Understanding and engineering the enantioselectivity of candida antarctica lipase b towards sec-alcohols
The kinetic resolution of sec-alcohols catalyzed by Candida antarctica lipase B
(CALB) was investigated. High enantioselectivity was achieved with some
aliphatic, allylic, propargylic and halogenated sec-alcohols. Steric and non-steric
interactions were found to be important in the chiral recognition.
Empirical rules for qualitative prediction of the enantioselectivity of CALB towards
sec-alcohols were defined. High reaction rate and enantioselectivity can be
expected for substrates with the following requirements at the stereocenter: (i)
large substituent equal to or larger than a propyl group or containing a halogen
atom (ii) medium substituent smaller than a propyl group and without one
Parameters influencing the enantioselectivity in organic solvents were
investigated and reviewed. The enantioselectivity was affected by many
parameters, such as temperature, solvent, acyl-donor and enzyme preparation.
Particular attention was given to the large differences in enantioselectivity and in
reaction rate among the lipase preparations used. Mass-transport limitations were
the main causes of the difference in effectiveness between preparations.
Diffusion limitations were detrimental to the reaction rate and the
enantioselectivity and, thus, should be avoided. Lipase immobilizations and high
substrate concentrations reduced mass-transport problems in organic solvents. E-
values varied between 60 and 200 for the resolution of 3-methyl-2-cyclohexen-1ol
The molecular basis of the enantioselectivity of CALB towards sec-alcohols was
studied by means of molecular modeling and experimental kinetic data. The
origin of the chiral recognition was traced to the permutation of the large and the
medium substituents of the alcohol enantiomers in the active site i.e. the slowreacting
enantiomer placed its large substituent in a pocket of limited volume,
whereas the fast-reacting enantiomer placed its medium substituent in this
Rational protein engineering, based on the above model, allowed the creation of
one synthetically useful mutant as well as one mutant with annihilated
enantioselectivity towards two target 1-halo-2-octanols. These results supported
our model concerning the molecular recognition of sec-alcohol enantiomers by
Key words: kinetic resolution, protein engineering, mass-transport limitations,
organic media, halohydrins, chiral recognition, empirical rules, biocatalysis.
List of articles
I. Candida antarctica lipase B catalysed kinetic resolutions: substrate structure
requirements for the preparation of enantiomerically enriched secondary alcanols
C. Orrenius, N. Öhrner, D. Rotticci, A. Mattson, K. Hult, and T. Norin,
Tetrahedron: Asymmetry, 1995, 6, 1217.
II. Enantiomerically enriched bifunctional sec-alcohols prepared by Candida
antarctica lipase B catalysis. Evidence of non-steric interactions
D. Rotticci, C. Orrenius, K. Hult, and T. Norin, Tetrahedron: Asymmetry, 1997,
III. Chiral recognition of alcohol enantiomers in acyl transfer reactions catalysed
by Candida antarctica lipase B
C. Orrenius, F. Hæffner, D. Rotticci, N. Öhrner, T. Norin, and K. Hult, Biocatal.
Biotransform., 1998, 16, 1.
IV. Molecular recognition of sec-alcohol enantiomers by Candida antarctica
D. Rotticci, F. Hæffner, C. Orrenius, T. Norin, and K. Hult, J. Mol. Catal. B:
Enzym., 1998, 5, 267.
V. Candida antarctica lipase B: a tool for the preparation of optically active
D. Rotticci, J. Ottosson, T. Norin, and K. Hult, in Enzymes in non-aqueous
media, (Eds.: P. Halling, E. Vulfson, J. Woodley, B. Holland), Totowa, NJ,
Humana Press. In press.
VI. Mass-transport limitations reduce the effective stereospecificity in enzymecatalyzed
D. Rotticci, T. Norin, and K. Hult, Org. Lett., In press.
VII. Improving the enantioselectivity of a lipase through rational protein
D. Rotticci, J. C. Mulder, S. Denman, T. Norin, and K. Hult, manuscript.
School:Kungliga Tekniska högskolan
Source Type:Doctoral Dissertation
Date of Publication:01/01/2000