New alkali tolerant β-galactosidase from Paracoccus marcusii KGP – A promising biocatalyst for the synthesis of oligosaccharides derived from lactulose (OsLu), the new generation prebiotics

Article abstract of DOI:10.1016/j.bioorg.2021.105207

The enzyme β-galactosidase can synthesise novel prebiotics such as oligosaccharides derived from lactulose (OsLu) which can be added as a supplement in infant food formula. In this study, the intracellular β-galactosidase produced by the alkaliphilic bacterium Paracoccus marcusii was extracted and purified to homogeneity using hydrophobic and metal affinity chromatography. The purification resulted in 18 U/mg specific activity, with a yield of 8.86% and an 18-fold increase in purity. The purified enzyme was a monomer with an 86 kDa molecular weight as determined by SDS PAGE and Q-TOF-LC/MS. β-Galactosidase was highly active at 50 °C and pH 6–8. The enzyme displayed an alkali tolerant nature by maintaining more than 90% of its initial activity over a pH range of 5–9 after 3 h of incubation. Furthermore, the enzyme activity was enhanced by 37% in the presence of 5 M NaCl and 3 M KCl, indicating its halophilic nature. The effects of metal ions, solvents, and other chemicals on enzyme activity were also studied. The kinetic parameters K_M and V_max of β-galactosidase were 1 mM and 8.56 μmoles/ml/min and 72.72 mM and 11.81 μmoles/ml/min on using oNPG and lactose as substrates. P. marcusii β-galactosidase efficiently catalysed the transgalactosylation reaction and synthesised 57 g/L OsLu from 300 g/L lactulose at 40 °C. Thus, in this study we identified a new β-galactosidase from P. marcusii that can be used for the industrial production of prebiotic oligosaccharides.

Full text of DOI:10.1016/j.bioorg.2021.105207

Bioorganic Chemistry 115 (2021) 105207
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
New alkali tolerant β-galactosidase from Paracoccus marcusii KGP – A
promising biocatalyst for the synthesis of oligosaccharides derived from
lactulose (OsLu), the new generation prebiotics
Pooja Kalathinathana, Krishnakanth Pulicherlab, Avtar Sainc,
Sankaranarayanan Gomathinayagama, Rama Jayarajd, Suresh Thangaraje,
,
*
Gothandam Kodiveri Muthukaliannana
a School of BioSciences and Technology, Vellore Institute of Technology, Tamil Nadu, India
b Department of Science and Technology, Government of India, New Delhi, India
c Centre for Bio-Separation Technology, Vellore Institute of Technology, Tamil Nadu, India
d Theme lead, Flinders NT, Flinders University, Northern Territory 0909, Australia
e Department of Chemistry, Bharathiar University, Coimbatore, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
The enzyme β-galactosidase can synthesise novel prebiotics such as oligosaccharides derived from lactulose
(OsLu) which can be added as a supplement in infant food formula. In this study, the intracellular β-galactosidase
produced by the alkaliphilic bacterium Paracoccus marcusii was extracted and purified to homogeneity using
hydrophobic and metal affinity chromatography. The purification resulted in 18 U/mg specific activity, with a
yield of 8.86% and an 18-fold increase in purity. The purified enzyme was a monomer with an 86 kDa molecular
weight as determined by SDS PAGE and Q-TOF-LC/MS. β-Galactosidase was highly active at 50 ◦C and pH 6–8.
The enzyme displayed an alkali tolerant nature by maintaining more than 90% of its initial activity over a pH
range of 5–9 after 3 h of incubation. Furthermore, the enzyme activity was enhanced by 37% in the presence of 5
M NaCl and 3 M KCl, indicating its halophilic nature. The effects of metal ions, solvents, and other chemicals on
enzyme activity were also studied. The kinetic parameters KM and Vmax of β-galactosidase were 1 mM and 8.56
Oligosaccharides synthesised from lactulose
(OsLu)
Prebiotics
β-galactosidase
Paracoccus marcusii
Phenyl hydrophobic chromatography
Immobilised metal affinity chromatography
Zymogram analysis
Enzyme characterisation
μ
moles/ml/min and 72.72 mM and 11.81 μmoles/ml/min on using oNPG and lactose as substrates. P. marcusii
β-galactosidase efficiently catalysed the transgalactosylation reaction and synthesised 57 g/L OsLu from 300 g/L
lactulose at 40 ◦C. Thus, in this study we identified a new β-galactosidase from P. marcusii that can be used for the
industrial production of prebiotic oligosaccharides.
