Multiobjective optimization of the operation of a liquid–solid circulating fluidized bed ion‐exchange system for continuous protein recovery

Like most real‐life processes, the operation of liquid–solid circulating fluidized bed (LSCFB) system for continuous protein recovery is associated with several objectives such as maximization of production rate and recovery of protein, and minimization of amount solid ion‐exchange resin requirement, all of which need to be optimized simultaneously. In this article, multiobjective optimization of a LSCFB system for continuous protein recovery was carried out using an experimentally validated mathematical model to find the scope for further improvements in its operation. Elitist non‐dominated sorting genetic algorithm with its jumping gene adaptation was used to solve a number of bi‐ and tri‐objective function optimization problems. The optimization resulted in Pareto‐optimal solution, which provides a broad range of non‐dominated solutions due to conflicting behavior of the operating parameters on the system performance indicators. Significant improvements were achieved, for example, the production rate at optimal operation increased by 33%, using 11% less solid compared to reported experimental results for the same recovery level. The effects of operating variables on the optimal solutions are discussed in detail. The multiobjective optimization study reported here can be easily extended for the improvement of LSCFB system for other applications. Biotechnol. Bioeng. 2009;103: 873–890. © 2009 Wiley Periodicals, Inc.     

Soy protein recovery in a solvent-free process using continuous liquid-solid circulating fluidized bed ion exchanger

Soy protein concentrates and soy protein isolates act as ingredients in bakery, meat and dairy products, baby formulas, starting materials for spun textured vegetable products, and other nutritional supplements. In this study, the effectiveness of a liquid-solid circulating fluidized bed (LSCFB) ion exchanger is demonstrated for the recovery of soluble soy proteins from full fat and defatted soy flour. Under steady-state operating conditions, about 50% of the proteins could be recovered from the feed streams entering the ion exchanger. The LSCFB was shown to be a promising system for the recovery of soy protein from both defatted and full fat soy flour solutions. As the ion exchange process captures dissolved proteins, the system may offer a less damaging form of processing compared with the acid precipitation process where soy protein aggregates form and functionality is affected. In addition, the LSCFB allows simultaneous adsorption and desorption of the proteins allowing for a continuous operation. No prefiltration of feed containing suspended particles is required as well, because fluidization is used in place of packed bed technology to improve on current ion exchange processes.     

Kinetic modeling for enzymatic hydrolysis of pretreated creeping wild ryegrass

A semimechanistic multi‐reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose‐to‐glucose and two heterogeneous reactions of cellulose‐to‐cellobiose and cellulose‐to‐glucose. Adsorption of cellulase onto pretreated CWR during enzymatic hydrolysis was modeled via a Langmuir adsorption isotherm. This is the first kinetic model which incorporated the negative role of lignin (nonproductive adsorption) using a Langmuir‐type isotherm adsorption of cellulase onto lignin. The model also reflected the competitive inhibitions of cellulase by glucose and cellobiose. The Matlab optimization function of “lsqnonlin” was used to fit the model and estimate kinetic parameters based on experimental data generated under typical conditions (8% solid loading and 15 FPU/g‐cellulose enzyme concentration without the addition of background sugars). The model showed high fidelity for predicting cellulose hydrolysis behavior over a broad range of solid loading (4–12%, w/w, dry basis), enzyme concentration (15–150 FPU/ g‐cellulose), sugar inhibition (glucose of 30 and 60 mg/mL and cellobiose of 10 mg/mL). In addition, sensitivity analysis showed that the incorporation of the nonproductive adsorption of cellulase onto lignin significantly improved the predictability of the kinetic model. Our model can serve as a robust tool for developing kinetic models for system optimization of enzymatic hydrolysis, hydrolysis reactor design, and/or other hydrolysis systems with different type of enzymes and substrates. Biotechnol. Bioeng. 2009;102: 1558–1569. © 2008 Wiley Periodicals, Inc.     

