Study on Thermodynamics and Adsorption kinetics of Purified endoglucanase (CMCase) from Penicillium notatum NCIM NO-923 produced under mixed solid-state fermentation of waste cabbage and Bagasse

In the current study, one thermostable endoglucanase was purified from Penicillium notatum NCIM NO-923 through mixed solid state fermentation of waste cabbage and bagasse. The molecular weight of the purified enzyme was 55kDa as determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had low activation energy (Ea) of 36.39KJ mol-1 for carboxymethyl cellulose hydrolysis and the enthalpy and entropy for irreversible inactivation was 87 kJ mol −1 and 59.3 J mol −1 K−1 respectively. The enzyme was quite thermostable with a Tm value of 62.2˚C. The pKa1 and pKa2 of ionizable groups of the active sites were 2.5 and 5.3 respectively. Apparent Km, Vmax and Kcat of the enzyme were found to be 5.2 mg mL-1, 80 U/gds and 322.4 sec-1 respectively. The enzyme showed about 1.4 fold increased activity in presence of 10mM MgSO4. Adsorption of endoglucanase on Avicel at wide pH range was studied at different temperatures. Langmuir type adsorption isotherm at 10˚C showed maximum adsorption strength of enzyme at pH 3.0, which was in a range of optimum pH of the enzyme.

Oxygen limitation favors the production of protein with antimicrobial activity in Pseudoalteromonas sp

This study examined the effect of dissolved oxygen concentration on the production of biomass and metabolites with antimicrobial activity of Pseudoalteromonas sp cultured at 0, 150, 250, or 450 revolutions per minute (rev. min-1). Dissolved oxygen (D.O) was monitored during the fermentation process, biomass was quantified by dry weight, and antimicrobial activity was assessed using the disk diffusion method. The bacterium Pseudoalteromonas reached similar concentration of biomass under all experimental agitation conditions, whereas antimicrobial activity was detected at 0 and 150 rev. min-1 registering 0% and 12% of D.O respectively corresponding to microaerophilic conditions. Antibiotic activity was severely diminished when D.O was above 20% of saturation; this corresponded to 250 or 450 rev. min-1. SDS-PAGE electrophoresis revealed a protein with a molecular weight of approximately 80 kilodaltons (kDa) with antimicrobial activity. Pseudoalteromonas is capable of growing under oxic and microaerophilic conditions but the metabolites with antimicrobial activity are induced under microaerophilic conditions. The current opinion is that Pseudoalteromonas are aerobic organisms; we provide additional information on the amount of dissolved oxygen during the fermentation process and its effect on antimicrobial activity.

Alkaline phosphatase isoenzymes in mouse plasma detected by polyacrylamide-gel disk electrophoresis.

Plasma alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. In the present study, we assessed the usefulness of a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying plasma ALP isoenzymes in mice of the Crlj:CD1 strain (ICR mice), which are commonly used in toxicity studies. We also examined age-related changes in plasma ALP isoenzymes in ICR mice. Electrophoresis was performed according to the manufacturer's instructions. In order to identify the origin of each ALP isoenzyme, in addition to plasma samples, tissue ALP extracts from the liver, bone and small intestine were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The kit revealed that main plasma ALP isoenzyme in intact ICR mice was bone-derived one, and it tended to decrease with age. On the other hand, liver-derived ALP isoenzyme greatly increased in plasma of cholestasis model mice induced by bile duct ligation. This model mouse had also a large molecular ALP detected in the stacking gel. This ALP was thought to be of intestinal origin because its activity remained even after levamisole inhibition. In addition, a minimum sample volume for sufficient resolution of plasma ALP isoenzymes was only 14µl. The results of this study suggest that the present method is a useful tool for detecting plasma ALP isoenzymes in mice and that pre-treatment of plasma with neuraminidase and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

[Prospects of using the electrophoresis technique for identification of the taxonomic status of earthworms (Lumbricidae)].

The polyacrylamide gel electrophoresis technique was used for comparison of common protein of body wall tissues in five species of Lumbricidae (Eisenia nordenskioldi, E.fetida, Lumbricus rubellus, Aporrectodea caliginosa, A. longa). The statistic processing of indexes of electrophoretic similarity revealed three levels of similarity: intraspecies, interspecies, and intergenus. It was discovered that the similarity of E. nordenskioldi and E.fetida proteins corresponded to the intergenus level. The use of this method for determination of the earthworm's taxonomic status is discussed.

