A downstream process with microemulsion extraction for microbial transformation in cloud point system

A downstream process strategy for a whole microbial transformation to produce l-phenylacetylcarbinol (PAC) in a nonionic surfactant Triton X mediated cloud point system was developed. By application of a Winsor I microemulsion, the product and the nonionic surfactant in the microbial transformation broth was separated successfully. Then the nonionic surfactant was recovered with a Winsor II microemulsion. In a single stage Winsor I microemulsion extraction process, the product recovery ratio 76.9% and the nonionic surfactant recovery ratio 66.5% were achieved. A discrete countercurrent extraction operation was also carried out to improve the separation efficiency. Finally, character of the recovery nonionic surfactant was also examined.     

Triton X-114 cloud point extraction to subfractionate blood plasma proteins for two-dimensional gel electrophoresis

A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS-PAGE to be a plasma protein concentration of about 10 mg/ml in 3% (w/v) Triton X-114. 2DE of proteins obtained by CPE of 400 μl of human plasma revealed about 200 spots constituting a spot pattern very different from the pattern of total plasma. The CPE procedure only had a limited contribution to the technical variation. Identification of about 60 spots, representing only 22 proteins, revealed that several proteins in the obtained subfraction were present in more isoforms or modifications. Among these were apolipoproteins (A-1, D, E, L1, and M), haptoglobin-related protein, phosphatidylcholine-sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins and their isoforms or modifications.     

The stabilization of beta-lactoglobulin by glycine and NaCl

Effect of glycine and NaCl on the thermal denaturation of beta-lactoglobulin was examined. The results showed that the transition temperature of beta-lactoglobulin is increased by the addition of glycine and NaCl at 0.5 and 1M. This observed stabilization by glycine and NaCl was interpreted in terms of their favorable interactions with the native state of beta-lactoglobulin and unfavorable interactions with the denatured state.     

Simultaneous determination of 3-nitro tyrosine, o-, m-, and p-tyrosine in urine samples by liquid chromatography-ultraviolet absorbance detection with pre-column cloud point extraction

Stable 3-nitro tyrosine (3-NO(2)-Tyr), o-, m-, and p-tyrosine isomers induced by oxidation of tyrosine residues in protein were considered important biomarkers for the existence of toxic oxidizing agents peroxynitrite (ONOO(-)) and OH*, which could lead to such diseases as acute lung injury, neurodegenerative disorders, atherosclerosis, cancers and many other diseases. Therefore, development of an accurate, simple and sensitive method to simultaneously detect o-, m-, and p-tyrosine and 3-NO(2)-Tyr is necessary. Fluorescence detection is highly sensitive to o-, m-, and p-tyrosine, but it cannot be used to detect 3-NO(2)-Tyr, due to the strong fluorescence-quenching characteristic of the NO(2) group. In this study, we developed a highly sensitive reversed HPLC-UV method, combined with pre-column cloud point extraction (CPE), to simultaneously determine o-, m-, and p-tyrosine and 3-NO(2)-Tyr. The procedure included derivatization of a sample with 6-aminoquinolyl-N-hydroxy-succinimidyl carbomate (AccQ) at 0.20 mol/l borate buffer (pH 8.80) for 30 min at 70 degrees C, and pre-concentration with surfactant cloud point extraction. The surfactant-rich phase was then diluted with deionized water and injected directly into the to HPLC column for analysis. A C(18) column (3.9 mm i.d. x 300 mm) was used for gradient elution separation at 25 degrees C and the detection wavelength was at 254 nm. Nineteen general amino acids showed no interference. The detection limits of p-, o-, m-Tyr and 3-NO(2)-Tyr were between 5 and 15 nmol/l. The linear range was from 0.05 to approximately 100 micromol/l.     