1. Introduction
digestibility of OsLu may be due to the greater endurance of galactosyl
fructose to host digestion compared to galactosyl glucose present in
Prebiotics are food ingredients that improve the host’s health by
selectively enhancing the growth and activity of a few colon bacteria
[1]. These prebiotic food ingredients are mainly confined to non-
digestible oligosaccharides, consisting of three and nine saccharide
units. Oligosaccharides derived from lactulose (OsLu) are gaining more
attention due to the growing interest in new prebiotic oligosaccharides.
OsLu was found to have remarkable beneficial effects on the gastroin-
testinal system. The trisaccharides of OsLu showed more resistance to
gut digestion than the trisaccharides derived from lactose [2]. The low
galactooligosaccharides (GOS) [3]. In addition to having a higher pre-
biotic index and bifidogenic properties, OsLu was also found to enhance
iron absorption and possess anti-inflammatory and immunomodulatory
properties.
OsLu is synthesised by the transgalactosylation activity of β-galac-
tosidase using lactulose as its substrate. During the transgalactosylation
reaction, the lactulose molecule covalently attaches to the β-galactosi-
dase and forms a galactose-enzyme intermediate by releasing one
molecule of fructose. The galactose-enzyme complex then reacts with
* Corresponding author at: 244, High-throughput Screening Lab, Department of Biotechnology, School of BioSciences and Technology, Vellore Institute of
Technology, Vellore, Tamil Nadu 632014, India.
E-mail address: k.m.gothandam@vit.ac.in (G. Kodiveri Muthukaliannan).
https://doi.org/10.1016/j.bioorg.2021.105207
Received 3 December 2020; Received in revised form 18 July 2021; Accepted 21 July 2021
Available online 26 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

P. Kalathinathan et al.
Bioorganic Chemistry 115 (2021) 105207
Fig. 1. A schematic representation of the catalytic mechanism of β-galactosidase in the synthesis of OsLu.
another molecule of lactulose to form a trisaccharide [4]. This trisac-
charide may also act as a substrate, leading to the formation of tetra-
saccharides and so on. Thus, oligosaccharides containing (galactosyl)n
lactulose units, with n varying from 2 to 10, are formed. A schematic
representation of the catalytic mechanism of β-galactosidase in the
synthesis of OsLu is shown in Fig. 1. The source of β-galactosidase is a
critical variable in the degree of polymerisation and yield of OsLu [4–6].
The increasing demand for prebiotics and the zest to synthesise new
prebiotic oligosaccharides has led to the search for novel β-galactosi-
dases of commercial importance. In our previous study, we isolated
β-galactosidase producing alkali tolerant Paracoccus marcusii KGP from
water samples collected along the coast of the Bay of Bengal. The bac-
terium KGP showed preliminary probiotic properties and was able to
utilise whey as a substrate for its growth with good β-galactosidase
production potential [7]. The bacterium was then optimised and char-
acterised for higher production of β-galactosidase. This β-galactosidase
was found to hydrolyse lactose into glucose and galactose efficiently and
synthesise GOS from lactose [8]. Furthermore, the β-galactosidase
enzyme extracted from P. marcusii KGP could hydrolyse 47% of lactose
in whey into sugars, glucose and galactose revealing the potential
application of this enzyme in whey bioremediation [7]. Considering its
industrial significance, the whole-genome of P. marcusii was sequenced
and submitted to DDBJ/ENA/GenBank under the accession number
VINR00000000 [9].
synthesise OsLu using lactulose as a substrate was evaluated.
2. Materials and methods
2.1. Bacterial strain and culture condition
P. marcusii KGP was initially grown in Zobell marine broth supple-
mented with 2% w/v lactose at 28 ◦C for 72 h and used as the starter
culture. 5% (v/v) inoculum was then transferred into a 3.1 L stirred tank
bioreactor containing 2 L production medium (12.5 g/L yeast extract,
12.5 g/L galactose, and 12.5 mM MgSO4), pH 7. The culture was
maintained at 28 ◦C with an agitation speed of 250 rpm and harvested
after 48 h to obtain cells growing in the exponential phase [8].
2.2. β-Galactosidase assay
β-Galactosidase activity was determined using ortho-nitrophenyl-β-D-
galactopyranoside (oNPG) as the substrate. 0.1 mL of enzyme solution
was added to 0.2 mL of oNPG (8.3 mM) and incubated at 50 ◦C for 5 min.
The reaction was then stopped by adding 0.5 mL of Na2CO3 (1 M), and
the absorbance was measured at 420 nm. One unit of β-galactosidase
activity is defined as the amount of enzyme that liberated 1 μmol of o-
nitrophenol (oNP) per minute under the reaction conditions. β-Galac-
tosidase activity was calculated using the following formula:
In the present work, the intracellular β-galactosidase from P. marcusii
KGP was extracted and purified to homogeneity using chromatographic
techniques. The β-galactosidase was then characterised extensively to
analyse the effect of various physical and chemical parameters on its
catalytic efficiency. The kinetic properties of β-galactosidase were also
analysed. Furthermore, the transgalactosylation ability of the enzyme to
Units/m L = (OD420 × dilution factor × RV) / (Є × t × v)
RV is the reaction volume in mL, v is the volume of culture in mL, t is
the incubation time in min, and Є is the molar absorptivity of oNP, which
is equal to 4.6 × 10-3 Mꢀ 1 cmꢀ 1
.
