Constitutive expression of hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris and its pre‐purification by foam separation during cultivation

Hydrophobins are small surface‐active proteins that have considerable potential for use in applications ranging from medical and technical coatings, separation technologies, biosensors, and personal care. Their wider use would be facilitated by the availability of recombinant tailor‐made hydrophobins. We successfully expressed the class II hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris under the control of the constitutive GAP (glyceraldehyde 3‐phosphate dehydrogenase) promoter. Avoiding the use of the AOX1 (alcohol oxidase 1) promoter prevents the costs and risks associated with the storage and delivery of methanol used as an inducer. Efficient secretion of hydrophobin was achieved using either the alpha‐factor prepro‐peptide or the native secretion signal of HFB1. The secreted hydrophobins have been isolated with a purity of up to 70% using in situ foam separation during the cultivation process. Coating experiments and surface pressure measurements demonstrated the activity of the hydrophobins. An immunodot assay showed the accessibility of carboxyterminally fused tags of the hydrophobin, which is necessary for potential applications using functionalized hydrophobins. The presented data show that Pichia pastoris is a suitable system for production of constitutively expressed and secreted active hydrophobin, allowing for in situ pre‐purification using foam separation.     

Technology of streptomycin sulfate separation by two-stage foam separation

Industrial discharges from manufacturing streptomycin sulfate (SS) are inhibitory to biological wastewater treatment and need to be stripped of residual SS. For effective SS recovery from the wastewater, a two-stage foam separation technology was investigated using a column with a vertical ellipsoid-shaped channel (VEC) and a conventional one, and sodium dodecyl sulfate (SDS) served as the collector. The mechanism of enhancing foam drainage by VEC was theoretically analyzed. In the first stage, the column with VEC was used and under the optimal conditions of the liquid-loading volume 300 mL, volumetric airflow rate 100 mL/min, the initial pH 7.0 and the molar ratio of SDS to SS 8.0, an improved SS enrichment ratio of 16.7 was obtained. In the second stage, a conventional column was used and with a volumetric airflow rate of 450 mL/min, the foamate had a SS concentration of about 0.5 g/L, so it was used as the feed solution of the first stage. By the two-stage technology, the total SS recovery percentage reached as high as 99.7%. Thus, it was significantly effective for the two-stage foam separation technology to recover SS from the simulative wastewater.     

Protein separation using metal ion-bound particles in aqueous two-phase system

Metal ion affinity partitioning of protein in aqueous two-phase systems was studied using Sepharose as ligand carrier as an integrated adsorption partitioning. Cu(II)-bound Sepharose was mixed with protein solution and an aqueous two-phase system. The affinity sorbent was distributed quantitatively to the upper side or the interface. The binding studies of lysozyme to copper-bound gel in PEG/dextran two-phase systems demonstrate the feasibility of this bioseparation process. PEG/dextran system did not affect binding and elution of lysozyme to and from the Cu(II)-Sepharose particles.     

Purification effect of artificial chaperone in the refolding of recombinant ribonuclease A from inclusion bodies

Artificial chaperone (AC) containing cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD) has been used to refold recombinant ribonuclease A (RNase A) from inclusion bodies (IBs). At low urea concentration (0.8 M), the AC could enhance the refolding yield of RNase A by effectively suppressing its intermolecular interaction-induced aggregation. As a result, 0.9 mg/mL RNase A could be 77% refolded, which was a 57% increase as compared to that without the AC. At high protein concentration range (0.9–2.3 mg/mL in total protein concentrations) and 1.6 M urea, CTAB selectively precipitated contaminant proteins distinctly, so a purification effect was achieved. For example, 1.5 mg/mL RNase A could be 62% refolded and recovered at a purity of 87%, which was a 34% increase in purity as compared to that in IBs (65%). The precipitation selectivity was considered due to the differences in the hydrophobicity of the proteins. The work indicates that by using the AC, RNase A could be efficiently refolded at low urea concentration and purified at high urea concentration from IBs at high protein concentrations.     

