Production and purification of a novel exopolysaccharide from lactic acid bacterium Streptococcus phocae PI80 and its functional characteristics activity in vitro

Optimum culture conditions which ease the synthesis of a novel exopolysaccharide (EPS) from a potent marine strain Streptococcus phocae was proposed in this study. The strain grows well at 35 °C, pH 7.0 and NaCl (2%) with lactose and yeast extract as best carbon and nitrogen sources. The maximum yield of EPS (11.75 and 12.14 g/L) was obtained in the presence of lactose and yeast extract at a concentration of 20 g/L respectively. EPS was refined by gel filtration chromatography using phenyl Sepharose column which revealed the presence of arabinose, fructose and galactose sugar units with molecular mass about 2.8 × 10⁵ Da. Emulsifying and flocculating stability of EPS compared with three commercial hydrocolloids. EPS exhibited better activities which are similar to that of commercial hydrocolloids. Both crude and purified EPS exhibited strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Antibiofilm activity by inhibition of Gram positive and Gram negative biofilm forming bacteria was evident in our studies. Potential antioxidant activity and biofilm inhibiting property of EPS may lead to the development of novel food grade adjuncts.     

Intra-Tumoral Heterogeneity in Metastatic Potential and Survival Signaling between Iso-Clonal HCT116 and HCT116b Human Colon Carcinoma Cell Lines

Background

Colorectal cancer (CRC) metastasis is a leading cause of cancer-related deaths in the United States. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. Recent reports have stressed the importance of intra-tumoral heterogeneity in the development of a metastatic phenotype. The purpose of this study was to characterize the intra-tumoral phenotypic heterogeneity between two iso-clonal human colon cancer sublines HCT116 and HCT116b on their ability to undergo metastatic colonization and survive under growth factor deprivation stress (GFDS).

Materials and Methods

HCT116 and HCT116b cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into other male BALB/c nude mice using microsurgical techniques. Animal tissues were studied for metastases using histochemical techniques. Microarray analysis was performed to generate gene signatures associated with each subline. In vitro assessment of growth factor signaling pathway was performed under GFDS for 3 and 5 days.

Results

Both HCT116 and HCT116b iso-clonal variants demonstrated 100% primary tumor growth, invasion and peritoneal spread. However, HCT116 was highly metastatic with 68% metastasis observed in liver and/or lungs compared to 4% in HCT116b. Microarray analysis revealed an upregulation of survival and metastatic genes in HCT116 cells compared to HCT116b cells. In vitro analysis showed that HCT116 upregulated survival and migratory signaling proteins and downregulated apoptotic agents under GFDS. However, HCT116b cells effectively showed the opposite response under stress inducing cell death.

Conclusions

We demonstrate the importance of clonal variation in determining metastatic potential of colorectal cancer cells using the HCT116/HCT116b iso-clonal variants in an orthotopic metastatic mouse model. Determination of clonal heterogeneity in patient tumors can serve as useful tools to identify clinically relevant biomarkers for diagnostic and therapeutic assessment of metastatic colorectal cancer.     

Rapid purification of a prekallikrein from rat pancreas

A prekallikrein from rat pancreas was purified 1500-fold with an overall yield of 20% using a rapid, simple procedure. DEAE-Sephadex A-50 chromatography permitted the separation of two prekallikrein components present in rat pancreatic homogenates; the major fraction was further purified by Sephadex G-100 gel filtration and immunoadsorption chromatography. The zymogen is a single-chain molecule with pI 4·35. Apparent M_r values of 38,000 and 37,000 were determined by sodium dodecyl sulfate-polyacrylamide gradient electrophoresis and gel filtration, respectively.     

Molecular Cloning, Co-expression, and Characterization of Glycerol Dehydratase and 1,3-Propanediol Dehydrogenase from Citrobacter freundii

1,3-Propanediol (1,3-PD), an important material for chemical industry, is biologically synthesized by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDOR). In present study, the dhaBCE and dhaT genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase respectively were cloned from Citrobacter freundii and co-expressed in E. coli. Sequence analysis revealed that the cloned genes were 85 and 77 % identical to corresponding gene of C. freundii DSM 30040 (GenBank No. U09771), respectively. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration, and recombinant GDHt and PDOR were characterized by activity assay, kinetic analysis, pH, and temperature optimization. This research may form a basis for the future work on biological synthesis of 1,3-PD.     

