Nanoparticles decorated carbon nanotubes as novel matrix: A comparative study of influences of immobilization on the catalytic properties of Lens culinaris β-galactosidase (Lcβ-gal)
Introduction
Nowadays, enzymologist have shown their exceptional interest in enzyme catalysis because of their diverse applications in various fields such as food industry [1], textile [2], paper pulp [3], cosmetics [4], fine chemical synthesis [5], and pharmaceuticals industries [6], [7] etc. Major advantage of enzyme catalysis is that it plays a crucial role towards green and sustainable industrial development [8], [9]. But, soluble enzymes possess some serious drawbacks that limits their applications such as they are extremely sensitive to industrial conditions like higher temperature, pH variations, shearing forces, and organic solvents, which affects their bioconversion efficiency as compared to in vivo conditions [10], [11]. Immobilization technology provides a potential platform to re-engineer the biocatalyst with enhanced properties which tremendously increases the thermal stability, broadens the operational pH and temperature working range. It boosts resistance to various organic solvents and also prevents product inhibition etc. Apart from these properties, it also increases the reusability of the expensive biocatalyst which allows simplistic separation of products from reaction mixture [12], [13].
Nanotechnological approach to immobilize biocatalyst on diverse scaffolds such as graphene, graphene oxide, iron oxide, carbon nanotubes, and nanowire has gained momentum because of their widespread applications in the field of biomedical engineering, tissue engineering, drug delivery [14], gene therapy [14], and biosensor technology [15], [16]. Recently, carbon nanotubes (CNTs) and many other two dimensional (2D) transition metals such as tin oxide (SnO2), tungsten disulfide (WS2), molybdenum disulfide (MoS2), and manganese dioxide (MnO2) have grabbed attention due to their high chemical and mechanical stability, large surface area, biocompatibility and enhanced electrical properties [17], [18]. In the past, our laboratory explored nanomaterials such as graphene nanosheets [19], MoS2 nanosheets [20], iron oxide, and graphene oxide [21] with successful immobilization of enzymes onto them. But, these 2D nanomaterial showed some shortcomings such as poor solubility in water and buffers, and often tend to aggregate due to their high active surface area which further reduced the surface to volume ratio and hence reduces enzyme loading capacity [22]. Furthermore, aggregation of 2D nanomaterial leads to structural deformation, resulting in reduced electrical conductivity [22].
Three dimensional (3D) nanocomposites have emerged as a solution to conquer the inadequacies associated with these nanomaterials. 3D nanocomposites have been synthesized in which CNTs were incorporated in between nanoparticles/nanorods to prevent the alteration in volume change. Recently, we have successfully immobilized Lens culinaris β-galactosidase onto MWCNT-MoS2 nanocomposite with immobilization efficiency of 93% [23]. Asmat and Husain [24] have also successfully fabricated various nanocomposites such as nanocellulose fused polypyrrole/graphene oxide nanocomposites, poly(o-toludine) functionalized nanocomposites [25], polyamine grafted MWCNTs impregnated with cobalt [26], and polyprrole-methyl anthranilate functionalized worm like titanium oxide [27] for immobilization of lipase. In all the cases, exquisite stability and catalytic performance were observed. These nanocomposites contain porous network structure, which provide larger surface area for attachment of biocatalyst and also facilitates the diffusion of substrate to the enzyme and release of product. These nanocomposites due to their excellent electrical property are more acquiescent for fabrication of biosensor for sensitive detection and recognition of different biomolecules.