Gram scale separation of casein proteins from whole casein on a Source 30Q anion-exchange resin column utilizing fast protein liquid chromatography (FPLC)

Casein is used as an additive in binders or paints and as such exhibits unique properties which might be based on the properties of certain subproteins in the complex whole casein mixture. Therefore, the separation of whole casein (CN) from cow milk was performed on a gram scale in order to yield sufficient amounts of the protein subfractions α-, β-, and κ-casein for further testing utilizing fast protein liquid chromatography (FPLC) and preceding enrichment in the case of κ-casein. Construction chemical grade casein, which differs in quality from dairy grade casein, was used for separation because of our interest in the proteins responsible for plastification of cementitious systems such as mortar. The solubilized proteins were separated chromatographically via ion exchange chromatography (IEX) and the subsequently desalted protein fractions were tested for purity by isoelectric focusing (IEF).     

Lysis-free separation of hybridoma cells by continuous disc stack centrifugation

The non-destructive removal of hybridoma cells from fermentation broth with an improved disc stack centrifuge (CSA1, Westfalia Separator AG, Oelde, Germany) was investigated. The centrifuge was equipped with a hydrohermetic feed system, which allowed a gentle, shearless acceleration of the cells inside the bowl. No significant cell damage was observed during the separation of hybridoma cells from repeated batch fermentation in 100 liter scale. In the clarified liquid phase there was no increase in Lactate-Dehydrogenase (LDH) activity. Consequently, there was no increased exposure of the product to intracellular components.Due to continuous operation with a periodic and automatic discharge of sediment, a high throughput was achieved without any considerable loss of product. The clarification for mammalian cells was in the range of 99% to 99.9%, depending on the operating conditions. The content of cell debris and other small particles decreased about 30 to 50%, depending on the particle load in the feed stream. The centrifuge was fully contained; cleaning and sterilizing in place possible. Therefore, the decice could be integrated easily into the fermentation process.     

Foam fractionation of protein: correlation of protein adsorption onto bubbles with a pH-induced conformational transition

A foam fractionation apparatus was prepared to aid protein separation at the gas–liquid interface. Using lysozyme as a model protein, we investigated the alteration of enzymatic and optical activities through foaming. The lysozyme transferred to the gaseous nitrogen phase after 5 min of bubbling with no exogenous detergent. The bacteriolytic and optical activities of lysozyme from the foamate were nearly equivalent to those of the original lysozyme. This result indicated that lysozyme did not irreversibly denature during foam fractionation. We then performed protein separation using binary mixtures of lysozyme and α-amylase. When the two proteins were dissolved in bulk solution of pH 10.5, which is close to the isoelectric point (pI) of lysozyme (10.7), selective fractionation of lysozyme from the foam was observed. Indeed, this fractionation was identical to that from a single component solution of lysozyme. Similarly, selective fractionation of α-amylase was achieved in pH 3.0 buffer. Furthermore, circular dichroism (CD) and subsequent model fitting revealed that the protein had a reduced or nearly complete absence of α-helical content, whereas the amount of β-sheet structure and random coil was elevated in the buffer conditions that promoted protein adsorption. These results indicate that a pH-induced conformational transition might correlate with protein foaming.     

Atomic simulation of Xe and Kr separation in silica-templated amorphous mesoporous carbons CMK-3 and CMK-5

We perform a molecular simulation study on adsorption and separation of the noble gases Xe and Kr in silica-templated amorphous mesoporous carbons (CMK) materials. We generate the atomic models of CMK-3 and CMK-5 materials by adsorbing carbon in a model MCM-41 pore. Our carbon structures can capture the surface roughness and the disordered nature of the carbon rods and carbon pipes as reported in the experiment. The adsorption isotherms and isosteric heats of pure gases have been examined further. We find that the existence of the carbon interconnections between nanorods for CMK-3 and between nanopipes for CMK-5 will reduce the excess uptakes of the noble gases, whereas the isosteric heats are favoured in the materials with interconnections. The carbon interconnections are not advantageous to the adsorption storage of pure gases, but they can improve the separation ability of Xe for gas-mixture adsorption. The effects of temperature and concentration on the Xe separation are investigated and it is shown that the selectivities of Xe in the CMK-5 materials are insensitive to the two factors. We also find that both gas storage and separation of CMK materials are comparable to IRMOF-1 and UMCM-1 metal-organic frameworks.     

