, KH
2
PO were acquired from Fluka
4
(Buchs, Switzerland). Amino and epoxy-butyl methacrylate resins used as immobilization
supports were a generous donation of Purolite (Llantrisant, South Wales, UK). A mixture of
oligosaccharides from corn syrup was provided by Phenomenex (Torrance, CA, USA), allowing
the identification of oligosaccharides up to 9 units.
2
.2. Preparation of the magnetic particles
The polycrystalline powders of Ni0.4Co0.2Fe
chemical coprecipitation route [20]. For the synthesis analytical grade precursors of
Ni(NO ·6H O, Zn(NO ·6H O, Co(NO ·6H O and Fe(NO ·9H O were used, all
purchased from VWR International (Radnor, PA, USA). The precipitated powders were dried at
05°C, and then calcined at 1000°C for 2 h. The size distribution of the obtained powders was
determined by a HORIBA LA 950 A2 laser particle size analyzer (HORIBA Scientific, USA).
.3. Immobilization of β-galactosidase and glucose oxidase
2
O
4
and Ni0,5Zn0,5Fe
2
O were synthesized by the
4
3
)
2
2
3
)
2
2
3
)
2
2
3
)
3
2
1
2
All immobilization and biocatalysis experiments were carried out using an Eppendorf
Thermomixer Comfort heating shaker (Eppendorf, Hamburg, Germany).

onto magnetic particles
β-GALs from Kluyveromyces lactis, Aspergillus oryzae and glucose oxidase (GOX) from
Aspergillus niger were immobilized by covalent binding onto Co0.4Ni0.2Zn0.4Fe and
magnetic particles, which were previously activated by using 3-aminopropyl-
trimethoxysilane (NH -TMOS), or 3-aminopropyl-triethoxysilane (NH -TEOS). The
2
O
4
Ni0.5Zn0.5Fe
2
O
4
2
2
immobilization was performed using a method adopted from the Sigma-Aldrich protocol (I 7643)
for magnetic particles functionalized with amino groups. The magnetic particles were
equilibrated by washing three times with coupling buffer 0.01 M pyridine, pH 6.0, then the
supernatant was aspirated by magnetic separation. The coupling agent, 5% glutaraldehyde
solution, was added, and the magnetic particles were subjected to continuous stirring for 3 hours
at 1000 rpm and 25°C. The glutaraldehyde activated particles were then washed three times with
the same coupling buffer. To the activated magnetic particles 1 mL of enzymatic solution was
added, the mixture was stirred for 24 hours at 1000 rpm and 25°C, then the supernatant was
aspirated and washed three times with coupling buffer pH 6.00 and once with wash buffer pH
5

7
.40 (0.01 M Tris Base containing 0.15 M NaCl, 0.1% (w/v) bovine serum albumin, 0.001 M
EDTA, sodium salt and 0.1 % (w/v) sodium azide), by shaking and supernatant aspiration.
onto amino methacrylate beads

All three selected enzymes were immobilized through covalent binding onto amino
functionalized beads. The supports were washed two times with coupling buffer 0.01 M pyridine,
pH 6.00, through shaking and supernatant aspiration. The activation of the supports was
performed by using 1 mL of 5 % glutaraldehyde solution as a linker, followed by three hours
incubation at 25°C and 1000 rpm. The activated supports were further washed two times with
coupling buffer pH 6.00, and 1 mL of glucose oxidase solution was added. The immobilization
was carried out at 25°C and 1000 rpm for 24 hours. The supernatant was removed and the
immobilized enzyme was washed two times with the coupling buffer pH 6.00 and one time with
wash buffer pH 7.40 ( 0.01 M Tris Base containing 0.15 M NaCl, 0.1% (w/v) bovine serum
albumin, 0.001 M EDTA, sodium salt and 0.1 % (w/v) sodium azide).

onto epoxy-butyl methacrylate beads
-1
1
mL enzyme solution (0.75-3 mg ml ) in 50 mM phosphate buffer pH 7 was added to 25 mg
epoxy-butyl methacrylate resin. The mixture was incubated at 1000 rpm, 24 h, at 25ºC. The beads
were removed from the mixture by filtration, washed as described in the previous section and
stored at 4ºC until further use.
2
.4. Activity assay of immobilized β-galactosidase
Hydrolysis of the ONPG substrate

The catalytic activity of the immobilized β-GALs was assessed on the capacity of the enzymes to
hydrolyze the artificial substrate o-nitrophenyl-β-galactoside (ONPG), to galactose and o-
nitrophenol. In an Eppendorf tube 25 mg of immobilized enzyme, 900 μL phosphate buffer 0.02
M, pH 7.50 and 100 μL ONPG solution 30 mM were added. After 10 minutes of incubation of
the mixture at 37°C and 1000 rpm, the absorbance was measured at 420 nm.

