Process Optimisation Studies and Aminonitrile Substrate Evaluation of Rhodococcus erythropolis SET1, A Nitrile Hydrolyzing Bacterium

Article abstract of DOI:10.1002/open.202000088

A comprehensive series of optimization studies including pH, solvent and temperature were completed on the nitrile hydrolyzing Rhodococcus erythropolis bacterium SET1 with the substrate 3-hydroxybutyronitrile. These identified temperature of 25 °C and pH of 7 as the best conditions to retain enantioselectivity and activity. The effect of the addition of organic solvents to the biotransformation mixture was also determined. The results of the study suggested that SET1 is suitable for use in selected organo-aqueous media at specific ratios only. The functional group tolerance of the isolate with unprotected and protected β-aminonitriles, structural analogues of β-hydroxynitriles was also investigated with disappointingly poor isolated yields and selectivity obtained. The isolate was further evaluated with the α- aminonitrile phenylglycinonitrile generating acid in excellent yield and ee (>99 % (S) – isomer and 50 % yield). A series of pH studies with this substrate indicated pH 7 to be the optimum pH to avoid product and substrate degradation.

Full text of DOI:10.1002/open.202000088

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Process Optimisation Studies and Aminonitrile Substrate
Evaluation of Rhodococcus erythropolis SET1, A Nitrile
Hydrolyzing Bacterium
Tatenda M. Mareya+, Tracey M. Coady+, Catherine O’Reilly#, Michael Kinsella#, Lee Coffey#,
and Claire M. Lennon*[a]
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A comprehensive series of optimization studies including pH,
solvent and temperature were completed on the nitrile hydro-
lyzing Rhodococcus erythropolis bacterium SET1 with the
substrate 3-hydroxybutyronitrile. These identified temperature
ratios only. The functional group tolerance of the isolate with
unprotected and protected β-aminonitriles, structural analogues
of β-hydroxynitriles was also investigated with disappointingly
poor isolated yields and selectivity obtained. The isolate was
further evaluated with the α- aminonitrile phenylglycinonitrile
generating acid in excellent yield and ee (>99% (S) – isomer
and 50% yield). A series of pH studies with this substrate
indicated pH 7 to be the optimum pH to avoid product and
substrate degradation.
°
of 25 C and pH of 7 as the best conditions to retain
enantioselectivity and activity. The effect of the addition of
organic solvents to the biotransformation mixture was also
determined. The results of the study suggested that SET1 is
suitable for use in selected organo-aqueous media at specific
1. Introduction
enzymatic cascade (in the case of nitrile hydratase/amidase)
using milder, more efficient processes.[2] Various strains of
Rhodococci contain nitrilase, nitrile hydratase and amidase
enzymes and have been applied as efficient biocatalysts in
several key industrial processes.[3]
Because of the inherent enantioselectivity of enzymes and the
relatively benign reaction conditions used, biotransformations
are under active research by many academic researchers and
major pharmaceutical multinationals as routes to novel chiral
drug molecules.[1] An additional advantage of biocatalytic
synthesis is that it offers sustainable green technology with
reduced requirement for solvents and other reagents, helping
to decrease the environmental impact of the processes when
compared to traditional chemical syntheses. Nitriles as sub-
strates in biocatalytic processes can offer routes to synthetically
important acids and amides via hydrolysis reactions. Traditional
chemical methods of nitrile hydrolysis often utilize strong acids
and bases and employ high temperatures, which can lead to
degradation of other labile groups that may be present. Nitrile
hydrolyzing enzymes such as nitrilase, nitrile hydratase and
amidase offer the potential to generate such enantiopure acids
and amides via single step (in the case of nitrilase) or multi step
Enantiopure β-amino acids are useful precursor building
blocks for synthetic peptides, peptidomimetics, agrochemicals
and pharmaceuticals.[1e,4] In contrast to α-amino acids, β-amino
acids are not typically available in nature, the exceptions being
β-alanine and β-aminoisobutyric acid. In addition, β-peptides
can exhibit higher in vivo stability compared to α-peptides and
when β-residues are present in mixed α/β-peptides they can
protect closely situated amides from proteolytic cleavage. This
offers promise for the development of novel peptide-based
drugs which are not rejected or degraded in the human body.[5]
The synthesis of enantiopure β-amino acids can be difficult
and currently several types of biocatalysts are employed, for
example, transaminase and reductase enzymes.[6] Nitrile metab-
olizing enzymes also offer the potential for the synthesis of
single enantiomer β-amino acids from β-amino nitriles and
hence this prompted the work undertaken by our group.[2l,7]
Such enzymes have also been used in tandem with other
enantioselective enzyme systems in a non-asymmetric hydrol-
ysis step.[8]
The ability to generate single enantiomer a-amino acids,
highly important building blocks in organic synthesis, medicinal
chemistry and pharmacology remains a challenge with methods
such as diastereomeric salt resolution, the use of chiral
auxiliaries and enzymatic synthesis as options.[9] Nitrile hydro-
lyzing enzymes can also be highly efficient in generating
enantiopure α-amino acids and their derivatives.[10] There are
limited examples of nitrilases being used in such routes
however,[11] although they offer great potential to generate
highly selective reaction products and the ability for possible
[a] T. M. Mareya,+ Dr. T. M. Coady,+ Dr. C. O’Reilly,# Dr. M. Kinsella,#
Dr. L. Coffey,# Dr. C. M. Lennon
Department of Science
Waterford Institute of Technology
Cork Road
Waterford X91K0EK (Ireland)
E-mail: clennon@wit.ie
[+] These authors contributed equally to this work.
[#] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.202000088
Biosynthesis
© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This
is an open access article under the terms of the Creative Commons Attri-
bution Non-Commercial NoDerivs License, which permits use and distribu-
tion in any medium, provided the original work is properly cited, the use is
non-commercial and no modifications or adaptations are made.
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°
additional in-situ racemization of the unwanted isomer. Both
Chaplin and Wei have reported high yield synthesis of D-
phenylglycine and its derivatives by nitrilase mediated dynamic
kinetic resolution.[11b] This prompted us to also evaluate this
substrate as a model for the synthesis of α-amino acids.
biotransformation at 30 C caused an approximate 63% loss of
the initial enantioselectivity with a further decrease to 84% at
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9
°
40 C as shown in Figure 1.
When compared to other nitrile metabolising strains, SET1
appears to follow the trend that biotransformations at higher
temperatures (30–40 C) can result in higher activity.[15]
°
A strategy for the rapid identification of isolates demon-
strating nitrile-hydrolysing activity was previously published by
our research group[7h] and a whole cell catalyst Rhodococcus
erythropolis SET1 was found to catalyse the hydrolysis of 3-
hydroxybutyronitrile with remarkably high enantioselectivity.
