The mechanism of flocculation of a Saccharomyces cerevisiae cell homogenate using polyethyleneimine

The effects of polyethyleneimine, electrolyte concentration and pH on the electrophoretic mobility values for whole yeast, yeast homogenate and borax clarified homogenate were observed. The polyethyleneimine concentration at which there is optimum clarification of the borax-clarified homogenate corresponded to a mobility value of zero. Increased floc size up to the point of zero mobility was obtained by use of polyethyleneimine of larger molecular weight and also higher concentration. The effect of ageing under shear on the size of the flocs was examined and indicated reversibility of break-up at low shear rates while at higher shear rates the flocs were irreversibly broken. Such growth and ageing observations suggest floc formation comprises a two-stage process; the initial formation of primary particles by polymer bridging, and the subsequent formation of larger flocs as these primary particles are brought together by charge neutralisation.UCL is the Biotechnology and Biological Science Research Council sponsored Advanced Centre for Biochemical Engineering and we are grateful to the Council for financial support.     

Selective flocculation and precipitation for the improvement of virus-like particle recovery from yeast homogenate

The purification of an intracellular product from a complex mixture of contaminants after cell disruption is a common problem in processes downstream of fermentation systems. This is particularly challenging for the recovery of particulate (80 nm in diameter) multimeric protein products, named virus-like particles (VLPs), from cell debris and other intracellular components. Selective flocculation for debris removal followed by selective precipitation of the target protein can be used as a preclarification step to aid purification. In this paper, selective borax flocculation of cell debris in yeast homogenate, followed by selective poly(ethylene glycol) precipitation of VLPs are defined with a view to demonstrating their potential in aiding the initial clarification stages of the purification sequence. The translation from laboratory scale to pilot scale operation is addressed, demonstrating the challenge of scale-up of solid-liquid separation stages for biological particle processing.     

Precipitation of nucleic acids with poly(ethyleneimine).

Removal of nucleic acids from cell extracts is a common early step in downstream processing for protein recovery. We report on the precipitation of nucleic acids from a homogenate of Saccharomyces cerevisiae by addition of the cationic polyelectrolyte poly(ethyleneimine) (PEI), focusing on the effect of PEI dosage on particle size, protein loss, and extent of nucleic acid removal in both batch and continuous mode. Better than 95% removal of nucleic acids from yeast homogenates was achieved by means of precipitation with PEI with protein losses of approximately 15% with or without previous removal of cell debris. The coprecipitated protein is predominately large molecular weight material and exhibits both low and high isoelectric points. Such treatment does not aggregate the cell debris; size distribution of the precipitated particles from a continuous precipitator is very similar to that for protein precipitation.     

Effect of chronic ethanol, catalase inhibitor 3-amino-1,2,4-triazole and clofibrate treatment on lipid peroxidation in rat myocardium

1. The effect of chronic alcohol consumption, catalase inhibitor 3-amino-1,2,4-triazole (amino-triazole) and peroxisome proliferator clofibrate on the level of Fe/ADP-ascorbate-induced lipid peroxidation has been studied in the rat myocardium. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde formation. 2. Combined us well as separate treatment with ethanol (36% of dietary calories) and aminotriazole caused elevation of the rate of lipid peroxidation in the nuclear-free homogenate or total particulate fraction of the rat heart. The most pronounced effect was noted during combined application of ethanol and aminotriazole. 3. Prolonged clofibrate treatment significantly increased the level of nonenzymatic lipid peroxidation in the rat myocardium. 4. Peroxidative alteration of the myocardial lipids in vivo was evaluated by measurement of conjugated dienes (absorbance at 233 nm). Separate ethanol, aminotriazole or clofibrate treatment did not affect the level of u.v. absorption of lipids from the total particulate fraction. However, when ethanol and aminotriazole were administered simultaneously an increase of conjugated diene formation was observed. 5. The data obtained confirm the hypothesis that ethanol or clofibrate-induced activation of the myocardial lipid peroxidation may be due to the increase of hydrogen peroxide-generating capacity of the heart microperoxisomes.     

