Purification and Characterization of Î²-Galactosidase Synthesized from

Article pubs.acs.org/IECR

Purification and Characterization of β‑Galactosidase Synthesized from Bacillus safensis (JUCHE 1) Arijit Nath,† Sudip Chakrabarty,†,‡ Shubhrajit Sarkar,† Chiranjib Bhattacharjee,*,† Enrico Drioli,‡,§ and Ranjana Chowdhury† †

Chemical Engineering Department, Jadavpur University, Kolkata 700032, West Bengal, India Department of Chemical Engineering and Materials, L'Istituto per la Tecnologia delle Membrane (ITM-CNR), Cubo-44C, 87036 Rende (CS), Italy § WCU Energy Engineering Department, Hanyang University, Seoul 133-791, South Korea ‡

ABSTRACT: In the present investigation, the purification of β-galactosidase synthesized from Bacillus safensis (JUCHE 1), a new strain isolated from casein whey, has been considered. Broth harvested after 28 h (100.8 × 103 s) was first centrifuged, and the sedimented cell pellet was sonicated to isolate intracellular β-galactosidase. After sonication, the intracellular fluid was subjected to microfiltration (MF) (0.2 × 10−6 m) and ultrafiltration (UF) (300 kg mol−1) in series using a flat-sheet poly(ether sulfone) (PES) membrane in a novel rotating-disk membrane module. In the present investigation, four-stage discontinuous diafiltration (DD) at a constant volume concentration factor (VCF = 2) was carried out. Hydrodynamic studies were conducted to determine the appropriate stirrer speed, transmembrane pressure (TMP), and membrane rotation speed. Finally, β-galactosidase was purified based on the principle of salting-out upon addition of 40% (w/v) ammonium sulfate, followed by dialysis using a 1 kg mol−1 membrane. Subsequently, purified β-galactosidase was characterized using ortho-nitrophenyl-β-galactoside (ONPG) and lactose as substrates.

1. INTRODUCTION Extensive research is now being carried out in the field of bioprocess engineering, including downstream processing. Various works have been carried out to obtain high-resolution separation of proteins with the objective of obtaining high-purity enzymes. In the present century, microbial-synthesized therapeutic biopharmaceuticals are attracting worldwide attention.1,2 The lactose hydrolyzing enzyme, β-galactosidase (β-galactosidase galacto hydrolysase, trivially lactase) has long been accepted as an important ingredient in food processing industries. β-Galactosidase catalyzes hydrolysis of lactose to produce glucose and galactose, and in some cases, it takes part in the transgalactosylation reaction that produces galacto-oligosaccharide (GOS) [for example, Gal (β1 → 3) Gal (β1 → 4) Gal (β1 → 6)].3 In the dairy industry, β-galactosidase has been used to prevent crystallization of lactose; to improve sweetness; to increase the solubility of milk product; to prepare low-lactose-containing food products for lactose-intolerant people; and to use up cheese whey, which would otherwise be an environmental pollutant.4−9 Therefore, it is obvious that the purification of β-galactosidase from crude extract will boost both the dairy and pharmaceutical industries. A variety of strategies have been developed for protein purification. These strategies address different requirements of downstream applications, including scale and throughput. Ultrafiltration (UF) is primarily a size-exclusion-based pressuredriven membrane separation process that is an accepted and well-practiced processing operation in the dairy industry for whey treatment. Depending on the molecular weight distribution of solute molecules in the feed mixture, an appropriate UF membrane is generally chosen for the separation process based on its molecular weight cutoff (MWCO). During UF, © 2013 American Chemical Society

high-molecular-weight components, such as proteins, and suspended solids are rejected, whereas low-molecularweight components such as monosaccharides, disaccharides, salts, amino acids, and organic and inorganic acids pass through the membrane freely. In the UF technique, the pressure applied to the working fluid provides the driving potential to force the solvent and low-molecular-weight solutes to flow through the membrane. Solutes that are rejected by the membrane accumulate on the membrane surface and form a concentration polarization layer. At the steady state, the quantity of solutes conveyed by the solvent to the membrane surface is equal to the amount that diffuses back for a dead-end UF membrane module or the amount that is convectively carried away by the flowing fluid for a cross-flow UF membrane module. For protein purification, UF is often carried out in diafiltration (DF) mode, where more purification of the higher-molecular-weight fraction can be achieved because of the continuous washing effect of the low-molecular-weight solutes.10,11 Different separation techniques have already been attempted for the purification of β-galactosidase from crude extracts. Details of these works are presented in Table 1. Bacillus safensis (JUCHE 1) is a unique microorganism, isolated in our laboratory, that is capable of producing thermostable β-galactosidase, amylase, cellulase, and alkaline phosphatase. In the present investigation, Bacillus safensis (JUCHE 1) was cultivated in modified de Man−Rogosa−Sharpe (MMRS) medium in a 5 × 10−3 m3 jar fermentor (working volume of 2 × 10−3 m3) and Received: Revised: Accepted: Published: 11663

March 17, 2013 June 10, 2013 June 19, 2013 June 19, 2013 dx.doi.org/10.1021/ie4008584 | Ind. Eng. Chem. Res. 2013, 52, 11663−11672

11664

gel permeation chromatography, affinity chromatography, and ammonium sulfate precipitation ammonium sulfate precipitation and gel permeation chromatography protamin sulfate precipitation, ammonium sulfate precipitation, and ion-exchange chromatography heat treatment, ammonium sulfate precipitation, ion exchange, and gel filtration chromatography ion-exchange chromatography on Ni Sepharose 6 fast-flow column and ultrafiltration ion-exchange chromatography on DEAE−Sepharose, affinity chromatography on PABTG-Sepharose, and gel filtration on Sephacryl S-300 Q fast-flow chromatography and gel chromatography on a Superose 6 HR column

Bacillus circulans

Streptococcus thermophilus

Streptococcus lactis

Bacillus stearothermophilus (expressed in Bacillus subtilis)

Bacillus licheniformis (cloned and expressed in Escherichia coli)

Arthrobacter sp.

