Liquid-liquid extraction of commercial glucose oxidase by reversed micelles

Aim of this work was to establish the optimum operating conditions for the extraction and recovery by cationic reversed micelles of commercial glucose oxidase (GOX) from Aspergillus niger, in view of possible application to raw cell homogenates. The influence of pH, temperature, electric conductivity and solvent/co-solvents ratio on the extraction was investigated by a fractional factorial design of 2(3-1) type, conjugated with a mixture experimental design, using the residual enzyme activity to evaluate the results. The best conditions for GOX extraction were ensured using isooctane as solvent and hexanol and butanol as co-solvents at 76/6/18 volume ratio, pH 6.0, 0.2 M cetyl trimethylammonium bromide (CTAB) as cationic surfactant, and electrical conductivity (kappa) of 4.8 mS cm-1. The highest yield of GOX activity recovery (about 90%) was in fair accordance with the value predicted by the model.     

Separation and purification of lipase using reverse micellar extraction: Optimization of conditions by response surface methodology

Downstream processing of lipase involving reverse micellar extraction of lipase using cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated. Effect of various process parameters on both forward and backward extraction of lipase from crude extract was studied to optimize its yield and purity. Complex interaction of salt concentration (0.05∼0.15M), surfactant concentration (0.10∼0.30 M), and pH (6.0∼9.0) for forward extraction, as well as, salt concentration (0.5∼1.5 M) and pH (6.0∼9.0) for backward extraction have been studied using response surface methodology. Optimum processing conditions, namely, salt concentration 0.16M, surfactant concentration 0.20 M, and pH 9.0 for forward extraction, as well as, salt concentration 0.80 M and pH 7.23 for backward extraction, fulfill the conditions to obtain activity recovery of lipase ≥78% and purification factor of lipase ≥4.0. The study demonstrated that response surface methodology can be used for optimization of the conditions for reverse micellar extraction of lipase.     

Reverse micellar extraction for downstream processing of lipase: Effect of various parameters on extraction

Reverse micellar extraction of lipase using cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated. The effect of various process parameters on both forward and backward extraction of lipase from crude extract was studied to optimize its yield and purity. Forward extraction of lipase was found to be maximum using Tris buffer at pH 9.0 containing 0.10 M NaCl in aqueous phase and 0.20 M CTAB in organic phase consisting of isooctane, butanol and hexanol. In case of backward extraction, lipase was extracted from the organic phase to a fresh aqueous phase in 0.05 M potassium phosphate buffer (pH 7.0) containing 1.0 M KCl. The activity recovery, extraction efficiency and purification factor of lipase were found to be 82.72%, 40.27% and 4.09-fold, respectively. The studies also indicated that the organic phase recovered after back extraction could be reused for the extraction of lipase from crude extract.     

Downstream processing of soy hull peroxidase employing reverse micellar extraction

Phase transfer studies were conducted to evaluate the solubilization of soy hull peroxidase (SHP) in reverse micelles formed in isooctane/butanol/hexanol using the cationic surfactant cetyltrimethylammonium bromide (CTAB). The effect of various parameters such as pH, ionic strength, surfactant concentration of the initial aqueous phase for forward extraction and buffer pH, type and concentration of salt, concentration of isopropyl alcohol and volume ratio for back extraction was studied to improve the efficiency of reverse micellar extraction. The active SHP was recovered after a complete cycle of forward and back extraction. A forward extraction efficiency of 100%, back extraction efficiency of 36%, overall activity recovery of 90% and purification fold of 4.72 were obtained under optimised conditions. Anionic surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) did not yield good results under the conditions studied. The phase transfer of soy hull peroxidase was found to be controlled by electrostatic and hydrophobic interactions during forward and back extraction respectively.     