Hydrolysis of the lactose substrate
The hydrolysis reaction was carried out in 0.02 mol/L potassium phosphate buffer pH 6 at 30ºC,
00 rpm, adding 50 mmol/L lactose and 25 mg preparate. The mixtures were incubated for 1 h
3
and the hydrolyzed lactose was quantified as glucose release. The amount of D-glucose in the
presence of D-galactose was measured in each sample spectrophotometrically, using the coupled
enzyme assay with GOD and POD, as described in section 2.5.
6

2
.5. Activity assay of glucose oxidase
The activity of immobilized glucose oxidase was assayed according to the method of Lott and
Turner [21], adding to 25 mg of immobilized biocatalyst 920 μL phosphate buffer 0.02 M, pH
7
.50, 160 μL phenol 0,128 M, 160 μL 4-aminoantipyrine 20 mM, and 100 μL glucose 25 mM.
The mixture was stirred at 25 °C and 1000 rpm. After 20 min the supernatant was separated from
the immobilized glucose oxidase and 160 μL peroxidase solution 1 mg/mL was added.
Subsequent to 1 h incubation, the absorbance was measured at 505 nm.
2
.6. HPLC analysis
The analysis of mono-, di- and oligosaccharides, as well of GA, was carried out by HPLC (Jasco
HPLC system, equipped with PU-2089 Plus quaternary pump and RI-2031 Plus Intelligent
detector, Jasco International Co., Japan), using a Rezex RSO Ag+ 4%, LC Column, 200x10mm
oligosaccharides column (Phenomenex, Torrance, CA, USA). The elution was performed by
water as mobile phase, at 0.3 ml/min flow rate and 85C column temperature. This column
allowed the separation and identification of all components of the reaction system, lactose,
glucose, galactose, GA and GOS. Sugar and oligosaccharide standards (as stated in the materials
part) were used for the qualitative identification and quantitation.
2
.7. Influence of the enzyme loading
The correlation between the catalytic activity and the amount of the protein subjected to
immobilization was examined by immobilizing different amounts of native enzymes, at initial
protein amounts varying between 3-12% (mg protein/ mg support). The activity assays were
performed as described in sections 2.4 (ONPG substrate) and 2.5. The protein loading was
calculated as the percentage ratio of the immobilized protein (protein subjected to immobilization
x immobilization yield) and the amount of the immobilized support. The protein content in both
the solution subjected to immobilization and the solution remaining after the immobilization was
determined by the Bradford method. Unloaded protein was detected only at the highest amount of
enzymes subjected to immobilization (120 mg protein/ g support). In all other cases, the
immobilization yield was considered 100%.
2
.8. pH profiles of the native and immobilized enzymes
The influence of pH on the activity of native and immobilized β-GAL and GOX was evaluated in
the pH range 4-9 in 9 steps, by using a broad range pH buffer solution containing citric acid,
7

boric acid and trisodium phosphate [22]. The activity assays were performed as described in
sections 2.4 (ONPG substrate) and 2.5.
2
.9. Temperature profiles of the native and immobilized enzymes
Incubation of the native/immobilized enzymes was carried out for 1 h in the absence of the
substrate at different temperatures, in the 30-60ºC range, followed by cooling on ice for 10 min.
The residual activity of the enzymes was determined as described in sections 2.4 and 2.5.
2
.10. Reuse of the biocatalysts
The activity of immobilized β-GAL and GOX was determined after repeated use of the
biocatalyst at 30ºC. For β-GAL the reusability studies were performed for two substrates, ONPG
and lactose. For GOX the reactions were performed using glucose as substrate. After each cycle,
the immobilized enzyme was separated and washed 3 times with the buffers. Fresh buffer and
substrate were added, the activity of the reused biocatalyst was assayed as described in sections
2
.5 and 2.6, and the resulted value was compared to the first run (defined as 100%).
2
.11. The influence of H
2
O
2
concentration on β-GAL activity
The effect of different H
2
O
2
concentrations on β-GAL activity was evaluated for the native
enzyme and the enzyme immobilized on magnetic supports functionalized with amino groups.
The recovered activity of the native and immobilized enzyme was determined as described in
section 2.4 (ONPG).
2
.12. Initial-rate studies and steady-state kinetic analysis
The reactions were performed at 30°C in phosphate buffer, at optimal pH values, and were
monitored by the product formation (glucose and gluconic acid), in discontinuous assays. The
typical concentrations of β-GAL and GOX were 5 mg/mL and 10 mg/mL, respectively, and 1 h
incubation time was used. The substrate concentrations were varied in a suitable range: lactose (1
-
600 mM), glucose (1- 600 mM). Control reactions lacking the enzyme or one of the substrates
were performed in all cases, and the initial rates were corrected as required. Kinetic parameters
(Vmax, K
m
) were obtained from non-linear fits of data to equation (1). kcat is defined in equation
(2).
S