Saccharomyces lactis

Kluyveromyces marxianus

Kluyveromyces lactis

ammonium sulfate fractionation, column chromatography on Sephadex G-100 and DEAE−Sephadex A-50, 78-fold purification

gel filtration, anion-exchange chromatography on DEAE− Sepharose CL-6B, hydrophobic chromatography on octylSepharoseCL-4B, and cation-exchange chromatography on CMSepharose CL-6B precipitation with ammonium sulfate, ion-exchange chromatography on DEAE−Sephadex, affinity chromatography, and chromate focusing gel filtration on a Superose 12 PC 3.2/30 column and ionexchange chromatography on a Mono Q PC 1.6/5 column using a fast protein liquid chromatography system cell disruption, DEAE−Sephadex ion-exchange chromatography, and chromatography on hydroxylapatite extraction with 2% chloroform, acetone, and ammonium sulfate precipitation

Aspergillus niger

Penicillium chrysogenum

2-propanol fractionation, column chromatography on DEAE− Sephadex A-50 and Sephadex G-200

Aspergillus oryzae

Bifidobacterium longum CCRC 15708

ammonium sulfate precipitation, hydrophobic interaction chromatography, and affinity chromatography

Lactobacillus reuteri

purification procedure ZnCl2 precipitation, ion-exchange membrane chromatography, hydrophobic interaction chromatography, and gel filtration chromatography

Bacillus sp. 3088

source

Table 1. Different Methodologies for β-Galactosidase Purification enzyme characteristics

MW was 135 kg mol−1; optimum pH was 7.25; Km values were (12−17) × 10−3 M for lactose and 1.6 × 10−3 M for ONPG; metal activators were Na+ and Mn2+ MW was 280 kg mol−1; optimum temperature and pH were 318.15−325.15 K and 6.2, respectively; inactivated in 240 s at 329.15 K; Km values were 3.1 × 10−3 and 25 × 10−3 M for ONPG; inhibition constants were 58 × 10−3, 110 × 10−3, 111 × 10−3, and 52 × 10−3 M for ONPG, galactose, ribose, and lactose, respectively; inhibitors were p-chloromercuribenzoate and dithiothreitol optimum pH was 7.2; metal activator was Mg2+; Km was 1.18 × 10−3 M for ONPG

MW was 484 kg mol−1, associated with subunits of 115, 86.5, 72.5, 45.7, and 41.2 kg mol−1; optimum pH and temperature were 8 and 333.15 K, respectively; isoelectric point was 6.2; Km values were 6.34 × 10−3 and 6.18 × 10−3 M for ONPG and lactose, respectively; inhibitors were galactose, divalent Hg, Cu, and Ag; metal activator was divalent Mg; ethylenediaminetetraacetic acid did not affect the enzyme activity heterodimer enzyme; MW was 105 kg mol−1 (monomers of 72 and 35 kg mol−1); isoelectric points were 4.6−4.8 and 3.8−4.0 for Lactobacillus reuteri L103 and Lactobacillus reuteri L461, respectively; optimum pH was 6−8; inhibitor was D-glucose; activators were Na+, Mn2+, and K+ MW was 212 kg mol−1, consisting of 145 and 86 kg mol−1; Km values for the three subunits were 3.6 × 10−3, 5.0 × 10−3, and 3.3 × 10−3 M for ONPG as a substrate and 3.7 × 10−3, 2.94 × 10−3, and 2.71 × 10−3 M for lactose as a substrate intracellular; MW was 530 kg mol−1; optimum pH was 7.1; galactose was a competitive inhibitor (Ki 60 × 10−3 M), Km values for ONPG and lactose were 0.98 × 10−3 M and 6.9 × 10−3 M, respectively enzyme was most labile when suspended in cold (278.15 K) phosphate buffer; in the presence of a high concentration ammonium sulfate (0.85 M), the enzyme was highly active and stabilized intracellular; MW was 70 kg mol−1; isoelectric point was 5.1; optimum temperature and pH were 343.15 K and 7, respectively; kinetics of thermal inactivation and half-lives at 338.15 and 343.15 K were 18 × 104 and 324 × 102 s, respectively; Km and vmax were 2.96 × 10−3 M and 0.11 × 10−3 kmol s−1 (kg of protein)−1, respectively; inhibitors were Fe2+, Zn2+, Cu2+, Pb2+, Sn2+, and thiol agent homodimeric; optimum temperature and pH were 323.15 K and 6.5, respectively; Km values for lactose and ONPG were 169 × 10−3 and 13.7−3 M, respectivel; inhibitors were glucose and galactose; metal activators were Na+, K+, Mg2+, Mn2+, and Ca2+ intracellular; homodimeric; MW of each subunit was 116 kg mol−1; optimum pH and temperature were 6−8 and 298.15 K, respectively; activators were thiol compounds, Na+, and K+; inactivated by 4-chloromercuribenzoic acid, Pb2+, Zn2+, and Cu2+ specific activity was 168.6 × 103 U (kg of protein)−1; MW was 357 kg mol−1; optimum pH and temperature were 7 and 323.15 K, respectively; Km and vmax were 0.85 × 10−3 M and 7.67 × 10−3 U (kg of protein)−1, respectively; inhibitors were lactose, fructose, Fe2+, Co2+, Cu2+, Ca2+, Zn2+, Mn2+, Mg2+, and Fe3+; metal activators were Na+ and K+ extracellular; optimum pH values for ONPG and lactose were 4.5 and 4.8, respectively; optimum temperature was 319.15 K; Km values were 7.2 × 10−4 and 1.8 × 10−2 M for ONPG and lactose, respectively; inhibitors were Hg2+, Cu2+, N-bromosuccinimide, and sodium laurylsulfate; apparent MW was 105 kg mol−1. glycoprotein in nature; associated with three subunits, with MWs of 124, 150, and 173 kg mol−1; isoelectric point was 4.6; optimum pH was 2.5−4.0; heat-stable up to 333.15 K; Km values were (85−125) × 10−3 M for lactose and 2.4 × 10−3 M for ONPG; vmax values at 303.15 K were 104 × 103 and 121 × 103 (unit of enzyme) (kg of protein)−1 for ONPG and lactose, respectively intracellular; specific activity was 5.84 × 103 U (kg of protein)−1; optimum temperature and pH were 303.15 K and 4 respectively; Km and isoelectric point were 1.81 × 10−3 M and 4.6, respectively, for ONPG; multimeric enzyme; MW was 270 kg mol−1; MW of a single monomer was 66 kg mol−1 intracellular; MW of a single monomer was 124 kg mol−1

