Continuous production of enantiopure 1,2-epoxyhexane by yeast epoxide hydrolase in a two-phase membrane bioreactor

A two-phase membrane bioreactor was developed to continuously produce enantiopure epoxides using the epoxide hydrolase activity of Rhodotorula glutinis. An aqueous/organic cascade, hydrophilic, hollow-fiber membrane bioreactor was used: (1) to carry out large-scale resolution of epoxides, (2) to continuously extract residual enantiopure epoxides from the aqueous phase, and (3) to separate inhibitory formed diol from the yeast cells contained in the aqueous phase. Dodecane was employed to dissolve-feed epoxide as well as to extract residual epoxide. 1,2-Epoxyhexane was used as a model substrate. By use of this membrane bioreactor, enantiopure (S)-1,2-epoxyhexane (>98% enantiomeric excess) was obtained with a volumetric productivity of 3.8 g l⁻¹ h⁻¹. The continuous-production system was operated for 12 days and resulted in 38 g enantiopure (S)-1,2-epoxyhexane. Received: 14 February 2000 / Received revision: 15 June 2000 / Accepted: 18 June 2000     

Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that the epoxide hydrolase of R. glutinis also belongs to the α/β hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min⁻¹ (mg protein)⁻¹ towards 1,2-epoxyhexane. Received: 2 August 1999 / Received revision: 4 October 1999 / Accepted: 10 October 1999     

Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis

Major characteristics, substrate specificities and enantioselectivities of epoxide hydrolases from various sources are described. Epoxide hydrolase activity in yeasts is discussed in more detail and is compared with activities in other microorganisms. Constitutively produced bacterial epoxide hydrolases are highly enantioselective in the hydrolysis of 2,2- and 2,3-disubstituted epoxides. A novel bacterial limonene-1,2-epoxide hydrolase, induced by growth on monoterpenes, showed high activities and selectivities in the hydrolysis of several substituted alicyclic epoxides. Constitutively produced epoxide hydrolases are found in eukaryotic microorganisms. Enzymes from filamentous fungi are useful biocatalysts in the resolution of aryl- and substituted alicyclic epoxides. Yeast epoxide hydrolase activity has been demonstrated for the enantioselective hydrolysis of various aryl-, alicyclic- and aliphatic epoxides by a strain of Rhodotorula glutinis. The yeast enzyme, moreover, is capable of asymmetric hydrolysis of meso epoxides and performs highly enantioselective resolution of unbranched aliphatic 1,2-epoxides. Screening for other yeast epoxide hydrolases shows that high enantioselectivity is restricted to a few basidiomycetes genera only. Resolution of very high substrate concentrations is possible by using selected basidiomycetes yeast strains.     

Epoxidation of 1,7‐octadiene by Pseudomonas oleovorans in a membrane bioreactor

A growing cell culture of Pseudomonas oleovorans was used to biotransform 1,7‐octadiene to 1,2‐epoxy‐7,8‐octene in a continuous‐flow bioreactor with an external membrane module. A dense silicone rubber membrane was used to contact an organic phase, containing both the reactant (1,7‐octadiene) and the growth substrate (heptane), with an aqueous biomedium phase containing the biocatalyst. Heptane and octadiene delivery to the aqueous phase, and epoxide extraction into the solvent, occurred by diffusion across the dense membrane under a concentration‐driving force. In addition, a liquid feed of heptane and octadiene was pumped directly into the bioreactor to increase the rate of delivery of these compounds to the aqueous phase. In this system 1,2‐epoxy‐7,8‐octene accumulated in a pure solvent phase, thus, product recovery problems associated with emulsion formation were avoided. Furthermore, no phase breakthrough of either liquid across the membrane was observed. In this system, the highest volumetric productivity obtained was 30 U.L⁻¹, and this was achieved at a dilution rate of 0.07 h⁻¹, 70 m².m⁻³ of membrane area, and a steady‐state biomass concentration of 2.5 g.L⁻¹. The system was stable for over 1250 h. Decreasing the dilution rate led to an increased biomass concentration, however, the specific activity was significantly reduced, and therefore, an optimal dilution rate was determined at 0.055 h⁻¹. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 601–611, 1999.     

