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Biomedical subjects

Bernhard Hauer

Publications and source records attributed to Bernhard Hauer.

At least 19 recordsLinked to original sources
PubMed2006

The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation.

Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 micromol/min/liter aqueous phase (with an average of 8.4 g/L(aq) x h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products.

Biotransformation↗
PubMed2006

Sequential kinetic resolution catalyzed by halohydrin dehalogenase.

A sequential kinetic resolution catalyzed by halohydrin dehalogenase was employed for the synthesis of two valuable enantiopure building blocks. Resolution of methyl 4-chloro-3-hydroxybutanoate methylester ((R,S)-2) with use of a Trp249Phe mutant of halohydrin dehalogenase yielded methyl 4-cyano-3-hydroxybutanoate methylester ((S)-4) with 96.8% ee (40% yield) and (S)-2 with 95.2% ee (41% yield). This reaction is carried out in aqueous solution under mild conditions and provides access to a useful statin side-chain building block.

Catalysis↗
PubMed2006

Identification of novel enzymes with different hydrolytic activities by metagenome expression cloning.

Metagenome cloning has become a powerful tool to exploit the biocatalytic potential of microbial communities for the discovery of novel biocatalysts. In a novel variant of direct expression cloning, metagenomic DNA was isolated from compost by a modified direct lysis method, purified by size exclusion chromatography and cloned into an expression vector allowing bidirectional transcription. Transformation of Escherichia coli DH5alpha resulted in a metagenomic expression library with an average insert size of 3.2 kb. To estimate the functional diversity of the constructed library, it was screened by different approaches based on functional heterologous expression. A large number of active clones were identified, including lipolytic enzymes, amylases, phosphatases and dioxygenases. Molecular analysis of one important class of industrial biocatalysts, the lipolytic enzymes, confirmed the novelty and dissimilarity of all recovered genes, which exhibited only limited similarity to known enzymes. Equally, the novelty of another three genes encoding phosphatase or dioxygenase activity, respectively, was shown. These results demonstrate the suitability of this direct cloning approach, which comprised a dual-orientation expression vector and a simple one-step DNA purification method, for the efficient discovery of numerous active novel clones. By this means it provides an efficient way for the rapid generation of large libraries of hitherto unknown enzyme candidates which could be screened for different specific target reactions.

Amino Acid Sequence↗
PubMed2006

Crystal structure and enzyme kinetics of the (S)-specific 1-phenylethanol dehydrogenase of the denitrifying bacterium strain EbN1.

(S)-1-Phenylethanol dehydrogenase (PED) from the denitrifying bacterium strain EbN1 catalyzes the NAD+-dependent, stereospecific oxidation of (S)-1-phenylethanol to acetophenone and the biotechnologically interesting reverse reaction. This novel enzyme belongs to the short-chain alcohol dehydrogenase/aldehyde reductase family. The coding gene (ped) was heterologously expressed in Escherichia coli and the purified protein was crystallized. The X-ray structures of the apo-form and the NAD+-bound form were solved at a resolution of 2.1 and 2.4 A, respectively, revealing that the enzyme is a tetramer with two types of hydrophobic dimerization interfaces, similar to beta-oxoacyl-[acyl carrier protein] reductase (FabG) from E. coli. NAD+-binding is associated with a conformational shift of the substrate binding loop of PED from a crystallographically unordered "open" to a more ordered "closed" form. Modeling the substrate acetophenone into the active site revealed the structural prerequisites for the strong enantioselectivity of the enzyme and for the catalytic mechanism. Studies on the steady-state kinetics of PED indicated a highly positive cooperativity of both catalytic directions with respect to the substrates. This is contrasted by the behavior of FabG. Moreover, PED exhibits extensive regulation on the enzyme level, being inhibited by elevated concentrations of substrates and products, as well as the wrong enantiomer of 1-phenylethanol. These regulatory properties of PED are consistent with the presence of a putative "transmission module" between the subunits. This module consists of the C-terminal loops of all four subunits, which form a special interconnected structural domain and mediate close contact of the subunits, and of a phenylalanine residue in each subunit that reaches out between substrate-binding loop and C-terminal domain of an adjacent subunit. These elements may transmit the substrate-induced conformational change of the substrate binding loop from one subunit to the others in the tetrameric complex and thus mediate the cooperative behavior of PED.

Bacteria↗
PubMed2006

Altering the regioselectivity of cytochrome P450 CYP102A3 of Bacillus subtilis by using a new versatile assay system.

