Enzymatic resolution of racemic phenyloxirane by a novel epoxide hydrolase from Aspergillus niger SQ-6 and its fed-batch fermentation

A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum temperature and pH for stereospecific vicinal diol production were 35°C and 7.0, respectively. After a 24-h conversion, the enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30°C for 44 h, glycerol (20 g L⁻¹) and corn steep liquor (CSL) (30 g L⁻¹) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of sucrose (2 g L⁻¹ h⁻¹) and continuous feeding of CSL (1 g L⁻¹ h⁻¹) at a fermentation time of 28 h. After optimization, the maximum dry cell weight achieved was 24.5±0.8 g L⁻¹; maximum EH production was 351.2±13.1 U L⁻¹ with a specific activity of 14.3±0.5 U g⁻¹. Partially purified EH exhibited a temperature optimum at 37°C and pH optimum at 7.5 in 0.1 M phosphate buffer. This study presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of epoxide intermediates.     

Production of epoxide hydrolases in batch fermentations of <Emphasis Type="Italic">Botryosphaeria rhodina</Emphasis>

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.     

Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici

Cells of the propionate-tolerant strain Propionibacterium acidipropionici P200910, immobilized in calcium alginate beads, were tested for propionic and acetic acid production both in a semidefined laboratory medium and in corn steep liquor in batch, fed-batch, and continuous fermentation. Cell density was about 9.8 × 10⁹ cells/g (wet weight) of beads, and beads were added to the medium at 0.1 g (wet weight) beads/ml. Beads could be reused for several consecutive batch fermentations; propionic acid production in the tenth cycle was about 50%–70% of that in the first cycle. In batch culture complete substrate consumption (glucose in semidefined medium, lactate in corn steep liquor) and maximum acid production were seen within 36 h, and acid yields from the substrate were higher than in free-cell fermentations. Fed-batch fermentations were incubated up to 250 h. Maximum propionic acid concentrations obtained were 45.6 g/l in corn steep liquor and 57 g/l in semidefined medium; this is the highest concentration achieved to date in our laboratory. Maximum acetic acid concentrations were 17 g/l and 12 g/l, respectively. In continuous fermentation of semidefined medium, dilution rates up to 0.31 h^–1 could be used, which gave higher volumetric productivities (0.96 g l^–1 h^–1 for propionic acid and 0.26 g l^–1 h^–1 for acetic acid) than we have obtained with free cells. Corn steep liquor shows promise as an inexpensive medium for production of both acids by immobilized cells of propionibacteria.Journal paper no. J- 15614 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project no. 3122     

Production of Oenococcus oeni biomass to induce malolactic fermentation in wine by control of pH and substrate addition

To increase the commercial production of Oenococcus oeni strains to be used for biological deacidification of wines, substrates addition and pH control have been optimized. The highest biomass yield of Oenococcus oeni (Y=6.9 mg mmol–1 sugar) was obtained when 55 mmol glucose l–1 and 30 mmol fructose l–1 were added both to the culture medium, and the pH was controlled at 4.8. Fructose was used as carbon and energy source, but also as electron acceptor improving the ability to reoxidize NAD(P)H.     

A low-cost medium for mannitol production by Lactobacillus intermedius NRRL B-3693

