Cell-free extracts have been researched and continuously streamlined for around 50 years. It is b... more Cell-free extracts have been researched and continuously streamlined for around 50 years. It is believed that these extracts work best when routinely obtained from exponentially growing cells to capture the most active translation system. Here we report on an active cell-free extract derived from E. coli A19 that was harvested under nongrowing, stressed conditions. Although this process is based on the conventional routine process for the production of S30-extracts, our process is less labor intensive and reduces variability between extracts.
<p>Intact and cleaved 16S rRNA were purified from a 2% agarose gel. Primers specific to reg... more <p>Intact and cleaved 16S rRNA were purified from a 2% agarose gel. Primers specific to regions 1470 to 1491 bp (P1), 895 to 915 bp (P2), 431 to 450 bp (P3) were annealed and cDNA synthesis was performed. RNA was digested and cDNA was visualized on a 1% agarose gel.</p
Applied Microbiology and Biotechnology, Mar 26, 1999
Hydantoinases are valuable enzymes for the production of optically pure D-and L-amino acids. They... more Hydantoinases are valuable enzymes for the production of optically pure D-and L-amino acids. They catalyse the reversible hydrolytic ring cleavage of hydantoin or 5¢-monosubstituted hydantoins and are therefore classi®ed in the EC nomenclature as cyclic amidases (EC 3.
A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escheri... more A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g L −1 cell dry weight and 3.8 g L −1 of recombinant L-Ncarbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins.
A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of... more A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of natural and unnatural chiral D- and L-amino acids from chemically synthesized hydantoin derivatives. Potentially, 100% conversion and 100% optically pure amino acids can be obtained at the same time if racemic substrates are used. Recent research activities concentrate on newly isolated or improved enzymes and include directed evolution techniques, structure elucidation, studies of fusion proteins and the use of specially designed whole cell biocatalysts.
The aim of this study was to engineer Escherichia coli strains that efficiently produce succinate... more The aim of this study was to engineer Escherichia coli strains that efficiently produce succinate from glycerol under anaerobic conditions after an aerobic growth phase. We constructed E. coli strain ss195 with deletions of pykA and pykF, which resulted in slow growth on glycerol as sole carbon source. This growth defect was overcome by the selection of fast-growing mutants. Whole-genome resequencing of the evolved mutant ss251 identified the mutation A595S in PEP carboxylase (Ppc). Reverse metabolic engineering by introducing the wild-type allele revealed that this mutation is crucial for the described phenotype. Strain ss251 and derivatives thereof produced succinate with high yields above 80% mol mol(-1) from glycerol under nongrowth conditions. The results show that during the aerobic growth of ss251, the formation of pyruvate proceeds via the proposed POMP pathway, starting with the carboxylation of PEP by Ppc. The resulting oxaloacetate is reduced by malate dehydrogenase (Mdh) to malate, which is then decarboxylated back to pyruvate by a malic enzyme (MaeA or MaeB). Mutation of ppc is crucial for fast growth of pykAF mutants on glycerol. An E. coli mutant that is capable of achieving high yields of succinate (a top valued-added chemical) from glycerol (an abundant carbon source) was constructed. The identified ppc mutation could be applied to other production strains that require strong PEP carboxylation fluxes.
Journal of Molecular Catalysis B-enzymatic, Sep 1, 1998
The hydantoin amidohydrolase hydantoinase from Arthrobacter aurescens DSM 3745 was purified to ho... more The hydantoin amidohydrolase hydantoinase from Arthrobacter aurescens DSM 3745 was purified to homogeneity and Ž. subjected to metal analysis under atomic absorption spectrometry AAS and inductive coupled plasma-atomic emission Ž. spectrometry ICP-AES. Three independent preparations of homogeneous enzyme indicated that 1 mol of the active enzyme contains 10 mol zinc ions. This corresponds to 2.5 mol zinc per mol subunit, since the hydantoinase consists of four identical subunits. Only trace amounts of manganese, magnesia, nickel and cobalt were detected. Other metals were either absent or existed below detection levels.
