Acrylamide Gel Electrophoresis Research Articles

A new Rose Bengal glycopolymer: Photosensitization in two stages

Antimicrobial photodynamic therapy is a promising alternative to deal with antimicrobial resistance. However, both the low specificity and low local oxygen molecular concentrations decrease the antimicrobial efficiency limiting its use. An interesting approach to the problem is the use of molecules that can react reversibly with singlet oxygen by the formation of reversible endoperoxides, such as naphthalene, anthracene and pyridone derivatives. Particularly, the use of these molecules with mannosyl derivatives allow the interaction with adhesins presented on pili and fimbriae improving the localization near to bacteria. In this work, we synthesized polymeric nanoparticles able to generate singlet oxygen (under both irradiation and dark conditions) in the vicinity of a center capable of recognizing mannose and oxidize nearby biomolecules. Rose Bengal was used as photosensitizer due to its attractive photophysical properties (vis absorption, high singlet oxygen generation) and biocompatibility. The polymeric nanoparticles were obtained by radical polymerization using polyvinyl alcohol as a template, showing sizes around 300 nm with negative zeta potential by dynamic light scattering. The singlet oxygen generation was monitored following DPBF consumption and showed to be dependent on the amount of pyridone in the feed of polymers. In addition, the release of singlet oxygen was also dependent on pyridone concentration showing a slower rate constant at 40 % pyridone, while for contents of 10 % and 60 % higher rate constants were observed. The specific interaction of glycopolymers with Concanavalin A was demonstrated by successful agglutination assays, but also a low participation of unspecific interactions for polymers without mannosyl derivatives was observed. On the other hand, the oxidation of amino acids of Concanavalin A was monitored by acrylamide gel electrophoresis. Type I and Type II photosensitization were observed with the formation of dimers and fragments with lower molecular weight, while in dark conditions only products with lower molecular weight were observed, result consistent with singlet oxygen released by pyridone endoperoxides.

Phleum pratense-pollen adaptive variations and pollen microbiome investigation under different climatic regions and prospects of allergenicity.

Phleum pratense is an allergenic grass that pollinates in spring in Pakistan. Databases Allergenonline.org and Allergen.org record ten P. pratense allergens and their isoforms. Phl P 1, Phlp 5, and Phl p 11 are major P. pratense-pollen allergens with demonstrated basophil activity and skin test reactivity. Little is known about P. pratense pollen adaptive variations in different climatic regions and pollen-associated microbial diversity. In this study, we collected P. pratense-pollen and soils in the spring season 2022. Samples were collected from three climatic regions in Pakistan (R1, R2 and R3) with differences in mean monthly air temperature, mean monthly precipitation and elevation. The morphology of pollen was observed by light microscopy, scanning electron microscopy (SEM), biochemical fingerprint analysis, and composition of pollen were investigated by fourier-transform infrared spectroscopy (FTIR). The pollen-associated bacterial populations were identified through a Biolog GEN III microplate system. The pollen water-soluble proteins were isolated and stabilized in phosphate buffer saline (PBS) and tested for allergenicity responses through dot blots and western blots analysis. The morphology study found difference in pollen biochemical composition. Biolog identified Brevibacterium epidermidis and Pantoea agglomerans from P. pratense pollen. Protein extract quantification and sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) gel found decreased protein expression in R1 region pollen compared to R2 and R3 region pollen. Allergenicity studies found differential expression of beta-expansin and profilin allergens in pollen obtained from the three regions. Beta-expansin and profilin were suppressed in R1 pollen and expressed in compared to R2 and R3 pollen. This is the first study to identify B. epidermidis and P. agglomerans growth on P. pratense pollen. Variable allergen expression in P. pratense pollen has also been observed in different regions. Soil pH, an increase in mean monthly temperature and a decrease in mean monthly precipitation correlated with pollen biochemical composition, and reduced beta-expansin and profilin expression involved in pollen growth and development. The findings of this research are unique, which enhances basic knowledge and understanding of P. pratense-pollen associated microbiota and climate change impacts on the pollen allergen expression.