This was subsequently screened against 34 related nitriles.[12]
The aim of the work presented in this publication was to
optimise reaction conditions with 3-hydroxynitrile and inves-
tigate the solvent tolerance of the bacterial isolate with this
substrate. In addition, we wanted to assess the functional group
tolerance of the isolate with β-aminonitriles, which are struc-
tural analogues of β-hydroxynitriles and which can upon
reaction with the enzyme potentially generate enantiopure β-
amino acids. Finally, as D-phenyl glycine is an important
intermediate in the synthesis of medicinal products and in
particular antibiotics,[13] and its precursor nitrile is a further
amino analogue of previously studied substrates mandelonitrile
and 2-phenylpropionitrile, we wanted to examine the reaction
of the isolate with phenylglycinonitrile as a model α-amino
nitrile. This would further expand the substrate scope of this
isolate.
Although higher temperatures resulted in higher activity,
enantioselectivity was lost, however. When comparing the
results of this particular study with initial screening work and
substrate evaluation as described in our earlier papers, which
were performed at 25 C, enantioselectivity at this temperature
was also >99% towards 3-hydroxybutyronitrile following
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°
°
incubation of cells at 25 C for 24 hours. Since optimum activity
and enantioselectivity seemed to be almost identical at 20 and
°
25 C, with an apparent significant drop in ee when temperature
rose above this we deemed 25 C to be optimum for further
substrate studies and to allow direct comparisons.
The alkaline and acid tolerance of R. erythropolis SET1, were
further determined with the results presented in Figure 2.
Activity was maximal at pH 7.0, falling sharply at both higher
and lower values. Isolate SET1 was found to have a pH optimum
°
2. Results and Discussion
2.1. Effect of Temperature and pH
The effect of temperature on SET1 activity and enantioselectiv-
ity towards 3-hydroxybutyronitrile 1 was first investigated,[14] as
shown in Scheme 1. Relative activity was calculated based on
the activity observed and concentration of ammonia produced
during the hydrolysis of nitrile (10 mM) in potassium phosphate
Figure 1. Effects of temperature on the nitrilase activity of R. erythropolis
SET1. Reactions were run for 24 hours in potassium phosphate (100 mM,
pH 7.0) at 10, 20, 30 and 40 C. Activity was determined in triplicate using
the Nesslers microscale colorimetric assay. Enantioselectivity was determined
in triplicate by HPLC analysis using a chiral column.
°
°
buffer at pH 7 at 25 C. Observations from this work were that a
minor decrease in activity was observed when the temperature
°
°
was reduced from 25 C to 10 C with 82% relative activity
maintained, while 104% relative activity was retained when the
temperature was increased from 25 C to 30 C. A further
increase in temperature from 30 C to 40 C significantly
increased the relative activity to 132%.
Although lower temperature (10 C) was successful in
maintaining the enantioselectivity, lower solubility of the nitrile
was observed, and the reaction activity decreased. In contrast,
°
°
°
°
°
°
above 30 C the nitrilase enantioselectivity was rapidly lost. The
Figure 2. Effect of pH on the nitrilase activity of R. erythropolis SET1.
Reactions were run for 24 hours at 25 C in potassium phosphate (100 mM)
buffered at pH 4, 6, 7, 9 and 11. Activity was determined in triplicate using
the Nessler’s microscale colorimetric assay. Enantioselectivity was deter-
mined in triplicate by HPLC analysis using a chiral column.
°
Scheme 1. An investigation into the effect of temperature and pH on the
SET1 nitrilase catalysed hydrolysis of 3-hydroxybutyronitrile.
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of 7, with maximum activity and enantioselectivity both
retained. When the pH was reduced to 6, 86% of the maximum
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5
°
activity relative to that observed at pH 7 (25 C) was maintained.
A further decrease of pH resulted in a further loss of activity
with 67% relative activity observed at pH 4.
A similar trend is observed with biotransformations carried
out under alkaline conditions where activity is lost at higher pH
values. However, the drop in activity is not as sharp under basic
conditions, with 89% relative activity at pH 9 and 76% at pH 11.
The enantioselectivity of the biotransformation however was
less dramatically influenced by acid pH; >99.9% ee was
retained under acidic conditions even at pH 4. A significant
decrease in ee was found however under alkaline conditions,
with approximately 83% and 66% of the initial enantioselectiv-
ity remaining at pH 8 and pH 9 respectively. To compare isolate
SET1 with other nitrilases, the pH optimum of nitrilases can lie
within the range of 7.5 to 9. In contrast for example, R.
rhodochrous K22 demonstrated a maximum activity in the more
acidic region of pH 5.5 towards crotononitrile.[15a,c,16] Therefore,
the relatively high activity observed under acidic pH conditions
with SET1, which enabled the biotransformation to be per-
formed without any substantial loss in enantioselectivity, is
promising for biotransformations of nitrile analogues which
may decompose at a higher pH.
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Figure 3. Effects of monophasic solvents on the nitrilase activity of R.
erythropolis SET1. Reactions were run in duplicate for 24 hours in potassium
°
phosphate (100 mM, pH 7.0) solvent mixture at 25 C. Activity was
determined in triplicate using the Nesslers microscale colorimetric assay
Whole cells of R. erythropolis SET1 were incubated with 3-
hydroxybutyronitrile dissolved in aqueous buffer with various
additional co-solvents at quantities between 5 and 50% (v/v).
°
The reaction mixture was incubated at 25 C for 24 hours. The
appropriate cell blanks in the various solvent ratios allowed for
careful determination of the nitrilase activity. Each biotransfor-
mation and subsequent activity assay were performed in
triplicate. Relative activity was calculated based on the activity
observed from the standard reaction conditions without added
solvent, using the concentration of ammonia produced during
the hydrolysis of 3-hydroxybutyronitrile (10 mM) in potassium
phosphate buffer at pH 7.
2.2. Effect of Organic Solvents on Nitrilase Activity of SET1
Aqueous media can limit the use of nitrile hydrolysing enzymes
in the hydration of inherently hydrophobic nitriles, though
added quantities of organic solvents can be used to aid
substrate availability to the enzyme in some cases.[17] Several
solvent based approaches have been reported in the literature
including monophasic systems with solvents such as DMSO[18]
and biphasic systems such as toluene/H2O and hexane/H2O[1d]
and some systems can operate in both buffer-organic solvent
monophasic or biphasic mixtures.[18] In several cases,
enhancement of the enzyme activity and enantioselectivity of
hydrolysis has also been observed in organic solvent
systems.[1f,6e,19] However it has been noted that the mechanism
of solvent interaction with nitrile hydrolysing reactions is much
less studied than in the case of other hydrolytic enzymatic
systems.[7b]
In order to assess the solvent tolerance of SET1 and
determine an optimum solvent system, a series of solvent
studies were carried out. An important criterion for the
selection of a potential organic solvent is its biocompatibility
towards the enzyme. Eight organic solvents were examined in
this work. This included toluene, hexane, ethyl acetate, butanol,
IPA, ethanol, DMSO and THF. Initial work examined water
miscible organic solvents, such as IPA, DMSO, ethanol and THF
for the effect on the activity retention of the whole cells and
results are presented in Figure 3. In this study we chose to focus
on the effect of activity of the enzyme in the various solvent
mixtures monitoring this using the technique of Nesslerisation
for ease of reaction handling.
