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network; F315I was chosen because it is volumetrically analo-
gous to the wild-type enzyme. The triple mutant still exhibits
higher amidase activity than the wild-type, however mutation
F315I seems to negate most of the beneficial effect of the
other two mutations (Table 1). According to these results, the
F315 mutation seems to be crucial for the stabilisation of loop
314–324 through the p–p network.
amidase catalytic behaviour, compared to the wild-type BS2.
The enhancement is proven with three different substrates,
which leads us to the conclusion that the observed increase in
the promiscuous activity is not substrate dependent and is
more related to the intrinsic catalytic mechanism. A p–p net-
work is responsible for the enhancement of the amidase activi-
ty, which positions loops 314–324 and 265–275 in a more fa-
vourable orientation for the stabilisation of the tetrahedral in-
termediate. If we take into consideration the fact that substrate
structure can significantly affect the amide hydrolysis[9] or the
reverse reaction,[11] it seems necessary for any further studies
on the field that the substrate scope of the promiscuous activi-
ty should be studied, so that the effect of the mutations can
be evaluated irrespectively of the substrate used.
The network generated for the double mutant suggests that
the aromatic ring of the substrate is also a part of the p–p net-
work (Figure SI-1b). To evaluate if the presence of the phenyl
ring is crucial for the enhancement of the promiscuous activity,
butyl butyrate and butyl butanamide were used as aliphatic
substrates. The ester is an easily accepted substrate for both
enzyme variants (100% hydrolysis in less than 1 h). The amide
hydrolysis is much slower, as with the phenolic substrates.
After 24 h of biocatalysis, the wild-type BS2 gene hydrolysed
only 2.1Æ0.1% of the amide, whereas the double mutant hy-
drolysed 16.4Æ0.3% at the same time. This shows that the
beneficial effect of the two mutations is not restricted only to
phenolic substrates, but is extended also to aliphatic com-
pounds. The promiscuous activity of the double mutant on the
aliphatic substrate is 7.7 fold higher than the wild-type, which
is in the same order of magnitude that was observed for the
aromatic substrates. These results underline that the enhance-
ment of the amidase activity depends on the p–p network of
the double mutant and not on the phenolic ring of the
substrate.
Experimental Section
General
All chemicals were purchased from Fluka (Buchs, Switzerland),
Sigma (Steinheim, Germany), Carl-Roth (Karlsruhe, Germany) and
Merck (Darmstadt, Germany), unless stated otherwise. p-Nitroani-
lide butyrate (pNAB) was synthesised according to previous
work.[12] N-Butyl butanamide was purchased from Enamine (Kiev,
Ukraine). Restriction enzymes and polymerases were obtained from
New England Biolabs (Frankfurt am Main, Germany). Primers were
synthesised by Invitrogen (Darmstadt, Germany). Sequencing was
performed by Eurofins MWG operon (Ebersberg, Germany).
The enhancement of the promiscuous activity reported here
can be considered high, because it is comparable to the best
results in this field so far. Nakagawa and co-workers increased
the amidase activity of a Pseudomonas aeruginosa lipase
28 fold.[3a] More interesting are the results on CALB. Syrꢀn and
co-workers found a 7.3 fold higher amidase activity for the
I189Y mutant of CALB in the hydrolysis of pNAB.[3c] In a later
work, Jung and co-workers showed a 24 fold increase in the
hydrolysis of a smaller substrate, namely p-nitroanilide aceta-
te.[3b] However, Suplatov and co-workers could not reproduce
the enhancement of CALB amidase activity reported for muta-
tions at position I189 by using another aromatic substrate,
namely 2-chloro-N-benzyl acetamide.[16] Moreover, they also
tried to incorporate in CALB the beneficial mutations found in
Pseudomonas aeruginosa lipase,[3a] but yielded a mediocre in-
crease in the amidase activity of only 10%.[16] By taking all of
these factors into consideration, the fact that the enhancement
of the amidase activity of BS2 by incorporation of I270F/
F314Y is certified by three structurally different substrates im-
plies a more generalized amidase character of this specific
variant.
PCR protocols
The BS2 gene (1.5 KDa) was available in our laboratory in the
pET28b vector with a C-terminal His6-tag (pET28b_BS2). The epPCR
protocol was established with the GeneMorph II Random Mutagen-
esis Kit (Stratagene, Santa Clara, USA). The protocol was fine-tuned
to yield approximately three mutations per gene. The amplified
gene was then purified with a QIAEX II Gel Extraction kit (QIAGEN,
Venlo, Netherlands) and was cloned into the pET28b vector by
using the Megaprimer PCR of whole plasmid (MEGAWHOP) ap-
proach.[21] After the MEGAWHOP reaction, the parental vector was
removed by digestion with the DpnI enzyme. The product was
transformed in Escherichia coli TOP10 cells to repair the nicks. Fi-
nally, the plasmids were isolated and used to transform BL21(DE3)
for protein expression. For site-directed mutagenesis a modified
QuikChange PCR protocol was used, according to the literature,[22]
and the product was used directly to transform Escherichia coli
BL21(DE3) cells. A complete list of the primers used in the present
study can be found in the Supporting Information (Table SI-2).
Protein expression and purification
Kanamycin was used in all cultivations for selection. In a typical
procedure, Escherichia coli BL21(DE3) cells transformed with
pET28b_BS2 were inoculated in terrific broth medium from an
overnight culture (1:100 v/v) and were grown at 308C until they
reached an optical density at l=600nm (OD600) of 0.5–0.8. The
protein expression was initiated by the addition of isopropyl b-D-
1-thiogalactopyranoside (IPTG) (1 mm). Twenty hours after induc-
tion, the culture was harvested (6000 g, 48C, 10 min) and re-sus-
pended in sodium phosphate buffer (50 mm, pH 7.5) that con-
tained NaCl (0.3m). The cells were lysed with sonication and centri-
Conclusions
The rational design of a carboxylic ester hydrolase that would
trade its original functionality for the promiscuous amidase ac-
tivity is still a challenging milestone. In the present work sever-
al techniques are used to enhance the amidase activity of BS2,
an esterase that exhibits a low amidase activity. The best
mutant, I270F/F314Y shows a 7.5 fold enhancement of its
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