ASSESSMENT OF HIGH MOLECULAR WEIGHT PROTEINS IN SELECTED INDIGENOUS BREAD WHEAT VARIETIES OF PAKISTAN

Higher molecular weight gluten subunits (HMW-GS) articulate variation in allelic frequency, reflecting a strong correlation with bread quality. In order to determine this quality trait, 23 different Pakistani indigenous wheat varieties were screened that may be used as selection criteria in a breeding program. For detecting variations in HMW-GS, sodium dodecyl sulfate acrylamide gel electrophoresis was used. In all, 77 Glu-1 were identified including 25 at Glu-A1, 26 at Glu-B1 and 26 at Glu-D1. Alleles at Glu-1 were 2* with a frequency of 53.3% and N with 60% frequency at Glu-A1, 6+8 with 60%, 17+18 with 45 % at Glu-B1 and 5+10 with 60% and 72% at Glu-D1. The highest frequency of subunits 20 and 5+10 were found at Glu-B1-e and Glu-D1 (a), followed by a frequency of 2* and Null at Glu-A1 (b, c), which is then followed by 2+12 at GluD-1 (a). The lowest frequency of 13+16, 17+18 and 6+8 were observed at Glu-B1 (f, i, d), respectively. Glu-A1, Glu-B1, and Glu-D1 alleles showed genetic index (i.e. 0.587, 0.54), (0.587. 0.065 and 0.48, 0.39, respectively) with an average of 0.55 and 0.37. The glu-D1 locus has a lower genetic index comparatively. The composition N, 20, 5+10 was found in maximum number (4) in Fakhr-e-Sarhad, Pasban, Pirsabak-2004 and Tatara genotypes, followed by the composition of 2*, 20, 2+12 in one check (Marvi-2000) and two varieties i.e. Chakwal-97 and Bakhtawar-92. The results describing the allelic frequency and composition of bread wheat would benefit further breeding plans for wheat to select good quality parameters.

Atomically precise Pdm(SR)n nanoclusters for efficient hydrogen evolution reaction

Thiolate (SR) protected noble metal nanoclusters have grasped immense attention because of their promising applications in electrocatalysis, electrochemical sensors, photoluminescencse, supercapacitor and energy production. Among the noble metals, numerous reports have highlighted interesting results for Au25(SR)18, Au22(SR)18 and Ag44(SR)30 clusters but Pd and Pt nanoclusters are less addressed. Room temperature ionic liquids (RTIL) show enhanced electrocatalytic activity and stability as a binding matrix as compared to commercial binders like Nafion, Polyvinylidene fluoride (PVDF) and so on. Here, we have synthesised water-soluble thiolate 3-mercaptopropyl sulfonate (MPS) capped Palladium nanoclusters (Pd-MPS NCs). Further, Poly Acrylamide Gel Electrophoresis (PAGE), a separation technique was used to isolate size-dependent NCs and their electrocatalytic activity for the hydrogen evolution reaction (HER) with room temperature ionic liquid as a binding matrix was demonstrated. The electron microscope images of isolated Pd-MPS NCs from these three bands (obtained from PAGE separation) resulted with size ranges of 0.8-1.8 nm, 2-4 nm and 4.2-8 nm respectively. Among the three bands obtained from PAGE gel from the crude Pd NCs, the ultrasmall Pdm(SR)n NCs was isolated from the lower band resulted in high HER activity with less overpotential of 197 mV and a Tafel slope of 125.2 mV dec-1 as compared to Pd-MPS NCs obtained from the other two distinctive bands.

