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Biochemical Engineering Journal (v.38, #3)
Kinetic modeling of lactic acid production from molasses using Enterococcus faecalis RKY1 by Anjana D. Nandasana; Surendra Kumar (pp. 277-284).
A kinetic model has been developed for batch fermentation of cane-sugar molasses for lactic acid production by Enterococcus faecalis RKY1. Parameters of the kinetic model have been determined based on experimental data by using genetic algorithm. The values of key kinetic constants are maximum specific growth rate ( μmax), 1.6h−1, growth-associated constant for lactic acid production ( α), 0.26gg−1; maximum specific lactic acid production rate ( qp,max), 3gg−1h−1; maximum specific sugar utilization rate ( qs,max), 3.33gg−1h−1. It has been observed that the growth of biomass and lactic acid production are affected by lactic acid inhibition and it has been taken into account with an exponential term. However, effects of substrate limitation and substrate inhibition have been found to be relatively small. When compared with batch experimental data, the model provides good predictions for growth of biomass, sugar consumption and lactic acid production profiles on media with initial molasses concentration ranging from 130 to 333g/l with few exceptions. Effect of pH on kinetic parameters has also been studied. It has been found that the growth of biomass and lactic acid production are strongly influenced by pH. Optimum pH is found to be 7.
Keywords: Enterococcus faecalis; Fermentation; Growth kinetics; Lactic acid; Modeling; Molasses
Optimization of cultivation conditions for the production of 1,3-dihydroxyacetone by Pichia membranifaciens using response surface methodology by Zhiqiang Liu; Zhongce Hu; Yuguo Zheng; Yinchu Shen (pp. 285-291).
By isolating soil samples, we obtained a novel Pichia strain (sp. ZJB-0009) capable of producing 1,3-dihydroxyacetone from glycerol. Based on the physiological, biochemical characteristics, and ITS rDNA gene sequence analysis, the strain was identified as Pichia membranifaciens and subsequently named P. membranifaciens ZJB-0009. A mathematical model was developed to investigate the influences of various fermentation parameters and to predict the optimum fermentation conditions for 1,3-dihydroxyacetone production. The maximal concentration of 1,3-dihydroxyacetone (12.91g/L) was predicted to occur when the pH was 6.74, the incubation temperature was 29.6°C, and the growth time was 48.8h. A repeat fermentation of 1,3-dihydroxyacetone by P. membranifaciens ZJB-0009 was carried out in a 15-L fermenter under the optimized conditions for the verification of optimization. The maximum concentration of 1,3-dihydroxyacetone was 13.57g/L, which was significantly higher than that obtained under unoptimized conditions.
Keywords: Pichia membranifaciens; 1,3-Dihydroxyacetone; Identification; Medium optimization; Response surface methodology; Box–Behnken design
Biological manganese removal from potable water using trickling filters by A.G. Tekerlekopoulou; I.A. Vasiliadou; D.V. Vayenas (pp. 292-301).
Two pilot-scale trickling filters were constructed and tested for manganese removal from potable water, using different fractions of silicic gravel as support media (mono- and multilayer filter). Manganese oxidation in drinking water was found to be cause by both biological oxidation and heterogeneous catalytic paths. Mixed culture populations were used to inoculate the trickling filters and the feed manganese concentrations and volumetric flow rates (VFRs) were between 0.6–2.0mg/l and 500–2000ml/min, respectively. The monolayer filter was flooded for high VFRs, and it was very effective for all conditions tested (100% removal efficiency, up to 2850mgMn/day). The multilayer filter was less effective for high manganese concentrations but it could remove up to 3250mgMn/day. A new mathematical model was developed assuming heterogeneous autocatalytic and biological as the main oxidation manganese paths. First order kinetics was used to describe the heterogeneous catalytic oxidation, while Monod-type kinetics was used to describe the net biological manganese oxidation. The simplicity of the pilot-scale design, the lack of need for an external mechanical aeration source and the ability to predict operation of the system offers a very attractive solution for manganese removal from potable water.
Keywords: Manganese removal; Biofilm; Modeling; Trickling filter; Potable water; Support media
Stability of sludge flocs under shear conditions by Guo-Ping Sheng; Han-Qing Yu; Xiao-Yan Li (pp. 302-308).
