Recovery Of Lysozyme Research Articles

Purification of lysozyme by affinity-based reversed micellar two-phase extraction

A dye-affinity reversed micellar system was used for lysozyme purification from a crude solution of chicken egg white. The dye-affinity reversed micelles consisted of Cibacron Blue F-3GA (CB; 0.1 mM) modified lecithin (50 g/l) in n-hexane. Starting with a crude egg white solution containing lysozyme of 0.0381 mg/mg protein, lysozyme purity was increased by 16 to 20 times, reached 0.62 to 0.76 mg/mg protein. The affinity micellar system was recycled and used three times. Addition of polyoxyethylene (20) sorbitan trioleate (Tween 85) as a cosurfactant could increase the capacity of the affinity-based reversed micelles. A lysozyme recovery yield of over 70% was obtained at a forward aqueous phase pH of 9.16 using the reversed micelles additionally containing 20 g/l of Tween 85.

Stabilization of proteins against methylene chloride/water interface-induced denaturation and aggregation

The purpose of this study was to stabilize proteins during the commonly used microencapsulation process of methylene chloride/water emulsification. The model proteins used in this study included bovine serum albumin (BSA), S-carboxymethylated BSA (CM-BSA), reduced- S-carboxymethylated BSA (RCM-BSA), ovalbumin, and lysozyme. Emulsification of a 0.5-mg/ml protein solution in methylene chloride brought about changes in the composition of water-soluble species and interfacial coagulation. As a result, 37.8, 71.8 and 98.7% of ovalbumin, lysozyme and BSA were recovered from the aqueous phase after emulsification, respectively. Experiments with BSA, CM-BSA, and RCM-BSA demonstrated that a free thiol group and/or disulfide bond participated in interfacially induced dimerization and polymerization of proteins. Interfacial reactions that led to the aggregation of ovalbumin and lysozyme were inhibited by adding hydroxypropyl-β-cyclodextrin or BSA into their aqueous solutions. Moreover, such beneficial effect of the excipients was observed to be concentration dependent. Under our experimental conditions, the recovery of ovalbumin and lysozyme was improved up to 97.7 and 95.6%, respectively. This study substantiated that an adequate formulation could overcome denaturing effects of the methylene chloride/water interface upon a protein of interest to be encapsulated into microspheres.

Separation of Egg White Lysozyme by Anionic Polysaccharides

ABSTRACTAnionic polysaccharides (Na‐alginate, Na‐carboxyl‐methyl‐cellulose, high methoxy pectin and κ‐carrageenan) were evaluated for their suitability towards simple and more efficient separation of lysozyme from salted or fresh duck eggs and hen egg whites for use in foods. Only κ‐carrageenan interacted and formed precipitates with lysozyme. Recovery of lysozyme from fivefold diluted salted duck egg whites was 60–65% and 78–81% from fresh duck and hen egg whites using 0.7%κ‐carrageenan. The recovered lysozyme from the salted duck egg whites was stable during storage at 4°C for 35 days.

Macroporous chitin affinity membranes for lysozyme separation

Macroporous chitin membranes with high, controlled porosity and good mechanical properties have been prepared using a technique developed in this laboratory based on silica particles as porogen. They were employed for the affinity separation of lysozyme. Chitin membranes (1 mm thickness) can be operated at high fluxes (≥1.1 mL/min/cm2) corresponding to pressure drops ≥2 psi. Their adsorption capacity for lysozyme (∼50 mg/mL membrane) is by an order of magnitude higher than that of the chitin beads employed in column separation. In a binary mixture of lysozyme and ovalbumin, the membranes showed very high selectivity towards lysozyme. The effect of some important operation parameters, such as the flow rates during loading and elution were investigated. Lysozyme of very high purity (>98%) was obtained from a mixture of lysozyme and ovalbumin, and from egg white. The results indicate that the macroporous chitin membranes can be used for the separation, purification, and recovery of lysozyme at large scale. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 610–617, 1997.