The formation of cyclic inositol 1,2-monophosphate, inositol 1-phosphate, and glucose 6-phosphate by brain preparations stimulated with deoxycholate and calcium: a gas chromatographic study

A gas chromatographic method has been developed for the separation and isolation of water-soluble phosphates as trimethylsilyl ethers. With this method cyclic inositol 1,2-monophosphate and inositol 1-phosphate, derived from endogenous phosphatidylinositol, have been shown to increase when a particulate portion of brain homogenate is stimulated with deoxycholate and Ca⁺⁺, confirming earlier observations of Lapetina and Michell (1). Concomitant with the appearance of inositol phosphates is the stimulated formation of glucose 6-phosphate in the whole homogenate. Although ATP can replace deoxycholate and Ca⁺⁺ in a dialyzed homogenate, glucose 6-phosphate apparently does not arise by any known metabolic pathway but from another unidentified source.     

Integrated removal of nucleic acids and recovery of LDH from homogenate of beef heart by affinity precipitation

Lactate dehydrogenase (LDH) was purified from beef heart homogenate by affinity precipitation. The protein purification was integrated with nucleic acid removal and was done by precipitation of nucleic acids by addition of poly(ethylene imine) PEI onto which a ligand, Cibacron blue, had been coupled. The yield of LDH after elution from the precipitate was 63%, the purification factor 6.9 and the nucleic acid content was reduced by 98%. The capacity of the affinity polymer Cibacron blue-PEI is dependent on the nucleic acid concentration in the homogenate. The beef heart homogenate had an unfavourable ratio of nucleic acids to LDH. Precipitation with recirculated Cibacron blue-PEI, already complexed with some nucleic acids, improved the yield of the enzyme to 74%. The loss of Cibacron blue-PEI, when recirculated, was less than 1% after each cycle. This revised version was published online in July 2006 with corrections to the Cover Date.     

Origin, biochemical composition and vertical flux of particulate organic matter under the pack ice in Terra Nova Bay (Ross Sea, Antarctica) during late summer 1995

Water samples and particulate materials settling under the pack ice were collected in an ice-covered area near the Terra Nova Bay Italian Station during late summer 1995, in order to study short-term changes in the biochemical composition of particulate organic matter. At the end of the study period the phytoplankton biomass increase (up to >3.0 μg chlorophyll-a l⁻¹) was probably related to the intrusion under the pack ice of chlorophylls-enriched surface waters coming from the near ice-free area. Such increase was associated also with a notable increase in particulate organic matter concentrations, as well as in particulate organic matter vertical fluxes (up to >100 mg C m⁻² day⁻¹). Proteins were the most abundant biochemical class of particulate organic matter (on average about 49%), followed by lipids (29%) and carbohydrates (22%). By contrast, organic matter collected in the sediment trap was characterized by the dominance of lipids (about 55% of the total biopolymeric carbon flux) over carbohydrates (28%) and proteins (17%). The hydrolizable particulate biopolymeric carbon accounted for about 23% of total biopolymeric carbon. This value was about one-half of that found in ice-free waters, suggesting that the suspended particulate organic material under the pack ice was less digestible than in ice-free waters or was already partially digested. Despite this, and the decay of labile organic compounds in the sediment trap during the deployment, material settling towards the sea bottom under the pack ice in Terra Nova Bay, owing to its high lipid content, might represent an important high-quality food source for benthic consumers. Finally, assuming as possible the intrusion under sea ice of primary organic matter-enriched waters, we hypothesize the occurrence of a “fertilization” effect deriving from ice-melting areas towards under-ice waters, supplying the latter with an additional rate of primary organic matter. Accepted: 18 February 1999     

Effect of lipid peroxidation on Na+,K(+)-ATPase, 5'-nucleotidase and CNPase in mouse brain myelin

In the presence of H2O2, solutions of Fe2+ were applied to brain homogenate and isolated myelin from adult SWV control mice and the shiverer dysmyelinating mutant mouse as a source of a reactive oxygen species (Fenton reaction). Under these conditions, lipid peroxidation was initiated and measured as thiobarbituric acid-reactive oxidation products (TBAR). This was accompanied by 85% inhibition of myelin-associated Na+,K(+)-ATPase and 25% inhibition of 5'-nucleotidase. In contrast, CNPase activity was not altered. Studies on the shiverer mutant brain revealed that in spite of hypomyelination and prevalence of premature, myelin-like membranes in the homogenate, the myelin-related enzymes reacted as normal enzymes to peroxidation. Differences in the resistance of Na+,K(+)-ATPase to peroxidation in the brain homogenate and myelin suggest that the myelin enzyme is extremely sensitive to reactive oxygen toxicity.     

Total and hydrolizable particulate organic matter (carbohydrates, proteins and lipids) at a coastal station in Terra Nova Bay (Ross Sea, Antarctica)

We analysed quantity and quality of particulate organic matter during the austral summer 1994/1995 at a coastal station in Terra Nova Bay (Ross Sea, Antarctica). Our main aims were to investigate the origin and biochemical composition of particulate organic matter (POM), to measure its availability for consumers through the study of its digestible fraction (measured by using different enzymes separately) and to highlight the role of hydrolizable compounds in the organic matter diagenesis in the coastal waters at Terra Nova Bay. Temporal and spatial patterns of chlorophyll-a concentrations were reflected by the particulate organic carbon, nitrogen and total biopolymeric carbon concentrations, suggesting that most POM originated directly from phytoplankton. The most evident feature of POM in the coastal waters at Terra Nova Bay was the dominance of proteins (on average 57% of total biopolymeric particulate carbon), followed by carbohydrates (25%) and lipids (18%). We found that about 30% of the refractory particulate organic carbon (assumed to be present only after the complete exploitation of particulate organic nitrogen) did not originate from biopolymeric carbon (as sum of carbohydrate, protein and lipid carbon). This allows us to suggest the use of the digestible fraction of particulate biopolymeric carbon as a more accurate measure of the food availability of POM for consumers. In Terra Nova Bay coastal waters, most of the particulate protein pool was associated with large phytoplankton cells or phytodetritus. As a result, the protein pool appeared less available (i.e. less digestible) than the one present in oligotrophic waters where, conversely, most particulate organic nitrogen is sequestered into bacteria. The relative low availability of the protein pool, together with the rapid sinking of POM and the low remineralization rates of benthic heterotrophic microbes, are suggested as possible factors in determining the “inefficiency” in organic matter recycling of coastal waters at Terra Nova Bay, which behaves as a “loss type” system. Received: 17 June 1997 / Accepted 25 September 1997     

Experimental autoallergic sialadenitis in the LEW rat. II. Target antigens are associated with cell surface and intracellular particulate fractions derived from the submandibular gland

Experimental autoallergic sialadenitis (EAS) in the LEW rat is an autoimmune lymphocytic destruction of the submandibular gland (SMG) induced by sensitization with an SMG homogenate emulsified in adjuvant. Here we report that the antigens in the SMG homogenate involved in the induction of EAS can be localized to both cell-surface and intracellular particulate fractions. Differential centrifugation procedures were used to isolate 10,000g, 25,000g, 40,000g, and 100,000g particulate fractions from the SMG of LEW or WF rats. The particulate SMG fractions were used to sensitize female LEW rats for EAS induction. Since the protein concentrations of the fractions varied as a percentage of their composition in the whole homogenate, additional experiments were performed using equivalent protein concentrations for sensitization. Two weeks after sensitization, the SMGs of the sensitized animals were recovered and processed for histologic examination. Samples of the SMG fractions used for sensitization were also examined by electron microscopy. The 25,000g and 100,000g fractions consistently induced extensive mononuclear cell infiltrates and exocrine gland destruction in the SMGs while the 10,000g and 40,000g fractions caused infrequent and minimal inflammatory cell infiltrates in the glands. These data, combined with the electron microscopic analysis of the fractions, suggest that the target antigens in EAS reside on particulate fractions from the SMG that have the characteristics of the cell surface (25,000g) and microsomes (100,000g).     

