A facile process for adipic acid production in high yield by oxidation of 1,6-hexanediol using the resting cells of Gluconobacter oxydans.

BACKGROUND: Adipic acid (AA) is one of the most important industrial chemicals used mainly for the production of Nylon 6,6 but also for making polyurethanes, plasticizers, and unsaturated polyester resins, and more recently as a component in the biodegradable polyester poly(butylene adipate terephthalate) (PBAT). The main route for AA production utilizes benzene as feedstock and generates copious amounts of the greenhouse gas NO2. Hence, alternative clean production routes for AA from renewable bio-based feedstock are drawing increasing attention. We have earlier reported the potential of Gluconobacter oxydans cells to oxidize 1,6-hexanediol, a potentially biobased diol to AA. RESULTS: The present report involves a study on the effect of different parameters on the microbial transformation of 1,6-hexanediol to adipic acid, and subsequently testing the process on a larger lab scale for achieving maximal conversion and yield. Comparison of three wild-type strains of G. oxydans DSM50049, DSM2003, and DSM2343 for the whole-cell biotransformation of 10 g/L 1,6-hexanediol to adipic acid in batch mode at pH 7 and 30 °C led to the selection of G. oxydans DSM50049, which showed 100% conversion of the substrate with over 99% yield of adipic acid in 30 h. An increase in the concentrations of the substrate decreased the degree of conversion, while the product up to 25 g/L in batch and 40 g/L in fed-batch showed no inhibition on the conversion. Moreover, controlling the pH of the reaction at 5-5.5 was required for the cascade oxidation reactions to work. Cell recycling for the biotransformation resulted in a significant decrease in activity during the third cycle. Meanwhile, the fed-batch mode of transformation by intermittent addition of 1,6-hexanediol (30 g in total) in 1 L scale resulted in complete conversion with over 99% yield of adipic acid (approximately 37 g/L). The product was recovered in a pure form using downstream steps without the use of any solvent. CONCLUSION: A facile, efficient microbial process for oxidation of 1,6-hexanediol to adipic acid, having potential for scale up was demonstrated. The entire process is performed in aqueous medium at ambient temperatures with minimal greenhouse gas emissions. The enzymes involved in catalyzing the oxidation steps are currently being identified.

Immobilization to Positively Charged Cellulose Nanocrystals Enhances the Antibacterial Activity and Stability of Hen Egg White and T4 Lysozyme.

Antibacterial bionanostructures were produced from cellulose nanocrystals (CNC) with immobilized lysozyme from hen egg white (HEW) and T4 bacteriophage, respectively. The nanocrystals were prepared from microcrystalline cellulose by ammonium persulfate oxidation with a yield of 68% and having an average size of 250 nm and low polydispersity index. HEW lysozyme (HEWL) and T4 lysozyme (T4L) were immobilized to CNC by different mechanisms including adsorption and covalent coupling to carbodiimide-activated carboxylate groups and to glutaraldehyde-activated aminated CNC (Am-CNC), respectively. The effect of immobilization on the enzymatic activity (both lytic and hydrolytic) and antibacterial activity of the lysozymes was studied using different methods. Am-CNC-lysozyme conjugates retained the highest lytic activity, 86.3% and 78.3% for HEWL and T4L, respectively. They also showed enhanced bactericidal activity with high potency against Gram-positive as well as Gram-negative bacteria in a relatively shorter time as compared to the free enzymes and resulted in extensive cellular damage, as shown by transmission electron microscopy. The enhanced antibacterial activity was correlated with the increase in zeta potential of Am-CNC-lysozyme conjugates. The immobilized lysozyme preparations further exhibited enhanced storage stability at 4 and 22 °C.

Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects.

Biodegradable materials with plastic or elastomeric properties are in great demand for a variety of applications. Polyhydroxyalkanoates (PHAs), polyesters synthesized by microorganisms, possess such desired features. Industrial production of PHAs is currently achieved using recombinant Escherichia coli. Nevertheless, recent research on halophiles, salt requiring microorganisms, has shown a remarkable potential for biotechnological production of PHAs. The halophilic archaeon Haloferax mediterranei accumulates a co-polymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in large amounts using glucose, starch, and hydrolyzed whey as carbon sources. Chemical composition and molecular weight of PHAs produced by H. mediterranei can be modified depending on the substrate utilized as precursor. Phylogenetic studies on haloarchaeal enzymes able to polymerize the components of PHAs (i.e., PHA synthases) reveal a novel cluster, with a close relationship with PHA polymerases of bacteria and archaea found in marine-related niches. On the other hand, sequences of PHA synthases of two halophilic bacteria are more closely affiliated to synthases of Proteobacteria. Several bacterial species of the family Halomonadaceae accumulate PHAs. Halomonas boliviensis reached PHA yields and volumetric productivities close to the highest reported so far. Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process.

