Roland Ulber

Fluorescence and bioluminescence reporter functions in genetically modified bacterial sensor strains

Genetically modified bacteria, engineered to generate a quantifiable signal in response to pre-determined sets of environmental conditions, may serve as combined sensing/reporting elements in whole-cell biosensors. We have compared two of the several available reporter genes in such cells: green fluorescent proteins (GFPs) (Aquorea victoria gfp) and bioluminescence (Vibrio fischeri luxCDABE) genes, fused to either SOS (recA) or heat shock (grpE) promoters. In both cases, bacterial bioluminescence allowed faster and more sensitive detection of the model toxicants; the fluorescent reporter proteins were much more stable, and following long-term exposure allowed detection at levels similar to that of the bioluminescent sensors. From the two green fluorescent proteins tested, enhanced GFP (EGFP) displayed a more rapid response and higher signal intensity than GFPuv. To combine the advantages of both reporter functions, representatives of both types were jointly encapsulated in a sol‐gel matrix and immobilized onto a glass surface, to generate a bioluminescent toxicity and a fluorescent genotoxicity sensor. The dual-function sensor detected both toxic and genotoxic model compounds with no interference from the co-immobilized member. # 2003 Elsevier Science B.V. All rights reserved.

Future aspects of bioprocess monitoring.

Nature has the impressive ability to efficiently and precisely control biological processes by applying highly evolved principles and using minimal space and relatively simple building blocks. The challenge is to transfer these principles into technically applicable and precise analytical systems that can be used for many applications. This article summarizes some of the new approaches in sensor technology and control strategies for different bioprocesses such as fermentations, biotransformations, and downstream processes. It focuses on bio- and chemosensors, optical sensors, DNA and protein chip technology, software sensors, and modern aspects of data evaluation for improved process monitoring and control.

Bioanalytics: detailed insight into bioprocesses

The principles of bioanalytical systems for an on-line bioprocess monitoring are described within this paper. These sensor systems can be interfaced to the bioprocess in different ways according to the needs of the single bioprocess. Modular systems are necessary, which can fit exactly to the needs of the single process. Invasive as well as non-invasive bioanalytical tools are described and discussed in detail. Immunosensors give the possibility to monitor high molecular weight components within short time intervals. Non-invasive optical sensors allow the direct monitoring of various analytes such as oxygen pH for the complex fluorescence behavior of the bioprocess medium. These so-called fluorescence sensors offer the possibility to monitor intra- as well as extracellular components without interfering with the bioprocess. An industrial example for the application of bioanalytical tools for a process optimization are presented in this application. Here a biosensor system is used to optimize the downstreaming of molasses on a technical scale. The economic as well ecological advantages are discussed.

Challenges in integrating biosensors and FIA for on-line monitoring and control

Several obstacles prevent the increased use of biosensors in industrial bioprocess monitoring and control. Among these are the absence of robust and reliable biosensors with the required specificity and sensitivity for use in complex media, and the absence of adequate sampling techniques. Progress with the integration of amperometric and potentiometric biosensors into flow injection analysis (FIA) systems may provide a route to developing automated, reliable, on-line systems for bioprocess control.

Immobilized Peroxidase as a Valuable Tool in the Remediation of Aromatic Pollutants and Xenobiotic Compounds: A Review

The authors attempted to survey literature based on the role of immobilized peroxidases in the treatment and remediation of various types of organic pollutants present in wastewater. Peroxidases from fungal (Caldariomyces fumago, Lentinula edodes, Phanerochaete chrysosporium, Pleurotus pulmonarius, Aspergillus oryzae, Trametes versicolor, Chrysonilia sitophila [TFB-27441]) and plant sources (horseradish, turnip, tomato, soybean, bitter gourd, white radish chayote, and Saccharum uvarum) were immobilized by using different methods of enzyme immobilization (adsorption, covalent attachment, entrapment and micro-encapsulation). Various types of organic and inorganic supports have been considered for the immobilization of peroxidases. These immobilized peroxidase preparations were employed for the oxidative degradation and removal of organic compounds such as phenols, derivative of phenols, anilines, anthracene, benzidine, dyes, 2,4-dimethyldibenzothiophene, 2-chlorobiphenyl, chlorolignins, 4,6-dimethyldibenzothiophene chlorpromazine, 2,4,6-trinitrotoluene, and hydroxylated aromatic compounds in batch processes as well as in different types of continuous reactors at a large scale.