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P. Kalathinathan et al.
Bioorganic Chemistry 115 (2021) 105207
2.3. Extraction and fractionation of β-galactosidase
incubated in Z-buffer containing 1 mg/mL X-gal at 50 ◦C for 60 min or
until the appearance of a blue colour band, which corresponds to the
enzyme β-galactosidase [14]. The proteins can be visualised on the
native page by running the replica of the same gel along with a standard
protein and staining with CBB R-250.
P. marcusii bacterial cells from 2 L of culture broth were harvested by
centrifuging the cells for 10 min at 10,000 rpm. The cells were sus-
pended in 400 mL of 50 mM phosphate buffer at pH 7. The pelleted cells
were disrupted using a sonicator at an amplitude of 25% for 5 min. Cell
debris was pelleted, and cell-free supernatant was used as a crude
enzyme solution [10].
2.6. Determination of molecular weight using QTOF LC/MS
The crude enzyme was fractionated in a two-step ammonium sul-
phate precipitation process carried out at 4 ◦C with continuous stirring
overnight. In the first step, 25% salt was added to the crude enzyme and
the precipitated proteins were removed by centrifugation. In the next
step, 50% ammonium sulphate was added to the supernatant, and the
precipitated β-galactosidase was re-suspended in 50 mL phosphate
buffer. The ammonium sulphate salt in the protein suspension was
removed by dialysing the sample against the same buffer [11].
The molecular weight of the purified β-galactosidase was confirmed
by QTOF LC/MS. The gel slices of β-galactosidase were excised from a
Coomassie-stained SDS-PAGE gel and sent to Agilent Research Labora-
tories, India. The sample was dissolved in 0.1% formic acid in water,
vortexed, centrifuged, and injected into a 1290 Infinity II UHPLC using
an Agilent PLRP-S and Advance Bio Peptide Map column and MS Source
and Acquisition parameters.
2.7. Determination of factors influencing enzyme activity and stability
2.4. Purification of β-galactosidase
The factors affecting β-galactosidase activity, such as temperature,
pH, NaCl, metal ions, protein denaturants, hydrolytic products, and
solvents, were analysed using oNPG as the substrate. In each assay, the
relative enzyme activity was determined by comparing the β-galactosi-
dase activity with that of the control [15].
The fractionated protein sample was purified by hydrophobic
interaction chromatography and immobilised metal affinity chroma-
tography (IMAC) using an automated, fast pressure liquid chromatog-
raphy (FPLC) system (Akta Purifier, GE Healthcare). 1 ml of the protein
sample was loaded onto a Hi-Trap Phenyl FF hydrophobic column (1
mL, GE Healthcare). The column was pre-equilibrated with phosphate
buffer (50 mM, pH 7) with 0.75 M ammonium sulphate. Unbound
proteins were eluted in the flow-through using the same equilibration
buffer. The bound proteins were eluted using phosphate buffer (50 mM,
pH 7) without salt in a stepwise gradient. A flow rate of 1 mL/min was
maintained throughout the experiment. The flow-through and elution
were collected in 1 mL fractions, and the β-galactosidase activity of each
fraction was analysed. The fractions showing enzyme activity were
pooled, de-salted, and subjected to the second step of purification using
2.7.1. Effect of temperature
The optimum temperature for β-galactosidase activity was deter-
mined by incubating the enzyme and substrate (oNPG) mixture for 5 min
over a range of temperatures (10–100 ◦C) in increments of 10 ◦C. The
thermostability was studied by pre-heating β-galactosidase at different
◦
temperatures (30–100 C) for different periods (1–100 h), and the re-
sidual β-galactosidase activity was calculated.
2.7.2. Effect of pH
The optimum pH was determined by measuring the activity of
β-galactosidase diluted in buffers with different pH values (citrate buffer
(100 mM, pH 3–5), phosphate buffer (100 mM, pH 6–8), carbonate
buffer (100 mM, pH 9–11), and KCl/NaOH buffer (100 mM, pH 12)).
The pH stability of β-galactosidase was analysed by incubating the
enzyme in the buffers mentioned above (pH 3–11) for 3 h at 37 ◦C.
Samples were collected at different time periods, and the residual
β-galactosidase activity was measured.
a Hi-Trap IMAC HP column (5 mL, GE Healthcare) charged with Cu2+
.
