Enzyme and Microbial Technology 55 (2014) 40–49

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Synthesis and characterization of thermo-responsive poly-N-isopropylacrylamide bioconjugates for application in the formation of galacto-oligosaccharides Tapas Palai a , Ashok Kumar b,∗∗ , Prashant K. Bhattacharya a,∗ a b

Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, India

a r t i c l e

i n f o

Article history: Received 30 August 2013 Received in revised form 28 November 2013 Accepted 5 December 2013 Keywords: Bioconjugate Responsive polymer Enzyme immobilization Galacto-oligosaccharide Poly-N-isopropylacrylamide

a b s t r a c t The study demonstrates the properties of conjugation of ␤-galactosidase with a thermo-responsive polymer, poly-N-isopropylacrylamide (PNIPAAm) in comparison to a non-responsive polymer, polyacrylamide (PAAm). The maximum formation of bioconjugate (PNIPAAm-␤-galactosidase) was 75% (yield) with 50% chemically modified enzyme (using itaconic anhydride). The process of bioconjugation (bioconjugate concentration: 7.4%) decreases lower critical solution temperature from 32.5 ◦ C (with pure PNIPAAm) to 26.5 ◦ C. The effect of temperature on the activities of PNIPAAm-␤-galactosidase, PAAm-␤-galactosidase and native enzyme was also compared. At 70 ◦ C, the maximum activity was observed for PNIPAAm-␤-galactosidase while for others it was at 60 ◦ C. However, the effect of pH was insignificant on activities of both the bioconjugates than the native enzyme. The addition of ethylene glycol (20%, v/v) enhances the activity (by 45%) of PNIPAAm-␤-galactosidase with no loss in stability; however; the trend is reversed with the addition of ethanol. Further, employing bioconjugates even up to 24 cycles of precipitation (at 40 ◦ C) followed by re-dissolution (4 ◦ C) around 90% of activity could be retained by PNIPAAm-␤-galactosidase. The PNIPAAm-␤-galactosidase also showed much-improved thermal and storage stabilities. A lower Michaelis–Menten constant (Km ) was estimated with the PNIPAAm-␤-galactosidase than the native enzyme as well as PAAm-␤-galactosidase. Finally, PNIPAAm-␤-galactosidase was tested to synthesize galacto-oligosaccharides from lactose solution. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Enzyme immobilization has always received a great deal of attention in research for its use in industrial-scale enzymatic processes. Enzyme immobilization may overcome the drawbacks associated with enzymatic processes under free conditions [1]. The advantages of immobilization include easy separation of the enzyme from the reaction mixture [2], reuse of the enzyme, continuous product formation, better product quality, and improved stability [3]. Extensive studies have been reported in relation to enzyme immobilization on various solid supports [2–5], however, with certain drawbacks [6]. Incorporating the concept of soluble–insoluble polymer bioconjugates may eliminate the problem associated with immobilization solid supports [7,8]. Responsive polymers, also known as smart/intelligent/environmentally

∗ Corresponding author. Tel.: +91 512 2597093; fax: +91 512 2590104. ∗∗ Corresponding author. Tel.: +91 512 2594051; fax: +91 512 2594010. E-mail addresses: [email protected] (A. Kumar), [email protected] (P.K. Bhattacharya). 0141-0229/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2013.12.003

sensitive/reversible polymers, have recently received a great deal of attention from researchers regarding their applications to the fields of biotechnology, medicine, and engineering [9–12]. Responsive polymers respond to slight changes in environmental stimuli such as pH, temperature, and ionic strength. The polymers then regain their previous form when the stimulus is removed [13,14]. Thermo-responsive polymers are the most widely studied class of responsive polymers [15,16]. A slight change in temperature around the lower critical solution temperature (LCST) causes the polymer to undergo a reversible phase change from soluble to insoluble or vice versa [13]. Several polymers with different LCSTs show thermo-responsive properties, such as poly-N-isopropyl-acrylamide (PNIPAAm; LCST: ∼32 ◦ C) [8], poly-N-vinyl-caprolactam (LCST: 31–37 ◦ C) [17], poly-N,N-diethylacrylamide (LCST: 32–34 ◦ C) [18], poly-acrylicacid-co-acrylamide (LCST: 25 ◦ C) [19], and poly-N-ethyl-oxazoline (LCST: 62 ◦ C) [16]. PNIPAAm is the most preferred thermo-responsive polymer [20–24] because of its solubility in water, sharpness of phase transition (coil-globule conformation) at its LCST (32 ◦ C), and easy separation from the solution phase by the addition of salt or surfactants above the LCST [25]. Owing to its soluble–insoluble property,

T. Palai et al. / Enzyme and Microbial Technology 55 (2014) 40–49

Notations and abbreviations Michaelis–Menten constant Km Mw weight-average molecular weight Mn number-average molecular weight Mz Z-average molecular weight unit of enzyme U Vm maximum reaction rate AAm acrylamide ammonium persulphate APS BCA bicinchoninic acid DC denaturation capacity EG ethylene glycol GOS galacto-oligosaccharides gel permeation chromatography GPC HPLC high performance liquid chromatography LCST lower critical solution temperature NIPAAm N-isopropylacrylamide ONP o-nitro phenol o-nitrophenyl-␤-d-galactopyranoside ONPG PAAm poly-acrylamide PDI polydispersity index PNIPAAm poly-N-isopropylacrylamide TEMED N,N,N ,N -tetra-methylethylenediamine THF tetrahyrofuran

