Melloney J. Dröge

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.

Directed Evolution of Bacillus subtilis Lipase A by Use of Enantiomeric Phosphonate Inhibitors: Crystal Structures and Phage Display Selection

Phage display can be used as a protein‐engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2‐O‐isopropylidene‐sn‐glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)‐(+)‐ and (R)‐(−)‐IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis–Menten parameters. The 3D structures of the Sc and Rc inhibitor–lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.

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.

Extracellular lipases from Bacillus subtilis: regulation of gene expression and enzyme activity by amino acid supply and external pH

Bacillus subtilis secretes two lipases LipA and LipB into the culture medium. Both enzyme genes were differentially expressed depending on the growth conditions as determined by activity assays and Western blotting in B. subtilis mutant strains lipA, lipB, and the corresponding lipA/lipB double mutant. In minimal medium, LipA was produced at wild-type level in a lipB mutant, however, no LipB protein was detected in a lipA mutant. Interestingly, LipA was produced and secreted at wild-type level in rich medium, but the enzyme remained enzymatically inactive, presumably being caused by a shift of the growth medium to acid pH. Furthermore, expression of the lipase genes was studied using transcriptional fusions with the lacZ reporter gene. The expression of lipA was repressed by high amino acid concentrations, whereas the lipB gene expression remained unaffected.

Loop grafting of Bacillus subtilis lipase A : Inversion of enantioselectivity

Lipases are successfully applied in enantioselective biocatalysis. Most lipases contain a lid domain controlling access to the active site, but Bacillus subtilis Lipase A (LipA) is a notable exception: its active site is solvent exposed. To improve the enantioselectivity of LipA in the kinetic resolution of 1,2-O-isopropylidene-sn-glycerol (IPG) esters, we replaced a loop near the active-site entrance by longer loops originating from Fusarium solani cutinase and Penicillium purpurogenum acetylxylan esterase, thereby aiming to increase the interaction surface for the substrate. The resulting loop hybrids showed enantioselectivities inverted toward the desired enantiomer of IPG. The acetylxylan esterase-derived variant showed an inversion in enantiomeric excess (ee) from -12.9% to +6.0%, whereas the cutinase-derived variant was improved to an ee of +26.5%. The enantioselectivity of the cutinase-derived variant was further improved by directed evolution to an ee of +57.4%.

Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor

Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.

Immobilization of chiral enzyme inhibitors on solid supports by amide-forming coupling and olefin metathesis

Abstract The question whether phage display can be used as a selection method in the directed evolution of enantioselective enzymes has not been answered satisfactorily to date. In order to be able to test this in a specific case, namely in the hydrolytic kinetic resolution of the acetate derived from α,β-isopropylideneglycerol (IPG) catalyzed by the lipase from Bacillus subtilis, suicide enzyme inhibitors anchored on porous glass or polymer beads were designed and synthesized. These are immobilized phosphonates, which bear a leaving group and also contain the chiral substrate ( d ) and ( l )-IPG. Modified SIRAN® (porous glass) and Tentagel® (polymer) were chosen as carriers, attachment occurring via amide-forming coupling or Ru-catalyzed olefin metathesis. Initial lipase inhibition studies are also reported.

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

ABSTRACT Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

Arachidonic acid mediates non-capacitative calcium entry evoked by CB1-cannabinoid receptor activation in DDT1 MF-2 smooth muscle cells.

Cannabinoid CB1‐receptor stimulation in DDT1 MF‐2 smooth muscle cells induces a rise in [Ca2+]i, which is dependent on extracellular Ca2+ and modulated by thapsigargin‐sensitive stores, suggesting capacitative Ca2+ entry (CCE), and by MAP kinase. Non‐capacitative Ca2+ entry (NCCE) stimulated by arachidonic acid (AA) partly mediates histamine H1‐receptor‐evoked increases in [Ca2+]i in DDT1 MF‐2 cells. In the current study, both Ca2+ entry mechanisms and a possible link between MAP kinase activation and increasing [Ca2+]i were investigated. In the whole‐cell patch clamp configuration, the CB‐receptor agonist CP 55, 940 evoked a transient, Ca2+‐dependent K+ current, which was not blocked by the inhibitors of CCE, 2‐APB, and SKF 96365. AA, but not its metabolites, evoked a transient outward current and inhibited the response to CP 55,940 in a concentration‐dependent manner. CP 55,940 induced a concentration‐dependent release of AA, which was inhibited by the CB1 antagonist SR 141716. The non‐selective Ca2+ channel blockers La3+ and Gd3+ inhibited the CP 55,940‐induced current at concentrations that had no effect on thapsigargin‐evoked CCE. La3+ also inhibited the AA‐induced current. CP 55,940‐induced AA release was abolished by Gd3+ and by phospholipase A2 inhibition using quinacrine; this compound also inhibited the outward current. The CP 55,940‐induced AA release was strongly reduced by the MAP kinase inhibitor PD 98059. The data suggest that in DDT1 MF‐2 cells, AA is an integral component of the CB1 receptor signaling pathway, upstream of NCCE and, via PLA2, downstream of MAP kinase.

(Endo)cannabinoids mediate different Ca2+ entry mechanisms in human bronchial epithelial cells

In human bronchial epithelial (16HBE14o−) cells, CB1 and CB2 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 ([Ca2+]i) evoked by Δ9-tetrahydrocannabinol (Δ9-THC), CP55,940, and virodhamine in 16HBE14o− cells. Δ9-THC induced [Ca2+]i increase and a large transient [Ca2+]i mobilization, the latter probably reflecting store-depletion-driven capacitative Ca2+ entry (CCE). In contrast, CP55,940 induced a rather moderate Ca2+ influx and a sustained [Ca2+]i mobilization. CP55,940-induced Ca2+ influx was inhibited by Ni2+, 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 μM. Interestingly, higher virodhamine induced an additional Ca2+ entry, insensitive to Ni2+, but sensitive to the TRPV1 antagonist capsazepine, the TRPV1-TRPV4 inhibitor ruthenium red, and the non-CCE (NCCE) inhibitors La3+ and Gd3+. 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 Ca2+ entry. Virodhamine also enhanced arachidonic acid release, which was insensitive to cannabinoid receptor antagonism, but sensitive to the phospholipase A2 inhibitor quinacrine, and to capsazepine. Arachidonic acid induced [Ca2+]i increase similar to virodhamine. Collectively, these observations suggest that [Ca2+]i alterations induced by Δ9-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.