The column was equilibrated with phosphate buffer (50 mM, pH 7), with
0.5 M NaCl at 1 mL/min. The sample (5 mL) was loaded onto the col-
umn, and unbound proteins were collected using the equilibration
buffer. The bound proteins were eluted using an imidazole gradient in
the same buffer. The 1 mL fractions collected from the flow-through and
elution were assayed for β-galactosidase activity and total protein con-
centration (OD280 nm). Fractions exhibiting β-galactosidase activity
were combined and dialysed.
2.7.3. Effect of salt
The total protein concentration was measured using the Bradford
assay [12]. The specific activity, yield, and purification fold were
calculated using the following formulae:
The effect of salt was investigated by adding β-galactosidase to a
phosphate buffer (pH 7) containing different concentrations of NaCl
(1–5 M) and KCl (1–3 M). Enzyme activity was measured, and activity
without any salt was used as a control. The salt stability of β-galactosi-
dase was evaluated by incubating the enzyme in a buffer containing 1 M
to 5 M NaCl and buffer without salt for 24 h. The residual β-galactosidase
activity was assayed using a buffer without salt as a control.
Specific activity (U/m g) = β ꢀ Galactosidase activity
/Total protein concentration
Yield(%) = (Total enzym e activity of sam ple
/Total enzym e activity of crude enzym e) × 100
2.7.4. Effect of metal ions
The effect of metal ions on β-galactosidase activity was evaluated in
the presence of the corresponding chloride ions at concentrations of 10
mM and 100 mM. Metal chlorides of ammonium, barium, calcium, co-
balt, cadmium, lithium, magnesium, manganese, sodium, and potassium
were prepared in phosphate buffer (50 mM, pH 7) and used in this study.
Purification fold = Specific activity of the sam ple
/Specific activity of the crude enzym e
2.5. SDS PAGE, native PAGE, and zymogram
10 microlitres of enzyme was added to 90
was performed as described previously.
μL of metal ions, and the assay
The purity of the enzyme at each purification step was analysed by
10% SDS-PAGE as described by Laemmli [13]. The molecular weight of
β-galactosidase was determined using a standard molecular weight
marker (10–250 kDa). β-Galactosidase activity was analysed by zymo-
gram analysis. The samples were loaded onto 10% native gel. After
completion of electrophoresis, the native gel was immersed in Z-buffer
(0.06 M Na2HPO4⋅7H2O, 0.04 M NaH2PO4⋅H2O, 0.01 M KCl, 0.001 M
MgSO4⋅7H2O, and 0.05 M β-mercaptoethanol) for 1 h. The gel was then
2.7.5. Effect of protein denaturants
Chemicals such as EDTA, SDS, urea, guanidium hydrochloride,
β-mercaptoethanol, and thiourea at concentrations of 100 mM and 1 M
were used to determine their influence on β-galactosidase activity. The
chemicals were suspended in a phosphate buffer (50 mM, pH 7). The
assay in the absence of chemicals was used as a control.
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P. Kalathinathan et al.
Bioorganic Chemistry 115 (2021) 105207
Table 1
Purification summary of β-galactosidase from P. marcusii KGP.
Purification step
Total activity (U)
Total protein (mg)
Specific activity (U/mg)
Yield (%)
Purification fold
Crude extract
194.43 ± 0.76
159.6 ± 4.07
33.89 ± 0.58
17.23 ± 0.11
191.96 ± 8.5
74.02 ± 2.05
6.11 ± 0.24
0.94
1.01 ± 0.04
2.15
100
1
Ammonium sulphate precipitation
Hydrophobic chromatography
Metal affinity chromatography
82.08 ± 1.77
17.43 ± 0.23
8.86 ± 0.09
2.12 ± 0.1
5.47 ± 0.38
18.06 ± 0.99
5.54 ± 0.12
18.29 ± 0.12
2.7.6. Effect of hydrolytic products
higher oligosaccharide production [2].
The inhibitory effects of the hydrolysis products of lactose on
β-galactosidase activity were analysed. 0.2 M to 1 M concentrations of D-
glucose and D-galactose were added individually to β-galactosidase, and
the activity was assayed.
The presence of oligosaccharides was examined qualitatively by
loading the samples onto a thin layer chromatography (TLC) plate. A
mobile phase consisting of n-propanol, ethyl acetate, and water at a
volume ratio of 7:1:2, was used to separate the carbohydrates. The
components were then detected using 10% H2SO4 followed by heating at
◦
95 C [17]. The spots developed were identified using glucose, galac-
2.8. Enzyme kinetics
tose, lactose, fructose, and lactulose as standards.
