Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

Bioorganic Chemistry 77 (2018) 176–189

Contents lists available at ScienceDirect

Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg

Purification and characterization of b-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis Preeti Chanalia, Dimpi Gandhi, Pooja Attri, Suman Dhanda ⇑ Department of Biochemistry, Kurukshetra University, Kurukshetra, India

a r t i c l e

i n f o

Article history: Received 21 July 2017 Revised 26 December 2017 Accepted 2 January 2018 Available online 4 January 2018 Keywords: b-galactosidase Probiotic Milk lactose P. acidilactici Lactic acid bacteria Galactooligosaccharide

a b s t r a c t b-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of b-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8–7.0 with more than 97% activity. Purified b-galactosidase was optimally active at 50 °C. Kinetic parameters Km and Vmax for purified enzyme were 400 µM and 1.22
101 U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca2+, Mg2+ and Mn2+ slightly activated the enzyme whereas NH+4, Co2+ and Fe3+ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that b-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min1 and t1/2 of 14.74 min. This is better than other studied b-galactosidases. Both sonicated Pediococcus acidilactici cells and purified b-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of b-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca2+ shows that it is suitable for milk processing. Ó 2018 Elsevier Inc. All rights reserved.

1. Introduction b-galactosidase (EC. 3.2.1.23) is an exoglycosidase that has health benefits and industrial applications. It catalyses the hydrolysis of lactose and structurally related galactosides and also transglycosylation reactions [1]. Study of b-galactosidase production from microorganisms is not new but many of them were not approved because they were not obtained from food grade microorganisms. Microbes are preferred source and yeasts are major commercial source of this enzyme. Moreover, the yield from studied microorganisms is also low that limits their use for commercial purpose. Therefore, b-galactosidase from food grade probiotic microorganisms are safe for human use. Probiotic bacteria producing high level of b-galactosidase is very significant. ⇑ Corresponding author. E-mail addresses: [email protected] (P. Chanalia), dimpigandhi88@ gmail.com (D. Gandhi), [email protected] (P. Attri), [email protected] (S. Dhanda). https://doi.org/10.1016/j.bioorg.2018.01.006 0045-2068/Ó 2018 Elsevier Inc. All rights reserved.

Lactic acid bacteria (LAB) are considered as good source of enzyme because of their GRAS status. Pediococcus acidilactici is a LAB that possessed all the probiotic attributes [2] and also effectively tenderised meat [3]. It possesses a spectrum of enzyme activities [4] and b-galactosidase activity is 22 times higher than other studied strains even in the absence of inducer [2]. Pediococcus acidilactici is also a good source of commercial b-galactosidase because dairy environment is its natural habitat. b-galactosidase finds prominent place in pharmaceutical industry such as in development of digestive supplements (prebiotics) and treatment of disorders (lactose intolerance). Prebiotics are non-digestible food ingredients that have gained interest because they affect the host beneficially by selectively stimulating growth of indigenous microflora. b-galactosidases are currently being used for galactooligosaccharide (GOS) synthesis i.e. prebiotics. Lactose intolerance is reported in 70% of world population. b-galactosidase is also used in dairy industry for avoiding crystallization of lactose in concentrated frozen dairy products such as

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

condensed milk, and ice creams, and in solving the problem of whey disposal by converting whey into lactic acid [5]. This strain under study produced lactic acid using different waste resources [6]. Lactose intolerance, relatively low solubility and sweetness of lactose and GOS synthesis has led to increased demand and development of non-immunogenic and thermostable b-galactosidases of commercial importance. This study reports the purification and characterization of b-galactosidase from probiotic P. acidilactici which exhibited high yield, high rate of milk lactose hydrolysis and also synthesised GOS. This is expected that it will reduce the operating cost. Thermal and kinetic properties of the purified enzyme are also discussed.

177

1 ml of 1 M Na2CO3. The absorbance was read against a suitable blank at 420 nm. One unit of enzyme activity was defined as amount of enzyme that liberated 1 µmole of ONP from the substrate per minute under assay conditions. 2.5. Effect of carbohydrate source on b-galactosidase production

2. Materials and methods

Sterilized modified MRS media without any carbon source was prepared. Different carbon source i.e. maltose, sucrose, fructose, lactose, galactose and glucose were added separately at a concentration of 2% each. The media without any carbon source was used as control. Then each media was inoculated with P. acidilactici and incubated at 32 °C for 24 h. Cells were harvested at 9000 g and enzyme activity was determined in Miller units. Highest activity was taken as 100%.

2.1. Source of study

2.6. Extraction of b-galactosidase

P. acidilactici earlier purchased from NCDC (National collection of Dairy Cultures), National Dairy Research Institute (NDRI), Karnal is being maintained in our lab.

P. acidilactici cells were incubated for 24 h and then harvested by centrifugation at 9000 rpm for 25 min. Intracellular bgalactosidase was extracted using following different protocols to optimize the extraction:

2.2. Collection of biomass P. acidilactici was cultured in MRS medium for 24 h at 32 °C under shaking conditions at 250 rpm and then harvested by centrifugation at 9000 rpm for 25 min. 2.3. Materials o-nitrophenyl-b-d-galactopyranoside (ONPG), MRS media and other chemicals used were from Hi media, Mumbai, India. Chromatographic slurries were from Sigma Aldrich. The reagents used were of analytical grade unless stated otherwise. 2.4. b-galactosidase assay 2.4.1. b-galactosidase assay of whole cells Bacterial cell pellets were harvested, washed twice and suspended in same volume of Z buffer (One litre contains: 16.1 g Na2HPO47H2O (0.06 M), 5.5 g NaH2PO4H2O (0.04 M), 0.75 g KCl (0.01 M), 0.246 g MgSO47H2O (0.001 M), 2.7 ml b-mercaptoethanol (0.05 M), pH 7.0). Absorbance of cell suspension was measured at 600 nm against Z buffer. For each reaction mixture 0.1 ml cells were diluted to 1 ml with Z buffer. Diluted cells were permeabilized by adding 100 µl chloroform and 50 µl 0.1% SDS. The tubes were vortexed for 30 s and equilibrated for 5 min in water bath at 28 °C. The reaction was started by adding 0.2 ml ONPG (4 mg/ml) substrate followed by incubation at 28 °C for 10 min. The reaction was stopped by adding 0.5 ml of 1 M Na2CO3 and contents were centrifuged to remove debris and chloroform. OD was recorded at 420 nm and 550 nm. Miller units for b-galactosidase were calculated using following formula.