Dual-responsive pegylated polypeptoids with tunable cloud point temperatures

A series of pegylated polypeptoids have been readily synthesized by a strategy combining ring-opening polymerization (ROP) and thiol-yne photoaddition. The polypeptoids simultaneously incorporated branched oligo(ethylene glycol) (OEG) units and thioether bonds in the side-chains. All the polypeptoids are readily soluble in aqueous solution and show reversible thermo-responsive properties. The cloud points (CPs) were demonstrated to be readily tunable in the range of ~25 °C-60 °C by varying the chemical composition, OEG chain length and the degree of polymerization. Attractively, the chemical compositions of the side chains are readily tunable via adjusting the molar ratios of a mixture of thiol terminated OEG molecules, which avoid synthesizing new monomers or copolymerization of different monomers. Further, the oxidation/reduction of thioether groups shows significant influence on the CPs, providing a second stimulus to tune the phase transition behaviors. Considering the biocompatibility and degradability, the dual-responsive polypeptoids are potential candidates for various biomedical or biotechnological applications.     

Whole cell microbial transformation in cloud point system

Cloud point system, consisting of nonionic surfactant in an aqueous solution, has been developed as a novel medium for whole cell microbial transformation. The basic properties of cloud point system including phase separation and solubilization are introduced. The application of cloud point system for extractive microbial transformation is different from that of water-organic solvent two-phase partitioning system or aqueous two-phase system are discussed, which mainly focus on the biocompatibility of microorganism in a cloud point system and a downstream process of microbial transformation in cloud point system with oil-water-surfactant microemulsion liquid-liquid extraction for surfactant recovery and product separation. Finally, examples of whole cell microbial transformation in cloud point systems, especially in situ extraction of moderate polar substrate/product, are also presented.     

Stripping of nonionic surfactants from the coacervate phase of cloud point system for lipase separation by Winsor II microemulsion extraction with the direct addition of alcohols

Extractive microbial fermentation of lipase by Serratia marcescens ECU1010 in cloud point system was previously carried out in the cloud point system. The direct addition of different alcohols, including iso-butanol, 2-phenylethanol and 1-octanol, into the coacervate phase of the clear supernatant of the fermentation broth formed microemulsion, where the nonionic surfactants and lipase were unevenly partitioned between the different phases in the microemulsion system. The polarity of alcohols strongly affected the microemulsion type at room temperature condition. The results indicated that the Winsor II microemulsion, formed by the addition of iso-butanol or 2-phenylethanol as the organic solvent, favored the stripping of the nonionic surfactant into the O_m phase, whereas the lipase was left in the excess aqueous phase. However, the Winsor I microemulsion, formed by the addition of 1-octanol as the organic solvent, failed to separate the lipase from the nonionic surfactant in the coacervate phase of cloud point system, because the nonionic surfactant and lipase were partitioned into the W_m phase at the same time. Moreover, in the Winsor II microemulsion extraction with 2-phenylethanol as the organic solvent, in which case the protein–surfactant complexes were absent at the interface between the O_m phase and the excess aqueous phase, the high lipase recovery (above 80%) and good nonionic surfactant removal were achieved. The effect of nonionic surfactants on lipase activity was also presented.     

Large scale extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey by precipitation with polyethylene glycol and partitioning in aqueous two-phase systems.

The milk proteins alpha-lactalbumin and beta-lactoglobulin have been isolated from bovine whey by fractional precipitation with polyethylene glycol (PEG) and hydrophobic partitioning in aqueous PEG-hydroxypropylstarch two-phase systems using PEG-bound palmitate as hydrophobic ligand. The possible use of this combination for large scale purification of these whey proteins is discussed.     

Extractive fermentation in cloud point system for lipase production by Serratia marcescens ECU1010

Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase (coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus, a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation process. This is the first report about extractive fermentation of proteins in cloud point system.     

Extractive biodecolorization of triphenylmethane dyes in cloud point system by Aeromonas hydrophila DN322p

The biological treatment of triphenylmethane dyes is an important issue. Most microbes have limited practical application because they cannot completely detoxicate these dyes. In this study, the extractive biodecolorization of triphenylmethane dyes by Aeromonas hydrophila DN322p was carried out by introducing the cloud point system. The cloud point system is composed of a mixture of nonionic surfactants (20 g/L) Brij 30 and Tergitol TMN-3 in equal proportions. After the decolorization of crystal violet, a higher wet cell weight was obtained in the cloud point system than that of the control system. Based on the results of thin-layer chromatography, the residual crystal violet and its decolorized product, leuco crystal violet, preferred to partition into the coacervate phase. Therefore, the detoxification of the dilute phase was achieved, which indicated that the dilute phase could be discharged without causing dye pollution. The extractive biodecolorization of three other triphenylmethane dyes was also examined in this system. The decolorization of malachite green and brilliant green was similar to that of crystal violet. Only ethyl violet achieved a poor decolorization rate because DN322p decolorized it via adsorption but did not convert it into its leuco form. This study provides potential application of biological treatment in triphenylmethane dye wastewater.     