using these polymers as supports for enzyme immobilization does not impose any mass transfer limitations on the substrate and products [8]. Furthermore, the LCST of PNIPAAm is almost independent of the concentration or molecular weight of the polymer [16]. PNIPAAm-based bioconjugates are most suitable for biomedical applications because their LCST is close to normal human body temperature [14], which is the optimum temperature for most enzymes. ␤-Galactosidase (EC 3.2.1.23) is an important and wellestablished enzyme for the production of galacto-oligosacchatides (GOS) [3,26–28]. The enzyme ␤-galactosidase converts lactose via two simultaneous reactions: the trans-galactosylation reaction, which produces GOS; and the hydrolysis reaction, which forms glucose and galactose [2,29,30]. GOS is an important nutraceutical with prebiotic properties. It has useful applications in the dairy, food, pharmaceutical, and beverage industries. Synthesis of GOS with enzymes under free condition [29] provides maximum conversion (GOS yield: 39.0%, lactose conversion: 60.0% with lactose concentration of 525 g/L, enzyme concentration 2.27 kU/L at 6-pH and 40 ◦ C), however, with this approach one additional step is required for separating enzymes from reaction mixture. In this regard, immobilization of enzymes saves such an additional step, though, the conversion may get affected [2]; still, this approach may be a preferred mode. Most studies utilize solid supports which have mass transfer limitations [6] under immobilization. It is in this regard, the present work was conceived with a belief that bioconjugates may prove useful because of its special properties. Relating to our earlier work [2,29], it is felt that bioconjugates in its soluble state may be considered equivalent to freeing enzyme [29] while in its insoluble state it may be considered equivalent to enzymes under immobilized state [2]. Though, similar to any kind of immobilization approach, the conjugation of enzymes may also reduce the activity of enzymes. Still, there may be distinct possibility of retaining enzyme activity for repeated use as compared to immobilization on membrane as support [2] where a maximum of 3-times the membrane could be utilized. Further, the bioconjugates can be separated easily from the reaction mixture by heating above its LCST.

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Bioconjugates are generally synthesized through covalent or non-covalent approaches by using different physical forms such as soluble cross-linked gels, and nanoparticles. To achieve better conjugation, the polymer, enzyme, or both need to be modified. Random conjugation is the most frequently used technique in covalent approaches, where enzymes are randomly attached to the polymer backbone via covalent bonding [31]. Modifying the enzyme with itaconic anhydride introduces an acryl amide group, which is stable over a wide pH (1–12) range [32]. Enzymes generally possess lysine residues, which are the easiest and most preferred site for conjugation with polymers through the amine terminal [33]. The choice of the conjugation site of the enzyme depends on several factors such as the pH, temperature, and ionic strength of the medium [31]. Random conjugation provides structural rigidity to the enzyme by multipoint attachment. Structural conformations of enzymes are flexible in an aqueous solution and rigid in an organic medium [34]. Studying the change in properties of PNIPAAm-␤galactosidase compared to the native enzyme and the enzyme conjugated with non-responsive poly-acrylamide (PAAm) would be of interest. The present study focused on the synthesis and characterization of thermo-responsive PNIPAAm-␤-galactosidase to produce GOS from lactose. The properties of PNIPAAm-␤-galactosidase were compared with those of the native enzyme as well as those of PAAm-bioconjugates. The bioconjugates were synthesized from their respective monomers using the copolymerization approach. The effects of operating parameters such as the pH, temperature, and organic solvent on the catalytic activity of enzyme were studied. The PNIPAAm-␤-galactosidase was utilized to form GOS from lactose under soluble conditions. 2. Experimental 2.1. Materials and chemicals N-Isopropylacrylamide (NIPAAm; assay 97%), 2,4,6trinitrobenzenesulfonic acid (TNBS; 5% aqueous solution), and o-nitrophenyl-␤-d-galactopyranoside (ONPG; assay 98%) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Itaconic anhydride (assay 97%) was obtained from Alfa Aesar (Heysham, Lancashire, UK). Acrylamide (AAm) was supplied by Sisco Research Laboratories (Mumbai, India). Lactose (monohydrate), d-galactose (assay 99%), and N,N,N ,N -tetra-methylethylenediamine (TEMED) were procured from Loba Chemie Pvt. Ltd. (Mumbai, India). Ammonium persulphate (APS) and d-glucose (anhydrous) was purchased from Qualigens Fine Chemicals (Mumbai, India). Commercial grade ␤-galactosidase (EC 3.2.1.23; commercial name: Biolacta FN5) from Bacillus circulans was provided by Daiwa Kasei K.K. (Osaka, Japan). Bicinchoninic acid (BCA) protein reagent kit was purchased from Novagen (WI, USA). Ethanol and ethylene glycol (EG) were procured from Marck KGaA (Darmstadt, Germany). Milli-Q (Millipore Corporation, India) water was utilized for the preparation of all solutions. All of the chemicals were used without any further purification. 2.2. Enzyme assay and protein estimation The activities of both the bioconjugates and native enzyme were measured by using ONPG as the substrate [29]. The reaction was carried out by mixing 0.5 mL of 10 mM ONPG with 400 mM citrate buffer (pH 6.0) and 0.1 mL enzyme solution. The mixture was incubated at 30 ◦ C for exactly 10 min and to stop the reaction 3.0 mL of 200 mM borate buffer (pH 9.8) was added. The amount of onitrophenol (ONP) released by the reaction between the enzyme and ONPG was estimated by measuring the absorbance at 410 nm in an UV–vis spectrophotometer (Parkin Elmer, Lambda 35). The

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protein content of all enzyme preparations was measured according to the BCA method [35] by using bovine serum albumin as a standard protein. The BCA working reagent was prepared by mixing BCA reagents A and B in the volume ratio of 50:1. A mixture of 3.0 mL working reagent and 0.15 mL enzyme solution was incubated at 37 ◦ C for 30 min, and the absorbance was measured at 562 nm with the UV–vis spectrophotometer. One unit (U) of enzyme was defined as the amount of enzyme that produces 1.0 ␮mol of ONP from ONPG per minute at pH 6.0 and 30 ◦ C. The specific activity of the commercial-grade enzyme was determined to be 0.52 U/mglyophilized powder and 3.1 U/mg-protein.

yellow-colored compound that was detected by measuring its absorbance at 335 nm using the UV–vis spectrophotometer [36]. The degree of modification was defined as the ratio of modified enzyme to amount of enzyme taken for modification; it was calculated using Eq. (1) from Shetty et al. [37].



