Show/Hide Menu
Hide/Show Apps
Logout
Türkçe
Türkçe
Search
Search
Login
Login
OpenMETU
OpenMETU
About
About
Open Science Policy
Open Science Policy
Open Access Guideline
Open Access Guideline
Postgraduate Thesis Guideline
Postgraduate Thesis Guideline
Communities & Collections
Communities & Collections
Help
Help
Frequently Asked Questions
Frequently Asked Questions
Guides
Guides
Thesis submission
Thesis submission
MS without thesis term project submission
MS without thesis term project submission
Publication submission with DOI
Publication submission with DOI
Publication submission
Publication submission
Supporting Information
Supporting Information
General Information
General Information
Copyright, Embargo and License
Copyright, Embargo and License
Contact us
Contact us
Purification and characterization of cytoplasmic and proteasome associated chymotrypsin-like proteases from Thermoplasma volcanium
Download
index.pdf
Date
2003
Author
Özdemir, F. İnci
Metadata
Show full item record
Item Usage Stats
140
views
0
downloads
Cite This
In this study, two novel cytoplasmic serine proteases were isolated and characterized from thermophilic archaea Thermoplasma volcanium. The first protease was purified by ion exchange and affinity chromatographies and identified as a chymotrypsin-like serine protease mainly based on its substrate profile and inhibition pattern. The presence of protease activity was analyzed by gelatin zymography which was detected as a single band (35 kDa). Optimum temperature was found to be 60oC for azocasein hydrolysis and 50oC for N-Suc-Phe-pNA hydrolysis. Optimum activity was observed in the pH range of 6.0-8.0 with a maximum value at pH 7.0. The Km and Vmax values for the purified protease were calculated to be 2.2 mM and 40 æmoles of p-nitroanilide released min-1.ml-1, respectively, for N-Suc-Phe-PNA as substrate. Ca2+ and Mg2+ at 4 mM concentrations were the most effective divalent cations in activating the enzyme. In the second stage of this study, 20S proteasome of Tp. volcanium with substantial chymotrypsin-like activity was purified and characterized. This enzyme complex was purified with 19.1 U/mg specific activities from cell free extract by a four-step procedure. SDS-PAGE analysis revealed two strong bands with relative molecular masses of 26 kDa (a-subunit) and 21.9 kDa (β-subunit). Tp. volcanium 20S proteasome predominantly catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residue Phe (chymotrypsin-like activity) in short chromogenic peptides. Low-level hydrolyzing activity was also detected carboxyl to basic residue Arg (trypsin-like activity). Chymotrypsin-like activity of Tp. volcanium 20S proteasome was significantly inhibited by chymotrypsin specific serine protease inhibitor chymostatin. When N-CBZ-Arg was used which is a substrate for trypsin, 20S proteasome was strongly inhibited by TLCK. The
Subject Keywords
Serine proteinases
,
Chymotrypsin
,
Proteinase
URI
http://etd.lib.metu.edu.tr/upload/3/1137629/index.pdf
https://hdl.handle.net/11511/13477
Collections
Graduate School of Natural and Applied Sciences, Thesis
Suggestions
OpenMETU
Core
Identification and characterization of hydrolytic enzymes from the midgut of the cotton bollworm, Helicoverpa armigera Hubner (Lepidoptera: Noctuidae)
Ozgur, Ebru; Yucel, Meral; Öktem, Hüseyin Avni (2009-01-01)
Midgut hydrolytic enzymes of Helicoverpa armigera Hubner (Lepidoptera: Noctuidae) were identified and partially characterized. K-m, V-max, optimum pH, and specific activity were determined for proteolytic enzymes and alpha-amylases. All hydrolytic enzyme activity had an optimum pH value in the alkaline pH range. We observed major serine protease activity, together with minor cysteine-like activity, the former being significantly inhibited by soybean trypsin inhibitor (SBTI) and aprotinin. Moreover, differen...
Characterization of extracellular beta-lactamases from penicillin G-resistant cells of Streptococcus thermophilus
Chirica, LC; Güray, Nülüfer Tülün; Gültekin, Güzin Candan; Bozoglu, F (International Association for Food Protection, 1998-07-01)
In this study, biochemical properties of two extracellular beta-lactamases produced by penicillin-resistant Streptococcus thermophilus cells were investigated. Both beta-lactamases showed specificity for penicillins but not for cephaloridins. The p-lactamases exhibited different affinities for penicillin G. The one with the higher molecular weight (F1) had a K(m) value of 3.44 mu M and a V(max), value of 8.33, mu mol/min/mg of protein, whereas the beta-lactamase with the lower molecular weight (FII) had a K...
Identification and characterization of hydrolytic enzymes from the midgut of Sunn Pest of wheat (Eurygaster integriceps)
Ogur, E.; YÜCEL, MUSTAFA; Öktem, Hüseyin Avni (Informa UK Limited, 2009-01-01)
To help in the development of Sunn Pest-resistant transgenic plants employing protease or alpha-amylase inhibitors, midgut hydrolytic enzymes of Sunn Pest (Eurygaster integriceps, Put.) (Heteroptera: Scutelleridae) were identified and characterized biochemically. We observed levels of very low proteolytic activity of trypsin (3 nmoles/min/mg), elastase (0.66 nmoles/min/mg) and leucine aminopeptidase-like (14.4 nmoles/min/mg) proteases, but no chymotrypsin and papain-like activity. Proteolytic activities wer...
Characterization and functional analysis of a novel multicopper oxidase and associated polyketide biosynthesis gene cluster of aspergillus fumigatus
Metin, Banu; Ögel, Zümrüt Begüm; Department of Food Engineering (2007)
In this study, novel polyketide biosynthesis gene cluster of Aspergillus fumigatus was characterized and functionally analyzed. Analysis of the newly sequenced A. fumigatus genome for laccases, which are involved in melanin biosynthesis and detoxification in fungi, resulted in several putative laccase and multicopper oxidase gene sequences, one of which, Afu4g14490 (tpnJ), was selected for further characterization. The predicted amino acid sequence TpnJp showed 63% identity with the dihydrogeodin oxidase of...
Synthesis and characterization of a new soluble conducting polymer and its electrochromic device
Varis, Serhat; Ak, Metin; Tanyeli, Cihangir; Akhmedov, Idris Mecidoglu; Toppare, Levent Kamil (Elsevier BV, 2006-12-01)
A mixture of isomers 2,5-di(4-methyl-thiophen-2-yl)-1-(4-nitrophenyl)-1H-pyrrole, 2-(4-methyl-thiophen-2-yl)-5-(3-methyl-thiophen-2-yl)1-(4-nitrophenyl)-1H-pyrrole and 2,5-di(3-methyl-thiophen-2-yl)-1-(4-nitrophenyl)-1H-pyrrole (Me-SNS(NO2)) were synthesized. Resulting monomers were polymerized chemically, producing soluble polymers in common organic solvents. The average molecular weight has been determined by gel permeation chromatography (GPC) as Mn = 5.6 x 10(3) for the chemically synthesized polymer. T...
Citation Formats
IEEE
ACM
APA
CHICAGO
MLA
BibTeX
F. İ. Özdemir, “Purification and characterization of cytoplasmic and proteasome associated chymotrypsin-like proteases from Thermoplasma volcanium,” Ph.D. - Doctoral Program, Middle East Technical University, 2003.