Plant proteases and anti-bacterial substances in Allium sativum L. varieties

Allium sativum L. protease still remains largely understudied although new varieties of garlic appear quite often, e.g., lanang garlic. This study tested the antibacterial effect of garlic and the effectiveness of various A. sativum proteases as meat tenderizers. 
 The research involved powder extracts of four varieties of A. sativum: kating, lanang, black garlic, and sin-chung. The degradation kinetics was defined based on the Lineweaver-Burk equation. The degradation zones were measured using sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE). Scan electron microscopy served to test the changes in meat connective tissue.
 Lanang demonstrated the largest inhibition zones against Escherichia coli (9.75 ± 0.15 mm) and Staphylococcus aureus (1.04 mm). Sin-chung protease degraded beef protein with the highest Vmax of 0.1818 μg/μL/min at 10–22 KDa (small peptide, troponin C, and troponin I), 25–40 KDa (myosin light chain, troponin T, α and β tropomyosin, actin), and 100–140 KDa (protein C). The same garlic variety degraded mutton meat protein at 10–17 KDa (small peptide) and 25–40 KDa (myosin light chain, troponin T, α and β tropomyosin, actin) with Vmax of 0.1135 μg/μL/min.
 All four A. sativum proteases proved to be quite effective meat tenderizers.

Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

A CTB-SARS-CoV-2-ACE-2 RBD Mucosal Vaccine Protects Against Coronavirus Infection.

Mucosal vaccines protect against respiratory virus infection by stimulating the production of IgA antibodies that protect against virus invasion of the mucosal epithelium. In this study, a novel protein subunit mucosal vaccine was constructed for protection against infection by the beta coronavirus SARS-CoV-2. The vaccine was assembled by linking a gene encoding the SARS-CoV-2 virus S1 angiotensin converting enzyme receptor binding domain (ACE-2-RBD) downstream from a DNA fragment encoding the cholera toxin B subunit (CTB), a mucosal adjuvant known to stimulate vaccine immunogenicity. A 42 kDa vaccine fusion protein was identified in homogenates of transformed E. coli BL-21 cells by acrylamide gel electrophoresis and by immunoblotting against anti-CTB and anti-ACE-2-RBD primary antibodies. The chimeric CTB-SARS-CoV-2-ACE-2-RBD vaccine fusion protein was partially purified from clarified bacterial homogenates by nickel affinity column chromatography. Further vaccine purification was accomplished by polyacrylamide gel electrophoresis and electro-elution of the 42 kDa chimeric vaccine protein. Vaccine protection against SARS-CoV-2 infection was assessed by oral, nasal, and parenteral immunization of BALB/c mice with the CTB-SARS-CoV-2-ACE-2-RBD protein. Vaccine-induced SARS-CoV-2 specific antibodies were quantified in immunized mouse serum by ELISA analysis. Serum from immunized mice contained IgG and IgA antibodies that neutralized SARS-CoV-2 infection in Vero E6 cell cultures. In contrast to unimmunized mice, cytological examination of cell necrosis in lung tissues excised from immunized mice revealed no detectable cellular abnormalities. Mouse behavior following vaccine immunization remained normal throughout the duration of the experiments. Together, our data show that a CTB-adjuvant-stimulated CTB-SARS-CoV-2-ACE-2-RBD chimeric mucosal vaccine protein synthesized in bacteria can produce durable and persistent IgA antibodies in mice that neutralize the SARS-CoV-2 subvariant Omicron BA.1.1.

Técnicas para el análisis de poblaciones de la molécula de fibrinógeno

The purpose of this article is to analyze the integrity of fibrinogen purified by electrophoresis in a group of healthy people to obtain a reference standard considered normal to make comparisons possible with the results from people with coronary heart disease. An explanatory identification research was carried out with an experimental design based on the application of laboratory techniques for the analysis of various types of populations of the fibrinogen molecule in healthy people. The purification of the molecule was achieved by the method of Kazal (1963) with the subsequent performance of electrophoresis in acrylamide gel SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis by the technique of Laewmli (1970), obtaining the visualization of the main characteristic chains of the normal fibrinogen molecule (α, β, and γ). It is concluded that SDS-PAGE acrylamide gel electrophoresis allows the visualization of the main characteristic chains of the normal fibrinogen molecule (α, β, and) for later comparison with pathological patterns.

