Enantiocomplementary inverting sec-alkylsulfatase activity in cyano- and thio-bacteria Synechococcus and Paracoccus spp.: selectivity enhancement by medium engineering

Article abstract of DOI:10.1016/j.tetasy.2009.01.007

Whole resting cells of cyano- and thio-bacteria Synechococcus and Paracoccus spp. were shown to possess inverting alkylsulfatase activity for a broad spectrum of sec-alkylsulfate esters, which furnished either (R)- or (S)-sec-alcohols from the correspondi

Full text of DOI:10.1016/j.tetasy.2009.01.007

Tetrahedron: Asymmetry 20 (2009) 115–118
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Tetrahedron: Asymmetry
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Enantiocomplementary inverting sec-alkylsulfatase activity in cyano- and
thio-bacteria Synechococcus and Paracoccus spp.: selectivity enhancement
by medium engineering
Petra Gadler a, Tamara C. Reiter a, Kathrin Hoelsch b, Dirk Weuster-Botz b, Kurt Faber a,
*
a Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
b Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Boltzmannstrasse 15, D-85748 Garching, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Whole resting cells of cyano- and thio-bacteria Synechococcus and Paracoccus spp. were shown to possess
inverting alkylsulfatase activity for a broad spectrum of sec-alkylsulfate esters, which furnished either
(R)- or (S)-sec-alcohols from the corresponding rac-sulfate esters in an enantiocomplementary fashion.
Low enantioselectivities (E-values 1–4) could be dramatically improved by the addition of lower alcohols
(e.g., t-BuOH) or by using a biphasic medium containing t-BuOMe (E >200).
Received 23 October 2008
Accepted 19 January 2009
Available online 14 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
and the asymmetric reduction of halogenated carbonyl com-
pounds.17,18 In addition, facultatively lithoautotrophic neutrophilic
Sulfatases are a heterogenic group of hydrolytic enzymes,1
which catalyse the cleavage of the sulfate ester bond to yield the
corresponding alcohol (or phenol) and inorganic hydrogen sul-
fate.2,3 Depending on the subtype of enzyme, the reaction may pro-
ceed via cleavage of the S–O or the C–O bond, which leads to either
retention or inversion of configuration at the stereogenic carbon
centre.4 Whereas the mechanism of action for retaining sulfatases
has been studied in great detail mainly on aryl sulfatases, such as
human aryl sulfatase A,5 nothing is known about the catalytic
mechanism of inverting sulfatases.6,7 This latter activity is particu-
larly intriguing, since the SN2-type base-induced (chemocatalytic)
hydrolysis of sulfate esters with concomitant inversion of configu-
ration at carbon is practically impossible,8 which is in contrast to
facile acid-catalysed hydrolysis.9 Intrigued by the possibility of cat-
alysing a ‘chemically impossible’ reaction by an enzyme, our search
for alkyl sulfatases was driven by the selection of microbial strains
possessing unusual sulfur metabolic pathways. In this context,
inverting sulfatases were identified in Rhodococcus,7 Sulfolobus10
and Pseudomonas spp.,11 whereas retaining sulfatase activities
were identified in marine bacteria.12
(thio)bacteria of the genus Paracoccus are included in this study
due to their ability to oxidise sulfur species in low oxidation states
(mainly elemental S08 or hydrogen sulfide) to form sulfuric acid.19
2. Results and discussion
Following
a
previously established protocol,11 lyophilised
whole (resting) cells of Synechococcus and Paracoccus spp. were
used in an initial screening for alkyl sulfatase activity using sub-
strates rac-1a–6a, while the formation of the corresponding sec-
alcohol 1b–6b was monitored (Scheme 1).
We were able to see that among the range of strains tested,20
our metabolism-based concept for the strain-selection was suc-
cessful: For the first time, alkylsulfatase activity was detected in
the lithoautotrophic (thio)bacterium Paracoccus denitrificans DSM
6392 and in cyanobacteria Synechococcus spp. PCC 7942 and RCC
556 (Table 1). In contrast to Rhodococcus and Sulfolobus spp., which
were restricted to linear and non-functionalised sec-alkylsulfate
esters, the newly detected strains showed a broad substrate toler-
ance by accepting substrate rac-6a. Whereas the activities of Syn-
echococcus sp. RCC 556 were low, those of Synechococcus sp. PCC
7942 and Paracoccus DSM 6392 were useful for further studies.
For both organisms, the stereochemical course of sulfate ester
hydrolysis was shown to proceed via inversion of configuration
by using (R)- and (S)-2a as substrates, which gave (S)- and (R)-
2b, respectively. Despite these encouraging activities, the enanti-
oselectivities were disappointingly low and did not exceed E-val-
ues of about 4.33
Prompted by reports on the regulation of the complex (four-com-
ponent) periplasmic sulfate transport system of unicellular cyano-
bacteria Synechococcus spp.,13,14 we speculated about the
occurrence of (alkyl) sulfatase activities in these obligate photoauto-
trophs.15 Based on their unique light-dependent metabolism, these
organisms have recently gained increasing interest in biotechnolog-
ical applications, in particular for the bioremediation of pesticides16
In whole-cell transformations of alkylsulfate esters, poor
enantioselectivities are often caused by competing (iso)enzymes
* Corresponding author. Tel.: +43 316 380 5332; fax: +43 316 380 9840.
E-mail address: Kurt.Faber@Uni-Graz.at (K. Faber).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.01.007

