Co-immobilized coupled enzyme systems on nylon mesh capable of gluconic and pyruvic acid production

Summary The coupled enzyme systems glucose oxidase-catalase and lactate oxidase-catalase have been immobilized in a cross-linked gelatin matrix on a nylon mesh fabric. These immobilized enzyme systems can be used for the production of gluconic and pyruvic acids, respectively.     

On the evolution of prebiotic enzyme coupled macromolecular systems

In this paper we consider the evolution of prebiotic macromolecular systems in which a coding and replicating machinery has come into existence. We introduce a specific enzyme coupling model and discuss the formation, selection and survival of mutants and hypercycles. Implications of the model for the selection of a universal genetic code are discussed.     

Multifunctional cargo systems for biotechnology

One of the challenges in the field of bio-nanotechnology is the development of nano-sized delivery systems comprising different functionalities. These systems should carry bioactive substances to predefined site and unload them in controlled manner. Capsules assembled layer-by-layer have been intensively studied in the past few years owing to their ability to be modified, their capacity to encapsulate a wide range of chemicals, their responsiveness to different factors, and the variety of functionalities with which they can be enhanced. Current research focuses on the development of carriers with remote guiding and activation (optical, magnetic or ultrasound), thereby addressing unique in vivo applications with multifunctional biomaterials. Submicron-sized capsules are good models to mimic biochemical processes in a confined geometry that imitates cell organelles. Moreover, the cellular and tissue-targeted delivery of the capsules might serve as an intracellular reporter or enzymatic reactor. However, several obstacles still have to be overcome before capsule technology can be implemented. This article discusses possible solutions as well as promising applications.     

Applications of aqueous two-phase systems in biotechnology

Aqueous two-phase systems can be employed in several areas of biotechnology including the purification of biomolecules, cells and organelles and increasing the speed of product-inhibited fermentations such as the production of acetone, butanol and ethanol. Furthermore, separations in aqueous two-phase systems have been successfully applied in binding assays.     

Biotransformations by microbial Baeyer-Villiger monooxygenases stereoselective lactone formationin vitro by coupled enzyme systems

Summary The regio- and stereoselective biotransformation of bicyclo (3. 2. 0) hept-2-en-6-one by the NADH-dependent Baeyer-Villiger monooxygenase from camphorgrownPseudomonas putida NCIMB 10007 has been shown to yield a chiral lactone not accessible by curently-used biocatalysts. The biotransformation can be conductedin vitro using two alternative coupled enzyme systems (alcohol dehydrogenase and monooxygenase: formate dehydrogenase and monooxygenase) within situ recycling of NAD⁺/NADH.     

Emergent properties of coupled enzyme reaction systems 1. Switching and clustering behaviour

The dynamics of locally and globally coupled cells that convert a substrate to a product via an uncompetitive substrate-inhibition mechanism is studied. When the cell-cell coupling strength is below a threshold value, the coupled system exhibits a large number of steady states; however, all cells cluster to one state when coupling exceeds the threshold value. The coupled system also exhibits a buffering capacity that maintains low and almost constant intracellular and extracellular substrate levels; however, there exists a threshold value on the influx rate of extracellular substrate beyond which the system switches to higher substrate levels. This transition becomes sharper as the number of coupled cells increases. Propagation failure of concentration fronts between adjacent cells is also exhibited by the system.     

Kinetics of coupled enzyme reactions

A theory has been developed for the kinetics of coupled enzyme reactions. This theory does not assume that the first reaction is irreversible. The validity of this theory is confirmed by a model system consisting of enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) with 2,4-decadienoyl coenzyme A (CoA) as a substrate. This theory, in contrast to the conventional theory, proves to be indispensible for dealing with coupled enzyme systems where the equilibrium constant of the first reaction is small and/or the concentration of the coupling enzyme is higher than that of the intermediate. Equations derived on the basis of this theory can be used to calculate steady-state velocities of coupled enzyme reactions and to predict the time course of coupled enzyme reactions during the pre steady state.     

Inducible gene expression systems and plant biotechnology

Plant biotechnology relies heavily on the genetic manipulation of crops. Almost invariantly, the gene of interest is expressed in a constitutive fashion, although this may not be strictly necessary for several applications. Currently, there are several regulatable expression systems for the temporal, spatial and quantitative control of transgene activity. These molecular switches are based on components derived from different organisms, which range from viruses to higher eukaryotes. Many inducible systems have been designed for fundamental and applied research and since their initial development, they have become increasingly popular in plant molecular biology.This review covers a broad number of inducible expression systems examining their properties and relevance for plant biotechnology in its various guises, from molecular breeding to pharmaceutical and industrial applications. For each system, we examine some advantages and limitations, also in relation to the strategy on which they rely. Besides being necessary to control useful genes that may negatively affect crop yield and quality, we discuss that inducible systems can be also used to increase public acceptance of GMOs, reducing some of the most common concerns. Finally, we suggest some directions and future developments for their further diffusion in agriculture and biotechnology.     