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Industrial & Engineering Chemistry Research Article

dx.doi.org/10.1021/ie4008584 | Ind. Eng. Chem. Res. 2013, 52, 11663−11672

Industrial & Engineering Chemistry Research

Article

the culture harvested after 100.8 × 103 s was considered for extraction and purification of intracellular β-galactosidase. The intracellular fluid of the microbial biomass was obtained by sonication, and the sonication time was optimized. The optimum sonication time was considered with respect to highest enzymatic activity. Size-exclusion-based UF followed by salting-out using ammonium sulfate was employed for the purification of β-galactosidase. In the present investigation, a rotating-disk membrane module equipped with a flat-sheet poly(ether sulfone) (PES) UF membrane of the desired MWCO was used for β-galactosidase separation. In the UF process, operating parameters such as the transmembrane pressure (TMP) (98.07−392.27 kPa), stirrer speed (0.83−3.33 rps), and membrane rotation speed (0.67−1.67 rps) were varied. In the UF process, a constant volume concentration factor (VCF = 2) was maintained. In the present investigation, a fourstage discontinuous diafiltration (DD) process was carried out for the purification of β-galactosidase. Hydrodynamic studies of a rotating-disk membrane module under different operating conditions were also performed. The basic philosophy of using a rotating-disk membrane, a high-shear device, is to reduce concentration polarization, a characteristic of pressure-driven membrane separation processes, thus reducing the fouling and increasing the throughput of the membrane module. Conventional cross-flow UF with such a complex protein mixture in many cases does not provide satisfactory results. Many different process intensification strategies have been employed in many different applications, but not much success has been recorded in the case of protein separation and purification.28,29 In 2013, Suárez et al. discussed the performance of a membrane emulsification unit using flat membranes in a stirred-tank reactor where the rotating speed within the reactor had an effect on the process optimization.30 The results of that study indicated that UF with a rotatingdisk membrane module could provide an efficient route for the purification of β-galactosidase with higher flux and reduced equipment footprint, an essential prerequisite for process intensification. Finally, the purified β-galactosidase was characterized with respect to the maximum reaction velocity (vmax), the Michaelis−Menten constant (K m), optimum reaction temperature, and pH considering ortho-nitrophenyl-β-galactoside (ONPG) and lactose as substrates.

acid or sodium hydroxide with the help of a digital pH probe. Temperature was maintained by a hot water bath and a digital temperature sensor. The aeration rate in the fermentor was maintained by a digital dissolved oxygen (DO) probe and an associated controller. A schematic diagram of the fermentor setup is shown in Figure 1.

Figure 1. Schematic diagram of the fermentor and its components.

An indigenous rotating-disk dead-end membrane module (Gurpreet Engineering Works, Uttar Pradesh, India) made of stainless steel (SS316) that had an 82 × 10−3 m outer diameter, a 4 × 10−3 m thickness, and a 3.50 × 10−4 m3 working volume was used in these experiments. The module was equipped with two motors with speed controllers to provide rotation of the stirrer (ac motor, 1/20 HP, maximum 66.67 rps) and the membrane housing (dc motor, 0.25 HP, maximum 25 rps). The module had the facility to rotate the membrane and the stirrer in opposite directions to provide maximum shear in the vicinity of the membrane surface. The membrane module was capable of withstanding a maximum pressure of 980 kPa. An air compressor associated with a digital pressure gauge was used for the pressurization of the cell so as to provide the desired TMP across the membrane surface. Membranes were placed properly within the membrane module using a gasket, and the gap between the end of stirrer and the membrane surface was 5 × 10−3 m. The membrane module was fitted with four baffles at 90° spacing to avoid the formation of vortexes in the working fluid. The module could withstand high temperature as it was made of SS316, but the maximum temperature during the run was fixed according to the membrane characteristics.31 In preliminary experiments (gel permeation chromatography using Sephadex G-200 and gel electrophoresis), it was observed that the molecular weight of β-galactosidase was 420 kg mol−1. Therefore, to purify β-galactosidase, a PES membrane with a 300 kg mol−1 MWCO (76 × 10−3 m diameter, 280 × 10−6 m thickness) was used. The membrane module was operated in dead-end mode. The same module was used for microfiltration (MF) prior to UF using a 0.2 × 10−6 m PES membrane with a diameter of 76 × 10−3 m. As PES membranes exhibit no hydrophilic interactions, they are usually preferred for their low fouling characteristics, broad pH range, and durability. Schematic diagrams of the experimental setup and membrane module are presented in Figure 2. A biochemical oxygen demand (BOD) incubator cum shaker (S. C. Dey & Co., Kolkata, India), a UV laminar flow hood,

