Synthesis and Characterization of Thermo-Responsive Bioconjugates: Immobilized Enzyme for Galacto-Oligosaccharide Formation

Synthesis and characterization of thermo-responsive bioconjugates: immobilized enzyme for galacto-oligosaccharide formation

 

Tapas Palai¹, Ashok Kumar² and Prashant K. Bhattacharya^1*

 

¹          Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur-208016, India.

²          Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur-208016, India.  

* Corresponding author: E-mail: pkbhatta@iitk.ac.in Tel.: +91-512-2597093; Fax: +91-512-2590104  

Abstract

Thermo-responsive polymers, also known as smart/intelligent/environmentally sensitive/and reversible polymer, have received much attention in biotechnology, medicine and engineering applications. With a change in temperature around its lower critical solution temperature (LCST), the polymer undergoes a reversible phase change form soluble to insoluble form or vice versa [1].  This property was considered in immobilizing enzymes on such reversible polymers which are known as polymer-protein bioconjugate and the attachment of enzymes may be via physical or chemical means. Further, such bioconjugates may effectively carry out enzymatic reactions under immobilized states [2]. In order to test one such enzymatic reaction, the formation of galacto-oligosaccharides from lactose [3] was considered with β-galactosidase (EC 3.2.1.23; commercial name: Biolacta FN5) on reversible polymer, poly-N-isopropylacrylamide (PNIPAAM). In the present study, apart from PNIPAAM, a non-thermo-responsive polymer poly-acrylamide (PAAM), was chosen as control polymer [4]. Experimental evidences too confirm the formations of PNIPAAM-β-galactosidase bioconjugates, exhibiting thermo-responsive behavior, whereas PAAM-β-galactosidase bioconjugates do not exhibit such thermo-response. Oligosaccharides and their derivatives are important nutraceuticals and prebiotic food ingredient, having a range of important functions in biological systems. The utility of thermo-responsive bioconjugates help in carrying out the reaction under homogeneous condition (below LCST) and effectively help in separating enzymes above LCST.

The synthesis of bioconjugates was carried out in three steps [4] using commercial grade β-galactosidase from Bacillus circulans. Firstly, acryl group was introduced to enzyme by treating with itaconic anhydride in phosphate buffer (pH 6.0). Furthermore, unreacted itaconic anhydride was removed by dialysis. The extent of enzyme modification was estimated by 2,4,6-trinitrobenzenesulphonic acid (TNBS) method [5].

Then, PNIPAAM and PAAM-β-galactosidase bioconjugates were copolymerized from their respective monomers (N-isopropylacrylamide (NIPAAM) and acrylamide (AAM)). The reaction was catalyzed and initiated with N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulphate (APS), respectively. Finally, the bioconjugates thus formed were separated from their respective monomers by salt-induced thermal precipitation (for PNIPAAM) and dialysis (for PAAM). The activities of native enzyme, PNIPAAM and PAAM bioconjugates were determined using o-nitro-phenyl-β-D-galactopyranoside (ONPG) as substrate at 40^oC and pH 6.0. [3]. Bioconjugation yield was estimated by measuring the amount of conjugated protein using bicinchoninic acid method. The yield of PNIPAAM- β-galactosidase bioconjugate was measured as 75% at an enzyme modification level of 50%. A decreased LCST of 29.5^oC, in comparison to 32.5^oC for the pure PNIPAAM polymer, was observed for PNIPAAM-enzyme bioconjugate. This decrease may be due to the increase in hydrophobicity of the polymer during bioconjugation with enzyme [4].