Modification (%) = 1 −



Amod Anat

× 100

(1)

where, Amod and Anat represent the absorbance values of modified and native enzyme, respectively. 2.5. Synthesis of PNIPAAm-ˇ-galactosidase and PAAm-ˇ-galactosidase bioconjugates

2.3. Chemical modification of enzyme In order to introduce an acrylic group, the ␤-galactosidase enzyme was treated with itaconic anhydride [8,32] at 4 ◦ C. The enzyme (20 mg/mL) was dissolved in 50 mM phosphate buffer (pH 6.0). Itaconic anhydride (50 mg/mL) was slowly added to the enzyme solution along with addition of 1.5 M glucose. The reaction was allowed to continue for 2 h under mild stirring conditions at 4 ◦ C. The unreacted itaconic anhydride was removed from the mixture by performing dialysis using 12.4 kDa molecular weight cut-off dialysis tube for 24 h at 4 ◦ C; 50 mM phosphate buffer (pH 7.0) was used as the dialysate. 2.4. Degree of modification The degree of acrylation of ␤-galactosidase was estimated using the TNBS method [8,32]. Free amine groups of both native and modified enzymes were reacted with TNBS to form a

NIPAAm and AAm monomers were copolymerized with modified ␤-galactosidase to synthesize PNIPAAm-␤-galactosidase and PAAm-␤-galactosidase bioconjugates (Fig. 1) [8,10]. NIPAAm and AAm monomers (200 mg) were dissolved in 10 mL phosphate buffer of pH 7.0; the modified enzyme was then added. The mixture was then purged with nitrogen gas for 20 min to remove the dissolved oxygen, which may have interfered with the polymerization reaction. The reaction was catalyzed by TEMED (7 ␮L) and initiated by the addition of APS (10 mg); the solution was kept at 4 ◦ C for 12 h. Finally, the PNIPAAm-␤-galactosidase bioconjugates were separated through salt-induced thermal precipitation achieved by adding 0.05 M NaCl at 40 ◦ C. PAAm-␤-galactosidase bioconjugates were separated through dialysis for 24 h in 50 mM phosphate buffer (pH 7.0). PNIPAAm polymer was synthesized and separated by following same procedure, however, without using modified enzyme.

O

O OH

+

O

E

H2N

E

NH O

O

Acrylated enzyme

Enzyme

Itaconic anhydride

(a) COOH O OH

O

+

HN

E

HN

O

NIPAAm

*

n

NH

CH3

H3C

*

H3C

Acrylated enzyme

m

O

O NH

CH3

E

PNIPAAm-enzyme (b) COOH

O OH

O H2N

*

+

NH

H2N O

AAm

*

n

E

m

O

O NH

Acrylated enzyme

E

PAAm-enzyme

(c) Fig. 1. Schematic presentation of (a) enzyme modification with itaconic anhydride (b) synthesis of PNIPAAm-␤-galactosidase bioconjugate, and (c) synthesis of PAAm-␤galactosidase bioconjugate.

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2.6. Influences of operating variables on catalytic activity

2.9. Gel permeation chromatography

2.6.1. Effect of temperature The catalytic activities of the PAAm-␤-galactosidase, PNIPAAm␤-galactosidase bioconjugates, and the native ␤-galactosidase were measured at various temperatures (20–80 ◦ C) in 50 mM phosphate buffer (pH 6.0); ONPG was used as the substrate.

The molecular weights of the bioconjugates were estimated using gel permeation chromatography (GPC). The GPC system (Waters® ) was equipped with a refractive index detector (Waters 2414, RID-10A), two pumps (Waters 515), a column heater, an autosampler (Waters 2707), an in-line degasser (DG2), and Empower-2 data processing software. A bank of three columns (Styragel HT 6E, HT 5, and HT 3; dimensions: 4.6 mm × 300 mm) was employed for analysis. The samples were dissolved in HPLC-grade tetrahydrofuran (THF) and filtered through a 0.45-␮m filter. The temperatures of both the column and detector were maintained at 35 ◦ C. THF was used as a mobile phase at a flow rate of 1.0 mL/min. The molecular weights of the bioconjugates and pure PNIPAAm were estimated with the help of a universal calibration graph generated by utilizing Eq. (2) for polystyrene standards.

2.6.2. Effect of pH In order to observe the pH dependency of the PAAm␤-galactosidase, PNIPAAm-␤-galactosidase, and the native ␤galactosidase, the activities were tested at different pH (4–8) at 40 ◦ C; ONPG was used as the substrate. Separate buffers were employed depending on the pH (pH 4–5: 50 mM acetate buffer (acetic acid/sodium acetate), pH 6–7: 50 mM phosphate buffer (Na2 HPO4 /NaH2 PO4 ), pH 8: 50 mM tris–HCl buffer (trishydroxymethyl aminomethane/HCl)). 2.6.3. Effect of organic solvent The activities of bioconjugates were also tested in the presence of organic solvents. The PNIPAAm-␤-galactosidase and native ␤galactosidase was incubated with six varying concentrations (2, 5, 10, 15, 20 and 25%, v/v) of organic solvents such as EG and ethanol for 24 h at 30 ◦ C. The residual activity was measured at regular intervals of time; ONPG was used as the substrate. The solution without organic solvent was taken as the control for reference. 2.7. LCST determination The LCSTs of both the PNIPAAm-␤-galactosidase bioconjugate and PNIPAAm polymer were estimated by observing the change in absorbance at 450 nm using the UV–vis spectrophotometer [8]. Each solution was kept at a particular temperature for 10 min, and the absorbance was measured. Isothermal heating was provided using a circulatory water bath. The procedure was repeated by increasing the temperature stepwise in increments of 0.5 ◦ C. The LCST may depend on the polymer or bioconjugate concentration and the presence of salt. The LCST was thus determined at varying concentrations of the PNIPAAm-␤-galactosidase bioconjugate and PNIPAAm polymer, as well as in the presence of NaCl and lactose. 2.8. Functional stability The functional stabilities (thermal, pH, storage, and operational) of the bioconjugates and native enzyme were estimated. The thermal stability was determined in two different ways. First, enzyme solutions were incubated at different temperatures (20–80 ◦ C) for 15 min followed by residual activity measurement. Second, the thermal stability over prolonged heating was investigated by incubating the enzyme solutions at 55 ◦ C for 90 min; the residual activity was measured at regular time intervals. The pH stabilities of the bioconjugates and native enzyme were tested by incubating the solutions in 50 mM buffers with different pH values (pH 4–5: acetate buffer, pH 6–7: phosphate buffer, pH 8: tris–HCl) at 30 ◦ C for 30 min; activities were measured at 30 ◦ C under assay conditions. The storage stability was monitored by keeping the bioconjugates and native enzyme dissolved in 50 mM phosphate buffer (pH 6.0) at 20 ◦ C for 30 days. Residual activity was measured with a spectrophotometer using ONPG at 30 ◦ C. The operational stabilities of the bioconjugates and native enzyme were studied by heating the enzyme solutions at 40 ◦ C for 10 min followed by cooling at 4 ◦ C for 10 min. The process was repeated for 24 cycles. The residual activity was measured at an interval of every four cycles.

