Loop Grafting of Bacillus subtilis Lipase A: Inversion of Enantioselectivity

Abstract

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%.

Fitness Loss and Library Size Determination in Saturation Mutagenesis

“…In two unrelated experiments, each involving randomization of a single NNS position, Boersma et al [42] screened 1,250 variants and Maeda et al [43] screened 360 variants, where ∼80 variants seem to suffice. Similarly, Champion et al [44] screened ∼8,000 variants when randomizing two NNN positions, where ∼2,000 seem to suffice.…”

Site‐Selective Ser‐Hydrolase Labelling with a Luminescent Organometallic NCN–Platinum Complex

“…BSLA was provided by Prof. Wim J. Quax and co-workers. [69] CALB (batch PPW 4534) was purchased from Novo Nordisk A/S, Copenhagen, Denmark. Electrospray ionization mass spectra and high-resolution electrospray ionization mass spectra were recorded with a Micromass LCT spectrometer.…”

“…Encouraged by these results, it was decided to investigate the labelling properties of 6 towards other proteins [22,23] and to assay the formation and stability of the covalent protein-6 adducts by performing gel electrophoresis. For this study, the serine hydrolases Bacillus subtilis lipase A (BSLA) [69] (19 kDa), commercially available Candida antarctica lipase B (CALB) (33 kDa) and cutinase as reference protein were chosen.…”

A Combination of Computational and Experimental Approaches to Investigate the Binding Behavior of B.sub Lipase A Mutants with Substrate pNPP

“…Figure 3. The van der Waals effect between Ile12 and ligand detected by PROBE, [29] the van der Waals effect are characterized by blue dots. Models are based on lipase A crystal structure (PDB entry 1r4z) and were prepared with the PyMol program.…”