AOT/异辛烷反胶束萃取纯化胰蛋白酶及酶变性失活问题的解决

用反胶束技术分离纯化蛋白质,具有高选择性、易于大规模操作等优点,具有良好的工业应用前景。但是离子型表面活性剂形成的反胶束体系萃取蛋白质容易引起蛋白质的变性,这是由于离子型表面活性剂的强电荷作用所导致的。对用AOT/异辛烷反胶束体系从胰酶粗提物中萃取胰蛋白酶进行了研究,通过在反胶束相加入乙醇,解决了反胶束萃取蛋白质时蛋白质变性失活的问题。并且由于乙醇的加入大大减少了分相的时间,简化了实验步骤,优化了实验方法,使此技术在工业上的大规模应用成为可能。通过优化各种实验条件,胰蛋白酶的前萃取率达到90%,反萃取率接近100%。最终得率为88%。纯化后的比活提高了5倍多,从300U/mg左右提高到了1800U/mg。     

Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes

Reverse micellar systems of CTAB/isooctane/hexanol/butanol and AOT/isooctane are used for the extraction and primary purification of bromelain from crude aqueous extract of pineapple wastes (core, peel, crown and extended stem). The effect of forward as well as back extraction process parameters on the extraction efficiency, activity recovery and purification fold is studied in detail for the pineapple core extract. The optimized conditions for the extraction from core resulted in forward and back extraction efficiencies of 45% and 62%, respectively, using reverse micellar system of cationic surfactant CTAB. A fairly good activity recovery (106%) and purification (5.2-fold) of bromelain is obtained under these conditions. Reverse micellar extraction from peel, extended stem and crown using CTAB system resulted in purification folds of 2.1, 3.5, and 1.7, respectively. Extraction from extended stem using anionic surfactant AOT in isooctane did not yield good results under the operating conditions employed.     

Study of the factors affecting the extraction of soybean protein by reverse micelles

In this work, the forward and back extraction of soybean protein by reverse micelles was studied. The reverse micellar systems were formed by anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT), isooctane and KCl solution. The effects of AOT concentration, aqueous pH, KCl concentration and phase volume ratio on the extraction efficiency of soybean protein were tested. Suitability of reverse micelles of AOT and Triton-X-100/AOT mixture in organic solvent toluene for soybean protein extraction was also investigated. The experimental results lead to complete forward extraction at the AOT concentration 120 mmol l⁻¹, aqueous pH 5.5 and KCl concentration 0.8 mol l⁻¹. The backward extraction with aqueous phase (pH 5.5) resulted in 100% extraction of soybean protein from the organic phase.     

Protein extraction by reverse micelles: studies on the recovery of horseradish peroxidase

Phase transfer studies were carried out on the solubilization of horseradish peroxidase (HRP) (E.C. 1.11.1.7) in reverse micelles formed in isooctane using the anionic surfactant, aerosol OT, at concentrations between 50 and 110mM. The selectivity of this methodology was tested, because the HRP used comprised a mixture of seven different isoenzymes with a wide range of isoelectric points. Forward and backward transfers were carried out in wellstirred vessels until equilibrium was reached. Significant protein partitioning could only be obtained by using NaCl to adjust ionic strength in pH range between 1.5 and 3.5, with a maximum at pH 3. The back transfer process was best at pH 8 with 80mM phosphate buffer and 1 M KCI. A loss of 1% to 3% of the surfactant through precipitation at the interface at pH<4 was observed, which may be due to instability in this pH region, because, even without protein, a similar precipitate was noticed. Protein partitioning was approximately constant when the ionic strength was increased up to 1 MNaCl at pH 3, but protein recovery in back transfer decreased accordingly. Hydrophobic interactions together with association between the protein and surfactant might be responsible for that behavior. Protein partitioning remained the same when the surfactant concentration was decreased to 50 mM, at the expense of higher variability. HPLC chromatograms showed no apparent damage to the protein after reverse micellar extraction. Protein partitioning is best when the temperature is kept at 25xC. The amount of protein and specific activity recovered strongly depends on the phase ratio used during forward transfer. Overall activity recovery varied from 87% to 136% when the phase ratio was increased from 1:1 to 30:1 in forward transfer. This behavior may be due to a change in the ratio of the three isoenzymes recovered after the backward transfer process, with the most active one being increasingly enriched at higher phase ratios. (c) 1994 John Wiley & Sons, Inc.     