 Vmax

(1)
(2)
K  S
m
Vmax
k 
cat
E
8

where ν is the observed reaction rate [mM/min], Vmax is the maximal initial rate [mM/min], S is
the substrate concentration [mM], K is the apparent Michaelis-Menten constant [mM], kcat is the
m
-1
catalytic constant [s ] and E is the total molar enzyme concentration based on a molecular mass
of 105 kDa for the β-GAL [23] and 160 kDa for GOX [24].
2
.13. Experimental design and data evaluation
The experimental design and the analysis of the results were carried out using the Unscrambler
.2 and Statistica 10 software package (CAMO Software AS, Oslo, Norway), and the Box-
9
Behnken method [14], choosing three center samples. For each variable 3 different levels (the
lowest, the central and highest of their fixed domain) were given.
2
.14 Time course of the one-pot lactose conversion process
The reactions were performed in 2 mL Eppendorf tubes using native and immobilized β-GAL
and GOX in phosphate buffer at optimal reactions conditions determined based on the
experimental design results: temperature, 43.5C; pH, 6.7; β-GAL amount (expressed as units),
1
7.62 U; GOX amount (expressed as units), 15U. Six different lactose concentrations were tested
40-165 mM) and samples were taken after 3, 6, 20, 24, 48, 72 h. The lactose conversion,
gluconic acid and oligosaccharides formations were monitored by HPLC as described in section
.6. All the reactions were performed in duplicate and the results were given as mean value. The
standard deviations values do not exceed 5%. Control reactions were performed in all cases.
. Results and discussion
(
2
3
There is increasing interest toward valorization of sugars, demonstrated by the high number of
patents and publications in the field. This research was carried out to convert lactose into
valuable products, GA and GOS, using β-GAL and GOX. Both enzymes were demonstrated to be
versatile biocatalysts for the individual reactions, opening the possibility for their utilization in a
one-pot system (Fig. 1). However, determination of the optimal reaction conditions for the
simultaneous action of these enzymes, as well as specific optimization of the immobilization
conditions, were firstly needed.
3
.1. Optimization of lactose conversion using native β-galactosidase and glucose oxidase by
experimental design.
Optimization of the parameters that influence the lactose hydrolysis and glucose oxidation in the
one-pot system was performed using the Box-Behnken design. Six independent variables
9

(temperature, pH, reaction time, substrate concentration, β-galactosidase and glucose oxidase
amounts) were selected and the maximum and minimum limits were set based on previous
experimental results in the batch mode. For each variable 3 different levels (the lowest, the
central and highest of their fixed domain) were given. The resulted set of 51 experiments is
presented in Table 1S (Supplementary material). The selected response variables were the
conversion of lactose and the gluconic acid yield (the results are shown in Table 1S, as well).
The obtained experimental data were subjected to statistical analysis by the ANOVA technique,
analysis of effects, and response surface analysis. Among the predefined models available in the
Unscrambler package, the higher multiple correlation value of 0.917 was achieved for the
quadratic model. The results of the statistical analysis (p-values, t-test and the confidence limits
for each effect) when productivity was selected as response variable (Table 2S, Supplementary
material) indicate p-values lower than 0.05 for the GOX activity values, demonstrating that the
model is statistically significant, and the variables are correlated. The p-value for the lack of fit
was found to be 0.051, which indicated that lack of fit was not significant and showed the
goodness of fit.
The pure error, i.e. the mean square of deviations of the gluconic acid yield values, measured at
the central points from their mean value, was 1.19 from 3 repetitions, corresponding to a √1.19 =
1
.09% standard deviation measured at the central point of variables, a satisfactory value.
The normal probability plot of residuals (Figure 1S, Supplementary material) show that the data
are normally distributed, indicating acceptable goodness of fit.
Response surface analysis. The regression surface was plotted, in order to determine the
influence of time, pH and temperature on the lactose conversion and gluconic acid yield. The
response surface analysis (Fig. 2, Fig 2S - 4S, Supplementary material) indicates that the optimal
values for the synthesis of gluconic acid as main product in the one-pot system, using native β-
GAL and GOX, are: temperature, 43.5C; pH, 6.7; initial lactose concentration, 63.75 mM;
reaction time, 3.16 h; β-GAL amount (expressed as units), 17.62 U; GOX amount (expressed as
units), 15U.
3
.2. Increasing the stability of the biocatalysts by tailored immobilization
Beyond the possibility of multiple enzyme utilization, immobilization can lead to enhancement of
the activity, as a result of stabilization of the protein conformation. Therefore, the appropriate
selection of both support and immobilization parameters became increasingly important [9].
1
0

