Kinetic Resolution of Aromatic β-Amino Acids Using a Combination of Phenylalanine Ammonia Lyase and Aminomutase Biocatalysts

Article abstract of DOI:10.1002/adsc.201600894

An enzymatic strategy for the preparation of (R)-β-arylalanines employing phenylalanine aminomutase and ammonia lyase (PAM and PAL) enzymes has been demonstrated. Candidate PAMs with the desired (S)-selectivity from Streptomyces maritimus (EncP) and Bacillus sp. (PabH) were identified via sequence analysis using a well-studied template sequence. The newly discovered PabH could be linked to the first ever proposed biosynthesis of pyloricidin-like secondary metabolites and was shown to display better β-lyase activity in many cases. In spite of this, a method combining the higher conversion of EncP with a strict α-lyase from Anabaena variabilis (AvPAL) was found to be more amenable, allowing kinetic resolution of five racemic substrates and a preparative-scale reaction with >98% (R) enantiomeric excess. This work represents an improved and enantiocomplementary method to existing biocatalytic strategies, allowing simple product separation and modular telescopic combination with a preceding chemical step using an achiral aldehyde as starting material. (Figure presented.).

Full text of DOI:10.1002/adsc.201600894

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Kinetic Resolution of Aromatic b-Amino Acids Using a
Combination of Phenylalanine Ammonia Lyase and Aminomutase
Biocatalysts
Nicholas J. Weise,a Syed T. Ahmed,a Fabio Parmeggiani,a and Nicholas
J. Turnera,*
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Manchester Institute of Biotechnology & School of Chemistry, Faculty of Science & Engineering, University of
Manchester, 131 Princess Street, M1 7DN, Manchester, United Kingdom
Phone: +44 161 306 5173
Fax: +44 (161) 275 1311; e-mail: nicholas.turner@manchester.ac.uk
17 Received: September 13, 2016; Revised: January 5, 2017; Published online: && &&, 0000
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Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201600894
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characterised enzymes from various species of yew
tree, all involved in the production of the cytotoxic
agent taxol[1] and distant relatives associated with
Abstract: An enzymatic strategy for the prepara-
tion of (R)-b-arylalanines employing phenylalanine
aminomutase and ammonia lyase (PAM and PAL)
enzymes has been demonstrated. Candidate PAMs
with the desired (S)-selectivity from Streptomyces
maritimus (EncP) and Bacillus sp. (PabH) were
identified via sequence analysis using a well-studied
template sequence. The newly discovered PabH
could be linked to the first ever proposed biosyn-
thesis of pyloricidin-like secondary metabolites and
was shown to display better b-lyase activity in many
cases. In spite of this, a method combining the
higher conversion of EncP with a strict a-lyase
from Anabaena variabilis (AvPAL) was found to be
more amenable, allowing kinetic resolution of five
racemic substrates and a preparative-scale reaction
with >98% (R) enantiomeric excess. This work
represents an improved and enantiocomplementary
method to existing biocatalytic strategies, allowing
simple product separation and modular telescopic
combination with a preceding chemical step using
an achiral aldehyde as starting material.
antibiotic synthesis in two distinct bacteria.[2–4]
A
PAM-like enzyme is also proposed to form part of the
biosynthesis of the fungal toxin cyclochlorotine in
Talaromyces islandicum based on identification of a
candidate predicted open reading frame within the
biosynthetic gene cluster.[5] The lack of character-
isation with this class of enzyme is surprising given the
detailed structural and biochemical knowledge of
known enzymes[6–10] and the wealth of organisms
shown to produce b-phenylalanine-containing com-
pounds.[2,11–14] Progress in this area is likely hindered
by the poor quality of enzyme sequence annotations
in biological databases – a fact which itself precludes
correct identification biosynthetic pathways for known
and novel bioactive molecules applicable to medical
research. This problem has already been highlighted
within this particular enzyme family by efforts to
uncover novel aminomutases specific to tyrosine
(TAMs).[15]
Despite the small number of investigations of
PAMs, these enzymes are of great interest in a
biocatalytic context where their isomerisation activity
has been used to access enantiopure b-arylalanines.