2.6.1. Sonication Bacterial cells were sonicated according to Feliu et al. [8] with slight modifications including standardization of time of sonication using 1–10 min of sonication period. Cell suspension (in 50 mM Na-Pohsphate buffer pH 7.0) was sonicated for 1–10 min in ice bath. The extract was then centrifuged at 15,000g for 10 min and assayed for enzyme activity. 2.6.2. Lysozyme-EDTA treatment Lysozyme solution was prepared by dissolving 50 mg of lysozyme in 1.5 ml Tris EDTA buffer pH 8.0. Seventy-five microlitre of this was added to one ml of cell suspension, then mixture was incubated for 30 min at room temperature and centrifuged at 15,000g for 10 min. b-galactosidase activity was determined in supernatant. 2.6.3. SDS-chloroform treatment One hundred microlitre of chloroform and 50 ll of 0.1% SDS were added to 10 ml of cell suspension and incubated for 30 min at room temperature under vortexing conditions. Then it was centrifuged at 15,000g for 10 min and assayed for enzyme activity. 2.6.4. Enzyme extraction by lysozyme Cell pellet was suspended in 5.0 ml of 0.05 M Na-phosphate buffer (pH 6.8) followed by vigorous vortexing. Lysozyme (10 mg ml1) was added to it and incubated at 37 °C for 15 min. Then 0.5 ml of 4 M NaCl was added and sample was again incubated at 37 °C for another 50 min. It was centrifuged at 10,000g for 15 min and enzyme was assayed in supernatant 2.7. Protein content

Miller units ¼ 1000
½ðOD420  1:75
OD550 Þ=ðT
V
OD600Þ where OD420 and OD550 are read from reaction mixture, OD600 is cell density in washed cell suspension, T-reaction time (min), V-culture volume (ml) used in assay. 2.4.2. b-galactosidase assay of cell free enzyme b-galactosidase activity was determined according to Bhomik and Marth [7]. Cell free extract (0.2 ml) was added to 1.6 ml Z buffer. The reaction was started by adding 0.2 ml of 10 mM o-nitrophe nyl-b-d-galactopyranoside (ONPG). Reaction mixture was incubated at 37 °C for 10 min and then reaction was stopped by adding

Protein content was determined by Lowry’s method [9] using bovine serum albumin as standard. 2.8. Purification Purification was achieved in three steps viz. ammonium sulphate fractionation and successive chromatographies on gel filtration and anion exchanger. Ammonium sulphate precipitation: The extracted proteins were concentrated by saturating the crude extract up to 80% by ammonium sulphate. The precipitates were re-dissolved in 4 ml of 50

178

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

mM Tris-HCl buffer (pH 7.0) and dialyzed against same buffer and then subjected to gel filtration chromatography. Gel filtration chromatography: Sample from previous step was loaded on Sephadex G-100 column pre-equilibrated with 50 mM Tris-HCl buffer of pH 7.0. Fractions containing b-galactosidase activity were pooled and concentrated on Amicon Diaflo using YM10 membrane (cut off 10 kDa). Concentrated enzyme was dialysed against 50 mM sodium phosphate buffer, pH 6.5. Anion exchange chromatography: Concentrated sample of gel filtration chromatography was loaded on Q-Sepharose column pre-equilibrated with 50 mM sodium phosphate buffer, pH 6.5. Unbound proteins were eluted isocratically using same buffer and bound proteins including b-galactosidase were eluted with linear gradient of 0.0–1.0 M NaCl. The fractions of 2 ml of each were collected and fractions having b-galactosidase enzyme were pooled and concentrated. 2.9. Davis gel electrophoresis Native PAGE (10%) was run according to Davis [10] and protein bands were fixed in a mixture of 40% methanol and 13.5% formaldehyde for 15 min then gel was washed with distilled water and incubated with 0.1% silver nitrate for 20 min followed by rinsing with water. Gel was then immersed in developer solution (3% sodium carbonate having 0.5% formaldehyde and 0.02% Na2S2O3) for 15 min. Reaction was stopped by immersing gel in stopping solution (25% isopropanol solution containing 10% acetic acid) for 5 min. 2.9.1. In situ gel assay Native PAGE (10%) was pre-run for 2 h at 4 °C and then run according to Davis [10] at 4 °C. After running one half of the gel was silver stained as described above whereas other half was stained with the substrate using ONPG under the assay conditions. 2.10. Molecular weight determination SDS-PAGE: Molecular weight was determined by 12% SDS PAGE. Purified enzyme (15 µl) was mixed with 10 µl of sample buffer (50 mM Tris-HCl buffer pH 6.8 containing 2% SDS, 0.1% bromophenol blue, 2 µl of 2-mercaptoethanol and 10% glycerol) and heated at 100 °C for 10 min. Samples were loaded on 12% SDS PAGE. The protein bands were stained with 0.25% Coomassie Brilliant Blue R250 in solution of methanol, water and acetic acid in a ratio of 45:45:10 followed by destaining in solution of methanol, water and acetic acid (45:45:10). Molecular weight was determined by comparing mobility with standard prestained markers of known molecular weight (11–245 kDa). MALDI-TOF: MALDI-TOF analysis was done at Centre for DNA Fingerprinting and Diagnostics (CDFD) Hyderabad. Trypsin digested b-galactosidase was treated with equal volume of matrix solution (a-cyano-4-hydroxy cinnamic acid (HCCA) (10 mg/ml) in 70% acetonitrile and 0.03% trifluoric acid) and dried at room temperature. Peptide mass spectra obtained by MALDI-TOF/TOF mass spectrometer (Bruker Ultraflex III TOF/TOF) was used to match protein identity. Sequence alignment and homology search was done with BLAST and Clustal W and peptide sequences were aligned with predicted b-galactosidase sequences of different P. acidilactici strains.

activity was calculated. All assays were performed in triplicate and mean value was reported. 2.12. Temperature optima and stability b-galactosidase was assayed at different temperatures: 0°, 5°, 10°, 20°, 30°, 37°, 40°, 45°, 50°, 55° and 60 °C with a control for each temperature. Temperature stability was determined by incubating the enzyme at different temperatures (0–60 °C) for 10 min and then assaying at optimum temperature. Assays were performed in triplicate and mean value was reported. 2.13. Kinetic characterization Purified enzyme was assayed at different concentrations of ONPG and Km and Vmax were calculated from Michaelis Menton Plot, Line Weaver Burk plot and Hanes Plot. Specificity constant i.e., Kcat/Km was also calculated. 2.14. Effect of enzyme inhibitors, thiol compounds and metal ions Effect of different inhibitors viz. PMSF, NEM, Iodoacetic acid, 1,4-Dithioerythritol, PCMB, AEBSF, 4-Nitrophenyl iodoacetate, Puromycin, EDTA, DEPC and thiol compounds including Cysteine, b-ME and DTT on b-galactosidase activity was studied by preincubating enzyme with different chemical reagents for 10 min at 37 °C. Effect of chloride salts of different metal ions on b-galactosidase activity was also studied by preincubating enzyme for 10 min with different dilutions of metal ion solutions against control. 2.15. Determination of thermodynamic parameters of purified b-galactosidase Thermal deactivation kinetics of purified b-galactosidase was studied by incubating the enzyme at different temperatures (40, 45, 50, 55 and 60 °C) in the absence of substrate. Aliquots were withdrawn at periodic intervals and cooled in ice bath prior to assay. The residual activity was expressed as a percentage of initial activity. The data obtained from the thermal stability profile was used to analyze thermodynamic parameters related to b-galactosidase activity. The experimental points were plotted according to the equation given below:

ln

A ¼ kd
t A0

where A0 is the initial activity, A is the residual activity after heat treatment, kd is thermal inactivation rate constant (min–1) and t is the exposure time (min). The half-life of b-galactosidase (t1/2/min–1) was determined using the following relationship:

t1=2 ¼

ln 2 kd

The D value (decimal reduction time or time required to preincubate the enzyme at a given temperature to maintain 10% residual activity) was calculated from the following relationship:

D¼

ln 10 kd

2.11. pH optima and stability

The activation enthalpy (D H) for each temperature was calculated was by

pH optima was determined in pH range of 4.5–11 by assaying enzyme in assay buffers of different pH. pH stability of enzyme was determined by incubating enzyme with buffers in pH range 4–10.5 at 37 °C for 10 min then assayed at optimum pH. Percent

DH ¼ Ea  RT where R is the universal gas constant (8.314 J mol1 K1), Ea is the activation energy and T is the absolute temperature in Kelvin.