Metabolic systems analysis to advance algal biotechnology

Algal fuel sources promise unsurpassed yields in a carbon neutral manner that minimizes resource competition between agriculture and fuel crops. Many challenges must be addressed before algal biofuels can be accepted as a component of the fossil fuel replacement strategy. One significant challenge is that the cost of algal fuel production must become competitive with existing fuel alternatives. Algal biofuel production presents the opportunity to fine-tune microbial metabolic machinery for an optimal blend of biomass constituents and desired fuel molecules. Genome-scale model-driven algal metabolic design promises to facilitate both goals by directing the utilization of metabolites in the complex, interconnected metabolic networks to optimize production of the compounds of interest. Network analysis can direct microbial development efforts towards successful strategies and enable quantitative fine-tuning of the network for optimal product yields while maintaining the robustness of the production microbe. Metabolic modeling yields insights into microbial function, guides experiments by generating testable hypotheses, and enables the refinement of knowledge on the specific organism. While the application of such analytical approaches to algal systems is limited to date, metabolic network analysis can improve understanding of algal metabolic systems and play an important role in expediting the adoption of new biofuel technologies.     

Plasmid addiction systems: perspectives and applications in biotechnology

Biotechnical production processes often operate with plasmid-based expression systems in well-established prokaryotic and eukaryotic hosts such as Escherichia coli or Saccharomyces cerevisiae, respectively. Genetically engineered organisms produce important chemicals, biopolymers, biofuels and high-value proteins like insulin. In those bioprocesses plasmids in recombinant hosts have an essential impact on productivity. Plasmid-free cells lead to losses in the entire product recovery and decrease the profitability of the whole process. Use of antibiotics in industrial fermentations is not an applicable option to maintain plasmid stability. Especially in pharmaceutical or GMP-based fermentation processes, deployed antibiotics must be inactivated and removed. Several plasmid addiction systems (PAS) were described in the literature. However, not every system has reached a full applicable state. This review compares most known addiction systems and is focusing on biotechnical applications.     

蔗糖酶和GOD的共固定化研究

本文研究了用吸附交联技术共固定化蔗糖酶和葡萄糖氧化酶（ＧＯＤ）的方法,考查了共固定化酶的动力学性质。试验结果表明：与溶液酶相比较,固定化蔗糖酶和ＧＯＤ的响应滞迟期分别为３分钟和２分钟,４态响应时间增加６分钟和４分钟,Ｋｍ值增大,ｐＨ─活力曲线变宽,最适ｐＨ值分别增大０．７和０．６４,最适温度则降低７．３℃和１６℃。以活性氧化铝作载体,戊二醛作交联剂制备的共固定化蔗糖酶和ＧＯＤ,其蛋白质固定化率为９２．９％,分解葡萄糖的总速度为４４１．６ＩＵ,当蔗糖浓度在０．２％以内时其反应速度与蔗糖浓度呈正相关（ｒ＝０．９９６）,使用半衰期１６２３次,在４℃下保存１２０天活力残存为８３．７％。     

An enzyme biotechnology for the total utilization of leather wastes

Summary The isolation of protein and fat fractions from a waste of the leather industry is discussed. This represents the main waste of leather production and gives rise to problems of ecological and economical nature. A middle capacity factory deposits about 30 tones of the so called carrion, a source for the production of about 3 t useful products-protein and fat.     

Regulation of enzyme activity in adsorptive enzyme systems

The kinetic behaviour of adsorptive enzyme systems with free and adsorbed enzyme forms in rapid equilibrium has been analysed. It has been shown that the dependences of enzymic reaction rate on substrate or “adsorptive effector” concentrations reveal the deviations from simple kinetic laws of Michaelis-Menten type (positive or negative kinetic co-operativity). Such kinetic anomalies should be observed when adsorption of the enzyme results in the changing catalytic properties and when the state of the equilibrium between free and bound enzyme forms depends on the presence of low molecular substances (substrates, coenzymes and various cellular metabolites). The physiological significance of adsorption-desorption processes for the enzyme activity regulation has been emphasized.