Conformational changes of beta-lactoglobulin induced by anionic phospholipid

Conformational changes of beta-lactoglobulin (beta-LG) induced by anionic phospholipid (dimyristoylphosphatidylglycerol, DMPG) at physiological conditions (pH 7.0) have been investigated by UV-VIS, circular dichroism (CD) and fluorescence spectra. The experimental results suggest that beta-LG-DMPG interactions cause beta-LG a structural reorganization of the secondary structure elements accompanied by an increase in alpha-helical content, and a loosening of the protein tertiary structure. The interaction forces between beta-LG and DMPG are further evaluated by fluorescence spectra. The fluorescence spectral data show that conformational changes in the protein are driven by electrostatic interaction at first, then by hydrophobic interaction between a protein with a negative net charge and a negatively charged phospholipid.     

Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems

Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.     

Reduced immunogenicity of beta-lactoglobulin by conjugating with chitosan

Bovine beta-lactoglobulin (beta-LG) was conjugated with chitosan (CHS) by means of a water-soluble carbodiimide to reduce the immunogenicity of beta-LG. Each beta-LG-CHS conjugate was purified by ion-exchange chromatography and hydrophobic chromatography. The conjugation between beta-LG and CHS was confirmed by SDS-PAGE, the isoelectric point of the conjugate being higher than that of beta-LG. Two types of the beta-LG-CHS conjugate were obtained with molar ratios of beta-LG to CHS of 1:1 (F1) and 1:2 (F2). Structural analyses by fluorescence measurement, ELISA with monoclonal antibodies and retinol-binding activity indicated that the conjugates had almost maintained the native structure of beta-LG. The antigenicity of the beta-LG-CHS conjugates was similar to that of beta-LG in C3H/He mice. Reduction of the immunogenicity of beta-LG was achieved by conjugation with CHS. In particular, F2 showed very low immunogenicity. B cell epitopes of beta-LG and the conjugates recognized in C3H/He mice were determined with 15-mer multi-pin peptide; the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, while the antibody response to each epitope was dramatically reduced. Conjugation of beta-LG with chitosan was effective for reducing the immunogenicity of beta-LG.     

Promiscuous binding of ligands by beta-lactoglobulin involves hydrophobic interactions and plasticity

Bovine beta-lactoglobulin (betaLG) binds a variety of hydrophobic ligands, though precisely how is not clear. To understand the structural basis of this promiscuous binding, we studied the interaction of betaLG with palmitic acid (PA) using heteronuclear NMR spectroscopy. The titration was monitored using tryptophan fluorescence and a HSQC spectrum confirmed a 1:1 stoichiometry for the PA-betaLG complex. Upon the binding of PA, signal disappearances and large changes in chemical shifts were observed for the residues located at the entrance and bottom of the cavity, respectively. This observation indicates that the lower region makes a rigid connection with PA whereas the entrance is more flexible. The result is in contrast to the binding of PA to intestinal fatty acid-binding protein, another member of the calycin superfamily, in which structural consolidation occurs upon ligand binding. On the other hand, the ability of betaLG to accommodate various hydrophobic ligands resembles that of GroEL, in which a large hydrophobic cavity and flexible binding site confer the ability to bind various hydrophobic substrates. Considering these observations, it is suggested that, in addition to the presence of the hydrophobic cavity, the plasticity of the entrance region makes possible the binding of hydrophobic ligands of various shapes. Thus, in contrast to the specific binding seen for many enzymes, betaLG provides an example of binding with low specificity but high affinity, which may play an important role in protein-ligand and protein-protein networks.     