2. EXPERIMENTAL SECTION 2.1. Materials. Lactose, tryptone, yeast extract, and beef extract were obtained from HIMEDIA, Mumbai, India. Dipotassium hydrogen phosphate, sodium acetate, manganese sulfate, magnesium sulfate, Folin−Ciocalteau reagent, and acetonitrile were obtained from Merck, Mumbai, India. Ammonium sulfate, sodium hypochloride, ammonium citrate, sodium potassium tartarate, sodium carbonate, copper sulfate, hydrogen chloride, and sodium hydroxide were obtained from Ranbaxy, Mumbai, India. ONPG, sodium citrate buffer, sodium phosphate buffer, and Tris-HCl buffer were obtained from Sigma-Aldrich, St. Louis, MO. Deionized water, used in all experiments, was obtained from an Arium 611DI ultrapure water system (Sartorius AG, Göttingen, Germany). The flatsheet 300 kg mol−1 PES UF membrane (PMMK) was purchased from Millipore, Bedford, MA. 2.2. Equipment. In the present investigation, a 5 × 10−3 m3 jar fermentor (Eyla, Tokyo, Japan) was used. The working volume of the fermentor was 2 × 10−3 m3. The fermentor was associated with a suitable stirring arrangement (propeller-type blade) through a rotation-per-second (rps) indicator. The pH 7 of the growth medium was maintained with 0.1 N hydrochloric 11665



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Figure 2. (A) Experimental setup and (B) schematic diagram of the rotating-disk membrane module (not to scale).

conditions. Therefore, aerobic conditions were maintained during all experiments described in the next section. 2.5. Batch Culture of Microbial Growth. The batch culture for microbial growth was performed in a 5 × 10−3 m3 jar fermentor. The maintenance medium for microbial growth was MMRS medium, in which the carbohydrate source was lactose. The composition of the MMRS medium (kg m−3) was as follows: tryptone, 10.0 × 10−3; beef extract, 10.0 × 10−3; yeast extract, 5.0 × 10−3; ammonium citrate, 2.0 × 10−3; sodium acetate, 5.0 × 10−3; magnesium sulfate, 0.1 × 10−3; manganese sulfate, 0.5 × 10−3; dipotassium phosphate, 2.0 × 10−3; and lactose, 20.0 × 10−3. The pH of the medium was adjusted to 7.0 with 0.1 N sodium hydroxide and 0.1 N hydrochloric acid. Sterilization of all components of the growth medium was done in an autoclave at 394.15 K for 900 s. Lactose was sterilized using MF with a 0.2 × 10−6 m PES membrane (76 × 10−3 m diameter) because of its sensitivity to high temperature. Batch experiments were performed in 2 × 10−3 m3 of growth medium with 5% (v/v) inoculums. In the fermentation process, the rate of agitation, temperature, rate of aeration, and pH were

a water bath, a hot-air oven (Bhattacharya & Co., Kolkata, India), a magnetic stirrer, a sonicator (Sartorius AG, Göttingen, Germany), a cold centrifuge (C-24) (Remi Instruments Ltd., Mumbai, India), and an autoclave (G.B. Enterprise, Kolkata, India) were also used in the experiments. 2.3. Analytical Instruments. A digital pH meter, digital weighing machine (Sartorius AG, Göttingen, Germany), Varian UV−visible spectrophotometer (Cary50 Bio), and highperformance liquid chromatography (HPLC) instrument (Perkin-Elmer, Series 200) were used. The HPLC system was associated with a refractive index (RI) detector and a Spheri 5 amino column (5 × 10−6 m, 4.6 × 10−3 m × 220 × 10−3 m). The temperature of the HPLC column was maintained at 298 K. Acetonitrile 75% (v/v) was used as the mobile phase at a flow rate of 1.67 × 10−8 m3 s−1 for carbohydrate analysis.32 2.4. Microorganism. The isolated facultative anaerobic microbial consortium Bacillus safensis (JUCHE 1) was used in the present investigation. Although a facultative anaerobe, growth of Bacillus safensis (JUCHE 1) was favored under aerobic 11666



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Figure 3. Schematic flow diagram of the proposed process.

maintained at 2.83 rps, 310.15 K, 1.7 × 10−3 m3 m−3 s−1, and 7, respectively. 2.6. Methodology for β-Galactosidase Purification. In the present investigation, a culture broth that had been aged for 100.8 × 103 s was considered for enzyme purification. The harvested culture broth was centrifuged at 200 rps for 900 s at 277.15 K. The pellet of microbial biomass was washed twice with distilled water, and finally, the pellet was resuspended in 0.05 M sodium phosphate buffer at pH 7.0. Subsequently, sonication was used to obtain intracellular fluid from the cellular biomass. The cell pellet was sonicated at 16 kHz with a probe of 9.5 × 10−3 m outer diameter using a constant power source of 240 V for the optimum sonication time. The optimization of the sonication time is described in section 2.9. Cell debris was removed by centrifugation (166.67 rps for 1200 s), and the supernatant was collected. The intracellular fluid was subjected to MF with a 0.2 × 10−6 m membrane, followed in series by UF with a 300 kg mol−1 PES membrane. In the UF process, a constant volume concentration factor (VCF = 2) was maintained. During the UF process, operating parameters such as TMP (98.07−392.27 kPa), stirrer speed (0.83−3.33 rps), and membrane rotation speed (0.67−1.67 rps) were varied. In the present investigation, four-stage DD was carried out for the purification of β-galactosidase. During DD, 0.05 M sodium phosphate buffer (pH 7.0) was used for volume makeup. Permeate volumes of dead-end membrane module were collected at constant time intervals, and the permeate flux was calculated according to