Macroporous chitin affinity membranes for lysozyme separation

Macroporous chitin membranes with high, controlled porosity and good mechanical properties have been prepared using a technique developed in this laboratory based on silica particles as porogen. They were employed for the affinity separation of lysozyme. Chitin membranes (1 mm thickness) can be operated at high fluxes (>/=1.1 mL/min/cm(2)) corresponding to pressure drops >/=2 psi. Their adsorption capacity for lysozyme ( approximately 50 mg/mL membrane) is by an order of magnitude higher than that of the chitin beads employed in column separation. In a binary mixture of lysozyme and ovalbumin, the membranes showed very high selectivity towards lysozyme. The effect of some important operation parameters, such as the flow rates during loading and elution were investigated. Lysozyme of very high purity (>98%) was obtained from a mixture of lysozyme and ovalbumin, and from egg white. The results indicate that the macroporous chitin membranes can be used for the separation, purification, and recovery of lysozyme at large scale. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 610-617, 1997.

Protein refolding at high concentration using size‐exclusion chromatography

A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size-exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel-filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S-100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step. © 1996 John Wiley & Sons, Inc.

Protein refolding at high concentration using size-exclusion chromatography

A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size-exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel-filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S-100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step.

Novel function of guanidine hydrochloride in reverse micellar extraction of lysozyme from chicken egg white

The efficiency of guanidine hydrochloride (GuHCl) addition in the suppression of gel formation and the extraction of lysozyme during reverse micellar extraction from chicken egg white was investigated. A low concentration of GuHCl in the feed permitted the successful extraction of lysozyme in its native form without gel formation, which is perceived as a novel function of GuHCl. The highest recovery and specific activity of lysozyme were obtained at a GuHCl concentration of 0.06 M in 25 mM AOT reverse micellar extraction from 20-fold-diluted natural chicken egg white. Lysozyme and ovalbumin CD spectra in the corresponding GuHCl aqueous solutions revealed no changes in the higher order structures of the proteins. Furthermore, the specific activity of lysozyme in the feed was well preserved in the GuHCl system.

Removal of endotoxins by affinity sorbents.

Histidine, histamine and polymyxin B affinity sorbents were employed for the removal of Escherichia coli-derived endotoxins. Their effectiveness was compared with those of poly-L-lysine-Sepharose and DEAE-Sepharose. All sorbents reduced the concentration of endotoxins from an E. coli culture filtrate to tolerable levels. However, their effectiveness was not higher than that of the anion exchanger, which displayed clearance rates of up to 15,000. Endotoxin removal from protein solutions depended on the net charge of the desired protein. Lysozyme as a model for positively charged proteins enhanced endotoxin removal. In contrast, only low initial contamination levels (< 34 EU/ml) were reduced to tolerable levels from bovine serum albumin (BSA) as the negatively charged protein model owing to competition of BSA and endotoxins for adsorption sites. Hence also a low BSA recovery was observed after the treatment whereas the lysozyme recovery was almost 100%. At pH values below the isoelectric point of BSA, endotoxin removal was also more effective. The best conditions for the decontamination were found at neutral pH and low ionic strength (< or = 20 mM phosphate). Ionic forces between ligands and endotoxins are dominant at this ionic strength; hydrophobic interactions are not very effective. Hence the selectivities of all sorbents towards endotoxins are not exceptionally high. DEAE-anion exchangers are the most suitable sorbents for the removal of endotoxins from solutions accommodating positively charged proteins owing to their low cost and high capacity. Poly-L-lysine-Sepharose was most effective for the removal of small amounts of endotoxins from solutions of negatively charged proteins. The "affinity ligands" histamine, histidine and polymyxin B were effective for the removal of endotoxins from E. coli filtrate; however, their effectiveness decreased dramatically in the presence of BSA and it was lower than for poly-L-lysine- and DEAE-Sepharose in the presence of lysozyme.

