Showing papers in "Advances in Biochemical Engineering \/ Biotechnology in 2002"

Biosynthesis of triterpenoid saponins in plants.

Abstract: Many different plant species synthesise triterpenoid saponins as part of their normal programme of growth and development. Examples include plants that are exploited as sources of drugs, such as liquorice and ginseng, and also crop plants such as legumes and oats. Interest in these molecules stems from their medicinal properties, antimicrobial activity, and their likely role as determinants of plant disease resistance. Triterpenoid saponins are synthesised via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give primarily oleanane (β-amyrin) or dammarane triterpenoid skeletons. The triterpenoid backbone then undergoes various modifications (oxidation, substitution and glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyltransferases and other enzymes. In general very little is known about the enzymes and biochemical pathways involved in saponin biosynthesis. The genetic machinery required for the elaboration of this important family of plant secondary metabolites is as yet largely uncharacterised, despite the considerable commercial interest in this important group of natural products. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. Considerable advances have recently been made, however, in the area of 2,3-oxidosqualene cyclisation, and a number of genes encoding the enzymes that give rise to the diverse array of plant triterpenoid skeletons have been cloned. Progress has also been made in the characterisation of saponin glucosyltransferases. This review outlines these developments, with particular emphasis on triterpenoid saponins.

Bulk chemicals from biotechnology: the case of 1,3-propanediol production and the new trends.

Abstract: The need for a sustainable resource supply, the rapid advances in plant biotechnology and microbial genetics and the strategic shift of major chemical companies into the area of life sciences are some of the driving forces for renewed interest in producing bulk chemicals from renewable resources by biological processes. The microbial production of 1,3-propanediol as briefly reviewed in this article and compared with the competing chemical processes demonstrates the promise and constraints of bioprocesses for bulk chemicals. The new concept of biorefinery and biocommodity engineering and future research needs in this area are also outlined.

The use of MassARRAY technology for high throughput genotyping.

Abstract: This chapter will explore the role of mass spectrometry (MS) as a detection method for genotyping applications and will illustrate how MS evolved from an expert-user-technology to a routine laboratory method in biological sciences. The main focus will be time-of-flight (TOF) based devices and their use for analyzing single-nucleotide-polymorphisms (SNPs, pronounced snips). The first section will describe the evolution of the use of MS in the field of bio-analytical sciences and the protocols used during the early days of bioanalytical MALDI TOF mass spectrometry. The second section will provide an overview on intraspecies sequence diversity and the nature and importance of SNPs for the genomic sciences. This is followed by an exploration of the special and advantageous features of mass spectrometry as the key technology in modern bioanalytical sciences in the third chapter. Finally, the fourth section will describe the MassARRAY technology as an advanced system for automated high-throughput analysis of SNPs.

Sequencing by hybridization (SBH): advantages, achievements, and opportunities.

Abstract: Efficient DNA sequencing of the genomes of individual species and organisms is a critical task for the advancement of biological sciences, medicine and agriculture. Advances in modern sequencing methods are needed to meet the challenge of sequencing such megabase to gigabase quantities of DNA. Two possible strategies for DNA sequencing exist: direct methods, in which each base position in the DNA chain is determined individually (e.g., gel sequencing or pyrosequencing), and indirect methods, in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the DNA chain. One promising indirect method is sequencing by hybridization (SBH), in which sets of oligonucleotides are hybridized under conditions that allow detection of complementary sequences in the target nucleic acid. The unprecedented sequence search parallelism of the SBH method has allowed development of high-throughput, low-cost, miniaturized sequencing processes on arrays of DNA samples or probes. Newly developed SBH methods use DNA ligation to combine relatively small sets of short probes to score potentially tens of millions of longer oligonucleotide sequences in a target DNA. Such combinatorial approaches allow analysis of DNA samples of up to several kilobases (several times longer than allowed by current direct methods) for a variety of DNA sequence analysis applications, including de novo sequencing, resequencing, mutation/SNP discovery and genotyping, and expression monitoring. Future advances in biochemistry and implementation of detection methods that allow single-molecule sensitivity may provide the necessary miniaturization, specificity, and multiplexing efficiency to allow routine whole genome analysis in a single solution-based hybridization experiment.