Electrostatic and hydrophobic effects in affinity chromatography

A semi–quantitative theory is developed to explain the nonspecific binding of proteins to substituted affinity chromatography supports due to electrostatic and hydrophobic interactions. The equilibrium constant for the absorption of an enzyme to a solid support, and the rate of desorption of the enzyme are studied as functions of ionic strength. Experimental measurements were taken of the adsorption equilibrium constant and rate of desorption of E. coli β–galactosidase on Sepharose 4B substituted with 3, 3,-diaminodipropylamine in batch systems. It was found that the enzyme adsorption exhibits a hysteresis effect as the ionic strength is increased and then decreased. Furthermore, the adsorption of theenzyme becomes more reversible at the lower ionic strengths, while at the higher ionic strengths it is essentially irreversible. Using the measured equilibrium constants, and knowing the region of ionic strength where the adsorption becomes reversible, we were able to predict the desorption of enzyme in a continuous stirred tank as a function of time when a decreasing linear gradient of ionic strength was introduced into a slurry. It was found that the presence of another protein, hemoglobin, does not affect these results, and therefore can be separated from the enzyme.     

Alteration of bacterial surface electrostatic potential and pH upon adhesion to a solid surface and impacts to cellular bioenergetics

In our previous study [Hong Y, Brown DG (2009) Appl Environ Microbiol 75(8):2346–2353], the adenosine triphosphate (ATP) level of adhered bacteria was observed to be 2–5 times higher than that of planktonic bacteria. Consequently, the proton motive force (Δp) of adhered bacteria was approximately 15% greater than that of planktonic bacteria. It was hypothesized that the cell surface pH changes upon adhesion due to the charge‐regulated nature of the bacterial cell surface and that this change in surface pH can propagate to the cytoplasmic membrane and alter Δp. In the current study, we developed and applied a charge regulation model to bacterial adhesion and demonstrated that the charge nature of the adhering surface can have a significant effect on the cell surface pH and ultimately the affect the ATP levels of adhered bacteria. The results indicated that the negatively charged glass surface can result in a two‐unit drop in cell surface pH, whereas adhesion to a positively charged amine surface can result in a two‐unit rise in pH. The working hypothesis indicates that the negatively charged surface should enhance Δp and increase cellular ATP, while the positively charged surface should decrease Δp and decrease ATP, and these results of the hypothesis are directly supported by prior experimental results with both negatively and positively charged surfaces. Overall, these results suggest that the nature of charge on the solid surface can have an impact on the proton motive force and cellular ATP levels. Biotechnol. Bioeng. 2010;105: 965–972. © 2009 Wiley Periodicals, Inc.     

Bioproduction of the aroma compound 2‐Phenylethanol in a solid–liquid two‐phase partitioning bioreactor system by Kluyveromyces marxianus

The rose‐like aroma compound 2‐phenylethanol (2‐PE) is an important fragrance and flavor ingredient. Several yeast strains are able to convert l ‐phenylalanine (l ‐phe) to 2‐PE among which Kluyveromyces marxianus has shown promising results. The limitation of this process is the low product concentration and productivity primarily due to end product inhibition. This study explored the possibility and benefits of using a solid–liquid Two‐Phase Partition Bioreactor (TPPB) system as an in situ product removal technique. The system applies polymer beads as the sequestering immiscible phase to partition 2‐PE and reduce the aqueous 2‐PE concentration to non‐inhibitory levels. Among six polymers screened for extracting 2‐PE, Hytrel® 8206 performed best with a partition coefficient of 79. The desired product stored in the polymer was ultimately extracted using methanol. A 3 L working volume solid–liquid batch mode TPPB using 500 g Hytrel® as the sequestering phase generated a final overall 2‐PE concentration of 13.7 g/L, the highest reported in the current literature. This was based on a polymer phase concentration of 88.74 g/L and aqueous phase concentration of 1.2 g/L. Even better results were achieved via contact with more polymers (approximately 900 g) with the aqueous phase applying a semi‐continuous reactor configuration. In this system, a final 2‐PE concentration (overall) of 20.4 g/L was achieved with 1.4 g/L in the aqueous and 97 g/L in the polymer phase. The overall productivities of these two reactor systems were 0.38 and 0.43 g/L h, respectively. This is the first report in the literature of the use of a polymer sequestering phase to enhance the bioproduction of 2‐PE, and exceeds the performance of two‐liquid phase systems in terms of productivity as well as ease of operation (no emulsions) and ultimate product recovery. Biotechnol. Bioeng. 2009; 104: 332–339 © 2009 Wiley Periodicals, Inc.     