Recovery of proteins from reversed micelles using a novel ion-exchange material

The recovery of proteins solubilised inside reversed micelles is generally a low yielding process. We have studied three methods for recovery of lysozyme. The protein laden organic phase was contacted with solutions of varying pH. Greater than 95% recovery was achieved at pH values close to the isoelectric point (10.9). The use of 2.5 M KCl gave close to 100% recovery of lysozyme. The organic phase was passed onto a ion exchange matrix. This resulted in disruption of the reversed micelle releasing 60–80% of the protein into the eluent.

The role of the thiol/disulfide centers and peptide binding site in the chaperone and anti-chaperone activities of protein disulfide isomerase.

The complexity of protein folding is often aggravated by the low solubility of the denatured state. The inefficiency of the oxidative refolding of reduced, denatured lysozyme results from a kinetic partitioning of the unfolded protein between pathways leading to aggregation and pathways leading to the native structure. Protein disulfide isomerase (PDI), a resident foldase of the endoplasmic reticulum, catalyzes the in vitro oxidative refolding of reduced, disulfide-containing proteins, including denatured lysozyme. Depending on the concentrations of foldase and denatured substrate and the order in which they are added to initiate folding, PDI can exhibit either a chaperone activity or an anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem 269, 7764-7771). PDI's chaperone activity leads to quantitative recovery of native lysozyme. Its anti-chaperone activity diverts substrate away from productive folding and facilitates disulfide cross-linking of lysozyme into large, inactive aggregates that specifically incorporate PDI. A mutant PDI (NmCm-PDI), in which both the N- and C-terminal active site cysteines have been changed to serines, loses all chaperone activity and behaves as an anti-chaperone at all substrate and PDI concentrations tested. The dithiol/disulfide sites of PDI are essential for the chaperone activity observed at high PDI concentrations, but they are not required for the anti-chaperone activity found at low PDI concentrations. Inactivation of PDI's peptide/protein binding site by a specific photoaffinity label (Noiva, R., Freedman, R. B., and Lennarz, W. J. (1993) J. Biol. Chem. 268, 19210-19217) inhibits the disulfide isomerase and chaperone activity, but the protein still retains its anti-chaperone activity. In a glutathione redox buffer, lysozyme-PDI aggregates are disulfide cross-linked; however, disulfide cross-linking is not required for aggregate formation or for the incorporation of PDI into the aggregates. Although both the peptide binding site and the catalytic active sites of PDI are required for chaperone and disulfide isomerase activity, neither of these sites are involved in PDI's anti-chaperone activity. PDI's anti-chaperone activity could serve as a quality control device by providing an efficient mechanism to retain misfolded proteins in the endoplasmic reticulum (Marquardt, T., and Helenius, A. (1992) J. Cell. Biol. 117, 505-513).

Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme.

Reduced, denatured lysozyme tends to aggregate at neutral pH, and competition between productive folding and aggregation substantially reduces the efficiency of refolding (Goldberg, M.E., Rudolph, R., and Jaenicke, R. (1991) Biochemistry 30, 2790-2797). Protein disulfide isomerase (PDI), a catalyst of oxidative protein folding, has a variety of effects on the yield of native lysozyme during the oxidative refolding of the reduced, denatured protein. Depending on the concentration of lysozyme, the concentration of PDI, and the order in which lysozyme and PDI are added to initiate folding, PDI can produce a substantial increase or a substantial decrease in the recovery of native lysozyme, when compared with the uncatalyzed reaction. In the presence of a glutathione redox buffer, denatured lysozyme (1-10 microM) partitions almost equally between productive folding leading to native lysozyme (50-63%) and non-productive fates including the formation of disulfide cross-linked aggregates. At the higher lysozyme concentrations examined (5-10 microM), substoichiometric concentrations of PDI (0.5-1 microM) exhibit "anti-chaperone" activity; PDI actively diverts most of the denatured lysozyme away from productive folding so that only 17 +/- 9% of the lysozyme is recovered as native enzyme. PDI's anti-chaperone activity results in extensive intermolecular disulfide crosslinking of lysozyme into large, inactive aggregates. On the other hand, if PDI is initially present at a large molar excess (5-10-fold) when denatured lysozyme is diluted to initiate folding, PDI demonstrates a chaperone-like activity that prevents aggregate formation and promotes correct folding. When PDI's chaperone activity is dominant, virtually all of the denatured lysozyme is correctly folded. The schizophrenic chaperone/anti-chaperone nature of PDI activity accounts for a number of observations on in vivo protein folding, including the necessity for maintaining a high concentration of PDI in the endoplasmic reticulum and the formation of disulfide cross-linked aggregates in the endoplasmic reticulum during the expression of disulfide-containing proteins (deSilva, A., Braakman, I., and Helenius, A. (1993) J. Cell. Biol. 120, 647-655).