Metabolic Flux Analysis Using Mass Spectrometry

Abstract: Detailed knowledge on carbon flux distributions is crucial for the understanding and targeted optimization of cellular systems. Analytical methods to identify the topology of metabolic networks and to quantify fluxes through its different pathways are therefore in the core of metabolic engineering. An elegant approach for metabolic flux analysis is provided by tracer experiments. In such studies tracer substrates with stable isotopes such as 13C are applied and the labeling pattern of metabolites is subsequently measured. Detailed flux distributions can be obtained by a combination of tracer experiments and stoichiometric balancing. In recent years, mass spectrometry (MS) has emerged as an interesting method for labeling measurements in metabolic flux analysis and provided valuable insights into the cellular metabolism. The present review provides an overview on current experimental and modeling tools for metabolic flux analysis by MS. The application of MS for flux analysis is illustrated by examples from the literature for various biological systems, including bacteria, fungi, tissue cultures and in vivo studies in humans.

Cell Retention Devices for Suspended-Cell Perfusion Cultures

Abstract: Perfusion cultures of animal cells have several advantages over batch or fed-batch cultures. They give, for instance, higher productivities and a consistent product quality, and allow steady state operation and better cell physiology control. However, one of the main aspects limiting performance and scale-up of perfusion processes is the need for an adequate cell retention device. The devices currently in use for stirred perfusion bioreactors are continuous centrifuges, tangential flow membrane filters, dynamic filters, spin-filters, ultrasonic and dielectrophoretic separators, gravity settlers and, more recently, hydrocyclones. The advantages and disadvantages of each of these methods will be discussed.

Reverse micellar extraction for downstream processing of proteins/enzymes.

Abstract: New developments in the area of downstream processing are, hopefully, to fulfill the promises of modern biotechnology. The traditional separation processes such as chromatography or electrophoresis can become prohibitively expensive unless the product is of high value. Hence, there is a need to develop efficient and cost-effective downstream processing methods. Reverse micellar extraction is one such potential and a promising liquid-liquid extraction technique, which has received immense attention for isolation and purification of proteins/enzymes in the recent times. This technique is easy to scale-up and offers continuous operation. This review, besides briefly considering important physico-chemical and biological aspects, highlights the engineering aspects including mass transfer, mathematical modeling, and technology development. It also discusses recent developments in reverse micellar extraction such as affinity based separations, enzymatic reactions in reverse micelles coupled with membrane processes, reverse micellar extraction in hollow fibers, etc. Special emphasis has been given to some recent applications of this technique.

Short Monolithic Columns as Stationary Phases for Biochromatography

Abstract: Monolithic supports represent a novel type of stationary phases for liquid and gas chromatography, for capillary electrochromatography, and as supports for bioconversion and solid phase synthesis. As opposed to individual particles packed into chromatographic columns, monolithic supports are cast as continuous homogeneous phases. They represent an approach that provides high rates of mass transfer at lower pressure drops as well as high efficiencies even at elevated flow rates. Therefore, much faster separations are possible and the productivity of chromatographic processes can be increased by at least one order of magnitude as compared to traditional chromatographic columns packed with porous particles. Besides the speed, the nature of the pores allows easy access even in the case of large molecules, which make monolithic supports a method of choice for the separation of nanoparticles like pDNA and viruses. Finally, for the optimal purification of larger biomolecules, the chromatographic column needs to be short. This enhances the speed of the separation process and reduces backpressure, unspecific binding, product degradation and minor changes in the structure of the biomolecule, without sacrificing resolution. Short Monolithic Columns (SMC) were engineered to combine both features and have the potential of becoming the method of choice for the purification of larger biomolecules and nanoparticles on the semi-preparative scale.