The shear stability and surface characteristics of aerobic and anaerobic flocs that are formed in biological wastewater treatment processes were investigated. The aerobic flocs of activated sludge were found to be more stable during the shear test, with a low shear sensitivity of 0.032, than the anaerobic flocs of sludge digestion, which had a higher shear sensitivity of 0.088. In addition, the surface characteristics of the sludge flocs, such as hydrophobicity, surface charge and fractal dimension, changed rather differently for the aerobic and anaerobic flocs during the shear tests. The significant changes took place in the first 30min when the flocs were exposed to an elevated shear and little change was observed in the later stages of the shear tests. For quantitative description on the concentration of small primary particles in sludge solution, a modified adhesion–erosion model was used to describe the stability of the sludge flocs under shear conditions. The modification takes into account the effect of Brownian motion on particle dynamics in a sludge suspension, which effectively extends the application of the model to systems with a low shear intensity.
Keywords: Aerobic sludge; Anaerobic sludge; Shear sensitivity; Sludge flocs; Stability; Surface characteristics
Selection of recombinant antibody-producing E. coli cells by means of toxin conjugates by F. Sellrie; B. Micheel (pp. 309-313).
The toxicity of hapten–toxin conjugates can be neutralized by steric hindrance using antibodies or other molecules binding to the hapten part of the conjugates. This principle was proven by neutralization of the growth-inhibiting effect of fluorescein–ampicillin and biotin–ampicillin conjugates on E. coli cells after binding of the anti-fluorescein antibody B13-DE1 or streptavidin to the corresponding conjugates. In addition to that E. coli cells producing a single-chain B13-DE1 antibody fragment (scFv B13-DE1) are resistant to the growth-inhibiting effect of a fluorescein–ampicillin conjugate, and scFv B13-DE1-producing E. coli cells can be selected out of a majority of E. coli cells producing other single-chain antibody fragments. This principle offers therefore a general possibility for the selection of cells producing antibodies or other binding molecules.
Keywords: Antibody selection; Ampicillin; Fluorescein; Toxin; Toxin conjugate
The influence of copolymer ratio and drug loading level on the biocompatibility of P(3HB- co-4HB) synthesized by Cupriavidus sp. (USMAA2-4) by Jee-Wei Chee; A.A. Amirul; T.S. Tengku Muhammad; M.I.A. Majid; S.M. Mansor (pp. 314-318).
Poly(3-hydroxybutyrate- co-4-hydroxybutyrate) [P(3HB- co-4HB)] belongs to the members of polyhydroxyalkanoates (PHA) family with better degradation and biocompatibility properties. The biocompatibility of P(3HB- co-4HB) polyesters was evaluated in vitro. Mouse fibroblast cell line (L929) was inoculated on films made of P(3HB- co-4HB) of various compositions, various drug loading levels and poly(dl-lactide- co-glycolide) (50:50) (positive control). The effect of P(3HB- co-4HB) composition and surface morphology for both loaded and unloaded films was investigated. The low crystallinity degree of P(4HB) provided fairly regular and smooth surface in P(3HB- co-4HB) with higher 4HB composition. It was found that the cell growth was poor on PHB. As 4HB content increased, the surface morphology changed from a coralloid surface to a smoother one, leading to increasing cell viability from 2.7×105 to 12.2×105cells/ml. The surface morphology for P(3HB- co-4HB) loaded with Mitragyna speciosa crude extract also changed from smoother to a coralloid one, and even rougher when the drug loading level increased. MTS assay showed the cell growth on loaded films decreased with an increase in drug loading. This is due to the surface morphological changes as cells preferred smoother surfaces. Therefore, this demonstrated that the biocompatibility of P(3HB- co-4HB) was contributed by 4HB fraction as well as the drug loading level. The results also illustrated that the biocompatibility of P(3HB- co-4HB) was comparable with the positive control (PLGA), and thus can possibly be used as a matrix for drug carrier and tissue engineering applications.
Keywords: Polyhydroxyalkanoates; Poly(3-hydroxybutyrate-; co-; 4-hydroxybutyrate); P(3HB-; co-; 4HB); Surface morphology; Porosity; Biocompatibility
Kinetics modelling of biosorption by algal biomass from binary metal solutions using batch contactors by Vítor J.P. Vilar; Cidália M.S. Botelho; Rui A.R. Boaventura (pp. 319-325).