This class of compounds are known chiral precursors
in synthetic chemistry, where they are used to build
small molecule pharmaceuticals[16,17] as well as more
complex peptide-mimicking therapeutics.[18–20] Exten-
sive studies have been done with both the (S)-selective
Keywords: b-amino acids; biocatalysis; enzyme cas-
cades; aminomutases; ammonia lyases
Introduction
53 Phenylalanine aminomutase (PAM) enzymes, which AdmH from Pantoea agglomerans and (R)-selective
54 catalyse the a- to b-isomerisation of the proteinogenic TcPAM from Taxus canadensis starting with various
55 amino acid phenylalanine (Scheme 1), are seemingly unnatural and more readily-accessible a-phenylala-
56 rare in nature with only a handful of confirmed and nine derivatives to produce either enantiomer of
57 predicted examples reported. The former include various b-phenylalanine analogues.[9,10] Interestingly,
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In light of previous research, the goal of this work
was to expand the knowledge and application of (S)-
PAMs beyond AdmH whilst also developing a prepa-
rative kinetic resolution method complementary to
those already in existence. To do this, we sought to
find and investigate other enzymes in the family with
potentially useful (S)-PAM and/or b-lyase activity to
allow selective removal of a single enantiomer of
various b-arylalanines (S)-1 to yield their counterpart
cinnamic acids 3 with or without additional enzyme
partners (Scheme 1).
Results and Discussion
Scheme 1. Strategies for the kinetic resolution of b-phenyl-
alanine derivatives via selective conversion of a single
enantiomer to the corresponding acrylic acid.
As AdmH is the only enzyme whose (S)-selective
aminomutase activity has been exploited in a biocata-
lytic context, other family members were picked out
for investigation. This was done using the basic local
19 AdmH has been reported to produce small amounts alignment search tool (BLAST) to identify candidates
20 of cinnamate during interconversion of phenylalanine from publicly-available sequence data, using the
21 regioisomers, indicating a- and/or b-lyase side activ- AdmH primary sequence as a query. After sequence
22 ity.[3,10] The absolute requirement for enantiopure alignment, the hits were inspected for the function
23 starting material in these cases (due the strict discriminating residues F455 (reported to confer an
24 enantiopreference of aminomutase enzymes) has also (S)-PAM-specific substrate binding trajectory in
25 been addressed via combination of the (R)-PAM with AdmH)[6] and Q456 which is known to differentiate
26 a promiscuous alanine racemase from Pseudomonas histidine ammonia lyase (HAL) activity from all other
27 putida, allowing racemic substrates to be converted to family members.[15] Any sequences with an E aligning
28 the desired product.[21]
Similarly the near-identical PAM orthologue from HALs, leaving 19 potential mutases, all displaying F at
with position 456 in AdmH were discarded as putative
29
30 the Chinese subspecies of Taxus wallichiana has been the position homologous to 455. One of these was the
31 used in the opposite direction to remove (R)-b- phenylalanine ammonia lyase (PAL) EncP from
32 phenylalanine leaving the pure (S)-enantiomer. To Streptomyces maritimus – an enzyme known to possess
33 shift the equilibrium towards full kinetic resolution (S)-PAM activity[30] but that has never been used in
34 and remove the unwanted a-regioisomer, an ammonia biocatalytic isomerisation reactions. As all the other
35 lyase from Petroselinum crispum was employed in identified sequences were uncharacterised and anno-
36 tandem. This approach is particularly attractive for tated electronically as HALs, genetic context analyses
37 preparative applications as the remaining enantiopure were performed to add evidence to the simple func-
38 product can be simply separated from the cinnamic tional assignment used in this study.
39 acid by-product via acidification and/or ion ex-
The only natural products known to involve (S)-
40 change.[22] Despite this, there exists no PAM-mediated PAM activity in their metabolism are the polyketide
41 method for preparing the opposite enantiomer via (PK) stubomycin[4] and the non-ribosomal peptide
42 destruction of the (S)-form. An enantiocomplementa- (NRP) andrimid.[2] As such, the situation of many of
43 ry biocatalytic strategy does exist in which a b-amino our uncovered genes within operons resembling PK/
44 acid transaminase is employed to convert the un- NRP synthesis clusters was promising, adding weight
45 wanted isomer to the corresponding b-keto acid. This to predictions that they were all misannotated (SI).
46 method, however involves a co-substrate amine ac- The case of one such arrangement, from Bacillus sp.
