Proteome-Based Analysis of Colloidal Instability Enables the Detection of Haze-Active Proteins in Beer.

Colloidal haze is a serious quality defect of bright beers that considerably reduces their shelf life and is thought to be triggered by hordeins, a class of proline-rich barley proteins. In this work, the proteomes of fresh and old beers were investigated in bottled pilsners and compared to the protein inventory of haze to identify specific haze-active proteins. Haze isolates dissolved in rehydration buffer contained high concentrations of proteins and sugars but provided protein gels with weak spot signals.

GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris.

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin-auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol-based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost.

Fed-batch production and secretion of streptavidin by Hansenula polymorpha: Evaluation of genetic factors and bioprocess development.

Streptavidin - a protein secreted by the filamentous bacterium Streptomyces avidinii - is applied in a variety of methods, leading to numerous studies on its heterologous production. Development and characterization of a novel expression system for streptavidin genes by Hansenula polymorpha is described utilizing different target gene variants along with the two methanol-inducible promoters PMOX and PFMD. Extracellular product concentrations were higher for cultivation at 30 instead of 37°C.

Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli 'leaky mutant'.

Due to its various applications the protein streptavidin is a highly interesting target for heterologous production. This study focuses on different Escherichia coli-based constructs targeting a high-level expression and secretion of streptavidin to the medium. The effect of various promoters, variants of the target gene, leader sequences and host strains on expression and secretion into the culture broth was analyzed.

Applicability of Euglena gracilis for biorefineries demonstrated by the production of α-tocopherol and paramylon followed by anaerobic digestion.

In this study the use of Euglena gracilis biomass for α-tocopherol, paramylon and biogas production in a value-added chain was investigated. Therefore, we analyzed the dry cell weight and product concentrations at different growth phases during heterotrophic, photoheterotrophic and photoautotrophic cultivation in a low-cost minimal medium. Furthermore, the specific biogas yields for differently derived biomass with and without product recovery were investigated.

Antibiotic-free segregational plasmid stabilization in Escherichia coli owing to the knockout of triosephosphate isomerase (tpiA).

Background: Segregational stability of plasmids is of major concern for recombinant bacterial production strains. One of the best strategies to counteract plasmid loss is the use of auxotrophic mutants which are complemented with the lacking gene along with the product-relevant ones. However, these knockout mutants often show unwanted growth in complex standard media or no growth at all under uncomplemented conditions.

Extracellular recombinant protein production under continuous culture conditions with Escherichia coli using an alternative plasmid selection mechanism.

The secretion of recombinant proteins into the extracellular space by Escherichia coli presents advantages like easier purification and protection from proteolytic degradation. The controlled co-expression of a bacteriocin release protein aids in moving periplasmic proteins through the outer membrane. Since such systems have rarely been applied in continuous culture it seemed to be attractive to study the interplay between growth-phase regulated promoters controlling release protein genes and the productivity of a chemostat process.

Development of fed-batch strategies for the production of streptavidin by Streptomyces avidinii based on power input and oxygen supply studies.

Streptavidin is a tetrameric protein with an extremely high affinity to biotin and different biotin-like peptide-tags. This characteristic causes its widespread use in biotechnology. Streptavidin is produced by the fermentation of wild type Streptomyces avidinii or by recombinant Streptomyces lavendulae, Escherichia coli, and Bacillus subtilis strains.

Impact of profiling technologies in the understanding of recombinant protein production.

Since expression profiling methods have been available in a high throughput fashion, the implication of these technologies in the field of biotechnology has increased dramatically. Microarray technology is one such unique and efficient methodology for simultaneous exploration of expression levels of numerous genes. Likewise, two-dimensional gel electrophoresis or multidimensional liquid chromatography coupled with mass spectrometry are extensively utilised for studying expression levels of numerous proteins.

Efficient production of extracellular proteins with Escherichia coli by means of optimized coexpression of bacteriocin release proteins.

Aiming to facilitate the accessibility of recombinant proteins produced with Escherichia coli, extracellular expression may be achieved by means of bacteriocin release protein (BRP) coexpression. Different types of BRPs were tested in order to optimize protein secretion into the culture medium. Those included the well-studied BRPs of the Colicin E1 and Cloacin DF13 bacteriocins and variants thereof.

Screening for conditions of enhanced production of a recombinant beta-glucanase secreted into the medium by Escherichia coli.

The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength.

Generation of chromosomal DNA during alkaline lysis and removal by reverse micellar extraction.

The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of chromosomal from plasmid DNA.

Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein.

Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment.

A machine vision system for automated non-invasive assessment of cell viability via dark field microscopy, wavelet feature selection and classification.

Background: Cell viability is one of the basic properties indicating the physiological state of the cell, thus, it has long been one of the major considerations in biotechnological applications. Conventional methods for extracting information about cell viability usually need reagents to be applied on the targeted cells. These reagent-based techniques are reliable and versatile, however, some of them might be invasive and even toxic to the target cells.

