Munish Puri

Enzyme-assisted extraction of bioactives from plants

Demand for new and novel natural compounds has intensified the development of plant-derived compounds known as bioactives that either promote health or are toxic when ingested. Enhanced release of these bioactives from plant cells by cell disruption and extraction through the cell wall can be optimized using enzyme preparations either alone or in mixtures. However, the biotechnological application of enzymes is not currently exploited to its maximum potential within the food industry. Here, we discuss the use of environmentally friendly enzyme-assisted extraction of bioactive compounds from plant sources, particularly for food and nutraceutical purposes. In particular, we discuss an enzyme-assisted extraction of stevioside from Stevia rebaudiana, as an example of a process of potential value to the food industry.

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.

Production, purification, and characterization of the debittering enzyme naringinase

This review discusses the debittering enzyme naringinase and its essential role in the commercial processing of citrus fruit juice. Applications of this enzyme in other areas are identified. Characterization of the enzyme is detailed and its immobilized preparations are discussed. Production of microbial naringinase by fermentation is described.

Ribosome-inactivating proteins: current status and biomedical applications

Ribosome-inactivating proteins (RIPs) are mainly present in plants and function to inhibit protein synthesis through the removal of adenine residues from eukaryotic ribosomal RNA (rRNA). They are broadly classified into two groups: type I and type II. Type I RIPs are a diverse family of proteins comprising a single polypeptide chain, whereas type II RIPs are heterodimeric glycoproteins comprising an A-chain (functionally equivalent to a type I RIP) linked via a disulphide bond to a B chain, mediating cell entry. In this review, we describe common type I and type II RIPs, their diverse biological functions, mechanism of cell entry, stability in plasma and antigenicity. We end with a discussion of promising applications for RIPs in biomedicine.

Omega-3 biotechnology: Thraustochytrids as a novel source of omega-3 oils.

Thraustochytrids are large-celled marine heterokonts and classified as oleaginous microorganisms due to their production of docosahexaenoic (DHA) and eicosapentaenoic (EPA) ω-3-fatty acids. The applications of microbial DHA and EPA for human health are rapidly expanding, and a large number of clinical trials have been carried out to verify their efficacy. The development of refined isolation and identification techniques is important for the cultivation of thraustochytrids. With a high proportion of lipid biomass, thraustochytrids are also amenable to various production strategies which increase omega-3 oil output. Modifications to the existing lipid extraction methods and utilisation of sophisticated analytical instruments have increased extraction yields of DHA and EPA. Other metabolites such as enzymes, carotenoids and extracellular polysaccharides can also be obtained from these marine protists. Approaches such as the exploration for more diverse isolates having fast growth rates, metabolic engineering including gene cloning, and growing thraustochytrids on alternate low cost carbon source, will further enhance the biotechnological potential of thraustochytrids.

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.

Biochemical Basis of Bitterness in Citrus Fruit Juices and Biotech Approaches for Debittering

AbstractThis review discusses different facets of the subject of bitterness in, and debittering of, citrus juices. For this purpose, the tone is set up by giving an account of the biochemical properties of naringin, responsible for “immediate” bitterness, and of limonin, responsible for “delayed” bitterness. Their structures are ellucidated, and enzymes participating in their catabolic pathways are assigned. Thereafter, methods used to determine bitterness, based on the principles of colorimetry, chromatography, and biochemistry, are discussed, including their advantages and disadvantages. Finally, different physicochemical and biotechnological approaches for debittering are discussed.

Studies on the applicability of alginate-entrapped naringiase for the debittering of kinnow juice

Screening of matrices for the immobilization of naringinase demonstrated that 2% sodium alginate was an optimal matrix. Upon immobilization, 30 U of naringinase gave 82% naringin hydrolysis in 3 h, broadening of pH optima has attributed desirable flexibility for debittering kinnow juice of varying pHs, and temperature profiles indicated an improved thermostability which could be handy during the reduction in cost of debittering. Alginate permitted attainment of equilibrium readily with no hindrance in the inflow of naringin and the outflow of naringenin/prunin, and adequate mechanical stability, cumulatively indicating the feasibility of its commercial exploitation. The application of kinetic parameters optimized with pure naringin to kinnow juice resulted in 60% debittering. It suggested the desirability of ultrafiltration to minimize product inhibition in order to maximize naringin hydrolysis, and subsequent fortification by the permeate at the end of debittering to formulate sweetened kinnow juice with nourishing and natural characteristics. Efforts to change over from the batch process to a continuous column process are underway.

Ribosome Inactivating Proteins (RIPs) from Momordica charantia for Anti Viral Therapy

This review describes the nature and applications of ribosome inactivating proteins (RIPs) from Momordica charantia (bitter melon). RIPs from the plant kingdom have received much attention in biomedical research because they target conserved host protein synthesis machinery and show specificity towards human and animal cell targets. Recent studies aimed at unravelling the enzymatic activities of the M charantia RIPs provide a structural basis for their activities. It has been reported that RIPs are member of the single chain ribosome inactivating protein (SCRIP) family which act irreversibly on ribosome by removing adenine residue from eukaryotic ribosomal RNA. Various activities of RIPs include anti-tumor, broad anti-viral, ribonuclease and deoxyribonuclease. MAP30 (Momordica Anti-HIV Protein), alpha- and beta-momorcharins inhibit HIV replication in acutely and chronically infected cells and thus are considered potential therapeutic agent in HIV infection and AIDS. Further, MAP30 improved the efficacy of anti-HIV therapy when used in combination with other anti-viral drugs. MAP30 holds therapeutic promise over other RIPs because not only it is active against infection and replication of both HSV and HIV but is non toxic to normal cells. Here we review the nature, action, structure function relationship and applications of RIPs from Momordica charantia and evaluate their potential for anti-cancer and anti-viral therapy.

Downstream processing of stevioside and its potential applications

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana Bertoni, which is commercially produced by conventional (chemical/physical) processes. This article gives an overview of the stevioside structure, various analysis technique, new technologies required and the advances achieved in recent years. An enzymatic process is established, by which the maximum efficacy and benefit of the process can be achieved. The efficiency of the enzymatic process is quite comparable to that of other physical and chemical methods. Finally, we believe that in the future, the enzyme-based extraction will ensure more cost-effective availability of stevioside, thus assisting in the development of more food-based applications.