179

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

DG ¼ R
T
lnðkd
hP =KB
TÞ Activation entropy (D S) was calculated by:

DG ¼ DH  T DS where KB is the Boltzmann constant (1.38
1023 J/K), hP is the Planck’s constant (6.626
1034 J s), and T is the absolute temperature, kd (min1) the inactivation rate constant of each temperature. 2.16. Milk lactose hydrolysis by b-galactosidase Ten percent defatted milk powder was dissolved in distilled water. Soluble enzyme (3
101 U) was added to 5 ml of reaction mixture and incubated at 50 °C. Aliquots of 20 µl were withdrawn at regular intervals of time. Glucose oxidase peroxidase (Glucose (GO) Assay Kit sigma) were used to estimate the released glucose content. The percentage of unhydrolyzed lactose was calculated as % Lactose unhydrolyzed ¼

Glucose present beforeb-galactosidase treatment Glucose present afterb-galactosidase treatment

The rate constant of lactose hydrolysis was determined from the slope of plot between log of percent unhydrolyzed lactose versus time using the following formula:

Slope ¼

k 2:303 log 100 where rate constant k ¼ 2:303 t 100  x

where ‘x’ is the unhydrolyzed lactose. Therefore, time required for . the hydrolysis of 50% lactose could be calculated as t1=2 ¼ 0:693 k 2.17. Synthesis of galactooligosaccharides (GOS) GOSs were synthesised using purified b-galactosidase enzyme as well as sonicated cells of P. acidilactici. Reaction conditions for maximum GOS synthesis by sonicated P. acidilactici cells as well as purified b-galactosidase were optimized using response surface methodology (RSM). Lactose (10–50% w/v) was used as substrate and sonicated P. acidilactici cells and purified b-galactosidase were used as enzyme. Reactions were carried out at pH 7.0 in orbital shaker (150 rpm). Temperature (20–60 °C) and enzyme unit (0.05–5 units) for the reaction were also optimized. The best suited conditions for GOS synthesis using RSM were studied in vitro and results were analyzed on TLC. Aliquots of 100 µl were collected at regular interval of 4 h. Reaction was stopped by boiling the samples at 100 °C for 5 min and samples were then centrifuged at 5000 rpm. The samples were qualitatively analysed by TLC. TLC was carried out on silica gel plates (5
20 cm) using nbutanol:methanol:water (70:20:10 v/v/v) as a mobile phase. Test sample (1 µl) was spotted and dried on TLC plates along with standard glucose, galactose and lactose. After completing the run, plates were dried, sprayed with 35% H2SO4 in ethanol and dried in oven at 100 °C for 5 min to visualise the compounds. 3. Results and discussion b-galactosidase activity of P. acidilactici is advantageous because Pediococcus acidilactici is a probiotic which is also used as starter culture. There are few studies of b-galactosidase from Pediococcus and activity varied from strain to strain [11]. P. acidilactici under study expressed 65.08 ± 0.9 Miller Units of b-galactosidase activity which is 22 times higher (1.5–3.0 Miller units in uninduced lac+ operon) even under uninduced condition [2]. Therefore, b-galactosidase from probiotic P. acidilactici was purified and

characterized. Earlier only two strains of P. acidilactici were reported to have b-galactosidase activity but these were not further studied [7]. This enzyme was purified from P. pentosaceous only [12]. 3.1. Optimization of b-galactosidase production Several studies revealed that the role of carbon source in the biosynthesis of b-galactosidase varied greatly with the microorganism [13]. Production of b-galactosidase was optimized for carbohydrates and methods of extraction. The b-galactosidase activity of P. acidilactici under study, in the presence of different carbohydrates is shown in Fig. 1. Glucose (2%) was best carbon source that resulted in maximal b-galactosidase production followed by lactose (92.3%), galactose (74.12%), maltose (67.8%) and sucrose (32.8%). Fructose did not affect b-galactosidase production. Galactose is better inducer in Lactobacillus crispatus [14], whereas lactose was better for Lactobacillus reuteri. Aerobic cultivation of A. caviae in LB containing glucose, fructose, maltose and sucrose completely inhibited beta-galactosidase activity possibly due to acetic acid production while its growth with other carbon sources did not affect the enzyme activity and Arabinose, xylose and galactose induced the A. caviae beta-galactosidase activity by several folds and lactose moderately enhanced its activity [15]. Lactose was also reported as better inducer of this enzyme as compared to galactose and glucose in L. acidophilus [16]. Highest b-galactosidase activity was also detected in Bifidobacteria cultures containing lactose as sole carbon source, followed by galactose and the lowest activity with glucose [13]. Intracellular b-galactosidase of Kluyveromyces yeast was induced by galactose and repressed by glucose. There is no literature available on carbon source regulation for biosynthesis of this enzyme in P. acidilactici. Therefore, this study reveals a good understanding of the carbon source regulation of b-galactosidase in P. acidilactici. 3.2. Isolation b-galactosidase was assayed in extracellular fluid, intracellular fluid and sonicated membranes of P. acidilactici and the enzyme activity was only reported in intracellular fluid. Highest enzyme activity was observed after 24 h of incubation. Maximum enzyme extraction (90% total of enzyme activity) was achieved by sonication (Fig. 2). Therefore, isolation of b-galactosidase was done by sonication method. Optimum time of sonication was found to be 5 min. Other methods resulted in relatively poor release of b-galactosidase. Sonication was reportedly most effective method for extracting of b-galactosidase from B. animalis ssp lactis B12 [17].

120 100

% activity

The activation energy (Ea) was estimated by Arrhenius plot. The free energy of inactivation (DG) was determined according to the expression:

80 60 40 20 0

Maltose Sucrose Fructose Lactose Galactose Glucose

Control

Fig. 1. Effect of carbon sources on production of b-galactosidase.

180

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

Fig. 2. Isolation of b-galactosidase using different methods.

