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Protein Solubility (protein + solubility)
Selected AbstractsEffect of Actinidin on the Protein Solubility, Water Holding Capacity, Texture, Electrophoretic Pattern of Beef, and on the Quality Attributes of a Sausage ProductJOURNAL OF FOOD SCIENCE, Issue 3 2009M. Aminlari ABSTRACT:, The objective of this study was to study the effect of actinidin, a sulfhydryl protease from kiwi fruit, on the protein solubility (nitrogen solubility index [NSI]), water holding capacity (WHC), texture, and SDS,PAGE pattern of beef and to evaluate the effect of pretreatment of beef with actinidin on the quality attributes of a sausage product. Actinidin was partially purified by precipitation with ammonium sulfate, followed by DEAE-Sephadex column chromatography. Actinidin significantly (P < 0.05) increased NSI and WHC of beef; the highest NSI and WHC (approximately 20% and 8% increase, respectively) was observed when beef was incubated with 0.9 unit enzyme/g beef. Texture analysis indicated increased tenderization (10% decrease in shear force) when slices of cattle beef were treated with actinidin at 37 °C for 2 h. SDS,PAGE results indicated appearance of several low molecular weight bands (<10 kDa) after treating beef with different levels of actinidin for 30 or 60 min. Slight changes in protein band in the range of 100 to 120 kDa and 13 to 25 kDa were also observed. Use of actinidin-tenderized beef significantly improved emulsion stability, texture, and organoleptic properties of the sausage product. [source] Effect of lipid oxidation and frozen storage on muscle proteins of Atlantic mackerel (Scomber scombrus)JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 5 2002Suhur Saeed Abstract The effect of storage on the lipids and proteins in Atlantic mackerel stored for up to 24 months at ,20 and ,30,°C was studied. Traditional methods including the peroxide value, thiobarbituric acid-reactive substances (TBARS) and a reverse phase HPLC method were used to determine the primary and secondary lipid oxidation products. All tests showed an increase in lipid oxidation products with storage time and at a higher storage temperature of ,20,°C compared with samples stored at ,30,°C. Antioxidants had a significant effect (P,<,0.01) on the inhibition of lipid oxidation, as shown by the reduction in peroxide value and hydroxides, and malondialdehyde formation. Similarly, deterioration of protein structure and functionality in mackerel stored for 3, 6, 12 and 24 months was greater at ,20 than ,30,°C. ATPase activity in the myosin extract of Atlantic mackerel showed a significant decrease (P,<,0.01) with progressive frozen storage. Protein solubility in high salt concentration (0.6,M NaCl) decreased (P,<,0.01) during storage at both ,20 and ,30,°C but was greater at ,20,°C. Interestingly, antioxidants BHT, vitamin C and vitamin E protected the proteins against complete loss of ATPase activity and protein solubility to a significant level (P,<,0.01) for up to 1 year at ,20,°C compared with samples stored without antioxidants. This study confirms the deleterious effect of lipid oxidation products on protein structure and function in frozen fatty fish. © 2002 Society of Chemical Industry [source] Proteomic analysis of rat brain tissue: Comparison of protocols for two-dimensional gel electrophoresis analysis based on different solubilizing agentsELECTROPHORESIS, Issue 24 2002Lucia Carboni Abstract The present study reports a comparison of recently described solubilizing methods, to set up a simple protocol for obtaining two-dimensional (2-D) gel electrophoresis maps of brain tissue. Different protocols were used for preparing rat brain homogenates and the resulting maps were compared by image analysis. Three different detergents, two delipidation methods, and introduction of a fractionation step based on different protein solubility in surfactants, were evaluated. When using efficient zwitterionic detergents (3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, CHAPS; amidosulfobetaine 14, ASB-14), the patterns obtained by direct loading of total extracts were qualitatively overlapping with patterns obtained from fractionated samples. In contrast, a weaker nonionic agent (Nonidet P-40, NP-40) produced a different protein pattern in the collected fractions. Delipidation did not improve the results for all the different extraction methods. Immunoblots performed with antibodies recognizing cytosolic and membrane-spanning proteins, which were detected as nondegraded spots, showed that membrane proteins with intermediate molecular mass could be recovered. We suggest, as a simple and efficient method for preparing rat brain maps, the homogenization in a solution containing an efficient zwitterionic surfactant, which allows to solubilize cytosolic and membrane proteins in a single