47 ceptor pyruvate thus producing the a-amino acid strain LM 4–2, could even be linked via a retro-
48 alanine along with the desired product.[23] As amino biosynthetic strategy to the secondary metabolism of a
49 acid mixtures are difficult to separate, a route which pyloricidin-like secondary metabolite (Figure 1). Even
50 results in just the (R)-enantiomer and cinnamate though the narrow spectrum pyloricidin class of anti-
51 would be a desirable addition to the current suite of biotics was isolated from species of Bacillus over a
52 biocatalytic strategies. Additionally, although there decade ago,[11,12] its biosynthesis has since remained a
53 exist truly asymmetric methods for the biocatalytic mystery. Through inspection of the various open
54 production of b-amino acids,[24] these are often only reading frames surrounding the ‘PAM’ locus in strain
55 reported at small scale,[25–27] with low conversion[28] or LM 4–2, genes for the conversion of primary metabo-
56 require further chemical steps[29] to be viable.
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lites into amino acid precursors, construction of the
peptide and even processing and efflux of the final
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Figure 1. The proposed pyloricidin analogue biosynthesis (pab) gene cluster and biosynthetic pathway for pyloricidin-like
natural products from primary metabolites including phenylalanine (L-Phe) and uridine diphosphate N-acetylglucosamine
(UDP-GlcNAc).
25 antibiotic could be identified (SI). This included a ing an a-lyase from Anabaena variabilis (AvPAL)[31]
26 pathway from a nucleotide sugar to the unusual 5- to remove the contaminant 2 and displace the
27 amino-2,3,4-trihydroxy-6-oxohexanoic acid – a precur- equilibrium of the mutase reaction toward complete
28 sor which, to our knowledge, is unique in character- consumption of (S)-1.
29 ised secondary metabolic peptides. Given this in-
30 creased support of our initial assignment, the newly
Table 1. Comparison of the aminomutase and b-lyase pro-
31 dubbed PabH was chosen along with EncP for
pensities of PabH and EncP on racemic b-phenylalanine
32 investigation of their potential to resolve enantiomers
derivatives (rac-1a–m).
33 of b-phenylalanine derivatives.
34
The synthetic gene encoding codon-optimised
35 PabH was obtained and subcloned to a create a
36 construct analogous to that used in previous inves-
37 tigations with EncP.[27,30] Both plasmids were used to
38 obtain E. coli BL21(DE3) whole cell catalysts har-
39 bouring the relevant overproduced PAM. Biotransfor-
40 mations were performed with 5 mM rac-1 and 50 mg
41 whole cells in 1 mL. The reaction temperature was set
42 to 308C at pH 8.0, as these are conditions where EncP
43 is known to display mutase activity.[30] After 24 hours
44 the conversion of EncP was found to be superior to 1d
45 that of PabH in all cases, even achieving ~50%
46 conversion with substrates 1e and 1i (Table 1). In all
47 cases, however, the enzymes were found to act as both
48 aminomutases and b-lyases to varying degrees, con-
49 taminating all mixtures with a-amino acid 2. The most
50 promising reaction in this respect was 1h with PabH,
51 giving about half the conversion desired but a
52 mutase:lyase ratio of 12:88 – far lower than any such
53 ratio with EncP. In spite of this, attempts to improve
54 conversion and product ratios, such as additional
55 reaction time, cell loading and elevated temperature/
56 pH showed no improvement (see SI). As such the
57 cascade strategy was attempted with EncP by employ-
PabH
Conv.
[%][a]
EncP
Conv.
[%][a]
Subs.
R
Ratio
Ratio
2:3[a]
2:3[a]
1a
1b
1c
H
2-F
3-F
4-F
2-Cl
4-Cl
3-Br
4-Br
2-NO2
3-NO2
4-MeO
2-Me
3-Me
11
14
12
6
20
5
23
12
15
1
79:21
92:8
92:8
39
46
39
37
55
30
46
38
55
37
23
42
27
46:54
77:23
64:36
52:48
81:19
91:9
57:43
69:31
73:27
98:2
58:42
90:10
73:27
12:88
12:88
47:53
>99:1
12:88
23:77
39:61
1e
1f
1g
1h
1i
1j
1k
1l
3
8
2
46:54
62:28
63:37
1m
Expt. cond.: 5 mM 1a–m, 50 mgmLÀ1 wet cell weight,
100 mM borate buffer, pH 8.0, 308C, 24 h.