J=

1 dV A dt

water runs at different TMPs, including 98.07, 196.133, 294.2, and 392.27 kPa. 2.8. Membrane Cleaning. After each experiment, the membrane module was disassembled, and the membrane disk was thoroughly rinsed for 1200 s under deionized water in a laboratory wash basin. After water cleaning, the membrane was soaked in 1 × 10−3 m3 of 0.5 mM sodium hypochloride in 0.1 N sodium hydroxide for 1800 s in a beaker, and subsequently, it was thoroughly washed with deionized water for 1200 s. In each case, the water flux was found to regain more than 98% of its original value, suggesting the cause of flux decline to be limited by either osmotic pressure or formation of a reversible fouling layer. 2.9. Optimization of Sonication Time. The cell pellet was sonicated at 16 kHz with a probe of 9.5 × 10−3 m outer diameter using a constant power source of 240 V. Different sonication times in the range of 60−400 s were chosen for optimization of the process. Interstage cooling for 10 s was maintained after an operating time of 30 s. The sonication time corresponding to the maximum enzyme activity was designated as the optimum sonication time. 2.10. Effects of TMP. Different TMPs, ranging from 98.07 to 392.27 kPa, were used during experimentation to assess the effect of pressure on enzyme recovery and to characterize the membrane module from the hydrodynamic point of view. 2.11. Effects of Stirrer Speed. Different stirrer speeds, ranging from 0.83 to 3.33 rps, were used to provide the required turbulence in the membrane module (during UF) and to reduce the effects of concentration polarization. 2.12. Effects of Membrane Rotation. Different membrane rotation speeds, ranging from 0.67 to 1.67 rps, were used to provide the required turbulence in the membrane module (characteristics of a high-shear device) and to reduce the effects of concentration polarization. 2.13. β-Galactosidase Assay. β-Galactosidase was assayed according to the method described by Miller using ONPG and lactose as substrates.33 The protein concentration was estimated by Lowry’s assay method considering bovine serum albumin as a standard protein.34 The specific activity of β-galactosidase was determined from the total activity of the enzyme with respect to the total protein concentration.35 2.14. Estimation of the Kinetic Parameters of β-Galactosidase Activity. In the present investigation, the ONPG and lactose concentrations were varied in the range of (5−40) × 10−3 M. The initial reaction velocities corresponding to different initial lactose or ONPG concentrations were used to determine the kinetic parameters, namely, the maximum reaction velocity (vmax) and the Michaelis−Menten constant (Km). 2.15. Determination of the Optimum Temperature for Enzymatic Reaction. Specific enzyme activities were analyzed for pure β-galactosidase using ONPG and lactose as substrates

(1)

where J is the permeate flux (m3 m−2 s−1), V is the permeate volume (m3), A is the effective membrane area (m2), and t is the time (s). Additional purification of β-galactosidase was obtained by ammonium sulfate precipitation, followed by dialysis. Ammonium sulfate was added gradually to partially purified β-galactosidase under constant stirring at 1.67 rps and at a temperature of 277.15 K. The target enzyme pellet was dialyzed for 8.64 × 104 s on a 1.0 kg mol−1 dialysis membrane with a reference of 0.05 M sodium phosphate buffer (pH 7). A schematic diagram of the proposed working scheme is presented in Figure 3. 2.7. Membrane Compaction and Water Runs. Prior to experiments, the membrane was subjected to compaction for about 3600 s with ultrapure deionized water at a pressure of 392.27 kPa, the highest operating pressure to prevent any possibility of changing the membrane hydraulic resistance during UF. Once the water flux had become steady with no further decrease, it was concluded that full compaction of the membrane had taken place. After compaction, the membrane hydraulic resistance (Rm) was determined based on a series of 11667



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the enzyme activity decreased. This can be explained by the fact that, at a sonication time of 300 s, almost all of the cells had been lyzed, and after the critical sonication time, the enzyme was denatured by high shear, which resulted in a lower enzyme activity after 300 s. Therefore, the optimum sonication time for this process is considered to be 300 s. Interstage cooling for 10 s was maintained after an operating time of 30 s. Centrifugation (166.67 rps, 277.15 K, 900 s) followed by MF was employed to separate the cell suspension from the supernatant, and the permeate of the MF membrane was used as a feed for UF. 3.2. Effects of TMP. After membrane compaction, a series of water runs was performed to evaluate the membrane hydraulic resistance, Rm, which was found to be (5.05 × 108) ± (8.5 × 106) m−1 for a 300 kg mol−1 UF membrane. In Table 2, the final permeate fluxes at each stage of the DD process are described for different TMPs ranging from 98.07 to 392.27 kPa at a constant membrane rotation speed of 1.33 rps and a constant stirrer speed of 2.5 rps. It was observed that the permeate flux decreased with increasing DD stage because the high concentration of solute molecules in the feed side created concentration polarization on the membrane surface. With time, the concentration polarization layer became more compact and thicker, which can be attributed to the reduction of the flux until the third stage of the DD process. It was observed that the steady-state permeate fluxes in the third and fourth stages of the DD process were almost same (no significant drop in permeate flux), which might be due to the saturation of the concentration polarization layer that might lead to a “limiting flux” under fixed operating conditions. Because the concentration polarization layer became fully developed in the third stage of the DD process, the hydrodynamic behavior for the third stage of the DD process is elucidated in detail in the subsequent discussion (especially Figures 5, 6, and 8) to understand the effects of different operating parameters. In Figure 5, the time history of the permeate flux is plotted as a function of filtration time for the third stage of the DD process. In this case, the permeate flux was found to increase with increasing TMP (from 98.07 to 294.2 kPa), and at a TMP of 392.27 kPa, it became almost the same as the previous value. This can again be attributed to a fully developed concentration polarization layer at high TMP. As the pressure difference across the membrane surface increased, the convective flux increased because of the higher driving force. However, the higher flux resulted in more transport of solute molecules to the membrane surface and higher rejection. This resulted in more deposition and, hence, a higher polarized layer resistance. At higher TMP, the flux was generally found to attain a constant value, known as the limiting flux, as a result of the compensation of two effects: a higher driving force and a high polarized layer resistance. It was observed that the permeate flux decreased gradually and eventually became steady with time. From Figure 5, it can be concluded that the appropriate TMP is 294.2 kPa. In Table 3, the activities of β-galactosidase are reported for