3.3. Purification and characterization of b-galactosidase Though there are reports of b-galactosidase from fungi, yeasts, plants and a few bacteria. The enzyme was also purified from Bifidobacteria and Lactobacillus [18,19]. Pediococcus acidilactici is a

1

OD at 280nm

1.6

OD 280 nm

1.4

Activity(U×10-1)

1.2

0.8

1

0.6

0.8 0.6

0.4

0.4

0.2 0

0.2 1

3

5

7

9 11 13 15 17 19 21 23 25 27 29 31 33 35

Activity(U×10-1)

1.2

0

Fraction No. Fig. 3. Protein and b-galactosidase activity profile on gel filtration chromatography.

0.35 0.3

OD at 280nm

1.4

OD at 280 nm Activity (U×10-1)

1.2 1

0.25

0.8

0.2

0.6

0.15 0.1

0.4

0.05

0.2

0

1 6 111621263136414651566166717681869196

0

Activity (U×10-1)

0.4

Fraction No.

Fig. 4. Protein and chromatography.

b-galactosidase

activity

profile

on

anion

exchange

probiotic bacteria and this strain expressed 22 times higher activity even in the absence of inducer [2], therefore, this strain was used as source of b-galactosidase purification. Purification was achieved in three steps viz. Ammonium sulphate precipitation, gel filtration chromatography on Sephadex G-100 column (Fig. 3) and anion exchange chromatography on Q-Sepharose column (Fig. 4). Purification summary is shown in Table 1. b-galactosidase was 3.06-fold purified with specific activity of 0.883 U/mg and yield of 28.26%. Apparent homogeneity of purified enzyme was determined by 10% native PAGE comparative gel (Fig. 5a) and 10% native PAGE (Fig. 5b). Silver stained single band corresponded to activity stained band obtained in in-situ gel assay (Fig. 5b). This further confirmed purity and homogeneity. b-galactosidase from different source was purified to different extent. b-galactosidase of Kluyveromyces lactis was purified to 5.4-fold by Mazi et al. [20], b-galactosidase from L. brevis PLA28 was purified to 6.6-fold with a yield of 6% and specific activity of 4.0 U/mg [21], b-galactosidase from B. longum subsp. longum JCM 7052 was purified with a yield of 10% and specific activity of 7.42 U/mg [18] and b-galactosidase from Anoxybacillus sp. KP1 was purified with purification fold of 14.4, percent yield of 11.8 and specific activity of 1632.1 U/mg [22]. 3.4. Molecular weight determination Molecular weight of purified b-galactosidase was determined by SDS-PAGE and MALDI-TOF. SDS-PAGE: Studies revealed b-galactosidase to be a heterodimer (Fig. 6). The molecular weight determined from relative mobility was 35.3 kDa with constituent subunit mass of 19.9 and 15.4 kDa. MALDI-TOF: MALDI-TOF analysis of trypsin digested b-galactosidase revealed the molecular weight to be 39.07 kDa (Fig. 7). MALDI-TOF analysis also confirmed b-galactosidase as heterodimer of constituent subunits having molecular mass 15.55 and 19.58 kDa. Molecular weight analysis by both SDS-PAGE and

Table 1 Purification table of b-galactosidase. Purification step

Total activity (U)

Total protein (mg)

Specific activity (U/mg)

Percentage yield

Purification fold

Crude extract Ammonium sulphate Precipitation Gel Filtration Chromatography Anion Exchange Chromatography

75 ± 0.13 60 ± 0.05 40 ± 0.085 21.2 ± 0.1

260 ± 0.15 180 ± 0.12 51 ± 0.91 24 ± 0.81

0.288 ± 0.01 0.333 ± 0.01 0.784 ± 0.003 0.883 ± 0.01

100 80 ± 1 53.66 ± 0.9 28.26 ± 0.92

1 1.156 ± 0.02 2.722 ± 0.01 3.065 ± 0.01

Values are mean ± SD of three different experiments.

181

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

Fig. 6. SDS-PAGE (12%) of purified b-galactosidase (Lane A) with b-ME and Molecular weight markers (Lane B).

Fig. 5a. Lane 1 represents the purified b-galactosidase after anion exchange chromatography, lane 2 and 3 represents the proteins after gel filtration chromatography and ammonium sulphate precipitation respectively.

MALDI-TOF confirmed b-galactosidase as heterodimer. Mascot score Histogram and matching index is shown in Figs. 8a, 8b, 8c and 8d. The enzyme exhibited maximum identity with cytoplasmic domain of a Bacterial chemoreceptor (DBC) from Thermotoga maritima. Trypsin digested fragments of b-galactosidase are highlighted (Fig. 9). Peptide matching results and spectrum of peptides are shown in Figs. 9, 10a and 10b.

A

Purified β-galactosidase

Multiple sequence alignment of trypsin digested fragments of purified b-galactosidase with available predicted b-galactosidase sequences from different Pediococcus acidilactici strains was done using clustal omega, which revealed similarities and identities between them. This further confirmed the purity, homogenity and identity of the enzyme. Molecular mass of 41 kDa and 19 kDa were previously reported for b-galactosidase in Saccharomyces lactis [23]. Molecular weight of 56 kDa was reported for b-galactosidase from Pichia pastoris [24]. b-galactosidase from P. pentosaceus ATCC 25,745 had a molecular weight of 66 kDa [7]. The enzyme also appeared as high molecular weight protein of 102 kDa, 105 kDa and 107 kDa [25,26,19] respectively. b-galactosidase with very high molecular

B

Purified β-galactosidase

Fig. 5b. Davis Gel Electrophoresis of b-galactosidase (silver stained) (A) and in situ gel assay (B).

182

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

Fig. 7. Intact mass determination.

Fig. 8a. Mascot score Histogram of band corresponding to 19.9 kDa (on SDS-PAGE).

more than 90% in pH range of 6.0–7.0 and 80% activity was retained at pH 5.5 and 7.5. Optimum pH 6.0 was also observed for b-galactosidase of different species by Park and Oh [27] and Wu et al. [28]. Optimum pH of 7.5 was observed for b-galactosidase from Kluyveromyces marxianus [29], from L. plantarum [19], whereas optimum pH 7.0 was found for enzyme from K. fragilis [5]. b-galactosidase activity for ONPG shows a broader pH optimum (5.5–7.0). pH optimum of 6.5 was observed for b-galactosidase from L. plantarum HF571129 [25]. b-galactosidase under study was stable in a pH range of 5.8–7.0 with more than 97% activity (Fig. 11). More than 75% activity was retained at pH 5.5 and 7.5. Only 50% activity was retained at pH 5.0 and 8.0. b-galactosidase from Lactobacillus plantarum HF571129 was maximally stable at pH 8.0 and fairly stable at pH 6.5 and 7.5 [25]. b-galactosidase from Lactobacillus plantarum WCFS1 was most stable in pH range of 6.5–8.0 [19]. The enzyme from Teratosphaeria acidotherma AIU BGA-1 was stable in pH range 1.5–7.0 [30]. The pH optima and stability near neutral range makes this enzyme suitable for milk processing. 3.6. Temperature optima and stability