A novel monooxygenase (CYP102A3) has been discovered within the Bacillus subtilis genome that reveals a similarity of 76 % to the well-known cytochrome P450 BM-3 of B. megaterium (CYP102A1). Both enzymes are natural fusion proteins consisting of a heme domain and a FAD/FMN-reductase domain. Because of their high turnover rates, these biocatalysts are of special interest for industrial applications, but show only limited regioselectivity. In this work, the regioselectivity of CYP102A3 was changed by directed evolution and protein design to hydroxylate substrates not only in different subterminal, but also to a high extent, in terminal carbon chain positions. To enable a high-throughput screening procedure, a very versatile assay was developed that is capable of discriminating between terminal and subterminal hydroxylation of carbon chains. A double mutant of CYP102A3 was obtained that produces 48 % octan-1-ol as the main product of the reaction.

Bacterial Proteins↗
PubMed2006

A filter paper-based assay for laboratory evolution of hydrolases and dehydrogenases.

Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.

Alcohol Dehydrogenase↗
PubMed2005

pH shift enhancement of Candida utilis pyruvate decarboxylase production.

Pyruvate decarboxylase (PDC) catalyses the synthesis of asymmetric carbinols, e.g., chiral precursors for pharmaceuticals such as ephedrine and pseudoephedrine. The production of PDC by Candida utilis in a minimal medium was improved by manipulating the pH during fermentation in a 5 L bioreactor. At an aeration rate of 0.1 vvm with a stirrer speed of 300 rpm at constant pH 6, a specific PDC activity of 141 U/g dry cell weight (DCW) was achieved (average of two fermentations +/-13%). By allowing the yeast to acidify the growth medium from pH 6 to 2.9, the final specific PDC activity increased by a factor of 2.7 to 385 U/g DCW (average from 4 fermentations +/-16%). The effect of this pH drift on PDC production was confirmed by another experiment with a manual shift of pH from 6 to 3 by addition of 5 M sulfuric acid. The final PDC activity was 392 U/g DCW (average from two fermentations +/-5%). However, experiments with constant pH of 6, 5, 4, or 3 resulted in average specific activities of only 102 to 141 U/g DCW, suggesting that a transitional pH change rather than the absolute pH value was responsible for the increased specific PDC activity.

Bioreactors↗
PubMed2005

Enzymatic (R)-phenylacetylcarbinol production in a benzaldehyde emulsion system with Candida utilis cells.

Recent progress in enzymatic (R)-phenylacetylcarbinol (PAC) production has established the need for low cost and efficient biocatalyst preparation. Pyruvate decarboxylase (PDC) added in the form of Candida utilis cells showed higher stability towards benzaldehyde and temperature in comparison with partially purified preparations. In the presence of 50 mM benzaldehyde and at 4 degrees C, a half-life of 228 h was estimated for PDC added as C. utilis cells, in comparison with 24 h for the partially purified preparation. Increasing the temperature from 4 to 21 degrees C for PAC production with C. utilis cells resulted in similar final PAC levels of 39 and 43 g l(-1) (258 and 289 mM), respectively, from initial 300 mM benzaldehyde and 364 mM pyruvate. The overall volumetric productivity was enhanced 2.8-fold, which reflected the 60% shorter reaction time at the higher temperature. Enantiomeric excess values of 98 and 94% for R-PAC were obtained at 4 and 21 degrees C, respectively, and benzyl alcohol (a potential by-product from benzaldehyde) was not formed.

Acetone↗
PubMed2005

(R)-phenylacetylcarbinol production in aqueous/organic two-phase systems using partially purified pyruvate decarboxylase from Candida utilis.

Aqueous/organic two-phase systems have been evaluated for enhanced production of (R)-phenylacetylcarbinol (PAC) from pyruvate and benzaldehyde using partially purified pyruvate decarboxylase (PDC) from Candida utilis. In a solvent screen, octanol was identified as the most suitable solvent for PAC production in the two-phase system in comparison to butanol, pentanol, nonanol, hexane, heptane, octane, nonane, dodecane, methylcyclohexane, methyl tert butyl ether, and toluene. The high partitioning coefficient of the toxic substrate benzaldehyde in octanol allowed delivery of large amounts of benzaldehyde into the aqueous phase at a concentration less than 50 mM. PDC catalyzed the biotransformation of benzaldehyde and pyruvate to PAC in the aqueous phase, and continuous extraction of PAC and byproducts acetoin and acetaldehyde into the octanol phase further minimized enzyme inactivation, and inhibition due to acetaldehyde. For the rapidly stirred two-phase system with a 1:1 phase ratio and 8.5 U/mL carboligase activity, 937 mM (141 g/L) PAC was produced in the octanol phase in 49 h with an additional 127 mM (19 g/L) in the aqueous phase. Similar concentrations of PAC could be produced in the slowly stirred phase separated system at this enzyme level, although at a much slower rate. However at lower enzyme concentration very high specific PAC production (128 mg PAC/U carboligase at 0.9 U/mL) was achieved in the phase separated system, while still reaching final PAC levels of 102 g/L in octanol and 13 g/L in the aqueous phase. By comparison with previously published data by our group for a benzaldehyde emulsion system without octanol (50 g/L PAC, 6 mg PAC/U carboligase), significantly higher PAC concentrations and specific PAC production can be achieved in an octanol/aqueous two-phase system.