The production of mannitol by Lactobacillus intermedius NNRL B-3693 using molasses as an inexpensive carbon source was evaluated. The bacterium produced mannitol (104 g/l) from molasses and fructose syrups (1:1; total sugars, 150 g/l; fructose:glucose 4:1) in 16 h. Several kinds of inexpensive organic and inorganic nitrogen sources and corn steep liquor were evaluated for their potential to replace more expensive nitrogen sources derived from Bacto-peptone and yeast extract. Soy peptone D (5 g/l) and corn steep liquor (50 g/l) were found to be suitable substitutes for Bacto-peptone (5 g/l) and Bacto-yeast extract (5 g/l), respectively. The bacterium produced 105 g mannitol per liter from the molasses and fructose syrup (1:1, total sugars 150 g/l; fructose:glucose 4:1) in 22 h using a combination of soy peptone D (5 g/l) and corn steep liquor (50 g/l). This is the first report on the production of mannitol by fermentation using molasses and corn steep liquor.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Effect of an exponential feeding regime on the production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Aims: To evaluate the effect of and exponential feeding regime on the production of epoxide hydrolase (EH) enzyme in recombinant Yarrowia lipolytica in comparison to a constant feed strategy. Methods and Results: An exponential feed model was developed and fermentations were fed at six different exponential rates. A twofold increase in EH productivity and a 15% increase in volumetric EH activity was obtained by applying exponential glucose feed rates in fed‐batch cultivation. These responses were modelled to obtain a theoretical optimum feed rate that was validated in duplicate fermentations. The model optimum of 0·06 h^?1 resulted in a volumetric EH activity of c. 5500 U l^?1 h^?1 and a maximum activity of 206 000 U l^?1. This correlated well with model predictions, with a variance of <10%. Conclusions: The use of an exponential feed strategy at a rate of 0·06 h ^{? 1} yielded best results for all key responses which show a clear improvement over a constant feed strategy. Significance and Impact of the Study: The study was the first evaluation of an exponential feed strategy on recombinant Y. lipolytica for the production of EH enzyme. The results suggest a strategy for the commercial production of a valuable pharmaceutical enzyme.     

Comparison of esterification and transesterification of fructose by porcine pancreas lipase immobilized on different supports

The syntheses of fructose butyrate with five different immobilized porcine pancreas lipases were studied in acetonitrile with butyric acid and tributyrin as acyl donors. The hydrolytic activities of the enzymes were measured during the syntheses. The transesterification yield was the highest with the Celite-adsorbed enzyme: 57% (1.42 g fructose butyrate l–1), and this form exhibited the highest degree of hydrolytic activity preservation (57%) after operation for 20 days.     

Characterization of epoxide hydrolase activity in shape Alternaria alternata f. sp. shape lycopersici. Possible involvement in toxin production

Using trans-diphenylpropane oxide (tDPPO) as a substrate, we measured epoxide hydrolase (EH) activity in subcellular fractions of Alternaria alternata f. sp. lycopersici (Aal), a fungus that produces host-specific toxins. The activity was mainly (>99.5%) located in the soluble fraction (100,000 × g supernatant) with the optimum pH at 7.4. An increase of toxin production between days 3 and 9 found in a Aal liquid culture over a 15 days period was concomitant with a period of high EH activity. EH activity remained constant during the same period in an Alternaria alternata culture, a fungus which does not produce toxin. In vivo treatment of Aal culture with the peroxisome proliferator clofibrate stimulated EH activity by 83% and enhanced toxin production 6.3 fold. Both 4-fluorochalcone oxide (4-FCO) and (2S,3S)-(-)-3-(4-nitrophenyl)-glycidol (SS-NPG) inhibited EH activity in vitro with a IM₅₀f 23 ± 1 μM and 72 ± 19 μM, respectively. The possible physiological substrate 9,10-epoxystearic acid was hydrolyzed more efficiently by Aal sEH than the model substrates trans- and cis-stilbene oxide (TSO and CSO) and trans- and cis-diphenylpropane oxide (tDPPO and cDPPO). This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of aeration and of corn steep concentration on L-asparaginase production in Escherichia coli

Summary The production of L-asparaginase was investigated in Escherichia coli, growing under different conditions of aeration in a medium containing 2% or 6% corn steep. At both concentrations, excessive aeration decreased enzyme production. In the medium with 2% corn steep, L-asparaginase activity began to decline as soon as the oxygen absorption exceeded 0.22 mmol O₂ l^–1 min^–1, and when the oxygen absorption rate was 1.26 mmol O₂ l^–1 min^–1, enzyme activity reached only about 5% of maximum. In the medium with 6% corn steep, a decline of L-asperaginase activity did not appear until the oxygen absorption rate value exceeded 0.54 mmol O₂ l^–1 min^–1, at the oxygen absorption rate of 1.26 mmol O₂ l^–1 min^–1, the enzyme activity still reached about 50% of maximum.     

Isolation and Characterization of the Epoxide Hydrolase-Encoding Gene from Xanthophyllomyces dendrorhous

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the α/β hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.     