a [] = K · Mw

(2)

where, [] is intrinsic viscosity, Mw is the weight-average molecular weight; K and a are the Mark–Houwink parameters. The values of K and a for polystyrene (K = 11.0 × 10−3 mL/g, a = 0.725) and PNIPAAm (K = 9.59 × 10−3 mL/g, a = 0.65) in THF solvent were taken from the literature [38,39]. 2.10. HPLC analysis The quantitative analysis of the components in reaction mixture (lactose, glucose, galactose, and tri-, tetra-, and pentasaccharides) were performed by HPLC (UFLC model, Shimadzu Corporation® ) as per earlier method [2,29]. The HPLC system was consisted of two pumps (LC20-AD), a column (Sugar Pak I, Waters® , dimension: 6.5 mm × 300 mm), and a column oven (CTO-10AS VP), a refractive index detector (RID-10A), an autosampler (SIL-20A HT), a degasser (DGU-20A3), and LC-solution data processing software. The isocratic flow rate of mobile phase, 50 mg/L ethylenediamine-tetra-acetic acid calcium disodium in Milli-Q water, was maintained at 0.5 mL/min. The temperatures of the column oven and the detector during analysis were maintained at 75 ◦ C and 35 ◦ C, respectively. 2.11. GOS synthesis using PNIPAAm-ˇ-galactosidase bioconjugate GOS was synthesized from lactose using PNIPAAm-␤galactosidase (Fig. 2). Feed lactose (50 g/L) was dissolved in 50 mM phosphate buffer of pH 6.0, and the required amount of PNIPAAm-␤-galactosidase (1.9 kU/L) was added. The reaction was carried out under batch mode with a reaction volume of 25 mL at 30 ◦ C for 100 h. Samples were collected at regular time intervals and heated at 60 ◦ C for 5 min in a water bath. The mixture was then filtered through a 0.45-␮m filter under hot conditions, diluted as required, and analyzed through HPLC. 3. Results and discussion 3.1. Degree of modification The maximum enzyme modification of a 20 mg/mL enzyme solution in a 50 mM phosphate buffer of pH 6.0 was found to be 50% (calculated from Eq. (1)). 3.2. Bioconjugation yield The bioconjugation yield was defined as the fraction of modified enzyme conjugated to the polymer (Eq. (3)) [8]. It was estimated

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T. Palai et al. / Enzyme and Microbial Technology 55 (2014) 40–49

Fig. 2. Schematic presentation of lactose conversion using ␤-galactosidase (a) hydrolysis reaction forming galactose and glucose, and (b) trans-galactosylation reaction forming GOS.

Enzyme conjugated to polymer Bioconjugation yield (%) = × 100 Modified enzyme taken (3)

3.3. LCST determination The LCSTs of the PNIPAAm polymer and PNIPAAm-␤galactosidase bioconjugate were determined in the presence and absence of NaCl and lactose; the absorbance values were visualized at 450 nm. A sharp increase in absorbance value at a particular temperature indicates the LCST. The formation of intermolecular hydrogen bonds between the polymer and solvent water keeps the polymer in soluble form below the LCST. The polymer separates from the solution when the hydrogen bonds break down above the LCST [40]. The LCST of 1.5% (w/v) PNIPAAm was found to be 32.5 ◦ C, and it did not depend on the polymer concentration. Similar observations were also reported in the literature [16]. However, the LCST of 1.5% (w/v) PNIPAAm decreased to 31.5 ◦ C in the presence of 0.05 M NaCl (Fig. 3). The LCST of the PNIPAAm-␤-galactosidase bioconjugate decreased compared to that of the PNIPAAm polymer. The LCST of the bioconjugate was reduced from 32 ◦ C to 26.5 ◦ C when the concentration of PNIPAAm-␤-galactosidase was increased from 1.5% to 7.4% (w/v). Whereas, no such decrease in LCST was noted for PNIPAAm polymer with increasing concentration. The conjugation of acrylated ␤-galactosidase with PNIPAAm probably increased the hydrophobicity of the bioconjugate, which reduced the LCST [13]. Furthermore, the LCST of the bioconjugate also decreased in the presence of NaCl and lactose. Adding salt increased the ionic strength of the solution and hence decreased the charge repulsion between the polymer or bioconjugate molecules,

which facilitated the precipitation of the polymer or bioconjugate [20]. 3.4. Estimation of molecular weight Different types of molecular weights such as the weightaverage (Mw ), number-average (Mn ), and Z-average (Mz ) molecular weights of PNIPAAm polymer and PNIPAAm-␤-galactosidase bioconjugate were estimated through GPC. The polydispersity index (PDI = Mw /Mn ) was then calculated to understand the molecular weight distributions (Table 1) of the bioconjugate and polymer. The Mw and Mn values of PNIPAAm-␤-galactosidase were calculated to be 595 and 406 kDa, respectively, with a PDI of 1.46. Mw and Mn were higher for PNIPAAm-␤-galactosidase than for PNIPAAm polymer (Mw = 161 kDa, Mn = 119 kDa, PDI = 1.35) because of the higher