High-Density GeneChip Oligonucleotide Probe Arrays

Abstract: High-density DNA probe arrays provide a highly parallel approach to nucleic acid sequence analysis that is transforming gene-based biomedical research. Photolithographic DNA synthesis has enabled the large-scale production of GeneChip probe arrays containing hundreds of thousands of oligonucleotide sequences on a glass “chip” about 1.5 cm2 in size. The manufacturing process integrates solid-phase photochemical oligonucleotide synthesis with lithographic techniques similar to those used in the microelectronics industry. Due to their very high information content, GeneChip probe arrays are finding widespread use in the hybridization-based detection and analysis of mutations and polymorphisms (“genotyping”), and in a wide range of gene expression studies.

Integrated approach to explore the potential of marine microorganisms for the production of bioactive metabolites.

Abstract: During the last 10 years marine organisms have provided a large number of new natural products. Interesting compounds have mainly been derived from macroorganisms such as sponges, ascidians, corals and bryozoans. The number of secondary metabolites from marine microorganisms is smaller, but rapidly increasing. Because of the enormous difficulties involved in harvesting products from marine animals, and the fact that some of the bioactive compounds are produced by associated bacteria, the advantages of sustainable production of bioactive metabolites by bacteria or fungi, under the protection of natural resources, seem to be very attractive for the future. This review describes current progress in the isolation and identification of novel marine microorganisms, the discovery of new secondary metabolites, the biotechnological approaches to overproduce them, as well as the evaluation and characterization of their bioactivity.

Simulated moving bed chromatography (SMB) for application in bioseparation.

Abstract: Simulated Moving Bed (SMB) technology is of rising interest in the field of bioseparation. This is particularly due to its advantages such as reduction of solvent consumption, high productivity and final purities as well as low investment costs in comparison to eluent chromatography. SMB units can operate under high productivity overloaded conditions. This leads to nonlinear competitive adsorption behavior, which has to be accounted for when designing and optimizing new SMB separations. The so called “Triangle Theory”, which is briefly reviewed in this chapter, provides explicit criteria for the choice of the operating conditions of SMB units to achieve the prescribed separation of a mixture characterized by Langmuir, modified Langmuir and bi-Langmuir isotherms.

Production of core and virus-like particles with baculovirus infected insect cells

Abstract: In this paper the fundamental aspects of process development for the production of core and virus-like particles with baculovirus infected insect cells are reviewed. The issues addressed include: particle formation and monomer composition, chemical and physical conditions for optimal cell growth, baculovirus replication and product expression, multiplicity of infection strategy, and scale-up of the process. Study of the differences in the metabolic requirements of infected and non-infected cells is necessary for high cell density processes. In the bioreactor, the specific oxygen uptake rate (OURsp) plays a central role in process scale-up, leading to the specification of the bioreactor operational parameters. Shear stress can also be an important variable for bioreactor operation due to its influence on cell growth and product expression. The determination of the critical variables in process development is discussed, showing the relevance of the mathematical models that have been developed for the insect cells/baculovirus system in process implementation and control.

Enzyme catalysis in reverse micelles.

Abstract: Water in oil microemulsions with reverse micelles provide an interesting alternative to normal organic solvents in enzyme catalysis with hydrophobic substrates. Reverse micelles are useful microreactors because they can host proteins like enzymes. Catalytic reactions with water insoluble substrates can occur at the large internal water-oil interface inside the microemulsion. The activity and stability of biomolecules can be controlled, mainly by the concentration of water in these media. With the exact knowledge of the phase behaviour and the corresponding activity of enzymes the application of these media can lead to favourable effects compared to aqueous systems, like hyperactivity or increased stability of the enzymes.

The molecular mechanism of ATP synthesis by F1F0-ATP synthase: a scrutiny of the major possibilities.