The addition of 35% (v/v) IPA resulted in retention of 100%
relative activity. As the ratio of IPA increased (50% v/v) so too
did the activity (144%). This is in contrast to DMSO, ethanol and
THF, where the activity decreases when the percentage of
organic solvent is increased; this is also shown in Figure 3. The
addition of 5% ethanol and 5% THF resulted in a decrease in
relative activity of 54% and 47% respectively. The relative
activity decreased as the percentage of organic solvent
increased and the enzyme appeared to be inhibited (0%
relative activity) at concentrations of 50% v/v ethanol and THF.
This significant drop in enzyme activity may indicate that the
proteins became denatured in the presence of higher concen-
trations of these organic solvents.
Low concentrations of DMSO (5% v/v) resulted in retention
of 89% relative activity, while higher concentrations of DMSO
(50% v/v) further reduced the activity (4%). In summary,
although the presence of IPA (35% v/v) maintained the initial
activity, higher concentration of the solvent (50% v/v) increased
the activity to 144%. Other co solvents examined decreased the
relative activity of the reaction in all cases.
In order to relieve the biocatalyst from substrate inhibition
in the aqueous monophasic reaction system, a water organic
biphasic system may provide an attractive alternative. In this
case the hydrophobic substrate will be mainly retained in the
organic phase which can act as a reservoir for the toxic or
insoluble substrate, thus regulating the substrate concentration
around the biocatalyst and minimizing the substrate
inhibition.[7f,20] Taking into account the poor water solubility of
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some nitriles, this allows higher substrate concentrations and
also facilitates the product recovery.
Four water immiscible organic solvents; ethyl acetate,
toluene, butan-1-ol and hexane between 5–50% v/v were
examined for the effect on the activity retention of the whole
cells and the results are presented in Figure 4.
The highest relative activities were obtained in hexane and
toluene which had Log P values of 3.5 and 2.5 respectively. The
maximum activity retention was obtained in 50% (v/v) hexane/
aqueous which was 89% of that in neat aqueous buffer. This
may be due to the more rigid and stable conformational
structure of the enzyme in this solvent.[20] Although toluene has
a similar Log P value to hexane of 2.5, the activity in 50% (v/v)
solvent/aqueous decreased to 48%. It was observed that the
incorporation of low concentrations of ethyl acetate (5% v/v)
and butan-1-ol (5% v/v) decreased the relative activity of the
reaction to 4.8% and 9.7% respectively.
respectively) may inhibit the activity of SET1. The optimum
percentage of relative activity retained during the biotransfor-
mation of nitrile in the presence of various organic solvents
(water miscible and immiscible) is shown in Figure 5. The results
of the study suggested that SET1 may be suitable for use in
selected organo-aqueous media at selected ratios only.
Preliminary studies were carried out to probe the enantiose-
lectivity observed in the presence of organic solvents however,
it was difficult to extract the remaining nitrile and product from
the reaction solvents for derivatisation and analysis by normal
phase HPLC. This will be evaluated further in future studies.
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2.3. Further Substrate Evaluation of SET1 with β-Amino
Nitriles
Given that bacterial isolate SET1 could selectively hydrolyse β-
hydroxy nitriles to carboxylic acids and amides, with the
aliphatic substrate 3-hydroxybutyronitrile (3-HBN) giving high
yield and ee of acid product without a docking group, it was
thought this may be a promising isolate suitable for the
hydrolysis of analogous β-amino nitriles. In particular the
transformation of aliphatic β- amino nitriles is a much less
explored area and requires further knowledge. A series of
protected and unprotected aliphatic and aromatic β-amino
nitriles 2–9 Figure 6 were prepared for evaluation of SET1.
Both aliphatic and aromatic structures were included to
compare to previous work and thus offer comparisons to the
known preferences of isolate SET1. Commonly used amine
protecting groups were also evaluated to provide insight into
the conformation of the active site of the enzyme and also for
ease of recovery of the amino acid products.
This result indicates that a biphasic system Log P�2.5 is
preferable and that water immiscible solvents with low Log P
values such as ethyl acetate and butan-1-ol (0.68 and 0.88
The unprotected aliphatic, 3-aminobutyronitrile
2 was
Figure 4. Effects of biphasic solvents on the nitrilase activity of R. erythropolis
SET1. Reactions were run in duplicate for 24 hours in potassium phosphate
obtained commercially and protected amino acid derivatives
were prepared from this by employing standard protection
protocols from literature as outlined in the supplementary
information.[2i,7a,21] The aromatic analogue 3-amino-3-phenyl-
propionitrile 6 was prepared by adapting a procedure devel-
oped by Brady et al,[2i] to employ sodium triacetoxyborohydride
in place of sodium cyanoborohydride for enamine reduction
as.[2j] While this method did furnish 6 in appropriate quantities
for evaluation, reproducibility of the STAB reduction proved
difficult.
°
(100 mM, pH 7.0) and up to 50% (v/v) solvent mixture at 25 C. Activity was
determined in triplicate using the Nesslers microscale colorimetric assay.
In order to develop respective chiral HPLC analytical
methods quantities of amide, and acid standards were
Figure 5. Effects of organic solvents on the nitrilase activity of R. erythropolis
SET1. Reactions were run in duplicate for 24 hours in potassium phosphate
°
(100 mM, pH 7.0) solvent mixture at 25 C. Activity was determined in
Figure 6. Unprotected and protected aliphatic and aromatic β-aminonitrile
analogues evaluated with SET1.
triplicate using the Nesslers microscale colorimetric assay.
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synthesised by hydrolysis. In developing HPLC methods for ee
determination, for the majority of the compounds, including
the starting nitriles, derivatisation was required.
group. In these studies cells were induced on 3-hydroxybutyr-
onitrile prior to biotransformation.
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6
7
8
9
Unfortunately, disappointingly low yields and ee were
observed for the hydrolysis of unprotected 2 and 6 in all
reactions and no nitrile and only minor quantities of acid were
recovered after the biotransformation of 2, with no acid or
nitrile recovered in the case of the aromatic analogue 6
(Table 1, entries 1–3, entry 8). The result for 2 follows observa-
tions noted in prior work with SET1 and 3-hydroxybutyronitrile
where nitrile was not recovered post biotransformation. This
may be due to the nitrile becoming trapped within the cell or
in its outer membrane. Another possibility is the presence of
another enzymatic pathway that is competing for the nitrile
and causing degradation of the acid and nitrile products. In
addition, the recovery of both product and starting material
were hampered by their water solubility.
Initial work focused on the unprotected 3-aminobutyroni-
trile 2 and 3-amino-3-phenylpropionitrile 6 which we hoped to
transform without the need for a protecting group to ensure
maximum efficiency. In discussing the biotransformation the
high water solubility of both the unprotected acid and nitrile
must be highlighted. While this is desirable from a reaction
point of view, it severely hampered the recovery process by
solvent extraction and has been noted with unprotected 3-
aminobutyronitrile,[7a] and other aliphatic unprotected β-amino
nitriles and acids.[22] It must also be mentioned that the lack of a
UV- chromophore in both the starting material and product in
the case of 3-aminobutyronitrile also proved complex for HPLC
analytical method development and reaction determination
using TLC.