Enhancing productivity for cascade biotransformation of styrene to (S)-vicinal diol with biphasic system in hollow fiber membrane bioreactor

Biotransformation is a green and useful tool for sustainable and selective chemical synthesis. However, it often suffers from the toxicity and inhibition from organic substrates or products. Here, we established a hollow fiber membrane bioreactor (HFMB)-based aqueous/organic biphasic system, for the first time, to enhance the productivity of a cascade biotransformation with strong substrate toxicity and inhibition. The enantioselective trans-dihydroxylation of styrene to (S)-1-phenyl-1,2-ethanediol, catalyzed by Escherichia coli (SSP1) coexpressing styrene monooxygenase and an epoxide hydrolase, was performed in HFMB with organic solvent in the shell side and aqueous cell suspension in the lumen side. Various organic solvents were investigated, and n-hexadecane was found as the best for the HFMB-based biphasic system. Comparing to other reported biphasic systems assisted by HFMB, our system not only shield much of the substrate toxicity but also deflate the product recovery burden in downstream processing as the majority of styrene stayed in organic phase while the diol product mostly remained in the aqueous phase. The established HFMB-based biphasic system enhanced the production titer to 143 mM, being 16-fold higher than the aqueous system and 1.6-fold higher than the traditional dispersive partitioning biphase system. Furthermore, the combination of biphasic system with HFMB prevents the foaming and emulsification, thus reducing the burden in downstream purification. HFMB-based biphasic system could serve as a suitable platform for enhancing the productivity of single-step or cascade biotransformation with toxic substrates to produce useful and valuable chemicals.

     

Benzene/toluene/p-xylene degradation. Part I. Solvent selection and toluene degradation in a two-phase partitioning bioreactor

A two-phase organic/aqueous reactor configuration was developed for use in the biodegradation of benzene, toluene and p-xylene, and tested with toluene. An immiscible organic phase was systematically selected on the basis of predicted and experimentally determined properties, such as high boiling points, low solubilities in the aqueous phase, good phase stability, biocompatibility, and good predicted partition coefficients for benzene, toluene and p-xylene. An industrial grade of oleyl alcohol was ultimately selected for use in the two-phase partitioning bioreactor. In order to examine the behavior of the system, a single-component fermentation of toluene was conducted with Pseudomonas sp. ATCC 55595. A 0.5-l sample of Adol 85 NF was loaded with 10.4 g toluene, which partitioned into the cell containing 1 l aqueous medium at a concentration of approximately 50 mg/l. In consuming the toluene to completion, the organisms were able to achieve a volumetric degradation rate of 0.115 g l⁻¹ h⁻¹. This system is self-regulating with respect to toluene delivery to the aqueous phase, and requires only feedback control of temperature and pH. Received: 16 November 1998 / Received revision: 28 March 1999 / Accepted: 9 April 1999     

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.     

An optimization study of carotenoid production by Rhodotorula glutinis DBVPG 3853 from substrates containing concentrated rectified grape must as the sole carbohydrate source

A multivariate statistical approach was employed for the optimization of conditions for carotenoid production by Rhodotorula glutinis DBVPG 3853 from a substrate containing concentrated rectified grape must as the sole carbohydrate source. Several experimental parameters (carbohydrate, yeast autolysate and salt concentrations, and pH) were tested at two levels by following a fractional factorial design. Carotenogenesis was most sensitive to both initial pH and yeast autolysate concentration. A Central Composite Design experiment was then performed by obtaining both second-order polynomial models and isoresponse diagrams where initial pH and yeast autolysate concentration were considered as variables. In this way it was possible to determine the conditions (pH = 5.78, yeast autolysate = 4.67 g L⁻¹) which maximize both the concentration of total carotenoids and that of β-carotene (6.9 mg L⁻¹ and 1100 μg L⁻¹ of culture fluid, respectively, after 120 h of fermentation). Journal of Industrial Microbiology & Biotechnology (2000) 24, 41–45. Received 23 February 1999/ Accepted in revised form 14 September 1999     

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.     

Doubly entrapped baker's yeast survives during the long-term stereoselective reduction of ethyl 3-oxobutanoate in an organic solvent

To attain long-term bioreaction in organic solvents with living microorganisms, we tried to protect the microorganisms from the toxicity of the solvent by immobilization. In this study, baker's yeast, which is not tolerant to organic solvents such as isooctane, was selected as a model microorganism and the immobilized living yeast cells were examined for activity in the steroselective reduction of ethyl 3-oxobutanoate to ethyl (S)-3-hydroxybutanoate in isooctane; an activity that correlated well with the viability of the yeast cells. It was found that double entrapment, that is, further entrapment of calcium-alginate-gel-entrapped cells with a urethane prepolymer, made it possible for the yeast to remain viable in isooctane, although other conventional immobilization methods, such as single entrapment using polysaccharide or synthetic resin prepolymers, were insufficient for its protection. Furthermore, doubly entrapped living yeast cells could carry out the stereoselective reduction in isooctane repeatedly for a long period (more than 1200 h) with occasional cultivation. Thus, double entrapment enabled a microorganism sensitive to organic solvents to survive over long-term bioreaction in an organic solvent. Received: 29 August 1997 / Received last revision: 24 December 1997 / Accepted: 13 January 1998     