The samples showing the high intensity of the prebiotic spots in TLC
were selected and quantified using an HPLC system ((Shimadzu, Japan)
equipped with an autosampler and a refractive index detector (RID). A
Steady-state kinetic analysis was performed by varying the concen-
tration of substrates oNPG (0.2 mM to 25 mM) and lactose (20 mM to
500 mM) while maintaining a constant enzyme concentration (5 U).
Different concentrations of substrates were prepared using 50 mM
phosphate buffer, pH 7. The oNPG assay was performed as described
Shim-pack GIST NH2 column (250 × 4.6 mm, 5
μm) with an oven
temperature of 40 ◦C was used to separate the carbohydrates. A solution
of acetonitrile and water in a ratio of 75:25 (v/v) was used as the mobile
phase. The samples were injected and then eluted in isocratic mode at a
flow rate of 1 mL/min. The elution fractions were collected and stored at
4 ◦C until further characterisation studies. The carbohydrates were later
identified by comparing the retention times of the peaks with those of
standard sugars, and the amount of OsLu was quantified based on the
calibration curves of the standard sugars [2,18].
previously. In the case of lactose as substrate, 100
μL of β-galactosidase
was mixed with 900
μ
L of lactose solution and incubated at 50 ◦C for 15
min, and the samples were boiled for 3 min to stop the enzymatic re-
action. The amount of lactose present was then calculated by measuring
the amount of glucose liberated during the reaction using the GOD-POD
assay kit (ARKRAY, Inc.). The lactose hydrolysis rate was then calcu-
lated using the following formula [16]:
OsLu yield (g/L) = Initial lactulose – (rem aining lactulose + fructose +
% Lactose hydrolysed = [(CLi – CLf) / CLi] × 100
glucose).
where CLi is the initial lactose concentration in mg/L, and CLf is the final
lactose concentration in mg/L.
The oligosaccharides (OsLu) purified by HPLC from the reaction
◦
mixture incubated at 40 C for 24 h was analysed using (1D) proton
The data obtained were then fitted to the Michaelis-Menten equation
using non-linear regression using GraphPad Prism (GraphPad Software,
Inc., CA, USA).
NMR spectroscopy. The elution fractions corresponding to the oligo-
saccharide peak from multiple runs were pooled and lyophilised. The
lyophilised sample was initially exchanged with 300 µL of deuterated
water twice and then dissolved in 800 µL of deuterated water (D2O,
Sigma Aldrich). NMR spectra with water suppression were recorded
with a probe temperature of 25 ◦C on an Avance Neo Bruker 400 MHz
spectrometer and were visualised using Topspin software (v 4.1.1).
2.9. Synthesis and characterisation of OsLu
The transgalactosylation efficiency of β-galactosidase in producing
OsLu was determined by adding 200 μL of β-galactosidase (5 U) to 800
μ
L of lactulose (in phosphate buffer, pH 7) and incubating the reaction
2.10. Statistical analysis
mixture at an optimum temperature. The 1 mL reaction mixture was
then boiled at 100 ◦C for 3 min to stop the transgalactosylation reaction.
The samples were centrifuged at 13,000 rpm for 10 min and stored at 4
◦C for subsequent analysis. The process conditions, such as substrate
concentration (200–400 g/L lactulose), incubation temperature (40 ◦C
and 50 ◦C), and incubation time (12 and 24 h), were optimised for
All the assays were carried out in triplicate, and their mean, standard
deviation, and error bars were calculated, and the graphs were plotted
using GraphPad Prism™ 6.0 (GraphPad Software Inc., USA).
Fig. 2. (a) SDS PAGE (b) Native PAGE and (c) zymogram analyses of the fractions obtained during sequential purification of β-galactosidase from P. marcusii KGP.
Lane M - pre-stained protein molecular marker, lane 1 - cell lysate, lane 2 - ammonium sulphate precipitated fraction, lane 3 - fraction obtained from hydrophobic
chromatography, and lane 4 - fraction obtained from IMAC.
4

P. Kalathinathan et al.
Bioorganic Chemistry 115 (2021) 105207
Fig. 3. Intact mass determination of β-galactosidase using QTOF LC/MS.
Fig. 4. (a) Effect of temperature on β-galactosidase activity (b) Effect of temperature on β-galactosidase stability over different periods (c) Effect of pH on
β-galactosidase activity (d) Effect of pH on β-galactosidase stability (e) Effect of different concentrations of salts (NaCl and KCl) on β-galactosidase activity (b) Effect
of different concentrations of NaCl on β-galactosidase stability over 12 h and 24 h.