Fig. 8b. mascot score Histogram of band corresponding to 15.4 kDa (on SDS-PAGE).

weight of 630 kDa, and 550 kDa was reported in Saccharomyces lactis [23]. 3.5. pH optima and stability To exploit the commercial importance of the enzyme, pH optima and stability studies were performed. Optimum pH of purified b-galactosidase was found to be 6.0 (Fig. 11). Activity was

The enzyme was optimally active at 50 °C (Fig. 12). The enzyme exhibited 80% activity at 45° and 55 °C. However, beyond 55 °C, the enzyme activity decreased abruptly and no activity was left at 60 °C. Thermostability studies showed that purified enzyme retained about 90% activity up to 50 °C. There was a sharp decline in enzyme activity beyond 50 °C. At 55 °C, only 35% activity was left (Fig. 12). Similar optimum temperature was observed for b-galactosidase from Lactobacillus plantarum HF571129 [25] and Antartic arthrobacter sp. 32C [31]. Optimum temperature of 55 °C was reported for b-galactosidase from Lactobacillus plantarum WCFS1 [19]. Higher temperature optima and stability makes this enzyme industrially significant.

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

Fig. 8c. Matching index of band corresponding to 19.9 kDa.

Fig. 8d. Matching index of band corresponding to 15.4 kDa.

Fig. 9. Peptide matching.

183

184

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

Fig. 10a. Peptide spectrum of band corresponding to 19.9 kDa (on SDS-PAGE).

Fig. 10b. Peptide spectrum of band corresponding to 15.4 kDa (on SDS-PAGE).

120

% activity

100 80 60 40 20 0

0

2

4

6

8

10

12

pH

Fig. 11. pH optima and stability.

3.7. Kinetic characterization Kinetic parameters Km and Vmax of purified b-galactosidase as determined from Michaelis-Menten plot (Fig. 13a), Line Weaver

Burk plot (Fig. 13b) and Hanes plot (Fig. 13c) were 400 lM and 1.221
101 U respectively. The enzyme exhibited micromolar affinity for substrate under study. The Km value is relatively lower than studied b-galactosidases that show higher affinity of enzyme for its substrate. Km of 0.6 mM at 20 °C, 0.7 mM at 5 °C and 10 °C and 0.8 mM at 18 °C were reported for beta-galactosidase from Arthrobacter sp. [32]. However, Km for b-galactosidase in mM and µM range was also reported. There was a report of higher Km for b-galactosidase (s) from Lactobacillus plantarum WCFS1 (0.9 mM) [19] and uncultured soil bacterium (1.71 mM) [33]. Km and Vmax of purified b-galactosidase for ONPG were reported to be 8.33 mM and 6.6 U/mg protein respectively [21]. In Thalassospira sp. Km and Vmax values was reported as (1.2 mM and 1645.66 U/ml) and Lactobacillus pentosus (1.67 mM and 304 lmol min1 mg1) [34,26].

185

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

120

However, relatively lower Km of 0.083 mM was reported for silica matrix encapsulated enzyme from E. coli [35].

100 3.8. Effect of inhibitors

% activity

80 60 40 20 0

0

20

40

60

80

Temperature °C Temperature opma Temperature stability Fig. 12. Temperature optima and stability.

(a)

1.4 1.2

Table 2 Effect of inhibitors on activity of purified b-galactosidase.

1 Activity (U×10-1)

The effect of inhibitors was studied in order to investigate the active site machinery of enzyme and results are shown in Table 2. The enzyme was strongly inhibited by PMSF. More than 80% inhibition was observed at 1 mM PMSF which confirms the enzyme as a serine protease. Complete inhibition by 0.1% DEPC suggests the presence of His at active site and its participation in catalysis. Ser and His are reported at active site of several hydrolases. PCMB affected moderately (1 mM resulted in 62% inhibition). NEM inhibited up to 40.6% at 1 mM. Thiol compounds viz. DTE, DTT, cysteine, b-ME and some inhibitors viz AEBSF, iodoacetate, 4-Nitrophenyl iodoacetate and puromycin had no effect on enzyme activity. bgalactosidase of Thermus species was also inhibited by PMSF [36]. Though b-galactosidase of Pediococcus acidilactici exhibited moderate inhibition by PCMB, contrarily b-galactosidase of Bifidobacterium infantis [37] was strongly inhibited by PCMB. Inhibition of b-galactosidase derived from lactose fermenting yeasts Kluyveromyces marxianus, Torulopsis sphaerica and Torulopsis versatilis by NEM was reported by Itoh et al. [38]. b-galactosidase from different sources possessed different catalytic machinery. EDTA had no effect on activity of enzyme under study. Contrarily EDTA inactivated the b-galactosidase of Lactobacillus plantarum [19].

0.8

Inhibitors and thiol compounds

Concentration

% Inhibition

0.6

DEPC

0.10% 0.50% 0.1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 0.1 mM 1 mM

99.9 ± 0.85 100 ± 0.34 45.9 ± 1.87 80.02 ± 1.48 62.2 ± 2.2 40.6 ± 1.23 71 ± 2.3 0 0 0 0 0 0 0 0 0

0.4 PMSF

0.2 0

0

0.5

1

1.5

PCMB NEM AEBSF Iodoacetic acid 4-Nitrophenyl iodoacetate Puromycin 1,4 Dithioerythritol DTT Cysteine b-ME EDTA

2

Substrate (mM)

(b)

Values are mean ± SD of three different experiments.

Table 3 Effect of metal ions on b-galactosidase activity.

[S]/V

(c)

-0.8

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

0.2

[S]

1.2

2.2

Fig. 13. Determination of kinetic parameters (Km and Vmax) for purified b-galactosidase by Michaelis–Menten plot (a), Line weaver Burk plot (b) and Hanes Plot (c).

Metal ion

Concentration (mM)

Activity (%)

Control CaCl2 CuCl2 NH4Cl NaCl BaCl2 ZnCl2 HgCl2 MnCl2 MgCl2 CoCl2 KCl LiCl CdCl2 FeCl3 NiCl

0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

100 ± 0.04 109.28 ± 0.42 102.14 ± 0.1 89.28 ± 0.25 104.28 ± 0.12 107.85 ± 0.35 106.42 ± 0.54 107.85 ± 0.23 117.8 ± 0.33 110 ± 0.23 71.4 ± 0.65 100.7 ± 0.48 107.8 ± 0.37 109.28 ± 0.18 77.85 ± 0.45 120 ± 0.34

Values are mean ± SD of three different experiments.