Effect of mass transfer limitations on the enzymatic kinetic resolution of epoxides in a two-liquid-phase system

Optically active epoxides can be obtained by kinetic resolution of racemic mixtures using enantioselective epoxide hydrolases. To increase the productivity of the conversion of sparingly aqueous soluble epoxides, we investigated the use of a two-phase aqueous/organic system. A kinetic model which takes into account interphase mass transfer, enzymatic reaction, and enzyme inactivation was developed to describe epoxide conversion in the system by the epoxide hydrolase from Agrobacterium radiobacter. A Lewis cell was used to determine model parameters and results from resolutions carried out in the Lewis cell were compared to model predictions to validate the model. It was found that n-octane is a biocompatible immiscible solvent suitable for use as the organic phase. Good agreement between the model predictions and experimental data was found when the enzyme inactivation rate was fitted. Simulations showed that mass transfer limitations have to be avoided in order to maximize the yield of enantiomerically pure epoxide. Resolution of a 39 g/L solution of racemic styrene oxide in octane was successfully carried out in an emulsion batch reactor to obtain (S)-styrene oxide in high enantiomeric excess (>95% e.e.) with a yield of 30%.     

Caroteno-protein and exopolysaccharide production by co-cultures of Rhodotorula glutinis and Lactobacillus helveticus

The lactose-negative yeast Rhodotorula glutinis 22P and the homofermentative lactic acid bacterium Lactobacillus helveticus 12A were cultured together in a cheese whey ultrafiltrate containing 42 g L⁻¹ lactose. The chemical composition of the caroteno-protein has been determined. The carotenoid and protein contents are 248  μ g g⁻¹ dry cells and 48.2% dry weight. Carotenoids produced by Rhodotorula glutinis 22P have been identified as β-carotene 15%, torulene 10%, and torularhodin 69%. After separating the cell mass from the microbial association, the exopolysaccharides synthesized by Rhodotorula glutinis 22P were isolated from the supernatant medium in a yield of 9.2 g L⁻¹. The monosaccharide composition of the synthesized biopolymer was predominantly D-mannose (57.5%). Received 08 July 1996/ Accepted in revised form 11 December 1996     

Growth of Streptomyces hygroscopicus in rotating-wall bioreactor under simulated microgravity inhibits rapamycin production

Growth of Streptomyces hygroscopicus under conditions of simulated microgravity in a rotating-wall bioreactor resulted in a pellet form of growth, lowered dry cell weight, and inhibition of rapamycin production. With the addition of Teflon beads to the bioreactor, growth became much less pelleted, dry cell weight increased but rapamycin production was still markedly inhibited. Growth under simulated microgravity favored extracellular production of rapamycin, in contrast to a greater percentage of cell-bound rapamycin observed under normal gravity conditions. Received: 20 September 1999 / Received revision: 18 November 1999 / Accepted: 19 November 1999     

An extractive membrane biofilm reactor for degradation of 1,3-dichloropropene in industrial waste water

A bacterial biofilm, capable of mineralising a technical mixture of cis- and trans-1,3-dichloropropene (DCPE), was enriched on the biomedium side of an extractive membrane biofilm reactor (EMBR). The membrane separates the biomedium from the industrial waste water, in terms of pH, ionic strength and the concentration of toxic chemicals. The biofilm, attached to a silicone membrane, is able to mineralise DCPE after its diffusion through the membrane. Five bacterial strains with degradation capabilities were isolated from the metabolically active biofilm and further investigated in batch experiments. Two of them, Rhodococcus erythropolis strains EK2 and EK5, can grow with DCPE as the sole carbon source. Pseudomonas sp. EK1 utilises cis-3-chloroallylalcohol and cis-3-chloroacrylic acid, whereas the metabolite trans-3-chloroacrylic acid represents a dead-end product of the pathway of this strain. The other two strains, Delftia sp. EK3 and EK4, although unable to grow with DCPE as the carbon source, can transform DCPE and its upper-pathway intermediates at reasonable conversion rates. They may represent helper functions of the biofilm consortium, which mineralised up to 12.5 mmol DCPE per hour per gram of biomass protein. Higher feed rates in the EMBR (up to 15 mmol per hour per 100-l bioreactor volume) and shock loads corresponding to concentrations up to 1.8 mmol l⁻¹ led to a significant increase in the freely floating bacterial biomass in the reactor medium (OD₅₄₆= 0.2). At the standard operating feed rate of 1.8 mmol h⁻¹, the free biomass concentration was very low (OD₅₄₆= 0.04). Received: 23 April 1999 / Received revision: 1 July 1999 / Accepted: 5 July 1999     