step. Alternatively, a fractionation can be carried out on samples homogenized by a weak solubilizing agent, a more labor-intensive effort resulting in a larger number of proteins on two maps. [source] Functional and structural properties and in vitro digestibility of acylated hemp (Cannabis sativa L.) protein isolatesINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 12 2009Shou-Wei Yin Summary The effects of succinylation and acetylation on some functional, structural properties and in vitro trypsin digestibility of hemp protein isolate (HPI) were investigated. The extent of acylation gradually increased from 0 to 60,70%, with the anhydride-to-protein ratio increasing from 0 to 1.0 g g,1. Size exclusion chromatography showed that succinylation led to formation of more soluble protein aggregate than acetylation, especially at anhydride levels higher than 0.1 g g,1. Succinylation led to gradual increase in protein solubility (PS) from 30 to 85,90%, while in the acetylation case, the PS was improved only at low anhydride levels, increasing from 30 to about 50% with anhydride-to-protein ratio increasing from 0 to 0.2 g g,1. At neutral pH, the emulsifying activity indexes (EAI) of HPI was 22.1 m2 g,1, and the EAI linearly and significantly increased with the extent of acylation. The EAIs of succinylated and acetylated HPI (1.0 g g,1) were 119.0 and 54.4 m2 g,1, respectively. Differential scanning calorimetry (DSC) and intrinsic fluorescence spectrum analyses indicated gradual structural unfolding of proteins, or exposure of hydrophobic clusters to the solvent, especially at higher anhydride levels. Additionally, the in vitro trypsin digestibility was significantly improved by the succinylation. The results indicated that the chemical acylation treatment (especially succinylation) could be applied to modify some selected functional properties of hemp proteins, especially PS and emulsifying ability. [source] Functional properties of thermoformed wheat gluten/montmorillonite materials with respect to formulation and processing conditionsJOURNAL OF APPLIED POLYMER SCIENCE, Issue 1 2008Hélène Angellier-Coussy Abstract Wheat gluten (WG)/montmorillonite (MMT) films were prepared by a thermomechanical process consisting of first mixing the components in a two-blade, counter-rotating device and second thermoforming the obtained dough. A significant loss in protein solubility due to the formation and rearrangement of disulfide bonds was observed upon mixing and thermoforming. In the range of studied glycerol contents (25,42.8 wt %), it was shown that glycerol had no significant effect on the mechanical properties or water sensitivity of WG-based films. Increasing the thermoforming temperature from 60 to 120°C led to considerable improvements of the mechanical properties (increases in both the stress and strain at break) and a significant reduction of the water sensitivity. The introduction of MMT (up to 5 wt %) allowed the achievement of mechanical properties that were not possible by just the variation of the glycerol content and the processing temperature. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] RESPONSE SURFACE METHODOLOGY FOR STUDYING THE QUALITY CHARACTERISTICS OF COWPEA (VIGNA UNGUICULATA)-BASED TEMPEHJOURNAL OF FOOD PROCESS ENGINEERING, Issue 4 2010GEORGE AMPONSAH ANNOR ABSTRACT Response surface methodology was used to optimize the processing conditions in the preparation of cowpea tempeh. The independent factors studied were boiling time (varying from 5 to 30 min), incubation time (varying from 12 to 48 h) and incubation temperatures (varying from 25 to 50C), whereas the dependent factors were protein content, protein solubility, pH, titratable acidity and total color difference (using L, a* and b*). Regression models were generated and adequacy was tested with regression coefficients (R2) and the lack-of-fit tests. Optimum processing conditions were determined by method of superimposition. There was a strong and significant influence (P < 0.01) of the quadratic effect of the incubation time on the protein content of the cowpea tempeh, with similar significance (P < 0.01) noted in protein solubility with increasing boiling time. The optimum processing conditions observed for the preparation of cowpea tempeh were boiling time of about 20 min, incubation time of about 28 h and incubation temperature of about 37C. PRACTICAL APPLICATIONS Response surface methodology (RSM), as a statistical tool, has been effectively used in food process applications. This study embraced the use of RSM in the optimization of the processing conditions involved in the preparation of cowpea tempeh. Superimposition of the contour plots developed from the regression models indicated that cowpea with optimum quality characteristics should be processed at a boiling time of 20 min, incubation time of 28 h