Factors that influence the extracellular expression of streptavidin in Escherichia coli using a bacteriocin release protein.

Aiming to increase production of recombinant streptavidin in Escherichia coli, the effect of different leader sequences, different promoter strengths of the bacteriocin release protein (kil), host strain and medium composition on the expression and secretion into the medium was investigated. Expression vectors containing an expression or secretion unit were constructed with different combinations of leader sequence for the streptavidin gene and promoters for the kil gene and streptavidin gene. Results showed that a high-level extracellular production of streptavidin could be accomplished with E.

Integrated bioprocess for the production and purification of recombinant proteins by affinity chromatography in Escherichia coli.

In order to improve the effectiveness of the production of recombinant proteins in E. coli, integrated fermentation processes were developed. Therefore, expression vectors were constructed containing a strongly expressed gene for a beta-glucanase fused with a metal-chelating affinity tag and a leader peptide for directing the fusion protein into the periplasmic space.

Perceiving molecular evolution processes in Escherichia coli by comprehensive metabolite and gene expression profiling.

Background: Evolutionary changes that are due to different environmental conditions can be examined based on the various molecular aspects that constitute a cell, namely transcript, protein, or metabolite abundance. We analyzed changes in transcript and metabolite abundance in evolved and ancestor strains in three different evolutionary conditions - excess nutrient adaptation, prolonged stationary phase adaptation, and adaptation because of environmental shift - in two different strains of bacterium Escherichia coli K-12 (MG1655 and DH10B).

Results: Metabolite profiling of 84 identified metabolites revealed that most of the metabolites involved in the tricarboxylic acid cycle and nucleotide metabolism were altered in both of the excess nutrient evolved lines.

2DBase: 2D-PAGE database of Escherichia coli.

We present a web-based integrated proteome database, termed 2DBase of Escherichia coli which was designed to store, compare, analyse, and retrieve various information obtained by 2D polyacrylamide gel electrophoresis and mass spectrometry. The main objectives of this database are (1) to provide the features for query and data-mining applications to access the stored proteomics data (2) to efficiently compare the specific protein spots present in the comparable proteome maps and (3) to analyse the data with the integrated classification for cellular functions of gene products of E. coli.

Recycling of bacterial biomass in a process of L-threonine production by means of a recombinant strain of Escherichia coli.

The Gram-negative bacterium Escherichia coli B-3996 represents an interesting host organism for the production of the essential amino acid L-threonine. Microbial processes - especially those of aerobic cultivation - lead to the generation of considerable amounts of biomass, thus lowering the product yield. These are the reasons for studying methods for the recycling of biomass from E.

Reverse micellar extraction systems for the purification of pharmaceutical grade plasmid DNA.

Plasmid DNA as an active pharmaceutical ingredient (API) is gaining more and more importance. For the production of multigram quantities of this substance robust and scalable processes comprising several purification steps have to be designed. One main challenge is the initial separation of plasmid DNA and RNA in such a purification scheme.

Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter.

By using a beta-glucanase from Bacillus as a model protein, we investigated whether the secretion competence based on the action of the kil gene can be improved using stronger promoters for the expression of the kil gene. Since the production of extracellular target proteins also depends on the promoter strengths of the target gene, we constructed four expression vectors with all possible combinations of a weak and a strong stationary-phase promoter for the kil gene, and a weak and a strong constitutive promoter, respectively, for the beta-glucanase gene. The results of batch fermentations showed that the use of stronger promoters generally decreased the cell density.

Improvement of a beta-glucanase activity test by taking into account the batch reactor balance of the test system.

Activity tests of enzymes are often applied for determining their concentration. In the easiest case, just one product concentration is measured after a given time. This often leads to nonlinear dependences of the apparent activity with enzyme protein concentration.

The plasticity of global proteome and genome expression analyzed in closely related W3110 and MG1655 strains of a well-studied model organism, Escherichia coli-K12.

The use of Escherichia coli as a model organism has provided a great deal of basic information in biomolecular sciences. Examining trait differences among closely related strains of the same species addresses a fundamental biological question: how much diversity is there at the single species level? The main aim of our research was to identify significant differences in the activities of groups of genes between two laboratory strains of an organism closely related in genome structure. We demonstrate that despite strict and controlled growth conditions, there is high plasticity in the global proteome and genome expression in two closely related E.

Reagent-free automatic cell viability determination using neural networks based machine vision and dark-field microscopy in Saccharomyces cerevisiae.

Fermentation industries require in-situ real-time monitoring of cell viability during fermentation processes. For this purpose, reagent-free approaches are desired because they can be used for in situ analysis and reduce the system's complexity. We have developed an automatic way of determining cell viability via analysis of time-lapse image sequences taken by dark field microscopy without the aid of any additional reagents.