Benzene/toluene/p-xylene degradation. Part II. Effect of substrate interactions and feeding strategies in toluene/benzene and toluene/p-xylene fermentations in a partitioning bioreactor

A two-phase aqueous/organic partitioning bioreactor scheme was used to degrade mixtures of toluene and benzene, and toluene and p-xylene, using simultaneous and sequential feeding strategies. The aqueous phase of the partitioning bioreactor contained Pseudomonas sp. ATCC 55595, an organism able to degrade benzene, toluene and p-xylene simultaneously. An industrial grade of oleyl alcohol served as the organic phase. In each experiment, the organic phase of the bioreactor was loaded with 10.15 g toluene, and either 2.0 g benzene or 2.1 g p-xylene. The resulting aqueous phase concentrations were 50 mg/l, 25 mg/l and 8 mg/l toluene, benzene and p-xylene respectively. The simultaneous fermentation of benzene and toluene consumed these compounds at volumetric rates of 0.024 g l⁻¹ h⁻¹ and 0.067 g l⁻¹ h⁻¹, respectively. The simultaneous fermentation of toluene and p-xylene consumed these xenobiotics at volumetric rates of 0.066 g l⁻¹ h⁻¹ and 0.018 g l⁻¹ h⁻¹, respectively. A sequential feeding strategy was employed in which toluene was added initially, but the benzene or p-xylene aliquot was added only after the cells had consumed half of the initial toluene concentration. This strategy was shown to improve overall degradation rates, and to reduce the stress on the microorganisms. In the sequential fermentation of benzene and toluene, the volumetric degradation rates were 0.056 g l⁻¹ h⁻¹ and 0.079 g l⁻¹ h⁻¹, respectively. In the toluene/p-xylene sequential fermentation, the initial toluene load was consumed before the p-xylene aliquot was consumed. After 12 h in which no p-xylene degradation was observed, a 4.0-g toluene aliquot was added, and p-xylene degradation resumed. Excluding that 12-h period, the microbes consumed toluene and p-xylene at volumetric rates of 0.074 g l⁻¹ h⁻¹ and 0.025 g l⁻¹ h⁻¹, respectively. Oxygen limitation occurred in all fermentations during the rapid growth phase. Received: 16 November 1998 / Received revision: 29 March 1999 / Accepted: 9 April 1999     

Succinoglycan production by solid-state fermentation with Agrobacterium tumefaciens

Succinoglycan was produced by cultivating Agrobacterium tumefaciens on various solid substrates, including agar medium, spent malt grains, ivory nut shavings, and grated carrots, impregnated with a nutrient solution. Fermentations were performed on a laboratory scale, both under static conditions and with agitation, using bottles and a prototype horizontal bioreactor. Several fermentation parameters were examined and optimized, including carbon and nitrogen composition, water content and layer thickness of the substrate. The yields and rheological properties of the polymers obtained under different fermentation conditions were compared. The highest succinoglycan yield was achieved in static cultivation, reaching 42 g/l of impregnating solution, corresponding to 30 g/kg of wet substrate. The polymer production in the horizontal bioreactor was faster, but the final yield was lower (29 g/l of impregnating solution). Received: 26 January 1999 / Received revision: 20 April 1999 / Accepted: 23 April 1999     

Improvement of ferulic acid bioconversion into vanillin by use of high-density cultures of Pycnoporus cinnabarinus