Designing Biobased Recyclable Polymers for Plastics.

Several concurrent developments are shaping the future of plastics. A transition to a sustainable plastics system requires not only a shift to fossil-free feedstock and energy to produce the carbon-neutral building blocks for polymers used in plastics, but also a rational design of the polymers with both desired material properties for functionality and features facilitating their recyclability. Biotechnology has an important role in producing polymer building blocks from renewable feedstocks, and also shows potential for recycling of polymers. Here, we present strategies for improving the performance and recyclability of the polymers, for enhancing degradability to monomers, and for improving chemical recyclability by designing polymers with different chemical functionalities.

A process for the production of ectoine and poly(3-hydroxybutyrate) by Halomonas boliviensis.

The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L(-1)) at 10-15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L(-1)) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L(-1), respectively, were obtained at 5-7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed-the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH (4) (+) , and PO (4) (3-) . In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L(-1) h(-1) and ectoine concentration, content, and volumetric productivity of 4.3 g L(-1), 7.2 wt.%, and 2.8 g L(-1) day(-1), respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L(-1) day(-1).

Optimizing refolding and recovery of active recombinant Bacillus halodurans xylanase in polymer-salt aqueous two-phase system using surface response analysis.

An experimental design was used to determine optimal conditions for refolding of a recombinant thermostable and alkaline active xylanase from Bacillus halodurans in PEG-phosphate two-phase system. The influence of different experimental variables on the enzyme recovery has been evaluated. To build the mathematical model and minimize the number of experiments for the design parameters, response surface methodology with a face-centered central composite design (CCF) was defined based on the conditions found by preliminary tests that resulted in the highest refolding yield. The adequacy of the calculated model for the response was confirmed by means of variance analysis and additional experiments. Analysis of contours of constant response as a function of pH, polyethylene glycol (PEG) molecular weight and concentration, and salt concentration for different enzyme loads revealed different effects of these five factors on the studied parameters. Recovery of more than 92% active xylanase was predicted for a system with 18.3% (w/w) PEG 1000, 14.4% (w/w) phosphate at pH 8.5, and enzyme load corresponding to a protein concentration of about 0.05 mg/g system. The yield of the refolded enzyme was found to be optimal at 22 degrees C. The validity of the response model was verified by a good agreement between predicted and experimental results.

The role of poly(ethyleneimine) in stabilization against metal-catalyzed oxidation of proteins: a case study with lactate dehydrogenase.

The protection provided by poly(ethyleneimine) (PEI) to muscle lactate dehydrogenase (LDH) in metal-catalyzed oxidation (MCO) systems (CuSO(4) or FeCl(2) combined with H(2)O(2)) was studied, and comparisons were made with the chelators EDTA and desferal, respectively. The analytical chelating capacity of PEI was estimated to be around 1 mol Cu(2+)/10 mol ethyleneimine for all molecular weights of the polymer. The effect of [PEI monomer]/[metal ion] molar ratio on the oxidatively induced aggregation of LDH exhibited a similar trend as that of the other chelators; aggregation was enhanced at lower ratios and subsequently decreased until it was undetectable with increasing ratio. In contrast, the LDH activity showed a monotonic increase with increasing concentrations of the chelator. Total protection to the enzyme by PEI was provided at concentrations lower than that needed for full chelation of the copper ions, i.e. at [PEI monomer]/[Cu(2+)] ratio above 9 in case of PEI 2000, and above 7 for PEI 25000 and 2.6 x 10(6), respectively. The polymer also provided protection against oxidation in an iron-based MCO system. Hydroxyl radical formation during the MCO reaction was inhibited in the presence of PEI. The polymer of higher molecular weights also exhibited a stronger free radical scavenging effect.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by a moderate halophile Yangia sp. ND199 using glycerol as a carbon source.

