CellViCAM--Cell viability classification for animal cell cultures using dark field micrographs.

Online monitoring of cell density and cell viability is a challenging but essential task to control and optimize biotechnical processes and is of particular interest for the growing field of animal cell cultures. For this purpose, we introduce an optical approach for automated cell detection and viability classification of suspended mammalian cells. Our proposed system CellViCAM is capable of evaluating dark field micrographs by means of several image processing and supervised machine learning techniques without the use of any dyes or fluorescent labeling. Using a human cell line as the reference culture, an efficient cell detection procedure has been established also enabling a cell density estimation. Furthermore, a comprehensive but reagent-free viability analysis, based on a semi-automatic training data generation, has been developed. By means of an extensive validation dataset we can show that the CellViCAM approach can be considered as an equivalent to staining-based methods and moreover, how it provides a technical platform for a more differentiated cell state classification into living, necrotic, early and late apoptosis.

A rapid reporter system using GFP as a reporter protein for identification and screening of synthetic stationary-phase promoters in Escherichia coli.

To develop a rapid reporter system for the screening of stationary-phase promoters in Escherichia coli, the expression pattern of the green fluorescent protein (GFP) during bacterial cultivation was compared with that of the commonly used beta-galactosidase. Using GFP with enhanced fluorescence, the expression pattern of both reporter systems GFP and beta-galactosidase were similar and showed a typical induction of gene activity of the reporter genes, i.e. increase of expression at the transition from exponential to stationary phase. The expression was affected by the culture medium, i.e. in contrast to the complex medium (LB medium), the stationary-phase specific induction was only observed in synthetic medium (M9) when amino acids were added, whereas there was generally no induction in MOPS medium. To develop a rapid screening method on agar plates for stationary-phase promoters, a photographic approach was used, continued with computational image treatment. A screening method is presented which enables an on-line monitoring of gene activity.

Analysis of the behaviour of Dictyostelium discoideum in immobilised state by means of continuous cultivation.

The social amoeba Dictyostelium discoideum is amenable to cultivation in the immobilised state most simply by colonisation of porous supports. An analysis of the growth behaviour of D. discoideum in the immobilised state is reported. For this purpose, D. discoideum was cultivated in continuously operated reactors in a suspension culture (homogeneous system) and immobilised on a porous support (heterogeneous system). Thus, it is possible to compare homogeneous and heterogeneous systems under steady-state conditions. Immobilisation was achieved by the colonisation of porous glass beads (SIRAN). Simple models are applied in order to describe the growth behaviour of fractions of both the cells in free suspension and the cells inside the porous carrier. This analysis shows that D. discoideum inside the pores grows at a rate of only about 10% compared with that in free solution. The consequence of this behaviour is discussed in terms of reactor performance.

Optimization of the extracellular production of a bacterial phytase with Escherichia coli by using different fed-batch fermentation strategies.

The extracellular production of Escherichia coli phytase was studied in fed-batch fermentations. Two different feeding strategies were compared: control by keeping the glucose concentration constant, and control by keeping a low constant oxygen level in the medium. For the feeding control based on glucose concentration, a recently developed rapid glucose controlling system was tested for the first time in bacterial cultivations and used to establish the fermentative production of extracellular phytase with E. coli. High activity levels (120 U ml(-1)) at short cultivation times (14 h) were obtained. Even higher activity levels - albeit at longer cultivation times - were reached by applying a feeding control, the main characteristic of which was a constant low oxygen concentration. The optimum oxygen level for the production of phytase was in the range of 5-10% saturation.

Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors.

The gene for phytase from Escherichia coli was sequenced and compared with the appA gene. It was found to be a mutant derivative of the appA gene. After fusion with a C-terminal His-tag, phytase was purified by affinity chromatography and the enzymatic properties were analyzed. To develop a system for overexpression and extracellular production of phytase in E. coli, factors affecting the expression and secretion such as promoter type, host strain and selection pressure were analyzed. Using a secretion system based on the controlled expression of the kil gene, the expression of phytase was improved and the enzyme was released into the culture medium at a high level. An effective fermentation strategy based on fed-batch operation was developed.

Xanthomonas campestris pv. campestris secretes the endoglucanases ENGXCA and ENGXCB: construction of an endoglucanase-deficient mutant for industrial xanthan production.