Acetone↗
PubMed2005

Biocatalytic racemization of aliphatic, arylaliphatic, and aromatic alpha-hydroxycarboxylic acids.

[reaction: see text] Biocatalytic racemization of a range of aliphatic, (aryl)aliphatic, and aromatic alpha-hydroxycarboxylic acids was accomplished by using whole resting cells of a range of Lactobacillus spp. The mild (physiological) reaction conditions ensured an essentially "clean" isomerization in the absence of side reactions, such as elimination or decomposition. Whereas straight-chain aliphatic 2-hydroxycarboxylic acids were racemized with excellent rates (up to 85% relative to lactate), steric hindrance was observed for branched-chain analogues. Good rates were observed for aryl-alkyl derivatives, such as 3-phenyllactic acid (up to 59%) and 4-phenyl-2-hydroxybutanoic acid (up to 47%). In addition, also mandelate and its o-chloro analogue were accepted at a fair rate (45%). This biocatalytic racemization represents an important tool for the deracemization of a number of pharmaceutically important building blocks.

Animals↗
PubMed2005

Direct electrochemical regeneration of monooxygenase subunits for biocatalytic asymmetric epoxidation.

We report the first example of direct electrochemical regeneration of a flavin-dependent monooxygenase for asymmetric epoxidation catalysis. It is shown that electrochemical regeneration of the oxygenase subunit of the multicomponent styrene monooxygenase is sufficient to perform enantiospecific S-epoxidation of various styrene derivatives. Kinetic bottlenecks of the novel electroenzymatic reaction are identified. This work opens up new alternatives for asymmetric oxyfunctionalization catalysis.

Catalase↗
PubMed2005

Role of pyruvate in enhancing pyruvate decarboxylase stability towards benzaldehyde.

Biotransformation of benzaldehyde and pyruvate into (R)-phenylacetylcarbinol (PAC) catalysed by Candida utilis pyruvate decarboxylase (PDC) at low buffer concentration (20 mM MOPS) was enhanced by maintenance of neutral pH through acetic acid addition. PDC was very stable in this buffer (half-life 138 h at 6 degrees C), however a benzaldehyde emulsion (400 mM) caused rapid deactivation. The inclusion of 2M glycerol did not protect PDC from inactivation by benzaldehyde but initial rates were increased by 50% and the final PAC level was enhanced from 40 to 51 g l(-1). Low levels of by-products acetaldehyde (0.1-0.15 g l(-1)) and acetoin (1.1-1.3 g l(-1)) were formed in both the presence and absence of 2 M glycerol. Interestingly PDC was more stable towards benzaldehyde when pyruvate was present: no activity was lost during the first hour of biotransformation (2 M glycerol, benzaldehyde concentration decreased from 400 to 345 mM, pyruvate from 480 to 420 mM) but PDC was completely inactivated in less than 30 min when exposed to the same concentrations of benzaldehyde in the absence of pyruvate. Thus the enzyme in catalytic action was more stable than the resting enzyme.

Acetone↗
PubMed2005

Cells of Candida utilis for in vitro (R)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor.

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 degrees C, a screen of several 1-alcohols (C4-C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l-1 (PDC activity 2.5 U ml-1), PAC levels of 103 g l-1 in the octanol phase and 12.8 g l-1 in the aqueous phase were produced in 15 h at 21 degrees C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 degrees C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U-1 h-1) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l-1 h-1) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g-1).

Acetone↗
PubMed2004

Kinetic analysis and modelling of enzymatic (R)-phenylacetylcarbinol batch biotransformation process.

