Regiocomplementary O-Methylation of Catechols by Using Three-Enzyme Cascades.

S-Adenosylmethionine (SAM)-dependent enzymes have great potential for selective alkylation processes. In this study we investigated the regiocomplementary O-methylation of catechols. Enzymatic methylation is often hampered by the need for a stoichiometric supply of SAM and the inhibitory effect of the SAM-derived byproduct on most methyltransferases. To counteract these issues we set up an enzyme cascade. Firstly, SAM was generated from l-methionine and ATP by use of an archaeal methionine adenosyltransferase. Secondly, 4-O-methylation of the substrates dopamine and dihydrocaffeic acid was achieved by use of SafC from the saframycin biosynthesis pathway in 40-70 % yield and high selectivity. The regiocomplementary 3-O-methylation was catalysed by catechol O-methyltransferase from rat. Thirdly, the beneficial influence of a nucleosidase on the overall conversion was demonstrated. The results of this study are important milestones on the pathway to catalytic SAM-dependent alkylation processes.

TEMPO-Oxidized Nanofibrillated Cellulose as a High Density Carrier for Bioactive Molecules.

Controlled and efficient immobilization of specific biomolecules is a key technology to introduce new, favorable functions to materials suitable for biomedical applications. Here, we describe an innovative and efficient, two-step methodology for the stable immobilization of various biomolecules, including small peptides and enzymes onto TEMPO oxidized nanofibrillated cellulose (TO-NFC). The introduction of carboxylate groups to NFC by TEMPO oxidation provided a high surface density of negative charges able to drive the adsorption of biomolecules and take part in covalent cross-linking reactions with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) and glutaraldehyde (Ga) chemistry. Up to 0.27 µmol of different biomolecules per mg of TO-NFC could be reversibly immobilized by electrostatic interaction. An additional chemical cross-linking step prevented desorption of more than 80% of these molecules. Using the cysteine-protease papain as model, a highly active papain-TO-NFC conjugate was achieved. Once papain was immobilized, 40% of the initial enzymatic activity was retained, with an increase in kcat from 213 to >700 s(-1) for the covalently immobilized enzymes. The methodology presented in this work expands the range of application for TO-NFC in the biomedical field by enabling well-defined hybrid biomaterials with a high density of functionalization.

In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation.

BACKGROUND: Glycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from Campylobacter jejuni to the standard prokaryotic host Escherichia coli established a novel bioconjugation methodology termed bacterial glycoengineering. RESULTS: In this study, we report on the production of a new recombinant glycoconjugate vaccine against Shigella flexneri 2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that S. flexneri 2a O-polysaccharides can be transferred to a detoxified variant of Pseudomonas aeruginosa carrier protein exotoxin A (EPA) by the C. jejuni oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the in vivo production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg2+ ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production. CONCLUSION: The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli. This study is an important step towards this goal and provides a starting point for further optimization studies.

Cloning, expression and biochemical characterization of the cholesterol oxidase CgChoA from Chryseobacterium gleum.

BACKGROUND: Cholesterol oxidases are important enzymes for applications such as the analysis of cholesterol in clinical samples, the synthesis of steroid derived drugs, and are considered as potential antibacterial drug targets. RESULTS: The gene choA encoding a cholesterol oxidase from Chryseobacterium gleum DSM 16776 was cloned into the pQE-30 expression vector and heterologously expressed in Escherichia coli JM109 co-transformed with pRARE2. The N-terminally His-tagged cholesterol oxidase (CgChoA) was assigned to be a monomer in solution by size exclusion chromatography, showed a temperature optimum of 35°C, and a pH optimum at 6.75 using 0.011 M MOPS buffer under the tested conditions. The purified protein showed a maximum activity of 15.5 U/mg. CgChoA showed a Michaelis-Menten like kinetic behavior only when the substrate was dissolved in water and taurocholate (apparent K(m) = 0.5 mM). In addition, the conversion of cholesterol by CgChoA was studied via biocatalytic batches at analytical scale, and cholest-4-en-3-one was confirmed as product by HPLC-MS. CONCLUSION: CgChoA is a true cholesterol oxidase which activity ranges among the high performing described cholesterol oxidases from other organisms. Thus, the enzyme broadens the available toolbox of cholesterol oxidases for e.g. synthetic and biosensing applications.

Continuous monitoring of enzymatic reactions on surfaces by real-time flow cytometry: sortase a catalyzed protein immobilization as a case study.

