Madan L. Verma

Nanobiotechnology as a novel paradigm for enzyme immobilisation and stabilisation with potential applications in biodiesel production

Nanobiotechnology is emerging as a new frontier of biotechnology. The potential applications of nanobiotechnology in bioenergy and biosensors have encouraged researchers in recent years to investigate new novel nanoscaffolds to build robust nanobiocatalytic systems. Enzymes, mainly hydrolytic class of enzyme, have been extensively immobilised on nanoscaffold support for long-term stabilisation by enhancing thermal, operational and storage catalytic potential. In the present report, novel nanoscaffold variants employed in the recent past for enzyme immobilisation, namely nanoparticles, nanofibres, nanotubes, nanopores, nanosheets and nanocomposites, are discussed in the context of lipase-mediated nanobiocatalysis. These nanocarriers have an inherently large surface area that leads to high enzyme loading and consequently high volumetric enzyme activity. Due to their high tensile strengths, nanoscale materials are often robust and resistant to breakage through mechanical shear in the running reactor making them suitable for multiple reuses. The optimisation of various nanosupports process parameters, such as the enzyme type and selection of suitable immobilisation method may help lead to the development of an efficient enzyme reactor. This might in turn offer a potential platform for exploring other enzymes for the development of stable nanobiocatalytic systems, which could help to address global environmental issues by facilitating the production of green energy. The successful validation of the feasibility of nanobiocatalysis for biodiesel production represents the beginning of a new field of research. The economic hurdles inherent in viably scaling nanobiocatalysts from a lab-scale to industrial biodiesel production are also discussed.

Suitability of magnetic nanoparticle immobilised cellulases in enhancing enzymatic saccharification of pretreated hemp biomass

BackgroundPrevious research focused on pretreatment of biomass, production of fermentable sugars and their consumption to produce ethanol. The main goal of the work was to economise the production process cost of fermentable sugars. Therefore, the objective of the present work was to investigate enzyme hydrolysis of microcrystalline cellulose and hemp hurds (natural cellulosic substrate) using free and immobilised enzymes. Cellulase from Trichoderma reesei was immobilised on an activated magnetic support by covalent binding and its activity was compared with that of the free enzyme to hydrolyse microcrystalline cellulose and hemp hurds on the basis of thermostability and reusability.ResultsUp to 94% protein binding was achieved during immobilisation of cellulase on nanoparticles. Successful binding was confirmed using Fourier transform infrared spectroscopy (FTIR). The free and immobilised enzymes exhibited identical pH optima (pHxa04.0) and differing temperature optima at 50°C and 60°C, respectively. The KM values obtained for the free and immobilised enzymes were 0.87xa0mg/mL and 2.6xa0mg/mL respectively. The immobilised enzyme retained 50% enzyme activity up to five cycles, with thermostability at 80°C superior to that of the free enzyme. Optimum hydrolysis of carboxymethyl cellulose (CMC) with free and immobilised enzymes was 88% and 81%, respectively. With pretreated hemp hurd biomass (HHB), the free and immobilised enzymes resulted in maximum hydrolysis in 48xa0h of 89% and 93%, respectively.ConclusionThe current work demonstrated the advantages delivered by immobilised enzymes by minimising the consumption of cellulase during substrate hydrolysis and making the production process of fermentable sugars economical and feasible. The activity of cellulase improved as a result of the immobilisation, which provided a better stability at higher temperatures. The immobilised enzyme provided an advantage over the free enzyme through the reusability and longer storage stability properties that were gained as a result of the immobilisation.

Immobilization of β-glucosidase on a magnetic nanoparticle improves thermostability: application in cellobiose hydrolysis.

The objective of the present work was to develop a thermostable β-glucosidase through immobilization on a nanoscale carrier for potential application in biofuel production. β-Glucosidase (BGL) from Aspergillus niger was immobilized to functionalized magnetic nanoparticles by covalent binding. Immobilized nanoparticles showed 93% immobilization binding. Immobilized and free BGL were characterized using Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR) techniques. Free and immobilized enzyme exhibited different pH-optima at pH 4.0 and 6.0, respectively, but had the same temperature optima at 60 °C. Michaelis constant (KM) was 3.5 and 4.3mM for free and immobilized BGL. Thermal stability of the immobilized enzyme was enhanced at 70 °C. The immobilized nanoparticle-enzyme conjugate retained more than 50% enzyme activity up to the 16th cycle. Maximum glucose synthesis from cellobiose hydrolysis by immobilized BGL was achieved at 16 h.

Recent trends in nanomaterials immobilised enzymes for biofuel production

Abstract Application of nanomaterials as novel supporting materials for enzyme immobilisation has generated incredible interest in the biotechnology community. These robust nanostructured forms, such as nanoparticles, nanofibres, nanotubes, nanoporous, nanosheets, and nanocomposites, possess a high surface area to volume ratios that can cause a high enzyme loading and facilitate reaction kinetics, thus improving biocatalytic efficiency for industrial applications. In this article, we discuss research opportunities of nanoscale materials in enzyme biotechnology and highlight recent developments in biofuel production using advanced material supports for enzyme immobilisation and stabilisation. Synthesis and functionalisation of nanomaterial forms using different methods are highlighted. Various simple and effective strategies designed to result in a stable, as well as functional protein-nanomaterial conjugates are also discussed. Analytical techniques confirming enzyme loading on nanomaterials and assessing post-immobilisation changes are discussed. The current status of versatile nanomaterial support for biofuel production employing cellulases and lipases is described in details. This report concludes with a discussion on the likely outcome that nanomaterials will become an integral part of sustainable bioenergy production.