Identification of proteins differentially expressed between capillary endothelial cells of hepatocellular carcinoma and normal liver in an orthotopic rat tumor model using 2-D DIGE.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers with few treatment options. It is a hypervascular tumor in which angiogenesis plays a critical role in its progression. Tumor capillary endothelial cells (TECs) in HCC are known to originate from liver sinusoid endothelial cells, which then go through a capillarization process to become morphologically as well as functionally different TECs. In this work, we investigated proteins differentially expressed between freshly isolated TECs and sinusoid endothelial cells from well-formed rat HCC using 2-D DIGE coupled with MALDI-TOF/TOF MS. Thirty-eight unique proteins were identified to be differentially expressed more than twofold between the two endothelial cell types. Amongst the differentially expressed proteins, two novel endothelial markers, EH domain-containing protein 3 and galectin-3, were confirmed by Western blot and immunohistochemistry in both rat and human HCC samples. We showed that EH domain-containing protein 3 is significantly down-regulated in TECs, but galectin-3 is up-regulated. We propose possible roles of these two proteins in tumor vessel development in HCC.

Rapid and simple profiling of lipoproteins by polyacrylamide-gel disc electrophoresis to determine the heterogeneity of low-density lipoproteins (LDLs) including small, dense LDL.

This study aimed to explore the potential of polyacrylamide-gel disc electrophoresis (PAGE) for lipoprotein profiling in clinical practice. Blood samples were collected from 146 patients with type 2 diabetes mellitus and lipid parameters were assayed by PAGE, including small, dense low-density lipoprotein (LDL) (n = 41), and triglyceride-rich lipoprotein remnant cholesterol (n = 37). We also used a commercial kit to measure small, dense LDL (n = 41). By PAGE, we obtained the percentage of the area under the curve (AUC %) of each peaks and calculated respective AUC% x total cholesterol (AUC%xTC) values. The calculated values of LDL-AUC%xTC, small LDL-AUC%xTC, and HDL-AUC%xTC values were correlated well with values from homogeneous assay for LDL-cholesterol, small, dense LDL-cholesterol, and HDL-cholesterol assays (r = 0.94, 0.81, and 0.89, respectively). PAGE combined with measurement of total cholesterol and triglycerides provides a rapid evaluation of anti- or pro-atherogenic lipoproteins and a simple profiling system for both the "quantity" and "quality" of lipoproteins, allowing a better assessment of the risk of coronary artery diseases. This article discusses several methods for simple and rapid lipid profiling and outlines some recent patents relevant to the methods.

[Carbon dioxide of air inhibits the formation of silver nanoparticles initiated by proteins in polyacrylamide gel and in solution].

It was shown that the detection of proteins in polyacrylamide gel by silver is inhibited by contact with air of the ammonia complex with silver ions used at the first stage of detection. It was proved by experiments on the reduction of silver by ethanolamine from a complex with ethanolamine and by formaldehyde from a complex with ammonia that the formation of silver nanoparticles initiated by proteins is inhibited by air carbon dioxide. The participation of carbon dioxide in this process is discussed. It was found that even the breathing of an experimenter can induce variations in carbon dioxide concentration sufficient to adversely affect the reproducibility of the silver staining techniques. It was concluded that, for stable staining of proteins by silver in polyacrylamide gel, it is necessary to maintain a low concentration of carbon dioxide in air over the detection solutions.

[Specific aspects of detection of peroxidase isozymes and their genetic control in barley].

Identical specimens were separated by electrophoresis in two gels to detect and fix peroxidase isozymes. Both gels were stained by Coomassie brilliant blue for detecting proteins. One gel was previously incubated for detecting peroxidase activity. The differences in electrophoretic patterns between the gels indicate the zones of peroxidase activity. It has been shown that locus Prx 6H, controlling a low-mobility grain peroxidase (PRX 6H), is localized to barley chromosome 6. Two loci, Alb 4H and Alb 7H, controlling the biosyntheses of water-soluble proteins of barley endosperm, were localized to chromosomes 4 and 7. It has been demonstrated that barley culture is polymorphic at multiple molecular forms of peroxidase.

SDS disc electrophoresis of proteins in homogeneous, low-concentrated polyacrylamide gels.

In the attempt to separate in a single gel run low- and high-molecular-weight proteins, we present here a multiphasic buffer system designed for this purpose. It avoids the continuous stacking of SDS as it occurs in the 'classical' SDS-PAGE. The system allows complete stacking and destacking of proteins in the 3.5-250 kDa range at acrylamide concentrations as low as 4.5% T (total acrylamide concentration in %) and 2.6% C (degree of cross-linking in %). Taurine is used as the trailing ion in the cathode buffer and in the resolving zone of the gel, and two different counterions (Tris and imidazole) in the stacking zone. The gel system is easy to prepare and, due to the very low acrylamide concentrations, it is ideal for analytical as well as for preparative tasks.