Innovative Modular Membrane Adsorber System for High-Throughput Downstream Screening for Protein Purification

To develop the most efficient strategy for the purification of proteins, two types of adsorber membrane devices with different functionalities were designed and tested: 8‐strips and single spin columns. The most suitable type of membrane adsorber and the optimal chromatographic loading/elution conditions for several target proteins from different biological matrices could be determined simultaneously in microliter scale. Ion exchange (IEX), metal chelate (MC), and Concanavalin A (Con A) modified membrane types were tested in the devices. Bovine serum albumin (BSA) and lysozyme were used as model proteins for investigations of the binding capacity and protein recovery percentage of the 8‐strip anion exchange and the cation exchange membrane. The isolation of His6‐tagged proteins, Bgl‐His and GFP‐His from fermentation broth and lysate, respectively, was performed using an 8‐strip metal chelate affinity membrane loaded with different metal ions. Separation behavior of a ternary protein mixture (BSA, lysozyme, and Bgl‐His) was studied in 8‐strips IEX and metal chelate membrane chromatography. The Con A affinity devices were developed on the basis of metal chelate membrane spin columns loaded with Cu2+ ions and investigated using glucose oxidase (GOD) as model protein. In summary, the advantages of the membrane adsorber technology, such as fast processing and easy scale‐up, were utilized. The devices made it possible to load the membrane directly with preclarified fermentation broth or cell lysate and separate the protein of interest often in a single step.

Downstream Processing of Bovine Lactoferrin from Sweet Whey

The development of a downstream process for the isolation of bovine lactoferrin (bLF) from sweet whey is presented. Whey is a by-product from the cheese manufacturing process that is often used to produce whey protein concentrate powders for food applications. Besides the major whey proteins such as lactalbumin or BSA. minor whey proteins are present such as lactoperoxidase and bLF. In addition to the well-known biological functions as an antimicrobial and antiviral agent, bLF shows immunomodulatory functions in the host defence system. For the isolation of bLF, a two-step downstream process was developed based on membrane systems. This paper discusses the application of several membrane types for a crossflow filtration of sweet whey to remove insoluble particles and lipids from the whey with the aim of obtaining a permeate which can be directly used for down-streaming the minor component via ion exchange membrane adsorber systems. The application of such a membrane adsorber is demonstrated.

Determination of the minor whey protein bovine lactoferrin in cheese whey concentrates with capillary electrophoresis

In our present work we present the determination of bovine lactoferrin in whey concentrates as they are typically produced by milk and cheese industry after production of cheese. Due to the high total protein content the analysis of whey concentrate samples is difficult and even not possible by using capillary zone electrophoresis with UV detection. To enhance the detection sensitivity we applied a more promising approach by using affinity interactions in combination with laser-induced fluorescence detection. By mixing fluoresceine isothiocyanate (FITC)-conjugated polyanionic lipopolysaccharide with the mostly positively charged lactoferrin we found a significant migration time shift which is clearly dependent on the concentration of the added protein. In the second approach we developed an immunoassay using FITC-conjugated specific antibody against bovine lactoferrin. The results of the immunoassay measurements were compared with data obtained by standard enzyme-linked immunosorbent assay analysis.

Bioaffinity layering: A novel strategy for the immobilization of large quantities of glycoenzymes

A simple strategy for increasing considerably the quantities of glycoenzymes immobilized on insoluble supports is described. The strategy that we call bioaffinity layering makes use of the multivalent nature of concanavalin A (Con A) and the multiple oligosaccharide chains of most glycoenzymes to build alternating lectin and glycoenzyme layers on a Sepharose matrix with precoupled Con A. Using this procedure, it was possible to increase the amounts of several glycoenzymes immobilized on Sepharose and 19.0 mg glucose oxidase could be associated with one ml Sepharose matrix after seven Con A/glucose oxidase incubation cycles. Bioaffinity layered preparations of glycoenzymes exhibited high activities as indicated by very high effectiveness factor (eta) values and those of glucose oxidase and invertase exhibited a layer-by-layer increase in thermostability. The sensitivity of a flow-through glucose monitoring cartridge integrated into a flow injection analysis (FIA) system was enhanced significantly by increasing the amount of immobilized glucose oxidase via bioaffinity layering. A cartridge bearing six layers of glucose oxidase on Sepharose support was used effectively and repeatedly for analysis of medium glucose concentration during a fed-batch cultivation of the yeast Saccharomyces cerevisiae.

Production of l-lysine on different silage juices using genetically engineered Corynebacterium glutamicum

Corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. The resulting strain C. glutamicum lysC(fbr)dld(Psod)pyc(Psod)malE(Psod)fbp(Psod)gapX(Psod) was based on earlier work (Neuner and Heinzle, 2011). That mutant carries a point mutation in the aspartokinase (lysC) regulatory subunit gene as well as overexpression of D-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and malic enzyme (malE) using the strong Psod promoter. Here, we additionally overexpressed fructose 1,6-bisphosphatase (fbp) and glyceraldehyde 3-phosphate dehydrogenase (gapX) using the same promoter. The resulting strain grew readily on grass and corn silages with a specific growth rate of 0.35 h⁻¹ and lysine carbon yields of approximately 90 C-mmol (C-mol)⁻¹. Lysine yields were hardly affected by oxygen limitation whereas linear growth was observed under oxygen limiting conditions. Overall, this strain seems very robust with respect to the composition of silage utilizing all quantified low molecular weight substrates, e.g. lactate, glucose, fructose, maltose, quinate, fumarate, glutamate, leucine, isoleucine and alanine.