Application of reverse micelle extraction process for amylase recovery using response surface methodology

The effect of different process variables of reverse micelle extraction process like pH, addition of surfactant (AOT) concentration and potassium chloride (KCl) concentration on amylase recovery has been studied and analysed. Solid-state fermentation was used for the production of amylase enzyme. Response surface methodology (RSM) using central composite rotatable design (CCRD) was employed to analyse and optimize the enzyme extraction process. The regression analysis indicates that the effect of AOT concentration, and KCl concentration were significant, whereas the effect of pH was non-significant on enzyme recovery. For the maximum recovery of enzyme, the optimum operating condition for pH, AOT concentration (M) and KCl concentration were 10.43, 0.05 and 1.00, respectively. Under these optimal conditions, the enzyme recovery was 83.16%.     

Downstream antibody purification using aqueous two‐phase extraction

The extraction of antibodies using a polyethylene glycol (PEG)‐citrate aqueous two‐phase system (ATPS) was investigated. Studies using purified monoclonal antibody (mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cell protein (HCP) from clarified cell culture media was examined using statistical design of experiments (DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined. A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified as significant factors. To achieve the highest purity, the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm. An optimal ATPS consisting of 14.0% (w/w) PEG, 8.4% (w/w) citrate, and 7.2% (w/w) NaCl at pH 7.2 resulted in a product yield of 89%, an approximate 7.6‐fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%. A system consisting of 15% (w/w) PEG, 8% (w/w) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product‐related impurities (aggregates and low molecular product fragments) from ～40% to less than 0.5% while achieving 95% product recovery. At the experimental conditions that were optimized in the batch mode, a scale‐up model for the use of counter‐current extraction technology was developed to identify potential improvements in purity and recovery that could be realized in the continuous operational mode. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A Penicillium expansum glucose oxidase-encoding gene, GOX2, is essential for gluconic acid production and acidification during colonization of deciduous fruit

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO(4) chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum.     

Backward extraction of reverse micellar encapsulated proteins using a counterionic surfactant

The back-extraction of proteins encapsulated in AOT reverse micelles was performed by adding a counterionic surfactant, either TOMAC or DTAB. This novel backward transfer method gave higher backward extraction yields compared to the conventional method with high salt and high pH of the aqueous stripping solution. The protein activity was maintained in the resulting aqueous phase, which in this case had a near neutral pH and low salt concentration. A sharp decrease of the water content was observed in the organic phase corresponding to protein back-extraction using TOMAC. The backward transfer mechanism was postulated to be caused by electrostatic interaction between oppositely charged surfactant molecules, which lead to the collapse of the reverse micelles. The back-extraction process with TOMAC was found to be very fast; more than 100 times faster than back-extraction with the conventional method, and as much as 3 times faster than forward extraction. The formation of 1:1 complexes of AOT and TOMAC in the solvent phase was observed, and these hydrophobic complexes could be efficiently removed from the solvent using adsorption onto Montmorillonite in order for the organic solvent to be reused. A second cationic surfactant, DTAB, confirmed the general applicability of counterionic surfactants for the backward transfer of proteins.     

The recovery of the hepatitis B virus surface antigen (HBsAg) from a recombinant P. pastoris strain disruption and precipitation studies

Cell disruption studies for the extraction of HBsAg from a recombinant P. pastoris strain (r-HBsAg) were done using a bead mill disintegrator. Three sequential passages (4 min retention time each) were enough to disrupt the cells and extract most of the r-HBsAg and soluble proteins. An acid precipitation step was performed just after cell disruption to precipitate proteins together with the cell debris. Different precipitation pH values (2.5 to 6.0) were investigated. A pH value of 4.2 was selected as a compromise between recovery and improvement of specific activity. A 6 to 8-fold enhancement of the specific activity was obtained, having a r-HBsAg overall yield of about 80%. The influencing presence of a chaotropic salt (potassium thiocyanate) during the acid precipitation step was also studied.     

