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.     

Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor

The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity.     

Encapsulation of β-amylase in water-oil-water enzyme emulsion liquid membrane (EELM) bioreactor for enzymatic conversion of starch to maltose

Abstract

The β-amylase was encapsulated in emulsion liquid membrane (ELM), which acted as a reactor for conversion of starch to maltose. The membrane phase was consisted of surfactant (span 80), stabilizer (polystyrene), carrier for maltose transport (methyl cholate) and solvent (xylene). The substrate starch in feed phase entered into the internal phase by the process of diffusion and hydrolyzed to maltose by encapsulated β-amylase. Methyl cholate present in the membrane acts as a carrier for the product maltose, which helps in transport of maltose to feed phase from internal aqueous phase. The residual activity of β-amylase after the five-reaction cycle was found to decrease to ～70%, which indicated possibility to recycle the components of the emulsion and enzyme. The pH and temperature of the encapsulated enzyme were found to be optimum at 5.5 and 60?°C, respectively. The novelty of the present work lies in the development of Enzyme Emulsion Liquid Membranes (EELM) bioreactor for the hydrolysis of starch into maltose mediated by encapsulated β-amylase. The attempt has been made for the first time for the successful encapsulation of β-amylase into EELM. The best results gave the highest residual enzyme activity (94.1%) and maltose production (29.13?mg/mL).     

New reaction system for hydrocarbon oxidation by chloroperoxidase

A novel reaction system was developed to maximize the catalytic efficiency of chloroperoxidase (CPO, from Caldariomyces fumago) toward the oxidation of hydrocarbons. The reaction system consisted of an organic/aqueous emulsion comprising pure substrate and aqueous buffer supplemented with the surfactant dioctyl sulfosuccinate. The emulsion system attenuated not only the destabilizing effects of the substrate and product on the enzyme by emulsifying the compounds, but also oxidant toxicity (oxidative stress) by increasing substrate availability. As a result, CPO exhibited total turnover numbers (TTNs, defined as the amount of product produced over the catalytic lifetime of the enzyme) of ca. 20,000 mol product/mol enzyme for the oxidation of styrene, toluene, and o-, m-, p-xylenes. The TTNs are over 10-fold higher than those previously reported for the oxidation of benzylic hydrocarbons by CPO. This study represents a significant step toward the development of CPO as a practical catalyst for large-scale organic syntheses.     

Immobilization of Candida cylindracea lipase on colloidal liquid aphrons (CLAs) and development of a continuous CLA-membrane reactor

A novel technique is described for the immobilization of Candida cylindracea lipase in the soapy-shell of colloidal liquid aphrons (CLAs). CLAs consist of a micron-sized solvent droplet surrounded by a thin, aqueous, soapy-film and are stabilized by a mixture of nonionic and ionic surfactants. Retention of lipase within the CLAs is primarily determined by electrostatic interactions between the surface charges on the protein and those of the anionic surfactant used (SDS) because leakage of the lipase from dispersed CLAs was reduced at low continuous phase pH (相似文献   

Properties of glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate as an NADH-regeneration unit in enzyme reactors

Glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-regeneration unit were investigated. The conjugate has about two molecules of active and covalently linked NAD per tetramer. The specific activity of the enzyme moiety of the conjugate in the presence of exogenous NAD is about 77% of that of the native enzyme, and this decrease is mainly due to the decrease in the V_max value. The conjugate has the same pH-stability profile as the native enzyme and an internal activity of 0.26s⁻¹ (as a monomer); its NAD moiety has similar coenzyme activity to poly(ethylene glycol)-bound NAD. These results indicate that GlcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. The coupled reaction of GlcDH-PEG-NAD and lactate dehydrogenase was then studied. The specific activity of the conjugate is 1.1 s⁻¹ (as a tetramer), the recycling rate of the active NAD moiety is 0.54 s⁻¹, and the apparent K_m value for glucose is 24 mM. Kinetically, lactate dehydrogenase behaves like a substrate with an apparent K_m value of 1.8 units·ml⁻¹ in this coupled reaction system with low coenzyme concentration. l-Lactate was continuously produced from pyruvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDH-PEG-NAD and lactate dehydrogenase. GlcDH-PEG-NAD proved to be applicable in continuous enzyme reactors as an NADH-regeneration unit with a large molecular size.     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis–Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure–function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis–Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme–substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

Hydrolysis of olive oil catalyzed by surfactant-coated<Emphasis Type="Italic">Candida rugosa</Emphasis> lipase in a hollow fiber membrane reactor