Physico-chemical characterization, antioxidant and anticancer activities in vitro of a novel polysaccharide from Melia toosendan Sieb. Et Zucc fruit

A novel water-soluble polysaccharide pMTPS-3, obtained from Melia toosendan Sieb. Et Zucc fruit by hot-water extraction and ethanol precipitation, was fractionated by DEAE-52 cellulose anion-exchange and Sephadex G-100 gel filtration chromatography. Its primary structural features and molecular weight were characterized by Fourier infrared spectrometry (FTIR), gel permeation chromatography (GPC) and gas chromatography (GC). And the antioxidant activities of pMTPS-3 in vitro were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, superoxide radical scavenging assay and hydroxyl radical scavenging assay. The results suggested that pMTPS-3 was a heteropolysaccharide, composed of arabinose, glucose, mannose, and galactose in the molar ratio of 17.3:28.3:41.6:12.6 with molecular weight 26 100 Da. The purified pMTPS-3 was revealed to have notable scavenging activity against DPPH radical in a concentration-dependent manner and present a moderate inhibition of superoxide radicals with an IC₅₀ (5.6 mg/ml), and potent inhibiting power for hydroxyl radical compared with crude polysaccharide. Further, it exhibited strong inhibition effect in vitro on the growth of human gastric cancer BGC-823 cells. It is strongly evidenced that pMTPS-3 purified from the crude polysaccharides of Melia toosendan Sieb. Et Zucc could be explored as a potential antioxidant and therapeutics.     

Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of Bombyx mori, That Binds to Cry1A Toxins

We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and K_d constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.     

Purification and characterization of pectin lyase secreted by Aspergillus flavus MTCC 10938

An indigenously isolated fungal strain Aspergillus flavus MTCC 10938 was subjected to pectin lyase (PNL) production under submerged fermentation conditions. The enzyme was purified to homogeneity from the culture filtrate of the fungus involving concentration by ultrafiltration, anion exchange chromatography on DEAE cellulose and gel filtration chromatography on Sephadex G-100. The purified PNL gave a single protein band in SDS-PAGE analysis with a relative molecular mass corresponding to 50 kDa. Using citrus pectin as the substrate the K _m and k _cat values of the enzyme were obtained as 1.7 mg/ml and 66 s^?1, respectively. The optimum pH of the purified PNL from A. flavus MTCC 10938 was 8.0 and up to 90% of its activity retained in the pH range from 3.0 to 11.0 after 24 h incubation. The optimum temperature of the purified enzyme was revealed at 55°C and it was completely stable up to 40°C when exposed for 30 min. The purified A. flavus MTCC 10938 PNL showed efficient retting of Crotalaria juncea fibres.     

Purification and characterization of an α-d-galactosyl-binding lectin from Artocarpus lakoocha seeds

A lectin from Artocarpus lakoocha seeds has been purified by affinity chromatography on a melibiose-agarose column. The homogeneity of the purified lectin was tested by several criteria, viz., poly(acrylamide)-gel electrophoresis, ultracentrifugal analysis, and gel filtration. The molecular weight of the lectin was estimated to be ∼70,000 as determined by Sephadex gel filtration. SDS-poly-(acrylamide)-gel electrophoresis gave a single component of molecular weight 18,000, suggesting that the lectin is a tetramer composed of four apparently identical subunits. The lectin agglutinated human erythrocytes, regardless of blood group. Artocarpus lakoocha lectin is a glycoprotein, and contains 11.7% of carbohydrates, in which d-xylose (6%) is the main sugar, with smaller proportions of d-galactose, d-glucose, d-mannose, N-acetyl-d-glucosamine, and N-acetyl-d-mannosamine. Amino acid analysis of the lectin revealed a high content of acidic and hydroxylic amino acids, a relatively low proportion of basic amino acids, and a trace of cysteine and methionine. In hapten-inhibition assays with simple sugars, glycosides of α-d-galactopyranose and N-acetyl-d-galactosamine were potent inhibitors of the purified lectin.     