Yangia sp. ND199, a moderate halophile isolated from mangrove soil sample in Vietnam, was found to accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated carbon sources in a medium with 4.5% (w/v) NaCl. Cultivation with glycerol as carbon source and yeast extract as nitrogen source resulted in maximum cell dry weight of 5.7 g/l and PHBV content of 52.8 wt% (containing 2.9 mol% of 3HV) after 40 h. The 3HV content of the PHBV was the highest during initial stages of copolymer production and decreased with increase in the copolymer amount with time, but was not affected by changing the pH of the culture medium. Only homopolymer poly(3-hydroxybutyrate) was synthesized when monosodium glutamate was used as the nitrogen source. Fed-batch cultivation of Yangia sp. ND199 with glycerol and yeast extract gave PHBV content and productivity of 53.2 wt% and 0.44 g/l/h, respectively, which were reduced to 40.6 wt% and 0.25 g/l/h, respectively, with crude glycerol as carbon source. Both the copolymer content and productivity were improved to 56 wt% and 0.61 g/l/h, respectively, by using 1:1 mixture of crude glycerol and high fructose corn syrup. This is the first report of PHBV production by a wild-type halophilic bacterium using glycerol as carbon source.

Efficient poly(3-hydroxypropionate) production from glycerol using Lactobacillus reuteri and recombinant Escherichia coli harboring L. reuteri propionaldehyde dehydrogenase and Chromobacterium sp. PHA synthase genes.

Poly(3-hydroxypropionate), P(3HP), is a polymer combining good biodegradability with favorable material properties. In the present study, a production system for P(3HP) was designed, comprising conversion of glycerol to 3-hydroxypropionaldehyde (3HPA) as equilibrium mixture with 3HPA-hydrate and -dimer in aqueous system (reuterin) using resting cells of native Lactobacillus reuteri in a first stage followed by transformation of the 3HPA to P(3HP) using recombinant Escherichia coli strain co-expressing highly active coenzyme A-acylating propionaldehyde dehydrogenase (PduP) from L. reuteri and polyhydroxyalkanoate synthase (PhaCcs) from Chromobacterium sp. P(3HP) content of up to 40% (w/w) cell dry weight was reached, and the yield with respect to the reuterin consumed by the cells was 78%. Short biotransformation period (4.5h), lack of additives or expensive cofactors, and use of a cheap medium for cultivation of the recombinant strain, provides a new efficient and potentially economical system for P(3HP) production.

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts.

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for beta-galactosidase activity, were used for the synthesis of 1-hexyl-beta-d-galactoside and 1-octyl-beta-d-galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6-36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50mM lactose for at least a week.

Recovery of recombinant cutinase using detergent foam.

Foam generated by vigorous stirring of a nonionic detergent, Triton X-114, was used for the recovery of recombinant cutinase expressed by Saccharomyces cerevisiae. The enzyme with a hydrophobic fusion tag, (Trp-Pro)(4), was recovered with a higher yield as compared to the wild-type cutinase, indicating the involvement of hydrophobic interactions in protein isolation with the foam. The influence of various factors including volume, dilution, pH, different additives, and cell concentration in the medium on enzyme recovery was investigated. Interaction of the enzyme with detergent was monitored using fluorescence spectroscopy. No significant changes in protein conformation after the isolation procedure were observed using circular dichroism.

Semicarbazide-functionalized resin as a new scavenger for in situ recovery of 3-hydroxypropionaldehyde during biotransformation of glycerol by Lactobacillus reuteri.

3-Hydroxypropionaldehyde (3HPA), a potential C3-platform chemical for a biobased industry, is produced from glycerol using Lactobacillus reuteri through its glycerol dehydratase activity. However, the process is characterized by low yield and productivity due to toxic effects of 3HPA on the biocatalyst activity. In this study, a semicarbazide-functionalized resin was prepared, evaluated for adsorption and in situ recovery of 3HPA during biotransformation of glycerol. Adsorption of 3HPA onto the resin was characterized as "S-curve model", increasing with increasing initial 3HPA concentration, and reached a maximum of 9.48 mmol/g(resin) at 71.54 mM 3HPA used. Desorption of 3HPA was evaluated using water and different acids, and was enhanced by acetic acid with organic modifiers. Repeated adsorption­desorption of 3HPA in batch resulted in elution of 13­66.5% of the bound 3HPA during at least three sequential cycles using water and acetic acid, respectively as eluants. Using the resin for in situ product removal led to more than 2 times higher productivity of 3HPA.

Biotransformation of glycerol to 3-hydroxypropionaldehyde: improved production by in situ complexation with bisulfite in a fed-batch mode and separation on anion exchanger.