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

Effect of metal ions on purified b-galactosidase was studied and results are summarized in Table 3. NH+4 and metal ions Co2+ and Fe3+ resulted in slight decrease in enzyme activity and 89.28%, 71.4% and 77.85% activity was left respectively. Other studied metal ions either had no effect or slightly increased enzyme activity. Mn2+ and Mg2+ at 0.1 mM resulted in increase in 117.8% and 110% activity respectively. Ca2+ ions resulted in slight increase in b-galactosidase activity. This is beneficial for use of b-galactosidase in milk and dairy industry, whereas, there are reports of inhibition of b-galactosidase by Ca2+. Effect of Mono- and divalent cations on b-galactosidase activity has been well documented [39]. Divalent cations i.e. Mn2+ and Mg2+ enhanced enzyme activity, whereas monovalent cations may have positive or negative effect [40]. Divalent metal ions improved catalytic efficiency for most of studied b-galactosidases. Mg2+ is a physiological ion [41] and magnesium potential below 7, is associated with increased efficiency of b-galactosidases (Kcat rises) and thus increased affinity for substrate (Km falls) [41]. This activation of enzyme may be due to interaction of b-galactosidase with divalent ions. Studies in literature describe the interaction of b-galactosidase from Saccharopolyspora rectivirgula with divalent ions [42]. 3.10. Determination of thermodynamic parameters of purified b-galactosidase

to conformational changes occurring in the enzyme molecule during reaction. DH and DS parameters also provide a measure of the number of non covalent bonds broken and the net enzyme/solvent disorder changes related to the formation of the transition state. The results obtained have portrayed that enthalpy has increased in the range of 14.86–14.7 kJ/mol with increase in temperature from 40 to 60 °C. The Gibbs free energy (DG) measures the spontaneity of a reaction. The Gibb’s free energy decreased slightly with rise in temperature indicating that the enzyme undergoes slight thermal unfolding and is less stable at 60 °C and above. Negative entropy changes were found at all the studied temperatures. The possible reason for negative entropy could be formation of charged particles due to compaction of the enzyme around the enzyme molecules. 3.11. Milk lactose hydrolysis by b-galactosidase Rate of lactose hydrolysis and t1/2 were determined from plot of log% residual activity versus time. Results are shown in Figs. 14a and 14b. Rate of lactose hydrolysis and t1/2 were calculated to be 0.047 min1 and 14.74 min respectively. t1/2 is the time required for the hydrolysis of 50% lactose. t1/2 of the b-galactosidase under study is the best t1/2 observed so far amongst studied b-galactosidases. Lactose hydrolysis increased up to 65 min of reaction time and about 98% lactose was hydrolysed. Lactose hydrolysis rate of 0.0111 min1 and t1/2 of 62.4 min were reported for b-galactosidase from Lactobacillus plantarum HF571129. The

Linear plots were obtained when residual activity was plotted against incubation time, indicating that the inactivation of b-galactosidase could be expressed as first order kinetics in the temperature range of 40–60 °C (Table 4). Among stability properties of an enzyme at different temperatures, the half-life (t1/2) determinations are more reliable. With increase in temperature, the t1/2 (half life) and D value (Decimal retention time) decreased and the first order thermal deactivation rate constants (kd) increased. D value is the time needed for 90% reduction of the initial activity and is commonly used for the estimation of enzyme stability. A higher rate constant means the enzyme is less thermostable. These observations indicate the less thermostability of b-galactosidase at higher temperatures. D values for P. acidilactici b-galactosidase ranged between 14.2 and 2557 min. Enzyme stability increase with increase of D value. Maximum D value was reported at 40 °C while minimum D value was at 60 °C which confirmed its less thermostability at higher temperatures. The activation energy Ea for b-galactosidase was Ea = 17.47 kJ/mol (y = 2101.3x + 9.1944, r2 = 0.726). The study of thermodynamics parameters (DH, DS, DG and activation energy Ea) provides the understanding of the behaviour of molecules in different physiological conditions. There is no information of the thermodynamic parameters for P. acidilactici bgalactosidase enzyme. Thus, the enthalpy of activation (DH#), Gibbs free energy (DG#) and entropy of activation (DS#) were calculated (Table 4). The numerical values of thermodynamic properties are affected by two major factors, namely solvent effect due to presence of surrounding water molecules and structural effect due

Rate of lactose hydrolysis (%)

3.9. Effect of metal ions

120 100 80 60 40 20 0

0

20

40

60

80

100

Time (min)

Fig. 14a. Lactose hydrolysis by b-galactosidase in milk at 50 °C.

2.5

Log % lactose unhydrolyzed

186

2 1.5 1 0.5 0

0

20

40

60

80

Time (min) Fig. 14b. Rate of lactose unhydrolyzed.

Table 4 Thermodynamic parameters of b-galactosidase. Temperature °C

Kd (min1)

t1/2 (min)

D (min)

DH (kJ/mol)

DG (kJ/mol)

DS (J/mol K)

40 45 50 55 60

0.0009 0.0020 0.0096 0.0123 0.162

770 346.5 72 56.34 4.2

2557 1151 239.7 187.15 14.2

14.86 14.82 14.78 14.74 14.70

105.66 105.27 102.76 103.72 98.17

290 284.4 272.4 271.3 250.6

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189 Table 5 Set of experiments for optimization of GOS synthesis by RSM. S. No.

Lactose (%)

Temperature (°C)

Enzyme Unit

1 2 3 4 5 6 7 8

30 1.72 30 58.28 30 50 10 30

47.5 47.5 47.5 47.5 47.5 30 30 22.75

2.75 2.75 0.43 2.75 5.93 5 0.05 2.75

187

rate of lactose hydrolysis by b-galactosidase of L. plantarum was 0.023 and 0.04 min1, while t1/2 was calculated to be 30.13 and 17.325 min for batch and packed bed respectively [25]. This shows that b-galactosidase from P. acidilactici is more efficient and suited for milk lactose hydrolysis than other studied b-galactosidases. The usability of this enzyme in milk lactose hydrolysis also increases its importance in pharmaceutical industry for overcoming the problem of lactose intolerance. Milk lactose hydrolysis by this enzyme is also important for food industry for making lactose free dairy products. Being hygroscopic, lactose also absorbs flavours and

Fig. 15a. Thin layer chromatogram of GOS synthesis by purified b-galactosidase. Lane 1–8 correspond to the sample number 1–8 of Table 5 respectively. Sandard glucose (lane 9), standard glactose (lane 10), standard lactose (lane 11). Lane 12 represents the reaction mixture at the beginning of GOS synthesis.

Fig. 15b. Thin layer chromatogram of GOS synthesis by sonicated cells of P. acidilactici. Lane 1–8 correspond to the sample number 1–8 of Table 5 respectively. Sandard glucose (lane 9), standard glactose (lane 10), standard lactose (lane 11).

188

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189

odours. Lactose crystallizes in dairy foods and results in development of sandy or gritty texture due to deposit formation in refrigerated foods [5]. Hydrolysis of lactose increases its solubility from 18 to 55% (w/v) at 80% conversion which results in up to 70% increase in sweetness related to sucrose.

from probiotic organism with GRAS status is a promising venture for producing cost effective novel functional ingredients beneficial for human health.

3.12. Synthesis of galactooligosaccharides (GOS) using b-galactosidase

Preeti Chanalia is thankful to University Grant Commission (UGC), India for providing Rajiv Gandhi National Fellowship for financial assistance.