Only a few techniques, such as quartz crystal microbalance and surface plasmon resonance spectroscopy, enable the analysis of dynamic processes on solid supports. Here we have developed a straightforward assay based on flow cytometry to continuously follow enzymatic reactions directly on microparticle surfaces. We applied this real-time flow cytometry (RT-FCM) approach to study the covalent immobilization of green-fluorescent protein (GFPuv) on triglycine-modified polystyrene microbeads by the transpeptidase sortase A (SrtA) from Staphylococcus aureus. Though commonly treated as functionally identical catalysts, the SrtA variants SrtAΔ59 and SrtAΔ25, in which the N-terminal amino acid residues 1-59 and 1-25 of the native enzyme are truncated, were shown to perform very differently with regard to this particular immobilization reaction. While SrtAΔ59 efficiently catalyzed the covalent attachment of GFPuv to the surface (as indicated by a linear increase of microbead fluorescence), SrtAΔ25 was essentially inactive. Besides the length of the N-terminal amino acid extension on the SrtA construct, the position of the hexahistidine tag at either the N- or C-terminus affected the efficiency of enzymatic protein immobilization. Apart from three enzyme variants containing the native core structure of SrtA, we also included three recently evolved mutants of SrtA in this comparative study. With these mutants we observed a rapid initial attachment of the GFPuv target protein to the microbeads. However, with proceeding reaction time, cleavage of the covalently immobilized target protein from the surface prevailed over the coupling reaction, consequently causing a decline of microbead fluorescence. In general, the RT-FCM approach used herein represents a powerful analytical tool for qualitative dynamic studies of many heterogeneous enzymatic reactions or other binding events that influence the fluorescence properties of microparticle surfaces.

Improved productivity of poly (4-hydroxybutyrate) (P4HB) in recombinant Escherichia coli using glycerol as the growth substrate with fed-batch culture.

BACKGROUND: The most successful polyhydroxyalkanoate (PHA) in medical applications is poly(4-hydroxybutyrate) (P4HB), which is due to its biodegradability, biocompatibility and mechanical properties. One of the major obstacles for wider applications of P4HB is the cost of production and purification. It is highly desired to obtain P4HB in large scale at a competitive cost. RESULTS: In this work, we studied the possibility to increase P4HB productivity by using high cell density culture. To do so, we investigated for the first time some of the most relevant factors influencing P4HB biosynthesis in recombinant Escherichia coli. We observed that P4HB biosynthesis correlated more with limitations of amino acids and less with nitrogen depletion, contrary to the synthesis of many other types of PHAs. Furthermore, it was found that using glycerol as the primary carbon source, addition of acetic acid at the beginning of a batch culture stimulated P4HB accumulation in E. coli. Fed-batch high cell density cultures were performed to reach high P4HB productivity using glycerol as the sole carbon source for cell growth and 4HB as the precursor for P4HB synthesis. A P4HB yield of 15 g L-1 was obtained using an exponential feeding mode, leading to a productivity of 0.207 g L-1 h-1, which is the highest productivity for P4HB reported so far. CONCLUSIONS: We demonstrated that the NZ-amines (amino acids source) in excess abolished P4HB accumulation, suggesting that limitation in certain amino acid pools promotes P4HB synthesis. Furthermore, the enhanced P4HB yield could be achieved by both the effective growth of E. coli JM109 (pKSSE5.3) on glycerol and the stimulated P4HB synthesis via exogenous addition of acetic acid. We have developed fermentation strategies for P4HB production by using glycerol, leading to a productivity of 0.207 g L-1 h-1 P4HB. This high P4HB productivity will decrease the total production cost, allowing further development of P4HB applications.

High level production of tyrosinase in recombinant Escherichia coli.