In this study, we invetigated the hydrolysis of olive oil catalyzed by a surfactant-coatedCandida rugosa lipase in a hydrophilic polyacrylonitrile hollow fiber membrane reactor and then compared the results to those using the native lipase. The organic phase was passed through the hollow inner fibers of the reactor and consisted of either the coated lipase and olive oil dissolved in isooctane or the coated lipase dissolved in pure olive oil. The aqueous phase was pumped through the outer space. After 12 h and with conditions of 30°C, 0.12 mg enzyme/mL and 0.62 M olive oil, the substrate conversion of the coated lipase reached 60%. This was twice the conversion for the same amount of native lipase that was pre-immobilized on the membrane surface. When using pure olive oil, after 12 h the substrate conversion of the coated lipase was 50%. which was 1.4 times higher than that of the native lipase.     

Continuous hydrocarbon fermentation with colloidal emulsion feed. A kinetic model for two-liquid phase culture

Candida tropicalis was cultured in a chemostat-type fermentor with n-hexadecane, dispersed in water as submicron droplets, as the only carbon substrate. The emulsion as well as the aqueous medium were fed continuously into the fermentor. A Monod-type equation can correlate the specific group rate in the continuous fermentor with the concentration of submicron droplets. The same equation can also be fitted to the data for the conventional-type batch culture in the same fermentor in which an oil phase as well as an aqueous phase existed, if the hydrocarbon concentration in the aqueous phase excluding oil drops is employed as the substrate concentration. This demonstrates that Candida tropicalis takes up only submicron droplets of n-hexadecane as the carbon substrate.     

Systematic evaluation of the dihydrogen-oxidising and NAD+-reducing soluble [NiFe]-hydrogenase from Ralstonia eutropha H16 as a cofactor regeneration catalyst

Abstract

The oxygen-tolerant NAD⁺-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 has been described as a promising catalyst for cofactor regeneration in biocatalysed reductions. In this study, the actual potential of this enzyme for application in technical synthesis was evaluated. An overproduced, purified version of the enzyme was coupled to the carbonyl reductase from Candida parapsilosis (CPCR), where it allowed an almost quantitative conversion of the model substrate; total turnover numbers (TTN: n_product/n_enzyme) of up to 143,666 were achieved. This was distinctly superior to the commonly used NADH regenerating enzyme formate dehydrogenase (FDH) from Candida boidinii. In a systematic quantitative approach, maximum activity for NAD⁺ reduction was observed at 35 °C and pH 8, which corresponds to that of native SH. The half-life of the enzyme under these conditions was 5.3 hours. In the presence of sodium salts, distinct inhibitory effects were observed while ammonium and potassium ions increased the enzyme stability. Overall, a high but not unusual sensitivity of SH for changes in temperature, pH and mechanical stress in a reactor was found. Technical application in chemical synthesis can therefore be considered a feasible goal.     

A new dialysis fermentor for the production of high concentrations of extracellular enzymes

A new single-vessel dialysis fermentor is presented, which can be sterilized in situ and handled like the usual standard fermentors. The reactor is recommended for the production of high concentrations of extracellular enzymes. This is done in such a manner that the products are not spoiled through contact with the crude components of technical nutrient solutions. The reactor has been tested for the production of lipase with a culture of Staphylococcus carnosus. Sixty g·l⁻¹ of cellular dry mass and 227 mg·l⁻¹ lipase were produced within 46 h, using a continuously renewed high concentration of substrate. For planning the experiments and for the development of an optimal operation strategy, a mathematical model of the dialysis reactor system proved to be very helpful. The membrane permeability coefficient found for the limiting fraction of the yeast extract-peptone medium corresponds to a molecular weight between 300 and 400.     

Evaluation of kinetic data: What the numbers tell us about PRMTs

Protein arginine N-methyltransferase (PRMT) kinetic parameters have been catalogued over the past fifteen years for eight of the nine mammalian enzyme family members. Like the majority of methyltransferases, these enzymes employ the highly ubiquitous cofactor S-adenosyl-l-methionine as a co-substrate to methylate arginine residues in peptidic substrates with an approximately 4-μM median K_M. The median values for PRMT turnover number (k_cat) and catalytic efficiency (k_cat/K_M) are 0.0051 s⁻¹ and 708 M⁻¹ s⁻¹, respectively. When comparing PRMT metrics to entries found in the BRENDA database, we find that while PRMTs exhibit high substrate affinity relative to other enzyme-substrate pairs, PRMTs display largely lower k_cat and k_cat/K_M values. We observe that kinetic parameters for PRMTs and arginine demethylase activity from dual-functioning lysine demethylases are statistically similar, paralleling what the broader enzyme families in which they belong reveal, and adding to the evidence in support of arginine methylation reversibility.     

