180
J.A. Laszlo et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 175–180
VP OC 1600 used to immobilize wild-type CALB (Novozym 435)
butanol overcomes this effect [31,32]. However, this influence is
expected to be the same for either enzyme as they are immobilized
on the same support material. The substrate inhibition demon-
strated by cp283 (Fig. 3) thus likely reflects an altered sensitivity
of the enzyme to glycerol rather than due to the imposition of a
diffusion barrier, which would be the same for both enzymes.
into an otherwise very nonpolar ␣-helix (helix 17), which opens
up the substrate tunnel to the active site [29]. The increased
polarity of this sequence segment may account for cp283’s sus-
ceptibility to substrate inhibition by glycerol, and could also
contribute to the variant’s lower glycerol KmB (Table 3). However, the
commercially-sourced wild-type CALB (Novozym 435) and cp283
were not immobilized under identical conditions (i.e., differences
in co-adsorbates and surface coverage by lipase), which cannot be
precluded as contributing factors governing the kinetic responses
of the lipases to glycerol.
It could be a direct result of a change in the polar environment
near the enzyme’s active site, or may be related to the significantly
lower enzyme immobilization load for the CALB variant. Optimiza-
tion of the immobilization procedure and the support surface could
eliminate the undesirable effect. In future experiments, a targeted
screening of circular permutation libraries of CALB holds promise
for the identification of candidates with improved activity for ster-
ically challenged substrates and esters with unfavorable electronic
effects.
Acknowledgments
Leslie Smith (NCAUR) provided valuable technical support for
this research. This work was supported in part by funding from
the Biotechnology Research and Development Corporation (to J.A.L.
and D.L.C.), US National Science Foundation (CBET-0730312 to S.L.),
and the Petroleum Research Fund by the American Chemical Soci-
ety (PRF 47135-AC1 to S.L.). These organizations did not contribute
to the study design, data collection, analysis and interpretation, or
writing of the report.
site accessibility resulting from the termini relocation which led
to increased catalytic rates for bulky fatty acid esters hence did not
overcome the steric hindrance or energetic effects experienced by
arylaliphatic acids and esters [33]. Guyot et al. [27] first pointed out
the electron donating effect in cinnamic acid ester synthesis. The
electron donating effects intrinsically deactivate the electrophilic
carbon center of the carboxylic group for nucleophilic attack of
the alcohol group of the active site serine. The use of activated
esters (ethyl or vinyl leaving groups) partially counter this effect.
The greater active site accessibility in cp283 compared to wild-type
CALB which resulted in faster substrate binding and product release
for the fatty acids failed to do so for ferulic acid esters. Given the
ping-pong mechanism of the reaction, a possible reason for this
lack of improvement may be that there is a difference the in rate-
limiting step for the two types of substrates; the deacylation step
for fast substrates such as fatty acid esters, and active site (serine)
acylation for slow reacting ferulate esters [23].
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A detailed kinetic analysis demonstrated that the cp283 vari-
ant, which demonstrated very high catalytic performance relative
to wild-type CALB in hydrolytic reactions involving bulky leaving
groups [10], also retains this advantage for reactions of fatty acid
esters with glycerol. Cp283 displayed a sensitivity to substrate inhi-
bition by glycerol that was not evident in wild-type CALB. While
circular permutation has proven quite effective in identifying a
CALB variant with enhanced rates of ester and triglyceride hydrol-
ysis and synthesis, the same variant performed largely at levels
similar to wild type enzyme on sterically hindered arylaliphatic
esters such as ferulates. Whether these functional discrepancies
result from biases in the screening of circular permuted CALB for
tributyrin hydrolysis or whether these difference are a reflection of
fundamental aspects related to this protein engineering strategy is
not clear. Nevertheless, cp283 shows good activity for ␥-linolenate
which warrants further development of the engineered biocat-
alyst. Separately, the cause of the inhibitory effects of glycerol
observed for cp283 is unclear. Spectroscopic analysis indicated no
direct interaction between substrates and the termini of CP283 [9].