GOSs are selectively fermented ingredients which act as prebiotics by allowing specific changes in the composition and/or activity of gastrointestinal microbiota and confers health benefits to host. They are very useful for food and dairy industry for their prebiotic effects as well as their use as sweeteners in confectionery, beverages and fermented products [43]. The GOS products currently available in market are not rationally designed and thus there is need to study microbial sources of b-galactosidases with good transglycosylation activities [44]. GOS synthesis by sonicated cells of P. acidilactici and purified bgalactosidase was optimized using response surface methodology (RSM). Central composite design was employed and a set of experiments were run (Table 5). GOS synthesis was studied by purified b-galactosidase as well as sonicated P. acidilactici cells and results are shown in Figs. 15a and 15b respectively. Intense bands of GOS were observed in both samples i.e. purified b-galactosidase and sonicated P. acidilactici cells and results were comparable in both. Spots corresponding to standard lactose observed in lane 1, 4, 5, 6, 7, and 8 in Figs. 15a and 15b indicate unhydrolyzed lactose and spots at lower positions in these lanes as compared to standard lactose are the spots of GOSs. More than one spot of GOS in some lanes suggest that there is a mixture of different types of GOS. Intensity of GOS bands was almost same when 30% and 50% of lactose was used in both cases, while 1.72 and 10% lactose gave poor results. GOS synthesis at 47.5 °C gave good results as compared to at 22.75 °C. Intensity of GOS bands synthesised was also less at 30 °C and 22.75 °C (lane no. 6 and 8) as compared to at 47.5 °C (lane nos. 1, 4 and 5) in both the situations. Lactose concentration (30%), 47.5 °C and 2.75 enzyme units were determined as the best conditions for GOS synthesis in both cases (lane 1). Effective GOS synthesis by sonicated cells of P. acidilactici marks its importance in industries by avoiding the expensive and time consuming enzyme purification procedure and also enhances the probiotic value of this strain. Similar studies were also performed by Bhalla et al. [21]. b-galactosidase under study is found to be more efficient and suited for milk lactose hydrolysis than other studied b-galactosidases and its GOS synthesizing ability was studied as an additional application. It is difficult to make comparison of transgalactosylating properties of this enzyme with other because amount of GOS formed depends upon several factors viz. concentration of substrate and reaction conditions and reaction conditions used in other studies have been different. 4. Conclusion b-galactosidase was purified by 3.06-fold with 28.26% yield using ammonium sulphate precipitation, gel filtration chromatography and anion exchange chromatography. The enzyme was a heterodimer with molecular mass of 39.07 kDa. High activity near neutral pH, 50 °C, being of probiotic origin, efficient in milk lactose hydrolysis and transglycosylation activity makes it a suitable candidate for different industries. High b-galactosidase activity without inducer is significant for lactose intolerants. GOS synthesis by purified enzyme as well as sonicated Pediococcus acidilactici whole cells is a useful property. Research and development of this enzyme and synthesis of GOS using exogenous b-galactosidase

Acknowledgements

Conflict of interest It is submitted that there is no conflict of interest amongst the authors. References [1] B. Splechtna, T.H. Nguyen, M. Steinböck, K.D. Kulbe, W. Lorenz, D. Haltrich, Production of prebiotic galacto-oligosaccharides from lactose using bgalactosidases from Lactobacillus reuteri, J. Agric. Food Chem. 54 (2006) 4999–5006. [2] P. Attri, D. Jodha, D. Gandhi, P. Chanalia, S. Dhanda, In vitro evaluation of Pediococcus acidilactici NCDC 252 for its probiotic attributes, Int. J. Dairy Technol. 68 (2015) 533–542. [3] D. Gandhi, P. Chanalia, P. Attri, S. Dhanda, Dipeptidyl peptidase-II from probiotic Pediococcus acidilactici: purification and functional characterization, Int. J. Biol. Macromol. 93 (2016) 919–932. [4] P. Attri, D. Jodha, D. Gandhi, S. Dhanda, Screening of intracellular, extracellular and membrane bound exopeptidases in lactic acid bacteria (LAB), Milchwissenschaft 67 (4) (2012) 421–424. [5] C.R. Carrara, A.C. Rubiolo, Immobilization of b-galactosidase on chitosan, Biotechnol. Prog. 10 (1994) 220–224. [6] P. Chanalia, D. Gandhi, R. Kumar, P. Attri, S. Dhanda, Lactic acid production visa-vis biowaste management using lactic acid bacteria, World Appl. Sci. J. 34 (11) (2016) 1542–1552. [7] T. Bhowmik, E.H. Marth, b-galactosidase of Pediococcus species: induction, purification and partial characterization, Appl. Microbiol.Biotechnol. 33 (1990) 317–323. [8] J.X. Feliu, R. Cubarsi, A. Villaverrde, Optimized release of recombinant proteins by ultrasonication of E. coli, Biotechnol. Bioeng. 58 (5) (1998) 536–540. [9] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1) (1951) 265–275. [10] B.J. Davis, Disc electrophoresis II method and application to human serum proteins, Annals New York Acad Sci 121 (1964) 404–427. [11] T. Bhowmik, R. Riester, M.A.J.S. Van Boekel, E.H. Marth, Characterstics of lowfat Cheddar cheese made with added Micrococcus or Pediococcus species, Milchwissenschaft 45 (1990) 230–235. [12] J.Y. Lee, M.S. Kwak, J.B. Roh, K. Kim, M.H. Sung, Microbial b-galactosidase of Pediococcus pentosaceus ID-7: isolation, cloning and molecular characterization, J. Microbiol. Biotechnol. 27 (3) (2017) 598–609. [13] C.A. Hsu, R.C. Yu, C.C. Chou, Production of b-galactosidase by Bifidobacteria as influenced by various culture conditions, Int. J. Food Microbiol. 104 (2005) 197–206. [14] J.W. Kim, S.N. Rajagopal, Isolation and characterization of beta-galactosidase from Lactobacillus crispatus, Folia Microbiol. 45 (1) (2000) 29–34. [15] T. Karunakaran, B.G. Devi, BG, Factors influencing beta-galactosidase activity of Aeromonas caviae, J. Basic Microbiol. 34 (4) (1994) 245–252. [16] M. Carevic, M. Vukasinovic-Sekulic, S. Grbavcic, M. Stojanovic, M. Mihailovic, A. Dimitrijevic, D. Bezbradica, Optimization of b-galactosidase production from lactic acid bacteria, Hem. Ind. 69 (3) (2015) 305–312. [17] L.N. Prasad, B.C. Ghosh, F. Sherkat, N.P. Shah, Extraction and characterization of b-galactosidase produced by Bifidobacterium animalis spp. lactis Bb12 and Lactobacillus delbrueckii spp. bulgaricus ATCC11842 grown in whey, Int. Food Res. J 20 (1) (2013) 487–494. [18] N. Saishin, M. Ueta, A. Wada, I. Yamamoto, Properties of b-galactosidase purified from Bifidobacterium longum subsp. longum JCM 7052 grown on gum Arabic, J. Biol. Macromol 10 (1) (2010) 23–31. [19] S. Iqbal, T. Nguyen, T.T. Nguyen, T. Maischberger, b-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic glacto-oligosaccharides, Carbohydr. Res. 345 (2010) 1408–1416. [20] B.G. Mazi, H. Hamamci, D.M. Oqrydzial, S.R. Dungan, Single-step partial purification of intracellular b-galactosidase from Kluyveromyces lactis using microemulsion droplets, Appl. Biochem. Biotechnol. (2016), https://doi.org/ 10.1007/s12010-016-2148-y. [21] T.C. Bhalla, A. Devi, K. Angmo, N. Thakur, A. Kumari, b-Galactosidase from Lactobacillus brevis PLA28: purification, characterization and synthesis of galacto-oligosaccharides, J. Food Ind. Microbiol. 1 (2015) 104. [22] F.M. Bekler, S. Yalaz, O. Acer, K. Guven, Purification of thermostable bGalactosidase from Anoxybacillus sp. KP1 and estimation of combined effect of some chemicals on enzyme activity using semiparametric errors in variables model, Fresen. Environ. Bull. 26(3) (2017) 2251–2259.

P. Chanalia et al. / Bioorganic Chemistry 77 (2018) 176–189 [23] A. Mbuyi-Kalala, A.G. Schnek, J. Leonis, Separation and characterization of four enzyme forms of beta-galactosidase from Saccharomyces lactis, Eur. J. Biochem. 178 (2) (1988) 437–443. [24] X. Sun, X. Duan, D. Wu, J. Chen, J. Wu, Characterization of Sulfolobus solfataricus b-galactosidase mutant F441Y expressed in Pichia pastoris, J. Sci. Food Agric. 94 (7) (2013) 1359–1365. [25] E. Selvarajan, V. Mohanasrinivasan, Kinetic studies on exploring lactose hydrolysis potential of b-galactosidase extracted from Lactobacillus plantarum HF571129, J. Food Sci. Technol. (2015), https://doi.org/10.1007/ s13197-015-1729-z. [26] T. Maischberger, E. Leitner, S. Nitisinprasert, O. Juajun, M. Yamabhai, T.H. Nguyen, D. Haltrich, Galactosidase from Lactobacillus pentosus: purification, characterization and formation of galacto-oligosaccharides, Biotechnol. J. 5 (2010) 838–847. [27] A.R. Park, D.K. Oh, Effects of galactose and glucose on the hydrolysis reaction of a thermostable beta-galactosidase from Caldicellulosiruptor saccharolyticus, Appl. Microbiol.Biotechnol. 85 (2010) 1427–1435. [28] Y. Wu, S. Yuan, S. Chen, D. Wu, J. Chen, J. Wu, Enhancing the production of galacto-oligosaccharides by mutagenesis of Sulfolobus solfataricus betagalactosidase, Food Chem. 138 (2013) 1588–1595. [29] E. Jurado, F. Camacho, G. Luzon, J.M. Vicaria, Kinetic models of activity for bgalactosidases: influence of pH, ionic concentration and temperature, Enzyme Microb. Technol. 34 (2004) 33–40. [30] K. Isobe, M. Yamashita, S. Chiba, N. Takahashi, T. Koyama, Characterization of new b-galactosidase from acidophilic fungus, Teratosphaeria acidotherma AIU BGA-1, J. Biosci. Bioeng. 116 (3) (2013) 293–297. [31] P. Hildebrandt, M. Wanarska, J. Kur, A new cold-adapted beta-D-galactosidase from the Antarctic Arthrobacter sp. 32c – gene cloning, overexpression, purification and properties, BMC Microbiol. 9 (2009) 151. [32] J.A. Coker, P.P. Sheridan, J. Loveland-Curtze, K.R. Gutshall, A.J. Auman, J.E. Brenchley, Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an antartic Arthrobacter isolate, J. Bacteriol. 185 (2003) 5473–5482. [33] K. Wang, G. Li, S.Q. Yu, C.T. Zhang, Y.H. Liu, A novel metagenome-derived betagalactosidase: gene cloning, over expression, purification and characterization, Appl. Microbiol.Biotechnol. 88 (2010) 155–165.

189

[34] Z. Nagy, T. Kiss, A. Szentirmai, S. Biro, ß-Galactosidase of Penicillium Chrysogenum: production, purification, and characterization of the enzyme, Protein Expr. Purif. 21 (2001) 24–29. [35] M.C. Crescimbeni, V. Nolan, P.D. Clop, G.N. Marin, M.A. Perillo, Activity modulation and reusability of beta-D-galactosidase confined in sol-gel derived porous silicate glass, Colloids Surf.B Biointerfaces. 76 (2010) 387–396. [36] N. Ohtsu, H. Motoshima, K. Goto, F. Tsukasaki, H. Matsuzawa, Thermostable beta-galactosidase from an extreme thermophile, Thermus sp. A4: enzyme purification and characterization, and gene cloning and sequencing, Biosci. Biotechnol. Biochem. 62 (1998) 1539–1545. [37] M.N. Hung, B.H. Lee, Purification and characterization of a recombinant betagalactosidase with transgalactosylation activity from Bifidobacterium infantis HL96, Appl. Microbiol. Biotechnol. 58 (2002) 439–445. [38] T. Itoh, M. Suzuki, S. Adachi, Production and characterization of betagalactosidase from lactose-fermenting yeasts, Agric. Biol. Chem. 46 (1982) 899–904. [39] T. Vasiljevic, P. Jelen, Lactose hydrolysis in milk as affected by neutralizers used for the preparation of crude b-galactosidase extracts from Lactobacillus bulgaricus 11842, Innov. Food Sci. & Emerg. Technol. 3 (2002) 175–184. [40] L. Pivarnik, A.G. Rand, Assay conditions effect on b-galactosidase activity from Kluyveromyces lactis, J. Food Sci. 57 (4) (1992) 1020–1021. [41] G. Sutendra, S. Wong, M.E. Fraser, R.E. Huber, Beta galactosidase (Escherichia coli) has a second catalytically important Mg2+ site, Biochem. Biophys. Res. Commun. 352 (2) (2007) 566–570. [42] M. Martinez-Bilbao, M.T. Gaunt, R.E. Huber, E461H-betagalactosidase (Escherichia coli): altered divalent metal specificity and slow but reversible metal inactivation, Biochem. 34 (41) (1995) 13437–13442. [43] M. Barboza, D.A. Sela, C. Pirim, R.G. Locascio, S.L. Freeman, J.B. German, D.A. Mills, C.B. Lebrilla, Glycoprofiling bifidobacterial consumption of galactooligosaccharides by mass spectrometry reveals strain-specific, preferential consumptions of glycans, Appl. Environ. Microbiol. 75 (2009) 7319–7325. [44] A.C. Ouwehand, S. Salminen, E. Isolauri, Probiotics: an overview of beneficial effects, Antonie Leeuwenhoek 82 (1) (2002) 279–289.