Документ взят из кэша поисковой машины. Адрес оригинального документа : http://kodomo.cmm.msu.ru/~julia270692/term2/abstract.txt Дата изменения: Wed Mar 3 16:44:01 2010 Дата индексирования: Tue Oct 2 12:58:14 2012 Кодировка: Windows-1251

Выбрана статья: A novel genetic selection system for improved enantioselectivity of Bacillus subtilis lipase A.

1. Chembiochem. 2008 May 5;9(7):1110-5.

A novel genetic selection system for improved enantioselectivity of Bacillus
subtilis lipase A.

Boersma YL, DrГge MJ, van der Sloot AM, Pijning T, Cool RH, Dijkstra BW, Quax WJ.

Department of Pharmaceutical Biology, University of Groningen, Groningen, The
Netherlands.

In directed evolution experiments, success often depends on the efficacy of
screening or selection methods. Genetic selections have proven to be extremely
valuable for evolving enzymes with improved catalytic activity, improved
stability, or with altered substrate specificity. In contrast, enantioselectivity
is a difficult parameter to select for. In this study, we present a successful
strategy that not only selects for catalytic activity, but for the first time
also for enantioselectivity, as demonstrated by the selection of Bacillus
subtilis lipase A variants with inverted and improved enantioselectivity. A
lipase mutant library in an aspartate auxotroph Escherichia coli was plated on
minimal medium that was supplemented with the aspartate ester of the desired
enantiomer (S)-(+)-1,2-O-isopropylidene-sn-glycerol. To inhibit growth of less
enantioselective variants, a covalently binding phosphonate ester of the opposite
(R)-(-)-1,2-O-isopropylidene-sn-glycerol enantiomer was added as well. After
three selection rounds in which the selection pressure was increased by raising
the phosphonate ester concentration, a mutant was selected with an improved
enantioselectivity increased from an ee of -29.6 % (conversion 23.4 %) to an ee
of +73.1 % (conversion 28.9 %) towards the (S)-(+)-enantiomer. Interestingly, its
amino acid sequence showed that the acid of the catalytic triad had migrated to a
position further along the loop that connects beta7 and alphaE; this shows that
the position of the catalytic acid is not necessarily conserved in this lipase.

PMID: 18383241 [PubMed - indexed for MEDLINE]