Selection strategies for improved biocatalysts

Enzymes have become an attractive alternative to conventional catalysts in numerous industrial processes. However, their properties do not always meet the criteria of the application of interest. Directed evolution is a powerful tool for adapting the characteristics of an enzyme. However, selection of the evolved variants is a critical step, and therefore new strategies to enable selection of the desired enzymatic activity have been developed. This review focuses on these novel strategies for selecting enzymes from large libraries, in particular those that are used in the synthesis of pharmaceutical intermediates and pharmaceuticals

A Novel Genetic Selection System for Improved Enantioselectivity of Bacillus subtilis Lipase A

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 β7 and αE; this shows that the position of the catalytic acid is not necessarily conserved in this lipase.

(Endo)cannabinoids mediate different Ca(2+) entry mechanisms in human bronchial epithelial cells

In human bronchial epithelial (16HBE14o) cells, CB(1) and CB(2) cannabinoid receptors are present, and their activation by the endocannabinoid virodhamine and the synthetic non-selective receptor agonist CP55,940 inhibits adenylyl cyclase and cellular interleukin-8 release. Here, we analyzed changes in intracellular calcium ([Ca(2+)](i)) evoked by Delta(9)-tetrahydrocannabinol (Delta(9)-THC), CP55,940, and virodhamine in 16HBE14o(-) cells. Delta(9)-THC induced [Ca(2+)](i) increase and a large transient [Ca(2+)](i) mobilization, the latter probably reflecting store-depletion-driven capacitative Ca(2+) entry (CCE). In contrast, CP55,940 induced a rather moderate Ca(2+) influx and a sustained [Ca(2+)](i) mobilization. CP55,940-induced Ca(2+) influx was inhibited by Ni(2+), indicating CCE, possibly mediated by transient receptor potential channel TRPC1, the mRNA of which is expressed in 16HBE14o(-) cells. CP55,940-induced calcium alterations were mimicked by virodhamine concentrations below 30 mu M. Interestingly, higher virodhamine induced an additional Ca2+ entry, insensitive to Ni(2+), but sensitive to the TRPV1 antagonist capsazepine, the TRPV1-TRPV4 inhibitor ruthenium red, and the non-CCE (NCCE) inhibitors La(3+) and Gd(3+). Such pharmacological profile is supported by the presence of TRPV1, TRPV4, and TRPC6 mRNAs as well as TRPV1 and TRPC6 proteins in 16HBE14o(-) cells. Cannabinoid receptor antagonists increased virodhamine-induced Ca(2+) entry. Virodhamine also enhanced arachidonic acid release, which was insensitive to cannabinoid receptor antagonism, but sensitive to the phospholipase A(2) inhibitor quinacrine, and to capsazepine. Arachidonic acid induced [Ca(2+)](i) increase similar to virodhamine. Collectively, these observations suggest that [Ca(2+)](i) alterations induced by Delta(9)-THC, CP55,940 and by low concentrations of virodhamine involve mobilization and subsequent CCE mechanisms, whereas such responses by high virodhamine concentrations involve NCCE pathways

The Game of Health Search

Almost two of three Swedes use internet to search for health related information on diseases, treatments and care givers. Mobile devices such as smartphones and tablets are increasingly used to carry out these activities, and it raises the question on how a health information portal should behave to support the needs of today’s and tomorrow’s information seekers. In this thesis we present an analysis of the use of the official health information portals 1177.se and vardguiden.se with a focus on describing the relations between seekers and portals, as expressed by the language of queries and answers. Of special interest is the role of the language as a means to establish and maintain the seekers’ trust in a portal as a complement to doctor’s visits and calls. We present a number of principles of behaviour to which we believe a portal should adhere to be trustworthy in the eyes of the seekers. We also introduce a conceptual framework with a basis in game-theoretic models of rational behaviour, and the use of lingustic error analysis and stylistics, to provide a setting for analysis of information search