Production of L-tryptophan by enantioselective hydrolysis of d,l-tryptophanamide using a newly isolated bacterium

Article abstract of DOI:10.2478/s11696-013-0389-6

Bacterial strain ZJB-09211 capable of amidase production has recently been isolated from soil samples. The strain is able to asymmetrically hydrolyze l-tryptophanamide from d,l-tryptophanamide to produce l-tryptophan in high yield and with excellent stereoselectivity (enantiomeric excess > 99.9 %, and enantiomeric ratio > 200). Strain ZJB-09211 has been identified as Flavobacterium aquatile based on the cell morphology analysis, physiological tests, and the 16S rDNA sequence analysis. Optimization of the fermentation medium led to an about six-fold increase in the amidase activity of strain ZJB-09211, which reached 501.5 U L^-1. Substrate specifity and stereoselectivity investigations revealed that amidase of F. aquatile possessed a broad substrate spectrum and high enantioselectivity.

Full text of DOI:10.2478/s11696-013-0389-6

Chemical Papers 67 (10) 1262–1270 (2013)
DOI: 10.2478/s11696-013-0389-6
ORIGINAL PAPER
Production of L-tryptophan by enantioselective hydrolysis
of D,L-tryptophanamide using a newly isolated bacterium
a,b
a,bJian-Miao Xu, a,bBen Chen,
Yuan-Shan Wang, a,bYu-Guo Zheng*
aInstitute of Bioengineering, bEngineering Research Center of Bioconversion and Biopurification of Ministry of Education,
Zhejiang University of Technology, 310014 Hangzhou, China
Received 19 November 2012; Revised 3 February 2013; Accepted 5 February 2013
Bacterial strain ZJB-09211 capable of amidase production has recently been isolated from
soil samples. The strain is able to asymmetrically hydrolyze L-tryptophanamide from D,L-
tryptophanamide to produce L-tryptophan in high yield and with excellent stereoselectivity (enan-
tiomeric excess > 99.9 %, and enantiomeric ratio > 200). Strain ZJB-09211 has been identified
as Flavobacterium aquatile based on the cell morphology analysis, physiological tests, and the 16S
rDNA sequence analysis. Optimization of the fermentation medium led to an about six-fold increase
in the amidase activity of strain ZJB-09211, which reached 501.5 U L−1. Substrate specifity and
stereoselectivity investigations revealed that amidase of F. aquatile possessed a broad substrate
spectrum and high enantioselectivity.
c
ꢀ 2013 Institute of Chemistry, Slovak Academy of Sciences
Keywords: L-tryptophan, Flavobacterium aquatile, amidase, enantioselective biocatalysis
Introduction
ity and, therefore, have become promising tools for the
synthesis of chiral carboxylic acids and their deriva-
tives (Shaw et al., 2003; Martinkova & Mylerova, 2003;
Wang et al., 2010). In particular, amino-acid ami-
dases can steroselectively hydrolyze L- or D-amino-acid
amide to the corresponding chiral amino acids and
ammonia. Because racemic amino-acid amides can be
easily synthesized from aldehydes, hydrogen cyanides,
and ammonium (Komeda & Asano, 2008; Asano &
Yamaguchi, 2005a, 2005b), amidase-catalyzed asym-
metrical hydrolysis of amino-acid amides presents a
promising method for the production of optically pure
amino acids. Although various microbial amidases
have been reported, their enzymatic characteristics
vary remarkably (Wang et al., 2008). The amidase ac-
tivity is also significantly affected by the culture con-
ditions, which means that optimization of the culture
medium is a crucial requirement (A¸cıkel et al., 2010).
Here, isolation of a microorganism that can pro-
duce L-Trp in high yields and with excellent enan-
tioselectivity through asymmetric hydrolysis of the
L-enantiomer present in racemic tryptophanamide
L-Tryptophan (L-Trp) has a broad range of ap-
plications in the pharmaceutical and food industries,
and as a precursor in the preparation of other chiral
compounds (Azuma et al., 1993; Ko¸caba et al., 2006;
Mateus et al., 2004). Many chemical and biological
methods for L-Trp production have been developed.
Current biological methods, which include fermenta-
tion and enzymatic synthesis, are less economically at-
tractive than the chemical ones (Katsumata & Ikeda,
1993; Ikeda, 2006; Winnicka & Kan´ska, 2009; Eggers
et al., 1988). The fermentation route suffers from low
yields; whereas the enzymatic route requires a chiral
substrate as the starting material and is hampered
by the inhibitory activity of another substrate, indole.
However, the chemical methods usually result in the
formation of mixtures of L-Trp and D-tryptophan (D-
Trp).
Therefore, improved biological methods for L-Trp
production involving stereoselective enzymes are being
sought for. Amidases possess excellent enantioselectiv-
*Corresponding author, e-mail: zhengyg@zjut.edu.cn

