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Activity Staining (activity + staining)
Selected AbstractsDetection of Vacuum Infusion of Pectinmethylesterase in Strawberry by Activity StainingJOURNAL OF FOOD SCIENCE, Issue 3 2004P. B ANJONGSINSIRI ABSTRACT: Strawberry halves were infused with Valencia orange pectinmethylesterase (PME) under vacuum for 15 min at room temperature. Fruits were blotted onto pectin paper and stained for activity. Activity of PME-infused fruit was about twice (0.36 unit/mL or 0.86 unit/mg protein) that of noninfused control (0.19 unit/mL) or water-infused control (0.16 unit/mL). Instron firmness values were not significantly different (P± 0.05) between noninfused and PME-infused fruit. Firmness of PME-infused fruit was about twice that of water-infused control. Water-soluble pectin, chelator soluble pectin, alkaline soluble pectin, and total pectin ranged from 8.24 to 8.70, 3.24 to 4.56, 3.77 to 5.39, and 17.86 to 26.16 mg/100 mg alcohol insoluble solid (AIS), respectively, for all treatments. [source] Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilarisFEBS JOURNAL, Issue 16 2003Masahiro Sugimura A cellulase (endo-,-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 µmol·min,1·(mg protein),1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 µmol·min,1·(mg protein),1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods. [source] Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistanceJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2000J.P. Grill Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N,-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (Z,pH) was involved in Lactobacillus bile salt resistance. [source] Influence of culture conditions on laccase production and isozyme patterns in the white-rot fungus Trametes gallicaJOURNAL OF BASIC MICROBIOLOGY, Issue 3 2005Jia Li Dong Laccase production by the white-rot fungus Trametes gallica was studied, using twelve different media under static or shaking condition. The results indicated that organic nitrogen sources such as tryptone and peptone strongly improved laccase production. The application of an amino acid mixture and a lignin preparation also increased the formation of laccase, which was not observed in the presence of potato extract. Native polyacryl amide gel electrophoresis (PAGE) followed by laccase activity staining using guaiacol as the substrate was performed to analyze the laccase isozyme patterns under the different culture conditions employed. Zymograms revealed a total of twenty different laccase activity bands that appeared in individual patterns, dependent on the respective culture condition applied. This indicates that both the medium composition and the mode of incubation (static or shaking) influenced the laccase isozyme gene expression. This was the first time to report so many laccase isozymes in a fungus. Native PAGE with silver staining showed that laccases were the main protein productions in several media providing a potentially convenient way in purifying laccases from T. gallica. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] EFFECT OF SALTS AND POLYETHYLENE GLYCOLS ON THE PARTITIONING AND RECOVERY OF TRYPSIN FROM HYBRID CATFISH VISCERA IN AQUEOUS TWO-PHASE SYSTEMSJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2010SAPPASITH KLOMKLAO ABSTRACT The partitioning behavior of trypsin from hybrid catfish viscera in aqueous two-phase systems (ATPS) was studied. Factors such as polyethylene glycol (PEG) molecular mass and concentration, as well as types and concentration of salts, affected protein separation. Trypsin partitioned mainly in the top PEG-rich phase. ATPS formed by PEG of molecular weight 4,000 (20%, w/w) and NaH2PO4 (20%, w/w) showed the best capability for trypsin purification from hybrid catfish viscera. Under such conditions, the highest specific activity (30.05 units/µg protein) and purification (27.3-fold), were obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the enzyme after ATPS separation was near homogeneity and based on the activity staining, the band intensity of enzyme in ATPS fraction increased, indicating the greater specific activity of the viscera extract. The partitioned enzyme displayed optimal activity at pH 9.0 and 50C, respectively. The enzyme was stable up to 40C and within the pH range of 8,12. The enzyme exhibited a progressive decrease in activity with increasing NaCl concentration. PRACTICAL APPLICATIONS This paper describes the separation and recovery of trypsin from hybrid catfish viscera in ATPS and its properties. ATPS provides an efficient and attractive method for partitioning and recovery of trypsin from hybrid catfish viscera. Trypsins from various sources catalyze the hydrolysis of peptide bonds on the carboxyl sides of arginine and lysine. Therefore, it is expected that like other trypsins, trypsin after ATPS separation from hybrid catfish viscera could be useful in the biomedical, food and beverage industries. [source] Epiphytic bacteria on the Antarctic ice diatom Amphiprora kufferathii Manguin cleave hydrogen peroxide produced during algal photosynthesisPLANT BIOLOGY, Issue 4 2008M. Hünken Abstract The Antarctic ice diatom Amphiprora kufferathii Manguin is always accompanied by epiphytic bacteria in its natural habitat. To investigate the nature of this relationship, axenic cultures of A. kufferathii were obtained by ampicillin treatment. Diatom cultures without bacteria were less dense. The bacteria were shown to consume hydrogen peroxide produced by the diatom during photosysnthesis and algal photosynthesis after a hydrogen peroxide shock recovered faster in the presence of bacteria. Three proteobacterial strains isolated from a culture of A. kufferathii were phylogenetically affiliated with the alphaproteobacterial genus Sulfitobacter, the gammaproteobacterial genus Colwellia, and the genus Pibocella of the Bacteriodetes. Native protein gel electrophoresis and enzyme activity staining revealed the presence of superoxide dismutase and glutathione reductase in the isolated bacteria and in A. kufferathii cultures. Catalase was detected in bacterial extracts but not in axenic cultures of A. kufferathii. These observations indicate that the epiphytic bacteria make a significant contribution to the diatom's antioxidative defences. The relationship between the bacteria and A. kufferathii seems to be beneficial for both partners and enhances growth of Amphiprora in the sea ice. [source] | |||||||||||||