Protein instability during HIC: Evidence of unfolding reversibility,and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

A two‐conformation, four‐state model has been proposed to describe protein adsorption and unfolding behavior on hydrophobic interaction chromatography (HIC) resins. In this work, we build upon previous study and application of a four‐state model to the effect of salt concentration on the adsorption and unfolding of the model protein α‐lactalbumin in HIC. Contributions to the apparent adsorption strength of the wild‐type protein from native and unfolded conformations, obtained using a deuterium labeling technique, reveal the free energy change and kinetics of unfolding on the resin, and demonstrate that surface unfolding is reversible. Additionally, variants of α‐lactalbumin in which one of the disulfide bonds is reduced were synthesized to examine the effects of conformational stability on apparent retention. Below the melting temperatures of the wild‐type protein and variants, reduction of a single disulfide bond significantly increases the apparent adsorption strength (～6–8 kJ/mol) due to increased instability of the protein. Finally, the four‐state model is used to accurately predict the apparent adsorption strength of a disulfide bond‐reduced variant. Biotechnol. Bioeng. 2009;102: 1416–1427. © 2008 Wiley Periodicals, Inc.     

Continuous cephalosporin C purification: dynamic modelling and parameter validation

A mathematical kinetic model for the adsorption and desorption of cephalosporin C on Amberlite XAD-2 resin is proposed. The model can represent Langmuir, Freundlich or linear isotherms at equilibrium. The intrinsic kinetic parameters and adsorption isotherms as well as physical parameters such as the effective diffusivity and the external mass transfer coefficient were obtained at different temperatures and ethanol concentrations. An unfavourable cephalosporin C adsorption occurred when ethanol was present in the solution. It has been shown that at 25 degrees C the ethanol, at concentrations from 1.5% to 2.5%, decreases the cephalosporin C adsorption. However, this behaviour was not observed at 10 degrees C. The kinetic model fitted the experimental data well under different conditions. The model was validated in a continuous process of cephalosporin C purification using the same resin. The model with the validated parameters is able to predict the behaviour of the reactor system. The continuous process is composed of two stirred tank reactors with adsorber recycle. The adsorption occurs in the first stage, and elution of the product takes place in the second stage with ethanol as eluent. The dynamic behaviour of the process was described using the following parameters: hydraulic residence time for the first (theta(h1)) and second stage (theta(h2)), solid residence time (theta(s)), initial concentration of CPC (C(0)), inlet ethanol concentration (C(ET0)) and kinetics parameters.     

He–Ne laser (632.8 nm) pre‐irradiation gives protection against DNA damage induced by a near‐infrared trapping beam

We report the results of a study carried out to investigate the effect of He–Ne laser (632.8 nm) pre‐irradiation on DNA damage induced by continuous wave 1064 nm trapping beam exposure in MCF‐7 cells. A significant decrease in % tail DNA (p < 0.05) was observed in MCF‐7 cells pre‐exposed to He–Ne laser beam. The dependence of the induced protection against 1064 nm trapping beam irradiation induced DNA damage on the time interval between the two irradiations as well as the He–Ne laser pre‐irradiation parameters is presented. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Determination of trace α‐fetoprotein variant by affinity adsorption solid substrate‐room temperature phosphorimetry

The 3.5‐generation dendrimers (3.5G‐D)–porphyrin (P) dual luminescent molecule (3.5G‐D–P) was used to label concanavalin agglutinin (Con A); the product of the reaction is 3.5G‐D–P–Con A. A new method for the determination of trace AFP‐V by affinity adsorption solid substrate–room temperature phosphorimetry (AA‐SS–RTP) was established, based on the room temperature phosphorescence (RTP) property of the product on polyamide membrane (PAM) substrate and the specific affinity adsorption (AA) reaction between 3.5G‐D–P–Con A and α‐fetoprotein variant (AFP‐V), which caused the RTP of the system to be sharply enhanced, the ΔIp was linearly correlated to the content of AFP‐V. The sensitivity of the method was obviously high. It could accurately detect the content of AFP‐V in serum. The results were tallied well with those obtained by the ELISA method. Copyright © 2008 John Wiley & Sons, Ltd.     

