Linked Life Data

19631297

Source:http://linkedlifedata.com/resource/pubmed/id/19631297

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
11
pubmed:dateCreated
2009-9-23
pubmed:abstractText
Nattokinase (subtilisin NAT, NK) is a bacterial serine protease with strong fibrinolytic activity and it is a potent cardiovascular drug. In medical and commercial applications, however, it is susceptible to chemical oxidation, and subsequent inactivation or denaturation. Here we show that the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr(220) and Met(222), which were in the vicinity of the catalytic residue Ser(221) of the enzyme. Two nonoxidative amino acids (Ser and Ala) were introduced at these sites using site-directed mutagenesis. Active enzymes were successfully expressed in Escherichia coli with periplasmic secretion and enzymes were purified to homogeneity. The purified enzymes were analyzed with respect to oxidative stability, kinetic parameters, fibrinolytic activity and thermal stability. M222A mutant was found to have a greatly increased oxidative stability compared with wild-type enzyme and it was resistant to inactivation by more than 1 M H(2)O(2), whereas the wild-type enzyme was inactivated by 0.1 M H(2)O(2) (t(1/2) approximately 11.6 min). The other mutant (T220S) also showed an obvious increase in antioxidative ability. Molecular dynamic simulations on wild-type and T220S mutant proteins suggested that a hydrogen bond was formed between Ser(220) and Asn(155), and the spatial structure of Met(222) was changed compared with the wild-type. The present study demonstrates the feasibility of improving oxidative stability of NK by site-directed mutagenesis and shows successful protein engineering cases to improve stability of NK as a potent therapeutic agent.
pubmed:commentsCorrections
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0006-3002
pubmed:author
pubmed:issnType
Print
pubmed:volume
1794
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1566-72
pubmed:dateRevised
2011-4-14
pubmed:meshHeading
pubmed:year
2009
pubmed:articleTitle
Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli.
pubmed:affiliation
State Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't