and incubation temperature of 37C. These conditions could be adopted for the industrial production of cowpea tempeh. [source] PHYCOCYANIN CONTENT OF SPIRULINA PLATENSIS DRIED IN SPOUTED BED AND THIN LAYERJOURNAL OF FOOD PROCESS ENGINEERING, Issue 1 2008E.G. OLIVEIRA ABSTRACT The aim of this work was to study the drying of Spirulina platensis, evaluating the final product characteristics about its phycocyanin content and its protein solubility in water. Two drying techniques were used: the spouted bed and the thin layer. For drying in a spouted bed, the cone-cylindrical geometry was chosen, namely type conventional spouted bed (CSB) and jet-spouted bed, with a paste concentration of 5%. The thin-layer drying was performed at temperatures of 50 and 60C, with a load of material of 4 kg/m2 in the tray. The spouted bed dryer type CSB demonstrated good functionality, not presenting a collapse during the experiments. The solubility in aqueous mean was similar in all the drying techniques used, being the values found around 37%. The largest phycocyanin values were found in the thin-layer temperature of 50C and in the spouted bed type CSB; however, the thin layer was excluded so as not to reach the commercial moisture content. PRACTICAL APPLICATIONS Phycocyanin is the major phycobiliprotein in Spirulina. Phycocyanin has significant antioxidant, anti-inflammatory, hepatoprotective and radical scavenging properties. It is used as colorant in food and cosmetics. It was also shown to have therapeutic value (immunomodulating activity and anticancer activity). The drying operation is commonly used to prolong the shelf life of microbial biomasses. Preservation of cyanobacteria is a difficult process, since the cells are small and, moreover, the cultures are usually diluted. Drying of liquids and pastes in spouted beds with inert bodies has been presented as an alternative to spray drying in an attempt to obtain high-quality powdered products at a low cost. [source] EFFECTS of THERMAL TREATMENTS ON TEXTURE of SOY PROTEIN ISOLATE TOFUJOURNAL OF FOOD PROCESSING AND PRESERVATION, Issue 4 2000LEE KAH HUI Effects of three thermal treatments; boiling at 100C and retorting at 110 and 121C, on texture of soy protein isolate tofu were studied for a range of heating times. Retorted tofu showed a shrinkage of 18,25% of its original size and a loss of 6,12% of its original moisture. Tofu processed at high temperatures and long heating times developed higher values of texture profile parameters than tofu processed at lower temperature and shorter heating times. the thermal treatment of tofu at elevated conditions also caused an increase in sensory hardness and a decrease in moistness and overall acceptability of the tofu. the increase in texture profile values appeared to be mirrored by a decrease in protein solubility in 1% sodium dodecyl sulfate solvent and a reduction in moisture content of the tofu. the formation of disulfides linkages at elevated temperatures and during heating at 100C stabilized protein aggregates that were initially formed by hydrophobic interactions. Formation of the disulfides and shrinkage of tofu as a result of moisture loss were suggested as the major reason for decreased sensory values of thermally processed soy protein isolate tofu. [source] MANUFACTURING FUNCTIONALITY OF CHILLED VENISON AND BEEFJOURNAL OF FOOD QUALITY, Issue 5 2007M.M. FAROUK ABSTRACT The functional properties of venison and beef semimembranosus muscle stored at ,1.5C for 4 weeks were compared. Sarcoplasmic protein solubility (SPS), malondialdehyde (MDA) and reactive sulfhydryl (SH) contents were higher in venison compared with beef (P < 0.001). Cooked batter torsion stress, tensile strength, rigidity and yield were higher in beef compared with venison (P < 0.01). Venison and beef did not differ in total protein solubility (TPS), emulsion activity index (EAI) and emulsion stability (ES) (P > 0.05). TPS, EAI, ES, MDA, torsion stress and strain, tensile strength and extensibility in meats from both species increased, and SPS and SH decreased with chilled storage time (P < 0.01). Within the parameters of this study, chilled beef had a better manufacturing functionality compared with venison. Manufacturing functionality in meats from both species improved with chill storage time, but the improvement was more marked in beef compared with venison. PRACTICAL APPLICATIONS The following are some of the practical applications of this research: (1) processing parameters used to optimize the manufacturing functionality of beef are not suitable for venison because the latter tenderize much faster than beef with negative implications to manufacturing functionality; (2) the current practice of not vacuum packaging frozen manufacturing meat will have a more deleterious effect on the functionality of venison compared with beef because of the