3. Results
The results also indicated that P. marcusii β-galactosidase exists as a
monomer with an apparent molecular weight of 86 kDa. The QTOF LC/
MS spectrum showed an 86 kDa intact mass of the protein β-galactosi-
dase (Fig. 3), confirming the SDS-PAGE results. Nonetheless, the
β-galactosidase gene from Paracoccus sp. 32d, expressed in Escherichia
coli LMG194 cells, exists as a homodimer with a molecular weight of 160
KDa [19]. The literature reveals that β-galactosidase from different
sources exists in monomeric, dimeric, or oligomeric forms with different
molecular weights.
3.1. Purification of β-galactosidase and molecular weight determination
The alkali tolerant β-galactosidase was purified to homogeneity in 3
steps: initial fractionation by ammonium sulphate precipitation fol-
lowed by hydrophobic chromatography and metal affinity chromatog-
raphy. β-galactosidase in the crude enzyme extract had a specific activity
of 1 U/mg, and after purification, the β-galactosidase was 18-fold pu-
rified with a specific activity of 18.29 U/mg and a recovery rate of 8.8%.
The overall purification results are presented in Table 1.
3.2. Determination of factors influencing enzyme activity and stability
A single band of approximately 86 kDa was observed in SDS-PAGE
(Fig. 2 (a)), confirming the purity and homogeneity of β-galactosidase.
Furthermore, the β-galactosidase band on the native PAGE (Fig. 2(b))
developed a blue colour during the zymogram assay, indicating that the
purified β-galactosidase is in an enzymatically active form (Fig. 2(c)).
β-galactosidase was characterised in terms of its catalytic activity
and stability to exploit its commercial importance. The optimum tem-
◦
perature of β-galactosidase was found to be 50 C (Fig. 4(a)), while a
significant loss of catalytic activity occurred at temperatures higher than
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P. Kalathinathan et al.
Bioorganic Chemistry 115 (2021) 105207
Fig. 5. (a) Effect of different concentrations of (a) metal ions (b) hydrolytic products (glucose and galactose) and (c) protein denaturants.
60 ◦C. Interestingly, the enzyme exhibited 28% relative activity even at
10 ◦C, indicating the cold-active nature of the enzyme. In the stability
study, β-galactosidase was stable at 40 ◦C and below even after 24 h
(Fig. 4(b)), whereas the enzyme activity decreased rapidly at 50 ◦C after
15 min of incubation. Similarly, the β-galactosidase from Paracoccus sp.
32d exhibited maximum activity and stability at 40 ◦C and 30 ◦C,
respectively [19]. The thermal properties of P. marcusii KGP β-galacto-
sidase have been reported for enzymes from Pediococcus acidilactici and
Enterobacter ludwigii [20,21].
concentration of 4 M after 12 h of incubation, retaining high levels of
residual activity. However, after 24 h of incubation, the enzyme activity
was slightly reduced to 92% and 85% in the presence of 3 M and 4 M
NaCl, respectively. When the NaCl concentration was further increased
to 5 M, a decline in the enzyme activity of approximately 12% and 40%
was observed after 12 h and 24 h, respectively (Fig. 4(f)). Previous re-
ports have shown that β-galactosidases from Marinomonas sp. BSi20414
lost 70% of its initial activity within 1 h of incubation [24], whereas
β-galactosidase from Aspergillus tubingensis GR1 expressed halotolerance
by maintaining 100% relative activity at 5 M NaCl [25]. Thus, β-galac-
tosidase, isolated from P. marcusii KGP, is considered a salt-adapted
enzyme.
The pH profile of β-galactosidase showed that the enzyme exhibited
maximum activity in the pH range of 6–8. Even at pH 5 and 9, the
enzyme showed 78% and 90% relative activity, respectively (Fig. 4(c)).
Similarly, during the pH stability study, the enzyme retained more than
90% activity when incubated at pH 5–9 for 3 h but lost activity
completely under acidic conditions, even within 15 min of incubation
(Fig. 4(d)). The results suggest that β-galactosidase is appropriate for the
milk industry, where the pH of sweet whey and milk is between 6 and 7.
Compared to the same genus, the results are almost similar to that of
β-galactosidase from Paracoccus sp. 32d, except that our enzyme main-
tains more than 90% activity even at pH 9, exhibiting a higher alkali
tolerance [19]. A similar pH profile was also reported for β-galactosidase
from Teratosphaeria acidotherma, which has an optimum pH of 8 and is
found to be stable at pH 7.5–10, maintaining more than 80% activity
[22]. It was observed that most β-galactosidases from bacterial sources
had a neutral pH [23]. Due to this stable behaviour of P. marcusii KGP
β-galactosidase at the pH mentioned above, the enzyme is considered to
be alkali-tolerant. Therefore, β-galactosidase from P. marcusii KGP is one
of the very few alkali tolerant β-galactosidases reported to date.