[a] Conversion and product ratios determined by HPLC on a
non-chiral phase.
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Addition of 25 mg lyophilised AvPAL whole cells
to the reaction formula was shown to increase
conversion in most cases, with only 4 of the 13
substrates giving below 40% conversion and the best 5
biotransformations suggesting quantitative conversion
(Table 2). The purity of the amino acid component of
each reaction was also found to be greatly improved,
with b:a ratios all above 90:10. Unfortunately even
small traces of the unwanted regioisomer 2 in each
Table 3. Effect of pH on the kinetic resolution of racemic b-
phenylalanine (rac-1c) by AvPAL and EncP.
Conv.
[%][a]
ee of 1
[%][b]
Ratio
pH
1c:2c[a]
8.0
9.0
10.0
11.0
12.0
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50
32
31
>99 (R)
>99 (R)
>99 (R)
47 (R)
92:8
96:4
99:1
99:1
10 case was found to contravene chiral analysis of the
11 desired product (R)-1, giving overlapping peaks with
12 the HPLC method employed. As such, only the
13 enantiomeric excess value for the biotransformation
14 indicating complete consumption of the (S)-enantiom-
15 er and only a trace of a-amino acid could be
16 calculated (1c). This was found to be >99% (R) as
17 predicted. Subsequently, as an investigation of the
18 scalability of this reaction, the biotransformation was
19 repeated with 10 mM 1c. Although conversion was
20 unaltered there was evidence of accumulation of
21 amino acid 2c, again complicating calculation of
22 enantiomeric excess and indicating that the lyase
46 (R)
>99:1
Expt. cond.: 10 mM 1c, 50 mgmLÀ1 EncP wet cell weight,
25 mgmLÀ1 AvPAL dry cell weight, 100 mM borate buffer,
pH 8.0–12.0, 308C, 24 h.
[a] Conversion and product ratios determined by HPLC on a
[b]
non-chiral phase. Enantiomeric excesses determined by
HPLC on a chiral phase.
23 activity of the enzyme combination was insufficient to ducted. This revealed an increase in apparent lyase
24 remove this side product fully.
25
activity accompanied by a decrease in mutase activity
within the system with increasing pH (Table 4). The
two reactions were seemingly balanced at an inter-
mediate pH of 10 where the b:a ratio was better than
at lower values, but overall conversion was not
affected detrimentally by excessively alkaline condi-
tions. Having established a more optimal pH buffer
composition for the cascade, the intensification of the
reaction was continued via sequential biotransforma-
tions with increasing substrate concentration (Ta-
ble 3). Although conversion values continued to
indicate full kinetic resolution had been reached in all
experiments, chiral analysis of the reaction mix
revealed that at 100 mM traces of the unwanted (S)-
1c could be detected. As such no higher concentra-
tions were tested. In spite of this, the enantiomeric
excess values calculated even for this incomplete
reaction were found to be 98% (R).
Having demonstrated promising intensification of
the kinetic resolution strategy, a preparative scale
synthesis was attempted with 100 mg rac-1c, 300 mg
EncP wet whole cells and 150 mg AvPAL lyophilisate
in 6 mL pH 10 borate buffer. Following isolation after
a 24 hour incubation at 308C, 40.5 mg product was
obtained and shown to be >95% pure with an ee
>98% (R) by HPLC. This represents a 25-fold
improvement in space-time yield over the previously
reported, enantiocomplementary PAM-PAL cascade,
requiring one third the reaction time and just a fifth of
the volume (achieved by increasing the starting
material 100 mg vs. 50 mg and catalyst loading
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27
28
Table 2. Kinetic resolution of racemic b-phenylalanine de-
rivatives (rac-1a–m) via an EncP-AvPAL cascade.
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30
31
32
Subs.
R
Conv.