Cryoprotective properties and preliminary characterization of exopolysaccharide (P-Arcpo 15) produced by the Arctic bacterium Pseudoalteromonas elyakovii Arcpo 15

Twenty-two bacterial strains that secrete exopolysaccharides (EPS) were isolated from marine samples obtained from the Chukchi Sea in the Arctic Ocean; of these, seven strains were found to be capable of producing cryoprotective EPS. The ArcPo 15 strain was isolated based on its ability to secrete large amounts of EPS, and was identified as Pseudoalteromonas elyakovii based on 16S rDNA analysis. The EPS, P-ArcPo 15, was purified by protease treatment and gel filtration chromatography. The purified EPS (P-ArcPo 15) had a molecular mass of 1.7 × 10⁷ Da, and its infrared spectrum showed absorption bands of hydroxyl and carboxyl groups. The principal sugar components of P-ArcPo 15 were determined to be mannose and galacturonic acid, in the ratio of 3.3:1.0. The cryoprotective properties of P-ArcPo 15 were characterized by an Escherichia coli viability test. In the presence of 0.5% (w/v) EPS, the survival percentage of E. coli cells was as high as 94.19 ± 7.81% over five repeated freeze–thaw cycles. These biochemical characteristics suggest that the EPS P-ArcPo 15 may be useful in the development of cryoprotectants for biotechnological purposes, and we therefore assessed the utility of this novel cryoprotective EPS.     

Purification of a proteinase and proteinase inhibitor from Tetrahymen a pyriformis

• 1.1. A cysteine proteinase and cysteine proteinase inhibitor have been purified from Tetrahymena.
• 2.2. The proteinase was purified by ammonium sulphate fractionation, gel filtration, ion exchange chromatography and affinity chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 28,000). It hydrolysed BAPNA, degraded azocasein, and converted 80S ribosomes to subunits. Thiol reagents inhibited these activities.
• 3.3. The inhibitor was purified by heat treatment, ammonium sulphate fractionation and ion exchange chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 12.500). The inhibitor was heat stable and it inhibited papain, as well as the Tetrahymena proteinase.

     

Purification of a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) by chromatography and its use as an affinity ligand

An ammonium sulphate fraction (20–60%) of bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) was purified by affinity chromatography to give 6.59-fold purity with 81.48% yield. The same ammonium sulphate fraction was also subjected to ion exchange chromatography and was purified 4.28-fold with 75.95% yield. The ion exchange fraction was subjected to gel filtration and the inhibitor was purified to 6.67-fold with 67.36% yield. Further sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the homogeneity of purified amylase/trypsin inhibitor obtained through affinity, ion exchange and gel chromatography. The molecular weight of the inhibitor was found to be 14 kDa. This purified inhibitor was used as affinity ligand for the purification of a commercial preparation of pancreatic amylase.     

Enzymatic clarification of fruit juices (Apple, Pineapple, and Tomato) using purified Bacillus pumilus SV-85S xylanase

The xylanase obtained from a hyper-producer Bacillus pumilus SV-85S was purified and characterized to evaluate its potential in industrial applications. Xylanase was purified to homogeneity 25.3-fold with 63.2% recovery using cation-exchange chromatography through CMSephadex C-50. The purified xylanase showed a single band on Native-PAGE and a single peak in RP-HPLC confirming its homogeneity. The purified enzyme revealed a single band on SDS-PAGE with a molecular mass of 23.6 kDa, which was confirmed with gel filtration chromatography through Sepharose 6B. The K_m and V_max of the purified xylanase was 1.0 mg/mL and 333.3 IU/mL, respectively. The temperature and pH profiles of the purified xylanase revealed that it was thermo and alkali stable. In recent years due to the overall increase in natural fruit juice consumption, juices have become important from a consumption point of view. However, raw juice is turbid and viscous which tends to settle during storage. Therefore, it must be clarified before commercialization. The efficacy of absolutely purified xylanase was studied on juice enrichment of apples (Malus domestica), pineapples (Ananas comosus L.) and tomatos (Lycopersicum esculentum). The treatment with xylanase lead to an increased juice yield by 23.53% (apple), 10.78% (pineapple), and 20.78% (tomato) as well as having a significant effect on juice clarity by an increase of % transmittance of 22.20, 19.80, and 14.30, respectively. The turbidity and viscosity was also decreased without affecting acid neutrality significantly.     

