Abstract：
:Threonine aldolase (TA) catalyzes a reversible reaction, in which threonine is decomposed into glycine and acetaldehyde. The same enzyme can be used to catalyze aldol reaction between glycine and a variety of aromatic and aliphatic aldehydes, thus creating various alpha-amino-alcohols. Therefore, TA is a very promising enzyme that could be used to prepare biologically active compounds or building blocks for pharmaceutical industry. Rational design was applied to thermophilic TA from Thermotoga maritima to improve thermal stability by the incorporation of salt and disulfide bridges between subunits in the functional tetramer. An activity assay together with CD analysis and Western-blot detection was used to evaluate mutants. Except one, each of the designed mutants preserved activity toward the natural substrate. One of the 10 proposed single point mutants, P56C, displayed significantly enhanced stability compared to the wild type (WT). Its initial activity was not affected and persisted longer than WT, proportionally to increased stability. Additionally one of the mutants, W86E, displayed enhanced activity, with stability similar to WT. Higher activity may be explained by a subtle change in active site availability. Salt bridge formation between glutamic acid at position 86 and arginine at position 120 in the neighboring chain may be responsible for the slight shift of the chain fragment, thus creating wider access to the active site both for the substrate and PLP.
journal_name
J Biotechnoljournal_title
Journal of biotechnologyauthors
Wieteska L,Ionov M,Szemraj J,Feller C,Kolinski A,Gront Ddoi
10.1016/j.jbiotec.2015.02.013subject
Has Abstractpub_date
2015-04-10 00:00:00pages
69-76eissn
0168-1656issn
1873-4863pii
S0168-1656(15)00067-Xjournal_volume
199pub_type
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