[%][a]
Ratio
33
34
35
36
37
38
39
40
1:2[a]
1a
1b
1c
1d
1e
1f
H
2-F
3-F
4-F
2-Cl
4-Cl
3-Br
4-Br
2-NO2
3-NO2
4-MeO
2-Me
3-Me
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43
51
53
45
47
54
55
17
15
15
45
24
98:2
97:3
99:1
97:3
99:1
98:2
91:1
96:4
93:7
92:8
93:7
92:8
99:1
41 1g
1h
1i
1j
1k
1l
1m
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43
44
45
46
47
48 Expt. cond.: 5 mM 1a–m, 50 mgmLÀ1 EncP wet cell weight,
25 mgmLÀ1 AvPAL dry cell weight, 100 mM borate buffer,
49
pH 8.0, 308C, 24 h.
50
51
52
[a] Conversion and product ratios determined by HPLC on a
non-chiral phase.
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54
55
To ascertain whether the decrease in amino acid 4.5 mgmgÀ1 vs. 0.15 mgmgÀ1).[22]
56 purity was due to the suboptimal conditions with
Furthermore, we sought to investigate the in situ
57 regard to AvPAL activity,[31] a pH profile was con- chemo-enzymatic combination of substrate synthesis
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fashion, with no need for isolation of the intermediate
rac-1c (as already demonstrated previously with the
one-pot telescopic synthesis of a-amino acids by
coupling of cinnamic acids synthesis and PAL-medi-
ated amination [33–35]).
Table 4. Effect of varying substrate concentration on the
kinetic resolution of rac-1c by AvPAL and EncP.
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8
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It is worth noting that the synthesis of rac-1c from
4 produces considerable amounts of the corresponding
cinnamic acid 3c as a by-product, and to minimise this
side-reaction different ammonium sources, substrate
concentrations and temperatures were tested (the best
conditions were found to be 1 M starting material,
2 equiv. of ammonium formate, 608C, 6 h), giving a
ratio of 88:12 of rac-1c:3c. With complete kinetic
resolution of this mixture upon dilution (to 20 mM) in
the EncP-AvPAL tandem biotransformation, a yield
of 44% (of a possible 50%) could be achieved. Other
dilutions (to 50, 75 and 100 mM) were found to give
significant amounts of contaminating 2c following
kinetic resolution, possibly due to the sensitivity of
AvPAL to a component of the chemical reaction
mixture. Nevertheless, our formal asymmetric syn-
thesis (over the two steps) is a particularly elegant
combination as the by-product of the chemical and
initial enzymatic transformations are identical to the
product of the final biocatalyst. In this way, no further
Substrate conc.
[mM]
Conv.
[%][a]
ee of 1c
[%][b]
Ratio
1c:2c[a]
10
20
35
50
60
75
100
51
50
50
51
50
50
49
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
98 (R)
99:1
99:1
99:1
99:1
97:3
98:2
98:2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Expt. cond.: 10–100 mM 1c, 50 mgmLÀ1 EncP wet cell
weight, 25 mgmLÀ1 AvPAL dry cell weight, 100 mM borate
buffer, pH 10.0, 308C, 24 h.
[a] Conversion and product ratios determined by HPLC on a
non-chiral phase.
[b] Enantiomeric excesses determined by HPLC on a chiral
phase.
26 and kinetic resolution, with the advantage of starting complexity is introduced to the general work-up
27 from the inexpensive and commercially available procedure required to isolate the enantiopure b-amino
28 benzaldehydes. However, since the synthesis of b- acid.
29 amino acids requires higher temperatures and an
30 organic solvent (typically refluxing EtOH),[32] a tele-
31 scopic approach was required to overcome the issue of
Conclusion
32 incompatible conditions for the two steps (Scheme 2). In summary, we have reported the investigation of two
33 Using again the preparation of (R)-1c as a model enzymes with respect to their previously unexploited
34 reaction, rac-1c was prepared from m-fluorobenzalde- (S)-selective aminomutase/b-lyase activity, as applied
35 hyde 4, malonic acid and an ammonium source in to the kinetic resolution of racemic b-arylalanines.
36 ethanol, then the crude mixture was diluted with the The biocatalysts chosen for comparison were EncP, a
37 biotransformation buffer (to ~6 mM final concentra- known ammonia lyase with unexplored (S)-PAM side
38 tion of rac-1c), cells were added and the mixture activity, and PabH, an enzyme discovered, linked to
39 incubated at 308C until complete resolution. This antibiotic synthesis and characterised for the first time
40 approach was shown to afford (R)-1c with an enantio- in this work. An initial comparison of the two
41 meric excess of 99% from 4, demonstrating the simple candidates revealed PabH to be a better b-lyase than
42 combination of the two steps in a one-pot telescopic EncP making it a more promising candidate for single
43
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45
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50
51
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53
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biocatalyst kinetic resolution methods. However, low-
er activity and unsuccessful optimisation attempts led
to the use of EncP instead as part of a two enzyme
cascade. This system was shown to be applicable
across a of range amino acid starting materials,
tunable in terms of amino acid product ratios and
scalable up to 100 mM substrate concentration and
100 mg substrate loading with excellent resolution in
most cases. This highly selective method represents an
improved and complementary route to previously
reported PAM-PAL combinations and can be easily
integrated with a chemical step in a one-pot fashion to
produce enantiopure b-amino acid product from a
simple achiral starting material. In addition to the
biotransformation strategies reported, the method
Scheme 2. Synthesis of (R)-1c from the corresponding ben-
zaldehyde 4, via a one-pot telescopic chemo-enzymatic
cascade with 3c as the only by-product.
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(100 mM, pH 8.0) for the biotransformation, performed as
described in the previous section.
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used to mine sequence databases in this study could
be taken further to uncover more of nature’s elusive
(S)-PAMs, potentially with improved biocatalytic
properties and associated with additional, as of yet
undiscovered biosynthetic pathways for bioactive
secondary metabolites.
Purification of amino acid (R)-1c. The reaction mixture was
acidified to pH <2.0 by addition of aqueous H2SO4 (10% w/
v) and centrifuged (4000 rpm, 10 min, 48C) to remove cells
and insoluble components. Dowexꢁ 50WX8 hydrogen form
(2.5 g) was washed with deionised water (50 mL) and
aqueous H2SO4 (25 mL, 10% w/v). The acidified supernatant
from the biotransformation was loaded onto the resin
(1 mLminÀ1). The resin was washed repeatedly with deion-
ised water (until pH ~7.0) and the product was eluted with
aqueous NH4OH (30 mL, 10% w/v). Fractions containing the
Experimental Section
Bacterial transformations. Plasmids pET-28a-EncP and pET-
16b-AvPAL (containing codon-optimised genes encoding
EncP and AvPAL, respectively) were used as previously
described.[27,34] The codon-optimised gene encoding PabH
was ordered from Thermo Fisher Scientific with the inclusion
of an upstream NdeI and downstream XhoI endonuclease
restriction site. The gene was then subcloned into a pET-28a
expression vector following a procedure used previously to
form the pET-28a-EncP construct.[30] All three plasmids were
then used as previously described to transform Escherichia
coli BL21(DE3) in accordance with the manufacturer’s
protocol, producing single colonies on LB-agar microbiolog-
ical plates supplemented with the appropriate antibiotic
product were pooled and evaporated in
a centrifugal
evaporator, to afford the title compound (30.6 mg, 44%)
Acknowledgements
This work was funded by the European Union’s 7th Frame-
work program FP7/2007-2013 under grant agreement
no. 289646 (KYROBIO). S. T. A. and F. P. were supported
by the Biotechnology and Biological Sciences Research
Council (BBSRC) and Glaxo-SmithKline (GSK) under the
Strategic Longer and Larger (sLoLa) grant initiative ref. BB/
K00199X/1. N. J. T. thanks the Royal Society for a Wolfson
Research Merit Award. Thanks must also go to Daniel Ward
for research assistance in the laboratory.
22 (kanamycin for EncP and PabH, ampicillin for AvPAL).
23
24
25
Preparation of whole cell biocatalysts. LB medium (5 mL,
supplemented with the appropriate antibiotic) was inocu-
lated with a single colony of E. coli BL21(DE3) containing
the suitable plasmid and grown for 16 h at 378C and
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250 rpm. This starter culture was then used to inoculate LB-
based autoinduction medium[36] (800 mL, supplemented with
the appropriate antibiotic), which was incubated at 188C and
250 rpm for 4 days. The cells were pelleted by centrifugation
(4000 rpm, 2 min) and separated from the supernatant for
storage of the wet cell mass at À208C until further use. In
the case of AvPAL, a lyophilised dry cell powder formulation
was used as reported previously.[34]
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Kinetic Resolution of Aromatic b-Amino Acids Using a
Combination of Phenylalanine Ammonia Lyase and
Aminomutase Biocatalysts
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