Phase separation rates of aqueous two-phase systems: correlation with system properties

The kinetics of phase separation in aqueous two-phase systems have been investigated as a function of the physical properties of the system. Two distinct situations for the settling velocities were found, one in which the light, organic-rich (PEG) phase is continuous and the other in which the heavier, salt-rich (phosphate) phase is continuous. The settling rate of a particular system is a crucial parameter for equipment design, and it was studied as a function of measured viscosity and density of each of the phases as well as the interfacial tension between the phases. Interfacial tension increases with increasing tie line length. A correlation that describes the rate of phase separation was investigated. This correlation, which is a function of the system parameters mentioned above, described the behavior of the system successfully. Different values of the parameters in the correlation were fitted for bottom-phase-continuous and top-phase-continuous systems. These parameters showed that density and viscosity play a role in the rate of separation in both top continuous- and bottom continuous-phase regions but are more dominant in the continuous top-phase region. The composition of the two-phase system was characterized by the tie line length. The rate of separation increased with increasing tie line length in both cases but at a faster rate when the bottom (less viscous) phase was the continuous phase. These results show that working in a continuous bottom-phase region is advantageous to ensure fast separation.     

Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.     

Cloacal gland foam enhances motility and disaggregation of spermatozoa in Japanese quail (Coturnix japonica)

The adult male Japanese quail produces white foam from the cloacal gland, which is transferred to the female proctodeum during natural mating. The physiological role of foam on quail spermatozoa is still unclear. Therefore, attempts have been made to understand the effect of cloacal foam on motility and metabolism of quail spermatozoa. The profile of various biochemical constitutes in the foam extract was investigated. The addition of foam extract to neat semen completely disaggregated the clumps of spermatozoa leading to vigorous motility. The metabolic rate (MBRT) of the spermatozoa was significantly increased with the addition of foam extract. The foam extract was sub fractionated into seven different fractions by using the molecular cut off devices. Among all the seven sub-fractions from the foam extract, the addition of < 1 KDa sub-fraction contained lactate and has enhanced sperm motility and metabolism. Another fraction (3-10 KDa) has non-protein and non-heparin components which completely disaggregated the clumped quail spermatozoa. However, the remaining fractions did not show any effect on quail spermatozoa. It can be concluded from the present investigation that the lactate present in foam might be a fuel for sperm metabolism and motility. Furthermore, low molecular weight (3-10 KDa) components in the foam may responsible for sperm disaggregation.     

Influence of foam thickness on the control of EMG activity during a step-down task in females

Compliant foams can be used to mitigate ground reaction forces. However, it is unknown how foam surfaces influence the modulation of leg muscle activity. Thus, the current study aimed to investigate how the neuromuscular system managed changes in expected loading due to various thickness of foam placed on the landing surface during a step down task. The surface electromyographic signal (sEMG) pre-activation duration and the root mean square (RMS) amplitude of tibialis anterior (TA), lateral gastrocnemius (LG), and vastus medialis (VM) of 10 active females were measured as they stepped-down with a single leg onto polyurethane foam slabs of varying thickness (0–50 mm). Pre-activation duration was not affected by the thickness of the foam padding. LG RMS amplitude was less in the foam conditions than the control (no– foam) condition, with the greatest reduction observed for the 50 mm foam condition. In some trials, the muscles remained active throughout the step-down task. In such instances, a sEMG onset time and thus a pre-activation duration could not be determined. All foam conditions significantly increased the odds of continuous muscle activity above that of the no-foam condition. The results indicate that foam surfaces may alter the modulation of muscle activity during step-down tasks.     

Slow irreversible unfolding of Pyrococcus furiosus triosephosphate isomerase: separation and quantitation of conformers through a novel electrophoretic approach