at different reaction temperatures in the range of 293.15− 333.15 K. 2.16. Determination of the Optimum pH for Enzymatic Reaction. Specific enzyme activities were also analyzed for pure β-galactosidase with respect to ONPG and lactose as substrates at different pH values of the reaction medium. The pH of the enzymatic reaction was varied in the range of 4−12. Sodium citrate buffer (pH 4−6), sodium phosphate buffer (pH 7−8.5), and Tris-HCl buffer (pH 9−12) were used separately to control the pH. All experiments were performed in triplicate, and average values are reported.

3. RESULTS AND DISCUSSION In the present investigation, the economical production of a biopharmaceutical, namely, β-galactosidase, with respect to isolation and purification from crude extract was attempted. The intracellular fluid of the microbial biomass was obtained by sonication, and the sonication time was optimized. A novel dead-end rotating-disk membrane module equipped with a magnetic drive stirrer and membrane rotating device and pressurized with a nitrogen gas cylinder was used for the experiments. Bacterial intracellular fluid is a mixture of different enzymes, metabolites, and their derivatives. Therefore, purification of a particular enzyme fraction requires a very fine-tuned high-precision technique. Different processes including sizeexclusion-based UF, salting-out, and dialysis were selected for this operation to provide a satisfactory β-galactosidase activity. Purification characteristics and β-galactosidase activity were investigated under different process parameters. 3.1. Optimization of the Sonication Procedure and Feed Pretreatment. In Figure 4, the β-galactosidase activity

Figure 4. β-Galactosidase activity [kmol (kg of protein−1) s−1] as a function of sonication time (s).

[kmol (kg of protein)−1 s−1] is plotted against sonication time (s). It was observed that the enzyme activity increased with increasing sonication time, and after a certain limit (300 s),

Table 2. Steady-State Permeate Flux (J, 106 m s−1) through a 300 kg mol−1 UF Membrane at Different TMPs (kPa) and Stages of the DD Process for a Membrane Rotation Speed of 1.33 rps and a Stirrer Speed 2.5 rps TMP (kPa) stage of the DD process

98.07

196.133

294.2

392.27

first second third fourth

1.75 ± 0.02 0.808 ± 0.03 0.455 ± 0.03 0.455 ± 0.02

2.1 ± 0.03 1.763 ± 0.03 1.381 ± 0.02 1.381 ± 0.03

3.202 ± 0.04 2.79 ± 0.02 2.497 ± 0.03 2.497 ± 0.02

3.202 ± 0.02 2.79 ± 0.03 2.497 ± 0.03 2.497 ± 0.04

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Figure 6. Time history of the permeate flux through a 300 kg mol−1 UF membrane for the third stage of the DD process at a membrane rotation speed of 1.33 rps, a TMP of 294.2 kPa, and different stirrer speeds: (⧫) 0.83, (■) 1.67, (▲) 2.5, and (●) 3.33 rps.

−1

Figure 5. Time history of the permeate flux through a 300 kg mol UF membrane for the third stage of the DD process at a membrane rotation speed of 1.33 rps, a stirrer speed of 2.5 rps, and different TMPs: (⧫) 98.07, (■) 196.133, (▲) 294.2, and (●) 392.27 kPa.

Table 3. Specific Activity of β-Galactosidase [10−3 kmol (kg of protein)−1 s−1] at Different TMPs (kPa) and Stages of the DD Process for a Membrane Rotation Speed of 1.33 rps and a Stirrer Speed of 2.5 rps

The stirrer speed provides the required turbulence in the membrane module with the objective of reducing the concentration polarization. From Figure 6, it can be observed that the permeate flux increased gradually with increasing stirrer speed from 0.83 to 2.5 rps, but at 3.33 rps, it remained almost the same as the value at 2.5 rps. Further, the rate of flux decline was higher at low stirrer speeds. The permeate flux was found to increase with increased stirring because of the enhanced turbulence at the membrane surface. The possible cause of the higher flux and lower rate of flux decline at high stirrer speeds is the higher sweeping action, which is responsible for solute removal from the membrane surface, resulting in a reduction of concentration polarization along with an increase in the rate of permeation. It can also be observed that the permeate flux decreased gradually and eventually became asymptotic with the time axis, a characteristic of typical pressure-driven membrane separation techniques. In Figure 7, the activity of β-galactosidase is plotted as a function of stirrer speed, and it can be observed that the activity of β-galactosidase increased with increasing stirrer speed, from 0.83 to 2.5 rps, at a constant TMP of 294.2 kPa and a constant membrane rotation speed of 1.33 rps This can be explained by the fact that at, high stirrer speed, lower-molecular-weight protein molecules and metabolites passed through the 300 kg mol−1 UF membrane, so that the retained β-galactosidase was more pure. Thus, purification of β-galactosidase was found to increase with increasing stirrer speed. 3.4. Effects of Membrane Rotation Speed. In the present investigation, the effects of membrane rotation speed on β-galactosidase purification were considered to be a great challenge. In Table 5, the final permeate flux (steady-state flux) at each stage of the DD process is presented for different membrane rotation speeds, ranging from 0.67 to 1.67 rps, at a constant stirrer speed of 2.5 rps and a constant TMP of