Amino acid incorporation by cell fractions from the oviduct of the laying hen and the synthesis of egg-white proteins

1. Homogenates of the magnum of the hen oviduct have been fractionated by differential centrifuging. 2. Centrifuging at 600g for 10min. gave a pellet containing most of the particulate material of the cell, but on washing this fraction some particles were removed from it. The washed 600g pellet contained most of the DNA of the homogenate. 3. Centrifuging the 600g supernatants at 10000g for 10min. gave particulate fractions (I particles) richer in RNA than other fractions which were active in incorporating amino acids into protein in isolation. When minced tissue was incubated with radioactive amino acids before homogenizing these particles were the most radioactive of the cell fractions. 4. The pellet obtained by centrifuging the 10000g supernatant at 105000g for 60min. was very small; its RNA/protein ratio was raised compared with that of the homogenate. It only incorporated amino acids in isolation to a small extent or not at all. 5. The 105000g supernatant contained a large proportion of the protein of the homogenate. 6. The I particles could be subfractionated by layering over denser sucrose to give a pellet with lower RNA content and incorporating activity, and also suspended material richer in both these properties. 7. Treatment of the I particles with deoxycholate gave rise to particles sedimenting at 105000g for 60min. containing most of the RNA of the original particles, but only about 34% of the protein, and with a high activity in incorporating amino acids. 8. The I particles, or particles derived from them by deoxycholate treatment, required GTP and phosphoenolpyruvate for the incorporation of free amino acids. The omission of ATP reduced the incorporation to varying extents. Chicken-liver cell sap stimulated the activity. 9. Radioactive amino acids linked to transfer RNA were transferred to protein by I particles. GTP and phosphoenolpyruvate were required for this transfer. The phosphoenolpyruvate requirement could not be replaced by increasing the GTP concentration. ATP partially inhibited the transfer. 10. After incorporation by the cell-free system, the hot-trichloroacetic acid extract, but not the lipid extract, was radioactive. Ribonuclease and puromycin inhibited at low concentrations. Lecithinase-C was much less inhibitory. Transfer of amino acid, from a radioactive lipid-amino acid complex prepared from hen oviduct, was not detected. 11. After short periods of incubation of minced tissue with [(14)C]lysine some of the radioactive protein of the isolated I particles behaved as ovalbumin. The distribution of radioactivity in the protein resembled that in ovalbumin in soluble extracts of the tissue obtained after longer periods of incubation.     

Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified,long shelf-life polyethylenimine

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.     

A monolith purification process for virus-like particles from yeast homogenate

Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. The monolith structure provides a more open pore structure to improve accessibility for these materials and better mass transport from convective flow and reduced pressure drops. To examine the performance of this format for bioprocessing we selected the challenging capture of a VLP from clarified yeast homogenate. Using a recombinant Saccharomyces cerevisiae host it was found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity was shown to be three-fold superior for the monolith with equivalent 90% recovery of the VLP. To understand the impact of the crude feed material confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. It was seen that the lipid formed a layer on top of the column, even after regeneration of the column with isopropanol, resulting in increasing pressure drops with the number of operational cycles. Removal of the lipid pre-column significantly reduces the amount and rate of this fouling process. Using Amberlite/XAD-4 beads around 70% of the lipid was removed, with a loss of VLP around 20%. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11 mg/mL column to 0.25 mg/mL column.     

Notes and queries

If asymmetrical free-floating cells or cell elements are mixed with a material which can be extruded as a fiber, a preparation can be made in which the long axes are parallel to one another. For example, a 10% polyvinyl alcohol (Du Pont Elvanol 71-30) solution is mixed with a cell suspension, and extruded through a nozzle (60-300 β in diameter) into an aqueous solution of sodium sulfate (4%) and borax (0.2%). The resulting fiber is then lifted out, stretched and dried, after washing if necessary.     

The identification of thyroglobulin within a rat thryroid subcellular fraction

A particulatte fraction obtained from a rat thyroid homogenate by centrifugation at 27 000 × g was compared to the soluble fraction used for the preparation of thyroglobulin. By sodium dodecyl sulfate polycrylamide gel electrophoresis analysis after reduction, the soluble fraction inculded, mainly, three high molecular weight componenents which became strongly radioactive after an in vivo radioiodine injection. The particulate fraction on the same gels after reduction inlcude several low molecular weight components and a single high molecular weight component containing little radioiodine and showing, by electron microscopy, at least three types of structure: rough surfaced and smooth surfaced vesicles and apical vesicles (the secretory granules of thyroid follicular cells). The substrate present in the particulate fraction behaved antigenically like thyrolobulin. It is concluded that the particulate fraction contained nascent thyroglobulin inits undegraded form and that this material is probably the precursor to the various components encountered in the soluble fraction.     

A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds

Summary Sunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles.     