1.5% PNIPAAm-β-galactosidase 3.7% PNIPAAm-β-galactosidase 3.7% PNIPAAm-β-galactosidase +0.05 M NaCl 3.7% PNIPAAm-β-galactosidase +0.05 M lactose 3.7% PNIPAAm-β-galactosidase + 0.1 M NaCl 7.4% PNIPAAmβ-galactosidase

3.5 3.0

Absorbance (at 450 nm)

by measuring the protein content of the bioconjugates, with the respective polymers taken as the control for reference. The yield of the PNIPAAm-␤-galactosidase with an enzyme modification level of 50% was measured to be 75%.

2.5 2.0 1.5 1.0

1.5% PNIPAAm 1.5% PNIPAAm + 0.05 M NaCl

0.5 0.0 24

26

28

30

32

34

36

o

Temperature (C) Fig. 3. Lower critical solution temperature of PNIPAAm polymer and PNIPAAm-␤galactosidase bioconjugate.

T. Palai et al. / Enzyme and Microbial Technology 55 (2014) 40–49 Table 1 Estimated molecular weight of PNIPAAm polymer and PNIPAAm-␤-galactosidase bioconjugate (Mw : weight-average molecular weight, Mn : number-average molecular weight, Mz : Z-average molecular weight, PDI: polydispersity index). Mw (Da)

Mn (Da)

Mz (Da)

PDI

PNIPAAm PNIPAAm-␤-galactosidase

161,610 595,862

119,792 406,355

220,978 913,008

1.35 1.46

molecular weight of the ␤-galactosidase enzyme (205 kDa). The PDI values clearly showed that a wide range of molecular weights for both polymer and bioconjugates was obtained because of radical uncontrolled polymerization [8]. 3.5. Effect of parameters on catalytic activity of bioconjugates 3.5.1. Effect of temperature The effect of temperature on the catalytic activity of bioconjugates and the native enzyme was investigated at different temperatures (20–80 ◦ C); ONPG was used as the substrate. The maximum obtained values were: PNIPAAm-␤galactosidase-0.18 U/mL, PAAm-␤-galactosidase-0.14 U/mL, and native ␤-galactosidase-0.20 U/mL. Subsequent values were presented in related percentage to these maximum values. The activities of all three enzyme preparations increased with the temperature up to 60 ◦ C; the activities of the PAAm-␤-galactosidase bioconjugate and native enzyme then decreased (Fig. 4). On the other hand, the activity of the PNIPAAm-␤-galactosidase bioconjugate increased up to 70 ◦ C and then started to decrease. 3.5.2. Effect of pH Each enzyme has an optimum pH value where it shows maximum activity. Fig. 5 shows the change in catalytic activities of the bioconjugates and native enzyme on the ONPG substrate with respect to changes in the assay pH at 30 ◦ C. The maximum activity values (PNIPAAm-␤-galactosidase: 0.09 U/mL, PAAm-␤galactosidase: 0.10 U/mL, and native ␤-galactosidase: 0.15 U/mL) were normalized to 100%. The native enzyme showed maximum activity at pH 6. The optimum pH of both the PAAm-␤-galactosidase and PNIPAAm-␤-galactosidase shifted to pH 5. Furthermore, the PNIPAAm-␤-galactosidase showed a broad pH profile, which signified that it was less sensitive to pH variation than its native form. This may be attributed to the change in pH value around the active sites of the bioconjugates by polymer support. The

100

Relative activity (%)

Polymer/bioconjugate

45

80

60 PNIPAAm-β-galactosidase PAAm-β-galactosidase Native β-galactosidase

40

20 4

5

6

7

8

pH Fig. 5. Effect of pH on enzyme activity at 30 ◦ C. The maximum values (PNIPAAm-␤-galactosidase: 0.09 U/mL, PAAm-␤-galactosidase: 0.10 U/mL, and native ␤-galactosidase: 0.15 U/mL) were normalized as 100%.

interaction (electrostatic or hydrophobic) between molecular species in solution and support matrix changed the microenvironment of the active sites of the enzyme [41]. 3.5.3. Effect of organic solvent The presence of various organic solvents may change the activity and stability of the enzyme, depending on the nature of the enzyme and organic solvents [42]. PNIPAAm-␤-galactosidase and native ␤-galactosidase were tested in the presence of varying concentrations of EG and ethanol in an aqueous medium to observe the changes in the activity and stability. The solutions without organic solvent were taken as the control for reference, and their activities (PNIPAAm-␤-galactosidase: 0.034 U/mL and native ␤galactosidase: 0.24 U/mL) were normalized to 100%. Fig. 6 shows the change in enzyme activity in the presence of ethanol and EG. The activity of both the native ␤-galactosidase and PNIPAAm␤-galactosidase increased with the EG concentration up to 20% (v/v) and then decreased slightly. On the other hand, the activity of PNIPAAm-␤-galactosidase decreased gradually with increasing ethanol concentration; no such change was observed for the native enzyme.

Relative activity (%)

100

80

60 Native β -galactosidase PAAm-β -galactosidase PNIPAAm-β -galactosidase

40

20 10

20

30

40

50

60

70

80

o

Temperature (C) Fig. 4. Effect of temperature on enzyme activity in phosphate buffer of pH 6. The maximum values (PNIPAAm-␤-galactosidase: 0.18 U/mL, PAAm-␤-galactosidase: 0.14 U/mL, and native ␤-galactosidase: 0.20 U/mL) were normalized as 100%.