Immobilization by covalent bonding offers important advantages compared to other methods
concerning the stability against leaching, which can be an essential issue during long-term
exploitation [10].
3
.2.1. Screening of supports for covalent immobilization
Four solid supports were selected: two magnetic supports containing different amounts of Co, Ni
and Zn (not reported as immobilization supports up to now), and two methacrylate resins bearing
amino and oxirane functional groups. Even though several supports were used for β-GAL
immobilization, there are few reports related to covalent immobilization onto magnetic particles.
Compared to the well-known Fe
3
O
4
magnetic particles, these Ni-Zn or Ni-Zn-Co based magnetic
particles spinel ferrites (MFe
2
O
4
) with different metallic cations (M: Zn, Mn, Co, Cr, Ni) have
attracted interest due to their magnetic properties like superparamagnetism, spin glass behavior,
with a range of applications in different fields such as magnetic recording, high frequency
electronic cores, biomedical applications [25].
The magnetic particles were obtained by the co-precipitation method, as described in section 2.2.
The size distribution of the particles was measured by laser particle size analyzer (Figure 5S,
Supplementary material), showing that for both samples the particles average diameter was less
than 10 µm. The magnetic parameters of the powders, measured by vibrating sample
magnetometer (VSM), are presented in Table 1. The VSM values indicate that for the Co
containing samples the magnetic parameter values are slightly higher.
To be suitable for immobilization, the magnetic particles were functionalized with amino surface
groups, using 3-aminopropyl trymethoxysilane. Glutaraldehyde was used as linker for all amino
activated supports. The immobilization experiments were carried out for all three selected
enzymes, and the resulted activities are presented in Table 2. Among β-GALs, the highest
activity values were obtained for the β-GAL from Aspergillus oryzae covalently attached to the
magnetic supports, while for GOX, the activity of the immobilized biocatalyst was higher when
the enzyme was attached onto the epoxy-functionalized support. The results confirm the different
“affinity” of the biocatalyst for the tested supports. The somewhat surprising low activity of β-
GAL immobilized on supports functionalized with epoxy groups was reported by other groups,
too [26]. Immobilization onto supports with epoxy functional groups involves a covalent binding
preceded by a hydrophobic adsorption which might develop a certain inactivation of the
hydrophilic native enzyme or destabilization due to some negative interactions may take place.
1
1

Therefore, β-GAL immobilized on amino functionalized Ni-Zn magnetic particles and GOX
immobilized onto epoxy-functionalized methacrylate resin beads were selected for the following
experiments. These solid supports will also allow an easy individual separation of the
immobilized catalysts from the reaction system.
All data were obtained based on duplicate values at least. Since the activities were lower and the
reproducibility of the experiments was not adequate in case of the β-GAL from Kluyveromyces
lactis, the immobilized β-GAL from Aspergillus oryzae was selected for the following
experiments. Although the catalytic activity is not directly influenced by the magnetic core, the
stability and long-term efficiency can be improved by using mixed spinnel ferrites, therefore
these results are promising for the future utilization of such magnetic particles.