Two-step modification of silica for efficient adsorption of phosphatidylcholine in water and application in phospholipase D-catalyzed transphosphatidylation

An efficient and green aqueous–solid system was introduced for phospholipase D-mediated transphosphatidylation. γ-(2,3-epoxypropoxy) propytrimethoxysilane was covalently bound to silica and esterified by acetic acid, which acted as an anchor molecule to facilitate the adsorption of phosphatidylcholine (PC) in aqueous solutions. Obtained silica-adsorbed PC was successfully used for transphosphatidylation to produce phosphatidylserine (PS). The PC loading and PS yield reached 98.8 and 98.3%, respectively. A new model was proposed to illustrate the adsorption and enzymatic processes. Moreover, this aqueous–solid system provides a promising way for the continuous production. Four kinds of phospholipids were biosynthesized in the pack-bed reactor. The stability of the aqueous–solid system was excellent, as demonstrated by its use 30 times without any loss of the productivity. The product was eluted by coconut oil and manufactured into microcapsules. Toxic agents were completely avoided in the whole production process.     

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

Impact of carbon to nitrogen ratio on nutrient removal in a liquid–solid circulating fluidized bed bioreactor (LSCFB)

The performance of a liquid–solid circulating fluidized bed bioreactor (LSCFB) with anoxic and aerobic beds and lava rock as a biofilm carrier media was used to investigate the impact of the COD/N ratio on the process performance, with particular focus on total nitrogen removal. Three different COD/N ratios of 10:1, 6:1 and 4:1 were tested at an empty bed contact time of 0.82 h. More than 90% of the influent organic matter was removed throughout the study with 58% removal in the anoxic column in Phase III. Total nitrogen removal efficiencies in Phases I–III were 91%, 82% and 71% and simultaneous nitrification–denitrification (SND) occurred in the aerobic downer. The LSCFB demonstrated tertiary effluent quality at COD/N ratio of 10:1 and 6:1 with soluble biochemical oxygen demand (SBOD) <10 mg l^?1 and total nitrogen (TN) <10 mg l^?1.     

Synthesis and characterization of core‐shell magnetic molecularly imprinted polymers for selective extraction of allocryptopine from the wastewater of Macleaya cordata (Willd) R. Br.

A novel type of magnetic molecularly imprinted polymers (MMIP) as the solid‐phase extraction sorbent was prepared, which can extract effectively the allocryptopine from the waster of Macleaya cordata (Willd) R. Br. In this study, MMIP was synthesized by using Fe₃O₄@SiO₂, 4‐vinyl‐pyridine, ethylene glycol dimethacrylate, and allocryptopine, and these ingredients worked as magnetic core, functional monomer, cross‐linker, and template, respectively. Concluded by the calculation of Gaussian 09 software, different ratio models of 4‐vinyl‐pyridine and allocryptopine were simulated, and the optimal ratio was 1:5 and the energy was ?2205.34 kJ/mol. Transmission electron microscopy, vibration sample magnetometry, X‐ray diffraction, Fourier transform infrared spectroscopy, and thermogravimetric analysis were used to determine the morphology and structure of MMIP. Furthermore, the results of adsorption experiments indicated that MMIP had high selectivity, excellent recyclability, and good adsorption performance (9.86 mg/g, 298 K). The adsorption process was consistent with the Langmuir adsorption isotherm (R² > 0.98, 298 K) and pseudo‐second‐order kinetics model (R² > 0.99, 298 K). After six times adsorption‐desorption experiments, the adsorption amount of MMIP only reduced to 8.5%. In the experiments of selective adsorption, MMIP has better adsorption properties for allocryptopine (ALL, C₂₁H₂₃NO₅) than those having the same functional group. The limit of detection (LOD) was 0.4 μg/mL. The relative standard deviation ranged from 0.09% to 0.72%. The recovery of allocryptopine in samples ranged from 93.60% to 106.19%. In addition, the synthesized complex had a certain adsorption effect on allocryptopine separating from the wastewater of Macleaya cordata (Willd) R. Br.     

A comprehensive model of simultaneous denitrification and methanogenic fermentation processes

The denitrification process was incorporated into the IWA Anaerobic Digestion Model No. 1 (ADM1) in order to account for the effect of denitrification on the methanogenic fermentation process. The model was calibrated and optimized using previously published experimental data and kinetic parameter values obtained with a mixed, mesophilic (35°C) methanogenic culture. Model simulations were used to predict the effect of nitrate reduction on the methanogenic fermentation process in batch, semi‐continuous, and continuous flow reactors experiencing operational changes and/or system disturbances. The extended model clearly revealed the importance of substrate competition between denitrifiers and non‐denitrifiers as well as the impact of N‐oxide inhibition on process interactions between fermentation, methanogenesis, and denitrification. Biotechnol. Bioeng. 2010;105: 98–108. © 2009 Wiley Periodicals, Inc.     