The thermostability of hyperthermophile proteins is not easily studied because such proteins tend to be extremely recalcitrant to unfolding. Weeks of exposure to structurally destabilizing conditions are generally required to elicit any evidence of conformational change(s). The main reason for this extreme kinetic stability would appear to be the dominance of local unfolding transitions that occur within different parts of the structures of these molecules; put differently, local sub structural unfolding transitions that occur autonomously and reversibly are thought to fail to cooperate to bring about global unfolding in a facile manner, leading to a low overall observed rate of unfolding. For reasons that are not yet fully understood, unfolding is also reported to occur irreversibly in hyperthermophile proteins. Therefore, conventional experimental approaches are often unsuited to the study of their unfolding. Here, we describe a novel electrophoretic approach that facilitates separation, direct visualization, and quantitation of the folded, partially folded, and unfolded forms of the hyperthermophile protein triosephosphate isomerase from Pyrococcus furiosus, produced in the course of its irreversible structural destabilization by the combined action of heat and chemical agents. Our approach exploits (i) the irreversibility of global unfolding effected by heat and denaturants such as urea or guanidine hydrochloride, (ii) the stability of the native form of the protein to unfolding by the anionic detergent sodium dodecyl sulfate, (iii) the differential susceptibilities of various protein conformations to being bound by SDS, and (iv) the differential electrophoretic migration behavior displayed as a consequence of differential SDS binding.     

Biosorption and desorption of lanthanum(III) and neodymium(III) in fixed-bed columns with Sargassum sp.: perspectives for separation of rare earth metals

Rare earth (RE) metals are essentials for the manufacturing of high-technology products. The separation of RE is complex and expensive; biosorption is an alternative to conventional processes. This work focuses on the biosorption of monocomponent and bicomponent solutions of lanthanum(III) and neodymium(III) in fixed-bed columns using Sargassum sp. biomass. The desorption of metals with HCl 0.10 mol L(-1) from loaded biomass is also carried out with the objective of increasing the efficiency of metal separation. Simple models have been successfully used to model breakthrough curves (i.e., Thomas, Bohart-Adams, and Yoon-Nelson equations) for the biosorption of monocomponent solutions. From biosorption and desorption experiments in both monocomponent and bicomponent solutions, a slight selectivity of the biomass for Nd(III) over La(III) is observed. The experiments did not find an effective separation of the RE studied, but their results indicate a possible partition between the metals, which is the fundamental condition for separation perspectives.     

X-ray diffraction and foam film investigations of PC head group interaction in water/ethanol mixtures

The influence of ethanol on single phospholipid monolayers at the water/air interface and in foam films has been investigated. Grazing incidence X-ray diffraction investigations (GIXD) of Langmuir monolayers from 1,2-distearoyl-phosphatidylcholine (DSPC) spread on water subphases with different amounts of ethanol were performed. The thickness and free specific energy of formation of foam films stabilized by 1,2-dimyristoyl-phosphatidylcholine (DMPC) at different concentrations of ethanol in the film forming dispersions were measured. The GIXD investigations show that the tilt angle of the alkyl chains in the PC lipid monolayer decreases with increasing concentration of ethanol caused by a decrease of the diameter of the head groups. With increasing ethanol content of the solution also the thickness of the aqueous core of PC lipid foam films decreases. We assume that ethanol causes a decreasing probability for the formation of hydrogen bonds of water molecules to the PC head groups. The distinct difference between the effects of ethanol on lipid bilayers as described in the literature and on monolayers and foam films found in this study is discussed. Whereas PC monolayers at the water/air interface become unstable above 25 vol.% ethanol, the PC foam films are stable up to 50 vol.% ethanol. This is related to the decrease of the surface excess energy per lipid molecule by the interaction between the two film surfaces.     

Enrichment of diazotrophic bacteria from rice soil in continuous culture

Summary A chemostat was used as a model system to study competitive interactions of diazotrophic microorganisms. Enrichment experiments were carried out under microaerobic conditions (8.7 mol O₂/l) with malate as the sole carbon source. The starting material was a Korean rice soil including intact root pieces. The enrichment process was governed by the dilution rate. High dilution rates resulted in the enrichment ofAzospirillum lipoferum, whereas low dilution rates led to the predominance of an unidentified organism, named Isolate R. Dilution rates were set in the range from D=0.005 to D=0.1 h^–1. The growth kinetics of both organisms followed Monod's model in the enrichment culture. From the experiments, the maximum specific growth rate ofA. lipoferum and Isolate R were 0.069 h^–1 and 0.025 h^–1, respectively. The corresponding K_s-values were 8.4 and 0.9 (mg. 1^–1). The point of theoretical coexistence of both organisms was calculated to occur at a substrate concentration of s=3.0 (mg.l^–1) with a growth of rate =0.018 h^–1. Hence the preset nutritional niches occupied by at least two organisms.Azospirillum lipoferum seems to represent the copiotroph microflora and Isolate R is of the oligotroph type. In addition to its high substrate affinity Isolate R liberatedca. 75% of the fixed nitrogen into the medium, which indicates its potential role for mutualistic interactions in the rhizosphere.     

