Actinobacterial chitinase-like enzymes: profiles of rhizosphere versus non-rhizosphere isolates

2003 ◽  
Vol 49 (11) ◽  
pp. 683-698 ◽  
Author(s):  
Ana C Gonzalez-Franco ◽  
Lee A Deobald ◽  
Aaron Spivak ◽  
Don L Crawford
Keyword(s):  
Cell Wall ◽  
Gel Electrophoresis ◽  
Rhizosphere Soil ◽  
Polyacrylamide Gel Electrophoresis ◽  
Hydrolytic Enzymes ◽  
Fungal Cell ◽  
Native Page ◽  
Substrate Preferences ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Over Time

The objective of this study was to determine if antifungal actinomycetes isolated from rhizosphere and non-rhizosphere soils exhibit different chitinase-like production and (or) induction patterns. Selected isolates from both habitats were compared. Chitinase-like levels and isoform characteristic patterns were evaluated over time in culture fluids of isolates grown on media containing different combinations of colloidal chitin and fungal cell wall (FCW) preparation. Supernatants were also subjected to native and non-native polyacrylamide gel electrophoresis (PAGE), using glycol chitin amended gels. For non-native PAGE, protein samples were denatured by two different approaches. Multiple active bands, ranging from 20 to 53 kDa and present in varying amounts, were detected in gels for most strains. Different substrate preferences were observed among strains, and different chitinase-like enzymes were produced, depending upon the substrate combinations used. The presence of FCW in the medium induced specific chitinase-like enzymes not observed otherwise. Enzymatic activities and profiles of the isolates, however, were strain and substrate specific rather than habitat specific. However, a sagebrush rhizosphere soil had a larger actinomycete community with higher chitinolytic activities than the nearby bulk soil. The use of PAGE to compare chitinase-like proteins induced in media with and without FCW was useful for identifying chitinase-like enzymes potentially involved in antifungal activity.Key words: chitinase, actinomycetes, hydrolytic enzymes, rhizosphere, antifungal.

scholarly journals APPLICATION OF GRAPHENE OXIDE ON APTAMER-BASED BIOSENSOR DEVELOPMENT FOR AUTHENTICATION OF GELATIN

2019 ◽  
pp. 254-258
Author(s):  
HARI WIDADA ◽  
ABDUL ROHMAN ◽  
RIRIS ISTIGHFARI JENIE ◽  
SISMINDARI .
Keyword(s):  
Graphene Oxide ◽  
Gel Electrophoresis ◽  
Dna Analysis ◽  
Selection Process ◽  
Polyacrylamide Gel Electrophoresis ◽  
Pcr Amplification ◽  
Native Page ◽  
Sharp Separation ◽  
Aptamer Selection ◽  
Native Polyacrylamide Gel Electrophoresis

Objective: The objective of this study was to perform aptamer selection using systematic evolution of ligands by exponential enrichment (SELEX) method which assisted by graphene oxide against target of porcine gelatin (non-halal gelatin). Methods: The aptamer selection was carried out using SELEX method without target immobilization. Selection of aptamer capable of binding porcine gelatin by applying grafen oxide (GO) was known as GO-SELEX. The selection process was initially carried out by incubation of single-stranded DNA (ssDNA) libraries targeting on porcine gelatin with the addition of graphene oxide. The selected ssDNA was then purified by several stages namely; symmetric PCR amplification, purification of products with DNA purification kits, asymmetric PCR amplification, and continued purification of DNA with native PAGE. The analysis of each stage was done by agarose gel electrophoresis. Results: the results showed that aptamer targeting porcine DNA could be selected. This was indicated by the results of DNA analysis using native polyacrylamide gel electrophoresis (PAGE) in which sharp separation band with a base length equivalent to the marker of the ssDNA library (about 80 base pair) was obtained. Conclusion: Aptamer targeting on porcine gelatin has been successfully developed using GO-SELEX method. GO can increase selectivity in developing aptamer which will be used as a biosensor to detect porcine gelatin. The method could be proposed as a standard of apatamer based method for porcine gelatin detection on halal products authentication.

scholarly journals Genetic Variation of Isozyme Polyphenol Oxidase (PPO) Profiles in Different Varieties of Capsicum annuum L.