J. M. Xu et al./Chemical Papers 67 (10) 1262–1270 (2013)
1263
Fig. 1. Production of L-Trp by amidase-mediated stereospecific hydrolysis of D,L-tryptophanamide.
is reported (Fig. 1). Also, taxonomic study of the
microorganism and the optimization of the culture
medium for amidase production are presented. In ad-
dition, the substrate specificity of amidases was inves-
tigated.
The resulting culture broth was transferred to a fresh
enrichment medium with the inoculum size of 5 vol. %.
After the enrichment procedure was performed three
times, the resulting cultures were diluted, spread on
the enrichment agar medium and incubated at 30◦C
for three days.
Experimental
Strains growing on the enrichment agar medium
were three times purified by sub-culturing on the en-
richment agar medium and then cultured aerobically
in the fermentation medium at 30◦C for 48 h. The
culture broth was centrifuged at 4◦C, 10000 min−1
for 10 min, and the pellets were washed with saline
(0.85 mass % NaCl). The washed cells were used in
the biotransformation.
General
Sixty soil samples collected from different loca-
tions in China, were used for amide-degrading bacte-
ria screening. D,L-tryptophanamides were purchased
from HanHong Chemicals (China). L-Trp, D-Trp,
and racemic tryptophan were from J&K Chemicals
(China). Other chemicals were of analytical reagent
grade and obtained from various commercial sources.
Taxonomic characterization of strain
ZJB-09211
Media and culture conditions
Cell morphology of strain ZJB-09211 was inves-
tigated using a light microscope (Olympus CH20,
Japan) and a transmission electron microscope (Phi-
lips-XL30, The Netherlands). Physiological character-
ization of the strain was carried out by indole produc-
tion, starch hydrolysis, and nitrate reduction testing
as well as by examining the carbon source utilization
using a standardized micromethod (ATB Expression
ID32 GN, bioMérieux, France).
Components of the enrichment medium were as fol-
lows: 1.0 g L−1 of K2HPO4, 1.0 g L−1 of KH2PO4,
0.2 g L−1 of MgSO4 ·7H2O, 0.01 g L−1 of FeSO4 ·
7H2O, 1.0 g L−1 of NaCl, and 3.0 g L−1 of D,L-
tryptophanamide. The enrichment agar medium con-
tained 20 g L−1 of agar. pH was adjusted to 7.5 and
the medium was then autoclaved at 121◦C for 20 min.
Components of the fermentation medium were as
follows: 10.0 g L−1 of glucose, 2.0 g L−1 of pepton,
5.0 g L−1 of beef extract, 1.0 g L−1 of azepan-2-one
(caprolactam), 1.0 g L−1 of K2HPO4, 1.0 g L−1 of
KH2PO4, 0.2 g L−1 of MgSO4 · 7H2O, 0.01 g L−1
of FeSO4 · 7H2O, and 1.0 g L−1 of NaCl. pH of the
fermentation medium was adjusted to 7.5 and the
medium was then autoclaved as above.
16S rDNA sequence determination and phylo-
genetic analysis
Chromosomal DNA of a pure colony of stain ZJB-
09211 was extracted according to the slightly mod-
ified procedure of Wilson et al. (1997). The PCR
amplification of 16S rDNA was carried out with
a TProfessional/Standard thermocycler (Biometra,
Germany) using a universal primer set: p16S-8 (5’-
AGAGTTTGATCCTGGCTCAG-3’) and p16S-1510
(5’-AAGGAGGTGATCCAGCCGCA-3’) (Shigema-
tsu et al., 2003). The PCR products were cloned into
the pMD-18T vector (Takara, Japan) using the T/A
cloning procedure (Fournand et al., 1998; Jakoby &
Fredericks, 1964). Related sequences were retrieved
Isolation of tryptophanamide-degrading
bacteria
Microorganisms were isolated from soil samples by
an enrichment method. Briefly, the soil samples were
suspended in sterile water and then cultured on a ro-
tary shaker at 30◦C, and 150 min−1 for three days.

