Biomedical subjects

U T Bornscheuer

Publications and source records attributed to U T Bornscheuer.

16 recordsLinked to original sources

PubMed2002

Esterases from Bacillus subtilis and B. stearothermophilus share high sequence homology but differ substantially in their properties.

A novel esterase from Bacillus subtilis (BsubE) was cloned, functionally expressed in Escherichia coli and biochemically characterized. BsubE shows high homology (74% identity, >95% homology) to an esterase from the thermophilic B. stearothermophilus (BsteE). Both enzymes were efficiently expressed in E. coli, using a L-rhamnose-expression system [11,500 units/l (BsteE), 3,400 units/l (BsubE)] and were purified by Ni-nitrilotriacetic acid chromatography, yielding specific activities of 70 units/mg (BsteE) and 40 units/mg (BsubE), as determined by the hydrolysis of p-nitrophenyl acetate. Despite the high homology, both esterases revealed remarkable differences in their properties. As expected, the esterase from the thermophilic organism showed significantly higher temperature stability. Whereas BsteE showed highest activity at 65-70 degrees C, BsubE was almost inactivated at 50 degrees C. Moreover, both enzymes showed quite different substrate patterns in the hydrolysis of various esters. Whilst the B. subtilis esterase accepted esters with a branched alcohol moiety well, the B. stearothermophilus esterase was more useful in the hydrolysis of substrates with a sterically demanding carboxylic acid group. BsteE showed excellent enantioselectivity ( E>100) in the kinetic resolution of menthyl acetate and even accepted the bulky menthyl benzoate as substrate ( E=19). In contrast, BsubE converted 1-phenethylacetate with higher selectivity ( E>150 vs E=8).

Amino Acid Sequence↗

PubMed2002

Cloning, functional expression and biochemical characterization of a stereoselective alcohol dehydrogenase from Pseudomonas fluorescens DSM50106.

Sequencing of a genomic library prepared from Pseudomonas fluorescens DSM 50106 identified an orf showing 29% identity to a C alpha-dehydrogenase of Pseudomonas paucimobilis and high homology to several sequences with unknown functions derived from genome projects. The corresponding gene adhF1 encodes a dehydrogenase of 296 amino acids with a calculated molecular mass of 31.997 kDa. The gene was functionally expressed in E. coli using a rhamnose inducible expression system. The resulting recombinant enzyme was active in the pH range 6-10 (best pH 8) and at 5-25 degrees C. This dehydrogenase converts cyclic ketones to the corresponding alcohols utilizing the cofactor NADH. The highest activity was found for cyclohexanone. The enzyme also exhibits high stereoselectivity in the desymmetrization of the prochiral ketone acetophenone, producing optically pure ( R)-alpha-phenyl ethanol (>99%ee) at high conversion (95%).

Alcohol Dehydrogenase↗

PubMed2001

Cloning, functional expression, and characterization of recombinant pig liver esterase.

The N-terminal amino acid sequence of pig liver esterase (PLE) from a commercial sample was determined and shown to match closely to a published sequence encoding a proline-beta-naphthylamidase from pig liver. Next, mRNA isolated from pig liver was transcribed into cDNA and primers deduced from the N-terminal sequence were used to clone the 1698 base pairs of PLE cDNA. Initial attempts to express the cDNA in Escherichia coli and Pichia pastoris with different expression vectors and secretion signal sequences failed. Only after deletion of the putative C-terminal sequence His-Ala-Glu-Leu, usually considered as an endoplasmic reticulum retention signal, could heterologous expression of PLE be readily achieved in the methylotrophic yeast P. pastoris. Recombinant PLE (rPLE) was secreted into the medium and exhibited a specific activity of approximately 600 Umg(-1) and a Vmax/Km value of 139 micromolmin(-1)mM(-1) with p-nitrophenyl acetate as a substrate. Activity staining of renatured sodium dodecylsulfate-polyacrylamide gels gave a single band with esterolytic activity for rPLE, whereas several bands are visible in crude commercial PLE preparations. This was confirmed by native gels, which also show that rPLE is active as a trimer. Biochemical characterization of the recombinant enzyme and comparison with properties of commercial PLE preparations as well as with published data confirmed that we expressed a single PLE isoenzyme which showed a high preference for proline-beta-naphthylamide. This is a substrate specificity for the so-called gamma subunit of PLE. The optimum pH value and temperature for the recombinant PLE were 8.0 and 60 degrees C, respectively. The determined molecular weight of the secreted enzyme was approximately 61-62 kDa, which closely matches the calculated value of 62.419 kDa. The active site residues are located at Ser203, His448, and Asp97, and the typical consensus sequence motif for hydrolases was found around the active site serine (Gly-Glu-Ser-Ala-Gly).