Cooperative alpha-helix formation of beta-lactoglobulin and melittin induced by hexafluoroisopropanol.

Alcohols denature the native state of proteins, and also stabilize the alpha-helical conformation in unfolded proteins and peptides. Among various alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) are often used because of their high potential to induce such effects. However, the reason why TFE and HFIP are more effective than other alcohols is unknown. Using CD, we studied the effects of TFE and HFIP as well as reference alcohols, i.e., methanol, ethanol, and isopropanol, on the conformation of bovine beta-lactoglobulin and the bee venom melittin at pH 2. Upon addition of alcohols, beta-lactoglobulin exhibited a transformation from the native state, consisting of beta-sheets, to the alpha-helical state, whereas melittin folded from the unfolded state to the alpha-helical state. In both cases, the order of effectiveness of alcohols was shown to be: HFIP > TFE > isopropanol > ethanol > methanol. The alcohol-induced transitions were analyzed assuming a two-state mechanism to obtain the m value, a measure of the dependence of the free energy change on alcohol concentration. Comparison of the m values indicates that the high potential of TFE can be explained by the additive contribution of constituent groups, i.e., F atoms and alkyl group. On the other hand, the high potential of HFIP is more than that expected from the additive effects, suggesting that the cooperative formation of micelle-like clusters of HFIP is important.     

Abnormal solubility behavior of beta-lactoglobulin: salting-in by glycine and NaCl

The causes of the salting-in of beta-lactoglobulin by glycine and NaCl, a solubility behavior contrary to expectations, were probed by a detailed study of the interactions between these solvent components and the protein. The preferential interactions of beta-lactoglobulin with solvent components in aqueous glycine and NaCl systems have been compared with those of bovine serum albumin and lysozyme. At neutral pH, beta-lactoglobulin exhibited insignificant preferential interactions in glycine and NaCl at low cosolvent concentrations and an increasing preferential hydration at higher concentrations, the levels approaching the values expected from the other two proteins. These results indicate considerable binding of the electrolytes to beta-lactoglobulin, sufficient to compensate for the exclusion due to perturbation of the solvent surface tension. The difference between the preferential interactions of beta-lactoglobulin and the other proteins with these two solvent additives was shown to be the cause of the increase of beta-lactoglobulin solubility even at high concentrations of the additives, at which they have salting-out effects on the other proteins. The preferential interactions of NaCl with the three proteins were examined as a function of pH. The results showed no pH dependence of the preferential hydration for bovine serum albumin and lysozyme, while this parameter increased significantly for beta-lactoglobulin at lower pH. This suggests that the binding of electrolytes to beta-lactoglobulin is due to a unique charge distribution on the surface of the protein around neutral pH, which imparts to this protein a large dipole moment.     

Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran

We prepared two beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates (Conj. 10A and Conj. 10B) by using a water-soluble carbodiimide to decrease the immunogenicity of beta-LG. The molar ratios of beta-LG to CMD in the conjugates were 5:1 (Conj. 10A) and 2:1 (Conj. 10B). The beta-LG-CMD conjugates maintained the retinol-binding activity of native beta-LG. Intrinsic fluorescence study indicated that shielding of the surface of beta-LG by CMD occurred in each conjugate, which was eminent in Conj. 10B. A local conformational change around (125)Thr-(135)Lys (alpha-helix) in each conjugate was detected by ELISA with monoclonal antibodies. The denaturation temperature of beta-LG evaluated by differential scanning calorimetry was greatly enhanced in each conjugate. The anti-beta-LG antibody response was markedly reduced after immunization with the beta-LG-CMD conjugates in BALB/c, C57BL/6, and C3H/He mice. We determined the B cell epitopes of beta-LG and each conjugate recognized in these mice and found that the linear epitope profiles of the beta-LG-CMD conjugates were similar to those of beta-LG, while the antibody response for each epitope was dramatically reduced. The reduced immunogenicity of beta-LG was most marked in the case of Conj. 10B, which contained more CMD than Conj. 10A, and was effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible for the decreased immunogenicity of the beta-LG in these conjugates.