Effect of the silane precursors and the coating time.
The aim of this study was to increase the immobilized enzyme activity, investigating the effect of
the silane precursors and the coating time. Two aminopropyl silanes, NH -TMOS and NH
TEOS, were considered for surface coating of the magnetic particles. The particles were
2
2
-
incubated in the presence of the silane at 70C, for 24 h and 48 h. Longer incubation time led to
the visible agglomeration of the particles. The immobilization of β-GAL onto the amino coated
supports was performed as described in section 2.3. The resulted activity values, determined
using ONPG as substrate (Table 3S, Supplementary material) indicate that the activity was about
5
5% higher when the coating time was longer (48 h). Thus, 48 h coating time and NH
coating reagent were selected for the following experiments.
.2.2. Influence of the loaded protein
2
-TMOS as
3
The optimal amount of protein subjected to immobilization was evaluated by using five different
protein amounts, resulting in 3 to 12% percentage of protein related to the support. Among the
previously tested supports, Ni0.5Zn0.5Fe
2
4
O was used for the immobilization of β-GAL from
Aspergillus oryzae, and the epoxy-terminated beads for GOX. The enzyme loading capacity was
evaluated by calculation of the immobilization yield, based on the initial protein amount and the
protein amounts found after the immobilization in the supernatant and in the washing solutions.
The results are presented in Table 3. In almost all cases the protein immobilization yield (assayed
by the Bradford method) was higher than 99%. A slight decrease of the immobilization yield was
observed only at 12% protein subjected to immobilization, (96.4% for β-GAL and 94.3% for
GOX). An increase of the activity values was observed up to 10% protein loading, directly
1
2

correlated to the protein amount. Only at protein loadings above 11%, a small increase of the
activity was observed for β-GAL and even a decrease for GOX, probably due to the multipoint
immobilization effect.
3
.2.3. Influence of immobilization on the pH profile of -GAL and GOX
Both pH and temperature studies were carried out with the immobilized enzymes which
demonstrated the highest activities in the previous studies, -GAL covalently attached to
Ni0,5Zn0,5Fe
2
O
4
magnetic particles, surface-coated with NH
MG2-βGAL), and GOX covalently attached to butyl-epoxy polymethacrylate beads, at 10%
protein loading (EPX-GOX), respectively.
2
-TMOS, at 10% protein loading
(
pH is one of the most important parameters influencing the enzymatic activity, particularly at
extreme pH values. As a result of pH changes, the protein structure may suffer general unfolding,
or the ionic state of amino acids catalytic triad could be affected. Usually, native enzymes present
activity in a narrow pH range, but this inconvenience could be solved by immobilization. In this
study, the pH profiles of native and immobilized -GAL and GOX were assayed in the pH unit
ranges of 4 to 8, and 4 to 9, respectively.
The results indicate the improvement of the pH profiles for both biocatalysts subsequent to
immobilization (Fig. 3a and 3b). The immobilized β-GAL maintained its highest activity up to
5
.5, and at pH 6.5 the activity was 55% of the maximal value. The covalent binding had a
positive effect for GOX, too. In a broad pH range (from 5.5 to 9) the activity of the immobilized
EPX-GOX was at least 80% of the maximal value. Similar effect of pH on enzyme activity was
reported when β-GAL from Aspergillus oryzae was immobilized onto chitosan magnetic
particles. The enhanced stability in alkaline conditions was explained by multipoint covalent
immobilization when the β-GAL was fixed on the surface of carriers, increasing the tolerability
of enzyme to pH variance in surroundings [27]. The GOX immobilization was not intensively
studied and there are only a few reports related to the full characterization of the immobilized
biocatalyst. However, compared to the previously reported data related to GOX immobilization
by covalent binding onto amino-supports [28], and onto poly(2-hydroxyethyl methacrylate)
(pHEMA) membranes [29] the immobilized GOX obtained in this study presents higher stability
in a broader range of pH values. A similar trend of pH stability was observed by Jaquish et al. for
GOX covalently attached onto silica magnetic particles [30].
3
.2.3. Influence of immobilization on the temperature profile of -GAL and GOX
1
3