Abstract: A critical goal of metabolism in living cells is the synthesis of adenosine triphosphate (ATP). ATP is synthesized by the enzyme F1F0-ATP synthase. This enzyme, the smallest-known molecular machine, couples proton translocation through its membrane-embedded, hydrophobic domain, F0, to the synthesis of ATP from adenosine diphosphate (ADP) and inorganic phosphate (Pi) in its soluble, hydrophilic headpiece, F1. Animals, plants and microorganisms all capture and utilize energy by this important chemical reaction. How does it occur? The binding change mechanism and the torsional mechanism of energy transduction and ATP synthesis are two mechanisms that have been proposed in the literature. According to the binding change mechanism (which considers reversible catalysis and site-site cooperativity), energy is required primarily for release of synthesized ATP, but not for its synthesis. On the other hand, according to the torsional mechanism (which considers an irreversible mode of catalysis and absence of cooperativity), all the elementary steps require energy, and the ion-protein interaction energy obtained from the ion gradients is used to synthesize ATP, for Pi binding, and for straining the β-e bond in order to enable ADP to bind. The energy to release preformed ATP from the tight catalytic site (βdp) is provided by the formation of the β-e ester linkage. First, the central features of these mechanisms are clearly delineated. Then, a critical scrutiny of these mechanisms is undertaken. The predictions of the torsional mechanism are listed. In particular, how the torsional mechanism deals with the specific difficulties associated with other mechanisms, and how it seeks to explain a wealth of structural, spectroscopic, and biochemical data is discussed in detail. Recent experimental data in support of the mechanism are presented.

Porous polymer monoliths: an alternative to classical beads.

Abstract: Porous polymer monoliths are a new category of materials developed during the last decade. These materials are prepared using a simple molding process carried out within the confines of a closed mold. Polymerization of a mixture that typically contains monomers, free-radical initiator, and porogenic solvent affords macroporous materials with large through-pores that enable flow-through applications. The versatility of the preparation technique is demonstrated by its use with hydrophobic, hydrophilic, ionizable, and zwitterionic monomers. The porous properties of the monolith can be controlled over a broad range. These, in turn, determine the hydrodynamic properties of the devices that contain the molded media. Since all the mobile phase must flow through the monolith, the mass transport within the molded material is dominated very much by convection, and the monolithic devices perform well even at very high flow rates. The applications of monolithic materials are demonstrated on the chromatographic separation of biological compounds and synthetic polymers, electrochromatography, gas chromatography, enzyme immobilization, molecular recognition, and in advanced detection systems. Grafting of the pore walls with selected polymers leads to materials with completely changed surface chemistries.

Combinatorial algorithms for design of DNA arrays.

Abstract: Optimal design of DNA arrays requires the development of algorithms with two-fold goals: reducing the effects caused by unintended illumination (border length minimization problem) and reducing the complexity of masks (mask decomposition problem). We describe algorithms that reduce the number of rectangles in mask decomposition by 20–30% as compared to a standard array design under the assumption that the arrangement of oligonucleotides on the array is fixed. This algorithm produces provably optimal solution for all studied real instances of array design. We also address the difficult problem of finding an arrangement which minimizes the border length and come up with a new idea of threading that significantly reduces the border length as compared to standard designs.

In-situ-fluorescence-probes: a useful tool for non-invasive bioprocess monitoring.

Abstract: Optical sensors appear to be very promising for different applications in modern biotechnology. They offer the possibility to interface all the well known optical analysis techniques to bioprocesses via fiber optical cables. Thus, high sophisticated and sensitive optical analysis techniques can be coupled to a bioprocess via these light signal transporting fibers. A wide variety of sensor types for application in biotechnology has been described [1-4]. Normally these sensors are non-invasive and the response times are nearly instantaneous. In particular, the use of glass fiber technology makes these sensors small, robust and reduces their costs.

Protein array technology: the tool to bridge genomics and proteomics.

Abstract: The generation of protein chips requires much more efforts than DNA microchips While DNA is DNA and a variety of different DNA molecules behave stable in a hybridisation experiment, proteins are much more difficult to produce and to handle Outside of a narrow range of environmental conditions, proteins will denature, lose their three-dimensional structure and a lot of their specificity and activity The chapter describes the pitfalls and challenges in Protein Microarray technology to produce native and functional proteins and store them in a native and special environment for every single spot on an array, making applications like antibody profiling and serum screening possible not only on denatured arrays but also on native protein arrays

Bioreactor developments for tissue engineering applications by the example of the bioartificial liver