The biosorption from four binary metal solutions, Pb2+/Cu2+, Pb2+/Cd2+, Pb2+/Zn2+ and Cd2+/Zn2+, onto algae Gelidium, algal waste and composite material, at pH 5.3 and 20°C, were studied in batch adsorber. A mass transfer model based on a one-dimensional intraparticle diffusion rate, combined with a binary Langmuir isotherm, was applied to describe the overall biosorption rate of binary metal solutions mixture in flat seaweed biomass. A good correlation has been found between mass transfer model and experimental data for all systems. Model equations were solved numerically yielding the metal ions homogeneous diffusion coefficients. The biosorbents showed a preference for sorbing Pb over Cd, Zn and Cu even for low lead initial concentrations. Cu and Pb show a similar competition for the binding sites, although a higher uptake was found for copper ions, due to the higher initial copper concentration.
Keywords: Algae; Algal waste; Modelling; Two-metal systems; Biosorption; Kinetic parameters
Effect of osmolyte or GdnHCl on volumetric properties of aqueous solutions containing cyclic dipeptides by Pannur Venkatesu; Ming-Jer Lee; Ho-Mu Lin (pp. 326-340).
To understand the biomolecular interactions between osmolytes or guanidine hydrochloride (GdnHCl) with proteins, we have measured the densities ( ρ) of a homologous series of cyclic dipeptides (CDs) in water and in aqueous solutions of osmolytes or GdnHCl through the high precision vibrating tube digital densitometer up to saturated state, as a function of solute concentrations at 25°C under atmospheric pressure. The materials investigated in the present study included the CDs of cyclo(Gly-Gly), cyclo(Ala-Gly), cyclo(Ala-Ala), cyclo(Leu-Ala) and cyclo(Val-Val), the osmolytes of trimethylamine N-oxide (TMAO), sarcosine, betaine, proline and sucrose, and the denaturant of GdnHCl. In this series of measurements, the aqueous samples were prepared with various concentrations of the CDs, up to saturated conditions, and as a function of osmolyte or GdnHCl concentrations. The density increments resulting from the addition of the osmolyte or GdnHCl and different model compounds of CDs were investigated, respectively. An empirical linear combination equation with an augmented term to account the interactions between CDs and osmolyte or GdnHCl was used to quantitatively correlate the experimental densities over the entire concentration ranges. The addition of this augmented term significantly improved the correlation of the density data for those systems which were not accurately represented by the linear combination equation.
Keywords: Densities; Cyclic dipeptides; Osmolytes; GdnHCl; Molecular interactions
Refolding of recombinant homodimeric malate dehydrogenase expressed in Escherichia coli as inclusion bodies by Xiao-Yan Dong; Min-Ling Fu; Yan Sun (pp. 341-348).
Escherichia coli malate dehydrogenase (eMDH) is a homodimeric enzyme composed of identical subunits, and the catalytically active form of the enzyme is a dimer. Herein, a recombinant eMDH was overexpressed in E. coli as inclusion bodies (IBs). The purified eMDH IBs were solubilized by urea for refolding studies. It was found that refolding at low temperature (4°C) enhanced the refolding yield of the homodimeric enzyme. Some representative folding aids were examined, of which glycerol, sucrose and adenosine triphosphate were found to be effective. However, the refolding method with an artificial chaperone (AC) was the most efficient one that gave a specific activity recovery as high as 155U/mg, about two times higher than a simple dilution. Analyses by high-performance size-exclusion chromatography, fluorescence spectroscopy and circular dichroism (CD) spectroscopy have well explained the reason for the high activity yield of the AC approach. That is, although the enzyme subunit was well folded to a tertiary structure close to its native state by both the simple dilution and the AC method, refolding by simple dilution led to less formation of dimeric protein, so its enzyme activity was much lower than that obtained by the AC method. CD spectra analysis indicates that there was higher α-helix fraction in the eMDH refolded by the AC method, and higher α-helix fraction is considered to be significant for the dimer formation of the enzyme.
Keywords: Malate dehydrogenase; Expression; Inclusion bodies; Refolding; Folding aids; Artificial chaperone
Integrated process for production of surfactin by Ludovic Montastruc; Tao Liu; Frédérique Gancel; Ling Zhao; Iordan Nikov (pp. 349-354).