Type-specific separation of animal cells in aqueous two-phase systems using antibody conjugates with temperature-sensitive polymers.

A new type of aqueous two-phase system (ATPS) has been developed in which a temperature-sensitive polymer, poly-N-isopropylacrylamide [poly (NIPAM)] was used as a ligand carrier for the specific separation of animal cells. Monoclonal antibodies were modified with itaconic anhydride and copolymerized with N-isopropylacrylamide, and the ligand-conjugated carriers were added to the polyethylene glycol 8000-dextran T500 aqueous two-phase systems. The antibody-polymer conjugates were partitioned to the top phase in the absence or presence of 0.15 M NaCl. When ligand-conjugated carriers were used, more than 80% of the cells were specifically partitioned to the top phase in the presence of NaCl up to 0.1 M. The cells were partitioned almost completely to the bottom phase at 0.1 M NaCl or above, when no antibody-conjugate was added in the ATPS. As a model system, CD34-positive human acute myeloid leukemia cells (KG-1) were specifically separated from human T lymphoma cells (Jurkat) by applying anti-CD34 conjugated with poly-N-isopropylacrylamide in the aqueous two-phase system. By the temperature-induced precipitation of the polymer, about 90% of the antibody-polymer conjugates were recovered from the top phase, which gave approximately 75% cell separating efficiency in the next cycle of reuse.     

Efficient phase separation and product recovery in organic‐aqueous bioprocessing using supercritical carbon dioxide

Biphasic hydrocarbon functionalizations catalyzed by recombinant microorganisms have been shown to be one of the most promising approaches for replacing common chemical synthesis routes on an industrial scale. However, the formation of stable emulsions complicates downstream processing, especially phase separation. This fact has turned out to be a major hurdle for industrial implementation. To overcome this limitation, we used supercritical carbon dioxide (scCO₂) for both phase separation and product purification. The stable emulsion, originating from a stereospecific epoxidation of styrene to (S)‐styrene oxide, a reaction catalyzed by recombinant Escherichia coli, could be destabilized efficiently and irreversibly, enabling complete phase separation within minutes. By further use of scCO₂ as extraction agent, the product (S)‐styrene oxide could be obtained with a purity of 81% (w/w) in one single extraction step. By combining phase separation and product purification using scCO₂, the number of necessary workup steps can be reduced to one. This efficient and easy to use technique is generally applicable for the workup of biphasic biocatalytic hydrocarbon functionalizations and enables a cost effective downstream processing even on a large scale. Biotechnol. Bioeng. 2010;107:642–651. © 2010 Wiley Periodicals, Inc.     

A novel twin‐column continuous chromatography approach for separation and enrichment of monoclonal antibody charge variants

Downstream processing of mAb charge variants is difficult owing to their similar molecular structures and surface charge properties. This study aimed to apply a novel twin‐column continuous chromatography (called N‐rich mode) to separate and enrich acidic variants of an IgG1 mAb. Besides, a comparison study with traditional scaled‐up batch‐mode cation exchange (CEX) chromatography was conducted. For the N‐rich process, two 3.93 mL columns were used, and the buffer system, flow rate and elution gradient slope were optimized. The results showed that 1.33 mg acidic variants with nearly 100% purity could be attained after a 22‐cycle accumulation. The yield was 86.21% with the productivity of 7.82 mg/L/h. On the other hand, for the batch CEX process, 4.15 mL column was first used to optimize the separation conditions, and then a scaled‐up column of 88.20 mL was used to separate 1.19 mg acidic variants with the purity of nearly 100%. The yield was 59.18% with the productivity of 7.78 mg/L/h. By comparing between the N‐rich and scaled‐up CEX processes, the results indicated that the N‐rich method displays a remarkable advantage on the product yield, i.e. 1.46‐fold increment without the loss of productivity and purity. Generally, twin‐column N‐rich continuous chromatography displays a high potential to enrich minor compounds with a higher yield, more flexibility and lower resin cost.