Xanthomonas campestris pv. campestris secretes at least two cellulose-degrading endoglucanases. One of these endoglucanases is encoded by the engXCA gene of X. c. pv. campestris 8400 that was previously characterized by Gough et al. [Gene (1990) 89: 53-59]. An additional endoglucanase encoded by the engXCB gene was identified in X. c. pv. campestris 8400 and FC2. The engXCB gene product that was grouped into the endoglucanase family E contains a putative N-terminal signal peptide, suggesting a secretion by the type II secretion system. The ENGXCB protein contributed approximately 8% to the cellulase activity in xanthan preparations. Deletion of engXCA and engXCB resulted in a fivefold reduction of the cellulose-degrading activity in xanthan preparations. The cellulase activity determined in xanthan preparations of the engXCA-engXCB mutant was only slightly higher than the activity found in preparations that were subjected to heat treatment. Mutations in engXCA and engXCB did not affect the growth rate and xanthan production of X. c. pv. campestris FC2 under several cultivation conditions. The engXCA-engXCB deletion mutant is markerless, which makes this mutant a valuable strain for xanthan production and approaches aimed at inactivating further genes encoding extracellular enzymes.

Plasmid DNA for pharmaceutical applications.

With the advent and progress of recombinant DNA technology into a variety of fields such as medical therapy, preventive or curative vaccination or the induction of regeneration, the demand for large quantities of highly purified DNA is increasing. Traditional methods of purifying plasmids usually require sophisticated methodology if the DNA is to be separated from RNA and other contaminating organic components. In particular, methods for obtaining supercoiled covalently closed circular (CCC) plasmid DNA in pure form, cope with the requirement that other plasmid topologies also produced have to be separated from the final product. The innovative technology of capillary gel electrophoresis (CGE) contributes a sensitive tool to the short list of applicable quality control assays for clinical grade plasmid DNA.

Alfalfa and tobacco cells react differently to chitin oligosaccharides and sinorhizobium meliloti nodulation factors

Alfalfa (Medicago sativa L.) suspension cultures respond to yeast elicitors with a strong alkalinization of the culture medium, a transient synthesis of activated oxygen species, and typical late defence reactions such as phytoalexin accumulation and increased peroxidase activity. The alkalinization reaction as well as the oxidative burst were also observed when tobacco (Nicotiana tabacum L. ) cell-suspension cultures were treated with yeast elicitors. Depending on the degree of polymerization, N-acetyl chitin oligomers induced the alkalinization response in both plant cell-suspension cultures, while only tobacco cell cultures developed an oxidative burst. Suspension-cultured tobacco cells responded to Sinorhizobium meliloti nodulation factors with a maximal alkalinization of 0.25 pH units and a remarkable oxidative burst. In contrast, addition of Sinorhizobium meliloti nodulation factors to suspension-cultured alfalfa cells induced a slight acidification of the culture medium, instead of an alkalinization, but no oxidative burst.

Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis.

Plasmids may appear in different forms: circular with different degrees of coiling, partially cleaved or linear, and multimeric as concatamers or catenates. Capillary gel electrophoresis (CGE) of plasmid samples allows the determination of plasmid form distribution. Monomeric and dimeric plasmid DNA forms were separated by both CGE and agarose gel electrophoresis (AGE). The pattern of isoform bands from AGE was compared to the corresponding peak pattern from CGE, and differences in the relative mobility of the plasmid forms between the two methods were found. The comparison of AGE and CGE allows the assignment of AGE bands to CGE peaks. Additionally, the different isoforms can now be quantified by CGE. Routine plasmid form analysis by CGE may be automated, allowing easy, fast, and highly reliable quantification. CGE also offers high resolution and the amount of DNA required is very low. Therefore this method is very useful for the analysis of therapeutics based on plasmid DNA during their production, isolation, and formulation.

High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium.

The Tn5-based transposon Tn5-KIL3 (Miksch et al. 1997c) bearing the kil gene of the ColE1 plasmid of Escherichia coli, which mediates controlled export of periplasmic proteins into the culture medium, was stably integrated into the chromosome of Klebsiella planticola with high transposition frequency. A Bacillus hybrid beta-glucanase located on an RSF1010-derived plasmid was mobilized from E. coli to K. planticola and used as a reporter protein to select strains with high expression and secretion competence. During fermentation experiments it was shown that the production of beta-glucanase in K. planticola was improved to an unexpectedly high level when the enzyme was secreted into the medium. Due to the stationary-phase promoter used for the expression of the kil gene the secretion of beta-glucanase into the medium started at the transition from the exponential to the stationary phase, as in E. coli, and the fraction of secreted protein reached 90%. The results showed that K. planticola may represent an interesting organism for the production of heterologous proteins.

