CM-cellulose Column Research Articles

A peptide fraction of Olive Flounder (Paralichthys olivaceus) Skin Hydrolysate Inhibits Amyloid-β Generation in SH-SY5Y Cells via Suppression of BACE1 Expression

The prevalence of degenerative neuro-diseases such as Alzheimer’s disease has increased with an increase in the life expectancy especially as seen in developed countries. According to the Amyloid Hypothesis, β-secretase leads to the onset of Alzheimer's disease though the generation of amyloid-β by proteolysis of amyloid precursor protein. Amyloid-β aggregates to form oligomers that build up to form plaques. Olive flounder (Paralichthys olivaceus) is high in protein content, with a higher protein content in skin (27.65%) compared to muscle (19.65%) according to proximate analysis results. Enzyme hydrolysates were prepared from skin and muscle using several enzymes. The neutrase skin hydrolysate showed the highest β-secretase inhibitory activity IC50 0.61 mg/mL. Purification steps were performed using ion exchange chromatography on QMA and CM-cellulose columns and SPE C18 column to produce an active fraction QMA-F1 with an IC50 value of 32.80 µg/mL. QMA-F1 was non-cytotoxic to SH-SY5Y cells, reduced BACE1 overexpression and attenuated amyloidogenesis. This suggests that the active β-secretase inhibitory peptides from olive flounder skin hydrolysates can be utilized as an ingredient in development of new pharmaceutical and nutraceutical therapeutic agents for Alzheimer’s Disease.

A lectin with anti-microbial and anti proliferative activities from Lantana camara, a medicinal plant

BackgroundLectins are known to possess interesting biological properties such as anti microbial, nematicidal, anti tumor and anti viral activities. Lantana camara from verbenaceae family is a medicinal plant known for possessing anti oxidant and anticancer activities. Since anticancer activity is reported in plant lectins, leaves of Lantana camara was used to check the presence of lectin. Methods and resultsHere we report the purification, characterization and biological properties of a lectin from Lantana camara (LCL) leaves. LCL was purified by ion exchange chromatography on CM-cellulose column followed by affinity chromatography on mucin coupled Sepharose 4B column and gel filtration chromatography on Superdex G75 column. LCL is a glycoprotein with 10% of the carbohydrate and is blood group non specific. SDS-PAGE analysis of affinity purified LCL showed two proteins with apparent molecular weight of 14.49 kDa and 17.4 kDa which were subsequently separated by Gel filtration chromatography on Superdex G75 column. Hapten inhibition studies of LCL revealed its highest affinity for Chitin, Milibiose, α-D-Methyl galactopyranoside and glycoproteins like mucin, asialomucin. LCL showed strong binding to human colon adenocarcinoma HT29 cells with MFI of 242 which was effectively blocked by 68.1 and 62.5% by both mucin and milibiose. LCL showed dose and time dependent growth inhibitory effects on HT29 cells with IC50 of 3.75 μg/ml at 48 h. LCL has potent antibacterial and anti fungal activity. ConclusionLCL can be explored for its clinical potential.

Selective Cytotoxicity of Staphylococcal α-Hemolysin (α-Toxin) against Human Leukocyte Populations.

Staphylococcus aureus produces a variety of exoproteins that interfere with host immune systems. We attempted to purify cytotoxins against human leukocytic cells from the culture supernatant of S. aureus by a combination of ammonium sulfate precipitation, ion-exchange chromatography on a CM-cellulose column and HPLC on a Mono S 5/50 column. A major protein possessing cytotoxicity to HL60 human promyelocytic leukemia cells was purified, and the protein was identified as α-hemolysin (Hla, α-toxin) based on its molecular weight (34 kDa) and N-terminal amino acid sequence. Flow cytometric analysis suggested differential cytotoxicity of Hla against different human peripheral blood leukocyte populations. After cell fractionation with density-gradient centrifugation, we found that peripheral blood mononuclear cells (PBMCs) were more susceptible to Hla than polymorphonuclear leukocytes. Moreover, cell surface marker analysis suggested that Hla exhibited slightly higher cytotoxicity against CD14-positive PBMCs (mainly monocytes) than CD3- or CD19-positive cells (T or B lymphocytes). From these results, we conclude that human leukocytes have different susceptibility to Hla depending on their cell lineages, and thereby the toxin may modulate the host immune response.