Egg White Lysozyme Purification by Ultrafiltration and Affinity Chromatography

ABSTRACTIsolation and purification of lysozyme from hen egg white was studied using a two‐step procedure. The egg white was diluted 5‐ to 9‐fold with sodium phosphate buffer, and then processed by sequential dilution diafiltration using a UF membrane (molecular weight cut‐off 300,000 dalton). The membrane process increased the specific activity of lysozyme 6‐fold, and recovered 96% of lysozyme activity. The permeate from diafiltration was further purified by affinity chromatography using chitin as adsorbent. The second step of the process yielded a product of specific activity of 70,400 units/mg protein. The overall lysozyme recovery was 79%.

Continuous protein separations in a magnetically stabilized fluidized bed using nonmagnetic supports

Continuous protein separations were performed using a magnetically stabilized fluidized bed (MSFB) and a commercially available affinity adsorption resin that contained no magnetically susceptible material. These nonmagnetic materials can be stabilized at relatively low fields (<75 G requiring <30 W) if sufficient magnetically susceptible particles are also present in the stabilized bed. The minimum amount of magnetic particles necessary to stabilize the bed is as low as 20% by volume and is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. Advantages of these beds for performing separations include true continuous, countercurrent liquid-solids contact, mass-transfer efficiencies nearly equal to that of packed beds, and the ability of handle suspended cells or cell debris. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations; results are presented for the adsorption and recovery of lysozyme from an aqueous mixture of lysozyme and myoglobin using an affinity resin.

Simultaneous Isolation of Avidin and Lysozyme from Egg Albumen

ABSTRACTA single column cation exchange method was developed which allowed simultaneous recovery of lysozyme and avidin from undiluted egg white. A unique application‐elution sequence was developed, involving accumulation of avidin on the column through several cycles of egg white application and lysozyme elution. Lysozyme was recovered with higher yields than reported for the isoelectric precipitation methods often used in the industry (86% vs 60–80%). Lysozyme peaks appeared homogeneous on SDS‐PAGE. Avidin recovery was also as good or better than that of previously reported ion exchange methods (74%–80%). The purity of the avidin fraction (up to 40.9%) was superior to that of other reported primary avidin fractions.

Lysozyme Separation from Egg White by Cation Exchange Column Chromatography

ABSTRACTCation exchange column chromatography was investigated for simple and efficient separation of lysozyme from homogenized egg white. A macroporus weak acid type resin, Duolite C‐464, was selected based on high lysozyme recovery in the90–95% range, retention of whipping and gelling properties in the lysozyme‐separated egg white, and ease of column operation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated high purity of the recovered lysozyme and simultaneous recovery of avidin under the eluting conditions used. The process for lysozyme separation from egg white using Duolite C‐464 is suitable for operation in an automated, continuous mode.

Isolation of lysozyme on chitosan.

Lysozyme has been immobilized on chitosan, a polyaminosaccharide, without using any intermediate reagent. The best pH conditions for operating the chitosan columns have been determined and the best eluting agent was found to be a 2% solution of propylamine. The lysozyme activity was determined after reacting lysozyme with the product of glycolchitin and Remazol Brilliant Blue R. The recovery of lysozyme from chicken egg white yields lysozyme with 55% activity.