Controlled secretion into the culture medium of a hybrid beta-glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5-derived transposon.

A Tn5-based transposon bearing the kil gene (killing protein), mediating controlled export of periplasmic proteins into the culture medium, was constructed (Tn5-KIL3). This transposon contained the kil gene of the ColEl plasmid under the growth-phase-dependent promoter of the fic gene (filamentation induced by cAMP) of Escherichia coli, an interposon located upstream of kil, a kanamycin/neomycin-resistance gene, a multiple cloning site and the mob site. The transposition of Tn5-KIL3 to Acetobacter methanolicus showed a moderate transposition frequency (10(-5) -10(-6). By insertion of a Bacillus hybrid beta-glucanase (bgl) as a model protein into the transposon (Tn5-LF3) it was shown that the secretion function as well as the gene of the target protein had been transferred to and stably integrated into the chromosome of A. methanolicus, and that the transposition of Tn5-LF3 was non-specific. beta-Glucanase was highly overexpressed and secreted into the medium during stationary phase. Total and extra-cellular production of beta-glucanase varied depending on the integration site of the transposon. The viability of the bacterial cells was not affected, and cell lysis did not occur.

Extracellular production of a hybrid beta-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors.

Cultivation conditions for the extracellular production of a hybrid beta-glucanase from Bacillus were established by using Escherichia coli JM 109 carrying the plasmid pLF3. This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid beta-glucanase gene of Bacillus. When controlled by the fic promoter, the kil gene led to a higher total production of beta-glucanase and a higher protein secretion than when it was under control of the bolA promoter. When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion. With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed. In defined media the secretion of beta-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation. The strain with the highest production and secretion yield of beta-glucanase [E. coli JM 109(pLF3)] was tested on the fermenter scale.

The green fluorescent protein is a versatile reporter for bioprocess monitoring.

The green fluorescent protein (GFP) of Aequorea victoria has become a convenient and versatile tool as a reporter protein in molecular cell biology and developmental biology. Here, it is shown that GFP may advantageously be used as a reporter system for bioprocess monitoring as well. Examples are given for monitoring fermentation as well as downstream processes for protein recovery. Thus, separation processes based on the application of affinity-fusion tags may be optimized in terms of the operational conditions by using GFP as a model target protein owing to facile screening by simple visual inspection. This item is discussed together with the presentation of a novel fusion tag with strong affinity for metal-chelate ligands: hisactophilin, a histidine-rich protein of Dictyostelium discoideum. This tag is of particular interest for affinity separation processes requiring multiple sites of interaction like aqueous and reverse micellar two-phase extraction as well as precipitation.

Production of nodulation factors by Rhizobium meliloti: fermentation, purification and characterization of glycolipids.

Lipooligosaccharides, synthesized by soil bacteria of the genera Rhizobium, are known to have multifunctional effects on a wide variety of plants as signal substances in symbiosis initiation, cell response elicitation and growth regulation. These so called nodulation (Nod-) factors represent interesting biotechnological products with respect to fundamental studies of symbiotic interactions as well as for potential applications. Therefore, a batch fermentation process on a scale of 30 l has been developed by means of the Rhizobium meliloti strain R.m. 1021 (pEK327) strongly overexpressing the genes for the synthesis of Nod factors. Induction by the flavone luteolin led to growth associated production of the lipooligosaccharides. Ultrafiltration was used for separating the biomass from the filtrate containing the extracellular Nod factors. Simultaneously, ultrafiltration reduced the amount of lipophilic substances, which would otherwise interfere with processes downstream. The second separation step consisted in adsorption on XAD-2, a nonspecific hydrophobic adsorptive resin. Adsorption of Nod factors was carried out by batch operation of a stirred tank. Desorption was performed by elution with methanol in a fixed bed column. A semi-preparative reversed phase HPLC (Polygoprep 100-30 C18) was chosen as the final purification step. The Nod factors were obtained after evaporation and lyophilization. Thus, about 600 mg of Nod factors were produced from 20 l of fermentation broth. The Nod factors produced by Rhizobium meliloti R.m. 1021 (pEK327) were identified by liquid secondary ion mass spectrometry and by reversed-phase HPLC as fluorescent derivatives of 2-aminobenzamide. The biological activity of the products was demonstrated by means of the root hair deformation (HAD-) assay.

Rapid determination of plasmid copy number.