Affinity purification and characterization of a yeast epoxide hydrolase

Purification of the membrane-associated epoxide hydrolase from the yeast Rhodosporidium toruloides CBS 0349 to electrophoretic homogeneity was achieved in a single chromatographic step employing the affinity ligand adsorbent Mimetic Green. More than 68% of the total epoxide hydrolase activity present in the whole cells was recovered from the membrane fraction. The enzyme was purified 26-fold with respect to the solubilized membrane proteins and was obtained in a 90% yield. The purified epoxide hydrolase has an apparent monomeric molecular weight of 54 kDa, and a pI of 7.3. The enzyme was optimally active at 30–40 °C, and pH 7.3–8.5. The enzyme is highly glycosylated with a carbohydrate content >42%. The specific activity of the purified enzyme for (±)-1,2-epoxyoctane is 172 mol min^–1 mg protein^–1. The amino acid composition of the protein was determined. This is the first report of a yeast epoxide hydrolase purified to homogeneity in milligram amounts.     

Immobilization of the Solanum tuberosum epoxide hydrolase and its application in an enantioconvergent process

Five supports have been evaluated for the immobilization of the epoxide hydrolase from Solanum tuberosum (StEH) by adsorption. The highest immobilization yield (90-99%) and the maximum EH (epoxide hydrolase) activity (0.6 U g^-1 wet support) were obtained by ionic adsorption onto DEAE-cellulose. Although the activity recovered upon immobilization of StEH onto DEAE-cellulose was low, a notable stabilization factor of 6.9 at 65°C was obtained. In addition, the immobilized StEH showed a higher temperature for maximal activity (57°C) and the optimal pH (5.0) was shifted one unit towards the acidic region as compared to the free enzyme. Immobilized StEH was successfully reused in six consecutive hydrolytic kinetic resolutions of rac-pCSO without noticeable loss in activity. Finally, the sequential use of immobilized StEH with the immobilized EH from Aspergillus niger (AnEH) in a repeated batch reactor, operated for five cycles, enabled the enantioconvergent preparation of the corresponding (R)-diol, which was thus obtained with an ee of 89% and an overall yield of 100%.     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

Preparative-scale kinetic resolution of racemic styrene oxide by immobilized epoxide hydrolase

Epoxide hydrolase from Aspergillus niger was immobilized onto the modified Eupergit C 250 L through a Schiff base formation. Eupergit C 250 L was treated with ethylenediamine to introduce primary amine groups which were subsequently activated with glutaraldehyde. The amount of introduced primary amine groups was 220 μmol/g of the support after ethylenediamine treatment, and 90% of these groups were activated with glutaraldehyde. Maximum immobilization of 80% was obtained with modified Eupergit C 250 L under the optimized conditions. The optimum pH was 7.0 for the free epoxide hydrolase and 6.5 for the immobilized epoxide hydrolase. The optimum temperature for both free and immobilized epoxide hydrolase was 40 °C. The free epoxide hydrolase retained 52 and 33% of its maximum activity at 40 and 60 °C, respectively after 24h preincubation time whereas the retained activities of immobilized epoxide hydrolase at the same conditions were 90 and 75%, respectively. Immobilized epoxide hydrolase showed about 2.5-fold higher enantioselectivity than that of free epoxide hydrolase. A preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide using immobilized preparation was performed in a batch reactor and (S)-styrene oxide and (R)-1-phenyl-1,2-ethanediol were both obtained with about 50% yield and 99% enantiomeric excess. The immobilized epoxide hydrolase was retained 90% of its initial activity after 5 reuses.     

Preparative-scale kinetic resolution of racemic styrene oxide by immobilized epoxide hydrolase

Epoxide hydrolase from Aspergillus niger was immobilized onto the modified Eupergit C 250 L through a Schiff base formation. Eupergit C 250 L was treated with ethylenediamine to introduce primary amine groups which were subsequently activated with glutaraldehyde. The amount of introduced primary amine groups was 220 μmol/g of the support after ethylenediamine treatment, and 90% of these groups were activated with glutaraldehyde. Maximum immobilization of 80% was obtained with modified Eupergit C 250 L under the optimized conditions. The optimum pH was 7.0 for the free epoxide hydrolase and 6.5 for the immobilized epoxide hydrolase. The optimum temperature for both free and immobilized epoxide hydrolase was 40 °C. The free epoxide hydrolase retained 52 and 33% of its maximum activity at 40 and 60 °C, respectively after 24 h preincubation time whereas the retained activities of immobilized epoxide hydrolase at the same conditions were 90 and 75%, respectively. Immobilized epoxide hydrolase showed about 2.5-fold higher enantioselectivity than that of free epoxide hydrolase. A preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide using immobilized preparation was performed in a batch reactor and (S)-styrene oxide and (R)-1-phenyl-1,2-ethanediol were both obtained with about 50% yield and 99% enantiomeric excess. The immobilized epoxide hydrolase was retained 90% of its initial activity after 5 reuses.     