The catalytic activities of PAAM, PNIPAAM-enzyme bioconjugates and native enzyme were measured at varying temperature (20-80^oC) and pH (4.0-6.0) using ONPG as substrate. The activities of all the three preparations were observed to increase with increasing temperature till 60^oC and thereafter it a decrease was observed only for PAAM-enzyme bioconjugate and native enzyme. On the other hand, the activity of PNIPAAM-enzyme bioconjugate increased till 70^oC and thereafter it starts decreasing. Furthermore, it was observed that the activities of both the PAAM, PNIPAAM bioconjugates were less sensitive to pH variation than its native form. Thermal stability was also performed; in two different ways. In the first case, enzyme solutions were incubated at different temperature (20-80^oC) for 15 min, followed by residual activity measurement. PNIPAAM-enzyme bioconjugates retained ~100% activity at 50^oC; however, it is around 50% of initial activity at 80^oC. On the other hand, PAAM-enzyme retained ~60% and ~8% activity at 50^oC and 80^oC, respectively; whereas, the native enzyme retained ~60% and 0% at 50^oC and 80^oC, respectively. In the second case, the enzyme solutions were heated continuously at 55^oC for 90 min and residual activity was measured at regular time intervals. The PNIPAAM-bioconjugate offered a maximum resistance to thermal deactivation by keeping 67.5% of initial activity after 90 min of incubation as compared to PAAM-enzyme (24%) and native enzyme (16.5%). Furthermore, study on operational stability showed the residual activities of PNIPAAM-enzyme, PAAM-enzyme and native enzyme after 24 cycles were 91%, 88.5% and 86%, respectively. The effect of organic solvents (ethylene glycol and ethanol) on activity of PNIPAAM-bioconjugate was studied by keeping it at varying concentrations of organic solvents (2-25% v/v) for 24 h at 30^oC. Residual activity was measured at a regular interval of time. It was observed that the activity of PNIPAAM-bioconjugate got increased by 45% with 20% (v/v) ethylene glycol; whereas, with ethanol, it showed negative trend.

The storage stability was monitored by keeping the enzyme solution at 20^oC for 30 days. PNIPAAM-bioconjugate retained ~90% of its initial activity; whereas, PAAM-bioconjugate retained ~80% till 30 days. The native enzyme however lost its full activity within 20 days.

Further, Michaelis-Menten constant (K_m) and maximum reaction rate (V_m) values were also estimated (from Lineweaver-Burk method) using ONPG as substrate at 30^oC.  The values were found to be: K_m = 1.1 mM, V_m = 2.61 x 10^-5 mM s^-1.

Fig. 1: Time-course reaction mixture composition

Finally, the synthesis of GOS from lactose was carried out with the application of PNIPAAM-β-galactosidase bioconjugates. Utilizing such bioconjugates for the enzymatic formation of GOS, thus considered the system would provide not only a homogeneous system also an immobilized system too. Accordingly, the enzyme β-galactosidase converts lactose via two simultaneous reactions; trans-galactosylation reaction, which produces GOS and hydrolysis reaction, which forms glucose and galactose [2]. The reaction was carried out under simple batch mode at 30^oC and pH 6.0 for 100 h. Samples were collected at a regular interval and analyzed through high performance liquid chromatography (HPLC). A maximum of 29.5% GOS (dry basis) with 44.0% of lactose conversion was obtained after 100 h reaction time at 50 g/L initial lactose (Fig. 1). The equilibrium reaction mixture contained 7.0% tetra-saccharide, 22.5% tri-saccharide, 12.0% glucose, 2.5% galactose along with 56.0% unreacted lactose.

Keywords: Bioconjugate, Responsive polymer, enzyme immobilization, Galacto-oligosaccharide, Lactose

                                                                                

References:

[1] A. Kumar, A. Srivastava, I.Y. Galaev, B. Mattiasson, Smart polymers: Physical forms and bioengineering applications, Progress in Polymer Science 32 (2007) 1205-1237.

[2] T. Palai, P.K. Bhattacharya, Kinetics of lactose conversion to galacto-oligosaccharides by β-galactosidase immobilized on PVDF membrane, Journal of Bioscience and Bioengineering 115 (2013) 668-673.

[3] T. Palai, S. Mitra, P.K. Bhattacharya, Kinetics and design relation for enzymatic conversion of lactose into galacto-oligosaccharides using commercial grade β-galactosidase, Journal of Bioscience and Bioengineering 114 (2012) 418-423.

[4] A.K. Shakya, P. Sharma, A. Kumar, Synthesis and characterization of thermo-responsive poly(N-isopropylacrylamide)-bovine liver catalase bioconjugate, Enzyme and Microbial Technology 47 (2010) 277-282.

[5] L. Fischer, F. PeiBker, A covalent two-step immobilization technique using itaconic anhydride, Applied Microbiology and Biotechnology 49 (1998) 129-135.