New zeolite adsorbents for downstream processing of polyphenols from renewable resources

Commercial materials with polyvinylpolypyrrolidone and polymeric amberlites (XAD7HP, XAD16) are commonly used for the adsorptive downstream processing of polyphenols from renewable resources. In this study, beta‐zeolite‐based adsorbent systems were examined, and their properties were compared to organic resins. Batch adsorption experiments were conducted with synthetic solutions of major polyphenols. Adsorption isotherms and desorption characteristics of individual adsorbent were determined based on these results. Maximum adsorption capacities were calculated using the Langmuir model. For example, the zeolites had capacities up to 203.2 mg/g for ferulic acid. To extend these results to a complex system, additional experiments were performed on rapeseed meal and wheat seed extracts as representative renewable resources. HPLC analysis showed that with 7.5% w/v, which is regarded as the optimum amount of zeolites, zeolites A and B could bind 100% of the major polyphenols as well as release polyphenols at high yields. Additionally, regeneration experiments were performed with isopropyl alcohol at 99°C to evaluate how zeolites regenerate under mild conditions. The results showed only a negligible loss of adsorption capacity and no loss of desorption capacity. In summary, it was concluded that beta‐zeolites were promising adsorbents for developing new processes to isolate polyphenols from renewable resources.     

Adsorption of chemically synthesized mussel adhesive peptide sequences containing DOPA on stainless steel

The adsorption of proteins at solid–liquid interfaces is important in biosensor and biomaterial applications. Marine mussels affix themselves to surfaces using a highly cross‐linked, protein‐based adhesive containing a high proportion of L‐3,4‐dihydroxyphenylalanine (DOPA) residues. In this work, the effect of DOPA residues on protein adhesion on stainless steel surfaces was studied using a quartz crystal microbalance with dissipation system. The adsorption of two repetitive peptide motifs, KGYKYYGGSS and KGYKYY, from the mussel Mytilus edulis foot protein 5 on stainless steel was studied before and after chemo‐enzymatic modification of tyrosine residues to DOPA using mushroom tyrosinase. Conversion from tyrosine to DOPA, evaluated by HPLC, was in the range 70–99%. DOPA‐modified sequences showed fourfold greater adhesion than unmodified M. edulis foot protein 5 motifs. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag

The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91–95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023–1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203–273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1–60, 203–273) and L2 (203–273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203–273) fusion protein on diatomite was shorter than that of L2 (1–60, 203–273) fusion protein. The maximum adsorption capacity of L2 (203–273) fusion protein was larger than that of L2 (1–60, 203–273) fusion protein. In order to study whether the L2 (203–273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203–273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203–273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.     

Antibody purification with protein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel

Immunoglobulin G (IgG) purification from human plasma with protein A attached supermacroporous poly(hydroxyethyl methacrylate) [PHEMA] cryogel has been studied. PHEMA cryogel was prepared by bulk polymerization which proceeds in aqueous solution of monomer frozen inside a plastic syringe (cryo-polymerization). After thawing, the PHEMA cryogel contains a continuous matrix having interconnected pores of 10–200 μm size. Protein was covalently attached onto the PHEMA cryogel via cyanogen bromide (CNBr) activation. The maximum IgG adsorption on the PHEMA/protein A cryogel was found to be 83.2 mg/g at pH 7.4 from aqueous solutions. The non-specific IgG adsorption onto the PHEMA cryogel was about 0.38 mg/g. The macropore size of the cryogel makes it possible to process blood cells without blocking the column. Higher adsorption capacity was observed from human plasma (up to 88.1 mg/g). Adsorbed IgG was eluted using 0.1 M glycine–HCl buffer (pH 3.5) with a purity of 85%. PHEMA–protein A cryogel was used for repetitive adsorption/desorption of IgG without noticeable loss in IgG adsorption capacity after 10 cycles. PHEMA–protein A cryogel showed several advantages such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption–desorption cycles.