116
P. Gadler et al. / Tetrahedron: Asymmetry 20 (2009) 115–118
-
-
SO3
SO3
R2
OH
O
O
R1
R2
R1
R2
R1
Paracoccus sp.
Synechococcus sp.
DSM 6392
pcc 7942
(S)-1b-6b
(R)-1a-6a
+
Aqu. Buffer pH 7.5
Organic co-solvent
Aq. Buffer pH 7.5
Organic co-solvent
+
+
-
-
SO3
SO3
OH
O
O
R1
R2
1b-6b
R1
R2
R1
R2
(S)-1a-6a
(R)-
1a-6a
rac-
R
1 = large group, R2 = small group
Substrate
1a,b
n-C5H11
CH3
2a,b
3a,b
n-C7H15
CH3
4a,b
n-C5H11 n-C4H9 (CH2)2-CH=C(CH3)2
C2H5 n-C3H7 CH3
5a,b
6a,b
R1
R2
n-C6H13
CH3
Scheme 1. Enantio- and stereoselective microbial hydrolysis of sec-alkylsulfate esters rac-1a–6a (cf. Table 2).
Table 1
Substrate tolerance of Synechococcus spp. PCC 7942, RCC 556 and Paracoccus denitrificans DSM 6392
Substrate
Synechococcus sp. RCC 556
Synechococcus sp. PCC 7942
Paracoccus denitrificans DSM 6392
c (%)
<1
1
1
1
8
eeP (%)
Ea
c (%)
eeP (%)
Ea
c (%)
eeP (%)
Ea
rac-1a
rac-2a
rac-3a
rac-4a
rac-5a
rac-6a
—
—
4
11
24
11
11
9
53 (R)
29 (R)
3 (S)
16 (S)
12 (S)
49 (R)
3
2
5
4
8
7
1
6
50 (S)
28 (S)
<1
<1
7 (S)
22 (S)
3
2
ꢀ1
ꢀ1
ꢀ1
2
n.d.
n.d.
n.d.
<1
n.d.
n.d.
n.d.
ꢀ1
—
ꢀ1
ꢀ1
ꢀ1
4
n.c.
—
n.c. = no conversion; n.d. = not determined due to low conversion; c = conversion; eeP = enantiomeric excess of product 1b–6b.
a
E-value;33 time: RCC 556 = 96 h; PCC 7942 = 72 h; DSM 6392 = 24 h.
possessing lower (or even) opposite stereo- and/or enantioprefer-
ences6 by acting through retention or inversion of configuration,
or by preferring opposite substrate enantiomers. This assumption
was supported by the fact that depending on the substrate, Syn-
echococcus sp. PCC 7942 produced the opposite enantiomeric prod-
ucts (R)-1b, (R)-2b, (R)-6b and (S)-3b–5b in low to moderate
enantiomeric excesses (Table 1). Since the overall activities of Syn-
echococcus sp. PCC 7942 and P. denitrificans DSM 6392 were satis-
factory, we considered improving their enantioselectivities by
medium engineering (Table 2).
In order to improve insufficient regio- or stereoselectivities of
biocatalysts, various techniques have been developed, which the
aim of (i) the modification of the substrate21 or cosubstrate struc-
ture22 (substrate engineering); (ii) changing the enzyme by chem-
ical23 or genetic methods24 (enzyme engineering); and (iii) tuning
of the reaction medium (medium engineering). The latter tech-
nique makes use of various components, such as carbohydrates
(e.g., cyclodextrins), PEG, detergents25 and metal ions26 which
are added in low amounts and are believed to act as enantioselec-
tive inhibitors.27 Alternatively, the bulk-solvent as a whole may be
Table 2
Selectivity enhancement for Paracoccus denitrificans DSM 6392 and Synechococcus sp. PCC 7942
Entry
Substrate
Strain
Additivea
c (%)
eeP (%)
E
1
2
3
rac-6a
rac-6a
rac-6a
Synechococcus PCC 7942
Synechococcus PCC 7942
Synechococcus PCC 7942
None
EtOH 10%
i-PrOH 10%
2
<1
<1
71 (R)
n.d.
n.d.
6
—
—
4
5
6
7
8
rac-6a
rac-6a
rac-6a
rac-6a
rac-6a
rac-6a
rac-6a
Paracoccus denitrificans DSM 6392
Paracoccus denitrificans DSM 6392
Paracoccus denitrificans DSM 6392
Paracoccus denitrificans DSM 6392
Paracoccus denitrificans DSM 6392
Paracoccus denitrificans DSM 6392
Paracoccus denitrificans DSM 6392
None
6
11
9
9
3
22 (S)
48 (S)
85 (S)
93 (S)
>99 (S)
92 (S)
>99 (S)
2
3
13
31
>200
25
>200
FeCl3 5 mM
MeOH 10%
EtOH 10%
EtOH 20%
i-PrOH 10%
t-BuOH 10%
9
10
5
7
11
12
rac-2a
rac-2a
Synechococcus PCC 7942
Synechococcus PCC 7942
None
t-BuOMe 50%
3
3
42 (R)
89 (R)
3
17
13
14
rac-6a
rac-6a
Paracoccus denitrificans DSM 6392
Synechococcus PCC 7942
t-BuOMe 50%
t-BuOMe 50%
3
3
>99 (S)
>99 (R)
>200
>200
a
Concentrations are denoted as v:v; time: 24 h.

