Linked Life Data

11115391

Source:http://linkedlifedata.com/resource/pubmed/id/11115391

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:dateCreated
2001-2-12
pubmed:abstractText
Immobilization of proteins usually leads to random orientation of the molecules on the surface of the carrier material, whereby mechanistic interpretations of changes in properties, such as thermal stability, become very difficult. Recently, we have prepared several mutant enzymes of the thermolysin-like neutral protease from Bacillus stearothermophilus, containing cysteine residues in different positions on the surface of the protein molecule. These enzymes allowed site-specific immobilization to Activated Thiol-Sepharose and showed that stabilization effects strongly depend on the position of attachment [Mansfeld, Vriend, Van den Burg, Eijsink and Ulbrich-Hofmann (1999) Biochemistry 38, 8240-8245]. The greatest stabilization was achieved after immobilization of the mutant enzymes S65C and T56C/S65C within the structural region (positions 56-69) where unfolding is initiated. In this study thermal inactivation kinetics of these two mutant enzymes, as well as those of the pseudo-wild-type enzyme and thermolysin, were compared for different types of immobilization. Besides site-specific immobilization via thiol groups, the enzymes were bound randomly via their amino groups or by mixed-type binding. The basic matrix was Sepharose 4B in all carriers. Whereas the enzymes bound site-specifically to Activated Thiol-Sepharose showed clear first-order inactivation kinetics like the soluble enzymes, the other immobilized enzyme preparations were characterized by distinct biphasic inactivation kinetics reflecting the heterogeneity of enzyme molecules on the carrier with respect to thermal unfolding. Site-specific binding resulted in stronger stabilization than the mixed binding type. However, immobilization to a highly functionalized carrier via amino groups increased stability further, suggesting that multiple fixation outside of the unfolding region 56-65 is able to increase stability of the enzyme molecules additionally.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0885-4513
pubmed:author
pubmed:issnType
Print
pubmed:volume
32 ( Pt 3)
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
189-95
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
2000
pubmed:articleTitle
Site-specific and random immobilization of thermolysin-like proteases reflected in the thermal inactivation kinetics.
pubmed:affiliation
Martin-Luther University Halle-Wittenberg, Department of Biochemistry/Biotechnology, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany. mansfeld@biochemtech.uni-halle.de
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't