Comparison of statistical cluster methods in electrophoretic protein pattern analysis.

Standard electrophoresis methods were used in the qualitative and quantitative protein analyses of cerebrospinal fluid (CSF). Disc electrophoresis was carried out for detection of oligoclonal IgG bands in cerebrospinal fluid on polyacrylamide gel. Pairs of CSF and serum were taken from 30 patients, mainly with multiple sclerosis and other central nervous system dysfunctions, polyradiculoneuritis, known as Guillain-Barre syndrome, encephalitis, paraproteinemia, and analyzed. ImageMaster 1D Elite and GelPro specialized software packages were used for fast accurate image and gel analysis. The results obtained from different hierarchic cluster analysis methods were compared. In some cases, despite substantial similarities between electropherograms, different cluster methods produced different dendrograms. Therefore, the cluster analysis should be used cautiously. It offers only additional diagnostic information on the inflammatory conditions of the central nervous system.

Electrophoresis: the march of pennies, the march of dimes.

The present review encompasses ca. 65 years of history of developments in electrokinetic separations, taking as a starting point the year 1937, i.e. the official launching of Tiselius' moving boundary electrophoresis (MBE). The 1950s have been particularly rich in introducing novel methodologies in zone electrophoresis (ZE), thus bringing about the decline of MBE. Among them of extraordinary importance was the development of electrophoresis on agar gels coupled to immuno-diffusion at right angles, which brought a big revolution not only in biochemistry but also in clinical chemistry. Also the by now forgotten paper electrophoresis was a landmark in separation science, in that it implemented, in its "fingerprinting" version, the first genuine two-dimensional (2D) map, coupling orthogonally a charge to a hydrophobic scale separation, while permitting for the first time the detection of spot mutations, i.e. single amino acid replacements in a polypeptide chain, that paved the way to modern genetic analysis. Equally important was the introduction of starch-block electrophoresis, that brought about the notion of sieving and the first discontinuous buffers, refined, in the 1960s, by Ornstein and Davies with their classical papers combining multiphasic buffer systems to polyacrylamide gels, that went down to history as disc-electrophoresis. The 1960s also contributed with two fundamental techniques, isoelectric focusing (IEF) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) that permitted to discriminate proteins solely on the basis of surface charge and molecular mass, respectively. The 1970s gave other fundamental contributions, such as isotachophoresis, the first example of a fully instrumental approach to electrophoresis, both in its analytical and preparative version (Tachophor and Tachofrac), 2D maps combining IEF to SDS-PAGE at right angles and silver staining techniques, that incremented sensitivity by 3 orders of magnitude. The 1980s generated immobilized pH gradients and capillary zone electrophoresis (CZE), two big players that dominated the electrokinetic horizon for all the 1990s and still in vigorous use in present days. The review terminates with a glimpse, in the third millennium, onto microchip technology and hyphenated techniques, notably direct interfacing of various electrophoretic separation methods with mass spectrometry (MS).

[Population analysis of electrophoretic variation in blood serum albumins of European (Acipencer ruthensis L.) and Siberian (A. ruthensis marsiglii Brandt) sterlet].

We studied blood serum albumins in European (Acipencer ruthensis L.) and Siberian (A. ruthensis marsiglii Brandt) sterlet using disk electrophoresis in polyacrylamide gel. The albumins were shown to be controlled by three codominant alleles of a single locus (ALB*a, b, c). In European sterlet, all three theoretically possible genotypes were described, one of which (ALB*c/c) occurred extremely rarely (one individual). Siberian sterlet was found to be monomorphic for albumins: all fish examined had the ALB*a/a genotype. There was no correlation between albumin patterns and fish fatness. In a number of samples from the Volga River basin, spatial and temporal differentiation was found and analyzed. The results suggest that construction of hydroelectric plants may provoke massive and prolonged starlet migrations.

Enzymatic studies of riboflavin oversynthesis in Eremothecium ashbyii.