Previous work has presented kinetics of pure surfactin adsorption onto activated carbon. Being an efficient biosurfactant, the lipopeptide surfactin has been produced in a bioprocess supported by the strain Bacillus subtilis ATCC 21332. This work is aimed at studying surfactin recovery directly from the culture medium. A thermodynamic study is carried out. Referring to adsorption capacity, the thermodynamic study confirmed that the adsorption of pure surfactin is an exothermic process. The capacity of surfactin adsorption from culture media shows that the activated carbon could be used as efficient adsorbent for surfactin recovery in an integrated process. The study shows the importance of the temperature for process control. Aimed at fixed bed column design, surfactin adsorption modelling on a single microporous pellet is demonstrated.
Keywords: Adsorption; Surfactin; Thermodynamics; Kinetics
Patch controlled protein adsorption in mixed-mode chromatography with benzylamine as functional ligand by Dong Gao; Dong-Qiang Lin; Shan-Jing Yao (pp. 355-361).
The adsorption mechanisms of bovine serum albumin (BSA) on mixed-mode adsorbents with benzylamine as the functional ligand were discussed in terms of the isotherm adsorption behavior and chromatographic retention under various conditions. The adsorbents showed the salt-tolerant and pH-dependent properties for protein binding. The results indicated that the electrostatic interactions might be an important contributor to the protein adsorption under the conditions of electrostatic attractive protein–ligand interactions. While there are some amounts of electrostatic repulsion protein–ligand interactions, it seems that the adsorption process is patch controlled, and the specific salt concentration to result in a minimum adsorption capacity could be found. Accordingly the isocratic retention factors have a minimum value under a certain salt concentration, and showed the typical “U” shape curve as the function of salt concentration. In addition, the total number of released water molecules during protein adsorption was analyzed using the preferential interaction theory, and was found to be consistent with the estimated hydrophobic contact area between the bound protein and adsorbent surface.
Keywords: Mixed-mode chromatography; Protein adsorption; Patch controlled adsorption; Retention models; Preferential interaction model
Purification and properties of an extracellular cold-active protease from the psychrophilic bacterium Pseudoalteromonas sp. NJ276 by Quan-Fu Wang; Yan-Hua Hou; Zhong Xu; Jin-Lai Miao; Guang-You Li (pp. 362-368).
The extracellular cold-active protease from the psychrophilic bacterium Pseudoalteromonas sp. NJ276 was purified by 22.5-fold using precipitate of saturation (NH4)2SO4, DEAE-Sephadex A50 and Sephadex G-75. It was shown that purified enzyme was homogeneous in terms of SDS-PAGE with molecular mass estimate of 28kDa. The protease depicted an optimal pH of 8.0 and was stable at pH 7.0–9.0, and its optimal temperature was at 30°C. The protease was completely inhibited by PSFM. It was partially inhibited by metal salts, especially, had high tolerance to a wide range of NaCl concentrations (0–3M NaCl). The highest kcat and kcat/ Km values were observed at 35°C, and 35 and 54% of their highest values retained at 0°C, respectively. Milk protein treated by this protease released more free amino acids than those treated by mesophilic papain at 4°C. These results suggested that Pseudoalteromonas sp. NJ276 protease had broad substrate specificities and potential application in low-temperature food processing.
Keywords: Cold-active protease; Purification; Psychrophilic bacterium; Pseudoalteromonas; sp.
Enhancing enzymatic digestibility of switchgrass by microwave-assisted alkali pretreatment by Zhenhu Hu; Zhiyou Wen (pp. 369-378).
Pretreatment of lignocellulosic materials to disrupt their recalcitrant structures is a crucial step in cellulosic ethanol production. Most pretreatment processes require a high temperature reaction, which is often achieved through conventional heating. In this study, microwave-based heating was used to pretreat switchgrass, which was then hydrolyzed by cellulase enzymes. When switchgrass was soaked in water and treated by microwave, total sugar (xylose+glucose) yield from the combined treatment and hydrolysis was 34.5g/100g biomass, equivalent to 58.5% of the maximum potential sugars released. This yield was 53% higher than that obtained from conventional heating of switchgrass. To further improve the sugar yield, switchgrass was presoaked in different concentrations of alkali solutions and then treated by microwave or conventional heating. With alkali loading from 0.05 to 0.3galkali/g biomass, microwave pretreatment resulted in a higher sugar yield than from conventional heating, with the highest yield (90% of maximum potential sugars) being achieved at 0.1g/g of alkali loading. Scanning electron microscope images revealed that the advantage of microwave over conventional heating was due to the disruption of recalcitrant structures. Finally, the effects of temperature, solid content, and treatment time on microwave pretreatment of switchgrass were investigated. At optimal conditions of 190°C, 50g/L solid content, and 30min treatment time, the sugar yield from the combined pretreatment and hydrolysis was 58.7g/100g biomass, equivalent 99% of potential maximum sugars. The results demonstrate that microwave-assisted alkali treatment is an efficient way to improve the enzymatic digestibility of switchgrass.
