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)

Highlights

• MWCNT-WS₂ and MWCNT-SnO₂ were synthesized via eco-facile hydrothermal process.

• The nanocomposites were used to immobilize Lens culinaris β-galactosidase.

• Storage stability and reusability were superior in MWCNT-WS₂ than MWCNT-SnO₂ NC.

• AFM, FE-SEM, CLSM, and FTIR were employed for characterization of nanobiocatalyst.

Abstract

In the present study, Multiwalled carbon nanotubes (MWCNT) decorated with two different nanoparticles namely tungsten disulfide (WS₂) and tin oxide (SnO₂), nanocomposites (NCs) were synthesized via hydrothermal method. Spectroscopic studies showed that both synthesized NCs possess nearly same functional groups but MWCNT-SnO₂ NCs are rich in O-functional group. Microscopic studies revealed that both NCs have different morphological microstructure. Lens culinaris β-galactosidase (Lcβ-gal) was immobilized using glutaraldehyde cross-linker resulted in immobilization efficiency of 91.5% and 88% with MWCNT-WS₂ and MWCNT-SnO₂ NCs, respectively. Remarkable increase in rate of hydrolysis of whey lactose has been observed with both NCs i.e. Lcβ-gal immobilized MWCNT-WS₂ hydrolyzes the 97% whey lactose in 1.5 h while MWCNT-SnO₂ showed maximum 92% of whey hydrolysis in 2 h at optimum conditions. Both nanobiocatalyst could serve as a promising candidates for dairy industries and would offer a potential platform for enzyme based biosensor fabrication.

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.