Amino Acid Sequence↗

PubMed2001

Improved biocatalysts by directed evolution and rational protein design.

The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity. However, wild-type enzymes often need to be optimized to fulfill these requirements. Two rather contradictory tools can be used on a molecular level to create tailor-made biocatalysts: directed evolution and rational protein design.

Catalysis↗

PubMed2000

Epoxide hydrolase activity of Streptomyces strains.

The discovery of epoxide hydrolases within a Streptomyces sp. strain collection is described. Screening was performed in 96 well microtiter plates using a modified 4-(p-nitrobenzyl)pyridine assay with styrene oxide, 1,2-epoxy-hexane or 3-phenyl ethylglycidate (3-PEG) as substrates. Out of 120 strains investigated, S. antibioticus Tü4, S. arenae Tü495 and S. fradiae Tü27 exhibited epoxide hydrolase activity. These strains were further investigated by performing laboratory-scale biotransformations utilizing styrene oxide, 1,2-epoxy-hexane and 3-PEG followed by subsequent quantitative analysis employing chiral gas chromatography. The highest conversions were achieved with whole cells from S. antibioticus Tü4 in the presence of 10% (v/v) DMSO. However, enantioselectivity was only satisfying (E = 31) in the presence of 5% (v/v) acetone, which allowed isolation of optically pure non-hydrolyzed (R)-styrene oxide (99% enantiomeric excess (ee)) and (S)-phenyl-1,2-ethandiol (72% ee) at 55% conversion after 24 h. The resolution of 3-PEG proceeded with slightly lower enantioselectivity albeit higher reaction rates. With S. fradiae Tü27 and S. arenae Tü495 enantioselectivity towards styrene oxide was only E = 3-4.

Epoxide Hydrolases↗

PubMed2000

Screening of commercial hydrolases for the degradation of ochratoxin A.

Ochratoxin A (OTA) is a nephrotoxic and carcinogenic mycotoxin. The toxin is a common contaminant of various foods and feeds and poses a serious threat to the health of both humans and animals. A number of commercial hydrolases were screened for the ability to degrade OTA to nontoxic compounds. A crude lipase from Aspergillus niger (Amano A) proved to substantially hydrolyze OTA to the nontoxic OTalpha and phenylalanine, as confirmed by HPLC with fluorescence detection. The enzyme was purified by anion exchange chromatography to homogeneity. Activity staining of the purified enzyme with alpha-naphthyl acetate/Fast Red revealed only one band exhibiting hydrolytic activity. The specific activity of the purified enzyme toward OTA was 2.32 units/mg.

Aspergillus niger↗

PubMed2000

Purification and reconstitution of an integral membrane protein, the photoreaction center of Rhodobacter sphaeroides, using synthetic sugar esters.

Detergents are indispensable reagents for the extraction and solubilization of integral membrane proteins, but their removal from a reconstituted phospholipid-protein complex is usually desirable. In this paper, we describe a novel method in which the synthetic sugar esters 6-O-octanoyl-beta-D-glucose (OG) or 6-O-octanoyl-beta-D-mannose (OM) are used as detergents for both the isolation and the rapid reconstitution of the photosynthetic reaction center protein of Rhodobacter sphaeroides. Following solubilization of the reaction center with OG or OM and reconstitution of this protein in liposomes, a convenient removal of these detergents was achieved within less than two hours by hydrolytic cleavage of the sugar esters using immobilized lipases. Best results were achieved with lipase from Bacillus sp. immobilized on silica gel.