BACKGROUND: Tyrosinase is a bifunctional enzyme that catalyzes both the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the subsequent oxidation of the diphenols to o-quinones (diphenolase activity). Due to the potential applications of tyrosinase in biotechnology, in particular in biocatalysis and for biosensors, it is desirable to develop a suitable low-cost process for efficient production of this enzyme. So far, the best production yield reported for tyrosinase was about 1 g L(-1), which was achieved by cultivating the filamentous fungus Trichoderma reesei for 6 days. RESULTS: In this work, tyrosinase from Verrucomicrobium spinosum was expressed in Escherichia coli and its production was studied in both batch and fed-batch cultivations. Effects of various key cultivation parameters on tyrosinase production were first examined in batch cultures to identify optimal conditions. It was found that a culture temperature of 32 °C and induction at the late growth stage were favorable, leading to a highest tyrosinase activity of 0.76 U mL(-1). The fed-batch process was performed by using an exponential feeding strategy to achieve high cell density. With the fed-batch process, a final biomass concentration of 37 g L(-1) (based on optical density) and a tyrosinase activity of 13 U mL(-1) were obtained in 28 hours, leading to a yield of active tyrosinase of about 3 g L(-1). The highest overall volumetric productivity of 103 mg of active tyrosinase per liter and hour (corresponding to 464 mU L(-1) h(-1)) was determined, which is approximately 15 times higher than that obtained in batch cultures. CONCLUSIONS: We have successfully expressed and produced gram quantities per liter of active tyrosinase in recombinant E. coli by optimizing the expression conditions and fed-batch cultivation strategy. Exponential feed of substrate helped to prolong the exponential phase of growth, to reduce the fermentation time and thus the cost. A specific tyrosinase production rate of 103 mg L(-1) h(-1) and a maximum volumetric activity of 464 mU L(-1) h(-1) were achieved in this study. These levels have not been reported previously.

Poly(4-hydroxybutyrate) (P4HB) production in recombinant Escherichia coli: P4HB synthesis is uncoupled with cell growth.

BACKGROUND: Poly(4-hydroxybutyrate) (P4HB), belonging to the family of bacterial polyhydroxyalkanoates (PHAs), is a strong, flexible and absorbable material which has a large variety of medical applications like tissue engineering and drug delivery. For efficient production of P4HB recombinant Escherichia coli has been employed. It was previously found that the P4HB synthesis is co-related with the cell growth. In this study, we aimed to investigate the physiology of P4HB synthesis, and to reduce the total production cost by using cheap and widely available xylose as the growth substrate and sodium 4-hydroxybutyrate (Na-4HB) as the precursor for P4HB synthesis. RESULTS: Six different E. coli strains which are able to utilize xylose as carbon source were compared for their ability to accumulate P4HB. E. coli JM109 was found to be the best strain regarding the specific growth rate and the P4HB content. The effect of growth conditions such as temperature and physiological stage of Na-4HB addition on P4HB synthesis was also studied in E. coli JM109 recombinant in batch culture. Under the tested conditions, a cellular P4HB content in the range of 58 to 70% (w w(-1)) and P4HB concentrations in the range of 2.76 to 4.33 g L(-1) were obtained with a conversion yield (Y(P4HB/Na-4HB)) of 92% w w(-1) in single stage batch cultures. Interestingly, three phases were identified during P4HB production: the "growth phase", in which the cells grew exponentially, the "accumulation phase", in which the exponential cell growth stopped while P4HB was accumulated exponentially, and the "stagnation phase", in which the P4HB accumulation stopped and the total biomass remained constant. CONCLUSIONS: P4HB synthesis was found to be separated from the cell growth, i.e. P4HB synthesis mainly took place after the end of the exponential cell growth. High conversion rate and P4HB contents from xylose and precursor were achieved here by simple batch culture, which was only possible previously through fed-batch high cell density cultures with glucose.

Enzyme-catalyzed protein crosslinking.

The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different applications.

Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440.

BACKGROUND: Pseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain. RESULTS: The genes encoding xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h(-1) and a final cell dry weight (CDW) of 2.5 g L(-1) with a maximal yield of 0.5 g CDW g(-1) xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w(-1) of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under the employed conditions. CONCLUSIONS: Sequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.

Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB.

BACKGROUND: Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. RESULTS: In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette saturation mutagenesis. With the exception of I571C only hydrophobic residues were found in active variants. Variant I571V performed equally well as the wild type, cysteine at the same position reduced glycoprotein yield slightly, while a change to phenylalanine reduced activity by a factor of three. CONCLUSIONS: This study provides novel structure-function relationships for the periplasmic domain of the Campylobacter jejuni N-oligosaccharyltransferase PglB and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered E. coli system.

Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum.