Thermal inactivation of enzymes represents a major impediment for the industrial utilization of
biocatalysts. For several enzymes, the thermal stability has been improved by immobilization.
The irreversible activity losses at increasing temperature occur probably due to the
conformational changes at tertiary structure level. The effect of the temperature on the activity of
the native and covalently immobilized -GAL and GOX was investigated at temperatures in the
3
0-60°C range, by incubation for 2 h. The activities were expressed as percentage of the highest
value. The results (Fig. 4a and 4b) indicate a significant increase of the thermal stability for both
covalently attached biocatalysts (MG2-βGAL and EPX-GOX), particularly at temperatures
higher than 40C. At 50C, the native GOX lost 80% of the initial activity, while the immobilized
GOX still preserved about 75% of this value. In the case of β-GAL, the relative activity
percentages at the same temperature were 60% for the native and 95% for the immobilized
enzyme, respectively.
Previous reports about cross-linking of β-GAL from Aspergillus niger on magnetic beads indicate
comparable values at 60C for enzyme immobilized on magnetic Fe
3
4
O -chitosan nanoparticles.
Compared to other reported data concerning the thermal stability of GOX immobilized onto
epoxy resin [18], and onto poly(2-hydroxyethyl methacrylate) membranes [29], the thermal
stability of the immobilized GOX obtained in this study was about 20% and 60% higher,
respectively.
3
.2.4. Operational stability in repeated uses of the immobilized biocatalyst
The best-performing immobilized biocatalysts were used in consecutive reaction cycles, at the
optimal pH values for the ONPG and lactose hydrolysis (MG2-βGAL) and glucose oxidation
(EPX-GOX), respectively. The biocatalysts were recovered from the reaction mixture after each
batch reaction cycle through magnetic/filtration separation, were washed 4 times with phosphate
buffer, and reused in another reaction. Ten reaction cycles were performed using MG2-βGAL as
biocatalyst and ONPG as substrate. The results (Fig. 6S, Supplementary material) indicate
excellent stability of the biocatalyst, as about 85% of the activity was recovered after 10 reaction
cycles. The increase of the operational stability of MG2-βGAL in multiple uses was also
demonstrated for the lactose substrate (Fig. 5). After 5 reaction cycles, more than 90% of the
initial activity was recovered. The results are in concordance with previously reported values for
magnetic polysiloxane-polyvinyl alcohol particles, where the activity after 10 reaction cycles was
about 84% [10].
1
4

The operational stability of the EPX-GOX biocatalyst in repeated glucose oxidation reactions
was also excellent, the recovered activity values after 5 reaction cycles exceeding 90% (Fig. 5).
Other authors reported similar reusability results after 5 glucose oxidation cycles for Fe
3
O
4
-SiO -
2
GOX, obtained by covalently attachment through glutaraldehyde [30]. From our knowledge,
there are no other reports related to the operational stability of GOX immobilized on carriers with
pendant epoxy groups.
3
.2.5. The stability of -GAL in presence of H
2
O
2
Development of an efficient one-pot system for lactose conversion must also consider the
presence of hydrogen peroxide as the by-product of the glucose oxidation reaction and its
possible inhibitory effect on the other enzyme present in the reaction system. Therefore, the
effect of H
concentrations, in the range 25-100 mM. The results indicate an acceptable tolerability of the
native enzyme to H (less than 20% activity loss) and even an improvement of the stability in
the presence of H after immobilization compared to the native enzyme (Figure 7S,
Supplementary material). At higher H concentration (100 mM), the immobilized MG2-βGAL
lost only about 5% of the initial activity.
.2.6. Initial-rate studies and steady-state kinetic analysis
2
O
2
on the native and immobilized MG2-βGAL galactosidase was investigated at four
2
O
2
2
O
2
2
O
2
3
The kinetic parameters for the native and immobilized enzymes were considered useful to
evaluate the effect of the immobilization on the accessibility of the catalytic site for the substrate.
The initial rate values and Michaelis-Menten constants were determined for the natural substrates
of the investigated enzymes. The immobilized enzymes (MG2-βGAL and EPX-GOX) were
incubated at 30C in the presence of various initial concentrations of lactose and glucose,
respectively. The lactose concentration was varied in 20 levels and the glucose in 21 levels.
Based on the Michaelis-Menten plots, in all cases a saturated dependence of the initial rate on
substrate concentration was observed. Thus, an individual determination of the catalytic constant
k
1
cat and Michaelis constant K
m
was realized using the Linewearver-Burk plot and the SigmaPlot
1.0 software package. The K
m
values of 82.75 mM of native β-GAL from Aspergillus oryzae
and 58.68 for GOX from Aspergillus niger (Table 4) were in concordance with previous reports
for the lactose [31] and glucose substrates [30]. The K values increased after the
m
immobilization compared to the native enzymes, indicating that higher substrate concentrations
are probably needed due to conformation changes of the enzyme induced by the covalent binding
1
5