Detergents↗

PubMed1999

Highly selective synthesis of 1,3-oleoyl-2-palmitoylglycerol by lipase catalysis.

1,3-Oleoyl-2-palmitoylglycerol (OPO), an important structured triglyceride in infant nutrition, was synthesized by a two-step process in high yields and purity using sn1,3-regiospecific lipases. In the first step, tripalmitin (TP) was subjected to an alcoholysis reaction in an organic solvent catalyzed by sn1,3-regiospecific lipases yielding the corresponding 2-monopalmitin (2-MP). The 2-MP was isolated in up to 85% yield and >95% purity by crystallization and esterified in the second step with oleic acid using the same lipases to form the structured triglyceride OPO in up to 78% yield containing 96% palmitic acid in the sn2-position. Water activity, solvent, as well as carrier for lipase immobilization strongly influenced the yield and purity of products in both steps. The best results were achieved with lipases from Rhizomucor miehei and Rhizopus delemar immobilized on EP 100 and equilibrated to a water activity of 0.43. Special emphasis was given to develop this process in solvents that are allowed to be used in foodstuffs and to perform the second step in a solvent-free system.

Catalysis↗

PubMed1999

Solid-state NMR and FTIR studies on bilayer membranes from 1,2- dioctadec-(14-ynoyl)-sn-glycero-3-phosphatidylcholine.

Multilamellar dispersions from a new model phospholipid, 1, 2-dioctadec-(14-ynoyl)-sn-glycero-3-phosphatidylcholine (DO(14-yne)PC), bearing a triple bond in the fatty acid chains are studied by (2)H, (31)P NMR and Fourier transform infrared (FTIR) spectroscopy. The investigations are focused on the evaluation of the molecular properties of the lipid molecules as function of temperature and sample composition. Information about the fatty acid chain conformations are obtained from FTIR measurements by analysing the CH(2) wagging and stretching modes. (2)H NMR studies are performed on two selectively deuterated compounds that provide further insights into the molecular characteristics at two specific positions along the fatty acid chains. These studies demonstrate that the introduction of the triple bond is accompanied by a reduction of fatty acid chain order which holds for both the conformational and the orientational order. Likewise, (31)P NMR spectroscopy is used for the determination of the dynamics and ordering in the head group region. Here, particular emphasis is given to the evaluation of the lipid lateral motions that are quantified over a large temperature range within the liquid crystalline phase. It is found that the lateral mobility of the lipid molecules is almost unaffected by the triple bond in the fatty acid chains. The addition of cholesterol gives rise to a reduction in lateral mobility for DO(14-yne)PC, as can be followed by spin echo, 2D-exchange NMR and stimulated echo experiments.

Crystallization↗

PubMed1999

Directed evolution of an esterase: screening of enzyme libraries based on pH-indicators and a growth assay.

In order to resolve a sterically hindered 3-hydroxy ethyl ester, which was not accepted as substrate by 20 wild-type hydrolases, a directed evolution of an esterase from Pseudomonas fluorescens (PFE) was performed. Mutations were introduced using the mutator strain Epicurian coli XL1-Red. Enzyme libraries derived from seven mutation cycles were assayed on minimal media agar plates supplemented with pH indicators (neutral red and crystal violet), thus allowing the identification of active esterase variants by the formation of a red color caused by a pH decrease due to the released acid. A further selection criteria was introduced by using the corresponding glycerol estar, because release of the carbon source glycerol facilitates growth on minimal media. By this strategy, one double mutant (A209D and L181V) of PFE was identified, which hydrolyzed the 3-hydroxy ethyl ester in a stereoselective manner (25% ee for the remaining ester, E approximate to 5).

Cell Division↗

PubMed1999

Screening, nucleotide sequence, and biochemical characterization of an esterase from Pseudomonas fluorescens with high activity towards lactones.