Delineation of a Particulate Thyrotropin-Releasing Hormone-Degrading Enzyme in Rat Brain by the Use of Specific Inhibitors of Prolyl Endopeptidase and Pyroglutamyl Peptide Hydrolase

The degradation of thyrotropin-releasing hormone in rat brain homogenates was studied in the presence of N-benzyloxycarbonyl-prolyl-prolinal and pyroglutamyl diazomethyl ketone, specific and potent active-site-directed inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase, respectively. Substantial TRH degradation was observed, suggesting the presence of another thyrotropin-releasing hormone-degrading enzyme(s). Reports of a thyrotropin-releasing hormone-degrading enzyme with narrow specificity that cleaves the pGlu-His bond of this tripeptide led us to develop a coupled assay using pGlu-His-Pro-2NA as the substrate to measure this activity. Cleavage of the pGlu-His bond of this substrate under conditions in which pyroglutamyl peptide hydrolase is not expressed occurred in the particulate fraction of a rat brain homogenate. This particulate pyroglutamyl-peptide cleaving enzyme was not inhibited by pyroglutamyl diazomethyl ketone but was inhibited by metal chelators such as EDTA and o-phenanthroline. The particulate pyroglutamyl-peptide cleaving enzyme was found predominantly in the brain. Activity in brain regions varied widely with highest levels present in cortex and hippocampus and very low levels in pituitary. The data suggest that degradation of thyrotropin-releasing hormone by the particulate fraction of a brain homogenate is catalyzed mainly by an enzyme that cleaves the pGlu-His bond of thyrotropin-releasing hormone but is distinct from pyroglutamyl peptide hydrolase.     

Guanylate cyclase. Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.     

The Incorporation of d-Glucosamine into Glycolipids and Glycoproteins of Membrane Preparations from Phaseolus aureus Hypocotyls

Radioactivity from d-glucosamine-(14)C is incorporated into particulate fractions of hypocotyls of Phaseolus aureus (mung bean) seedlings. Polyacrylamide gel electrophoresis of the sodium dodecyl sulfate-solubilized materials revealed that several polypeptide components varying considerably in molecular weight had become radioactive during the incubation. A considerable amount of (14)C was also recovered in lipid. Equilibrium centrifugation of the particulate material, isolated by initial centrifugation at 100,000 times gravity on sucrose density gradients revealed that radioactivity was recoverable in all of the membrane fractions along the gradient. It is suggested that glycoproteins and glycolipids containing amino sugar are normal constituents of such membranes. The ability of the particulate preparations to catalyze the transfer of N-acetyl-d-glucosamine from uridine diphospho-N-acetyl-d-glucosamine to endogenous acceptor material was also tested. Transfer was optimal at around pH 9 and in the presence of 10 mm Mg(2+), and it occurred largely into an unidentified lipid fraction. After equilibrium centrifugation of crude membrane material on sucrose gradients, a number of distinct fractions could be detected which would catalyze the transfer reaction. Uridine diphospho-d-glucose transferase activity showed a similar but not identical distribution along the gradient.     

Retention and regeneration of native NAD(H) in noncharged ultrafiltration membrane reactors: application to L-lactate and gluconate production

NAD(H) was retained in a noncharged ultrafiltration membrane reactor for the simultaneous and continuous production of L-lactate and gluconate with coenzyme regeneration. Polyethyleneimine (PEI), a 50-kDa cationic polymer, achieved coenzyme retentions above 0.8 for PEI/NAD(H) molar ratios higher than 5. The ionic strength of the inlet medium caused a decrease of NAD(H) retention that can be counterbalanced by an initial addition of 1% bovine serum albumin (BSA). Continuous reactor performance in the presence of PEI and BSA showed that NAD(H), glucose dehydrogenase, and lactate dehydrogenase were retained by 10-kDa ultrafiltration membranes; L-lactate and gluconate were produced at conversions higher than 95%. PEI enhanced the thermal stability of the enzymes used and increased the catalytic efficiency of glucose dehydrogenase, while no effect was found on the kinetic parameters of lactate dehydrogenase. A model that implements the kinetic equations of the two enzymes describes the reactor behavior satisfactorily. In brief, the use of PEI to retain NAD(H) is a new interesting approach to be widely applied in continuous synthesis with the large number of known dehydrogenases.