Abstract: Tissue engineering is the application of the principles and methods of engineering and the life sciences towards the development of biological substitutes to restore, maintain or improve functions. It is an area which is emerging in importance worldwide. This article is to show the developments in tissue engineering research by the example of the bioartificial liver. As an alternative to liver transplantation, numerous researchers have been working towards the goal of development of a fully functional artificial liver. Liver support systems based on detoxification alone have proven ineffective because they cannot correct biochemical disorders. An effective artificial liver support system should be capable of carrying out the liver's essential processes, such as synthetic and metabolic functions, detoxification, and excretion. It should be capable of sustaining patients with fulminant hepatic failure and preparing patients for liver transplantation when a donor liver is not readily available. Although several hepatocyte-based liver support systems have been proposed, there is no current consensus on its eventual design configuration.

Microarray Data Representation, Annotation and Storage

Abstract: Management and analysis of the huge amounts of data produced by microarray experiments is becoming one of the major bottlenecks in the utilization of this high-throughput technology. We describe the basic design of a microarray gene expression database to help microarray users and their informatics teams to set up their information services. We describe two data models — a simpler one called ArrayExpressB and the complete model ArrayExpressC, and discuss some implementation issues. For latest developments see http: www.ebi.ac.uk/arrayexpress.

Cultivation of Hematopoietic Stem and Progenitor Cells: Biochemical Engineering Aspects

Abstract: The ex vivo expansion of hematopoietic cells is one of the most challenging fields in cell culture. This is a rapidly growing area of tissue engineering with many potential applications in bone marrow transplantation, transfusion medicine or gene therapy. Over the last few years much progress has been made in understanding hematopoietic differentiation, discovery of cytokines, isolation and identification of cellular subtypes and in the development of a variety of bioreactor concepts. All this has led to a number of (preliminary) clinical trials that gave a hint of the benefits that can be obtained from the use of expanded hematopoietic cells in therapy. Moreover, as we understand the complexity and the regulation of hematopoiesis, it becomes obvious that highly sophisticated cultivation techniques and bioreactor concepts are needed: a new challenge for bioprocess engineering in cell culture.

Chromatographic reactors based on biological activity.

Abstract: In the last decade there were many papers published on the study of enzyme catalyzed reactions performed in so-called chromatographic reactors. The attractive feature of such systems is that during the course of the reaction the compounds are already separated, which can drive the reaction beyond the thermodynamic equilibrium as well as remove putative inhibitors. In this chapter, an overview of such chromatographic bioreactor systems is given. Besides, some immobilization techniques to improve enzyme activity are discussed together with modern chromatographic supports with improved hydrodynamic characteristics to be used in this context.

Molecularly Imprinted Materials — Receptors More Durable than Nature Can Provide

Abstract: The chapter describes the concept of molecular imprinting. This technology allows the fabrication of artificial polymeric receptors applicable in many areas of biotechnology. Polymers imprinted with selected template molecules can be used as specific recognition elements in sensors or as selective stationary phases in affinity chromatography or in capillary electrochromatography. However, also in solid phase extraction or immunoassays these polymers (MIP) are able to compete with traditional materials such as biological antibodies. Furthermore, polymers molecularly imprinted with so-called transition state analogue templates can be applied as catalysts. In other words, these kind of polymers may be used as artificial antibodies (plastibodies) or biomimicking enzymes (plastizymes). Compared to their biological counterparts, MIP offer different advantages such as simplicity in manufacturing and durability. Thus, the author expects MIP to have a major impact on the whole area of biotechnology.

Capillary Electrochromatography: A Rapidly Emerging Separation Method

Abstract: This overview concerns the new chromatographic method — capillary electrochromatography (CEC) — that is recently receiving remarkable attention The principles of this method based on a combination of electroosmotic flow and analyte-stationary phase interactions, CEC instrumentation, capillary column technology, separation conditions, and examples of a variety of applications are discussed in detail

Microbial sensors on a respiratory basis for wastewater monitoring.

