In order to exploit fully the biotechnological opportunities afforded by nonaqueous enzymology, the issue of often drastically diminished enzymatic activity in organic solvents compared with that in water must be addressed and resolved. Recent studies have made great strides towards elucidating causes of this phenomenon of activity loss. None of these causes is insurmountable; by designing strategies that systematically target them, enzymatic activity in organic solvents can be readily enhanced by multiple orders of magnitude and ultimately brought to the aqueous-like level.
An important aspect of lipolytic enzymes is the unique physicochemical character of the reactions they catalyse at lipid-water interfaces, involving interfacial adsorption and subsequent catalysis . Lipases are now used as catalysts in aqueous as well as low-water media and accept various molecules as substrates. They were previously defined in kinetic terms, based on ‘interfacial activation’. This phenomenon was not found among esterases. Recently determined 3D structures of some, but not all, upases show a ‘lid’ controlling access to the active site. Thus, the presence of a lid, and ‘interfacial activation’, are unsuitable criteria for classifying specific esterases. Consequently, lipases can be pragmatically redefined as carboxyl-esterases acting on long-chain acylglycerols: they are simply fat-splitting ‘ferments’.
Cellulose is an important industrial raw material and a source of renewable energy. The crystalline structure of cellulose is biodegradable by the combined activities of many different enzymes. Cellobiohydrolases, the key cellulases, have extended tunnel-shaped active sites. Owing to the design of their active-site tunnels, they do not make cuts within cellulose chains but release cellobiose from the chain ends. Cellobiohydrolases may therefore prove to be useful for achieving subtle modifications of fiber properties in paper or textiles without the loss of fiber strength commonly observed with other cellulases.
Polyunsaturated fatty acids form a unique class of food constituents that show a wide range of functions in biological systems. Investigations over the past two decades have uncovered their roles and those of their eicosanoid metabolites, and have highlighted their homeostatic functions in mammals. A growing number of common human medical conditions are thought to be traceable to dysfunctions in the eicosanoid system, which could in turn be due to imbalances in the intake and/or metabolism of polyunsaturated fatty acids. This, together with medical advances, has spurred the introduction of biomédical products, nutritionals, fortified foods and health supplements.
Colloidal particles in the nanometre size range (less than 1 μm in diameter) can be engineered to provide opportunities for the site-specific delivery of drugs after injection into the general circulation or lymphatic systems. Targets include the liver (both Kupffer cells and hepatocytes), endothelial cells, sites of inflammation and lymph nodes. The size and surface of the particle are crucial factors in targeting, and the attachment of cell-specific ligands can lead to increased selectivity. The applications of such particle engineering are discussed in relation to conventional drugs as well as the emerging area of gene therapy.
Recent progress in the development of microelectrode structures has led to new techniques for the dielectrophoretic characterization and sorting of cells, microorganisms and other bioparticles using nonuniform AC electric fields. These methods utilize differences in the dielectric polarizabilities of cells for their effectiveness, and factors controlling such properties include the conductivity and permittivity of membranes and any cell walls, electrical double layers associated with surface charges, cell morphologies, and internal structures. Applications of dielectrophoresis have included the selective spatial manipulation and separation of mixtures of bacteria, viable and unviable cells, cancerous and normal cells, and red and white blood cells.
Polyunsaturated fatty acids form a unique class of food constituents that show a wide range of functions in biological systems, Investigations over the past two decades have uncovered their roles and those of their eicosanoid metabolites, and have highlighted their homeostatic functions in mammals, A growing number of common human medical conditions are thought to be traceable to dysfunctions in the eicosanoid system, which could in turn be due to imbalances in the intake and/or metabolism of polyunsaturated fatty acids. This, together with medical advances, has spurred the introduction of biomedical products, nutritionals, fortified foods and health supplements.
Traditionally, the intracellular fluxes of complex metabolic networks were quantified by isotopic-tracer experiments, but, owing to practical limitations, ‘metabolic-flux balancing’ is emerging as an alternative. This has become an important tool for the quantitative analysis of the physiology of microorganisms and mammalian cells. It has been successfully applied to finding potential sites for metabolic engineering, determining metabolic capabilities and designing optimal feeding strategies. However, it has the fundamental problem that metabolic networks, and cyclic metabolic pathways in particular, are underdetermined. The search for constraints that can be used to determine fluxes correctly for a range of different conditions is an exciting challenge.
Directed enzyme evolution has emerged in the past few years as a powerful alternative to rational approaches for engineering biocatalysts. Prerequisites for successful directed evolution are functional expression in a suitable microbial host, a rapid screen for the desired feature(s) and a well-thought-out working strategy for navigating protein landscapes. The rapidly growing body of literature on enzyme evolution includes techniques for creating and searching combinatorial enzyme libraries, as well as several successful examples of different evolutionary strategies being used.
Mitochondrial function can be manipulated selectively by targeting bioactive compounds to mitochondria in living cells. Current targeting mechanisms involve harnessing either the mitochondrial membrane potential or the mitochondrial protein-import machinery to take up molecules linked to lipophilic cations or mitochondrial signal peptides, respectively. Compounds localized like this have been used in studies of mitochondrial metabolism and show potential as anticancer drugs. In addition, because mitochondria play a role in many critical cell processes, mitochondrial targeting has the potential to protect, repair or kill cells selectively, and may become a key tool in the development of gene therapy for mitochondrial DNA diseases.