L'invention concerne un D-carbamoylase, et les sequences geniques qui codent cet enzyme prove... more L'invention concerne un D-carbamoylase, et les sequences geniques qui codent cet enzyme provenant de l'organisme Arthrobacter crystallopoietes DSM 20117. L'invention concerne egalement des plasmides, des vecteurs, des micro-organismes, en particulier des amorces, et des possibililtes d'utilisations specifiques relatives aux enzymes decrits. Enfin, l'invention concerne un procede relatif a la decouverte d'enzymes susceptibles d'etre utilises dans le cadre de la preparation d'acides D-amines a partir d'hydantoines substituees en 5'.
Applied and Environmental Microbiology, Sep 15, 2011
A novel technically compliant expression system was developed for heterologous protein production... more A novel technically compliant expression system was developed for heterologous protein production in Bacillus subtilis with the aim of increasing product yields at the same time as decreasing production costs. Standard systems involve the positively regulated manP promoter of the mannose operon, which led to relatively high product yields of 5.3% (5.3 g enhanced green fluorescent protein [eGFP] per 100 g cell dry weight [CDW]) but required large quantities of mannose to induce the reactions, thus rendering the system's technical application rather expensive. To improve this situation, mutant B. subtilis strains were used: the ⌬manA (mannose metabolism) strain TQ281 and the ⌬manP (mannose uptake) strain TQ356. The total amount of inducer could be reduced with TQ281, which, however, displayed sensitivity to mannose. An inducer-independent self-induction system was developed with TQ356 to further improve the cost efficiency and product yield of the system, in which glucose prevents induction by carbon catabolite repression. To create optimal self-induction conditions, a glucose-limited process strategy, namely, a fed-batch process, was utilized as follows. The initiation of self-induction at the beginning of the glucose-restricted transition phase between the batch and fed-batch phase of fermentation and its maintenance throughout the glucose-limiting fed-batch phase led to a nearly 3-fold increase of product yield, to 14.6%. The novel B. subtilis self-induction system thus makes a considerable contribution to improving product yield and reducing the costs associated with its technical application.
<p>(A) Sequencing of the final PCR products suggests a cleavage after C1400. (B) 16S rRNA s... more <p>(A) Sequencing of the final PCR products suggests a cleavage after C1400. (B) 16S rRNA secondary stucture [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168764#pone.0168764.ref050" target="_blank">50</a>]. The identified cleavage site is marked with an arrow.</p
Cell-free extracts have been researched and continuously streamlined for around 50 years. It is b... more Cell-free extracts have been researched and continuously streamlined for around 50 years. It is believed that these extracts work best when routinely obtained from exponentially growing cells to capture the most active translation system. Here we report on an active cell-free extract derived from E. coli A19 that was harvested under nongrowing, stressed conditions. Although this process is based on the conventional routine process for the production of S30-extracts, our process is less labor intensive and reduces variability between extracts.
<p>Intact and cleaved 16S rRNA were purified from a 2% agarose gel. Primers specific to reg... more <p>Intact and cleaved 16S rRNA were purified from a 2% agarose gel. Primers specific to regions 1470 to 1491 bp (P1), 895 to 915 bp (P2), 431 to 450 bp (P3) were annealed and cDNA synthesis was performed. RNA was digested and cDNA was visualized on a 1% agarose gel.</p
Applied Microbiology and Biotechnology, Mar 26, 1999
Hydantoinases are valuable enzymes for the production of optically pure D-and L-amino acids. They... more Hydantoinases are valuable enzymes for the production of optically pure D-and L-amino acids. They catalyse the reversible hydrolytic ring cleavage of hydantoin or 5¢-monosubstituted hydantoins and are therefore classi®ed in the EC nomenclature as cyclic amidases (EC 3.
A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escheri... more A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g L −1 cell dry weight and 3.8 g L −1 of recombinant L-Ncarbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins.
A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of... more A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of natural and unnatural chiral D- and L-amino acids from chemically synthesized hydantoin derivatives. Potentially, 100% conversion and 100% optically pure amino acids can be obtained at the same time if racemic substrates are used. Recent research activities concentrate on newly isolated or improved enzymes and include directed evolution techniques, structure elucidation, studies of fusion proteins and the use of specially designed whole cell biocatalysts.