Fig. 6. Effect of organic solvents (ethylene glycol and ethanol) on enzyme activity. The activity of ␤-galactosidase solution without organic solvent (PNIPAAm-␤galactosidase: 0.034 U/mL and native ␤-galactosidase: 0.24 U/mL) was normalized to 100%.

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T. Palai et al. / Enzyme and Microbial Technology 55 (2014) 40–49

140

120

Ethanol

Ethylene glycol PNIPAAm-β-galactosidase Native β-galactosidase

PNIPAAm-β-galactosidase Native β-galactosidase

Native β-galactosidase PAAm-β-galactosidase

100

PNIPAAm-β-galactosidase

Residual activity (%)

Residual activity (%)

120

100

80

80

60

40

20 60

0

5

10

15

20

0

25

20

30

Time (h)

40

50

60

70

80

o

Incubation temperature (C)

Fig. 7. Stability of native ␤-galactosidase and PNIPAAm-␤-galactosidase bioconjugate in organic solvents (ethylene glycol and ethanol) of concentration 25% (v/v). The initial activities of respective enzyme solutions (PNIPAAm-␤-galactosidase: 0.034 U/mL and native ␤-galactosidase: 0.24 U/mL) were normalized to 100%.

Fig. 8. Thermal stability of native ␤-galactosidase, PAAm-␤-galactosidase and PNIPAAm-␤-galactosidase at different incubation temperature. The initial activities at 20 ◦ C (PNIPAAm-␤-galactosidase: 0.047 U/mL, PAAm-␤-galactosidase: 0.052 U/mL, and native ␤-galactosidase: 0.053 U/mL) were normalized to 100%.

Furthermore, the stability of enzymes in an aqueous solution containing various amounts of ethanol or EG was monitored for 24 h at 30 ◦ C. The initial activities (PNIPAAm-␤-galactosidase: 0.034 U/mL and native ␤-galactosidase: 0.24 U/mL) were represented as 100%. Fig. 7 shows the stability profiles of the native and conjugated enzymes with the incubation time, for the cases when 25% (v/v) EG and 25% (v/v) ethanol are used. Both enzyme preparations showed much better stability in EG than in ethanol. It was also observed that both enzyme preparations showed muchimproved stability in EG with a maximum stability at 10% (native ␤-galactosidase) and 5% (PNIPAAm-␤-galactosidase) of EG (Supplementary material, Figs. S.1 and S.2). Beyond a certain ethanol concentration, they showed less stability in ethanol than in EG, and the values became even lower than the initial values (Supplementary material, Figs. S.3 and S.4). This may be explained by the change in hydrophobicity around the active site of the enzyme in the presence of organic solvent and the denaturation capacity (DC) of the solvents. Hydrophobicity is defined as the logarithm of the partition coefficient (log P) in the water/octanol system. DC is a relative measure of the denaturing tendency of different organic solvents. An organic solvent with a high DC value will have a strong denaturing tendency [43]. The organic solvent forms a layer around the active site, which provides conformational rigidity [44] to the enzyme and facilitates the smooth entry of the substrate molecule toward the active site. Owing to the higher hydrophobicity of EG (log P = −1.43) compared to ethanol (log P = −0.32) [43], the EG layer allows the substrate molecule to enter more easily than the ethanol layer. This results in higher activity for both the native enzyme and PNIPAAm-␤galactosidase in the presence of EG, compared with the case in which ethanol and a pure aqueous medium are used. The DC of ethanol (54.4) is higher than that of EG (18.7) [43], so the enzyme is less stable in ethanol.

PAAm-␤-galactosidase: 0.052 U/mL, and native ␤-galactosidase: 0.053 U/mL) were normalized to 100%. Fig. 8 shows that the PNIPAAm-␤-galactosidase bioconjugates retained ∼100% activity at 50 ◦ C; however, this decreased to around 50% of the initial activity at 80 ◦ C. The PAAm-␤-galactosidase retained ∼60% and ∼8% activities at 50 ◦ C and 80 ◦ C, respectively. The native enzyme retained ∼60% and 0% activities at 50 and 80 ◦ C, respectively. Second, the enzyme solutions were heated continuously at 55 ◦ C for 90 min, and the residual activity was measured at regular time intervals. The initial activities (PNIPAAm-␤-galactosidase: 0.038 U/mL, PAAm-␤-galactosidase: 0.039 U/mL, and native ␤-galactosidase: 0.039 U/mL) were normalized to 100%. Fig. 9 depicts that the PNIPAAm-␤-galactosidase offered the highest resistance to thermal deactivation and retained 67.5% of the initial activity after 90 min of incubation compared with the PAAm-␤-galactosidase (24%) and native ␤-galactosidase (16.5%). As the temperature of the PNIPAAm-␤-galactosidase increased to a value higher than the LCST, its conformation changed from a loose coil state (swollen state) to a globular state (precipitated state). Micro-environmental

Native β-galactosidase PAAm-β-galactosidase PNIPAAm-β-galactosidase

Residual activity (%)

100

80

60

40

20

3.6. Stability of bioconjugates 0

3.6.1. Thermal stability The thermal stabilities of the bioconjugates and native enzyme were investigated in two different ways. First, enzyme solutions were incubated at different temperatures (20–80 ◦ C) for 15 min followed by residual activity measurement. The initial activities at 20 ◦ C (PNIPAAm-␤-galactosidase: 0.047 U/mL,

0

20

40

60

80

100

Time (min) Fig. 9. Thermal stability of native ␤-galactosidase, PAAm-␤-galactosidase and PNIPAAm-␤-galactosidase with time at 55 ◦ C. The initial activities (PNIPAAm␤-galactosidase: 0.038 U/mL, PAAm-␤-galactosidase: 0.039 U/mL, and native ␤-galactosidase: 0.039 U/mL) were normalized to 100%.