Plasmid copy number, the number of expression vectors per host cell, is a key variable in recombinant microbial cultivation. Therefore, it would be very helpful, if the plasmid copy number could be determined during the operating process period. A rapid quantification of this important process variable would even open the possibility of its use in process control. However current assays like gel electrophoresis, CsCl-gradient centrifugation, HPLC and other methods are time consuming and difficult to quantify. Indirect methods, like the correlation of copy number with, e.g. the activity of an enzyme, coded on the plasmid, are prone to errors due to the production kinetics, turnover rate and protein denaturation. Here, a method is presented, which enables the plasmid copy number to be determined in less than 30 min. This novel procedure based on plasmid isolation by means of a commercial DNA-isolation kit and quantification by capillary electrophoresis, should allow the copy number to be used in process control.

Improvement of downstream processing of recombinant proteins by means of genetic engineering methods.

The rapid advancement of genetic engineering has allowed to produce an impressive number of proteins on a scale which would not have been achieved by classical biotechnology. At the beginning of this development research was focussed on elucidating the mechanisms of protein overexpression. The appearance of inclusion bodies may illustrate the success. In the meantime, genetic engineering is not only expected to achieve overexpression, but to improve the whole process of protein production. For downstream processing of recombinant proteins, the synthesis of fusion proteins is of primary importance. Fusion with certain proteins or peptides may protect the target protein from proteolytic degradation and may alter its solubility. Intracellular proteins may be translocated by means of fusions with signal peptides. Affinity tags as fusion complements may render protein separation and purification highly selective. These methods as well as similar ones for improving the downstream processing of proteins will be discussed on the basis of recent literature.

Transesterification of phenylalanine by means of chymotrypsin in a continuous fixed bed reactor.

An enzymic transesterification was carried out in a continuously operated fixed bed reactor. The reaction system consisted of immobilized alpha-chymotrypsin (E.C. 3.4.21.1) catalysing the transfer of the L-phenylalanine radical from the racemic propyl ester to 1,4-butanediol, yielding L-phenylalanine 4-hydroxybutyl ester. The desired reaction was accompanied by alcoholysis due to the presence of 1-propanol liberated during the reaction and by hydrolysis of both the propyl and the hydroxybutyl ester. The problem of shifting pH during the reaction due to ester hydrolysis was overcome by adjusting the initial pH of the substrate feed solution appropriately in order to obtain a sufficiently high buffer capacity provided by the free amino group of the esters. Thus, it was possible to work with shifting pH, an obvious disadvantage for operating reactors of low backmixing for this kind of reaction system. The overall reaction scheme was characterized by the appearance of a maximum ester yield as a function of the operating time in case of batch reactors. Surprisingly, the yield was found to become constant as a function of space-time for continuous operation due to a steeper pH drop. The maximum productivity achieved with respect to the hydroxybutyl ester was about 65 mol d-1 l-1 referred to the catalyst volume.

The production of alpha-cyclodextrin by enzymatic degradation of starch.

The maximum concentration of alpha-cyclodextrin for the enzymatic degradation of starch is limited to about 13.5 g X 1(-1). By addition of decanol, the equilibrium of the reaction system can be shifted towards an alpha-cyclodextrin yield of 50% even at high substrate concentrations. The main variables of the decanol process--pH, temperature, substrate quality, substrate, and enzyme concentration--have been studied. The cyclodextrin-glycosyltransferase from Klebsiella pneumoniae M5 al can preferentially be employed at pH 6 to 8, temperatures of 40 to 50 degrees C and a decanol concentration of 0.1 kg-1 starch. The dextrose equivalent of starch is important with respect to the maximum achievable starch concentration, but not with respect to the reaction. Under process conditions, the rate of alpha-cyclodextrin evolution is limited by the enzymatic reaction and not by mass transfer of decanol into the aqueous phase.

The kinetic of lactose hydrolysis for the beta-galactosidase from Aspergillus niger.

The kinetics of glucose liberation from lactose by means of the beta-glactosidase from Aspergillus niger has been studied in a wide range of the main variables. The analysis shows that the kinetic models proposed so far are not adequate. The main finding is that the reaction rate is not linearly correlated to the enzyme concentration-it increase more than proportionally. This nonlinear relationship results because this lactase can distinguish between alpha-and beta-galactose alpha-Galactose acts as competitive and anticompetitive inhibitor while beta-galactose is a competitive one. The competitive inhibition of the alpha-anomer is approximately 12 times more sever than that of the beta-anomer. The kinetics, including a simplified model for the mutarotation of galactose is given for a temperature of 50 degrees C at a pH of 3.5-the most likely conditions for the application of this lactase in acid whey treatment.