Isolation and characterization of the epoxide hydrolase-encoding gene from Xanthophyllomyces dendrorhous

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the alpha/beta hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.     

Biocatalytic resolution of benzyl glycidyl ether and its derivates by Talaromyces flavus: effect of phenyl ring substituents on enantioselectivity

Talaromyces flavus containing a constitutive epoxide hydrolase (EH) resolved racemic benzyl glycidyl ether and nine derivatives into their (R)-enantiomers. After optimization of the fermentation conditions, the specific EH activity and biomass concentration were improved from 13.5 U/g DCW and 14.8 g DCW/l to 26.2 U/g DCW and 31.3 g DCW/l, respectively, with final values for e.e. ( s ) of 96 % and E of 13 with (R)-benzyl glycidyl ether. Substituents on the phenyl ring, however, gave low enantioselectivities.     

High fructose formation from sugarcane syrup and molasses usingZymomonas mobilis mutants

High fructose recovery yields were obtained using sugarcane syrup and C-molasses (equal to blackstrap molasses) and a fructokinase negative mutant ofZymomonas mobilis. The fructose recovery was 95.7% with sugarcane syrup and 99.4% with 300 g/L C-molasses or mixtures of both. High fructose corn syrup of a 48/52 mixture of glucose and fructose gave only a 65–70% fructose recovery due to high sorbitol formation.     

Multiple Epoxide Hydrolases in Alternaria alternata f. sp. lycopersici and Their Relationship to Medium Composition and Host-Specific Toxin Production

The production of Alternaria alternata f. sp. lycopersici host-specific toxins (AAL toxins) and epoxide hydrolase (EH) activity were studied during the growth of this plant-pathogenic fungus in stationary liquid cultures. Media containing pectin as the primary carbon source displayed peaks of EH activity at day 4 and at day 12. When pectin was replaced by glucose, there was a single peak of EH activity at day 6. Partial characterization of the EH activities suggests the presence of three biochemically distinguishable EH activities. Two of them have a molecular mass of 25 kDa and a pI of 4.9, while the other has a molecular mass of 20 kDa and a pI of 4.7. Each of the EH activities can be distinguished by substrate preference and sensitivity to inhibitors. The EH activities present at day 6 (glucose) or day 12 (pectin) are concomitant with AAL toxin production.     

Epoxide hydrolase in the bulb mite Rhizoglyphus robini: properties and induction

Using styrene oxide as substrate, most of the epoxide hydrolase (EH) activity monitored in the bulb mite Rhizoglyphus robini was associated with the microsomal compartment. The microsomal and cytosolic EHs did not display any significant preference in hydrating trans stilbene oxide (TSO) and cis stilbene oxide (CSO). The microsomal EH, which has a Km value of 5×10^-5M and pH optimum of 7.8, was sensitive to ethanol and its activity was inhibited to a moderate extent by 4-fluorochalcone oxide, TSO, CSO and trans-chalcone oxide at a level of 10^-4M. Microsomal EH was considerably induced (4–5-fold) in mites feeding garlic and onion, or ingesting TSO-impregnated filter papers. Other epoxides like CSO, 2,4-dichlorostilbene oxide, methyl chalcone oxide and heptachlor epoxide displayed moderate induction levels (1.4–2.6-fold). Of the toxicants assayed only sodium phenobarbital was a potent inducer. Lindane, malathion and DDT did not stimulate EH activity and 3-methyl-cholanthrene was even inhibitory. A decrease in EH activity was observed with a number of phytochemicals tested such as sinigrin, flavone, menthol, trans--carotene, chalcone, allyl sulphide and trans-cinnamic acid.