Keywords: Microwave; Pretreatment; Lignocelluloses; Switchgrass; Enzymatic hydrolysis; Sugar yield
Improvement in anaerobic degradation of olive mill effluent (OME) by chemical pretreatment using batch systems by Nuri Azbar; Tugba Keskin; Ebru Cokay Catalkaya (pp. 379-383).
In this work, feasibility of using chemical pretreatment to improve the anaerobic biological degradation of industrial effluents containing high concentrations of phenolic compounds was investigated. For this purpose, chemical pretreatment of the wastewater generated from olive oil production process, namely olive mill effluent (OME), was conducted by means of acid cracking followed by coagulation–flocculation process using one of the coagulating agents such as Al2SO4, FeSO4 and FeCl3, respectively. Biochemical methane potential (BMP) assay was carried out for both crude OME and chemically pretreated OME samples in order to monitor and comparatively evaluate any increase in biogas production as the indicator of improvement in anaerobic biological degradation after chemical pretreatment. The experimental results obtained in this work suggest that pretreatment significantly enhances the biodegradability of OME which was much lower if it was digested alone (without pretreatment). Over 80% increase in biogas production was obtained when digesting OME after chemical pretreatment. It was concluded that among the alternative chemicals, Al2SO4 resulted in highest biogas production from OME, thereby the best results for the enhancement of anaerobic biodegradability of OME were also achieved. It was demonstrated that the biodegradability of OME could be significantly enhanced by chemical pretreatment and therefore anaerobic degradation after a suitable pretreatment could be considered as a safe disposal method for OME.
Keywords: Biogas; Bioenergy; Anaerobic digestion; Olive mill effluent; Chemical treatment
Production and characterization of terpolyester poly(3-hydroxybutyrate- co-4-hydroxybutyrate- co-3-hydroxyhexanoate) by recombinant Aeromonas hydrophila 4AK4 harboring genes phaPCJ by Wen Peng Xie; Guo-Qiang Chen (pp. 384-389).
Terpolyester consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyhexanoate (3HHx) abbreviated as P(3HB- co-4HB- co-3HHx), was produced by recombinant Aeromonas hydrophila 4AK4 harboring polyhydroxyalkanoate (PHA) synthesis genes phaPCJ. The monomer content of 4HB in the terpolyesters was dependent on the concentration of 1,4-butanediol, ranging from 4.3 to 8.5mol% in the presence of 5–25g/l 1,4-butanediol, respectively, accompanying by a decreasing 3HHx content from 22 to 17.6mol% and a constant 3HB content of 74mol% during growth in lauric acid containing mineral medium. Number average molecular weights of the terpolyesters were 440,000 to 550,000, much higher than 220,000 of the copolyester consisting of 3HB and 12mol%3HHx commonly produced by wild type A. hydrophila 4AK4. Thermal and mechanical properties of the above terpolyesters were better than those of homopolyester poly(3-hydroxybutyrate) (PHB) and its copolymers P(3HB- co-7mol%4HB), P(3HB- co-12mol%3HHx). Moreover, physical properties of the terpolyesters changed with variations of monomer composition. The terpolyesters were characterized using nuclear magnetic resonance (NMR), gas chromatography (GC), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and stress–strain measurements. The introduction of 4HB and 3HHx monomers into PHB enhanced the thermal stability, changed its crystallinity and flexibility compared with homopolyester PHB and their two monomer containing copolyesters.
Keywords: PHB; Aeromonas hydrophila; 1,4-Butanediol; Terpolyester; Polyhydroxyalkanoates; PHA
A general rate law equation for biosorption by Yu Liu; Liang Shen (pp. 390-394).