Attempts to recover and analyse the unprotected 3-amino-
butyric acid 2b and 3-aminobutyronitrile 2 products post
biotransformation via in-situ Cbz-protection with Cbz-OSu
generated complex mixtures of products not easily separated
by flash chromatography. For accurate yield determination it
was necessary to use HPLC with concentration of product
determined by comparison to standard curves prepared from
authentic standards completed in triplicate. For ee determina-
tion of the acid product due to availability of suitable
separating columns a GITC derivatisation method was devel-
oped which is outlined in the experimental section.
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It can be tentatively observed from these studies that as pH
increases that the enantioselectivity of isolate SET1 falls rapidly
(Table 1, entries 1, 2 and 3). This result further highlighted the
observations outlined in Section 1.1 above, that the optimum
°
temperature and pH for the microbial isolate are 25 C and
pH 7. Investigation of induction conditions of the isolate were
carried out in order to improve the activity and selectivity.[23]
Here, 2 was used to induce isolate SET1 rather than 1. The
biotransformation was subsequently carried out at pH 7 with 2
as substrate. In this instance again disappointingly low yield of
acid and nitrile was recovered. (Table 1, entry 4).
In the case of 2, it has been previously observed that N-
benzyl protection of the amino group significantly enhanced ee
and yield in a nitrile hydratase/amidase system after the
addition of the N-benzyl protecting group[7a] . As noted by
Klempier et al the choice of protecting group for β-aminonitriles
should be governed by the ease of protection and removal of
the group, the ability to improve analytical reaction monitoring
Protocols and biotransformation methods from the process
optimisation work outlined above were employed as a starting
point for investigation of the aminonitriles. Parameters that
°
were deemed optimum such as temperature of 25 C for
enantioselectivity and activity were retained. In terms of the pH
however, in the case of 2 biotransformations were carried out
at pH 7, 8 and 9 to investigate the effect of pH on the amino
Table 1. Results of the biotransformation of unprotected and protected racemic β-aminoalkane and β-aminoaromatic nitriles 2–9.
Entry
Substrate
pH
R1
R2
Recovered Nitrile
Yield (%)[b]
Product Amide b
Product Acid c
ee (%)[c]
Yield (%)[b]
ee (%)[c]
Yield (%)[b]
ee (%)[c]
1
2
3
4*
5
6
7
8
2
2
2
2
3
4
5
6a
7
7
8
9
7
7
7
7
7
7
7
7
H
H
H
H
Bn
Boc
Ts
H
Bn
Boc
Ts
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
C6H5
C6H5
–
–
–
8
–
–
–
2
–
–
–
–
3
–
9
–
–
–
–
–
–
–
–
90
–
1
–
–
–
–
<1
<1
<1
5
6
1
<1
–
–
–
29 (S)
18 (S)
5 (S)
7 (S)
75 (S)
10 (S)
>99 (S)[d]
–
-
-
55
74
17
78
66
96
79[d]
0
11
0
1
1
9
10
11
–
–
–
8
9
–
°
The biotransformations were carried out by incubating the nitrile (10 mmol/L) in a suspension of R. erythropolis SET1 (OD600nm=1) in phosphate buffer at 25 C.
[b] Isolated yield. [c] Determined by HPLC analysis using a chiral column. The configuration was determined by comparison of HPLC traces with prepared single
[d]
enantiomer standards and literature. N.d: not detected. Chiral HPLC analysis performed on the corresponding derivative. * Isolate induced on 3-
aminobutryonitrile.
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and product detection and also substrate solubility and these
were considered for this study.[22]
1
2
We chose to evaluate N-benzyl, N-Boc carbamate and N-
Tosyl derivatives for this work. We anticipated that some of the
N-protected nitriles would exhibit poor solubility in phosphate
buffer, in particular the N-Tosyl and N-Boc protected substrates
and measures were taken to ensure 100% availability of the
substrate to the bacterial isolate. The earlier organic solvent
studies shown above in Figure 5 suggest that the addition of
5% DMSO could allow for the retention of activity of SET1 with
3-hydroxybutyronitrile and this was chosen as a starting point
for this work. In all cases the pH was maintained at 7 for the
biotransformations. Each biotransformation was carried out in a
suspension of phosphate buffer (10 mM) with 5% DMSO,
containing induced cells (OD600nm =1) at pH 7 and enantiose-
lectivity was determined using HPLC employing a chiral column
with comparison to synthesised standards of acid and amide.
The optimum enantioselectivity was obtained with the N-
benzyl protected alphatic substrate 5 where ee of 75% of (S)-
acid was observed in the very low yield of 6% (Table 1 entry 5).
Again isolation of the product proved difficult and nitrile was
not recovered from the reaction mixture despite using
published methods. In addition a minor quantity of amide was
also recovered with ee of 90%. The N-Boc protected aliphatic
nitrile 4 did not yield any acid product after biotransformation
but did furnish enantioenriched starting nitrile (Table 1, entry 6).
In the case of the N-tosyl protected derivative a trace quantity
of acid was recovered with unreacted nitrile predominantly
recovered post biotransformation. The size of the protecting
group may have played a role in the lack of observed acid
product in this case. None of the protected aromatic variants 7–
9 yielded product from the reaction with racemic unreacted
starting material obtained. (Table 1, entries 9–11). The presence
of amide as product in two of the cases along with acid is not
unusual in reactions with this bacterial isolate. In our previous
work, we observed the formation of amide in cases where the
substrate contained an electron withdrawing group at the α
position to the nitrile, and this has also been noted in several
other similar studies.[12]
In a final separate study of the substrate scope of SET1, as
the bacterial isolate had shown activity and selectivity with
mandelonitrile in previous studies, we also preliminarily inves-
tigated the ability of the enzyme to transform the α-amino-
nitrile phenylglycinonitrile 10. Nitrilase enzymes have previously
been shown to successfully transform α-aminonitrile substrates
with relatively high selectivity[24,25,26]. Particular consideration
must be taken with phenylglycinonitrile as a substrate as it
undergoes spontaneous decomposition into benzaldehyde,
HCN and ammonia via retro-Strecker reaction. Meanwhile
mandelonitrile can arise from the spontaneous reaction of HCN
and benzaldehyde,[27] therefore, choice of reaction conditions
require careful consideration. To this end, the influence pH had
on the reaction outcome of SET1 with 2-phenylglycinonitrile
was examined. In these reactions, enzyme activity and
enantioselectivity were determined after 96 h incubation with
racemic 2-phenylglycinonitrile (10 mM) at pH ranging from 7 to
10 (Scheme 2) by chiral HPLC analysis following acidification
3
4
5
6
7
8
9
Scheme 2. Effect of pH on the nitrilase activity of R. erythropolis SET1
°
towards phenylglycinonitrile. Reactions were run for 96 hours at 25 C in
potassium phosphate (100 mM) buffered at pH 7, 8, 9 and 10. Activity and
enantioselectivity were determined by HPLC analysis using a CR+ chiral
column.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
and removal of the biomass by centrifugation. Standard curves
were completed with standard and runs in triplicate for acid,
amide and nitrile to allow for product quantitation.