3-Hydroxypropionaldehyde (3HPA) is an important C3 chemical that can be produced from renewable glycerol by resting whole cells of Lactobacillus reuteri. However the process efficiency is limited due to substrate inhibition, product-mediated loss of enzyme activity and cell viability, and also formation of by-products. Complex formation of 3HPA with sodium bisulfite and subsequent binding to Amberlite IRA-400 was investigated as a means of in situ product recovery and for overcoming inhibition. The adsorption capacity and -isotherm of the resin were evaluated using the Langmuir model. The resin exhibited maximum capacity of 2.92 mmol complex/g when equilibrated with 45 mL solution containing an equilibrium mixture of 2.74 mmol 3HPA-bisulfite complex and 2.01 mmol free 3HPA. The dynamic binding capacity based on the breakthrough curve of 3HPA and its complex on passing a solution with 2.49 mmol complex and 1.65 mmol free 3HPA was 2.01 mmol/g resin. The bound 3HPA was desorbed from the resin using 0.20 M NaCl with a high purity as a mixture of complexed- and free 3HPA at a ratio of 0.77 mol/mol. Fed-batch biotransformation of glycerol (818.85 mmol) with in situ 3HPA complexation and separation on the bisulfite-functionalized resin resulted in an improved process with consumption of 481.36 mmol glycerol yielding 325.54 mmol 3HPA at a rate of 17.13 mmol/h and a yield of 68 mol%. Also, the cell activity was maintained for at least 28 h.

Batch- and continuous propionic acid production from glycerol using free and immobilized cells of Propionibacterium acidipropionici.

Propionic acid production from glycerol was studied using Propionibacterium acidipropionici DSM 4900 cells immobilized on polyethylenimine-treated Poraver (PEI-Poraver) and Luffa (PEI-Luffa), respectively. Using PEI-Luffa, the average productivity, yield and concentration of propionic acid from 40 g L(-1) glycerol were 0.29 g L(-1) h(-1), 0.74 mol(PA) mol(Gly)(-1) and 20 g L(-1), respectively, after four consecutive recycle-batches. PEI-Poraver supported attachment of 31 times higher amounts of cells than PEI-Luffa and produced 20, 28 and 35 g L(-1) propionic acid from 40, 65 and 85 g L(-1) glycerol, respectively (0.61 mol(PA) mol(Gly)(-1)). The corresponding production rates were 0.86, 0.43 and 0.35 g L(-1) h(-1), which are the highest reported from glycerol via batch or fed-batch fermentations for equivalent propionic acid concentrations. Using a continuous mode of operation at a dilution rate of 0.1 h(-1), cell washout was observed in the bioreactor with free cells; however, propionic acid productivity, yield and concentration were 1.40 g L(-1) h(-1), 0.86 mol(PA) mol(Gly)(-1), and 15 g L(-1), respectively, using immobilized cells in the PEI-Poraver bioreactor. The choice of the immobilization matrix can thus significantly influence the fermentation efficiency and profile. The bioreactor using cells immobilized on PEI-Poraver allowed the fermentation of higher glycerol concentrations and provided stable and higher fermentation rates than that using free cells or the cells immobilized on PEI-Luffa.

Polyethyleneimine-protein interactions and implications on protein stability.

Protein stability assessment of seven model proteins in the presence of low molecular weight polyethyleneimine (PEI, MW 2000 Da) was performed. Thermodynamic stability, monitored by circular dichroism (CD) spectroscopy, showed that the polymer did not have a major effect on the melting temperature (T(m)) of the basic proteins - muscle lactate dehydrogenase (LDH), ribonuclease A, lysozyme and cutinase, while for the acidic ones - human growth hormone, human serum albumin and heart lactate dehydrogenase - there was a shift in T(m) towards lower temperatures. The secondary structures of the basic proteins were essentially the same, with none or a slight increase in the CD spectra, in presence of the polymer. In the case of the acidic proteins, the CD spectra were diminished mostly due to phase separation. Assuming a homogeneous distribution of the net charge on the protein surface a quantitative inverse relationship was established between surface charge density of the acidic proteins and the PEI(2000) concentration required for maximum flocculation. Despite lowering the thermal stability of acidic proteins, PEI(2000) was seen to protect heart LDH at an increasing oxidative stress.

Poly(3-hydroxybutyrate) production by Halomonas boliviensis in fed-batch culture.