The halotolerance study showed that β-galactosidase activity
increased with increasing salt concentration (Fig. 4(e)). The enzyme
activity was strongly enhanced by the salts NaCl and KCl, and it was
observed that at 3 M KCl and 5 M NaCl, the enzyme activity increased by
37%. The enzyme also displayed extraordinary stability even at a salt
Metal ions play a crucial role in maintaining the active configuration
of enzymes at higher temperatures. In this study, among the various
metal ions tested at a concentration of 10 mM, only Ba, Ca, and Mn
enhanced β-galactosidase activity by 21%, 29%, and 45%, respectively,
whereas Cd and Mg reduced the enzyme activity to 78% and 57%,
respectively. Other ions such as ammonium, cobalt, and lithium did not
significantly affect β-galactosidase activity. When the concentration of
metal ions was increased to 0.1 M, there was no significant effect on
β-galactosidase activity in the presence of potassium, lithium, sodium,
and ammonium ions, whereas all other ions completely arrested the
enzyme activity (Fig. 5(a)). Considering these results, it is essential to
reduce free Ca2+ levels in solutions during the application of enzymes in
the dairy industry. Unfortunately, there are no reports on the influence
of any chemicals or metal ions on the activity of β-galactosidases from
the same genus or species for comparison. However, the results can be
compared with those obtained using β-galactosidases from other bac-
terial sources. Similar to our study, the β-galactosidase from Exiguo-
bacterium acetylicum has a reduction in its activity in the presence of
Mg2+ ions [26], and the β-galactosidase from Teratosphaeria acidotherma
is reported to be enhanced by Mn2+ ions [22]. Alikunju et al. reported
that the 465 kDa β-galactosidase from Enterobacter ludwigii was inhibited
Fig. 6. Determination of kinetic parameters (KM and Vmax) of β-galactosidase by Michaelis –Menten plot with (a) oNPG as the substrate (b) lactose as the substrate.
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P. Kalathinathan et al.
Bioorganic Chemistry 115 (2021) 105207
Fig. 7. (a) TLC plate showing the synthesis of OsLu at different temperatures over different time intervals when n-propanol, ethyl acetate, and water (7:1:2) was used
as the mobile phase : (1–5: standard sugars) (1) glucose, (2) galactose, (3) lactose, (4) fructose, (5) lactulose; (6–9: samples) (6) 40 ◦C for 12 h, (7) 40 ◦C for 24 h, (8)
50 ◦C for 12 h, (9) 50 ◦C for 24 h; (b) Chromatographic profile of the carbohydrates present in the reaction mixture incubated at 40 ◦C for 24 h, as analysed by
HPLC-RID.
by Ca2+ ions and enhanced by Mn2+, Mg2+, and K+ ions [21]. Ding et al.
found that the activity of β-galactosidase from Marinomonas sp.
BSi20414 is stimulated up to 200% of its relative activity upon the
addition of Fe2+ ions [24].
with oNPG as the substrate were found to be 1 mM and 8.56 μmoles/ml/
min, respectively (Fig. 6(a)). Similarly, when lactose was used as the
substrate, the KM and Vmax of the enzyme were 72.72 mM and 11.81
μ
moles/ml/min, respectively (Fig. 6(b)).
A study on the effect of glucose and galactose on β-galactosidase
activity was assayed, and it was found that both glucose and galactose
are effective inhibitors of β-galactosidase. As shown in Fig. 5(b), enzyme
inhibition increased with an increase in sugar concentration, and
glucose was found to be a stronger inhibitor than galactose. The enzyme
lost 25% of its maximal activity at 400 mM glucose, whereas a similar
loss of activity was observed only at 600 mM galactose. The activity of
P. marcusii KGP β-galactosidase is more advantageous than the
β-galactosidase from Lactobacillus plantarum, which is much more sen-
sitive to these products [27].
The kinetic data from the present study indicate substrate inhibition
by oNPG. In contrast, enzyme activity was not inhibited by its natural
substrate lactose up to 300 mM. The β-galactosidase from P. marcusii has
a lower KM with oNPG than with lactose, indicating a higher enzyme
affinity for the synthetic substrate oNPG than the natural substrate
lactose. The KM value obtained using oNPG was within the range (less
than 15 mM) reported for most β-galactosidases belonging to the GH42
family. In contrast, the KM value reported using lactose is different from
that of β-galactosidases from other sources [28]. The KM value of the
enzyme for lactose was found to be higher than that of Bifidobacterium
and Arthrobacter species but comparatively lower than that of Bacillus
licheniformis, Aspergillus niger, and Picrophilus torridus [28–30]. This low
In the case of different additives on enzyme activity, there were no
significant changes in enzyme activity at lower concentrations (Fig. 5
(c)). However, at a concentration of 1 M, guanidium hydrochloride, and
thiourea decreased the enzyme activity to 59% and 53%, respectively.