Plasmid and Total Protein Analyses of Extended Spectrum Beta Lactamase Producing Bacteria from Periplaneta americana (Coackroaches)

Background: The importance of plasmid-encoded Extended Spectrum Beta Lactamase (ESBL) organisms in the dissemination of multi-drug resistance and complicated infections are contributory factors to treatment failure. Despite these, there is a paucity of information regarding plasmid-borne associated infection from primary sources including cockroaches while studies correlating total protein and plasmid-encoded ESBL are also limited especially in cockroaches. The objective of this study was therefore aimed at determining the presence of plasmid in the ESBL organisms from cockroaches as well as their total protein profiles.
 Materials and Methods: Bacterial isolation, characterization and molecular identification were carried out in our previous work following standard recommended techniques. These molecularly identified organisms which were delineated into twenty-two (22) were further subjected to plasmid and total protein analyses using alkaline lysis and Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) respectively.
 Results: Results obtained identifies 18(81.8%) of the ESBL isolates to be harbouring plasmids of different molecular weights (2-21.5kbp). The total protein profile of the plasmid-encoded ESBL and the non-plasmid encoded ESBL reveals differential protein expression patterns except for two non- plasmid encoded ESBL isolates that have similar patterns as that of plasmid-encoded ESBL organisms (isolates 2 and 3).
 Conclusion: There is a need to monitor the primary sources of infection in the epidemiological distribution of resistance. Improving environmental sanitation through proper disinfection of carrier insects may be a means to curtailing such spread.

The association between hordein polypeptide banding and agronomic traits in partitioning genetic diversity in six-rowed Ethiopian barley lines (Hordeum vulgare L.)

BackgroundEvaluation of the extent of genetic variation within and between the populations of crop genetic resources are of paramount importance in any breeding program. An experiment aimed at assessing the extent of variation among barley lines and the degree of association between hordein polypeptide and agronomic traits was hence executed.MethodsField experiment was conducted in six environments between 2017–2019 involving 19 barley lines. Hordein bands were separated using vertical Sodium Dodecyl Sulphate Poly- acrylamide Gel Electrophoresis (SDS-PAGE).ResultsThe analysis of variance revealed significant variation among lines and wider range units were observed for the agronomic traits. The line (Acc# 16,811–6) was superior, producing the highest grain yield (2.97 ton ha−1) across environments, 3.6 ton ha−1 at Holleta, and 1.93 ton ha−1 at Chefedonsa. At Arsi Negelle a different line Acc# 17146–9 was the highest yielding (3.15ton ha−1). SDS-PAGE-based analysis of barley lines separated 12 hordein bands between C (four bands) and B (eight bands) subunits. Interestingly bands 52, 46a, and 46b were uniquely conserved in the four naked barley lines (Acc#16809–14,16956–11, 17240–3, 17244–19). A considerably high proportion of genetic diversity within the populations than among the populations could be a repercussion of high gene flow which substantiates the longstanding and dominant informal seed exchange system among the farmers. The significant positive association between grain yield and band 50 evocates the expression of this allele may code for higher grain yield. The negative association between days to maturity and band 52 perhaps stipulates earliness in barely lines upon the manifestation of the band. Band 52 and 60 appeared to be associated with more than one agronomic trait (days to maturity and thousand kernel weight; grain filling period and grain yield respectively) and could be the result of pleiotropic characteristics of the genes residing in these banding regions.ConclusionThe barley lines exhibited substantial variation for hordein protein and agronomic traits. However, imparted the need for the implementation of decentralized breeding as a consequence of genotype-by-environment interaction. Significant hordein polypeptide and agronomic traits association advocated the utilization of hordein as a protein marker and perhaps consider them in the parental line selection.