The results in Figure 7 demonstrate the effect of pH on
nitrilase activity and enantioselectivity. In contrast to the results
for β-aminonitriles isolate SET1 was found to efficiently trans-
form the α-aminonitrile 2-phenylglycinonitrile 10 at pH 7, with
maximum yield and enantioselectivity obtained for the (S)-acid
product. In this case the bacterial isolate was extremely
selective generating acid in >99% ee and in addition nitrile
was detected in 10% yield and 44% ee. When the pH increased
the yield of acid product decreased from 51% at pH 7 to 36%
at pH 10, however, the high enantioselectivity was retained. The
decreasing acid yield may be due to enzyme inhibition caused
by benzaldehyde or deactivation of the nitrilase under alkaline
conditions. Detection of the remaining nitrile increased from
10% at pH 7 to 15% at pH 10, while the enantioselectivity data
for the nitrile along with low conversions indicate possible
partial racemisation of the aminonitrile as previously described
by Chaplin et. al..[28] They established that pH values >10
allowed for the rapid racemisation of amino nitriles. The loss in
mass balance for the nitrile and acid product may be attributed
to the decomposition of the starting material by such a
mechanism..[28] The yields and ee values are influenced by the
competition of the hydrolysis, decomposition and racemisation
and vary greatly depending on the reaction conditions. It is
plausible that spontaneous decomposition of the starting
material to produce benzaldehyde, HCN and ammonia may
cause enzyme inhibition. However, it is also likely that the
decreased quantity of remaining phenylglycinonitrile may also
Figure 7. Effect of pH on the nitrilase activity of R. erythropolis SET1 towards
°
phenylglycinonitrile Reactions were run for 96 hours at 25 C in potassium
phosphate (100 mM) buffered at pH 7, 8, 9 and 10. Yield and enantiose-
lectivity were determined by HPLC analysis using a CR+ chiral column.
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account for reduced yields, this may be supported by the loss Experimental Section
1
2
3
4
5
6
7
8
9
in mass balance for the nitrile and acid product.
In all reactions amide was not detected.[12] This is in contrast
to previous results obtained when examining the reaction of
SET1 with mandelonitrile, an α-hydroxy nitrile in which both
acid (8.2%) and amide (44.1%) were formed and thought to be
a result of the electron-withdrawing nature of the hydroxy
substituent (and proposed stabilisation of the tetrahedral
intermediate).[12] The sole formation of acid in the case of
phenylglycinonitrile may be due to the electronic and steric
interactions because of the NH2 and phenyl group (π-π
interactions). It is also interesting to compare the ability of the
isolate to selectively transform an unprotected α-aminonitrile
with the lack of detected selectivity with the analogous and
related unprotected β-aminonitrile substrates and with selectiv-
ity observed in β-hydroxyl derivatives. This further indicates
that both substrate functionality type and its distance from the
nitrile group, along with the presence of either aromatic or
aliphatic sterically demanding neighbouring groups may have
an effect on the selectivity of such biocatalytic reactions. This
final preliminary study also offers significant potential for the
transformation of other aminonitrile substrates, and the gen-
eration of further amino acid products and will be evaluated
further in future studies.
Materials
Racemic 3-hydroxybutyronitrile (1) and 2-phenylglycinonitrile were
purchased from Sigma-Aldrich and 3-aminobutyronitrile (2) was
purchased as
a hydrochloride salt from Enamine. All other
chemicals were of analytical grade and obtained from various
commercial sources. NMR spectra were recorded with a JEOL ECX
400 MHz spectrometer. The chemical shifts (expressed in ppm) of
1
the H and 13C NMR spectra are referenced to the solvent peaks.
TLC was carried out on aluminium-backed sheets with silica gel 60
F54 (Merck). Column chromatography was performed with silica gel
60 (0.04–0.063 mm, Merck). HPLC analysis was undertaken with a
Hewlett-Packard 1050 series instrument, LCMS was carried out with
an Agilent technologies 1200 series instrument with an LC/MSD
Trap XCT detector and GC-MS with a Varian 450 GC coupled with a
220 MS.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Bacterial Isolates
The bacterial isolate used in this study was previously described in
Coady et al.[7h] The isolate SET1 has been deposited in the National
Collection of Marine, Food and Marine Bacteria (NCIMB), Patent
Deposit number NCIMB 41986.
General Method for Nesslers Microscale Ammonia Assay
Isolate SET1 was examined in the hydrolysis of 3-hydroxybutyroni-
trile using the Nesslers colorimetric assay [5], in 96-well microtitre
plates (Sarstedt Ltd). Fresh cultures were grown in M9-minimal
media containing 10 mM nitrile (before washing 3 times with
500 μL of phosphate buffer). Each 150 μl reaction contained 10 mM
nitrile and cells (OD600nm =0.1) in potassium phosphate buffer
(100 mM, pH 7.0). Microtitre plates were sealed using adhesive film
(Sarstedt) and incubated at various temperatures at 250 rpm for
24 hours. The reaction was then quenched by adding 37.5 μL of
250 mM HCl. Plates were centrifuged at 500 g for 10 minutes to
pellet the cell debris. 20 μl of the supernatant was transferred to a
microtitre plate, 181 μL of assay mastermix was added (155 μL
deionised water, 1 μL 10 N NaOH, 25 μL Nesslers reagent (Merck)).
The reaction was allowed to stand for 10 minutes and the
absorbance was read at 425 nm. Cell blanks contained cells @
OD600nm =0.1 in phosphate buffer. Nitrile blanks contained 150 μL
of 10 mM nitrile in phosphate buffer.
3. Conclusion
We have presented detailed substrate optimisation studies on
the bacterial isolate R. erythropolis SET1 with 3-hydroxybutyroni-
trile 1 outlining the key parameters of pH, temperature and also
solvent compatibility for use with this substrate. The reaction
system was screened with a series of β- amino nitriles but does
yield sufficient isolated quantities of enantioenriched product. It
is clear that a protecting group is necessary for any appreciable
product recovery and detection in these reactions. Both the
yield and ee values for amide and acid product appear to be a
result of the structure and the size of the protecting group on
the amino moiety. This work also presents the inherent
difficulties associated with handling such β-aminonitrile sub-
strates in contrast to β-hydroxy analogues and indicates
possible alternative metabolic pathways as evident in the low/
unrecoverable yield of the starting substrate. In additional
studies, the position of the free amino group α- or β- to the
nitrile was observed to have a dramatic effect on the reaction
outcome. In contrast to the β-amino nitrile substrates the
biotransformation of α- substituted 2-phenylglycinonitrile with
SET1 resulted in excellent yields and ee of (S)-acid (51% yield,
>99% ee). This was observed to be pH dependent with lower
yield observed at higher pH. This offers significant potential for
the transformation of other amino nitrile substrates and the
generation of valuable amino acid products.