higher rate of lipid oxidation in venison; and (3) the best time to use chilled venison and beef for maximum functionality is at 2 or 3 weeks of storage at ,1.5C. [source] Effect of Actinidin on the Protein Solubility, Water Holding Capacity, Texture, Electrophoretic Pattern of Beef, and on the Quality Attributes of a Sausage ProductJOURNAL OF FOOD SCIENCE, Issue 3 2009M. Aminlari ABSTRACT:, The objective of this study was to study the effect of actinidin, a sulfhydryl protease from kiwi fruit, on the protein solubility (nitrogen solubility index [NSI]), water holding capacity (WHC), texture, and SDS,PAGE pattern of beef and to evaluate the effect of pretreatment of beef with actinidin on the quality attributes of a sausage product. Actinidin was partially purified by precipitation with ammonium sulfate, followed by DEAE-Sephadex column chromatography. Actinidin significantly (P < 0.05) increased NSI and WHC of beef; the highest NSI and WHC (approximately 20% and 8% increase, respectively) was observed when beef was incubated with 0.9 unit enzyme/g beef. Texture analysis indicated increased tenderization (10% decrease in shear force) when slices of cattle beef were treated with actinidin at 37 °C for 2 h. SDS,PAGE results indicated appearance of several low molecular weight bands (<10 kDa) after treating beef with different levels of actinidin for 30 or 60 min. Slight changes in protein band in the range of 100 to 120 kDa and 13 to 25 kDa were also observed. Use of actinidin-tenderized beef significantly improved emulsion stability, texture, and organoleptic properties of the sausage product. [source] Effect of Chemically Modified Soy Proteins and Ficin-tenderized Meat on the Quality Attributes of SausageJOURNAL OF FOOD SCIENCE, Issue 1 2003R. Ramezani ABSTRACT: The purpose of this investigation was to use ficin-tenderized meat and cysteine-modified soy proteins in the production of bologna and to evaluate the effect of these modifications on water-holding capacity (WHC), emulsion stability (ES), texture, and protein solubility. The effect of ficin on meat protein was also evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated that both ficin-tenderized meat and modified soy proteins substantially improved WHC, ES, and other quality factors. SDS-PAGE results showed the disappearance of several protein bands in ficin-treated meat. Solubility of meat proteins increased when ficin was used for meat tenderization. The results of this study indicated that some quality attributes of meat products can be improved by enzymatic and chemical modification of protein sources in the manufacture of meat products. [source] Texture and Chemical Characteristics of Soy Protein Meat Analog Extruded at High MoistureJOURNAL OF FOOD SCIENCE, Issue 2 2000S. Lin ABSTRACT: The relationships among extruder responses, texture, and protein solubility of soy protein meat analogs were studied. Soy protein isolate and wheat starch at 9:1 ratio were extruded at 60%, 65%, and 70% moisture contents and 137.8, 148.9, and 160°C cooking temperatures. The results showed that moisture content was a more important factor on the overall product texture than cooking temperature. Lower moisture content resulted in higher die pressure, harder texture, and lower total protein solubility. At a fixed moisture content, a higher cooking temperature resulted in a softer and less chewy product but only slightly changed the protein solubility. According to partial least square regression, the data from Texture Profile Analysis, protein solubility, and extruder responses correlated well and could be used to predict each other. [source] Interaction of phytate with protein and minerals in a soybean,maize meal blend depends on pH and calcium additionJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 10 2007Katrine Pontoppidan Abstract In order to investigate possible interactions of phytate with protein and minerals in simplified animal diets, studies were conducted on the solubility of endogenous phytate, protein and essential minerals in a soybean,maize meal blend within a physiological relevant pH range. The blend was mixed with water for 10 min and then allowed to incubate at 40 °C (30 min) after adjustment of the pH. Finally, soluble phytate, protein, zinc, manganese and iron were determined. Phytate and mineral solubility was highly influenced by pH whereas protein solubility was less affected. Addition of 5 g Ca2+ kg,1 drastically reduced the solubility of phytate, zinc, manganese and iron at pH above 4.4, indicating that the formation of insoluble phytate,mineral complexes is increased in the presence of calcium. The action of pepsin increased the solubility of protein and phytate at pH below 4, indicating that insoluble phytate,protein complexes are present at low pH. Calcium had the same solubilising effect as pepsin at pH 2,4 but