The aim of this study was to engineer Escherichia coli strains that efficiently produce succinate... more The aim of this study was to engineer Escherichia coli strains that efficiently produce succinate from glycerol under anaerobic conditions after an aerobic growth phase. We constructed E. coli strain ss195 with deletions of pykA and pykF, which resulted in slow growth on glycerol as sole carbon source. This growth defect was overcome by the selection of fast-growing mutants. Whole-genome resequencing of the evolved mutant ss251 identified the mutation A595S in PEP carboxylase (Ppc). Reverse metabolic engineering by introducing the wild-type allele revealed that this mutation is crucial for the described phenotype. Strain ss251 and derivatives thereof produced succinate with high yields above 80% mol mol(-1) from glycerol under nongrowth conditions. The results show that during the aerobic growth of ss251, the formation of pyruvate proceeds via the proposed POMP pathway, starting with the carboxylation of PEP by Ppc. The resulting oxaloacetate is reduced by malate dehydrogenase (Mdh) to malate, which is then decarboxylated back to pyruvate by a malic enzyme (MaeA or MaeB). Mutation of ppc is crucial for fast growth of pykAF mutants on glycerol. An E. coli mutant that is capable of achieving high yields of succinate (a top valued-added chemical) from glycerol (an abundant carbon source) was constructed. The identified ppc mutation could be applied to other production strains that require strong PEP carboxylation fluxes.
Journal of Molecular Catalysis B-enzymatic, Sep 1, 1998
The hydantoin amidohydrolase hydantoinase from Arthrobacter aurescens DSM 3745 was purified to ho... more The hydantoin amidohydrolase hydantoinase from Arthrobacter aurescens DSM 3745 was purified to homogeneity and Ž. subjected to metal analysis under atomic absorption spectrometry AAS and inductive coupled plasma-atomic emission Ž. spectrometry ICP-AES. Three independent preparations of homogeneous enzyme indicated that 1 mol of the active enzyme contains 10 mol zinc ions. This corresponds to 2.5 mol zinc per mol subunit, since the hydantoinase consists of four identical subunits. Only trace amounts of manganese, magnesia, nickel and cobalt were detected. Other metals were either absent or existed below detection levels.
L'invention concerne un D-carbamoylase, et les sequences geniques qui codent cet enzyme prove... more L'invention concerne un D-carbamoylase, et les sequences geniques qui codent cet enzyme provenant de l'organisme Arthrobacter crystallopoietes DSM 20117. L'invention concerne egalement des plasmides, des vecteurs, des micro-organismes, en particulier des amorces, et des possibililtes d'utilisations specifiques relatives aux enzymes decrits. Enfin, l'invention concerne un procede relatif a la decouverte d'enzymes susceptibles d'etre utilises dans le cadre de la preparation d'acides D-amines a partir d'hydantoines substituees en 5'.
Applied and Environmental Microbiology, Sep 15, 2011
A novel technically compliant expression system was developed for heterologous protein production... more A novel technically compliant expression system was developed for heterologous protein production in Bacillus subtilis with the aim of increasing product yields at the same time as decreasing production costs. Standard systems involve the positively regulated manP promoter of the mannose operon, which led to relatively high product yields of 5.3% (5.3 g enhanced green fluorescent protein [eGFP] per 100 g cell dry weight [CDW]) but required large quantities of mannose to induce the reactions, thus rendering the system's technical application rather expensive. To improve this situation, mutant B. subtilis strains were used: the ⌬manA (mannose metabolism) strain TQ281 and the ⌬manP (mannose uptake) strain TQ356. The total amount of inducer could be reduced with TQ281, which, however, displayed sensitivity to mannose. An inducer-independent self-induction system was developed with TQ356 to further improve the cost efficiency and product yield of the system, in which glucose prevents induction by carbon catabolite repression. To create optimal self-induction conditions, a glucose-limited process strategy, namely, a fed-batch process, was utilized as follows. The initiation of self-induction at the beginning of the glucose-restricted transition phase between the batch and fed-batch phase of fermentation and its maintenance throughout the glucose-limiting fed-batch phase led to a nearly 3-fold increase of product yield, to 14.6%. The novel B. subtilis self-induction system thus makes a considerable contribution to improving product yield and reducing the costs associated with its technical application.
<p>(A) Sequencing of the final PCR products suggests a cleavage after C1400. (B) 16S rRNA s... more <p>(A) Sequencing of the final PCR products suggests a cleavage after C1400. (B) 16S rRNA secondary stucture [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168764#pone.0168764.ref050" target="_blank">50</a>]. The identified cleavage site is marked with an arrow.</p
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