An alkali tolerant α-l-rhamnosidase from Fusarium moniliforme MTCC-2088 used in de-rhamnosylation of natural glycosides

Analkali tolerant α-l-rhamnosidase has been purified to homogeneity from the culture filtrate of a new fungal strain, Fusarium moniliforme MTCC-2088, using concentration by ultrafiltration and cation exchange chromatography on CM cellulose column. The molecular mass of the purified enzyme has been found to be 36.0 kDa using SDS-PAGE analysis. The Km value using p-nitrophenyl-α-l-rhamnopyranoside as the variable substrate in 0.2 M sodium phosphate buffer pH10.5 at50 °C was 0.50 mM. The catalytic rate constant was15.6 s−1giving the values of kcat/Km is 3.12 × 104M−1 s−1. The pH and temperature optima of the enzyme were 10.5 and 50 °C, respectively. The purified enzyme had better stability at 10 °C in basic pH medium. The enzyme derhamnosylated natural glycosides like naringin to prunin, rutin to isoquercitrin and hesperidin to hesperetin glucoside. The purified α-l-rhamnosidase has potential for enhancement of wine aroma.

Production, purification and biochemical characterization of an exo-polygalacturonase from Aspergillus niger MTCC 478 suitable for clarification of orange juice

Polygalacturonases (PG) represent an important member of pectinases group of enzymes with immense industrial applications. A fungal strain Aspergillus niger MTCC478 was used for the production of polygalacturonase both under submerged and solid-state fermentation condition. Further its production was optimized under solid-state fermentation condition with media comprising of wheat bran and tea extract. Purification of an exo-PG was achieved by acetone precipitation (60–90%) and CM-cellulose column chromatography revealing 15.28-fold purification with a specific activity of 33.47 U/mg protein and 1.2% yield. A relative molecular mass of purified PG was approximately 124.0 kDa. The pH and temperature optimum was found to be 4 and 50 °C, respectively. The kcat and Km value for degradation of PGA by the purified enzyme was found to be 194 s−1 and 2.3 mg/mL, respectively. Cu2+ was found to enhance the PG activity while Ag+ completely inhibited the enzyme activity. The application of the purified PG in orange juice clarification was elucidated.

Catalase from larvae of the camel tick Hyalomma dromedarii

Catalase plays a major role in protecting cells against toxic reactive oxygen species. Here, Catalase was purified from larvae of the camel tick Hyalomma dromedarii and designated TLCAT. It was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Sephacryl S-300 and CM-cellulose columns. Gel filtration and SDS-PAGE of the purified TLCAT indicated that the protein has a native molecular weight of 120kDa and is most likely a homodimer with a subunit of approximately 60kDa. The Km value of TLCAT is 12mM H2O2 and displayed its optimum activity at pH 7.2. CaCl2, MgCl2, MnCl2 and NiCl2 increased the activity of TLCAT, while FeCl2, CoCl2, CuCl2 and ZnCl2 inhibited the activity of TLCAT. Sodium azide inhibited TLCAT competitively with a Ki value of 0.28mM. The presence of TLCAT in cells may play a role in protecting H. dromedarii ticks against oxidative damage. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of untraditional methods to control them.

ISOLATION AND PROPERTIES OF POLYPHENOL OXIDASE FROM BASIDIOCARPS OF Lactarius pergamenus Fr. (Fr.) FUNGI.

Fresh juice of basidiocarps of Lactarius pergamenus Fr. (Fr.) fungi was subjected to ion exchange chromatography with used DEAE-toyopearl and CM-cellulose columns, as well as preparative electrophoresis in 7.5% polyacrylamide gels (pH 8.6). Three isoforms of polyphenol oxidase (PPO) were discovered and two isoforms (1-l and 1-2) were purified with a release of protein 0.42 mg/kg and 0.15 mg/kg of basidiocarps, respectively. These isoforms differ in the mobility at disc-electrophoresis in 7.5% PAGE in alkaline buffer system (pH 8.6). Specfic activity of isoform 1-2 is 4.8 times higher than that of the isoforms 1-1. The molecular weight determination by gel chromatography on the Toyopearl HW-55 demonstrated that both isoforms 1-1 and 1-2 have the same 64 ± 2 kDa molecular mass. Electrophoresis in 15% PAGE in the presence of sodium dodecylsulphate and β-mercaptoethanol revealed one band with molecular mass of 64 ± 1 kDa which suggests the presence of one polypeptide chain in the molecule ofthe enzyme. The enzyme has demonstrated the highest activity at pH 6.0 and temperature +10 °C, and at +70 °C the enzyme was inactivated. The PPO activity was the highest in young mushrooms and it decreased with their age and positively correlated with the content ofthe milky juice. Ortho-aminophenol was most effective among all the tested substrates to determine the activity of PPO (o-, m- and p-aminophenol, catechol, tyrosine, resorcinol, phloroglucinol) and its relative activity was 129% of the activity of catechol. Ascorbic acid was the most effective inhibitor of the polyphenol oxidase activity which was completely blocked at 1 mM concentration, whereas the same concentration of thiourea and sodium sulphite decreased the enzymatic activity by 40-45%. The PPO in L. pergamenus fungi basidiocarps was mainly localized in the mushroom milky juice where its high activity may be associated with protection of basidiocarps against various pathogens.