High-density cultures of Pycnoporus cinnabarinus were tested with a view to optimisation of ferulic acid bioconversion into vanillin. The dry weight was increased fourfold by using glucose, fructose or a mixture of glucose and phospholipids as carbon source instead of maltose, the carbon source previously used. 5 mmol l⁻¹ vanillin, i.e. 760 mg l⁻¹, was produced over 15 days with glucose-phospholipid medium. In contrast, formation of vanillin was lower using glucose or fructose compared to the maltose control. A bioreactor (2 l) with a glucose-phospholipid medium gave a molar yield of vanillin of 61% (4 mmol l⁻¹). An alternative strategy was to grow the fungus on a glucose or fructose medium for 3 days, then switch to maltose during the bioconversion phase: this method allowed 3.3 mmol l⁻¹ vanillin to be obtained in 10 days. Many by-products such as methoxyhydroquinone and vanillyl alcohol were also produced. Received: 19 February 1999 / Received revision: 4 June 1999 / Accepted: 4 June 1999     

Correlation between the physicochemical properties of organic solvents and their biocompatibility toward epoxide hydrolase activity in whole-cells of a yeast, Rhodotorula sp.

Epoxides are often highly hydrophobic substrates and the presence of an organic co-solvent within an aqueous bioreactor is in such cases indicated. The effect of 40 water-miscible and -immiscible organic solvents on epoxide hydrolase activity in whole-cells of the yeast Rhodotorula sp. UOFS Y-0448 was investigated. No formal correlation between solvent biocompatibility and physicochemical properties was deductible, although the introduction of hydroxyl groups increased biocompatibility. 1-Pentanol, 2-methylcyclohexanol and 1-octanol were the most biocompatible resulting in relatively low activity losses when used at up to 20% (v/v).     

A Glutathione S-Transferase with Activity towards cis-1,2-Dichloroepoxyethane Is Involved in Isoprene Utilization by Rhodococcus sp. Strain AD45

Rhodococcus sp. strain AD45 was isolated from an enrichment culture on isoprene (2-methyl-1,3-butadiene). Isoprene-grown cells of strain AD45 oxidized isoprene to 3,4-epoxy-3-methyl-1-butene, cis-1,2-dichloroethene to cis-1,2-dichloroepoxyethane, and trans-1,2-dichloroethene to trans-1,2-dichloroepoxyethane. Isoprene-grown cells also degraded cis-1,2-dichloroepoxyethane and trans-1,2-dichloroepoxyethane. All organic chlorine was liberated as chloride during degradation of cis-1,2-dichloroepoxyethane. A glutathione (GSH)-dependent activity towards 3,4-epoxy-3-methyl-1-butene, epoxypropane, cis-1,2-dichloroepoxyethane, and trans-1,2-dichloroepoxyethane was detected in cell extracts of cultures grown on isoprene and 3,4-epoxy-3-methyl-1-butene. The epoxide-degrading activity of strain AD45 was irreversibly lost upon incubation of cells with 1,2-epoxyhexane. A conjugate of GSH and 1,2-epoxyhexane was detected in cell extracts of cells exposed to 1,2-epoxyhexane, indicating that GSH is the physiological cofactor of the epoxide-transforming activity. The results indicate that a GSH S-transferase is involved in the metabolism of isoprene and that the enzyme can detoxify reactive epoxides produced by monooxygenation of chlorinated ethenes.     

Utilisation of saccharides in extruded domestic organic waste by Clostridium acetobutylicum ATCC 824 for production of acetone, butanol and ethanol

Domestic organic waste (DOW) collected in The Netherlands was analysed and used as substrate for acetone, butanol and ethanol (ABE) production. Two different samples of DOW, referred to as fresh DOW and dried DOW, were treated by extrusion in order to expand the polymer fibres present and to obtain a homogeneous mixture. The extruded material was analysed with respect to solvent and hot water extractives, uronic acids, lignin, sugars and ash. The total sugar content in the polymeric fractions of the materials varied from 27.7% to 39.3% (w/w), in which glucose represented the 18.4 and 25.1% of the materials, for fresh and dried DOW, respectively. The extruded fresh DOW was used as substrate for the ABE fermentation by the solventogenic strain Clostridium acetobutylicum ATCC 824. This strain was grown on a suspension of 10% (w/v) DOW in demineralised water without further nutrient supplement. This strain produced 4 g ABE/100 g extruded DOW. When C. acetobutylicum ATCC 824 was grown on a suspension of 10% (w/v) DOW hydrolysed by a combination of commercial cellulases and β-glucosidases, the yield of solvents increased to 7.5 g ABE/100 g extruded DOW. The utilisation of sugar polymers in both hydrolysed and non-hydrolysed DOW was determined, showing that only a small proportion of the polymers had been consumed by the bacteria. These results indicate that growth and ABE production on DOW is mainly supported by soluble saccharides in the medium. Received: 5 November 1999 / Received revision: 21 February 2000 / Accepted: 25 February 2000