Communications
for 16 h. The water activity of the batch was set (aw=0.11) over
LiCl (desiccator with LiCl (sat, aq), over 16 h).
In conclusion, it was experimentally verified that the truncat-
ed variant does not display interfacial activation. This strength-
ens the hypothesis that the mobile region functions as a flap
that governs the activation of the enzyme. The truncated
enzyme retained activity, and the stability was somewhat in-
creased (TM measurements, data not shown). The relative spe-
cificity constants for a chosen primary alcohol, and the enan-
tiomers of a secondary alcohol, were practically unchanged
compared to wild type. Furthermore, it was demonstrated that
the enantioselectivity in the alcohol- and the acyl-binding sites
was not influenced by the flap. This was shown for the alcohol
pocket by the use of 1-(naphth-2-yl)ethanol (1) and its ester 2,
and for the acyl pocket by the use of two esters (3 and 4)
chiral in the acid part. In all cases, the wild-type and the trun-
cated variant showed the same enantioselectivity.
Purification by hydrophobic interaction chromatography: Super-
natants from CalA variant culture (100 mL) was filtered (0.2 mm)
and ammonium sulfate was added (1m). The sample was loaded
onto a HiPrep Butyl FF 16/10 column (GE Healthcare) that was pre-
equilibrated in starting buffer (ammonium sulfate (1m) in potassi-
um phosphate (50 mm, pH 7.0)). Samples were loaded onto the
column, and eluted by a gradient of 100% starting buffer to 100%
elution buffer (ddH2O). Fractions containing the CalA variant (as
detected by absorbance at 280 nm and by monitoring the hydroly-
sis of 2-methylheptanoic acid p-nitrophenyl ester were concentrat-
ed on Amicon Ultra-15 10 kD columns, and the buffer was ex-
changed to potassium phosphate (100 mm, pH 8.0). BioRad Protein
Assay kit was used to quantify protein yield, using BSA as a stan-
dard.
The truncated variant showed increased specificity for the
dissolved substrate, but not when the substrate was present
as a dispersion. This is evident in the curve in Figure 2 and in
the lower kcat/KM (compared to wild type with concentrations
below the CMC). kcat was higher for the wild type, possibly due
to destabilizing structural distortion in the absence of the lid,
and most likely also a consequence of association to dispersed
oil droplets; this was not achieved by the truncated variant.
The higher activity of the wild-type might also arise from con-
formational change during interfacial activation, which might
involve catalytically active residues and/or residues in the pri-
mary sphere.[12] The truncated variant is a candidate for biocat-
alytic applications where interfacial activation is detrimental,
for example in transformations of completely dissolved sub-
strates.
Relative specificity constants: Benzyl alcohol (2.0 mm), racemic
1-phenylethanol (4.0 mm) and vinyl acetate (50 mm) dissolved in
iso-octane, with pentadecane as internal standard, were added to
immobilized preparation (30 mg). The reactions were monitored by
chiral GC.
Hydrolysis of naphth-2-yl butyrate (2): Supernatant containing
CalA (0.08 mgmLÀ1, 50 mL) was added to a solution of substrate 2
(2 mgmLÀ1; prepared from esterification of 1) in MOPS/NaOH
(8.5 mL, 50 mm, pH 7.5) with MeCN (1.5 mL). The reaction was run
on an orbital shaker (250 rpm) at 378C for 20 min. The reaction
was stopped by the addition of CH2Cl2 (1 mL) and acidified (to pH
~3–4, with 1m HCl). For conversions under 40%, the product was
worked up by extraction into CH2Cl2 (31 mL), analyzed by NMR
(conversion) and GC (ee). Reported E values are mean values of
three reactions.
Transacylation of 1-(naphth-2-yl)ethanol (1): Enzyme (5 mg on
Accurel pellets; 5% loading, calculated on enzyme supernatant)
It is possible that other lipases would retain activity without
the motile region that governs interfacial activation. Further
studies are required to find out how general this phenomenon
is.
was added to
1 (20 mm) in iso-octane with vinyl butyrate
(200 mm), and dodecane (20 mm) as an internal standard. The re-
action was run at 258C for 10 min on an orbital shaker (250 rpm).
Samples were directly analyzed by GC. Reported E values are mean
values of three reactions.
Experimental Section
Hydrolysis of p-nitrophenyl esters 3 and 4: Enzyme supernatant
(200 mL, 0.12 mgmLÀ1), was added to 3 or 4 (1.25 mL, 2 mgmLÀ1 in
MeCN, buffer up to 10 mL (50 mm Phosphate, pH 8.0). The reaction
was run on at 298C an orbital shaker (200 rpm). The reaction was
stopped by the addition of CH2Cl2 (1 mL) and acidified to pH 3–4,
with HCl (1m). For conversions under 40%, the product was
worked up by extraction into CH2Cl2 (31 mL), analyzed by NMR
(conversion) and GC (ee). Reported E values are mean values of
three reactions.
Construction of plasmid pBGP1 CalA was previously described.[26]
The flap region (residues 426–440) was removed by deletion PCR
(primers designed to exclude the terminal part of CalA). A flanking
region of the PCR primers consisted of a polyhistidine tag (6CAT)
for addition of this affinity tag. The PCR product was cleaned, and
the mother template removed by Dpn1 digestion. The product
was heat-shock transformed into E. coli DH5a, and verified by se-
quencing. The plasmid was transformed into Pichia pastoris by
electroporation, plated on YPD agar plates (yeast extract
(10 mgmLÀ1), peptone (20 mgmLÀ1), dextrose (20 mgmLÀ1), agar
(20 mgmLÀ1)), and colonies were cultivated (308C, 250 rpm) for 4–
8 days, during which time active enzyme was secreted into the
medium. The culture was harvested by centrifugation, and the
supernatant containing protein was used directly, immobilized, or
purified with hydrophobic interaction chromatography.
Synthesis of methyl 4-nitrophenyl hexylphosphonate (5): Tetra-
zole[29] (10 mg, 0.16 mmol) was dissolved in dry and degassed tolu-
ene (10 mL) under argon. The solution was cooled to 08C followed
by addition of methanol (64 mL, 1.6 mmol) and diisopropylethyl-
amine (310 mL, 1.8 mmol) with stirring. Hexylphosphonic dichloride
dissolved in dry and degassed toluene (5 mL) was added dropwise,
and the reaction was slowly heated to room temperature and
stirred 4 h. Next, a solution of diisopropylamine (310 mL, 222 mg
2.2 mmol) and p-nitrophenol (210 mg, 1.5 mmol) was added
(slightly exothermic reaction). The reaction mixture was stirred
overnight and then filtered through a fritted funnel. The filtrate
was concentrated in vacuo and 5 was isolated by column chroma-
tography (SiO2; gradient: 100% CH2Cl2 to CH2Cl2/EtOAc 98:2) as an
Immmobilization: Accurel MP 1000 pellets were wetted with EtOH
under vaccum. When the beads were no longer floating, they were
washed with MOPS buffer (3, 20 mm, pH 7.6) and added to
enzyme supernatant (0.1–1 mgmLÀ1 in MOPS (20 mm, pH 7.6)), and
mixed on an orbital shaker (250 rpm, 308C, 16 h). The beads were
then washed with MOPS (20 mm, pH 7.6) and dried under vacuum
ChemBioChem 2016, 17, 141 – 145
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