Initial rate and biotransformation studies were applied to refine and validate a mathematical model for enzymatic (R)-phenylacetylcarbinol (PAC) production from pyruvate and benzaldehyde using Candida utilis pyruvate decarboxylase (PDC). The rate of PAC formation was directly proportional to the enzyme activity level up to 5.0 U ml-1 carboligase. Michaelis-Menten kinetics were determined for the effect of pyruvate concentration on the reaction rate. The effect of benzaldehyde followed the sigmoidal shape of the Monod-Wyman-Changeux (MWC) model. The biotransformation model, which also included a term for PDC inactivation by benzaldehyde, was used to determine the overall rate constants for the formation of PAC, acetaldehyde, and acetoin. These values were determined from data for three batch biotransformations performed over a range of initial concentrations (viz. 50-150 mM benzaldehyde, 60-180 mM pyruvate, 1.1-3.4 U ml-1 enzyme activity). The finalized model was then used to predict a batch biotransformation profile at 120/100 mM initial pyruvate/benzaldehyde (initial enzyme activity 3.0 U ml-1). The simulated kinetics gave acceptable fitting (R2 = 0.9963) to the time courses of these latter experimental data for substrates pyruvate and benzaldehyde, product PAC, by-products acetaldehyde and acetoin, as well as enzyme activity level.

Acetone↗
PubMed2004

Biphasic aqueous/organic biotransformation of acetaldehyde and benzaldehyde by Zymomonas mobilis pyruvate decarboxylase.

Zymomonas mobilis pyruvate decarboxylase (PDC) transformed acetaldehyde and benzaldehyde into (R)-phenylacetylcarbinol (PAC), the precursor for the synthesis of ephedrine and pseudoephedrine. Organic solvents were screened for a biphasic biotransformation with the enzyme in an aqueous phase and the toxic substrates delivered through the organic phase. In the absence of substrates a second phase of 1-pentanol, hexadecane or MTBE (methyl tertiary-butyl ether) stabilized the PDC activity in comparison to a control without added solvent. Organic phase solvents for optimal PAC production had partitioning coefficient (log P) values between 0.8 and 2.8 (production of more than 8 mg PAC/ U PDC), however there was no correlation between enzyme stability and log P. Best PAC formation was observed with the eight tested alcohols, which in contrast to the other solvents allowed lower initial concentrations of toxic acetaldehyde (54-81 mM) in the aqueous phase. 1-pentanol, 1-hexanol, and isobutanol resulted in the highest specific PAC production of 11 mg PAC /U PDC. Without the addition of an organic phase, only 1.2 mg/U was formed.

Acetaldehyde↗
PubMed2004

Sequence and transcriptional analysis of a gene cluster of Pseudomonas putida 86 involved in quinoline degradation.

Although quinoline 2-oxidoreductase (Qor) and 1H-2-oxoquinoline 8-monooxygenase (OxoOR), which catalyse the first two steps of quinoline degradation by Pseudomonas putida 86, and their genes have been investigated in some detail, the genetic organization and regulation of the catabolic pathway are not known yet. A gene cluster involved in quinoline degradation was characterized. Upstream of oxoO encoding the oxygenase component of OxoOR, the gene oxoS coding for a XylS-type protein is located. The DNA region downstream of oxoO comprises potential open reading frames (ORFs) that may code for further catabolic enzymes (an alpha/beta-hydrolase fold protein, and an amidase), and for accessory proteins presumably required for the assembly of metal cofactor containing holoenzymes (XdhC-like protein, MoeC- and MobA-like protein(s), IscS and IscU). The potential iscU gene is followed by the genes qorMSL that encode the structural subunits of Qor. Three potential ORFs (ORFs7-9) are located between qorMSL and oxoR, which codes for the reductase component of OxoOR. ORFs7-9 have counterparts in the cox (CO oxidizing system) and nic (nicotine degradation) gene clusters. Transcription of all these genes and ORFs located downstream of oxoS is induced by quinoline or 1H-2-oxoquinoline. Insertional inactivation of oxoS abolished quinoline-induced transcription. However, weak transcription of ORFs7-9 also occurred independent of quinoline and OxoS. The typical tandem recognition site for a XylS-type transcriptional activator was identified in the putative promoter region of qorM, and archetypal XylS indeed was found to activate synthesis of Qor. Motifs corresponding to single half-sites of a XylS-type binding site are located upstream of oxoO, the xdhC-like gene, and oxoR. Putative quinoline-specific transcriptional start sites were identified for these genes, and for qorM. The gene cluster probably is transcribed from several promoters, resulting in multiple overlapping polycistronic mRNAs.

Base Sequence↗