2013 ◽  
Vol 5 (4) ◽  
pp. 454-457
Author(s):  
Owk ANIEL KUMAR ◽  
Tamminana RUPAVATHI ◽  
Sape SUBBA TATA
Keyword(s):  
Capsicum Annuum ◽  
Polyphenol Oxidase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Native Page ◽  
Chilli Pepper ◽  
Upgma Dendrogram ◽  
Low Genetic Diversity ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Genus Capsicum

The genus Capsicum commonly known as chilli pepper is a major spice crop and is of cosmopolitan in distribution. Native polyacrylamide gel electrophoresis (Native PAGE) was used to study the polyphenol oxidase (PPO) isozyme variation in 21 varieties of Capsicum annuum L. A maximum of 4 PPO bands were scored in five varieties i.e., Ca14, Ca15, Ca16, Ca19 & Ca20, while the minimum (2 bands) was observed in four varieties (Ca3, Ca10, Ca13 & Ca17). 15 pair wise combinations showed highest average per cent similarity (100%) and the UPGMA dendrogram represented low genetic diversity. The present study revealed that considerable intraspecific differences were found in the varieties. Thus the results obtained could be used in fingerprinting the genotypes.

scholarly journals Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes

2015 ◽  
Vol 81 (14) ◽  
pp. 4626-4633 ◽  
Author(s):  
Xiaoming Liang ◽  
Olivia Molenda ◽  
Shuiquan Tang ◽  
Elizabeth A. Edwards
Keyword(s):  
Specific Activity ◽  
Enrichment Culture ◽  
Polyacrylamide Gel Electrophoresis ◽  
Peptide Sequencing ◽  
Enzyme Assays ◽  
Content Type ◽  
Substrate Preferences ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
History Of ◽  
Different Strains

ABSTRACTMany reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediatescis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinatingGeobacterand severalDehalococcoidesstrains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase fromGeobacterwas only detected transiently at the beginning of TCE dechlorination. TheDehalococcoidesRDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. TheDehalococcoidesRDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity.trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains ofDehalococcoidesas a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.

scholarly journals A Novel Class of Ectomycorrhiza-Regulated Cell Wall Polypeptides in Pisolithus tinctorius

1999 ◽  
Vol 12 (10) ◽  
pp. 862-871 ◽  
Author(s):  
Pascal Laurent ◽  
Catherine Voiblet ◽  
Denis Tagu ◽  
Dulcinéia de Carvalho ◽  
Uwe Nehls ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Adhesion ◽  
Polyacrylamide Gel Electrophoresis ◽  
State Level ◽  
Root Surface ◽  
Pisolithus Tinctorius ◽  
Surface Proteins ◽  
Ectomycorrhizal Symbiosis ◽  
Fungal Cell ◽  
Fungal Hyphae

Development of the ectomycorrhizal symbiosis leads to the aggregation of fungal hyphae to form the mantle. To identify cell surface proteins involved in this developmental step, changes in the biosynthesis of fungal cell wall proteins were examined in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhizas by two-dimensional polyacrylamide gel electrophoresis. Enhanced synthesis of several immunologically related fungal 31- and 32-kDa polypeptides, so-called symbiosis-regulated acidic polypeptides (SRAPs), was observed. Peptide sequences of SRAP32d were obtained after trypsin digestion. These peptides were found in the predicted sequence of six closely related fungal cDNAs coding for ectomycorrhiza up-regulated transcripts. The PtSRAP32 cDNAs represented about 10% of the differentially expressed cDNAs in ectomycorrhiza and are predicted to encode alanine-rich proteins of 28.2 kDa. There are no sequence homologies between SRAPs and previously identified proteins, but they contain the Arg-Gly-Asp (RGD) motif found in cell-adhesion proteins. SRAPs were observed on the hyphal surface by immunoelectron microscopy. They were also found in the host cell wall when P. tinctorius attached to the root surface. RNA blot analysis showed that the steady-state level of PtSRAP32 transcripts exhibited a drastic up-regulation when fungal hyphae form the mantle. These results suggest that SRAPs may form part of a cell-cell adhesion system needed for aggregation of hyphae in ectomycorrhizas.

scholarly journals Analysis of Several Popular Cultivars of Madagascar Periwinkle (Catharanthus roseus (L.) G. Don.) using Biochemical Markers

2013 ◽  
Vol 5 (4) ◽  
pp. 458-461
Author(s):  
Owk ANIEL KUMAR ◽  
Sape S. TATA ◽  
Kancharla PAVAN KUMAR
Keyword(s):  
Catharanthus Roseus ◽  
Gel Electrophoresis ◽  
Biochemical Markers ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cultivar Differences ◽  
Madagascar Periwinkle ◽  
Large White ◽  
Electrophoretic Patterns ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Isozyme Polymorphism

Band designs of esterase (EST), peroxidase (PO) and polyphenol oxidase (PPO) isozymes in several selected cultivars of Catharanthus roseus by using native polyacrylamide gel electrophoresis (PAGE) were investigated in this study. It was confirmed that cultivar differences in isozyme polymorphism can be revealed by applied electrophoretic patterns. Three isozyme systems produced a total of 16 bands with polymorphism ranged from 66.6-100%. Considering the patterns of isozyme variations in the five cultivars of Catharanthus roseus, it is evident that the cultivar ‘First kiss coral’ displayed crimson red petal with large white eye’ displayed demarked profiles of EST, PO and PPO isozymes than other cultivars. This is the first report on isozyme polymorphism in members of the Cathanarathus roseus (L.) G. Don.

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