Mechanisms of riboflavin oversynthesis in a high flavinogenic mold, Eremothecium ashbyii, were examined in relation to growth, riboflavin formation and related synthases, and medium pH with increasing culture periods. Growth reached maximum at 1 d and then decreased, riboflavin formation proceeded rapidly up to 5 d and approached almost a plateau region. The medium pH reached minimum at 1 d and thereafter fairly rapidly increased until 3 d, then gradually increased to 7 d after cultivation. The crude enzyme solution from the mycelia at specified culture periods was run through a column of Sephadex G-200, indicating two riboflavin synthase activities on the chromatogram. The fluctuation of the growth and the specific activities of the two enzymes were examined with increasing culture periods, which showed that the heavy enzyme may be a constitutive one and that the light enzyme may be concerned with the oversynthesis of riboflavin in E. ashbyii. The heavy enzyme was then purified by 49-fold after dialysis of the ammonium sulfate precipitate by a series of column chromatographies with Sephadex G-200, hydroxyapatite, DEAE-Sepharose A-50 and DEAE-cellulose. The purified enzyme which was treated with weak alkaline solution was broken into the light enzyme, showing two bands on an acrylamide disc gel electrophoresis. The relation of the heavy and the light enzymes to the oversynthesis of riboflavin in E. ashbyii was discussed.

Lactate dehydrogenase isoenzymes in dental pulp of rats according to stage of root development

The objective of this study was to present a classification of the root development stage of female rat molar teeth and to evaluate the variation in the lactate dehydrogenase (LDH) activity and electrophoretic isoenzyme profile according to the stage of root development of the molar teeth. We also studied the LDH activity and isoenzymes of the pulp of incisor teeth. The stage of development of the rat first molar at the age of 15 days and that of the second molar at the age of 18 days was classified as the beginning of root formation. At the age of 15 days, the electrophoretic profile of the isoenzymes for the first molar showed a prevalence of LDH-1 followed by LDH-2. However, for the maxillary second molar there was a prevalence of LDH-4 followed by LDH-1, while for the mandibular second molar LDH-1 predominated followed by LDH-2 and LDH-4. From 18 days of age, the prevalence was always of LDH-1. The electrophoretic profile of LDH isoenzymes from the pulp of the incisor teeth at the ages studied (25 and 60 days) showed the following order of prevalence: LDH-1 > LDH-2 > LDH-3 > LDH-4 > LDH-5. These results suggest that there are variations in the prevalence of the various forms of LDH isoenzymes in the dental pulp of rats according to the developmental stage of the root

Lactate dehydrogenase isoenzymes in dental pulp of rats according to stage of root development.

The objective of this study was to present a classification of the root development stage of female rat molar teeth and to evaluate the variation in the lactate dehydrogenase (LDH) activity and electrophoretic isoenzyme profile according to the stage of root development of the molar teeth. We also studied the LDH activity and isoenzymes of the pulp of incisor teeth. The stage of development of the rat first molar at the age of 15 days and that of the second molar at the age of 18 days was classified as the beginning of root formation. At the age of 15 days, the electrophoretic profile of the isoenzymes for the first molar showed a prevalence of LDH-1 followed by LDH-2. However, for the maxillary second molar there was a prevalence of LDH-4 followed by LDH-1, while for the mandibular second molar LDH-1 predominated followed by LDH-2 and LDH-4. From 18 days of age, the prevalence was always of LDH-1. The electrophoretic profile of LDH isoenzymes from the pulp of the incisor teeth at the ages studied (25 and 60 days) showed the following order of prevalence: LDH-1 > LDH-2 > LDH-3 > LDH-4 > LDH-5. These results suggest that there are variations in the prevalence of the various forms of LDH isoenzymes in the dental pulp of rats according to the developmental stage of the root.

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis.

A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.

[Number, activity and thermostability of the electrophoretic forms of acid phosphatase in Amoeba proteus, cultured at different temperatures]. / Chislo, aktivnost' i termostabil'nost' élektroforeticheskikh form kisloi fosfatazy u ameb Amoeba proteus, kul'tiviruemykh pri razlichnykh temperaturakh.

In free-living amoebae (Amoeba proteus, strain B), cultured at 10 and 25 degrees C, we compared the number, activity, and thermostability of separate electromorphs of Triton-soluble acid phosphatase (AcP) revealed by disc-electrophoresis in polyacrylamide gel using 2-naphthyl phosphate (pH 4.0) as a substrate. No differences in the number of AcP electromorphs and their mobility were observed at both these temperatures. The total activity of AcP electromorphas per unit of cellular protein and their total thermostability were lower in amoebae acclimated to 10 degrees C than to 25 degrees C. The above decrease may be a consequence of a simultaneous decrease in the activity and thermostability of two tartrate-sensitive electromorphs, both being of lysosomal nature. The total activity and thermostability of tartrate-resistant AcP electromorphs did not differ in amoebae acclimated to the two above temperatures. In amoebae cultured at 10 degrees C the fall of activity and thermostability of lysosomal AcP correlates with the decrease in their primary cell thermoresistance and phagocytic activity. The obtained results confirm the earlier conclusion (Vysotskaya et al., 1994) that lysosomes may be involved in acclimation of electrothermal animals to changing environmental temperatures.