Immobilization of β-d-galactosidase from Kluyveromyces lactis on functionalized silicon dioxide nanoparticles: Characterization and lactose hydrolysis

β-D-Galactosidase (BGAL) from Kluyveromyces lactis was covalently immobilized to functionalized silicon dioxide nanoparticles (10-20 nm). The binding of the enzyme to the nanoparticles was confirmed by Fourier transform-infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Functionalized nanoparticles showed 87% immobilization yield. Soluble and immobilized enzyme preparation exhibited pH-optima at pH 6.5 and 7.0, respectively, with temperature optima at 35 and 40°C, respectively. Michaelis constant (K(m)) was 4.77 and 8.4mM for free and immobilized BGAL, respectively. V(max) for the soluble and immobilized enzyme was 12.25 and 13.51 U/ml, respectively. Nanoparticle immobilized BGAL demonstrated improved stability after favoring multipoint covalent attachment. Thermal stability of the immobilized enzyme was enhanced at 40, 50 and 65°C. Immobilized nanoparticle-enzyme conjugate retained more than 50% enzyme activity up to the eleventh cycle. Maximum lactose hydrolysis by immobilized BGAL was achieved at 8h.

Enzyme Immobilisation on Amino-Functionalised Multi- Walled Carbon Nanotubes: Structural and Biocatalytic Characterisation

Background The aim of this work is to investigate the structure and function of enzymes immobilised on nanomaterials. This work will allow better understanding of enzyme-nanomaterial interactions, as well as designing functional protein-nanomaterial conjugates. Methodology/Principal Findings Multiwalled carbon nanotubes (MWNTs) were functionalised with amino groups to improve solubility and biocompatibility. The pristine and functionalised forms of MWNTs were characterised with Fourier-transform infrared spectroscopy. Thermogravimetric analysis was done to examine the degree of the functionalisation process. An immobilised biocatalyst was prepared on functionalised nanomaterial by covalent binding. Thermomyces lanuginosus lipase was used as a model enzyme. The structural change of the immobilised and free lipases were characterised with transmission electron Microscopy, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy and Circular dichroism spectroscopy. Biochemical characterisation of immobilised enzyme showed broader pH and thermal optima compared to soluble form. Reusability of the immobilised enzyme for hydrolysis of long chain esters was demonstrated up to ten cycles. Conclusion/Significance Lipase immobilised on MWNTs has exhibited significantly improved thermal stability. The exploration of advanced nanomaterial for enzyme immobilisation support using sophisticated techniques makes nanobiocatalyst of potential interest for biosensor applications.

Enzyme immobilization on nanomaterials for biofuel production

The efficient immobilization of enzymes using nanostructured materials has recently been demonstrated. The materials used for this purpose, such as nanoparticles, nanofibers, nanotubes, nanoporous media, nanocomposites, and graphene all possess large surface areas that improve biocatalytic efficiency for industrial applications by increasing enzyme loading and facilitating reaction kinetics [1]. In this report, we present the research opportunities for nanoscale materials in enzyme biotechnology and highlight recent developments in biofuel production using more advanced material supports for enzyme immobilization and stabilization.

Exploring novel ultrafine Eri silk bioscaffold for enzyme stabilisation in cellobiose hydrolysis

The suitability of optimised ultrafine Eri silk microparticles as novel enzyme supports was studied for potential application in biofuel production. β-glucosidase (BGL) from Aspergillus niger was immobilised on Eri silk fibrion particles via an adsorption method resulting in a 62% immobilisation yield. Soluble and immobilised enzymes exhibited pH-optima at pH 4.0 and 5.0, respectively with optimum activity at 60°C. The Michaelis constant (K(M)) was 0.16 and 0.27 mM for soluble and immobilised BGL respectively. The immobilisation support has a protective effect on the enzyme by increasing rigidity; this is reflected by an increase in stability under thermal denaturation at 70°C. Immobilised enzyme retained more than 50% of initial activity for up to eight cycles. Maximum cellobiose hydrolysis by immobilised BGL was achieved at 20 h. Crystalline ultrafine Eri silk particles were found to be a promising viable, environmentally sound and stable matrix for binding BGL for cellobiose hydrolysis.

Enzymatic Nanobiosensors in the Agricultural and Food Industry

Detection of the environmental contaminants in the agricultural and food industries is a major challenge. Indeed, the widespread contamination of food by pesticides and other pollutants has raised concerns of the public. Fast, cheap and sensitive sensors are thus needed. The technology of enzymatic nanobiosensor offers a quick and cost-effective solution to the current concerns of agri-food industry. This article reviews recent trends in enzymatic nanobiosensor technology employed in agri-food industries, in particular the design of a bioconjugation strategy. Nanobiosensors offer ultrasensitivity and quick detection time for various pesticides and food-borne contaminants. The minimal detection limit of contaminant in soil samples by an enzymatic nanobiosensor is in the range of 50 picogram per litre, while the minimal contaminant detection limit in food samples is 1.6 nanomolar.

Nanobiotechnology advances in enzymatic biosensors for the agri-food industry

Early detection of contaminants is of prime importance in the agricultural and food industries. Until recently real-time analysis of contaminated samples has been limited due to non-portability of sophisticated laboratory instruments and need of skilled personnel. Therefore, cheap, portable and easy to use biosensors are needed. Nanotechnology provides advanced biosensors as a result of novel nanofabrication and nanobioconjugation techniques. In particular, enzymatic nanobiosensors display ultrasensitivity and quick detection time in real-time analysis. Reported detection limits are at the nanomolar to picomolar level for contaminants analysed by enzymatic nanobiosensors. This review summarises nanobiotechnological advances inxa0thexa0agricultural and food industry, with focus on the detection of pesticides and food-borne contaminants.