Effect of operating conditions on the extraction of ovomucin

Ovomucin, mainly responsible for the gelatinous property of egg white, has potential applications as a functional food and nutraceutical ingredient. A 2-step method for ovomucin preparation was recently developed. The purpose of this study was to determine the effects of various operating conditions, such as pH, NaCl concentrations and extraction volume at the second extraction, temperature, and centrifugation force, on the purity and yield of ovomucin. Our results showed that pH has a significant effect on the purity and yield of ovomucin extracts. Increasing the extraction pH from 4.0 to 5.0 could significantly (p < 0.05) increase the purity and yield of ovomucin; at pHs higher than 5.0, the purity was not affected but the yield was significantly decreased. The highest yield of ovomucin extract (308 mg/100 g of egg white) was achieved at pH 5.0 while the highest purity was achieved at pH 7.0. There is a trend that the purity of ovomucin increased (p < 0.05) but the yield of ovomucin decreased (p > 0.05) at increasing salt concentrations. Reducing extraction volume did not affect the yield of ovomucin whereas its purity was significantly decreased. The yield of ovomucin however was significantly increased at increasing settling time or centrifugation force, but the purity was less affected. Extraction of ovomucin at room temperature could significantly reduce the extraction yield compared to that at lower temperature (4 °C) but the purity was not affected.     

Reversed micellar extraction of trypsin: Effect of solvent on the protein transfer and activity recovery

By using trypsin as the model protein and AOT as the model surfactant, the effect of a variety of solvents on protein transfer and activity recovery during the liquid-liquid reversed micellar extraction was investigated. It was found that several solvents, including isooctane, octane, heptane, and kerosene, had a similar effect on the recovery of trypsin activity after a full cycle of forward and backward extraction, and could all be used as the solvents for AOT-reversed micelles in trypsin extraction. Two other solvents (hexane and cyclohexane), however, were not so efficient. (c) 1995 John Wiley & Sons, Inc.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.     

Selective separation of trypsin from pancreatin using bioaffinity in reverse micellar system composed of a nonionic surfactant

Selective separation of trypsin from a mixture involving many kinds of contaminating proteins, i.e., pancreatin, was achieved using trypsin inhibitor immobilized in the reverse micelles, which were composed of a nonionic surfactant, tetra-oxyethylene monodecylether. To determine the efficient operations throughout the whole separation process we examined the operating conditions, which affect the immobilization efficiency of trypsin inhibitor and also the forward and backward extractions of trypsin. Fifty percent of the recovery of trypsin from pancreatin was realized with no loss of activity of the recovered trypsin.     

Improvement in tannase recovery using enzymatic disruption of mycelium in combination with reverse micellar enzyme extraction

Summary A high activity tannase (tannin acyl hydrolase EC 3.1.1.20) is synthetized in high yield by Aspergillus niger LCF 8. At the production optimum, the tannase is strongly bound to the mycelium and detachment of the enzyme by classical physical and chemical means, largely failed. Enzymatic hydrolysis of cell walls using a chitinase from Streptomyces griseus followed by reverse micellar tannase extraction resulted in a recovery of 43% active enzyme, i.e. an improvement in yield of 2.5 from a previous process. Best conditions were enzymatic hydrolysis of mycelium with chitinase at pH 6.0 and 25°C for 2.5 h followed by tannase extraction at pH 7.5 with isooctane containing 80 mM cetyl trimethyl ammonium bromide and stripping at pH 4.0 in the presence of 0.35 M NaCl.     

Continuous extraction of a recombinant cutinase from Escherichia coli disrupted cells with reversed micelles using a perforated rotating disc contactor

This work reports on the continuous extraction of a recombinant cutinase with reversed micelles using a perforated rotating disc contactor. Intracellular cutinase was directly extracted at 20°C with 100 mM AOT in isooctane from complex biological media of Escherichia coli disrupted cells. The optimal conditions for the direct extraction of the enzyme from media containing cell debris led to an extraction yield of 54.4%.     

十二烷基硫酸钠辅助提取布渣叶中的黄酮类物质

以布渣叶为研究体系,采用表面活性剂辅助热回流法提取黄酮类物质.考察了表面活性剂种类及浓度、提取时间、液固比和溶剂pH等因素,并通过实验数据进行了正交试验设计,得到最优的工艺条件:表面活性剂选择十二烷基硫酸钠(SDS),质量浓度0.8 g/L,提取时间105 min,液固比45 mL/g,溶剂pH 6.3.在此条件下,布渣叶黄酮得率为15.73％,与其他方法相比具有显著优势.