p53-dependent change in replication timing of the human genome

The tumor suppressor gene p53 has roles in multiple cell-cycle checkpoints, including the G1/S transition, to prevent replication of cells with DNA damage. p53 is thought to be associated with regulation of replication timing during S-phase in the human genome. In the present study, we used p53-wild-type and p53-null HCT116 colon carcinoma cells to analyze p53-dependent changes in replication timing of the human genome. The percentage of HCT116 p53(−/−) cells in S-phase was higher than that of HCT116 p53(+/+) cells. We compared replication timing of human genes between the two cell lines using 25,000 human cDNA microarray. We identified genes that replicated earlier in HCT116 p53(−/−) cells than in HCT116 p53(+/+) cells. These genes included cell-cycle- and apoptosis-related genes. We propose that p53 plays a role in regulation of replication timing of the human genome through the control of cell-cycle checkpoints.     

Inhibition of the infectivity and cytopathic effect of human immunodeficiency virus by water-soluble lignin in an extract of the culture medium of Lentinus edodes mycelia (LEM)

Inhibition of the infectivity and cytopathic effect of human immunodeficiency virus type 1 (HIV-1) by the immunoactive fractions obtained from LEM, which is an extract of the culture medium of Lentinus edodes mycelia, is reported. A purified fraction, EPS4, obtained from LEM by ethanol precipitation followed by hydrophobic chromatography and gel filtration chromatography completely inhibited the HIV-1 induced cytopathic effect in vitro at concentrations of greater than or equal to 10 micrograms/ml. Chemical and spectral analysis revealed that EPS4 is composed of water-soluble lignins containing minor amounts of protein (3.2%) and sugars (12.2%). Taken together with the previously reported observation that EPS4 promotes the activation of macrophages and the proliferation of bone marrow cells, the fraction appears to possess both an immunostimulating activity and an anti-HIV effect in vitro.     

Interactions between Lactobacillus plantarum NCU116 and its environments based on extracellular proteins and polysaccharides prediction by comparative analysis

Lactic acid bacteria (LAB) play a significant role in food industry and artisan fermented-food. Most of the applicable LABs were commonly obtained from natural fermented food or human gut. And Lactobacillus plantarum NCU116 was screened from a LAB-dominated traditional Chinese sauerkraut (TCS). In order to comprehend the interaction between NCU116 and its environments, comparative genomics were performed to identify genes involved in extracellular protein biosynthesis and secretion. Four secretory pathways were identified, including Sec and FPE pathways, holins and efflux ABC transporter system. Then 348 potential secretory proteins were identified, including 11 alpha-amylases responsible for degradation of macromolecules, and 8 mucus binding proteins which attribute to adherence to intestine epithelium. Besides, EPS clusters of NCU116 (EPS116) were identified and analyzed by comparing to other strains, which suggested a novel genotype of EPS clusters. These findings could be critical to extend the application of NCU116 in food and pharmaceuticals industries.     

Isolation,purification, and some of the properties of hemi-cellulase from fusarium sp.

An extracellular hemicellulase from a soil fungus (Fusarium sp.) grown on a medium containing groundnut hemicellulose B was purified 76-fold by ammonium sulphate fractionation, chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. It was found to be homogeneous by disc-gel electrophoresis at pH 8. It showed optimal activity at pH 5.6 and 37°. It was observed that groundnut and sesame hemicellulose B were degraded considerably (~80 and 58%, respectively) by the purified hemicellulase, whereas glucomannan and xylan from groundnut were comparatively poorly hydrolysed (~30–40%).     