High poly(3-hydroxybutyrate) (PHB) content and volumetric productivity were achieved by fed-batch culture of Halomonas boliviensis using a defined medium. Initial shake flask cultivations in a minimal medium revealed that the growth of H. boliviensis was supported only when the medium was supplemented with aspartic acid, glycine, or glutamine. Addition of 0.1% (w/v) glutamine in the medium resulted in the highest cell dry weight (CDW; 3.9 g l(-1)). Glutamine was replaced by the less expensive monosodium glutamate (MSG) in the medium without any notable change in the final cell density. Effect of initial concentrations of NH(4)Cl and K(2)HPO(4) on cell growth and PHB accumulation by H. boliviensis was then analyzed using a fed-batch fermentation system. The best conditions for PHB production by H. boliviensis were attained using 0.4% (w/v) NH(4)Cl and 0.22% (w/v) K(2)HPO(4) and adding MSG intermittently to the fermentor. Poly(3-hydroxybutyrate) content and CDW reached 90 wt.% and 23 g l(-1), respectively, after 18 h of cultivation. In order to increase CDW and PHB content, MSG, NH(4)Cl, and K(2)HPO(4) were initially fed to the fermentor to maintain their concentrations at 2%, 0.4%, and 0.22% (w/v), respectively, and subsequently their feed was suppressed. This resulted in a CDW of 44 g l(-1), PHB content of 81 wt.%, and PHB volumetric productivity of 1.1 g l(-1) h(-1).

Optimizing conditions for poly(beta-hydroxybutyrate) production by Halomonas boliviensis LC1 in batch culture with sucrose as carbon source.

Halomonas boliviensis LC1 is able to accumulate poly(beta-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l(-1) CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l(-1) and 14 g l(-1), respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l(-1) in 17 h but reduced the PHB content in the cells to 52 wt.%.

Purification of plasmid DNA with polymer-salt aqueous two-phase system: optimization using response surface methodology.

An experimental design was used to optimize plasmid purification from an alkaline lysate of Escherichia coli cells using PEG-sodium citrate aqueous two-phase systems (ATPS), and to evaluate the influence of pH, PEG molecular weight, tie line length, phase volume ratio, and lysate load. To build the mathematical model and minimize the number of experiments for the design parameters, response surface methodology (RMS) with an orthogonal rotatable central composite design was defined based on the conditions found for the highest purification by preliminary tests. The adequacy of the calculated models for the plasmid recovery and remaining RNA were confirmed by means of variance analysis and additional experiments. Analysis of contours of constant response as a function of pH, PEG molecular weight, tie line length, and cell lysate load for three different phase volume ratios revealed different effects of these five factors on the studied parameters. Plasmid recovery of 99% was predicted for a system with PEG 400, pH 6.9, tie line length of 38.7%, phase volume ratio of 1.5, and lysate load of 10% (v/v). Under these conditions the predicted RNA removal was 68%.

Lactate production in an integrated process configuration: reducing cell adsorption by shielding of adsorbent.

The problem of binding of microbial cells to an adsorbent matrix during in situ recovery of bioproducts from a fermentation broth has been addressed by shielding the adsorbent with a thin layer of a non-ionic polymer. Extractive bioconversion of lactic acid by integrating ion-exchange adsorption with the fermentation stage was studied. The effect of coating of the ion-exchanger with agarose on product recovery and cell adsorption was evaluated. Extractive fermentation with both uncoated and coated resin resulted in an increase in reactor productivity as compared to the normal fermentation. The free cell density in the system with agarose-coated beads was similar to that in control fermentation, but was significantly lower in the system with the uncoated ion-exchanger. Electron microscopic scanning of the bead surface after passage of the fermentation broth showed cells attached to the native adsorbent but not to the coated one.

Selective recovery of lactate dehydrogenase using affinity foam.

Selective isolation of lactate dehydrogenase (LDH) from porcine muscle extract was studied using foam generated from the vigorous stirring of a non-ionic surfactant, Triton X-114 derivatized with Cibacron blue. The cloud point of the surfactant-dye conjugate was higher than that of the native Triton X-114, and also the foam prepared from the affinity surfactant was more rigid taking a longer time to collapse. The equilibrium dissociation constant between pure LDH and surfactant-dye conjugate was 5.0 microM as compared to the value of 2.2 microM for the enzyme and free dye as measured by differential spectroscopy. The isolation procedure involved mixing of the porcine muscle extract with the affinity foam, separating and collapsing the foam, and warming the solution formed to 37 degrees C to yield the surfactant-dye phase and an aqueous phase containing the enzyme. The effect of surfactant concentration and protein load on enzyme recovery and purification was investigated. Under optimal conditions, LDH was quantitatively recovered with high purification factor in a very short time. Both recovery and purification were higher when foam prepared from an equivalent mixture of surfactant-dye conjugate and unmodified surfactant was used. The selectivity of interaction between LDH and detergent-dye conjugate was confirmed by lowered recovery when NADH was included during the binding step.