General Procedure for Enantioselectivity Screening Towards
3-Hydroxybutyronitrile
Racemic nitrile (5.1 mg, 5.9μL, 10 mM) was added in one portion to
a solution of potassium phosphate buffer (0.1 M, 6 mL) containing
induced cells (OD600nm =1), and activated at the chosen temperature
for 30 minutes with orbital shaking (250 rpm). The reaction was
quenched after 24 hours by removal of the biomass by centrifuga-
tion at 3,000 g. The resulting aqueous solution was acidified by the
addition of 1 M HCl (200 μL). The aqueous portion was then
extracted with ethyl acetate, the extracts were dried over MgSO4
and the solvent removed under vacuum. The biotransformation
products were first derivatised to their corresponding β-benzylox-
yethers before analysis. Silver oxide (1 equiv, 0.06 mmol, 13.6 mg),
benzylbromide (4 equiv, 0.24 mmol, 28 μL) and dichloromethane
(2 mL) were added and the mixture stirred in the dark for 24 hours.
The reaction mixture was diluted with acetone and filtered through
a 0.45 μm filter and solvent was removed under vacuum. 1 mL of
mobile phase (90% hexane: 10% IPA and 0.1% TFA) was added
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before the solution was injected on the Chiral HPLC system.
Chiralcel ADÀ H stationary phase was used for the resolution of β-
hydroxyacids. Analytical conditions applied: 90% hexane, 10% IPA
and 0.1% TFA, with a flow rate of 0.8 mL/min and a detection
wavelength of 215 nm. All experiments were performed in tripli-
cate. % enantiomeric excess is calculated from the ratio of the
enantiomer by the difference in peak area divided by the sum of
the peak areas for the major and minor enantiomers.
Biotransformation Procedures for β-Aminonitrile Substrates
1
2
3
4
5
6
7
8
9
General Procedure for Biotransformations of β-Aminonitrile
Substrates
Each biotransformation was carried out in
a suspension of
phosphate buffer (100 mM) set to the required pH, containing SET1
cells, previously induced on 3-hydroxybutryonitrile (OD600nm =1).
Racemic β-aminobutyronitrile (10 mM) was added to the flask and
°
the mixture was incubated at 25 C for the required time with
Temperature Studies
mechanical shaking (200 RPM), and monitored by TLC. The
appropriate derivatisation, extractive work up or work-up with
cation exchange resin and semi-prep purification HPLC (Phenomen-
ex Jupiter C18, 10 μm), (gradient elution (ACN: H2O+0.1% formic
acid), 5 mL minÀ 1) as necessary was employed depending on the
substrate. Analysis was carried out by HPLC equipped with a chiral
column (Chiralpak IA, OJÀ H) or Waters Symmetry C18 after GITC
derivitisation. The products were confirmed and product yields
calculated by matching HPLC retention times to characterised
synthesised standards (run in triplicate), standard curves and by LC-
MS profiles. HPLC of single enantiomer acids and derivatives was
used in all cases to assign configuration of products.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Nitrilase activities towards 3-hydroxybutyronitrile (10 mM) was
assayed as described above using whole cells (SET1, OD600nm =1) in
°
KH2PO4 (100 mM, pH 7.0). The hydrolysis was performed at 0 C,
°
°
°
°
10 C, 20 C, 30 C, and 40 C. Chiral HPLC analysis as outlined in the
previous procedure was used to measure enantiomeric excess.
pH Studies
Nitrilase activities towards 3-hydroxybutyronitrile (10 mM) was
assayed as described above using whole cells (SET1, OD600nm =1) in
KH2PO4 (100 mM). The hydrolysis was performed at pH 4, 5, 7, 9 and
°
10 at 25 C. Chiral HPLC analysis as outlined in the previous
GITC Derivatisation Method for HPLC Analysis of Free Amine
Substrates[29]
procedure was used to measure enantiomeric excess.
Sequentially triethylamine (1.5 mmol) and GITC (1.5 mmol) were
added to a solution of the amine in acetonitrile (1 mL). The
Solvent Studies
°
Whole cells of R. erythropolis SET1 were incubated with 3-
hydroxybutyronitrile dissolved in aqueous buffer with various
additional co-solvents at quantities between 5 and 50% (v/v). The
resulting mixture was stirred at 40 C for 1 h. The mixture was
injected directly onto the chiral HPLC fitted with a C18 symmetry
column, with mobile phase MeOH: H2O+0.1% TFA and flow rate of
1.0 mL minÀ 1
.
°
reaction mixture was incubated at 25 C for 24 hours and enzyme
activity was monitored using the technique of Nesslerisation, along
with cell blanks and solvent blanks.
Studies on the Biotransformation of Phenylglycinonitrile
The activity of resting cells of SET1 with phenylglycinonitrile was
determined in reaction mixtures (1 mL) containing potassium
phosphate buffer (100 mM) buffered to pH 7, 8, 9 and 10 containing
2-phenylglycinonitrile (0.01 M). The reaction mixture was incubated
Synthesis of β- Aminonitrile Substrates
Compounds (3), (4), (5), (7), (8) and (9) were prepared using
literature procedures and are detailed in the supplementary
information.[7a,2i]
°
with resting cells of SET1 (OD=1) at 25 C for 96 hours and
quenched by the addition of HCl (200 μL) with biomass removed
via centrifugation. The reaction mixture was analysed immediately
to prevent further decomposition of the substrate.
3-Amino-3-Phenylpropionitrile (6a)
Solid NaBH4 (10 mmol, 0.39 g) was added portion-wise with stirring
A Chiralcel CR+ column was used for the resolution of both 2-
phenylglycinonitrile and 2-phenylglycine. Analytical conditions
applied: Mobile phase of perchloric acid (16.3 g in 1 L) adjusted to
pH 1, with a flow rate of 1.0 mL/min and a detection wavelength of
215 nm.% activity was calculated from the combined peak area of
the enantiomers and the equation of the line generated from a
series of standards for the nitrile and acid. Standard curves are
provided in the supplementary information.
°
to glacial acetic acid (0.18 mmol, 10 mL) at 10 C. The mixture was
stirred for 30 min. A solution of 3-amino-3-phenylacrylonitrile
(3.4 mmol, 0.49 g) in glacial acetic acid (3.4 mL) was added portion-
wise. The solution was left to stir at room temperature for 2 h. The
mixture was concentrated down in vacuo then NaOH (1 M, 40 mL)
was added. The mixture was extracted with EtOAc (3×40 mL) and
the combined organic extracts were washed with brine, dried over
Na2SO4, and the solvent removed in vacuo. The product was
purified by silica flash chromatography (Hex:EtOAc 70:30) to give
the title compound as a pale yellow oil (1.9 mmol, 0.27 g, 54%).1H
NMR (400 MHz, CDCl3), δ 7.36 (d, J=4.3 Hz, 4H, ArÀ H), 7.34–7.27 (m,
1H, ArÀ H), 4.31 (dd, J=7.2, 5.7 Hz, 1H, CH), 2.75–2.58 (m, 2H, CH2),
1.79 (s, 2H, NH2). 13C NMR (100 MHz, CDCl3), δ 142.5 (CN), 129.0
(CÀ Ar), 128.5 (CÀ Ar), 126.2 (CÀ Ar), 118.1 (C-5), 52.8 (C-4), 28.6 (C-
3).ESI-MS, low res, m/z 168.9 (M+Na+), 147.0 (M+H+), 130, C9H10N2
Acknowledgements
The authors would like to thank the Wales–Ireland Network for
Scientific Skills for financial support under the Ireland-Wales
2007–2013 INTERREG 4A programme granted to the PMBRC at
WIT. In addition, funding was gratefully received from Waterford
Institute of Technology as part of the WIT Postgraduate Scholar-
ship Scheme 2015–2019 and as part of the School of Science and
Computing Research Support Scheme 2020.
Acid and amide standards of all substrates were also prepared by
hydrolysis using methods provided in the literature,[7i] and are
detailed in the supplementary information along with chiral HPLC
methods for analysis of nitriles, amides and acids.
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Sartori, D. C. de Rezende, M. Alves Nogueira Diaz, Chirality 2019, 31,
Conflict of Interest
1
2
3
4
5
6
7
8
9
776–812; c) T. Narancic, S. A. Almahboub, K. E. O’Connor, World J.
Microbiol. Biotechnol. 2019, 35, 67; d) H. Pellissier, Adv. Synth. Catal.
2011, 353, 1613–1666; e) O. I. Afanasyev, E. Kuchuk, D. L. Usanov, D.
Chusov, Chem. Rev. 2019, 119, 11857–11911; f) S. M. So, H. Kim, L. Mui, J.
Chin, Eur. J. Org. Chem. 2012, 2012, 229–241; g) J. H. Schrittwieser, S.
Velikogne, W. Kroutil, Adv. Synth. Catal. 2015, 357, 1655–1685; h) E.
Ricca, B. Brucher, J. H. Schrittwieser, Adv. Synth. Catal. 2011, 353, 2239–
2262.
The authors declare no conflict of interest.
[1] a) R. de Souza, L. S. M. Miranda, U. T. Bornscheuer, Chem. Eur. J. 2017,
23, 12040–12063; b) P. N. Devine, R. M. Howard, R. Kumar, M. P.
Thompson, M. D. Truppo, N. J. Turner, Nat. Rev. Chem. 2018, 2, 409–421;
c) M. Honig, P. Sondermann, N. J. Turner, E. M. Carreira, Angew. Chem.
Int. Ed. 2017, 56, 8942–8973; d) Y. C. He, Z. J. Zhang, J. H. Xu, Y. Y. Liu, J.
Ind. Microbiol. Biotechnol. 2010, 37, 741–750; e) N. Layh, J. Parratt, A.
Willetts, J. Mol. Catal. B 1998, 5, 467–474; f) C. Vergne-Vaxelaire, F.
Bordier, A. Fossey, M. Besnard-Gonnet, A. Debard, A. Mariage, V.
Pellouin, A. Perret, J. L. Petit, M. Stam, M. Salanoubat, J. Weissenbach, V.
De Berardinis, A. Zaparucha, Adv. Synth. Catal. 2013, 355, 1763–1779.
[2] a) J. S. Gong, Z. M. Lu, H. Li, J. S. Shi, Z. M. Zhou, Z. H. Xu, Microb. Cell
Fact. 2012, 11; b) T. C. Bhalla, V. Kumar, V. Kumar, N. Thakur, Savitri,
Appl. Biochem. Biotechnol. 2018, 185, 925–946; c) J. S. Gong, J. S. Shi,
Z. M. Lu, H. Li, Z. M. Zhou, Z. H. Xu, Crit. Rev. Biotechnol. 2017, 37, 69–81;
d) J. S. Gong, H. Li, Z. M. Lu, J. S. Shi, Z. H. Xu, Prog. Chem. 2015, 27, 448–
458; e) R. Singh, R. Sharma, N. Tewari, Geetanjali, D. S. Rawat, Chem.
Biodiversity 2006, 3, 1279–1287; f) R. N. Thuku, D. Brady, M. J. Benedik,
B. T. Sewell, J. Appl. Microbiol. 2009, 106, 703–727; g) Y. P. Xue, Y. K.
Yang, S. Z. Lv, Z. Q. Liu, Y. G. Zheng, Appl. Microbiol. Biotechnol. 2016,
100, 3421–3432; h) O. K. L. Rapheeha, M. P. Roux-van der Merwe, J.
Badenhorst, V. Chhiba, M. L. Bode, K. Mathiba, D. Brady, J. Appl.
Microbiol. 2017, 122, 686–697; i) V. Chhiba, M. L. Bode, K. Mathiba, W.
Kwezi, D. Brady, J. Mol. Catal. B 2012, 76, 68–74; j) A. Dondoni, A. Massi,
E. Minghini, Synlett 2006, 539–542; k) M. X. Wang, Acc. Chem. Res. 2015,
48, 602–611; l) M. X. Wang, Chimia 2009, 63, 331–333.
[3] H. Busch, P. Hagedoorn, Hanefeld, Int. J. Mol. Sci. 2019, 20, 4787.
[4] a) U. Heinemann, C. Kiziak, S. Zibek, N. Layh, M. Schmidt, H. Griengl, A.
Stolz, Appl. Microbiol. Biotechnol. 2003, 63, 274–281; b) P. Kaul, A.
Banerjee, U. C. Banerjee, Biomacromolecules 2006, 7, 1536–1541.
[5] A. Banerjee, P. Kaul, R. Sharma, U. C. Banerjee, J. Biomol. Screening 2003,
8, 559–565.
[6] a) A. Stolz, S. Trott, M. Binder, R. Bauer, B. Hirrlinger, N. Layh, H. J.
Knackmuss, J. Mol. Catal. B 1998, 5, 137–141; b) J. Qiu, E. Z. Su, W.
Wang, D. Z. Wei, Catal. Commun. 2014, 51, 19–23; c) N. Layh, A. Stolz, J.
Bohme, F. Effenberger, H. J. Knackmuss, J. Biotechnol. 1994, 33, 175–182;
d) R. Bauer, B. Hirrlinger, N. Layh, A. Stolz, H. J. Knackmuss, Appl.
Microbiol. Biotechnol. 1994, 42, 1–7; e) N. Layh, A. Willetts, Biotechnol.
Lett. 1998, 20, 329–331; f) A. Bauer, N. Layh, C. Syldatk, A. Willetts,
Biotechnol. Lett. 1996, 18, 343–348; g) N. Layh, H. J. Knackmuss, A. Stolz,
Biotechnol. Lett. 1995, 17, 187–192; h) N. Layh, A. Stolz, S. Forster, F.
Effenberger, H. J. Knackmuss, Arch. Microbiol. 1992, 158, 405–411; i) M.