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Pigeon Pea (Cajanus cajan) Seeds

Glucose-6-phosphate dehydrogenase has been purified from pigeon pea (Cajanus cajan) seeds and subjected to characterization. The enzyme was purified 123.69 fold with a yield of 21.37% by ammonium sulphate fractionation, PEG-4000 precipitation, CM cellulose column chromatography and DEAE cellulose column chromatography. The catalytically active enzyme is a dimer of 113 KDa with a subunit molecular weight of 55 KDa. Thermal inactivation of enzyme follows first order kinetics at 30&#176C and 40&#176C with half life of 6 and 1.5 min respectively. Km value for glucose-6-phosphate and NADP+ was found to be 2.68 mM and 0.75 mM respectively whereas Vmax value was found to be 0.11 U/mL and 0.13 U/mL respectively. The enzyme shows more affinity towards NADP+ than glucose-6-phosphate. The pKa value was found to be 10.41 indicating that the amino acid residue at active site might be lysine. The enzyme exhibited maximum catalytic activity at pH 8.2. The enzyme was found to be highly thermosensitive with gradual loss of activity above 30&#176C temperature.

Bioactivities of Jc-SCRIP, a Type 1 Ribosome-Inactivating Protein fromJatropha curcasSeed Coat

In this study, a type 1 RIP, designated as Jc-SCRIP, was first isolated from the seed coat of Jatropha curcas Linn. It was purified by ammonium sulfate precipitation and chromatography on DEAE-Sephacel™ and CM-cellulose columns. Purification fold of Jc-SCRIP increased 113.8 times, and the yield was 1.13% of the total protein in the final step. It was shown to be a monomeric glycoprotein with a molecular mass of 38 938 Da, as determined by MALDI-TOF/MS. It exhibited hemagglutination activity and possessed strong N-glycosidase activity. The antimicrobial activity of Jc-SCRIP was tested against nine human pathogenic bacteria and one fungus; the most potent inhibitory activity was against Staphylococcus epidermidis ATCC 12228, with minimum inhibitory concentration value of 0.20 μm. Jc-SCRIP demonstrated in vitro cytotoxicity against human breast adenocarcinoma cell line (MCF-7), a colon adenocarcinoma (SW620), and a liver carcinoma cell line (HepG2), with IC50 values of 0.15, 0.25, and 0.40 mm, respectively. The results suggested that Jc-SCRIP may be a potential natural antimicrobial and anticancer agent in medical applications.

Isolation and characterization of novel protein with anti-fungal and anti-inflammatory properties from Aloe vera leaf gel

The Aloe protein of 14 kDa from the Aloe vera leaf gel was isolated by an ion exchange chromatography using DEAE-cellulose and CM-cellulose column. The purified Aloe protein exhibited a potent anti-fungal activity against Candida paraprilosis, Candida krusei and Candida albicans. In addition, the purified Aloe protein also showed an anti-inflammatory property against pure lipoxygenase and cyclooxygenase-2 with 84% and 73% inhibition, respectively, and was verified by binding with these proteins by real time method by the phenomenon of surface plasmon resonance. This Aloe protein is a novel protein possessing antifungal and anti-inflammatory properties and thus sets a platform to be used as a medicinal plant product.

Isolation and biochemical characterization of collagens from seaweed pipefish, Syngnathus schlegeli

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the seaweed pipefish (Syngnathus schlegeli) and partially characterized. The amount of collagens isolated in the subsequent treatments was 5.5% of ASC and 33.2% PSC on the basis of lyophilized pipefish body weight, respectively. According to the electrophoretic pattern and CM-cellulose column chromatogram, the collagens might be classified as type I collagens, containing α1 and α2 chain. The imino acid content of collagen from pipefish was lower than those of mammalian collagens as also were the denaturation temperatures (Td) of collagens were 34.8°C and 35.1°C, respectively. This study shows that there is a possibility to use pipefish collagen as the alternative source of collagen from industrial purposes and subsequently it may evaluate the economical value of the seaweed pipefish.