Isolation and purification of copeptin hormone from serum of patients with heart disease

This study included isolation and purification of copepin hormone from heart disease patients serum using different biochemical techniques. The study showed two proteinous peak has been isolated by gel filtration (Sephadex G-50) from the precipitation by cold acetone, the results predicted that (peak B) has a high concentration of copepin . Furthermore, the purity of the isolated copeptin ( peak B) had been identified by SDS-poly acryl amide gel electrophoresis and the reverse phase high performance liquid chromatography in which results showed agood identity in retention time between the standard and the isolated copeptin (peak B) .
 The approximate molecular weight of partially purified copeptin peak (B) was (4654±400 Dalton , ±4513400 Dalton) using gel filtration chromatography (Sephadex G-50) and (SDS-poly acryl amide gel electrophoresis) technique respectively .
 The study of secondary structure (α-helix) to copeptin hormone by the (Circular Dichroesim) showed the percentage of the (α-helix) between the standard solution of copeptin and sample solution (peak B) was good match.

OPTIMIZATION OF ASPERGILLUS TUBINGENSIS GROWTH FOR CELLULASE PRODUCTION

Celluloses are the basic component of plant cell walls which are difficult to break however, cellulase enzymes have the ability to degrade the cellulose into glucose. The use of celullase enzymes is spreading widely particularly in the paper, pulp and food industries to reduce biowaste. The purpose of this study was to optimize the production of cellulase enzymes by using fungal strain Aspergillus tubingensis. The fungal strain Aspergillus tubingensis was extracted from putrid fruits and vegetable samples collected from the local fruit market of Pattoki in the District Kasur. The identification of fungus was carried out using microscope. Corn Stover was used as a substrate for cellulase production. Different parameters were used for the optimization of fungus growth such as substrate level, substrate particle size, temperature, pH, moisture, urea and glucose. Cellulase enzymes were purified by salting out and gel filtration techniques. The optimum substrate level, substrate particle size, temperature, pH, glucose, urea and moisture were analyzed as 5g, 40mm, 40°C, 4.5pH, 0.5g, 0.04g and 60% respectively. The maximum activity reveled by cellulase enzyme at different optimized parameters were reported in U/mL/min. The cellulase molecular weight was determined 71 kDa by Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis. It was concluded that the study will help to a way for the scientists for the maximum production of cellulase from Aspergillus tubingensis using Corn stover as substrate and most cost-effective Solid State Fermentation method.

Bacterial Two Component Systems: Overexpression and Purification: In Vitro and In Vivo Inhibitor Screens.

Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy, such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses upon cell wall or cell membrane damage. However, (i) activity assays using truncated kinase proteins, that is, the cytoplasmic domains containing the conserved histidine residue for phosphorylation, have been shown to produce artifacts, and (ii) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from Gram-positive bacteria that do not harbor more than two trans-membrane domains in an Escherichia coli host. This guide also presents in vitro and in vivo phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases, either using a traditional radioactively labeled ATP assay to quantify histidine kinase phosphorylation or Phos-tag acrylamide gel electrophoresis to examine histidine kinase phosphorylation through mobility shift in the polyacrylamide gel. In addition, we describe the use of Phos-tag combined with a western blot approach to visualize the phosphorylation of a response regulator in vivo.

Isolation, characterization, bacteriocin production and biological potential of Bifidobacteria of ruminants

Bacteriocins exhibited a wide spectrum of antibacterial activity against different pathogens. The aim of current study was to characterize the bacteriocins produced by Bifidobacterium spp. isolated from ruminants. The Bifidobacterium isolates were identified as B. longum, B. pseudolongum, B. bifidum, B. thermophilum, B. boum, B. merycicum and B. ruminantium. Bacteriocins were found to be pH stable, heat resistant, highly diffusible, NaCl tolerant and resistant to UV radiations. SDS, EDTA and urea induced 14%, 21% and 24% bacteriocins activity loss. Modified MRS broth (1% tryptone, 1% yeast extract and 2% glucose) was found to be the best nutrient medium for optimal production of bacteriocins. Minimum inhibitory concentration (MIC) values varied from 300 μl/ml to 500 μl/ml and minimum bactericidal concentration (MBC) values ranged from 500 μl/ml to >500 μl/ml for E. coli and S. aureus respectively. The highest protein concentration (29.0248 mg/ml) was recorded for Bifidobacteria bacteriocin produced by B. longum. Tricine-Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) revealed that molecular weight of isolated bifidobacterial bacteriocins was in the range of 3.6 kDa–30 kDa. Current study indicated that bifidobacterial bacteriocins have considerable potential to be used as biopreservative.