to a lesser degree. Copyright © 2007 Society of Chemical Industry [source] Low-salt restructured fish products using microbial transglutaminase as binding agentJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 9 2002Simón J Téllez-Luis Abstract Low-salt restructured silver carp products were obtained using mechanically deboned fish meat from filleting wastes of silver carp (Hypophthalmichthys molitrix). The additives used were NaCl at three levels (0 (control), 10 and 20,g,kg,1) and microbial transglutaminase (MTGase) also at three levels (0 (control), 3 and 6,g,kg,1). The fish meat was massaged with the additives at <15,°C for 1,h. The massaged fish paste was then packed into steel stainless tubes and cooked at 40,°C for 30,min followed by 90,°C for 15,min. Changes in mechanical properties (texture profile analysis and punch test), solubility, electrophoretic profile and expressible water were evaluated. Hardness was in the range from 26.3 to 52.4,N, cohesiveness varied from 0.185 to 0.318 and springiness varied from 0.418 to 0.768. Increasing the amount of both additives improved the mechanical and functional properties of the restructured silver carp products. MTGase activity was associated with a decrease in protein solubility and a decrease in the myosin band (SDS-PAGE). Increasing NaCl decreased the amount of expressible water. The results indicated that it is feasible to obtain low-salt restructured silver carp products with improved mechanical and good functional properties using 3,g,kg,1 MTGase and 10,g,kg,1 NaCl. © 2002 Society of Chemical Industry [source] Effect of lipid oxidation and frozen storage on muscle proteins of Atlantic mackerel (Scomber scombrus)JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 5 2002Suhur Saeed Abstract The effect of storage on the lipids and proteins in Atlantic mackerel stored for up to 24 months at ,20 and ,30,°C was studied. Traditional methods including the peroxide value, thiobarbituric acid-reactive substances (TBARS) and a reverse phase HPLC method were used to determine the primary and secondary lipid oxidation products. All tests showed an increase in lipid oxidation products with storage time and at a higher storage temperature of ,20,°C compared with samples stored at ,30,°C. Antioxidants had a significant effect (P,<,0.01) on the inhibition of lipid oxidation, as shown by the reduction in peroxide value and hydroxides, and malondialdehyde formation. Similarly, deterioration of protein structure and functionality in mackerel stored for 3, 6, 12 and 24 months was greater at ,20 than ,30,°C. ATPase activity in the myosin extract of Atlantic mackerel showed a significant decrease (P,<,0.01) with progressive frozen storage. Protein solubility in high salt concentration (0.6,M NaCl) decreased (P,<,0.01) during storage at both ,20 and ,30,°C but was greater at ,20,°C. Interestingly, antioxidants BHT, vitamin C and vitamin E protected the proteins against complete loss of ATPase activity and protein solubility to a significant level (P,<,0.01) for up to 1 year at ,20,°C compared with samples stored without antioxidants. This study confirms the deleterious effect of lipid oxidation products on protein structure and function in frozen fatty fish. © 2002 Society of Chemical Industry [source] Low free energy cost of very long loop insertions in proteinsPROTEIN SCIENCE, Issue 2 2003Michelle Scalley-Kim Abstract Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with amino- and carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 × 108 sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60,80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures. [source] Laser-improved protein crystallization screeningACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Neela Yennawar Screening of proteins for crystallization under laser irradiation was investigated using six proteins: ribonuclease B, glucose dehydrogenase, lysozyme, sorbitol dehydrogenase, fructose dehydrogenase and myoglobin. Shining 532,nm green circularly polarized laser light with a picosecond pulse and 6,mW power for 30,s on newly set-up protein drops showed a marked improvement in the number of screen conditions amenable for crystal growth compared with control drops under identical conditions but without laser exposure. For glucose dehydrogenase and sorbitol dehydrogenase, larger and better quality crystals were formed and the resolution of X-ray diffraction was improved. The speed of crystallization increased in the case of ribonuclease B, lysozyme and sorbitol dehydrogenase. During laser irradiation, the amount of precipitation in the screened drops increased, indicating a transient decrease in protein solubility. At the optimized laser settings, there was no deleterious effect of the laser on crystal growth or on the protein. In the cases of ribonuclease B and lysozyme