Purification an α-galactosidase from Coriolus versicolor with acid-resistant and good degradation ability on raffinose family oligosaccharides

An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg^?1. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4–50 °C and at a pH range of 2–5. Among the metal ions tested, Cu²⁺, Cd²⁺ and Hg²⁺ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag⁺ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.     

Purification and characterization of dye degrading of veratryl alcohol oxidase from Pseudomonas aeruginosa strain BCH

In the present study we have purified the intracellular veratryl alcohol oxidase (VAO) enzyme from Pseudomonas aeruginosa strain BCH to evaluate its dye decolorizing potential. The enzyme was purified by ion exchange chromatography using DEAE cellulose followed by gel filtration chromatography using Biogel P-100. The molecular weight of the purified enzyme was estimated by polyacrylamide gel electrophoresis (PAGE) analysis. The VAO was purified up to 12 and 16.3-fold by ion exchange and gel filtration chromatography respectively. VAO was estimated to be about 85 kDa by SDS–PAGE. The optimum pH and temperature for purified VAO was 3 and 55°C respectively. The purified enzyme exerted its optimal activity with veratryl alcohol and also oxidized various other substrates, whereas diminished activity was noted in case of tryptophan and xylidine. The metal ions Mn⁺⁺ and Hg⁺⁺ were found to suppress the oxidase activity. The purified enzyme decolorized different dyes with variable decolorization rates and efficiencies. Decolorization mechanism of Remazol Black by purified enzyme was studies in detail using various analytical techniques like HPLC, GC–MS and FTIR. This study is useful for understanding the precise role of Pseudomonas aeruginosa strain BCH in the decolorization of textile dyes containing industrial wastewater.     

Isolation of corticotropin-β-lipotropin common precursor from ovine pituitary glands

Ovine corticotropin-β-lipotropin common precursor was purified to homogeneity from commercial frozen ovine pituitary glands. A crude preparation was obtained following a procedure published elsewhere (Lee, T.H. and Lee, M.S. (1977) Biochemistry 16, 2824–2829) and was purified by gel filtration on Sephadex G-200 in the presence of 0.5% SDS and 0.1% 2-mercaptoethanol, and under an atmosphere of nitrogen. The gel filtration was repeated once. The partially purified preparation obtained from the second Sephadex G-200 gel filtration was further fractionated by preparative SDS-acrylamide gel eletrophoresis, using immunoprecipitated and electrophoretically purified [¹²⁵I]corticotropin-β-lipotropin common precursor as a marker. The preparation was judged homogenous by the appearance of a single protein band in analytical SDS-acrylamide gel electrophoresis, which exhibited both corticotropin and β-lipotropin immunoreactivities, and a single symmetrical peak in high-pressure liquid chromatography on a reverse phase C18 column. The isolated ovine corticotropin-β-lipotropin common precursor possessed specific activities of 116 μg of immunoreactive corticotropin and 210 μg of immunoreactive β-lipotropin per mg of protein, equivalent to 89 and 62% of theoretical values, respectively. The amino acid composition of the homogeneous preparation was determined.     

Purification and properties of a high specific activity protein kinase from wheat germ

A protein kinase was extensively purified to near-homogeneity from wheat germ by a procedure involving affinity chromatography on casein-Sepharose 4B, gel filtration, and repeated chromatography on carboxymethyl-Sepharose CL-6B. The protein kinase preparations have the highest specific activities (up to 656 nanomoles phosphate incorporated per minute per milligram of protein) yet reported for plant protein kinases. The major polypeptides in purified preparations were revealed as two barely-resolved bands (molecular weight 31,000) on polyacrylamide gel electrophoresis in subunit-dissociating conditions. The molecular size of the protein kinase as determined from gel filtration is 30,000. The protein kinase catalyzes the phosphorylation of casein, phosvitin, and the wheat germ cyclic AMP-binding protein cABPII but not of bovine serum albumin and histones nor of the wheat germ cytokinin-binding protein CBP. The protein kinase has a pH optimum of 7.9 and a K_m value for ATP of 10 micromolar. The protein kinase differs from wheat germ CBP kinase in molecular weight, differential sensitivity to inhibitors, and in substrate specificity.