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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>A novel CotA-type laccase from <it>Bacillus pumilus </it>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 <it>K</it>_Mand <it>k</it>_catfor ABTS were 80 ± 4 μM and 291 ± 2.7 s^-1, for 2,6-DMP 680 ± 27 μM and 11 ± 0.1 s^-1and for SGZ only <it>k</it>_catcould 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 <it>B. pumilus </it>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.</p> <p>Conclusions</p> <p>The fully copper loaded, thermostable CotA laccase from <it>Bacillus pumilus </it>is a versatile laccase with potential applications as an industrial biocatalyst.</p

Periplasmic protein thiol:disulfide oxidoreductases of Escherichia coli

Disulfide bond formation is part of the folding pathway for many periplasmic and outer membrane proteins that contain structural disulfide bonds. In Escherichia coli, a broad variety of periplasmic protein thiol:disulfide oxidoreductases have been identified in recent years, which substantially contribute to this pathway. Like the well-known cytoplasmic thioredoxins and glutaredoxins, these periplasmic protein thiol:disulfide oxidoreductases contain the conserved C-X-X-C motif in their active site. Most of them have a domain that displays the thioredoxin-like fold. In contrast to the cytoplasmic system, which consists exclusively of reducing proteins, the periplasmic oxidoreductases have either an oxidising, a reducing or an isomerisation activity. Apart from understanding their physiological role, it is of interest to learn how these proteins interact with their target molecules and how they are recycled as electron donors or acceptors. This review reflects the recently made efforts to elucidate the sources of oxidising and reducing power in the periplasm as well as the different properties of certain periplasmic protein thiol:disulfide oxidoreductases of E. col

A heme tag for in vivo synthesis of artificial cytochromes

A genetic approach is described here that enables the specific covalent attachment of heme via a short C-terminal peptide tag to an otherwise non-heme-binding protein. Covalent attachment of heme to the apo-protein is catalysed by the cytochrome c maturation system of Escherichia coli. While its original enzymatic activity is retained, the resulting heme-tagged protein is red, has peroxidase activity and is redox active. The presence or absence of a C-terminal histidine tag results in low-spin heme iron with six- or high-spin heme iron with five coordinate ligands, respectively. The heme tag can be used as a tool for the rational design of artificial c-type cytochromes and metalloenzymes, thereby overcoming previous limitations set by chemical approaches. Moreover, the tag allows direct visualisation of the red fusion protein during purificatio

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 productio

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 application

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-w-undecenoate-co-3-hydroxy-w-nonenoate-co-3-hydroxy-w-heptenoate) (PHUE) and poly([R]-3hydroxy-w-undecenoate-co-3-hydroxy-w-nonenoate-co-3-hydroxyoctanoate- co-3-hydroxy-w-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 mm 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

Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity

DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of −131mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsb

Overexpression and characterization of medium-chain-length polyhydroxyalkanoate granule bound polymerases from Pseudomonas putida GPo1

<p>Abstract</p> <p>Background</p> <p>Polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA biosynthesis are PHA polymerases, which catalyze the covalent linkage of 3-hydroxyacyl coenzymeA thioesters by transesterification with concomitant release of CoA. <it>Pseudomonas putida </it>GPo1 and many other <it>Pseudomonas </it>species contain two different class II polymerases, encoded by <it>phaC1 </it>and <it>phaC2</it>. Although numerous studies have been carried out on PHA polymerases and they are well characterized at the molecular level, the biochemical properties of the class II polymerases have not been studied in detail. Previously we and other groups purified the polymerases, however, the activities of the purified enzymes were several magnitude lower than the granule-bound enzymes. It is problematic to study the intrinsic properties of these enzymes with such low activities, although they are pure.</p> <p>Results</p> <p>PHA polymerase 1 (PhaC1) and PHA polymerase 2 (PhaC2) from <it>P. putida </it>GPo1 were overexpressed in the PHA-negative host <it>P. putida </it>GPp104 and purified from isolated PHA granules. Only minor activity (two to three orders of magnitude lower than that of the granule bound proteins) could be recovered when the enzymes were purified to homogeneity. Therefore, kinetic properties and substrate ranges were determined for the granule bound polymerases. The polymerases differed significantly with respect to their association with PHA granules, enzyme kinetics and substrate specificity. PhaC2 appeared to bind PHA granules more tightly than PhaC1. When <it>R</it>-3-hydroxyoctanoic acid was used as substrate, the granule-bound PhaC1 exhibited a <it>Km </it>of 125 (± 35) μM and a <it>V</it>max of 40.8 (± 6.2) U/mg PhaC1, while a <it>Km </it>of 37 (± 10) μM and a <it>V</it>max of 2.7 (± 0.7) U/mg PhaC2 could be derived for the granule-bound PhaC2. Granule-bound PhaC1 showed a strong preference for medium chain length (mcl-) 3-hydroxyacly-CoAs, with highest affinity towards 3-hydroxydecanoyl-CoA (40 U/mg PhaC1). Granule-bound PhaC2 demonstrated a far broader specificity ranging from short chain length up to long chain length substrates. Activity increased with increasing chain length with a maximum activity for 3-hydroxyacyl-CoAs containing 12 or more C-atoms.</p> <p>Conclusion</p> <p>The kinetic properties and substrate ranges were determined for both granule bound polymerases. Evidence was provided for the first time that two PHA polymerases exhibited significant differences in granule release and in vitro activity profiles, suggesting that there are substantial functional differences between granule bound PhaC1 and PhaC2.</p

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

<p>Abstract</p> <p>Background</p> <p>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 β-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 <it>in vitro </it>activity measurements of PhaC and PhaZ in crude cell extracts are necessary.</p> <p>Results</p> <p>Two <it>in vitro </it>assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of <it>Pseudomonas putida </it>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.</p> <p>Conclusion</p> <p>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 <it>P. putida </it>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.</p

Production of glycoprotein vaccines in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>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. <it>In vivo </it>enzymatic coupling using the general glycosylation pathway of <it>Campylobacter jejuni </it>in recombinant <it>Escherichia coli </it>has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the <it>in vivo </it>biosynthesis of two novel conjugate vaccine candidates against <it>Shigella dysenteriae </it>type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.</p> <p>Results</p> <p>Two different periplasmic carrier proteins, AcrA from <it>C. jejuni </it>and a toxoid form of <it>Pseudomonas aeruginosa </it>exotoxin were glycosylated with <it>Shigella </it>O antigens in <it>E. coli</it>. 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 <it>E. coli</it>. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of <it>E. coli </it>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.</p> <p>Conclusions</p> <p>The presented data demonstrate that glycosylated proteins can be produced in recombinant <it>E. coli </it>at a larger scale. The described methodologies constitute an important step towards cost-effective <it>in vivo </it>production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.</p