stage of the DD process TMP (kPa) 98.07 196.133 294.2 392.27

first

second

third

fourth

± ± ± ±

1.18 ± 0.03 1.8 ± 0.02 2.31 ± 0.02 2.3 ± 0.03

2.36 ± 0.02 2.99 ± 0.02 3.542 ± 0.03 3.54 ± 0.03

2.36 ± 0.03 2.991 ± 0.03 3.541 ± 0.02 3.542 ± 0.02

0.78 1.42 1.97 1.96

0.03 0.02 0.02 0.03

different TMPs, and it can be observed that the activity of β-galactosidase increased with increasing TMP, from 98.07 to 294.2 kPa, at constant stirrer and membrane rotation speeds (2.5 and 1.33 rps, respectively). This can be explained by the fact that, at high TMP, lower-molecular-weight protein molecules and metabolites passed through the 300 kg mol−1 UF membrane. Therefore, the purification of β-galactosidase was enhanced at high TMP. 3.3. Effects of Stirrer Speed. In Table 4, the final permeate fluxes at each stage of the DD process are reported for different stirrer speeds, ranging from 0.83 to 3.33 rps, at a constant membrane rotation speed of 1.33 rps and a constant TMP of 294.2 kPa. It can be observed that the permeate flux decreased with increasing DD stage because of the concentration polarization of solute molecules on the membrane surface and there was hardly any variation in the steady-state permeate fluxes in the third and fourth stages of the DD process, which can be attributed to the limiting flux mentioned earlier. In Figure 6, the time histories of the permeate flux for the third stage of the DD process are shown for a 300 kg mol−1 membrane at different stirrer speeds, ranging from 0.83 to 3.33 rps.

Table 4. Steady-State Permeate Flux (J, 106 m s−1) through a 300 kg mol−1 UF Membrane at Different Stirrer Speeds (rps) and Stages of the DD Process for a TMP of 294.2 kPa and a Membrane Rotation Speed of 1.33 rps stirrer speed (rps) stage of the DD process

0.83


1.98 ± 0.03 1.616 ± 0.02 1.263 ± 0.04 1.263 ± 0.02

1.67

2.5

3.3

± ± ± ±

3.202 ± 0.04 2.79 ± 0.02 2.497 ± 0.03 2.497 ± 0.02

3.202 ± 0.02 2.79 ± 0.03 2.497 ± 0.03 2.497 ± 0.04

2.41 2.06 1.63 1.63 11669

0.02 0.03 0.06 0.06



Article

Figure 8. Time history of the permeate flux through a 300 kg mol−1 UF membrane for the third stage of the DD process at a stirrer speed of 2.5 rps, a TMP of 294.2 kPa, and different membrane rotation speeds: (⧫) 0.67, (■) 1, (▲) 1.33, and (●) 1.67 rps.

Figure 7. Specific activity of β-galactosidase [kmol (kg of protein)−1 s−1] at different stirrer speeds (rps) and stages of the DD process for a TMP of 294.2 kPa and a membrane rotation speed of 1.33 rps.

294.2 kPa. It can be observed that the permeate flux decreased with increasing DD stage because of the concentration polarization of solute molecules on the membrane surface, which is in agreement with earlier figures. Similar experiments can be cited to elucidate the observed phenomena. Figure 8 shows the variation of permeate flux as a function of time at different membrane rotation speeds, keeping all other parameters constant. The decline of the flux with time is typical for pressure-driven membrane separation processes such as UF, which is attributed to concentration polarization and fouling. The latter was found to be reversible in nature in this case. An enhancement in flux with increasing membrane rotation speed can be observed in the figure, which resulted from reduced concentration polarization due to the high shear induced by the rotating membrane disk. For the lower membrane rotation speeds, the concentration polarization and the resulting fouling became very severe as the UF process continued, which reduced the flux dramatically. From a comparison of Figures 5, 6, and 8, it can be concluded that membrane rotation is a more effective parameter than TMP and stirrer speed for enhancing the throughput from the membrane. In Figure 9, the time histories of the permeate flux in different DD stages are depicted at appropriate operating conditions. It can be observed that the permeate flux decreased with increasing DD stage as well as increasing process time. In Figure 10, the activities of β-galactosidase in different DD stages are plotted at different membrane rotation speeds, and it can be observed that the activity of β-galactosidase increased with increasing membrane rotation speed, from 0.67 to 1.33 rps, at a constant TMP of 294.2 kPa and a constant stirrer speed of 2.5 rps. This can be explained by the fact that, at high membrane rotation speeds, lower-molecular-weight protein molecules and metabolites passed through the 300 kg mol−1 UF membrane and the retained β-galactosidase became more concentrated.

Figure 9. Time histories of the permeate flux through a 300 kg mol−1 UF membrane for different stages of the DD process at a TMP of 294.2 kPa, a stirrer speed of 2.5 rps, and a membrane rotation speed of 1.33 rps.