(Table 4). The catalytic efficiency for both enzymes was about two-fold lower after
immobilization, but for GOX is at least two times higher compared to previously reported kinetic
parameters for covalently bound GOX [30].
3
.3. Time course of the one-pot lactose conversion process
Investigation of the time course of lactose conversion was considered important to determine the
reaction time needed to achieve the highest efficiency for production of GA as main reaction
product and GOS as secondary products. A previous report indicated that the presence of glucose
has inhibitory effect for GOS synthesis [32]. In this context, using GOX to convert the glucose to
gluconic acid could favor the GOS formation.
Previously, the ionic strength of the buffer was evaluated by adding different amounts of
gluconic acid to the buffer solution. The results (Figure 8S, Supplementary material),
demonstrated that important pH changes did not occur, as they were less than 0.2 pH units, at
gluconic acid concentrations up to 0.2 M.
The time-course studies of the one-pot reactions were performed at six different substrate
concentrations (from 40 to 165 mM), using native and immobilized biocatalysts. The reaction
parameters were set at the optimal values resulted from the experimental design.
Using the native enzymes, after 48 h the lactose was completely converted and the gluconic acid
yield exceeded 50%, at concentrations lower than 120 mM (Table 5). At higher lactose
concentrations the GOS yield increased and the degree of polymerization was also higher. The
highest gluconic acid yield was obtained at 40 mM, and even at this low concentration 3-unit
oligosaccharides were formed. The highest GOS yield was obtained at 165 mM lactose
concentration, when up to 6-unit oligosaccharides were synthesized. A similar increase of GOS
(tri- and tetra-saccharides) production at increasing lactose concentration was observed by Neri et
al. [10]. This effect probably corresponds to a reduced availability of water as alternative
galactosyl acceptor [32].
As it was expected, using the immobilized biocatalysts (MG2-βGAL and EPX-GOX) higher
reaction time was needed for lactose conversion, and high conversions were reached only after 20
h of reaction (Table 6). Compared to the native multienzyme system, the formation of GOS was
affected, but higher gluconic acid yields were achieved. At lower initial substrate concentrations,
up to 80 mM, higher than 90% lactose conversions were obtained and the gluconic acid yields
exceeded 80%, gluconic acid and galactose being the main products identified at the end of the
1
6

reaction. The presence of GOS was observed only in the experiments with higher initial substrate
concentration. The chain elongation was favored at increased reaction time, the presence of 4-unit
GOS being observed after 48 h. Based on the outcome of the time course study, the efficiency of
the immobilized biocatalyst was demonstrated, and the process can be considered able for
upgrading at continuous level.
The formation of GOS only at prolonged reaction time and the absence of oligosaccharides
higher than 4-units were also observed by Gaur et al, when β-GAL from A. oryzae immobilized
by covalent binding to chitosan was used [33].
These results demonstrate the viability of the one-pot concept, using immobilized biocatalysts,
for the studied system. Among the possible immobilization methods, covalent binding is more
suitable, because the reaction media is aqueous and leaching of the enzyme from the support is
avoided. Since the stability of the investigated enzymes is different, two different immobilization
supports were tested, allowing the selection of the best solution for every case. If the activity of
one enzyme is affected, this enzyme can be replaced, meanwhile the other can be further used,
allowing the development of a cost-effective biocatalytic process.
Future scale-up studies and will be connected with an appropriate downstream processing, which
needs a comprehensive investigation. At this moment, the eco-friendly production of high purity
gluconic acid still remains a challenge, due to the involvement of multiple downstream
processing units [34]. However, by coupling with GOS synthesis, the downstream separation
could have the advantage of less secondary products present in the system.
4
. Conclusions
Efficient immobilized biocatalysts, able to work in a one-pot system, were obtained by covalent
binding of β-GAL and GOX onto different supports. The covalent immobilization of β-GAL on
Ni-Zn and Ni-Co-Zn ferrite magnetic nanoparticles, surface-coated with amino-polysiloxane, was
achieved for the first time, opening the perspective of a new class of magnetic supports. Thermal,
pH and storage stabilities were improved following the immobilization. The immobilized
biocatalysts were successfully reused in seven reaction cycles, showing excellent operational
stability. One-pot biocatalytic systems are very flexible, allowing the possibility of maintaining
the concentration of the intermediate product at low level. The one-pot biocatalytic valorization
of lactose into GA and GOS was achieved with good yields, demonstrating the feasibility of this
approach. Moreover, the obtained results open the possibility to extend it to other biocatalytic
1
7

