TITLE:
Impact of Leucine 278 Residue on Fatty Acid Length Specificity of Candida antarctica Lipase B
AUTHORS:
Fanghua Wang, Shulin Hou, Qian Wang, Pu Wang, Jinsong Liu, Bo Yang, Yonghua Wang
KEYWORDS:
Candida antarctica Lipase B (CALB), Site-Directed Mutagenesis, Substrate Specificity, Stereospecific Blockade
JOURNAL NAME:
Advances in Microbiology,
Vol.5 No.7,
July
3,
2015
ABSTRACT: Structural analysis of Candida antarctica lipase B (CALB)
indicates that side chain of leucine at 278 site lies above the entrance of the
catalytic pocket, which prognosticates its potential role on substrate
specificity of the enzyme. To verify this presumption, shortened side chain of
glycine or proline was rational designed and mutants were constructed by
site-directed mutagenesis method. The colorimetric assay using p-nitrophenyl esters of fatty acids with
various chain-lengths was used to study the substrate preference of lipases.
Results indicated that L278G or L278P mutations both induced the drift of
substrate specificity of CALB from p-nitrophenyl
caprylate (pNP-C8) to longer carbon
chain length of p-nitrophenyl caprate
(pNP-C10). Meanwhile, Vmax value of two mutants to pNP-C10
was both higher than that of wild-type. Docking results also indicated that
shortened side chain of glycine or proline residues substitution at this site
could get rid of the space block present above the catalytic pocket, and made
longer chain substrate (pNP-C10)
enter into the catalytic pocket easier. The modulation of specificity observed
allowed for building substrate binding model and opened new possibilities for
designing ligand specific lipases.