Appl Microbiol Biotechnol
Table 1 Activities depending on
the scale of the shake flask
cultivation and the flask size
Entry
Flask size (L)
Medium/flask (L)
Total protein (mg/mL)
U/mL
U/mg
1
2
3
4
0.3[a]
1.0[a]
5.0
0.006
0.33
1.0
20.03
15.25
14.85
8.95
7.44
8.90
8.2
0.49
0.58
0.55
1.22
5.0
1.0
10.95
Cultivation conditions: LB-media, 200 μg L−1 AHTC, 30 °C, 21 h, 140 rpm
[a] Baffled flask
compared to the reaction without solvent (Fig. S11, gray
dashed line). Weakly water-miscible ethers, such as MTBE,
DIPE or diethyl ether, and the water-miscible aprotic solvent
DMSO, seemed to be slightly preferred, because the forma-
tion of 9 went almost equally well at 5 and 20 vol% concen-
tration, respectively. Larger differences with respect to con-
version and solvent concentration were obtained with the
water-miscible ones. Especially THF and, at higher concen-
trations, also DMF as well as several other protic and aprotic
co-solvents turned out to be rather detrimental to the ATase.
the ATase at 35 °C, the residual activity after 2 h was 68%
(Fig. 8, circle); however, at higher temperatures, such as 50 °C
(Fig. 8, square) or 60 °C (Fig. 8, triangle), the enzyme lost half
of its activity within 70 min. Prolonging the incubation time at
these temperatures up to 2 h resulted in residual activities of
approximately 40%. At even higher temperatures, severe heat-
deactivation was noticed. For instance, at 70 °C (Fig. 8,
diamond) the half-life was less than 10 min and consequently,
after 2 h the enzyme was not active anymore.
Inhibitors and additives
Storage and thermo-stability of different types
of enzyme preparation
The influence of commonly used inhibitors was studied in
order to gain more information about the ATase (Fig. 9). For
testing a small aliquot of cell-free E. coli extract containing the
recombinant ATase (50 μL) was pretreated with the respective
inhibitor/additive for 40 min at 28 °C: dithiothreitol (DTT, 0.5
or 2 mM), 2-mercaptoethanol (β-Met, 1 or 2 mM),
phenylmethanesulfonyl fluoride (PMSF, 1 mM), iodoacetic
acid (IAA, 1 or 2 mM), p-chloromecuribenzoic acid (pCMB,
1 mM), diethylpyrocarbonate (DEPC, 2 or 3 mM), EDTA
(5 mM), Triton-X100 (0.5 w/v%), or Tween-40 (0.5 w/v%).
All studied inhibitors affected the ATase activity, leading in
general to a loss of activity (Fig. 9). After treatment with
pCMP or IAA, which are known for their ability to covalently
bind to thiol moieties, the enzyme activity was abolished.
Furthermore DEPC, which is known to modify histidine res-
idues also led to a loss of conversion at 2 mM concentration.
DTT, β-Met, PMSF, and EDTA led to slightly reduced activ-
ities. Furthermore, the enzyme turned out to be sensitive to-
wards detergents (0.5% w/v, Triton-X100 and Tween-40).
Additionally, the enzyme was incubated with resorcinol
(10 mM), phloroglucinol (10 mM), DAPG (15 mM), and
MAPG (15 mM) prior to biotransformation. In case of resor-
cinol and phloroglucinol, a slight increase in residual activity
was observed (105 and 107% respectively, while for MAPG
and DAPG, a slight decrease was noticed, 92–93% (Fig. 9).
Enzyme activity was also investigated in the presence of
the chloride salts of Ca2+, Mg2+, Zn2+, Cu2+, Co2+, Mn2+,
Sr2+, or Ni2+ at 5 or 8 mM concentration and compared to a
control reaction in the absence of the metal (Fig. S9). To avoid
precipitation of the metal with a buffer component (e.g., phos-
phate), all reactions were performed in HEPES-buffer
To learn about the storage stability of the recombinant ATases,
batch activities of different types of enzyme preparations under
various conditions were monitored over 9 weeks (Fig. 7). To
test the residual activity of the enzyme batches, the
bioacetylation of 8 with DAPG was performed after 0, 14, 35,
or 63 days of storage. The impact of lyophilization and storage
temperature was evaluated. Additionally, the sensitivity to mo-
lecular oxygen was tested by storing the preparation under inert
atmosphere (argon). For instance, freeze-dried cells prepared in
KPi-buffer lost almost their entire activity after being stored for
9 weeks at 4 °C; inert storage under argon did not help to retain
activity (Fig. 7a and b, rows 1 and 2). However, if cells were
treated in salt-rich PBS-buffer instead of KPi-buffer prior to
lyophilization, the activity loss was significantly slower, indi-
cating that high salt concentrations contribute to the stabiliza-
tion of the enzyme (Fig. 7, row 3). If the ATase-containing cell-
free extract was lyophilized and stored at 4 °C, activity was lost
as well. However, the degradation was much slower than ob-
served for the lyophilized cells (Fig. 7, rows 4–5 vs. rows 1–2).
Interestingly, when the liquid cell-free extract was frozen or
simply stored at 4 °C instead of lyophilized, the entire batch
activity was retained for at least 9 weeks. Preparations stored at
+ 4 °C, − 20 or − 80 °C were equally active (Fig. 7, rows 6–8)
leading to 9 with 44–45% product yield.
Tolerance to an increased reaction temperature is a crucial
parameter having significant effects on both, the enzymatic
activity and its stability. The thermostability of PpATaseCH
was determined by heat-treatment of the ATase-containing
free extract assay at various temperatures. When incubating