cascade reactions. The best system performance for GA synthesis was obtained at lower substrate
concentrations.
CRediT author statement
All authors contributed to this research work, as follows:
Anamaria Todea: Conceptualization, Methodology, Investigation, Writing - Original Draft,
Writing - Review & Editing
Ioana Cristina Benea: Investigation, Writing - Original Draft
Ioan Bîtcan: Investigation, Methodology, Validation, Formal analysis
Francisc Péter: Conceptualization, Supervision, Resources, Validation Writing - Original Draft,
Writing - Review & Editing
Szilvia Klébert: Investigation, Methodology
Tivadar Feczkó: Resources, Validation, Formal analysis
Zoltán Károly: Investigation, Methodology
Emese Biró: Investigation, Validation, Writing - Original Draft, Writing - Review & Editing
Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by a grant of the Romanian Authority for Scientific Research and
Innovation, CNCS/CCCDI - UEFISCDI, project number PN-III-P2-2.1-PED-2016-0168, within
PNCDI III and partially supported by BIONANO_GINOP-2.3.2-15-2016-0-0017.
This work was supported by a grant of the Romanian Ministery of Research and Innovation,
project number 10PFE/16.10.2018, PERFORM-TECH-UPT, within Program 1- Development of
the national system of Research and Development, Subprogram 1.2 - Institutional Performance -
Institutional Development Projects - Excellence Funding Projects in RDI, PNCDI III
The authors acknowledge Dr. Simona Șerban from Purolite Ltd. for the kind donation of amino
methacrylate and epoxy-butyl methacrylate resins.
1
8

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2
0

Legend of Figures
Figure 1. Lactose conversion scheme into gluconic acid and galacto-oligosaccharides using a
combination of β-galactosidase and glucose oxidase in a one-pot system.
2
1

Figure. 2. (a) The combined effect of temperature and pH on the lactose conversion; (b) The
combined effect of temperature and pH on the gluconic acid yield
2
2

Figure 3. (a) pH profiles of native (black) and covalently immobilized (purple) β-GAL from
Aspergillus oryzae; (b) pH profiles of native (blue) and covalently immobilized (green) GOX
from Aspergillus niger
2
3

Figure 4. Thermal stabilities of (a) native (black) and covalently immobilized (purple) β-GAL
from Aspergillus oryzae (b) native (blue) and covalently immobilized (green) GOX from
Aspergillus niger
2
4

Figure 5. Stability of β-galactosidase immobilized on magnetic particles MG2-βGAL (violet)
and glucose oxidase immobilized on epoxy-functionalized methacrylate resin EPX-GOX (green),
in repeated batches of conversion of lactose and glucose, respectively
2
5

Table 1
Magnetic parameters of the magnetic ferrite nanoparticles containing Ni-Zn and Ni-Co-Zn,
measured by vibrating sample magnetometer
Particle type
Saturation
Remnant
Coericivity
magnetization,
Ms (emu g )
magnetization,
Mr (emu g )
-1
-1
Hc (Oe/g)
54
Ni0,5Zn0,5Fe
2
O
4
53
1,44
Ni0,4Co0,2Zn0,4Fe
2
O
4
56
2,10
110
Table 2
Influence of the support nature and functionalization on the activity of the immobilized enzymes
-1 -1
Entr Support
y
Immobilized enzyme activity [mol h g ]
β-GAL from β-GAL from GOX from A.
A. oryzae
K. lactis
niger
1
2
3
4
(Ni0,4Co0,2Zn 0,4 Fe
2
O
4
)-3NH
2
TMOS 146.6
37.07
41.92
(Ni 0,5Zn 0,5Fe
2
O
4
) -3NH
2
TMOS
321.8
222.7
136.5
30.15
42.16
28.99
30.35
62.04
92.60
NH
2
-functionalized beads
Epoxy-functionalized beads
2
6

Table 3
Influence of the protein loading on the activity of the immobilized biocatalysts. β-GAL from A.
oryzae was immobilized on Ni0.5Zn0.5Fe
2
4
O magnetic particles and GOX from A. niger was
immobilized on epoxy-butyl methacrylate beads
β-GAL from A. oryzae
GOX from A. niger
Immobilized enzyme
Protein
Immobilized enzyme Protein*
*
-1 -1
loading [%]
activity [µmol h g ] loading [%]
activity [µmol h-1g-1]
3
92.2
3
26.4
5
7
129.3
239.1
320.7
328.3
5
30.7
64.1
93.4
86.2
7
1
1
0
10
11.2
1.6
*
calculated as the percentage ratio of the immobilized protein (protein subjected to immobilization  immobilization yield) and the amount of the
immobilized support.
2
7

Table 4
Influence of immobilization on the kinetic parameters of the β-galactosidase and glucose oxidase
Kinetic parameter
Native β-GAL Immobilized
MG2-βGAL
Native GOX
Immobilized
EPX-GOX
156.07
K
m
[mM]
82.75
604
101.15
425
58.68
366.67
6.2
-
1
k
k
cat [s ]
cat/K
386.67
-
1 -1
m
[L mmol s ]
7.29
4.2
2.5
2
8