The pseudo first- and second-order equations have been most commonly applied to describe biosorption kinetics under various conditions. This study attempted to develop a general rate law equation for biosorption. It was shown that biosorption kinetics would be subjected to the universal rate law for a chemical process, and the pseudo first- and second-order kinetic equations are the special cases of the proposed general rate law equation for biosorption. As the reaction order cannot be theoretically calculated, it is not reasonable to preset the order of biosorption kinetics to be the first or second as commonly exercised in biosorption studies. The actual kinetic order of biosorption process should be estimated from the proposed general rate law equation without any preset constrain.
Keywords: Biosorption; Rate law; First-order kinetics; Second-order kinetics
Mechanistic model for evaluating the performance of suspended growth bioreactors for the off-gas treatment of VOCs by Sergio Bordel; Raúl Muñoz; Luis F. Díaz; Santiago Villaverde (pp. 395-405).
A mechanistic model capable to predict toluene removal in suspended growth bioreactors (SGRs) was proposed and validated. The model predicted with reasonable accuracy both toluene elimination capacities (11% of relative average error) and biomass concentration (6% of relative average error) in a Pseudomonas putida F1 culture growing in a lab-scale chemostat. Unlike previously reported mechanistic models this holistic model was capable to predict process performance under toluene, oxygen or nutrient limiting conditions. Mass transport, Henry law, and biomass yield coefficients were the most significant parameters influencing model predictions as shown by the sensitivity analysis performed. In addition, an innovative vector-based solving approach was proposed, which allowed reducing the complex systems of eight non-linear algebraic equations into a single equation and provided the dimensionless numbers necessary for bioreactor scale-up. Being based in general kinetic and mass transport equations it can be adapted to other processes where mass transport from a gaseous to an aqueous phase is involved.
Keywords: Biodegradation; Bioreactors; Gas–liquid mass transfer; Modelling; Pseudomonas putida; F1; Volatile organic compounds
Computational investigation of fluid dynamics in a recently developed centrifugal impeller bioreactor by Jian-Ye Xia; Si-Jing Wang; Si-Liang Zhang; Jian-Jiang Zhong (pp. 406-413).
Centrifugal impeller bioreactor (CIB) was recently developed for shear sensitive biological systems. To better understand its fluid velocity profile, liquid circulation flow, shear stress, and circulation time distribution (CTD), in this study a computational fluid dynamics (CFD) approach was taken to quantitatively assess the effects of major impeller designing and bioreactor operating parameters on those characteristics. Comparison of the simulated velocity profiles near the impeller tip with the experimental measurement results was carried out, and a good agreement was demonstrated. Based on the simulated circulation flow through the draft tube, a new empirical correlation of the circulation flow ( QL) with respect to the impeller designing and operating parameters was established. In addition, three distinct circulation cycles were identified and their time distribution was quantified. The above information obtained from the CFD simulation and analysis is helpful to future optimization and scale-up/scale-down of the CIB.
Keywords: CFD; Centrifugal impeller; Velocity profile; Shear force; Fluid circulation; Circulation time distribution
Integrated process for extraction and purification of alcohol dehydrogenase from Baker's yeast involving precipitation and aqueous two phase extraction by M.C. Madhusudhan; K.S.M.S. Raghavarao; Sanjay Nene (pp. 414-420).
The potential of aqueous two phase extraction for the purification of alcohol dehydrogenase (ADH) from Baker's yeast ( Saccharomyces cerevisiae) is demonstrated. An integrated process involving precipitation followed by aqueous two phase extraction (ATPE) is used for effective recovery of the enzyme. This is carried out in two schemes. In the first scheme, ammonium sulphate precipitation is carried out followed by ATPE of the precipitate after suspending it in buffer. While the second scheme involves polyethylene glycol precipitation followed by ATPE of the supernatant. The effect of various process parameters such as molecular weight of polymer, tie line length, phase volume ratio and neutral salt is investigated for enhancing the activity recovery of ADH. First scheme resulted in about 85% enzyme activity recovery of ADH with purification factor of 4.2 while the second scheme about 90% enzyme activity recovery with 6.6-fold purification.
Keywords: Alcohol dehydrogenase; Yeast; Precipitation; Enzyme extraction; Purification; Aqueous two phase extraction