Full Papers
er shake flask) was inoculated with an E. coli BL21(DE3) clone con-
taining the respective target plasmid from a fresh transformation
plate (LB agar). This pre-culture was grown overnight at 378C with
constant agitation at 130 rpm. With this culture, the main expres-
Photometric mandelonitrile cleavage assay for quantifica-
tion of HNL activity
Mandelonitrile cleavage reactions were performed as described
[25]
[34]
previously. In brief, the accumulation of benzaldehyde, which is
formed as one reaction product of the HNL-catalyzed cleavage of
rac-mandelonitrile, was photometrically monitored at 280 nm
sion culture (500 mL autoinduction medium in a 5 L shake flask)
was inoculated to an OD600 of 0.05. The employed autoinduction
medium consisted of premixed terrific broth (TB) medium (Roth,
Karlsruhe, Germany) supplemented with 0.4% (v/v) glycerol and
(
Spectramax, Molecular Devices, Sunnyvale, CA, USA). All measure-
À1
ments were carried out in 1 cm quartz cuvettes at 258C in assay
buffer (50 mm sodium acetate buffer) with a total assay volume of
glucose (0.5 gL ) to suppress gene expression in the first hours of
À1
incubation, and lactose (2 gL ) for the induction of expression in
1
mL. To start the reactions, rac-mandelonitrile was added (67 mm
a later phase. All media were supplemented with kanamycin
À1
in 50 mm citrate phosphate buffer, pH 3.5) to a final concentration
of 13.4 mm to the assay. The respective control in buffer (without
enzyme) was subtracted from all measurements to account for
non-enzymatic mandelonitrile cleavage. All measurements were
performed at least in triplicate. Enzymatic activity was calculated
(
50 mgmL ) for plasmid maintenance. Cultivation was performed
for 3 h at 378C and 130 rpm, followed by a prolonged incubation
for another 69 h at 158C and 130 rpm. After the cultivation, the
cells were harvested by centrifugation and were directly used for
cell disruption and purification or stored at À208C. Wild-type
[11]
by using the molar extinction coefficient of benzaldehyde (e
=
AtHNL was produced and purified as described previously.
280nm
À1
À1
1
.376 Lmmol cm ). One unit (U) of HNL activity is defined as the
amount of enzyme that converts 1 mmol (R)-mandelonitrile per
minute in the given buffer at 258C.
Cell disruption, fractionation, and purification of CatIBs
To measure the HNL activity of insoluble enzymes, such as AtHNL-
CatIBs, a modified endpoint-based mandelonitrile cleavage assay
was used. Because of insoluble material, all reaction tubes were
kept under constant agitation (1000 rpm) in a thermomixer (Ep-
pendorf, Hamburg, Germany). For the assay, the insoluble enzyme
was suspended in 50 mm citrate phosphate buffer, pH 5.5, diluted
by using assay buffer, and incubated at 258C. An aliquot (100 mL)
of this solution was transferred to a new reaction tube containing
assay buffer (700 mL) and the reaction was immediately started by
the addition of substrate solution (200 mL). After 2 min of reaction,
the tube was centrifuged (18890 g, RT) for 1 min, resulting in
a total reaction time of 3 min, and 700 mL of the supernatant was
immediately used for the absorbance measurement at 280 nm.
This value corresponds to the amount of benzaldehyde released
during the 3 min reaction time. To account for non-enzymatic man-
delonitrile cleavage, the respective control in buffer (without
enzyme) was subtracted from all measurements. Because mandelo-
nitrile is already slowly cleaved in the substrate stock solution, this
non-enzymatic cleavage had to be taken into account by measur-
ing the time-resolved decay of the substrate with the above men-
tioned assay. Moreover, to rule out measuring protein absorbance
at 280 nm, additional controls had to be prepared with AtHNL-
CatIBs and substrate buffer (50 mm citrate phosphate buffer,
pH 3.5) instead of substrate stock solution. By combining all con-
trols, the enzymatic activity could be calculated by using the molar
extinction coefficient of benzaldehyde as described above. All
measurements were performed at least in triplicate.
For the cell disruption, fractionation, and purification of CatIBs, ap-
propriate buffers for the different enzymes were used (BsLA buffer
and AtHNL buffer: 50 mm sodium phosphate, pH 8, 100 mm NaCl;
EcMenD buffer: 50 mm triethanolamine (TEA), pH 8, 2 mm MgSO4,
0
.1 mm thiamine diphosphate (ThDP)). For cell lysis, a 10% (w/v)
cell suspension in the appropriate buffer was prepared and frozen
overnight at À808C. The next day, the cells were thawed at room
temperature and disrupted by using a FrenchPress pressure ho-
mogenization system (Thermo Fisher Scientific, St. Leon-Rot, Ger-
many) by passing the suspension through the French press cell
three times at a constant pressure of 1000 psi (1103 bar internal
cell pressure). Directly after this step, the crude cell extract was
frozen at À808C overnight to enhance the efficiency of cell disrup-
tion. After thawing the extract at room temperature, a sample for
analysis of the protein content by SDS-PAGE and enzyme activity
was taken and the rest was used for fractionation into soluble and
insoluble parts by centrifugation (15000 g, 30 min). The obtained
pellet was resuspended in the initial volume of buffer and samples
for further analysis were withdrawn from both fractions. For further
purification of the CatIB-containing insoluble fraction, the pellet
was washed three times by using the buffers listed above, whereas
for the AtHNL-CatIBs, the buffer was changed in the last washing
step to 50 mm citrate phosphate buffer, pH 5.5, to reduce the non-
enzymatic side reaction in the synthesis reaction. After the last cen-
trifugation, the pelleted CatIBs were stored until further use at
À808C.
Lyophilization, quantification of CatIB yields, and protein
content
The wet weight of different CatIBs was determined directly after
purification by weighing the wet pellet. For lyophilization, a 10%
Influence of pH on the activity of AtHNL-CatIBs
(
w/v) CatIB suspension in MilliQ water was prepared, frozen in
The pH-dependent initial rate activities were determined by using
the endpoint-based mandelonitrile cleavage assay in the range
pH 3.5 to pH 5. For all measurements, one stock of resuspended
(50 mm citrate phosphate buffer, pH 5.5) AtHNL-CatIBs was pre-
pared and adjusted by dilution with buffer until the absorption
value at 280 nm after incubation at pH 5 and the following reac-
tion equaled 0.9. The AtHNL-CatIB stock was diluted at least 20-
fold with 50 mm acetate buffer of the respective pH value and in-
cubated for 5 min at 258C and 1000 rpm before withdrawing an
aliquot for the activity assay. All measurements as well as controls
without enzyme were performed in triplicate.
liquid nitrogen, stored overnight at À808C, and lyophilized (Lyovac
GT2, SRK Systemtechnik, Riedstadt-Goddelau, Germany). After-
wards, the dry weight was determined and the protein content of
a defined amount CatIBs was analyzed by denaturation in 6m gua-
nidine hydrochloride followed by measuring the absorbance at
2
80 nm. Protein concentrations were calculated by using the theo-
retical extinction coefficients of the respective fusion protein ob-
tained from the amino acid sequence by using the Protparam
Webservice.
[
37]
À1
À1
BsLA-CatIBs: 27.390 Lmol cm
;
AtHNL-CatIBs:
À1
À1
À1
À1
3
4.505 Lmol cm ; EcMenD-CatIBs: 110.265 Lmol cm
ChemCatChem 2016, 8, 142 – 152
149
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim