A new monooxygenase from: Herbaspirillum huttiense catalyzed highly enantioselective epoxidation of allylbenzenes and allylic alcohols

Article abstract of DOI:10.1039/d0cy00081g

Asymmetric epoxidation is a green route to enantiopure epoxides, but often suffers from low enantioselectivity toward unconjugated terminal alkenes. Mining of the NCBI non-redundant protein sequences with a reconstructed ancestral sequence based on six st

Full text of DOI:10.1039/d0cy00081g

View Article Online
View Journal
Catalysis
Science &
Technology
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: H. Lin, Y. Tang, S.
Dong, R. Lang and H. Chen, Catal. Sci. Technol., 2020, DOI: 10.1039/D0CY00081G.
Volume
Number
21 January 2017
Pages 313-534
7
2
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Catalysis
Science &
Technology
rsc.li/catalysis
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 2044-4761
PAPER
Chaoqiu Chen, Yong Qin et al.
Highly dispersed Pt nanoparticles supported on carbon
nanotubes produced by atomic layer deposition for hydrogen
generation from hydrolysis of ammonia borane
rsc.li/catalysis

Please do not adjust margins
Catalysis Science & Technology
Page 1 of 7
View Article Online
DOI: 10.1039/D0CY00081G
ARTICLE
A new monooxygenase from Herbaspirillum huttiense catalyzed
highly enantioselective epoxidation of allylbenzenes and allylic
alcohols
Received 00th January 20xx,
Accepted 00th January 20xx
Hui Lin, Yanhong Tang, Shuang Dong, Ruibo Lang and Hongge Chen *
DOI: 10.1039/x0xx00000x
Asymmetric epoxidation is a green route to enantiopure epoxides, but often suffers low enantioselectivity toward
unconjugated terminal alkenes. Mining of the NCBI non-redundant protein sequences with a reconstructed ancestral
sequence based on six styrene monooxygenases identified a monooxygenase (HhMo) from Herbaspirillum huttiense with
29.6-32.3% sequence identity to styrene monooxygenases, which was previously annotated as an alanine phosphoribitol
ligase. HhMo catalyzed the epoxidation of allylbenzenes with moderate to excellent enantioselectivity yielding the
corresponding epoxides in up to 99% ee. The HhMo-catalyzed epoxidation could also achieve the kinetic resolution of
racemic secondary allylic alcohols, yielding the corresponding epoxides with up to 50% yields, as well as excellent enantio-
and diastereo-selectivity (up to >99% ee and >99% de) within 20-60 min, making a greener strategy for the production of
valuable enantiopure glycidol derivatives in fine chemicals and pharmaceuticals.
allyl benzyl ethers, but most of the epoxides from the process
only with good enantioselectivity (70-90% ee) 10. Styrene
monooxygenase (SMO) is a two-component flavoprotein
composed of a FAD-dependent monooxygenase (StyA) and
NADH-dependent flavin oxidoreductase (StyB) that regenerates
the reduced FAD 11. SMOs are excellent biocatalysts for the
epoxidation of many aromatic conjugated and unconjugated
alkenes 4, 12, 13. SMO from Rhodococcus sp. ST-5 and ST-10
catalyzed the epoxidation of allylbenzene yielding the
Introduction
Chiral epoxides are versatile intermediates for the synthesis of
biologically active molecules and natural products 1-3. The
asymmetric epoxidation presents one of the most powerful
strategies for the synthesis of enantiopure epoxides, since it
supplies straightforward access to diverse chiral epoxides
4, 5
.
Much progress has been achieved in the field of asymmetric
epoxidation of alkenes, including many chemical and enzymatic
protocols that have been developed during the last few decades
corresponding
(S)-epoxide
with
only
moderate
enantioselectivities of 76% and 65% ee, respectively 14. SMO
from Rhodococcus opacus 1CP catalyzed the epoxidation of
allylbenzene giving enantiopure (S)-allylbenzene oxide (99%
ee), but producing only 54 mg/L epoxide 15. SMOs from marine
microbes Paraglaciecola agarilytica NO2 and Marinobacterium
litorale DSM 23545 were also reported to have the activity for
the asymmetric epoxidation of allylbenzene, while the
enantiomeric excess of the epoxide was only moderate (67% and
83% ee, respectively) 16. Previously, we used SMO from
Pseudomonas sp. LQ26 (StyAB2) to catalyze the epoxidation of
allylbenzene and produce the corresponding epoxide with only
36% ee 17. Moreover, a triple mutant of P450pyr monooxygenase
(P450pyr I83H/M305Q/A77S) could also catalyze the
epoxidation of allylbenzene but only yielded the (R)-epoxide
with 90% ee 18. Overall, six styrene monooxygenases have been
proved to have the catalytic activity on the epoxidation of
unconjugated terminal alkenes. However, they showed either
low enantioselectivity (36-83% ee) or poor activity.
2, 4
.
Although the chemo-catalyzed asymmetric epoxidations have
been extensively developed, the asymmetric epoxidation of
unconjugated terminal olefins, such as allylbenzene, usually
4
suffers insufficient enantioselectivities
. The Sharpless
epoxidation is the most efficient chemo-process for the
epoxidation of -hydroxyl substituted terminal olefins, which
offered the chiral epoxides with a high enantiomeric excess (ee)
of 90-98% 6, 7. However, the Sharpless epoxidation of secondary
allylic alcohols with phenyl substituents suffers low activity and
poor enantioselectivity. For example, the kinetic resolution of 1-
phenylallyl alcohol only yielded the corresponding oxide with
90% and 46% yield at -20 ºC after several days 8, 9
.
Several monooxygenases have been used to develop eco-
friendly enzymatic epoxidations of unconjugated terminal
olefins, while they suffer either low activity or poor
enantioselectivity. Pseudomonas oleovorans monooxygenase
catalyzed the asymmetric epoxidation of allyl phenyl ethers and
Typically, one of the known enzymes’ sequence was used as
the reference to mine potential enzymes from a database. In some
cases, some of the known enzymes may have high activity, some
of them may have high stability, and the others may have high
enantioselectivity. So it is difficult to identify new excellent
College of Life Sciences, Henan Agricultural University, No. 63 Nongye Road,
450002, Henan, People's Republic of China. Email: honggeyz@henau.edu.cn
Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x
Please do not adjust margins

Please do not adjust margins
Catalysis Science & Technology
Page 2 of 7
ARTICLE
Journal Name
enzymes from a database by using only one of the known containing motif 2 and with 31% identity with AnStyA (Table
View Article Online
DOI: 10.1039/D0CY00081G
enzymes as the reference. Here, we reconstructed an ancestral S1, Fig. S4).
protein from the 6 known SMOs and used it as the reference to Enzymatic epoxidation screening
identify new enzymes for the epoxidation of unconjugated
terminal alkenes with high activity and excellent
enantioselectivity. We identified one monooxygenase from
Herbaspirillum huttiense, which was previously annotated as
To create a functional catalytic system, the codon-optimized
genes of A. sacchari monooxygenase (designated as AsMO) and
the putative alaninephosphoribitol ligase from H. huttiense
(designated as HhL) were synthesized and cloned into plasmid
alanine phosphoribitol ligase, and the enzyme-catalyzed the
pETB, which carried the NADH-dependent flavin
epoxidation of allylbenzene derivatives and secondary allylic
oxidoreductase (PsStyB, GenBank No. ADE62391.1) to reduce
alcohols with high activity and yielded the epoxides with up to
the enzyme-bound FAD to FADH2 17. The resultant plasmids
>99% ee and >99% de.
were used to express AsMO or HhL including a C-terminal 6x
His tag, and PsStyB.
The E. coli cells expressing the candidate enzyme and PsStyB
Results and discussion
were used as the catalyst for the epoxidation of allylbenzene, and
Ancestral protein guided enzyme mining
the results showed that both enzymes could catalyze the
epoxidation of allylbenzene 1a. AsMo catalyzed the epoxidation
of allylbenzene and yielded the corresponding epoxide with 48%
yield and 50% ee, and HhL catalyzed the epoxidation of
allybenzene and yielded the corresponding epoxide with 30%
yield and >99% ee. It showed that AsMo containing motif 1 and
motif 2 had a higher catalytic epoxidation activity but moderate
enantioselectivity, while the HhL only containing motif 2 gave
Since no suitable protein reference sequence was available for
mining new enzymes, we reconstructed an ancestral protein from
the 6 styrene monooxygenases and used it as the reference. The
FASTML Server was used to reconstruct the ancestral protein
(designated as AnStyA, Fig. S1) of the
monooxygenases based on the maximum likelihood 19. The
6
styrene
AnStyA has 62-85% identities with the 6 SMOs.
excellent
enantioselectivity.
Here,
the
putative
To analyze the structural motifs in styrene monooxygenase,
MEME was used to discover the motifs in the 6 known styrene
monooxygenases 20. Three main motifs were identified with
motif 1 including site 1-69, motif 2 including site 92-180, and
motif 3 including site 193-405 (Fig. S2). Based on the structure
of styrene monooxygenase 21, it showed that motif 1 contained 2
amino acid residues in the FAD-binding site, motif 3 contained
3 residues in the FAD-binding site and the most of residues in
the substrate-binding site, while motif 2 did not contain any
residues either in the substrate-binding site or in the FAD-
binding site.
alaninephosphoribitol ligase from Herbaspirillum huttiense
(HhL) was renamed as monooxygenase (designated as HhMo)
because its functions as a monooxygenase.
HhMO was further tested for the epoxidation of a secondary
allylic alcohol, 1-phenylallyl alcohol, resulting in 100%
conversion of (S)-1-phenylallyl alcohol and yielding the epoxide
with >99% ee and >99% de. HhMo-catalyzed epoxidation had a
very high catalytic activity, excellent enantio- and diastereo-
selectivity on the secondary allylic alcohol compared to those of
9, 22
Sharpless epoxidation and StyAB2-catalyzed epoxidation
.
HhMo was identified as an excellent enzyme for the epoxidation
The AnStyA-catalyzed epoxidation of allylbenzene was first
investigated and it yielded the epoxide with only 44% ee (data
not shown). As we expected, AnStyA catalyzed the epoxidation
of unconjugated terminal alkene producing the epoxide in poor
enantioselectivity because of the high identity of AnStyA to
known SMOs (62-85%). We hypothesized that distantly related
monooxygenases might show higher enantioselectivity and high
catalytic activity. Here, AnStyA was submitted to a BLASTp
search against the entire NCBI nr protein database, returning the
500 sequences with E value < 4e-53. The returned sequences had
31-85% identities with AnStyA. Since the 6 known styrene
monooxygenases, having 62-85% identities with AnStyA, have
either low enantioselectivity or poor activity. Those sequences
having 30-50% identities with the AnStyA were considered as
candidates and were subjected to structural motifs analysis to
match the pattern in styrene monooxygenases. Finally, two
enzymes were selected to be studied. One was a putative
monooxygenase from Amycolatopsis sacchari (GenBank No.
WP_091504755) containing motif 1 and motif 2 and with 50%
identity with AnStyA (Table S1, Fig. S3), and the other one was
a flavin-containing putative alaninephosphoribitol ligase from
Herbaspirillum huttiense (GenBank No. WP_039783212) only
of unconjugated terminal alkenes, and it was used for further
analysis.
Optimization of the reaction conditions and enzyme kinetic
analysis
Here, the effects of temperature, co-solvent, and pH on the
HhMo-catalyzed epoxidation were carefully conducted. HhMo
exhibited high catalytic activities at the temperature between 35-
39 ºC and gave the highest yield of epoxide at 37 ºC (Fig. 1A).
The bi-phase system has been proved as an efficient condition
for the enzymatic epoxidation 23. When octane, cyclohexane,
toluene, or bis(2-ethylhexyl) phthalate (BEHP) was used as the
co-solvent, the results showed the system containing 10% octane
achieved the highest yield (Fig. 1B). BEHP, being the most
efficient co-solvent in bioepoxidation of styrene catalyzed with
other styrene monooxygenases 17, 23, resulted in a much lower
yield (Fig. 1B). As for the optimal pH, the HhMo-catalyzed
epoxidation of allylbenzene achieved the highest yield at pH 6.0
(Fig. 1C).
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 7
Catalysis Science & Technology
Please do not adjust margins
Journal Name
ARTICLE
Table 1. HhMo catalyzed epoxidation of unconjugated terminal
View Article Online
alkenes. a
DOI: 10.1039/D0CY00081G
Entry Substrate
Product yield (%) ee (%)
Fig. 1. The effects of temperature, co-solvent, and pH on the
HhMo-catalyzed epoxidation. Whole E. coli cells expressing
HhMo and PsStyB were used to convert allylbenzene, and the
yields of the product were measured by GC. Standard deviations
of triplicates are represented by error bars.
1
2
3
4
5
6
7
8
R = C6H5-, 1a
1b
2b
3b
4b
5b
6b
100
68
92
16
67
27
33
42
13
11
99
91
87
71
96
95
35
70
81
46
R = m-F-C6H5-, 2a
R = p-F-C6H5-, 3a
R = o-CH3-C6H5-, 4a
R = m-CH3-C6H5-, 5a
R = p-CH3-C6H5-, 6a
R = o-CH3O-C6H5-, 7a 7b
R = p-CH3O-C6H5-, 8a 8b
The HhMo protein was purified by Ni-resin, and the purity of
the sample was confirmed by SDS-PAGE (Fig. S5). The steady-
state kinetics of the epoxidation of 1-phenylallyl alcohol at 37 ºC
and pH 6.0 were measured (Fig. S6). The reactions were carried
out in a reconstituted system containing purified HhMo, PsStyB,
catalase, and formate dehydrogenase 18. Kinetic parameters of
HhMo were investigated at varying concentrations of 1-
phenylallyl alcohol. The Km value determined by non-linear
regression was 2.273 ± 0.679 M, which was lower than that of
SMOs catalyzing the epoxidation of styrene 11, 17. The turnover
number kcat of the enzymatic reaction was 0.0182 ± 0.006 min-1,
which was significantly lower than that of SMOA from
Pseudomonas sp. VLB120 and StyA from Pseudomonas sp.
LQ26 catalyzing the epoxidation of styrene 11, 17. In the activity
assay system, the purified HhMo aggregated in 2-3 min, which
might result in the lower kcat of the purified HhMo.
9
10
R = C6H5CH2-, 9a
R = C6H5-O-, 10a
9b
10b
b
11
-
, 11a
, 12a
12
-
a Reaction performed in 20 mL potassium phosphate buffer (pH 6.0, 100
mM), 8 mg alkene, 2.0 g wet cells, and n-octane (5% (v/v) at 37 °C, 220
rpm, 24 h. The ee of the epoxide was determined via chiral HPLC. b no
epoxide was detected.
Moreover, 4-phenyl-1-butene (9a) and allyl phenyl ether
(10a) also could be epoxidized by HhMo yielding the
corresponding epoxide with 81% ee (9b) and 46% ee (10b),
respectively (Table 1, Entries 9-10). However, the
enantioselectivity of the HhMo-catalyzed epoxidation of allyl
phenyl ether was lower than that of P. oleovorans
monooxygenase-catalyzed epoxidation 9. HhMo was further
used in the epoxidation of two alkenes (11a and 12a) with an
electron-withdrawing group. However, no epoxide products
were detected. The result showed that the electron-deficient
alkenes appeared unfavorable for the HhMo-catalyzed
epoxidation, which was consistent with those of other SMOs 24,
HhMo-catalyzed asymmetric epoxidation of allylbenzene
derivatives
Encouraged by the excellent enantioselectivity epoxidation of
allylbenzene 1a, more non-conjugated terminal alkenes were
investigated for the HhMo-catalyzed epoxidation. All the tested
allylbenzene derivatives could be epoxidized, while the yield and
enantioselectivity of epoxides varied greatly with the
substituents (Table 1). The HhMo catalyzed the epoxidation of
allylbenzene derivatives (2a-8a) producing the corresponding
epoxides with 16-92% yields, which were lower than that of
allylbenzene. HhMo may have a relatively small substrate
binding site, which led the enzyme to decrease the catalytic
activity to the substituted allylbenzenes. The epoxidation of
allylbenzene derivatives by HhMo yielded the epoxides in
moderate to excellent enantioselectivity. The epoxidation of the
substrates with fluoro- or methyl- on the phenyl moiety (2a-6a)
yielded the epoxides in good to excellent enantioselectivities
(71-95% ee), while the substrates harboring methyloxyl
substituent on the phenyl moiety (7a and 8a) decreased the
enantioselectivity of the epoxidation and produced the epoxides
only in 35% and 70% ee. The HhMo-catalyzed epoxidation of
the substrates with meta-substituent on the phenyl group (2a and
5a) had higher enantioselectivity and activity than those of the
substrates with ortho- or para-substituent on the phenyl group
(3a, 4a, and 6a-8a). HhMo had varied enantioselectivities of
substituent allylbenzenes, which was different from that of the
SMOs catalyzing the epoxidation of styrene derivatives.
Normally, SMOs always achieve excellent enantioselectivities
25
.
HhMo-catalyzed asymmetric epoxidation of secondary allylic
alcohols
First, the kinetic resolution of the racemic 1-phenylallyl alcohol
13a by HhMo was investigated (Fig. 2). About 50% (S)-13a
(25% conversion of total rac-13a) was converted into the (1R,
2R)-phenyl glycidol 13b after 6 min, and then about 80% (S)-
13a (40% conversion of total rac-13a) was converted after 10
min. The (S)-13a was totally epoxidized into 13b with excellent
enantioselectivity (ee >99%) and excellent diastereoselectivity
(de >99%) in 20 min, leaving the (R)-13a unreacted. The (R)-
13a could not be converted into epoxide by extending the
reaction time, which showed the enzyme had a specific activity
on the epoxidation of (S)-alcohol. Moreover, the (R)-13a was
used as the substrate for the freshly prepared resting E. coli cells
expressing HhMo, which resulted in no epoxide was detected by
HPLC. All the results indicated that HhMo had very high
stereospecificity of (S)-13a (E >200).
(99% ee) on different styrene derivatives 4, 12
.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Catalysis Science & Technology
Page 4 of 7
ARTICLE
Journal Name
by HhMo were higher than that by StyAB2 22, wVhiiecwhArrtieclqeuOinrleinde
180 min to finish the reaction, and yieldeDdOtIh: e10e.1p0o3x9/iDd0eCwY0it0h08916G-
98% de. The results showed that HhMo might be a strong
candidate for the preparation of enantiopure glycidol derivatives
More secondary allylic alcohols were investigated as the
substrate for the epoxidation by HhMo (Table 2). In most cases,
HhMo epoxidized (S)-allylic alcohols and yielded the
corresponding (R)-glycidols in excellent enantio- and diastereo-
selectivity (up to >99% ee, >99% de), but varied yield.
Substrates 14a and 15a are derived from 13a with fluoro-
substituent on the phenyl moiety. For both the mate-fluoro- and
para-fluoro-substituted
substrates,
HhMO-catalyzed
Fig. 2. Kinetic resolution of rac-1-phenylallyl alcohol by HhMo.
The conversion (cross, ✕), ee (filled circle, ●), and de (blank
triangle, △) were determined by chiral HPLC. Standard
deviations of triplicates are represented by error bars.
HhMo-catalyzed epoxidation gave the kinetic resolution of
rac-13a, resulting in much higher activity and
diastereoselectivity than those of chemo-catalytical epoxidation
8. For example, the famous Sharpless epoxidation and the
vanadium-based epoxidation needed up to 12 days for 50%
conversion but yielded the epoxide with lower ee (90-93% ee) 6,
9. The favored enantiomer of the epoxidation by HhMo is the
same as that of StyAB2 from Pseudomonas sp. LQ26, while the
activity and diastereoselectivity of the kinetic resolution of 13a
epoxidation had very high activity and excellent
enantioselectivity, producing the (R, R)-glycidol in 35 min and
30 min, respectively. For the substrate 16a and 17a containing
naphthyl group, HhMo had excellent enantio- and diastereo-
selectivity of the epoxidation of both 1-naphthyl and 2-naphthyl
substituted substrates, giving the corresponding epoxide with
>99% ee and >99% de. However, HhMo exhibited lower activity
in both 16a and 17a than that of 13a-15a, which showed the
naphthyl-substituent substrates might not easy to access the
substrate-binding site. Again, the results showed that HhMo
might have a relatively small substrate binding site, which led
the enzyme to have lower activity toward the larger substrates.
Table 2. HhMo-catalyzed epoxidation of racemic secondary allylic alcohols 13a–21a.a
epoxide 13b-21b
Entry
R =
t/min
drb
eec (%)
dec (%)
>99
96
>99
>99
>99
>99
>99
31
yield (%)
E d
>200
197
>200
>200
>200
>200
>200
2
1
2
3
4
5
6
7
8
9e
Ph, 13a
20
35
30
60
60
20
20
60
60
>99 :1
>99 :1
>99 :1
>99 :1
>99 :1
>99 :1
>99 :1
65:35
>99 :1
>99
>99
>99
>99
>99
>99
>99
>99
>99
50
50
50
8
m-FC6H4, 14a
p-FC6H4, 15a
1-Naphthyl, 16a
2-Naphthyl, 17a
2-Thienyl, 18a
3-Thienyl, 19a
Benzyl, 20a
5
50
50
12
50
Cyclohexyl, 21a
>99
>200
a Reaction performed in 20 mL phosphate buffer (pH 6.0, 100 mM), 10 mg alkene, 2.0 g wet cells, and n-octane (5% (v/v) at 37 °C, 220 rpm. The
absolute configuration of 13b was established by comparing the 1H-NMR spectrum and [ ]25 with that in the literature, and the others based on
the analogous chromatographic behavior of racemic mixtures. b Diasteroisomeric ratios (dr) determined via 1H NMR. c Determined via chiral HPLC
analysis. d Enantiomeric ratio E = ln[1-Yield(1 + de)]/ln[1-Yield(1 - de)]. e Determined by chiral GC.
The substrate range was extended to substrate 18a-20a with 2- of aliphatic substrate 21a yielded the corresponding epoxide 21b
thienyl, benzyl, and cyclohexyl group, which have been proved with >99% ee and >99% de. This is the first example of
to be the poor substrate for the StyAB2-catalyzed epoxidation 22
.
monooxygenase-catalyzed epoxidation of the aliphatic substrate
StyAB2-catalyzed epoxidation of 18a and 19a yielded the with excellent enantio- and diastereo-selectivity.
epoxide only with 31% de and 86% de, respectively. However,
HhMo catalyzed the epoxidation of 18a and 19a with very high
activity, excellent enantio- and diastereo-selectivity, converting
Conclusions
all (S)-18a and (S)-19a to the corresponding epoxide with 99%
ee and 99% de in 20 min. But the epoxidation of benzyl-
substituted substrate 20a by HhMo resulted in low yield and fair
diastereoselectivity (31% de) of the epoxide 18b, with the E
value of 2, which was similar to the StyAB2-catalyzed
epoxidation of 20a. Moreover, the HhMo-catalyzed epoxidation
In summary, we reconstructed an ancestral protein from all the 6
known styrene monooxygenases able to catalyze the
allylbenzene epoxidation and then used it as the reference for
mining new enzymes from the database. We successfully
identified a novel monooxygenase from H. huttiense (HhMo),
which was previously annotated as an alanine phosphoribitol
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 7
Catalysis Science & Technology
Please do not adjust margins
Journal Name
ARTICLE
ligase, for asymmetric epoxidation of allylbenzene derivatives and a flavin-containing putative alaninephosphoVriiebwitAortlicleliOgnalisnee
DOI: 10.1039/D0CY00081G
with excellent enantioselectivity (up to 99% ee) and secondary from Herbaspirillum huttiense (GenBank No. WP_039783212)
allylic alcohols with excellent enantio-and diastereo-selectivity only containing motif 2 and with 31% identity with AnStyA were
(99% ee and 99% de). HhMo-catalyzed epoxidation could give selected (Table S1, Fig. S4).
the kinetic resolution of secondary allylic alcohols, which Plasmids construction
only took 20-60 minutes to finish the reaction, yielding the
glycidol derivatives with contiguous stereogenic centers in
named as AsMo and WP_039783212.1, named as HhL) were
excellent enantioselectivity. The highly efficient, enantio- and
diastereo-selective HhMo-catalyzed epoxidation system
provides a green alternative to classic chemo-catalyzed synthesis
amplified by using the primers (HhMo-F and HhMo-R, AsMo-F
and has been shown its great potential in chiral synthesis.
The gene sequences of those two proteins (WP_091504755.1,
codon-optimized for the expression in E. coli, and synthesized
by Genewiz (Shenggong, Shanghai, China). These genes were
and AsMo-R) in Table S2, and the pET24a (Invitrogen, USA)
was amplified by the primers (24a-F and 24a-R) in Table S2.
The genes were inserted in the pET24a plasmid using the SLIC
Experimental
method 27. The constructed plasmids (pET24a-HhL and pET24a-
AsMO) were transferred into the E. coli DH5a competent cells.
Plasmids were extracted by Miniprep kit (Vezyme, Suzhou,
China), and the sequence was confirmed by sequencing
(Shenggong, Shanghai, China). To insert the StyB from
Pseudomonas sp. LQ26 into pET24a-AsMO and pET24a-HhL,
the StyB sequence was amplified from pETB using primers
pETB-F and pETB-R, and the pET24a-AsMO and pET24a-
HhMo plasmid were amplified with primers 24AsMo-F and
24AsMo-R, and 24HhMo-F and 24HhMo-R, respectively. The
resultant plasmid pET24a-HhL-StyB or pET24a-HhL-StyB was
constructed using the SLIC method, and the sequence was
confirmed by sequencing (Shenggong, Shanghai, China).
Protein expression and purification
Chemicals and general methods
Commercially available reagents were used without further
purification. Allylbenzene derivatives were purchased from
Sigma-Aldrich (St Louis, MO, USA) or Aladdin (Shanghai,
China). 1H NMR spectra were recorded on a Brucker-400
spectrometer in CDCl3, all signals were expressed as ppm down
field from tetramethylsilane. Optical rotations were measured
with a Perkin Elmer 341 polarimeter. Protein concentrations
were measured using the Bradford assay (Coomassie Brilliant
Blue G-250) and bovine serum albumin as the protein standard.
Protein purity was analyzed using SDS-PAGE using 4-12%
polyacrylamide gradient gel (SurePAGE, Genscript, Nanjing,
China) running in 50 mM MOPS, 50 mM Tris Base, 0.1% SDS,
1 mM EDTA, pH 7.7 and stained with Coomassie Blue.
Reconstruction of ancestral protein and searching motifs in
styrene monooxygenases
The constructed plasmids (pET24a-HhMo, pET24a-AsMO-
StyB, pET24a-HhL-StyB) were transferred into E. coli BL21
(DE3) competent cells. LB medium (10 mL) containing
kanamycin (50 ug/mL) in 100 mL flask was inoculated with a
single colony of E. coli BL21 (DE3) (pET24a-HhMo), E. coli
BL21 (DE3) (pET24a-AsMO-StyB), or E. coli BL21 (DE3)
(pET24a-HhL-StyB), cultured at 37 ºC and 220 rpm for
overnight. The overnight cultures were used as seeds to inoculate
LB medium (150 mL) containing kanamycin (50 ug/mL) in 500
Styrene monooxygenase from Pseudomonas sp. LQ26
(PsLQ26StyA, GenBank No. ADE62390), R. opacus 1CP
(RoStyA, GenBank No. AII82583), Rhodococcus sp. ST-5
(RsST5StyA, GenBank No. BAL04132), Rhodococcus sp. ST-
10 (RsST10StyA, GenBank No. BAL04129), P. agarilytica
(PaStyA, GenBank No. WP_008305084.1), and M. litorale
(MlStyA, GenBank No. WP_027855270.1) were downloaded
from Genbank. To reconstruct the ancestral protein of the 6
known styrene monooxygenases, MAFFT online server
(https://mafft.cbrc.jp/alignment/server/index.html) was used to
align the sequences and create the phylogenetic tree 26, and then
the ancestral protein (AnStyA, Fig. S1) was reconstructed in the
FASTML Server (http://fastml.tau.ac.il) based on the maximum
likelihood 19. To identify the motif of the six known styrene
monooxygenases, a structural motif search was conducted using
mL flasks, cultured at 37 ºC and 220 rpm for about 3 h (OD600
=
1.0). IPTG was added into the culture to the final concentration
of 0.05 mM, the cultures were incubated at 16 ºC and 220 rpm
for 24 h. Cells were harvested by centrifugation (5 min, at 4700
g) at 4 ºC, the cells were washed by phosphate buffer (0.1 M, pH
7.02) twice.
The collected cells (2.5 g) were resuspended in phosphate
buffer (10 mL, 0.1 M, pH 7.02) containing 20% glycerol,
potassium chloride (0.5 M), DTT (0.1 mM), 1mM phenylmethyl
sulfonylfluoride (PMSF), and imidazole (5 mM), and then the
cells were lysed by low-temperature ultra high-pressure
homogenizer JN-mini (JNBio, China) at 1000 bar and 4 ºC for 3
times. The lysis was centrifuged at 8000 rpm and 4 ºC for 30 min
to remove the cell debris. The supernatant was collected, and the
protein was purified by Ni2+-NTA agarose resin (Qiagen,
Germany), and the purity of the sample was checked by SDS-
PAGE (SurePAGE, Genscript, Nanjing, China) (Fig. S5),
pooled, concentrated and removed the salts by Amicon® Ultra-
15 Centrifugal Filter Unit (10 K, Merck Millipore) and used
freshly.
MEME (http://meme-suite.org/tools/meme) (Fig. S2) 20
Mining enzymes from the database
.
The reconstructed ancestral protein AnStyA (SI) was submitted
to a BLASTp search against the entire NCBI nr protein database.
The returned 500 sequences had 31-85% identities with AnStyA.
The proteins had 30-50% identities with AnStyA were subjected
to structural motifs analysis with the 6 known SMOs using
MEME. A putative monooxygenase from Amycolatopsis
sacchari (GenBank No. WP_091504755) containing motif 1 and
motif 2 and with 50% identity with AnStyA (Table S1, Fig. S3),
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Catalysis Science & Technology
Page 6 of 7
ARTICLE
Journal Name
Kinetic parameters assay
View Article Online
1. N. Wan, J. Tian, X. Zhou, H. Wang, B. Cui, W. Han and Y. Chen,
DOI: 10.1039/D0CY00081G
The kinetic parameters kcat and Km of HhMo were determined by
measuring the production of (S)-epoxide 13b by HPLC. The
reaction mixture containing 3 M of purified HhMo, 3 M of
Adv. Synth. Catal., 2019, 361, 4651-4655.
2. A. Fingerhut, O. V. Serdyuk and S. B. Tsogoeva, Green Chem.,
2015, 17, 2042-2058.
purified PsStyB, 0.5 U formate dehydrogenase (Sigma-Aldrich,
USA), 650 U catalase (Sigma-Aldrich, USA), 150 mM sodium
formate (Sigma-Aldrich, USA), 50 mM NADH (Sigma-Aldrich,
3. H.-B. Cui, L.-Z. Xie, N.-W. Wan, Q. He, Z. Li and Y.-Z. Chen, Green
Chem., 2019, 21, 4324-4328.
4. H. Lin, J.-Y. Liu, H.-B. Wang, A. A. Q. Ahmed and Z.-L. Wu, J. Mol.
Catal. B Enzym., 2011, 72, 77-89.
5. S. Hwang, C. Y. Choi and E. Y. Lee, J. Ind. Eng. Chem., 2010, 16,
1-6.
6. T. Katsuki and K. B. Sharpless, J. Am. Chem. Soc., 1980, 102,
5974-5976.
7. V. S. Martin, S. S. Woodard, T. Katsuki, Y. Yamada, M. Ikeda and
K. B. Sharpless, J. Am. Chem. Soc., 1981, 103, 6237-6240.
8. M. M. Heravi, T. B. Lashaki and N. Poorahmad, Tetrahedron
Asymmetry, 2015, 26, 405-495.
9. M. E. Jung and Y. H. Jung, Tetrahedron Lett., 1989, 30, 6637-
6640.
USA), 75
M FAD (Sigma-Aldrich, USA), 30% glycerol,
bovine serum albumin (BSA, 10 mg/mL) (NEB, USA) and
varying concentrations of 1-phenylallyl alcohol (from 100 uM in
DMSO), in 100 mM potassium phosphate buffer (5 mL, pH 6.0).
The mixture was incubated at 37 ºC and 220 rpm for 15 min. The
mixture was extracted by ether, and then the organic phase was
analyzed by Thermo Scientific TRACE 1300 gas chromatograph
equipped with TraceGOLD TG-5MS column (Thermo
Scientific, USA). The parameters were calculated by the Prism
program (Graphpad, San Diego, CA, USA) (Fig. S6).
10. H. Fu, M. Newcomb and C. H. Wong, J. Am. Chem. Soc., 1991,
113, 5878-5880.
11. K. Otto, K. Hofstetter, M. Röthlisberger, B. Witholt and A.
Schmid, J. Bacteriol., 2004, 186, 5292-5302.
12. T. Heine, A. Scholtissek, S. Hofmann, R. Koch and D. Tischler,
ChemCatChem, 2020, 12, 199-209.
13. S. Wu, Y. Zhou and Z. Li, Chem. Commun., 2019, 55, 883-896.
14. H. Toda, R. Imae, T. Komio and N. Itoh, Appl. Microbiol.
Biotechnol., 2012, 96, 407-418.
15. C. E. Paul, D. Tischler, A. Riedel, T. Heine, N. Itoh and F.
Hollmann, ACS Catal., 2015, 5, 2961-2965.
16. C. Cui, C. Guo, H. Lin, Z.-Y. Ding, Y. Liu and Z.-L. Wu, Enzyme
Microb. Technol., 2020, 132, 109391.
17. H. Lin, J. Qiao, Y. Liu and Z.-L. Wu, J. Mol. Catal. B Enzym., 2010,
67, 236-241.
18. A. Li, J. Liu, S. Q. Pham and Z. Li, Chem. Commun., 2013, 49,
11572-11574.
Preparation of enantiopure epoxides by asymmetric epoxidations
with recombinant E. coli and product assay
The harvested E. coli whole cells (2.0 g) were resuspended in
potassium phosphate buffer (20 mL, 0.1 M, pH 6.0), and were
transferred into a flask (100 mL), and then n-octane (5% (v/v) )
and alkene (10 mg) were added in the mixture. The reaction was
carried out at 37 ºC for 20 min with gyratory shaking at 220 rpm.
The mixture was extracted by ether (20 mL x 3). The organic
phase was combined, dried by anhydrous Na2SO4, and then the
solvents were removed under a certain vacuum. The crude
products were analyzed by HPLC or GC for analyzing the
conversion, or were further purified by silica gel
chromatography. The conversions of the reactions were
performed on a Thermo ScientificUltiMate3000 UHPLC system
equipped with diode array detector (DAD) and Kinetic C18
column (Phenomenex, USA). The Optical purities were
determined by chiral HPLC using CHIRALPAK AS-H column
(Daicel, Japan), or chiral GC using CHIRASIL-DEX CB column
(Agilent Technologies, USA).
19. H. Ashkenazy, O. Penn, A. Doron-Faigenboim, O. Cohen, G.
Cannarozzi, O. Zomer and T. Pupko, Nucleic Acids Res., 2012, 40,
W580-W584.
20. T. L. Bailey, N. Williams, C. Misleh and W. W. Li, Nucleic Acids
Res., 2006, 34, W369-W373.
21. U. E. Ukaegbu, A. Kantz, M. Beaton, G. T. Gassner and A. C.
Rosenzweig, Biochem., 2010, 49, 1678-1688.
22. H. Lin, Y. Liu and Z.-L. Wu, Chem. Commun., 2011, 47, 2610-
2612.
Conflicts of interest
There are no conflicts to declare.
23. S. Panke, M. Held, M. G. Wubbolts, B. Witholt and A. Schmid,
Biotechnol. Bioeng., 2002, 80, 33-41.
24. Y.-C. Liu and Z.-L. Wu, Chem. Commun., 2016, 52, 1158-1161.
25. S. Bernasconi, F. Orsini, G. Sello, A. Colmegna, E. Galli and G.
Bestetti, Tetrahedron Lett., 2000, 41, 9157-9161.
26. K. Katoh, J. Rozewicki and K. D. Yamada, Brief. Bioinform., 2019,
20, 1160-1166.
Acknowledgements
We are grateful to the National Natural Science Foundation of
China (31571775), Key Laboratory of Environmental and
Applied Microbiology of the Chinese Academy of Sciences
(KLCAS-2018-1), and Major Science and Technology Projects
in Henan Province (192102110163) for financial support, Prof.
Zhong-Liu Wu (Chengdu Institute of Biology, CAS) for
providing the plasmid pETAB2, pETB and helpful discussions,
and Prof. Romas Kazlauskas (UMN) for comments during the
writing of the manuscript.
27. M. Z. Li and S. J. Elledge, Methods Mol. Biol., 2012, 852, 51-59.
Notes and references
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 7
Catalysis Science & Technology
View Article Online
DOI: 10.1039/D0CY00081G
A novel monooxygenase (HhMo) from Herbaspirillum huttiense catalyzed the epoxidation of
allylbenzenes and secondary allylic alcohols yielding the epoxides with excellent enantio- and
diastereo-selectivity (up to >99% ee and >99% de). HhMo also catalyzed the epoxidation of
aliphatic alkene yielding the epoxide with >99% ee and >99% de.
6 styrene monoxygenases
Genbank
Database
Ancestral protein
as the reference
up to >99% ee
R
R
R
O
Herbaspirillum huttiense Monooxygenase
OH
OH
up to >99% ee, >99% de
R
O