P. Gadler et al. / Tetrahedron: Asymmetry 20 (2009) 115–118
117
altered by the addition of water-miscible or -immiscible organic
cosolvents, in order to furnish mono- or biphasic reaction media,
respectively. Although the molecular reasons of the selectivity
enhancement by medium engineering are still poorly understood,
they provide a powerful tool.28 As a rule of thumb, an increase in
stereoselectivity usually results in a loss of catalytic activity. In
general, water-miscible cosolvents, such as acetone, DMSO,29
THF,30 acetonitrile and lower alcohols,31 are often employed with
hydrolytic enzymes acting on substrates of medium polarity, such
as esterases and proteases, lipophilic organic (co)solvents to yield
biphasic media that are popular for enzymes acting on an interface,
such as lipases.
configuration of products 1b–6b was determined by coinjection
with an authentic reference material on GC using a chiral station-
ary phase. Achiral and chiral GC-analyses were performed as previ-
souly reported.11
Strains: Alcaligenes sp. DSM 2625, R. ruber DSM 44540, N. nova
DSM 43843, Ralstonia sp. DSM 6428 and P. denitrificans DSM
6392 were obtained from DSMZ (http://www.dsmz.de/) and were
grown for 72 h in media containing yeast extract (10 g/L), bacteri-
ological peptone (10 g/L), glucose (10 g/L), NaCl (2 g/L),
MgSO4Á7H2O (0.15 g/L), K2HPO4 (4.4 g/L) and NaH2PO4 (1.3 g/L) at
30 °C and 120 rpm in shaking flasks. Synechococcus sp. PCC 7942
and RCC 556 were provided by the Pasteur Culture Collection of
In our previous study on sec-alkylsulfatase RS2 from Rhodococ-
cus ruber DSM 44541, FeCl3 and hexadecyltrimethyl-ammonium
bromide turned out to be powerful selectivity enhancers.32
Although FeCl3 did not exhibit any effect on the enantioselectivity
of Paracoccus DSM 6392 (entry 5), lower alcohols such as MeOH,
EtOH, i-PrOH and t-BuOH had a strong impact. Although initial
tests showed that Synechococcus sp. PCC 7942 was very sensitive
towards these solvents (entries 1–3), they were very effective with
P. denitrificans DSM 6392 (entries 4–10). Variation of the nature
and concentration of alcohol revealed two trends:
Cyanobacteria
(http://www.pasteur.fr/recherche/banques/PCC/)
and Roscoff Culture Collection (http://www.sb-roscoff.fr), respec-
tively. Both cyanobacterial strains were cultivated according to
the method reported by Franco-Lara et al.37 on a 20 L-scale. For
the limnic strain Synechococcus sp. PCC 7942, BG-11 medium38
was used, whereas the marine Synechococcus sp. RCC 556 was
39
grown in PCR-Tu2 with artificial seawater as the base. After
264 h, cells were harvested by centrifugation (4528g, 30 min), they
were washed with Tris–HCl buffer (100 mM, pH 7.5) and
lyophilised.
(i) The selectivity enhancing effect increased with the steric
requirements of the alcohol, that is, t-BuOH was more effective
than MeOH (entries 6 and 10), with EtOH and i-PrOH as intermedi-
ates (entries 7 and 9); and (ii) increasing the amount of (ethyl)
alcohol led to enhanced selectivities with concomitant enzyme
deactivation (entries 7 and 8). No selectivity enhancement was de-
tected for Alcaligenes sp. DSM 2625, R. ruber DSM 44540, Norcadia
nova DSM 43843 or Ralstonia sp. DSM 6428 (data not shown).
Since water-immiscible organic cosolvents are generally better
tolerated by enzymes, a range of biphasic aqueous–organic solvent
systems were tested.34 Among them, t-BuOMe proved to be the
best. Substrate rac-2a, whose selectivity enhancement using alco-
hols failed with P. denitrificans DSM 6392, showed acceptable re-
sults (E-values from 3 to 17, entries 11 and 12), and rac-6a could
be hydrolysed with perfect enantioselectivity (E >200, entries 13
and 14) with both organisms in an enantiocomplementary fashion
to yield (R)- or (S)-6b in >99% ee using Synechococcus PCC 7942 or
P. denitrificans DSM 6392, respectively. The modest conversion may
be caused by solvent toxicity or by enzyme inhibition.
4.2. General screening-procedure for sec-alkylsulfatase activity
Lyophilised whole cells (50 mg) were rehydrated in 700
Tris–HCl buffer (100 mM, pH 7.5) for 1 h at 30 °C and shaking at
120 rpm. Next, 200 L of substrates 1a–6a from a stock solution
lL of
l
[30 mg/mL, in Tris-buffer (100 mM, pH 7.5) was added and the
reaction mixture was incubated at 30 °C with shaking at 120 rpm
for 24 and 72 h; in the case of Synechococcus sp., 96 h was required.
RCC 556. Work-up was performed by adding 600
centrifugation (13,000 rpm, 2 min, rt), the organic layer was dried
over Na2SO4 and 100 L of 1-decanol from a stock solution (10 mg/
lL of EtOAc. After
l
mL in EtOAc) was added as the internal standard for the determi-
nation of conversion. Samples were centrifuged again and sub-
jected to GC analysis on an achiral CP1301 or DB1701 column.
Positive hits were derivatised overnight using acetic anhydride
(60 lL) and catalytic 4-(dimethylamino)-pyridine (DMAP). After
quenching with water, the organic layer was treated as described
above without addition of internal standard. Derivatised samples
were measured on a chiral DEX-CB column, E-values were calcu-
lated from eeP and conversion.33
3. Conclusion
4.3. General procedure for selectivity enhancement of P.
denitrificans DSM 6392 and Synechococcus sp. PCC 7942
In conclusion, enantiocomplementary inverting sec-alkylsulfa-
tase activity has been detected for the first time in cyano- and
thio-bacteria Synechococcus and Paracoccus spp., which were pres-
elected for potential sulfatase activities due to their special sulfur-
metabolism. Low initial enantioselectivities (E-values up to 4) could
be improved by the addition of water-miscible organic cosolvents
(such as t-BuOH) or by using a biphasic medium containing t-BuOMe
(E >200). Isolation, characterisation and cloning of inverting sec-
alkylsulfatases are currently being undertaken to provide sufficient
amounts of proteins for the preparative-scale deracemisation sec-
alcohols via enantioconvergent chemoenzymatic hydrolysis of their
corresponding sulfate esters, either by employing a single inverting
sulfatase in combination with retaining chemical hydrolysis, or by
using a matching pair of an inverting and retaining enzyme.35
Lyophilised whole cells (50 mg) were rehydrated in 700 lL of
Tris–HCl buffer (100 mM, pH 7.5) for 1 h at 30 °C and shaken at
120 rpm. Water-miscible (10% v/v) or -immiscible organic cosol-
vents (50%) or FeCl3 (5 mM) or hexadecyltrimethyl-ammoniumbr-
omide (5 mM) was added. Then 200 lL of substrate from a stock
solution [30 mg/mL, rac-sulcatylsulfate 6a in Tris-buffer
(100 mM, pH 7.5) was added and the reaction mixture was incu-
bated at 30 °C with shaking at 120 rpm for 24 h. Work-up was per-
formed by adding 600 lL of EtOAc (in the case of water-miscible
cosolvents, FeCl3 and hexadecyltrimethyl-ammoniumbromide) or
the corresponding water-immiscible solvent, respectively After
centrifugation, work-up and analysis were carried out as described
above.
4. Experimental
4.1. General
Acknowledgement
Financial support by the Austrian Science Foundation (FWF,
project no. P 18689) is gratefully acknowledged.
Substrates rac-1a–6a and non-racemic reference compounds
1b–6b were synthesised as previously described.36 The absolute

118
P. Gadler et al. / Tetrahedron: Asymmetry 20 (2009) 115–118
20. No selectivity-enhancing effects through medium-engineering were found
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