Extraction and Purification of Phycocyanin from Arthrophira platensis Microalgae Using a Green Solid‐Liquid Extraction Technology (RSLDE)

AbstractSpirulina is a blue alga belonging to the class of Cyanobacteria. This alga is characterized by a high content of phycocyanin (PC), a blue photosynthetic pigment belonging to the phycobiliprotein family. As natural dye, PC has a lot of industrial applications, in several fields such as the cosmetic, food, and textile sectors. It is also used in the biomedical field as a fluorescent marker. Anti‐inflammatory, nephroprotective, and hepatoprotective properties are also reported, probably related to its antioxidant activity. However, the extraction of phycobiliproteins from cyanobacteria is a very complicated procedure due to the extreme resistance of the wall of the cellular aggregates that make up spirulina. In literature, several procedures have been used to cause rupture of cell envelopes, through cycles of freezing/thawing, sonication, and rupture of the cell wall with lysozyme. However, all these methods do not guarantee a high quality of the extract, due to the complexity of the mixture obtained. In order to improve the yield of PC and to reduce the extraction process time, in this work, the conventional extraction by sonication is compared with a green innovative extraction technique, the rapid solid liquid dynamic extraction (RSLDE). This technique can generate a pressure and a consequent depression on the matrix to be extracted. The final extract was immediately frozen at −20°C and then lyophilized. UV–Vis spectrophotometric analysis and acrylamide gel electrophoresis are performed to evaluate the extraction efficiency and purity of PC.

Highly selective titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles for the enrichment of intact phosphoproteins.

Phosphorylation is one of the most important protein post-translational modifications, which possesses dramatic regulatory effects on the function of proteins. In consideration of the low abundance and low stoichiometry of phosphorylation and non-specific signal suppression, efficient capture of the phosphoproteins from complex biological samples is critical to meet the need for protein profiling. In this work, a facile preparation of titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles was developed for the enrichment of intact phosphoproteins. The prepared magnetic nanoparticles were characterized by various instruments and had a spherical shape with an average diameter of 300 nm. The adsorption isotherms were investigated and the maximum capacity for β-casein was calculated to be 961.5 mg/g. Standard protein mixtures and biological samples (non-fat milk and human serum) were selected to test the enrichment performance. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated the excellent enrichment performance with high selectivity. With the superparamagnetic property, titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles were convenient for the practical application and clinical promotion, thus having a promising prospect in the field of phosphoprotein research.

Potential of whey protein isolate-lactose conjugates in the in-vitro infant digestion

Protein base modification is a notable potential method to alter the molecular structure and physicochemical and functional properties of the protein, thus affecting protein digestibility. This study investigated the protein digestibility of whey protein isolate– lactose (WPI-Lac) conjugates using an in-vitro infant gastric digestion static model. WPI was conjugated with lactose by dry Maillard reaction under optimised conditions. The following conditions were studied, WPI-Lac heated at 40°C, water activity aw = 0.80 and incubation time of 0, 1, 3, 5 and 7 days. Based on the brown colour and the conjugation rates in ortho-phthaldehyde analysis, the incubation time of day 3 of conjugation promises the extent of conjugation and prevents the formation of advanced Maillard reaction products (MRPs). Functional properties of glycated protein were found to significantly higher (p<0.05) in antioxidant activity and solubility compared with native WPI. In addition, Fourier Transform Infrared (FTIR) analysis indicated that the WPI was modified by dry MR with lactose. WPI-Lac day 3 was evaluated using the in-vitro gastric infant digestion model which undergo simulated gastric infant condition at pH 3 with 19 µL of 0.625mg/mL of pepsin. Sodium dodecyl sulfate acrylamide gel electrophoresis (SDSPAGE) analysis of digesta revealed that MRPs increased susceptibility to be hydrolysed by pepsin. The digestion product affirms dry MR conjugation can potentially improve WPI digestibility. Herein, this study of WPI-Lac conjugates contributes to an understanding of how protein–disaccharide glycates affect in-vitro infant gastric digestion.

THE USE OF BIO-MOLECULAR SUBSTANCE OF MARINE BIOTA AS AN ALTERNATIVE EARLY INDICATOR OF OIL POLLUTED ENVIRONMENT: A NEW APPROACH FOR MONITORING CONSIDERATION

THE USE OF BIO-MOLECULAR SUBSTANCE OF MARINE BIOTA AS AN ALTERNATIVE EARLY INDICATOR OF OIL POLLUTED ENVIRONMENT: A NEW APPROACH FOR MONITORING CONSIDERATION

Statistical Approach to Enhance α-Amylase Production from Bacillus licheniformis and Purification of the Enzyme

Background: The most widely used thermostable enzymes are the amylases in the starch industry. These are among the most important enzymes and are of great significance in present day biotechnology. Objective: The main objective of the present study was to enhance α-amylase production from Bacillus licheniformis using Response Surface Methodology (RSM) and the purification of the enzyme to homogeneity. Method: Bacterial culture producing α-amylase isolated from hot spring (Himachal Pradesh) was identified as Bacillus licheniformis using 16S rDNA gene sequencing (NCBI Accession No.: KR340466). Medium components and physical culture parameters viz. pH, temperature, inoculum size, peptone concentration and starch concentration were optimized using RSM. Among these five factors, three factors (starch concentration, peptone concentration and inoculum size) had a positive effect on amylase production. A 4.09-fold increase in the production of α-amylase from B. licheniformis was achieved using RSM as compared to One Factor At a Time. The enzyme was purified by using Diethylaminoethyl Cellulose column chromatography and subsequently by Sephadex G-75 gel filtration chromatography. Result: A purification fold of 23.39 and a yield of 12.12% were observed. A single band of 33 kDa was obtained using Sodium Dodecyl Sulphate (SDS) and native-Poly Acrylamide Gel Electrophoresis (PAGE), which indicated that the enzyme was purified to homogeneity and was a monomer. The enzyme showed stability at 50 and 65°C temperatures and at alkaline pH. Conclusion: The stability of purified enzyme at high temperatures and alkaline pH suggested its wide application in textile, detergent and paper industries.

Isoenzyme study of causative agents of leishmaniasis using glutamate dehydrogenase and hexokinase

Leishmania Spp. are a group of protozoan parasites which cause a wide range of human diseases from localized self-healing cutaneous lesions to fatal visceral infections. Isoenzyme characterization by using various enzyme systems is a reliable and important tool for differentiation between various species of protozoa especially family trypanosomatidae including genus Leishmania. In this study two enzyme systems were evaluated for identification of isolated organisms from patients with cutaneous leishmaniasis (CL) in endemic foci of Fars province South of Iran. In some cases, it was observed that some enzyme systems routinely used in Leishmania parasite identification centers do not differentiate well between species, so it was decided to use non routine enzyme systems. Isoenzyme pattern of two enzyme systems, Gloutamate Dehydrogenase (GDH) and Hexokinase (HK) were compared by Poly Acryl amide Gel Electrophoresis (PAGE) and Cellulose acetate Electrophoresis (CE) in characterization of Leishmania major, Leishmania tropica and Leishmania infantum. Our findings showed the enzymes GDH on PAGE could differentiate L. major from L. tropica and L. infantum. The HK enzymatic system used on CE in this study has ability to differentiate three species of L. major, L. tropica and L. infantum.