Antibodies are able to both bind antigens and trigger the responses that eliminate them from circulation. All antibodies are glycosylated at conserved positions in their constant regions, and the presence of carbohydrate can be critical for antigen clearance functions such as complement activation. The structure of the attached carbohydrate can also affect antibody activity. Antibody glycosylation can be influenced by the cell in which it is produced, the conformation of the antibody and cell culture conditions. These variables should be considered in the design and production of antibodies with selected specificity and function.
The processing of starches with biodegradable additives has made biodegradable plastics suitable for a number of applications. Starch plastics are partially crystalline as a result of residual crystallinity and the recrystallization of amylose and amylopectin. Such crystallinity is a key determinant of the product's properties. This article describes the influence of processing and storage conditions on starch crystallinity and offers possible explanations for the various properties of starch plastics, in particular for the problems associated with ageing, in terms of the different crystalline structures.
Expanded-bed adsorption for the capture of proteins from feedstocks containing cells or cell debris has been used frequently during the last few years. The main reason for the increased number of examples is the availability of purpose-designed adsorbents and equipment for expanded-bed processes, making the technique efficient and compliant with the requirements of the pharmaceutical industry. Examples of processes where expanded-bed adsorption has been used to recover products from several-cubic-metre volumes of feedstocks now exist but there is still a need for the development of equipment and adsorbents that can handle even larger feedstock volumes. There is clearly a need to develop new adsorbents with increased protein-binding capacity. The use of highly specific and stable ligands in combination with improved adsorbents will make expanded-bed adsorption even more attractive. Such improvements will allow the initial recovery of products in industrial bioprocessing to be carried out in a shorter time with a high product yield, even when very large feedstock volumes are used.
The commercial processing of starch to mono- and oligosaccharides depends on the availability of three major enzymes- glucoamylase, alpha- amylase and glucose isomerase. Each of these enzymes has a unique pH and temperature optimum for use, and so unit operations of a starch plant reflect the varying operating conditions for each individual enzymatic step. This article will discuss how recent advances in molecular biology and protein engineering have allowed enzymes with improved operating parameters to be introduced to commercial applications, providing the starch processor with enhanced plant efficiency, lower operating costs and higher-quality products.
Since its invention at the beginning of the 1990s, antibody phage display has revolutionized the generation of monoclonal antibodies and their engineering. It is now possible to create antibodies binding to any chosen target antigen without the use of laboratory animals or hybridomas, in a system that completely by-passes the immune system. Making antibodies from single-pot phage libraries, and improving their affinity up to the picomolar range if necessary, has never appeared easier. In this review, a variety of phage library-based strategies for the isolation of high-affinity antibodies are presented.
Heterologous surface display on Gram-negative bacteria was first described a decade ago and is now an active research area. More recently, strategies for surface display on Gram-positive bacteria have also been devised and these carry some inherent advantages. Bacterial surface display has found a range of applications in the expression of various antigenic determinants, heterologous enzymes, single-chain antibodies, polyhistidyl tags and even entire peptide libraries. This article explains the basis of bacterial surface display and discusses current uses and possible future trends of this emerging technology.
Homologous and heterologous protein production by filamentous fungi is often limited by the expression of proteases at high levels. By eliminating specific protease activities, protein production in Aspergillus niger can be improved considerably. Both classical mutagenesis and gene disruption techniques have yielded strains with reduced protease expression. Combinations of these mutations and disruptions result in a further reduction of protease activity. The coupling of efficient promoters to target genes allows their expression under conditions that repress the expression of several proteases, which further improves product yields. The strategies used have led to the development of a set of tester strains from which the appropriate genetic background for production can be selected.
Lactic acid bacteria are widely used in industrial food fermentations, contributing to flavour, texture and preservation of the fermented products. Here we describe recent advances in the development of controlled gene expression systems, which allow the regulated overproduction of any desirable protein by lactic acid bacteria. Some systems benefit from the fact that the expression vectors, marker genes and inducing factors can be used directly in food applications since they are all derived from food-grade lactic acid bacteria. These systems have also been employed for the development of autolytic bacteria, suitable for various industrial applications.
Mitogen-activated protein kinase (MAPK) cascades have essential roles in diverse intracellular signaling processes in plants, animals and yeasts. In plants, transcription of genes encoding protein kinases involved in MAPK cascades is upregulated by environmental stresses and plant hormones; in addition, MAPK-like kinase activities are transiently activated in response to environmental stresses. Consequently, MAPK cascades are now thought to have important roles in stress signal transduction pathways in higher plants.
Spontaneous mouse mutations that cause severe immunodeficiency or autoimmunity are invaluable tools with which to investigate the mammalian immune system. Mutations at the ‘motheaten’ locus result in severe immunological dysfunction due to disruption of the structural gene encoding Src-homology 2-domain phosphatase-1 (SHP-1). This natural model for a specific protein-tyrosine-phosphatase deficiency is being widely utilized to determine the role of SHP-1 in the negative regulation of multiple signaling pathways in a number of hematopoietic lineages.