BACKGROUND: Laccases are multi-copper oxidases that catalyze the one electron oxidation of a broad range of compounds. Laccase substrates include substituted phenols, arylamines and aromatic thiols. Such compounds are activated by the enzyme to the corresponding radicals. Owing to their broad substrate range laccases are considered to be versatile biocatalysts which are capable of oxidizing natural and non-natural industrial compounds, with water as sole by-product. RESULTS: A novel CotA-type laccase from Bacillus pumilus was cloned, expressed and purified and its biochemical characteristics are presented here. The molecular weight of the purified laccase was estimated to be 58 kDa and the enzyme was found to be associated with four copper atoms. Its catalytic activity towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine (SGZ) was investigated. The kinetic parameters KM and kcat for ABTS were 80 ± 4 µM and 291 ± 2.7 s(-1), for 2,6-DMP 680 ± 27 µM and 11 ± 0.1 s(-1) and for SGZ only kcat could be estimated to be 66 ± 1.5 s(-1). The pH optimum for ABTS was 4, for 2,6-DMP 7 and for SGZ 6.5 and temperature optima for ABTS and 2,6-DMP were found to be around 70°C. The screening of 37 natural and non-natural compounds as substrates for B. pumilus laccase revealed 18 suitable compounds. Three of them served as redox mediators in the laccase-catalyzed decolorization of the dye indigocarmine (IC), thus assessing the new enzyme's biotechnological potential. CONCLUSIONS: The fully copper loaded, thermostable CotA laccase from Bacillus pumilus is a versatile laccase with potential applications as an industrial biocatalyst.

Heme ladder, a direct molecular weight marker for immunoblot analysis.

Detection methods for immunoblot analysis are often based on peroxidase conjugates. However, molecular weight markers directly detectable for general use in such systems are not available. Here, we describe the preparation of a direct molecular weight marker consisting of heme-tagged proteins, whose enzymatic activities make them detectable simultaneously with the antigen in peroxidase-based immunoblot systems. The peroxidase activity results from the covalent attachment of heme to selected engineered periplasmic proteins, catalyzed by the cytochrome c maturation system of Escherichia coli. The newly designed heme-tagged proteins were combined with a previously constructed heme-tagged maltose-binding protein and cytochrome c. The resulting heme ladder was shown to be suitable as a protein standard for direct molecular weight estimation in immunoblot analysis due to the peroxidase activity of its constituents. The heme ladder consists of proteins between 12 and 85 kDa and can be produced at low cost. The marker was stable when kept at 4, -20, and -80°C for >6 months.

Haem-delivery proteins in cytochrome c maturation System II.

Cytochromes of the c-type function on the outer side of the cytoplasmic membrane in bacteria where they also are assembled from apo-cytochrome polypeptide and haem. Two distinctly different systems for cytochrome c maturation are found in bacteria. System I present in Escherichia coli has eight to nine different Ccm proteins. System II is found in Bacillus subtilis and comprises four proteins: CcdA, ResA, ResB and ResC. ResB and ResC are poorly understood polytopic membrane proteins required for cytochrome c synthesis. We have analysed these two B. subtilis proteins produced in E. coli and in the native organism. ResB is shown to bind protohaem IX and haem is found covalently bound to residue Cys-138. Results in B. subtilis suggest that also ResC can bind haem. Our results complement recent findings made with Helicobacter CcsBA supporting the hypothesis that ResBC as a complex translocates haem by attaching it to ResB on the cytoplasmic side of the membrane and then transferring it to an extra-cytoplasmic location in ResC, from where it is made available to the apo-cytochromes.

Influence of growth stage on activities of polyhydroxyalkanoate (PHA) polymerase and PHA depolymerase in Pseudomonas putida U.

BACKGROUND: Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the ß-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary. RESULTS: Two in vitro assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of Pseudomonas putida U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further. CONCLUSION: Using the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of PhaC.

Production of glycoprotein vaccines in Escherichia coli.

BACKGROUND: Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries. RESULTS: Two different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold. CONCLUSIONS: The presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.

Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives.

The growing awareness of the importance of chirality in conjunction with biological activity has led to an increasing demand for efficient methods for the industrial synthesis of enantiomerically pure compounds. Polyhydroxyalkanotes (PHAs) are a family of polyesters consisting of over 140 chiral R-hydroxycarboxylic acids (R-HAs), representing a promising source for obtaining chiral chemicals from renewable carbon sources. Although some R-HAs have been produced for some time and certain knowledge of the production processes has been gained, large-scale production has not yet been possible. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in biotechnological processes for R-HA production.

Application of activated charcoal in the downstream processing of bacterial olefinic poly(3-hydroxyalkanoates).

