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%.
Boersma
Ykelien L
YL
Department of Pharmaceutical Biology, Groningen University Institute for Drug Exploration (GUIDE), University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Pijning
Tjaard
T
Bosma
Margriet S
MS
van der Sloot
Almer M
AM
Godinho
Luís F
LF
Dröge
Melloney J
MJ
Winter
Remko T
RT
van Pouderoyen
Gertie
G
Dijkstra
Bauke W
BW
Quax
Wim J
WJ
D000115:Acetylesterase / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013237:Stereoisomerism; D013379:Substrate Specificity
D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D008049:Lipase; D000115:Acetylesterase; C081851:acetylxylan esterase