Novel strategy for enhancing productivity in l-DOPA synthesis: The electroenzymatic approach using well-dispersed l-tyrosine

Although l-DOPA (l-3,4-dihydroxyphenylalanine) is widely used as a drug for Parkinson's disease, there are critical drawbacks in the commercial synthetic method such as low conversion rate, poor productivity, and long operational time. In order to overcome these limitations, a novel electroenzymatic system using tyrosinase/carbon nanopowder/polypyrrole composite as a working cathode was reported with the outstanding conversion rate up to 95.9%. However, the productivity was still limited due to a low solubility of the substrate l-tyrosine in aqueous phase. Herein, we demonstrated a novel strategy for enhancing the productivity by employing well-dispersed l-tyrosine as the substrate. When using well-dispersed l-tyrosine, not only the concentration of the substrate was increased to 90.6 gL⁻¹ in aqueous phase but also the productivity was enhanced up to 15.3 gL⁻¹ h⁻¹. We also determined kinetic parameters in the electroenzymatic system and the kinetic results revealed that the outstanding conversion rate was based on the fast electrical reduction of the by-product to l-DOPA. Thus the electroenzymatic synthesis using well-dispersed l-tyrosine can be a potential candidate as a novel process for l-DOPA synthesis.     

Continuous ethanol production from raw starch using a reversibly soluble-autoprecipitating amylase and flocculating yeast cells

Direct ethanol production from raw starch was performed continuously using a combination of a reversibly soluble-autoprecipitating amylase (D-AS) in which Dabiase K-27 was immobilized covalently on an enteric coating polymer (hydroxypropyl methylcellulose acetate succinate, AS) as a carrier, and a flocculating yeast. Continuous production was carried out using a reactor equipped with a mixing vessel and a separation vessel. D-AS and the yeast were separated continuously from the product solution by self-sedimentation in the separation vessel and they were utilized repeatedly. In the continuous saccharification of raw starch by D-AS alone, the glucose productivity was about 3.6 g/l/h at a dilution rate (D) of 0.1 h⁻¹. In the continuous ethanol production from raw starch by a combination of D-AS and flocculating yeast cells, high ethanol productivity up to 2.0 g/l/h was achieved at D=0.1 h⁻¹. Although the enzymatic activity of D-AS is inactivated due to insolubilization of the enzyme by the accumulation of NaCl produced in controlling the pH in the reactor, it is possible to recover the D-AS enzymatic activity by removing the NaCl. This continuous fermentation system suggests a potential for effective ethanol production from raw starch, and it may be widely applicable in heterogeneous culture systems using solid substrates other than raw starch.     

Preparation of ACE-inhibitory peptides from milk protein in continuous enzyme membrane reactor with gradient dilution feeding substrate

In this study, a novel method of gradient dilution feeding substrate (GDFS) was established to improve the yield of angiotensin-converting enzyme (ACE) inhibitory peptides from milk protein. The hydrolysis process stability, enzymatic efficiency and kinetics of the method were studied and compared with traditional feeding modes, viz., adding water after feeding substrate or constant concentration feeding substrate. Results showed that the GDFS mode achieved the highest membrane flux and lowest fluctuation of protein concentration in the reactor. Moreover, the GDFS maximized protein conversion rate, yield of peptides, and ACE-inhibitory activity, with their values of 67.58 %, 138.51 g/(g*Neutrase), and 0.74 mg/mL (IC₅₀), respectively. In further study, the kinetic model of GDFS mode was successfully established with K_M of 69.481 g/L and V_max of 0.752 g·L⁻¹ min⁻¹. Based on the optimum condition of the kinetic model, the practical longest runtime was 720 min. Obtained results suggested that GDFS mode could be used as an alternative method in the preparation of high-yield bioactive peptides.     