T. Palai et al. / Enzyme and Microbial Technology 55 (2014) 40–49

100

100

80

80

Residual activity (%)

Residual activity (%)

47

60

Native β-galactosidase PAAm-β-galactosidase PNIPAAm-β-galactosidase

40

20

60

40

Native β-galactosidase PAAm-β-galactosidase PNIPAAm-β-galactosidase

20

0 0 4

5

6

7

8

pH Fig. 10. pH stability of native ␤-galactosidase, PAAm-␤-galactosidase and PNIPAAm-␤-galactosidase at different pH values at 30 ◦ C. The maximum activities (PNIPAAm-␤-galactosidase: 0.068 U/mL, PAAm-␤-galactosidase: 0.078 U/mL, and native ␤-galactosidase: 0.17 U/mL) were normalized to 100%.

stresses during this change lead to unfolding of the enzyme molecules. The polymer chain of the PNIPAAm-␤-galactosidase protects the enzyme from such stresses by creating a reversible soluble–insoluble shield around the active sites of enzyme [8]. Under isothermal heating conditions, the polymer moiety of a responsive polymer creates a hydrophobic environment around the enzyme active sites, which enhances the thermal stability [45] of PNIPAAm-␤-galactosidase. 3.6.2. pH stability The pH stabilities of the bioconjugates and native enzyme were tested by incubating the enzyme solutions at 30 ◦ C for 30 min and measuring the activity under assay conditions. The maximum activities (PNIPAAm-␤-galactosidase: 0.068 U/mL, PAAm-␤-galactosidase: 0.078 U/mL, and native ␤-galactosidase: 0.17 U/mL) were normalized to 100%. Fig. 10 depicts that the stability of the PAAm-␤-galactosidase shifted to a lower value (pH 5) than that of the native enzyme (pH 6). The pH stability profile of the PNIPAAm-␤-galactosidase remained similar to that of the native enzyme. 3.6.3. Storage stability The long-term storage stabilities of the bioconjugates and native enzyme were monitored by keeping the solutions at 20 ◦ C for 30 days. Initial activities of the respective enzyme solutions (PNIPAAm-␤-galactosidase: 0.14 U/mL, PAAm-␤-galactosidase: 0.078 U/mL, and native ␤-galactosidase: 0.29 U/mL) were taken as 100%. The PNIPAAm-␤-galactosidase retained ∼68% of the initial activity. The PAAm-␤-galactosidase retained ∼72% up to 30 days (Fig. 11). However, the native enzyme fully lost its activity within 20 days. The denaturation rate of the native protein was found to increase sharply after 10 days of storage. At low temperatures (below the LCST), both PNIPAAm and PAAm form a hydrated layer (protective colloid) over the protein molecules; the layer resists denaturation [45]. 3.6.4. Operational stability Most enzymes are very sensitive to changes in the surrounding environment (pH, temperature, etc.). However, actual practice conditions may change suddenly, so enzymes should have the ability to resist denaturation of protein under certain unfavorable conditions. To observe the change in activity of the conjugated enzymes during precipitation and re-dissolution, the enzyme

0

5

10

15

20

25

30

Storage time (day) Fig. 11. Storage stability of native ␤-galactosidase, PNIPAAm-␤-galactosidase and PAAm-␤-galactosidase. Initial activities of the respective enzyme solutions (PNIPAAm-␤-galactosidase: 0.14 U/mL, PAAm-␤-galactosidase: 0.078 U/mL, and native ␤-galactosidase: 0.29 U/mL) were taken as 100%.

solutions were repeatedly heated at 40 ◦ C for 10 min followed by cooling at 4 ◦ C. The residual activities of the PNIPAAm-␤galactosidase, PAAm-␤-galactosidase and native ␤-galactosidase after 24 cycles were 91%, 88.5%, and 86% of their initial activities, respectively. 3.7. Determination of Michaelis–Menten kinetic parameters To test the change in kinetic parameters of the conjugated enzyme, the Michaelis–Menten constant (Km ) and maximum reaction rate (Vm ) for the native enzyme and bioconjugates were estimated using the Lineweaver–Burk method at 30 ◦ C and 40 ◦ C; ONPG (range: 2.5–20 mM) was used as the substrate. The values are listed in Table 2. The estimated Km values of PAAm-␤-galactosidase and the native ␤-galactosidase were found to be same. The Km values of PNIPAAm-␤-galactosidase with respect to the native ␤galactosidase were reduced by ∼4.2 and 15 times at 30 ◦ C and 40 ◦ C, respectively. The decrease in Km signifies that the affinity of PNIPAAm-␤-galactosidase toward the ONPG substrate increased relative to that of the native enzyme. A five-fold decrease in Km of PNIPAAm-␤-galactosidase was observed when the phase changed from soluble (at 30 ◦ C) to insoluble (40 ◦ C). 3.8. GOS synthesis GOS was synthesized from lactose using PNIPAAm-␤galactosidase. Utilizing such bioconjugate for the enzymatic formation of GOS allows the system to provide not only a Table 2 Kinetic parameters of native enzyme, PAAm-␤-galactosidase and PNIPAAm-␤galactosidase bioconjugates.a Enzyme/bioconjugates

Temperature (◦ C)

Km (mM)

Vm × 105 (mM/s)

Native ␤-galactosidase

30 40

25.4 16.5

6.69 7.42

PAAm-␤-galactosidase

30 40

28.0 13.7

7.16 6.28

PNIPAAm-␤-galactosidase

30 40

6.0 1.1

2.31 2.45

a The parameters like Km and Vm were determined at two different temperatures where enzyme bioconjugate preparation using PNIPAAm was in soluble and insoluble form, respectively.