Extruction of keratinase from the local isolate Bacillus licheniformis and partially purified and characterized

The keratinase produced from local isolate Bacillus licheniformis was purified by two steps included precipitation by ammonium sulphate with 40% saturation; followed by ion exchange using CM-Cellulose column. The enzyme was purified to 12.6 times in the last step with an enzyme yield of 17%. Enzyme characterization results indicated that: The optimal pH for enzyme activity was 7.5 and it was stable at 7-9.5. The optimal temperature for enzyme activity was 50°C and it was stable for 30 min at 25-45 °C. Substrate specifity was tested using casein, Bovine serum albumin, gelatin, hooves, human hair, chicken feathers and wool; higher specifity was recorded using casein gave 0.6 unit /ml. The enzyme was inhibited by PMSF and metal ions like Hg+2, Fe+2, Cu+2 and Mn+2, and activated by Ca+2, Mg+2, Zn+2and Al+3.

Purification, Characterization, and Biochemical Properties of α-Amylase from Potato

Purification, characterization and biochemical properties of α-amylase from post harvest Bangladeshi Potato (Solanum tuberosum L.) were investigated. The α-amylase was purified by successive chromatography on DEAE and CM-cellulose columns with a yield of 24.24%. SDSPAGE showed a molecular weight of 44 kDa for the enzyme that contain 2.8% sugar. The enzyme lost total activity in the presence of the chelating agent EDTA, confirming it was an α-type amylase. The enzyme displayed optimum activity at pH 7.2 and 37°C, with an apparent Km value of 0.26% using starch as its substrate. The enzyme was strongly inhibited by Cu2+, Fe2+ and Zn2+; moderately by Li+, Hg+ and Cd2+; and slightly by Ag+, K+, Mn2+ and Mg2+. Conversely, Fe3+ and Na+ appreciably enhanced activity, while adding calcium ion nearly doubled enzyme activity. In addition, the activity of α-amylase gradually decreased with increasing concentrations of urea. Thus, potato α-amylase is an attractive target for study to better understand the structure-function relationships of α-amylases.

Ferromagnetic Levan Composite: An Affinity Matrix to Purify Lectin

A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A) and Cratylia mollis (Cramoll 1 and Cramoll 1, 4) did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column.

Isolation and characterisation ofBacillus amyloliquefaciens H2 glutamyl endopeptidase that is secreted in stationary phase of culture growth

The culture broth ofBacillus amyloliquefaciens H2 was used for isolation by chromatography on CM-cellulose and MonoS columns of proteinase that are secreted in early and late stationary phases of culture growth. Based on their inhibition by specific serine proteinase inhibitors and their action on specific chromogenic substrates and on oxidised B-chain of insulin, the enzymes were classified as glutamyl endopeptidases. The molecular weight of secreted enzyme is 26.5 kDa. We show that although the enzyme is secreted in different phases of culture growth, both enzyme fractions have similar enzymatic properties and amino acid content, but they were distinguished in.

Isolation and Characterization of Collagen from Skin of Bullfrog, Rana catesbeiana Shaw

In order to utilize skin of bullfrog (Rana catesbeiana Shaw) as an alternative source of collagen, we investigated and compared biochemical and physical properties of collagens isolated from bullfrog skin. Two kinds of collagen (BSASC; bullfrog skin acid-soluble collagen and BSPSC; bullfrog skin pepsin-solubilized collagen) were isolated by subsequent treatments with acetic acid and pepsin. The amounts of skin collagen isolated in the subsequent treatments were 7.3% BSASC and 18.2% BSPSC on the basis of lyophilized bullfrog skin weight, respectively. According to the electrophoretic pattern and CM-cellulose column chromatogram, the BSASC has the chain composition of <TEX>${\alpha}1{\alpha}2{\alpha}3$</TEX> heterotrimer, and the BSPSC consists of two <TEX>${\alpha}$</TEX> chains of <TEX>${\alpha}1{\alpha}2$</TEX>. In addition, the denaturation temperatures of all collagens tested were ranged from <TEX>$30^{\circ}C\;to\;38^{\circ}C$</TEX>. This study suggests that there is a possibility to use bullfrog skin collagen as an alternative source of collagen for industrial purposes, and subsequently it may increase the economical value of the bullfrog.