Interestingly, it was found that β-galactosidase activity was unaffected
by urea and β-mercaptoethanol, even at 1 M concentration. In addition
to protein denaturants, Tris buffer at a concentration of 0.1 M reduced
the enzyme activity by 46%.
K
M value of β-galactosidase from P. marcusii suggests the potential use of
this enzyme in the production of oligosaccharides and lactose
utilisation.
3.4. Synthesis and characterisation of OsLu
Lactose has been mainly used as a substrate for the production of
galactooligosaccharides. However, many studies have proven that lac-
tulose is also an excellent substrate for the enzymatic synthesis of oli-
gosaccharides using β-galactosidase. In this study, the capacity of the
β-galactosidase isolated from P. marcusii KGP to synthesise oligosac-
charides from lactulose was analysed. From the TLC plates, it was
inferred that OsLu could be produced at both 40 ◦C and 50 ◦C (Fig. 7(a)).
The topmost spot corresponds to the monosaccharides formed as a result
of lactulose hydrolysis, and the spots formed below the lactulose spots
3.3. Enzyme kinetics
The catalytic performance of an enzyme can be measured using the
kinetic parameters KM and Vmax. KM is the substrate concentration which
represents the affinity of the enzyme to the substrate. A lower KM value
indicates a higher affinity of the enzyme to its substrate and vice versa.
V
max represents the limiting velocity as the substrate concentration in-
creases. In this study, KM and Vmax of β-galactosidase from P. marcusii
7

P. Kalathinathan et al.
Bioorganic Chemistry 115 (2021) 105207
Fig. 8. Overlay of water suppressed NMR spectra of galactose (Red), lactulose (Green) and OsLu (Blue) obtained from fractions purified by HPLC from the reaction
mixture incubated at 40 ◦C for 24 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
might correspond to oligosaccharides OsLu with a DP ≥ 3. It was also
observed that the intensity of OsLu spots was almost the same when the
lactulose concentration was varied from 200 g/L to 400 g/L (data not
shown).
4. Conclusion
The alkali tolerant β-galactosidase from P. marcusii KGP has enzy-
matic properties that are more advantageous for industrial applications
than other enzymes as this enzyme can be utilised from mildly alkaline
to neutral pH regions, active at higher salt concentrations and tolerant to
various chemicals and reagents. The transgalactosylation efficiency of
the enzyme in producing OsLu from lactulose was also demonstrated.
Thus, this study reveals that the alkali tolerant β-galactosidase from
P. marcusii is a potent candidate for producing prebiotic carbohydrates
in industries. Further studies on the structure and properties of oligo-
saccharides may elucidate the novelty of these oligosaccharides.
On further quantitative analysis of the samples by HPLC, an un-
identified high retention time (13.4 min) oligosaccharide was detected
in the HPLC chromatogram, which was considered as OsLu. It was
observed that the lactulose concentration decreased, and the amount of
oligosaccharides increased in the reaction mixture as the reaction pro-
gressed for up to 24 h. The concentration of monosaccharides increased
simultaneously in the reaction mixture, wherein the amount of fructose
was higher than that of galactose. These results indicate that lactulose
was initially hydrolysed into galactose and fructose, and the released
galactose was used in the transgalactosylation reaction to form OsLu. It
was found that the highest amount of OsLu (57 g/L) was formed when
300 g/L lactulose was used at an incubation temperature of 40 ◦C after
24 h (Fig. 7(b)). Similarly, β-galactosidase from the bacteria Propioni-
bacterium acidipropionici and A. oryzae were able to synthesise 261 g/L of
OsLu using 300 g/L lactulose at 45 ◦C and 406 g/L OsLu at 50 ◦C,
respectively, after 24 h of incubation [18,31]. In another study by Cobas
et al., oligosaccharides with DP ≥ 3 were produced using β-galactosidase
from A. oryzae. A yield of 50% (w/v) total OsLu was obtained using 8 U/
mL enzyme and 450 g/L lactulose at an incubation temperature of 50 ◦C
[6].
Declaration of Competing Interest
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.
Acknowledgement
The authors would like to acknowledge Dr. Sanjit Kumar and Dr. C.
Rajasekaran, VIT, for their profound input and assistance with this
project.
Preliminary analysis with NMR revealed that there was a downfield
chemical shift in the OsLu spectrum with reference to the lactulose
spectrum (Fig. 8). Galactose-related signals were observed in both the
lactulose and OsLu spectra. The 2D NMR spectra of the latter would
enable us to assign residues of all chemical shifts in the signal, which
remains to be done extensively.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or non-profit sectors.
8

P. Kalathinathan et al.
Bioorganic Chemistry 115 (2021) 105207
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