A genomic library of Pseudomonas fluorescens DSM 50106 in a lambdaRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using alpha-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of P. fluorescens DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in E. coli. The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43 degreesC. The showed highest purified enzyme activities towards lactones. The activity increased from gamma-butyrolactone (18.1 U/mg) to epsilon-caprolactone (21.8 U/mg) to delta-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.

Amino Acid Sequence↗

PubMed1998

Cloning, expression, characterization and role of the leader sequence of a lipase from Rhizopus oryzae.

A lipase from Rhizopus oryzae DSM 853 (ROL) was cloned from a chromosomal gene bank, sequenced and overexpressed in Escherichia coli. ROL and its precursors ProROL and PreProROL were purified and their pH and temperature profile was determined. In contrast to ROL, ProROL and PreProROL had considerably higher thermostability and a slightly higher pH optimum. Moreover, it could be demonstrated by in vitro experiments that the natural leader sequence of ROL is able to inhibit the folding supporting properties of the prosequence, resulting in a retardation of folding. In addition, there is strong evidence that all different lipase forms derived from Rhizopus sp. described in the literature are a result of different proteolytic processing and originate from the same gene.

Amino Acid Sequence↗

PubMed1998

Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones.

The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated. Mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones. Screening was performed by plating esterase producing colonies derived from mutation cycles onto minimal media agar plates containing indicator substances (neutral red and crystal violet). Esterase-catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease caused by the released acid. Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester. One variant containing two mutations (A209D and L181V) stereoselectively hydrolyzed the ethyl ester resulting in 25% ee for the remaining ester.

Antineoplastic Agents↗

PubMed1998

Enantioselectivity of a recombinant esterase from Pseudomonas fluorescens towards alcohols and carboxylic acids.

A recombinant esterase from Pseudomonas fluorescens (PFE) was produced from E. coli cultures and the enantioselectivity towards a series of racemic substrates was investigated. PFE exhibited high rate and enantioselectivity in the acylation of alpha-phenyl ethanol with vinyl acetate in toluene (E > 100) and the hydrolysis of the corresponding acetate in phosphate buffer (E = 58). In sharp contrast, extremely low enantioselectivity (E from 1.1 to 7) was found for the acylation of a series of 1,2-O-protected glycerol derivatives and the hydrolysis of 3-phenylbutyric acid methylester. Almost no reaction occurred with alpha-phenyl propanol and its acetate and 2-phenylbutyric acid ethylester.

1-Propanol↗

PubMed1997

Lipase-catalysed resolution of gamma- and delta-lactones.

Lipase-catalysed stereoselective hydrolysis of a family of saturated gamma- and delta-lactones was investigated. Increasing the chain length from delta-octalactone to delta-dodecalactone leads to higher enantiomeric excess. Best results were found using a lipase from Pseudomonas species (KW51) yielding highest enantiomeric excesses (greater than 99% ee at 50% conversion, E > 100) at short reaction times (10 h) for delta-undecalactone and delta-dodecalactone. In contrast, gamma-lactones were resolved less efficiently. Highest enantioselectivities (70%ee at 50% conversion, E = 11) were found for gamma-nonalactone. Optimum reaction conditions were found at pH 8 and 12.5 degrees C.

Candida↗

PubMedDate not supplied

Directed evolution of an esterase from Pseudomonas fluorescens. Random mutagenesis by error-prone PCR or a mutator strain and identification of mutants showing enhanced enantioselectivity by a resorufin-based fluorescence assay.

The gene encoding an esterase from Pseudomonas fluorescens (PFE) was subjected to random mutagenesis by error-prone PCR or by using the mutator strain Epicurian coli XL1-Red. Enzyme libraries were then created in microtiter plates by expression of PFE-variants in E. coli. These were assayed for improved enantioselectivity in a Beckman robot system using optically pure (R)- or (S)-3-phenylbutyric acid resorufin esters, resulting in the identification of several mutants showing up to almost two-fold enantioselectivity (E(true) = 5.2 to 6.6) compared to wild-type PFE (E(true) = 3.5).

Directed Molecular Evolution↗