Fasciola gigantica: Enzymes of the ornithine–proline–glutamate pathway—Characterization of Δ 1-pyrroline-5-carboxylate dehydrogenase

Ornithine aminotransferase (OAT), proline oxidase (PO), Δ 1-pyrroline-5-carboxylate reductase (P5CR), and Δ 1-pyrroline-5-carboxylate dehydrogenase (P5CD) were assessed in Fasciola gigantica. All enzymes are involved in the conversion of ornithine into glutamate and proline. High levels of P5CD suggest that the direction of the metabolic flow from ornithine is more toward glutamate than proline. F. gigantica P5CD1 and P5CD2 were separated from the majority of contaminating proteins in crude homogenate using a CM-cellulose column. A Sephacryl S-200 column was employed for P5CD2 to obtain pure enzyme with increased specific activity. The molecular mass of P5CD2 was estimated to be 50 kDa using a Sephacryl S-200 column and SDS–PAGE. It migrated as a single band on SDS–PAGE, indicating a monomeric enzyme. P5CD2 had K m values of 1.44 mM and 0.37 mM for NAD and P5C, respectively. P5CD2 oxidized a number of aliphatic and aromatic aldehydes, where the aromatic compounds had higher affinity toward the enzyme. All amino acids examined had partial inhibitory effects on the enzyme. While 3 mM AMP caused 31% activation of enzyme, 3 mM ADP and ATP inhibited activity by 18% and 23%, respectively. Apart from Cu 2+, the divalent cations that were studied caused partial inhibitory effects on the enzyme.

Isolation of a novel Carica papaya α-amylase inhibitor with deleterious activity toward Callosobruchus maculatus

Cowpea ( Vigna unguiculata) is a subsistence crop for small and poor farmers from Latin America and Africa. This culture is commonly damaged by cowpea weevil ( Callosobruchus maculatus), which burrow into stored seeds to fed on. Due to impact of larval predation, several plant defense studies have been developed, indicating that α-amylase inhibitors are able to impede and/or reduce bruchids digestive process. In this report, a novel α-amylase inhibitor from papaya seeds ( Carica papaya) with activity against cowpea weevil enzymes was purified and biochemical characterized. Peeled seeds were macerated and extracted with a 0.6 M NaCl and 0.1% HCl solution. Crude extract was precipitated with ammonium sulphate (100%). After dialyses, this rich fraction was applied onto a CM-Cellulose column and retained peak was submitted to an analytic reversed-phase column HPLC (Vydac C-18TP) yielding several peaks. Only one fraction, with molecular mass of 4562 Da, showed significant inhibitory activity against C. maculatus α-amylases. Otherwise, no inhibitory activities against mammalian α-amylases were observed. Bioassays using artificial seeds containing C. papaya α-amylase inhibitor rich fraction (0.5% and 1.0%) were also conduced showing that α-amylase inhibitors were able to increase larval mortality (50%) and also decrease insect fecundity and adult longevity. These results showed the presence of an α-amylase inhibitor from C. papaya seeds with high specificity to insect enzymes, indicating that this inhibitor probably could be used, through genetic engineering, in the construction of transgenic plants with enhanced resistance toward cowpea weevil.

Absence of a Detectable Intermediate in the Compound I Formation of Horseradish Peroxidase at Ambient Temperature*

A microsecond-resolved absorption spectrometer was developed to investigate the elementary steps in hydrogen peroxide (H(2)O(2)) activation reaction of horseradish peroxidase (HRP) at ambient temperature. The kinetic absorption spectra of HRP upon the mixing with various concentrations of H(2)O(2) (0.5-3 mm) were monitored in the time range from 50 to 300 mus. The time-resolved spectra in the Soret region possessed isosbestic points that were close to those between the resting state and compound I. The kinetic changes in the Soret absorbance could be well fitted by a single exponential function. Accordingly, no distinct spectrum of the putative intermediate between the resting state and compound I was identified. These results were consistent with the proposal that the O-O bond activation in heme peroxidases is promoted by the imidazolium form of the distal histidine that exists only transiently. It was estimated that the rate constant for the breakage of the O-O bond in H(2)O(2) by HRP is significantly faster than 1 x 10(4) s(-1).

Purification and some properties of α-amylase from post-harvest Pachyrhizus erosus L. tuber

α-Amylase, a starch splitting enzyme, was purified to homogeneity from post-harvest Pachyrhizus erosus L. tuber by successive chromatography on DEAE- and CM-cellulose columns. Purification achieved was 110 fold from the crude extract with a yield of 22.8%. SDS-PAGE showed a molecular weight of 40 kDa for the enzyme. The enzyme is of α-type as it lost total activity in the presence of EDTA, a chelating agent. It is a glycoprotein that contains 2.6% sugar as estimated by the phenol-sulfuric acid method. The enzyme displayed optimum activity at pH 7.3 and 37 °C with an apparent K m value of 0.29% for starch as substrate. The enzyme was strongly inhibited by Cu 2+, Fe 2+ and Zn 2+, moderately by Li 2+, Hg + and Cd 2+ and slightly by Ag +, Mg 2+ and K +. Calcium ion almost doubled the activity whereas Fe 3+, Mn 2+ and Na + enhanced it appreciably.