β-Galactosidases (EC.3.2.1.23) also known as lactases are well-known for the hydrolysis of lactose sugar present in milk and other dairy products [28]. Milk and dairy products are essential component of human diet but when the consumption of milk exceeds over the residual activity of β-galactosidase left in small intestine where undigested lactose passes in large intestine and fermented by colonic microflora causing several symptoms like abdominal pain, loose stool, nausea etc. [28], [29]. According to a report, lactose intolerance prevails about 70% of world population. Therefore, lactose intolerant people are advised to take lactose reduced or lactose free dairy products. Hydrolytic property of β-galactosidase has been exploited by dairy industry as a solution for the production of lactose free/reduced dairy products [29], [30]. However, it also unravel the problem of formation of ice crystals in dairy products (ice cream) by addition of β-galactosidase and subsequently, it also enhances the sweetness and flavor of dairy products [31], [32]. It also owns the property of recycling the waste of cheese industry by bioconversion of whey in ethanol, and other value added products. Transgalactosylation property of β-galactosidase has been utilized by dairy industries in production of Galacto-oligosaccharides (GOS) which is of great biotechnological implications [33]. GOS act as prebiotic which help in maintaining the microflora of intestine and improve the immune system. The tremendous industrial applications of β-galactosidase has motivated us to choose this enzyme and followed by its immobilization onto nanocomposites (NCs) such as multiwalled carbon nanotubes-tungsten di sulfide (MWCNT-WS2) and multiwalled carbon nanotubes-tin oxide (MWCNT-SnO2).
In present work, two different NCs i.e. MWCNT-WS2 and MWCNT-SnO2 were synthesized via facile hydrothermal method. β-Galactosidase from Lens culinaris (Lcβ-gal) was immobilized onto these nanocomposites using glutaraldehyde cross-linker via covalent bond formation as shown in Scheme 1. Atomic Force Microscopy (AFM), Field Emission-Scanning Electron Microscopy (FE-SEM), Fourier Transformed Infrared region (FTIR), Confocal Laser Scanning Microscopy (CLSM) have been employed for characterization of both nanobiocatalyst (NBCs). In both cases, Response Surface Methodology (RSM) has been employed to optimize various parameters to get maximum immobilization. A comparative study of biochemical and kinetics parameters has been carried out for both nanobiocatalyst (NBCs). The NBCs, could be serve as a promising candidate for treatment of waste produced by dairy industry, before discharging into water bodies.
Section snippets
Experimental section
Tin chloride pentahydrate (SnCl4·5H2O), sodium hydroxide and iso-propyl alcohol were purchased from Molychem, India. Sodium tungstate dihydrate (Na2WO4·2H2O) and L-cysteine were purchased from Molychem, India. The chemical reagents used for buffer preparation throughout experiments were of analytical grade, and were procured from Merck Eurolab GmbH Darmstadt, Germany. All the chemicals used during experiments were purchased from Sigma-Aldrich (St. Louis, MO). Milli Q water (Millipore, Bedford,
Results and discussion
Lcβ-gal has been purified to electrophoretic homogeneity with 857 fold and a specific activity of 87 U/mg by using different fractionation and chromatography steps [23].
Conclusion
MWCNT-WS2 NC was found to be superior over MWCNT-SnO2 NC that may be due to heterofunctional nature. It is evident from reusability and whey hydrolysis test that Lcβ-gal immobilized MWCNT-WS2 showed better reusability and catalytic properties compared to MWCNT-SnO2. Thus, synthesized NBCs could serve as potential candidate to fulfil the requirement of dairy industries. Apart from this, we hope that present study will provide new insights to explore more novel heterofunctional material to
Acknowledgement
Anjali Yadav sincerely expresses her thanks to University Grant Commission, New Delhi for providing financial support in form of Junior and Senior Research Fellowship (F./2015-16/NFO-2015-17-OBC-UTT-32658/(SA-III-website). Anjali Yadav and Prof. Arvind M. Kayastha are indebted to Prof. S. M. Singh and Mr. Yugal Goel of Animal Cell Culture Laboratory of School of Biotechnology for carrying out cytotoxicity analysis of NBCs on the cell lines maintained in their laboratory. Anjali Yadav is also
Declaration of Competing Interest
Authors declare no competing financial interest.
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