Fed-batch hydrocarbon fermentation with colloidal emulsion feed

Candida tropicalis was cultured with n-hexadecane, dispersed in water as submicron droplets, as the only carbon substrate; the emulsion being fed continuously into a fermentor containing only an aqueous medium (fed-batch culture). The results have demonstrated that the organism takes up hydrocarbon accommodated in the aqueous phase as submicron droplets. The cell/substrate yield for the linear growth phase, where growth was limited by the supply of the substrate, was much higher than the yield for the exponential growth phase.     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-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 °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 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °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).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

Lipase-catalyzed kinetic resolution of (R,S)-1-phenylethyl propionate in an enzyme membrane reactor

Enzymatic stereoselective hydrolysis of (R,S)-1-phenylethyl propionate was performed in a stirred tank and in a biphasic enzyme membrane reactor. Lipase from Pseudomonas sp. was proved to be a good enantioselective catalyst for this reaction. The enzyme was covalently immobilized in a porous polyamide membrane (flat sheet as well as hollow-fibres) via glutaraldehyde. An influence of membrane hydrophobicity on reactor performance was observed. Initial lipase activity and productivity in the processes were equal to 1.05 × 10^?4, 1.3 × 10^?5 and 1.0 × 10^?5 mole/(h × mg of enzyme) in the case of native lipase, in the aromatic polyamide hydrophobic membrane reactor and in the hydrophilic polyamide-6 membrane reactor, respectively. The influence of some factors such as temperature, pH, buffer concentration, initial substrate concentration and addition of β-cyclodextrin derivatives on reaction rate and enantioselectivity was investigated and discussed. In the enzyme membrane reactor both organic and aqueous phases circulated countercurrently on both sides of the membrane. At a conversion degree of under 55–60%, pure enantiomer of the remaining ester (i.e. > 98%) was obtained.     

Calcium modulates membrane association,positional specificity,and product distribution in dual positional specific maize lipoxygenase-1

This study investigates how calcium modulates the properties of dual positional specific maize lipoxygenase-1, including its interaction with substrate, association with subcellular membrane and alteration of product distribution. Bioinformatic analyses identified Asp³⁸, Glu¹²⁷ and Glu²⁰¹ as putative calcium binding residues and Leu³⁷ as a flanking hydrophobic residue also potentially involved in calcium-mediated binding of the enzyme to subcellular membranes. Asp³⁸ and Leu³⁷ were shown to be important but not essential for calcium-mediated association of maize lipoxygenase-1 to subcellular membranes in vitro. Kinetic studies demonstrate that catalytic efficiency (V_max/K_m) shows a bell-shaped dependence on log of the molar ratio of substrate to unbound calcium. Calcium also modulates product distribution of the maize lipoxygenase-1 reaction, favoring 13-positional specificity and increasing the relative amount of (E,Z)-isomeric products. The results suggest that calcium regulates the maize lipoxygenase-1 reaction by binding to substrate, and by promoting binding of substrate to enzyme and association of maize lipoxygenase-1 to subcellular membranes.     

An integrated process for the production of toxic catechols from toxic phenols based on a designer biocatalyst

We describe the biocatalytic production of 3‐phenylcatechol from 2‐phenylphenol with the whole cell biocatalyst Escherichia coli JM101 (pHBP461). The recombinant produces 2‐hydroxybiphenyl 3‐monooxygenase, an enzyme from Pseudomonas azelaica HBP1. This enzyme introduces a hydroxyl‐group at the C₃‐position of a variety of 2‐substituted phenols, such as 2‐phenylphenol. This permits the biocatalytic production of 3‐substituted catechols, which are difficult to synthesize chemically. Both 2‐phenylphenol and 3‐phenylcatechol are highly toxic to E. coli. The toxic effects of 2‐phenylphenol were minimized by feeding this substrate to the reactor at a rate slightly below the maximum biooxidation rate. As a result, the substrate concentration in the reactor remained below toxic levels during the bioconversion. The toxic product formed was removed by continuous adsorption on the solid resin Amberlite™ XAD‐4. To this end the reaction mixture, containing the biocatalyst, was pumped continuously through an external loop with a fluidized bed of the resin. This resin efficiently and quantitatively adsorbed both 3‐phenylcatechol and the remaining trace amounts of 2‐phenylphenol. Consequently, the concentrations of these compounds were kept at subtoxic levels (below 100 mg L⁻¹) and gram amounts of 3‐phenylcatechol were produced with space–time yields of up to 0.39 g L⁻¹ h⁻¹. The product was recovered from the resin by acidic methanol elution and purified by recrystallization from n‐hexane resulting in overall yields exceeding 59%. The optimized system served as a surprisingly simple and efficient integrated process, that allows the bioconversion of toxic substrates to toxic products with whole cell biocatalysts. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 641–648, 1999.