Gao, D. X. Wang, Q. Y. Zheng, M. X. Wang, J. Org. Chem. 2006, 71, 9532–
9535.
[10] a) L.-B. Zhang, D.-X. Wang, L. Zhao, M.-X. Wang, J. Org. Chem. 2012, 77,
5584–5591; b) L.-B. Zhang, D.-X. Wang, M.-X. Wang, Tetrahedron 2011,
67, 5604–5609; c) M.-X. Wang, J. Liu, D.-X. Wang, Q.-Y. Zheng,
Tetrahedron: Asymmetry 2005, 16, 2409–2416; d) M.-X. Wang, S.-J. Lin, J.
Liu, Q.-Y. Zheng, Adv. Synth. Catal. 2004, 346, 439–445; e) M.-X. Wang,
Acc. Chem. Res. 2015, 48, 602–611.
[11] a) S. Y. Choi, Y. M. Goo, Arch. Pharmacal Res. 1986, 9, 45; b) J. Qiu, E. Su,
W. Wang, D. Wei, Tetrahedron Lett. 2014, 55, 1448–1451.
[12] T. M. Coady, L. V. Coffey, C. O’Reilly, C. M. Lennon, Eur. J. Org. Chem.
2015, 1108–1116.
[13] A. Bruggink, Synthesis of β-Lactam Antibiotics: Chemistry, Biocatalysis &
Process Integration, 2001.
[14] Preliminary results of these studies were presented in our previous
publication but these were not discussed in detail.
[15] a) K. Yamamoto, Y. Ueno, K. Otsubo, K. Kawakami, K. I. Komatsu, Appl.
Environ. Microbiol. 1990, 56, 3125–3129; b) M. Kobayashi, N. Yanaka, T.
Nagasawa, H. Yamada, J. Bacteriol. 1990, 172, 4807–4815; c) A. J. Hoyle,
A. W. Bunch, C. J. Knowles, Enzyme Microb. Technol. 1998, 23, 475–482.
[16] M. Kobayashi, N. Yanaka, T. Nagasawa, H. Yamada, Tetrahedron 1990,
46, 5587–5590.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
[17] G. W. Black, T. Gregson, C. B. McPake, J. J. Perry, M. Zhang, Tetrahedron
Lett. 2010, 51, 1639–1641.
[18] U. Heinemann, D. Engels, S. Burger, C. Kiziak, R. Mattes, A. Stolz, Appl.
Environ. Microbiol. 2003, 69, 4359–4366.
[19] P. Kaul, U. C. Banerjee, J. Ind. Microbiol. Biotechnol. 2008, 35, 713–720.
[20] J. Qiu, E. Z. Su, W. Wang, D. Z. Wei, Tetrahedron Lett. 2014, 55, 1448–
1451.
[21] D. Y. Ma, Q. Y. Zheng, D. X. Wang, M. X. Wang, Org. Lett. 2006, 8, 3231–
3234.
[22] M. Preiml, H. Honig, N. Klempier, J. Mol. Catal. B 2004, 29, 115–121.
[23] a) A. Banerjee, P. Kaul, U. C. Banerjee, Appl. Microbiol. Biotechnol. 2017,
101, 6299–6299; b) A. Banerjee, P. Kaul, U. C. Banerjee, Appl. Microbiol.
Biotechnol. 2006, 72, 77–87; c) S. C. Naik, P. Kaul, B. Barse, A. Banerjee,
U. C. Banerjee, Bioresour. Technol. 2008, 99, 26–31; d) V. P. Chhiba-
Govindjee, K. Mathiba, C. W. van der Westhuyzen, P. Steenkamp, J. K.
Rashamuse, S. Stoychev, M. L. Bode, D. Brady, Appl. Microbiol. Biotechnol.
2018, 102, 10055–10065.
[24] C. Kiziak, D. Conradt, A. Stolz, R. Mattes, J. Klein, Microbiology 2005, 151,
3639–3648.
[25] S. Y. Choi, Y. M. J. A. o. P. R. Goo, Arch. Pharmacol. Res. 1986, 9, 45.
[26] F. Bordier, M. Stam, E. Darii, S. Tricot, A. Fossey, J. Rohault, A. Debard, A.
Mariage, V. Pellouin, J.-L. Petit, A. Perret, D. Vallenet, M. Salanoubat, J.
Weissenbach, C. Vergne-Vaxelaire, V. de Berardinis, A. Zaparucha, J. Mol.
Catal. B 2014, 107, 79–88.
[27] D. Brady, A. Beeton, J. Zeevaart, C. Kgaje, F. van Rantwijk, R. A. Sheldon,
Appl. Microbiol. Biotechnol. 2004, 64, 76–85.
[28] J. A. Chaplin, M. D. Levin, B. Morgan, N. Farid, J. Li, Z. Zhu, J. McQuaid,
L. W. Nicholson, C. A. Rand, M. J. Burk, Tetrahedron: Asymmetry 2004, 15,
2793–2796.
[7] a) D. Y. Ma, D. X. Wang, J. Pan, Z. T. Huang, M. X. Wang, J. Org. Chem.
2008, 73, 4087–4091; b) B. Wilding, A. B. Vesela, J. J. B. Perry, G. W. Black,
M. Zhang, L. Martinkova, N. Klempier, Org. Biomol. Chem. 2015, 13,
7803–7812; c) Y. F. Ao, D. X. Wang, L. Zhao, M. X. Wang, J. Org. Chem.
2014, 79, 3103–3110; d) A. Banerjee, P. Kaul, U. C. Banerjee, Arch.
Microbiol. 2006, 184, 407–418; e) H. H. Kinfe, V. Chhiba, J. Frederick, M. L.
Bode, K. Mathiba, P. A. Steenkamp, D. Brady, J. Mol. Catal. B 2009, 59,
231–236; f) Z. J. Zhang, J. A. Pan, J. F. Liu, J. H. Xu, Y. C. He, Y. Y. Liu, J.
Biotechnol. 2011, 152, 24–29; g) A. F. Abdel-Magid, S. J. Mehrman, Org.
Process Res. Dev. 2006, 10, 971–1031; h) T. M. Coady, L. V. Coffey, C.
O’Reilly, E. B. Owens, C. M. Lennon, J. Mol. Catal. B 2013, 97, 150–155;
i) M. Winkler, L. Martinkova, A. C. Knall, S. Krahulec, N. Klempier,
Tetrahedron 2005, 61, 4249–4260.
[29] K. Mitsukura, M. Suzuki, K. Tada, T. Yoshida, T. Nagasawa, Org. Biomol.
Chem. 2010, 8, 4533–4535.
[8] N. Layh, B. Hirrlinger, A. Stolz, H. J. Knackmuss, Appl. Microbiol.
Biotechnol. 1997, 47, 668–674.
[9] a) M. A. Wegman, M. H. A. Janssen, F. van Rantwijk, R. A. Sheldon, Adv.
Synth. Catal. 2001, 343, 559–576; b) G. Diaz-Muñoz, I. L. Miranda, S. K.
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