Medium chain length poly(hydroxyalkanoates) (mcl-PHAs) are bacterial thermoplastic elastomers with a large potential in medical applications. The present study provides a novel process to isolate and purify poly([R]-3-hydroxy-omega-undecenoate-co-3-hydroxy-omega-nonenoate-co-3-hydroxy-omega-heptenoate) (PHUE) and poly([R]-3-hydroxy-omega-undecenoate-co-3-hydroxy-omega-nonenoate-co-3-hydroxyoctanoate-co-3-hydroxy-omega-heptenoate-co-3-hydroxyhexanoate) (PHOUE) from Pseudomonas putida species. Three different types of activated charcoal were compared with regard to their capability to selectively remove impurities. The product 'Charcoal activated, powder, pure' from Merck was found to be most suitable. Using ethyl acetate as solvent, the polyesters were extracted from freeze-dried biomass at room temperature and simultaneously purified by addition of activated charcoal at the beginning of the extraction. The period of extraction was one hour and the ratio solvent to biomass was 15:1 (vol/wt). After extraction, the solids were separated by pressure filtration through a metallic lace tissue. The filtrate was again passed through the previously accumulated filter cake, followed by a second filtration through a 0.45 microm membrane to remove finest coal particles. The resulting filtrate was concentrated, thus yielding polyesters whose quality and yield depended on the quantity of activated charcoal applied. For highly pure PHUE and PHOUE with low endotoxin levels, the optimum ratio of activated charcoal to solvent for extraction (V/V) was found to be 0.5 for PHUE and 0.25 for PHOUE. The yields with regard to the raw polymers amounted to 55 wt% for PHUE and 75 wt% for PHOUE, which are acceptable for polymers that can be used for medical applications.

Use of extracellular medium chain length polyhydroxyalkanoate depolymerase for targeted binding of proteins to artificial poly[(3-hydroxyoctanoate)-co-(3-hydroxyhexanoate)] granules.

Polyhydroxyalkanoates (PHA), which are produced by many microorganisms, are promising polymers for biomedical applications due to their biodegradability and biocompatibility. In this study, we evaluated the suitability of medium chain length (mcl) PHA as surface materials for immobilizing proteins. Self-stabilized, artificial mcl-PHA beads with a size of 200-300 nm were fabricated. Five of six tested proteins adsorbed nonspecifically to mcl-PHA beads in amounts of 0.4-1.8 mg m(-2) bead surface area. The binding capacity was comparable to similar-sized polystyrene particles commonly used for antibody immobilization in clinical diagnostics. A targeted immobilization of fusion proteins was achieved by using inactive extracellular PHA depolymerase (ePHA(mcl)) from Pseudomonas fluorescens as the capture ligand. The N-terminal part of ePhaZ(MCL) preceding the catalytic domain was identified to comprise the substrate binding domain and was sufficient for mediating the binding of fusion proteins to mcl-PHA. We suggest mcl-PHA to be prime candidates for both nonspecific and targeted immobilization of proteins in applications such as drug delivery, protein microarrays, and protein purification.

Simultaneous accumulation and degradation of polyhydroxyalkanoates: futile cycle or clever regulation?

The regulation of medium-chain-length polyhydroxyalkanoates (mcl-PHA) metabolism in Pseudomonas putida GPo1 was studied by analysis of enzymes bound to PHA granules and enzymes involved in fatty acid oxidation. N-terminal sequencing of granule-bound enzymes revealed the presence of PHA polymerase (PhaC) and PHA depolymerase (PhaZ) and an acyl-CoA synthetase (ACS1), which recently was found to be associated with PHA granules by in vivo study. The acs1 knockout mutant accumulated 30-50% less PHA than its parental strain, confirming the involvement of ACS1 in PHA metabolism. Isolated PHA granules showed both PhaC and PhaZ activities. PhaC activity was found to be sensitive to the ratio of [R-3-hydroxyacyl-CoA]/[CoA] in which free CoA was a mild competitive inhibitor. Fatty acid oxidation was regulated by the [acetyl-CoA]/[CoA] and [NADH]/[NAD] ratios, with high ratios resulting in accumulation and low ratios leading to rapid oxidation of 3-hydroxyacyl-CoA. These results suggest that PHA metabolism is likely to be controlled by the [acetyl-CoA]/[CoA] and [NADH]/[NAD] ratios. The physiological roles of simultaneous PHA accumulation and degradation are also discussed.