Abstract: In respect of their rapidity, their online capabilities, and their moderate costs, biosensing systems generally offer an attractive alternative to the existing methods of water analysis. Additionally, one particular advantage of microbial biosensors is the ability to measure direct effects on living cells,e.g., their respiratory activity and its alteration caused by environmental pollutants. It is true that microbial sensors, often do not provide the optimum solution for the determination of individual analytes when compared to established physico-chemical analysis methods. However, these biosensing devices are predestined for the summary determination of environmentally relevant compounds and their complex effects, respectively. For this reason, microbial sensors allow an integral evaluation of the degree of environmental pollution including the interaction of various compounds. Moreover, in some cases specific metabolic pathways in microorganisms are used, resulting in the development of microbial sensors for the more selective analysis for those compounds or pollutants, which cannot be measured by simple enzyme reactions, e.g., the determination of aromatic compounds and heavy metals.

Methods for biocatalyst screening.

Abstract: Biocatalysts are now widely accepted as useful alternative tools to classic organic synthetic techniques for the regio- and enantioselective synthesis under mild reaction conditions in many fields of chemistry. The development of techniques for the rational or evolutionary design of novel or modified enzymes has increased the need for fast and reliable methods for the identification of the most powerful catalysts. We present a short overview on screening techniques in this area. Beside classical methods such as spectrophotometry and fluorimetry, a number of new approaches like methods based on the measurement of pH changes or IR-thermography have been recently developed. Additionally the use of electrospray and matrix-assisted laser desorption/ionization mass spectrometry has gained increasing influence in this field of biotechnology.

Continuous annular chromatography.

Abstract: In recent years the demand for process scale chromatography systems in the industrial downstream process has been increasing steadily. Chromatography seems to be the method of choice when biological active compounds must be recovered from a mixture containing dozens of side products and contaminants as it is for example the case when processing fermentation broths. Since chromatography can solve almost any separation problem under mild operating conditions, a continuous chromatography system represents an extremely attractive and powerful option for such large-scale applications. The increasing number of biotechnological products forces system suppliers of the downstream processing side to develop new and improved high throughput purification technologies.

Three-Phase Oxygen Absorption and its Effect on Fermentation

Abstract: The absorption rate of oxygen in the presence of a second, dispersed, organic phase can be significantly increased due to the higher solubility and diffusivity of oxygen in the organic phase The oxygen supply of micro-organisms, which is very often a limiting factor during fermentation, can be improved, and the critical level of oxygen in the fermentation broth can be avoided by using dispersed organic phase This paper reviews the models of the enhanced absorption rates and their integration into mass balance equations of fermentation Several calculations were carried out to illustrate the effect of the dispersed organic phase and kinetic parameters on the absorption rates and on fermentation with double-substrate-limitation kinetics applying batch and continuous operation modes Using software taking from the literature, the effect of the organic phase on the baker’s yeast production is also presented in fed-batch mode

Oligonucleotide scanning arrays: application to high-throughput screening for effective antisense reagents and the study of nucleic acid interactions.

Abstract: Oligonucleotide scanning arrays are useful tools in the study of nucleic acid interaction Such arrays of oligonucleotides, corresponding to a full set of complements of a known sequence, can be readily made in a single series of coupling reactions, adding each nucleotide in the complement of the target sequence in turn The synthesis is carried out on the surface of a solid substrate such as glass or polypropylene that has been modified to allow nucleotide coupling A mask is used to apply the DNA synthesis reagents in a defined area and is moved by a fixed step size after each coupling reaction so that consecutive couplings overlap a portion of the previous one The size of the mask and the displacement at each coupling determine the length of the oligonucleotides

Perspectives in liquid membrane extraction of cephalosporin antibiotics.

Abstract: In this paper an overview of the developments in liquid membrane extraction of cephalosporin antibiotics has been presented. The principle of reactive extraction via the so-called liquid-liquid ion exchange extraction mechanism can be exploited to develop liquid membrane processes for extraction of cephalosporin antibiotics. The mathematical models that have been used to simulate experimental data have been discussed. Emulsion liquid membrane and supported liquid membrane could provide high extraction flux for cephalosporins, but stability problems need to be fully resolved for process application. Non-dispersive extraction in hollow fiber membrane is likely to offer an attractive alternative in this respect. The applicability of the liquid membrane process has been discussed from process engineering and design considerations.