the crystal packing did not change owing to the laser exposure. [source] Hydrophobic interaction chromatography in dual salt system increases protein binding capacityBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2009Anna M. Senczuk Abstract Hydrophobic interaction chromatography (HIC) uses weakly hydrophobic resins and requires a salting-out salt to promote protein,resin interaction. The salting-out effects increase with protein and salt concentration. Dynamic binding capacity (DBC) is dependent on the binding constant, as well as on the flow characteristics during sample loading. DBC increases with the salt concentration but decreases with increasing flow rate. Dynamic and operational binding capacity have a major raw material cost/processing time impact on commercial scale production of monoclonal antibodies. In order to maximize DBC the highest salt concentration without causing precipitation is used. We report here a novel method to maintain protein solubility while increasing the DBC by using a combination of two salting-out salts (referred to as dual salt). In a series of experiments, we explored the dynamic capacity of a HIC resin (TosoBioscience Butyl 650M) with combinations of salts. Using a model antibody, we developed a system allowing us to increase the dynamic capacity up to twofold using the dual salt system over traditional, single salt system. We also investigated the application of this novel approach to several other proteins and salt combinations, and noted a similar protein solubility and DBC increase. The observed increase in DBC in the dual salt system was maintained at different linear flow rates and did not impact selectivity. Biotechnol. Bioeng. 2009;103: 930,935. © 2009 Wiley Periodicals, Inc. [source] Rational improvement of simvastatin synthase solubility in Escherichia coli leads to higher whole-cell biocatalytic activityBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Xinkai Xie Abstract Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility of LovD without decreasing its catalytic activity can therefore elevate the performance of the whole-cell biocatalyst. Using a combination of homology structural prediction and site-directed mutagenesis, we identified two cysteine residues in LovD that are responsible for nonspecific intermolecular crosslinking, which leads to oligomer formation and protein aggregation. Replacement of Cys40 and Cys60 with alanine residues resulted in marked gain in both protein solubility and whole-cell biocatalytic activities. Further mutagenesis experiments converting these two residues to small or polar natural amino acids showed that C40A and C60N are the most beneficial, affording 27% and 26% increase in whole cell activities, respectively. The double mutant C40A/C60N combines the individual improvements and displayed ,50% increase in protein solubility and whole-cell activity. Optimized fed-batch high-cell-density fermentation of the double mutant in an E. coli strain engineered for simvastatin production quantitatively (>99%) converted 45 mM MJSS to SS within 18 h, which represents a significant improvement over the performance of wild-type LovD under identical conditions. The high efficiency of the improved whole-cell platform renders the biocatalytic synthesis of SS an attractive substitute over the existing semisynthetic routes. Biotechnol. Bioeng. 2009;102: 20,28. © 2008 Wiley Periodicals, Inc. [source] Lysozyme-lysozyme self-interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilizationBIOTECHNOLOGY JOURNAL, Issue 9 2009Virginie Le Brun Abstract The purpose of the presented study is to understand the physicochemical properties of proteins in aqueous solutions in order to identify solution conditions with reduced attractive protein-protein interactions, to avoid the formation of protein aggregates and to increase protein solubility. This is assessed by measuring the osmotic second virial coefficient (B22), a parameter of solution non-ideality, which is obtained using self-interaction chromatography. The model protein is lysozyme. The influence of various solution conditions on B22 was investigated: protonation degree, ionic strength, pharmaceutical relevant excipients and combinations thereof. Under acidic solution conditions B22 is positive, favoring protein repulsion. A similar trend is observed for the variation of the NaCl concentration, showing that with increasing the ionic strength protein attraction is more likely. B22 decreases and becomes negative. Thus, solution conditions are obtained favoring attractive protein-protein interactions. The B22 parameter also reflects, in general, the influence of the salts of the Hofmeister series with regard to their salting-in/salting-out effect. It is also shown that B22 correlates with protein solubility as well as physical protein stability. [source] | |||||||||||||