48

T. Palai et al. / Enzyme and Microbial Technology 55 (2014) 40–49

Table 3 Comparison of GOS yields: bioconjugate (PNIPAAm-␤-galactosidase) vs. native state (lactose concentration: 50 g/L, enzyme concentration: 1.9 kU/L, pH 6.0, 30 ◦ C). Reaction system

GOS yield (%)

Reaction mixture composition (%)

Native ␤-galactosidase PNIPAAm-␤-galactosidase

Total

Tri-

Tetra-

24.0 29.5

19.0 22.5

5.0 7.0

100

GOS Lactose Glucosse Galactose

80

60

40

20

0 0

20

40

60

80

100

Time (h) Fig. 12. Time-course reaction mixture composition under the initial conditions of 50 g/L lactose and 1.9 kU/L PNIPAAm-␤-galactosidase at pH 6.0 and 30 ◦ C.

homogeneous system but also an immobilized system. Again, the LCST of the bioconjugate was dependent on concentration. So, its concentration was taken in such a way that the reaction mixture remained homogeneous at 30 ◦ C. Fig. 12 shows a typical time-course composition of the reaction mixture. As expected, the lactose concentration decreased with time, whereas the product concentrations (GOS, glucose, and galactose) increased and reached a plateau after ∼60 h. A maximum yield of 29.5% GOS (dry basis) with 44.0% lactose conversion was obtained after 100 h of reaction time under the initial conditions of 50 g/L lactose and 1.9 kU/L conjugated enzyme. The reaction mixture at equilibrium contained 7.0% tetra-saccharide, 22.5% tri-saccharide, 12.0% glucose, 2.5% galactose, and 56.0% unreacted lactose. After the reaction was completed, the conjugated enzyme was easily separated by increasing the temperature to 40 ◦ C in the presence of 0.05 M NaCl. 3.8.1. Comparison of GOS yield: bioconjugate (PNIPAAm-ˇ-galactosidase) vs. native enzyme It was thought to carry out synthesis of GOS with bioconjugate (PNIPAAm-␤-galactosidase) as well as with native enzyme under similar conditions (lactose concentration: 50 g/L, enzyme 1.9 kU/L, pH 6.0 and 30 ◦ C) in order to compare results between GOS yield vis-à-vis yield of products out of lactose hydrolysis. It may be mentioned that such a reaction was not performed with PAAm-␤-galactosidase as the properties of this type conjugate (non-responsive) was considered unfavorable for enzymatic reaction. Table 3 summarizes such comparisons. It may be observed from Table 3 that under native state of ␤-galactosidase, only 24% of GOS yield (maximum) is observed with much higher release of products of lactose hydrolysis (galactose & glucose). However, the conversion of lactose is indeed higher under native state. Thus, the reaction with bioconjugates is proving to be better on both counts; higher GOS yield and lower monosaccharides. This clearly indicates that the desirable trans-galactosylation reaction is playing a dominant role with PNIPAAm-␤-galactosidase.

Glucose yield (%)

Galactose yield (%)

Lactose conversion (%)

19.0 12.0

6.0 2.5

49.0 44.0

In conclusion, PNIPAAm-␤-galactosidase bioconjugate was synthesized, characterized, and utilized for GOS formation. The yield of the PNIPAAm-␤-galactosidase was 75% at a 50% modified enzyme. The LCST of PNIPAAm-␤-galactosidase decreased compared to that of the PNIPAAm polymer. The activities of both the PAAm-␤-galactosidase and PNIPAAm-␤-galactosidase bioconjugates were less sensitive to pH variation than the native forms. The PNIPAAm-␤-galactosidase showed much improved thermal stability compared to the PAAm-␤-galactosidase and native enzyme. Addition of EG enhanced the activity of the PNIPAAm␤-galactosidase and native enzyme without any loss in stability; however, addition of ethanol decreased the stability. GOS was synthesized from lactose using PNIPAAm-␤-galactosidase bioconjugate. Thus, under native state of ␤-galactosidase, only 24% of GOS yield (maximum) is observed with much higher products of lactose hydrolysis (galactose & glucose). However, the conversion of lactose is indeed higher under native state. Thus, the reaction with bioconjugates is proving to be better on both counts; higher GOS yield and lower monosaccharides. Further research may be carried out to optimize the operating parameters for large-scale GOS formation. Acknowledgement One of the authors (PKB) gratefully acknowledges the Department of Biotechnology (DBT), Government of India for partial financial support against sanction order number: BT/PR14530/PID/06/599/2010. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.enzmictec. 2013.12.003. References [1] Panesar PS, Panesar R, Singh RS, Kennedy JF, Kumar H. Microbial production, immobilization and applications of ␤-d-galactosidase. J Chem Technol Biotechnol 2006;81:530–43. [2] Palai T, Bhattacharya PK. Kinetics of lactose conversion to galactooligosaccharides by ␤-galactosidase immobilized on PVDF membrane. J Biosci Bioeng 2013;115:668–73. [3] Albayrak N, Yang ST. Production of galacto-oligosaccharides from lactose by Aspergillus oryzae ␤-galactosidase immobilized on cotton cloth. Biotechnol Bioeng 2002;77:8–19. [4] Bayramoglu G, Tunali Y, Arica MY. Immobilization of ␤-galactosidase onto magnetic poly(GMA–MMA) beads for hydrolysis of lactose in bed reactor. Catal Commun 2007;8:1094–101. [5] Neri DFM, Balcão VM, Dourado FOQ, Oliveirad JMB, Carvalho Jr LB, Teixeira JA. Immobilized ␤-galactosidase onto magnetic particles coated with polyaniline: support characterization and galactooligosaccharides production. J Mol Catal B Enzym 2011;70:74–80. [6] Rios GM, Belleville MP, Paolucci D, Sanchez J. Progress in enzymatic membrane reactors – a review. J Membr Sci 2004;242:189–96. [7] Chen G, Hoffman AS. Preparation and properties of thermoreversible, phaseseparating enzyme-oligo(N-isopropylacrylamide) conjugates. Bioconjugate Chem 1993;4:509–14. [8] Shakya AK, Sharma P, Kumar A. Synthesis and characterization of thermoresponsive poly(N-isopropylacrylamide)-bovine liver catalase bioconjugate. Enzyme Microb Technol 2010;47:277–82. [9] Hoffman AS, Stayton PS. Bioconjugates of smart polymers and proteins: synthesis and applications. Macromol Symp 2004;207:139–52.

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