Thus, the purification of β-galactosidase increased at high membrane rotation speeds. From this investigation, it is clear that the appropriate TMP, stirrer speed, and membrane rotation speed in the UF process for this application are 294.2 kPa, 2.5 rps, and 1.33 rps, respectively. At the appropriate conditions for the UF process described in this study, a 45-fold increase in β-galactosidase purity with respect to the crude extract could be achieved, with an overall yield of 35%. 3.5. Salting-out and Dialysis. The retentate of the 300 kg mol−1 membrane at appropriate conditions was considered for the isolation of β-galactosidase by the salting-out method. Ammonium sulfate was added gradually to the partially purified β-galactosidase with constant stirring, and finally, β-galactosidase was purified by addition of 40% (w/v) ammonium sulfate. The target enzyme pellet was dialyzed for 8.64 × 104 s using a 1.0 kg mol−1 dialysis membrane with a reference of sodium phosphate buffer (pH 7). After this process, a 65-fold increase in

Table 5. Steady-State Permeate Flux (J, 106 m s−1) through a 300 kg mol−1 UF Membrane at Different Membrane Rotation Speeds (rps) and Stages of the DD process for a TMP of 294.2 kPa and a Stirrer Speed of 2.5 rps membrane rotation speed (rps) stage of the DD process

0.67


2.424 ± 0.02 2.06 ± 0.03 1.304 ± 0.04 1.304 ± 0.02

1 2.791 2.424 1.804 1.804 11670

± ± ± ±

0.03 0.02 0.02 0.04

1.33

1.67

3.202 ± 0.04 2.79 ± 0.02 2.497 ± 0.03 2.497 ± 0.02

3.202 ± 0.02 2.79 ± 0.03 2.497 ± 0.03 2.497 ± 0.04



Article

Figure 12. β-Galactosidase activity [kmol (kg of protein)−1 s−1] at different reaction pH values considering ONPG as a substrate.

Figure 10. Specific activity of β-galactosidase [kmol (kg of protein)−1 s−1] at different membrane rotation speeds (rps) and stages of the DD process for a stirrer speed of 2.5 rps and a TMP of 294.2 kPa.

protocol has been developed that is capable of giving a 65-fold purification of β-galactosidase with an overall yield of 25%. It is thus concluded that the proposed technology for the purification of β-galactosidase from the crude extract by UF using a rotating-disk membrane module could provide an efficient pathway for industrial production and a successful implementation of a process-intensification strategy.

β-galactosidase purity with respect to the crude extract was obtained, with an overall yield of 25%. 3.6. Characterization of β-Galactosidase. Purified β-galactosidase was characterized with respect to its maximum reaction velocity (vmax), Michaelis−Menten constant (Km), optimum reaction temperature, and optimum reaction pH considering ONPG and lactose as substrates. 3.6.1. Enzyme Kinetic Parameters. The concentrations of ONPG and lactose were varied in the range of (5−40) × 10−3 M to evaluate the kinetic parameters of the catalytic activities of purified β-galactosidase. The values of maximum reaction velocity (vmax), Michaelis−Menten constant (Km) were found to be 5.52 × 103 kmol (kg of protein)−1 s−1 and 20.16 × 10−3 M, respectively, considering ONPG as the substrate and 4 × 103 kmol (kg of protein)−1 s−1 and 20 × 10−3 M, respectively, considering lactose as the substrate. 3.6.2. Optimum Reaction Temperature. In Figure 11, enzymatic activity is plotted as a function of reaction temperature

4. CONCLUSIONS In the present investigation, the biopharmaceutical β-galactosidase was purified by a combination of UF, salting-out, and dialysis techniques. In the UF process, different operating parameters, including TMP (98.07−392.27 kPa), stirrer speed (0.83−3.33 rps), and membrane rotation speed (0.67−1.67 rps) were varied. In addition, four-stage discontinuous diafiltration (DD) was carried out at constant VCF for the purification of β-galactosidase. The appropriate TMP, stirrer speed, and membrane rotation were found to be 294.2 kPa, 2.5 rps, and 1.33 rps, respectively. It was observed that higher TMP, stirrer speed, and membrane rotation had a positive influence on permeation, as well as β-galactosidase activity. It was also found that membrane rotation was much more efficient than stirrer speed and TMP for diminishing concentration polarization. At appropriate conditions, the UF process could provide 45-fold β-galactosidase purification with respect to the crude extract, and finally, a 65-fold β-galactosidase purification with respect to crude extract was obtained with an overall yield of 25%. The values of maximum reaction velocity (vmax) and Michaelis− Menten constant (Km) were found to be 5.52 × 103 kmol (kg of protein)−1 s−1 and 20.16 × 10−3 M, respectively, considering ONPG as the substrate and 4 × 103 kmol (kg of protein)−1 s−1 and 20 × 10−3 M, respectively, considering lactose as the substrate. The optimum reaction temperature and pH of the enzymatic reaction were found to be 318.15 K and 7, respectively. The results could lead to further integration of different processes associated with the present investigation in process intensification strategies to reduce the equipment footprint while achieving a high product purity.

Figure 11. β-Galactosidase activity [kmol (kg of protein)−1 s−1] at different reaction temperatures considering ONPG as a substrate.

considering ONPG as the substrate, indicating that the optimum temperature of the enzymatic reaction is 318.15 K, as beyond this value, the enzyme activity declined. In Figure 12, enzymatic activity is plotted as a function of reaction pH considering ONPG as the substrate, indicating that the optimum pH of the enzymatic reaction is 7. Similar figures could be prepared for lactose as the substrate. Thus, in this study, appropriate conditions for β-galactosidase purification have been identified, and a suitable purification

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +91 98364 02118. Fax: +91 33 2414 6203. E-mail: [email protected], [email protected]. Notes

The authors declare no competing financial interest. 11671



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Article

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ACKNOWLEDGMENTS A.N. acknowledges the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for financial support as an SRF. The reported work is part of a University Grants Commission (UGC) Major Project, entitled “Production and Purification of β-Galactosidase from Milk Whey-Based Lactic Acid Bacteria Using Fermentation and Membrane-Based Separation Techniques”. The contribution of UGC is gratefully acknowledged. The authors also acknowledge the contribution of the Department of Science & Technology (DST) under the Ministry of Science & Technology, Government of India, New Delhi, India, for supporting the New INDIGO Collaborative Project with the European counterpart of ICRA, Girona, Spain, and UA, Antwerp, Belgium (Sanction Letter DST/TMC/ 2K11/345, dated May 17, 2012).

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