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J. M. Xu et al./Chemical Papers 67 (10) 1262–1270 (2013)
from GenBank (National Center for Biotechnology In-
formation, NCBI, USA) using the BLAST program,
and were aligned with the CLUSTAL W algorithm,
version 1.81 (Thompson et al., 1994). An unrooted
phylogenetic tree of homologous 16S rDNA sequences
was constructed by the neighbor-joining method using
MegAlign (DNASTAR, Madison, USA).
ions (Mg2+, Cu2+, C−o2+, Mn2+, Fe3+, Ag+, Ni2+
,
Ca2+, Ba2+, Fe2+, NO3 , Cl−, SO24−, HPO42−, H2PO4−,
CO23−, and SO23−).
HPLC analysis
D,L-Tryptophanamide and tryptophan were identi-
fied and quantified by HPLC (U3000, Dionex, USA)
using a C18 column (250 mm × 4.6 mm, Dionex) at
40◦C. The column was eluted using the following sol-
vents: acetonitrile/water (ϕr = 3 : 7) for 0–2 min;
acetonitrile/10 mM KH2PO4 (ϕr = 1 : 4) for 2–5 min;
and acetonitrile/water (ϕr = 3 : 7) for 5–12 min. The
flow rate was 1.0 mL min−1, and the detection wave-
length was 278 nm. The retention times for L-Trp and
D,L-tryptophanamide were 2.89 min and 5.42 min, re-
spectively. Separation of L-Trp was carried out in a
Chirobiotic column (250 mm × 4.6 mm, Astec, USA)
at 30◦C. The elution was performed with 80 vol. %
acetonitrile containing 0.2 vol. % of acetic acid at the
flow rate of 1.0 mL min−1. The retention times for
L-Trp and D-Trp were 9.28 min and 10.23 min, re-
spectively.
Preparation of amidase-containing bacterial
cells
Amidase-containing cells for biotransformation ex-
periments were prepared by growing strain ZJB-09211
in 250-mL flasks containing 50 mL of the fermentation
medium and 2 vol. % of inoculum. The flasks were in-
cubated at 30◦C and 150 min−1 for 48 h. The resulting
culture was centrifuged at 4◦C and 12000 min−1 for
15 min, and the cells were collected for further study.
Biotransformation
Hydrolysis of tryptophanamides to tryptophan was
carried out in 50 mL Erlenmeyer flasks with a screw
cap containing 10 mL of distilled water, 20 mM D,L-
tryptophanamide and amidase-containing cells (pre-
pared from a 30-mL culture as described above). The
flasks were incubated at 150 min−1 and 30◦C for 10
min, and the reaction was terminated by adding 20 µL
of 6 M HCl. The reaction mixture was centrifuged at
10000 min−1 and the tryptophan concentration in the
supernatants was quantified by HPLC.
Results and discussion
Screening of microorganisms
In recent years, the use of microbial amidases
asymmetrically hydrolyzing racemic amides has be-
come a promising method for the production of chi-
ral carboxylic acids (Wang et al., 2010; Zheng et al.,
2012; Yang et al., 2011). Amidase-producing microor-
ganisms have usually been isolated by enrichment
procedures using culture media containing a specific
amide (target substrate or a structural analogue) as
the sole source of nitrogen. In this study, a similar
strategy was followed by subjecting 60 soil samples to
enrichment cultivation in a medium containing D,L-
tryptophanamide (3 g L−1) as the sole nitrogen source.
The initial screening resulted in the isolation of three
amidase-producing microbial strains which were then
tested in biotransformation experiments. In these ex-
periments, the released L-Trp was monitored by HPLC
analysis. Strain ZJB-09211 showed the highest activ-
ity in the biotransformation assays (81.45 U L−1) and
was selected for further investigation.
One unit of the enzyme activity was defined as the
amount of cells required to form 1 µmol of L-Trp per
minute under the experimental conditions.
Substrate specificity
Amidase activity of the strain towards various
amides was examined in the biotransformation exper-
iments in which the substrate concentration and the
amount of wet cells were 20 mM and 0.5 g, respec-
tively. The reactions were carried out as described
above.
Optimization of fermentation medium
To select optimal composition of the fermenta-
tion medium for cell growth and amidase production,
variations in the following elements were tested: car-
bon sources (glucose, lactose, sodium citrate, sorbitol,
glycerin, starch, maltose, sucrose, mannitol, dextrin,
and gelatin, all in 1.0 mass %, or 1.0 vol. % concen-
tration), nitrogen sources (peptone, corn steep, beef
extract, yeast extract, glutamate, NH4Cl, (NH4)2SO4,
and NH4NO3, all in 0.7 mass %, or 0.7 vol. % concen-
tration), amidase inducers (caprolactam, phenylace-
tonitrile, acetonitrile, butyronitrile, acetamide, and
niacinamide, all in 1.0 g L−1 concentration) and
Identification of strain ZJB-09211
Colonies of the selected strain were primrose yel-
low, smooth, convex, translucent, and mucous on the
enrichment medium agar. The cells were short, and
rod-like, and their dimensions were 0.3–0.5 µm × 0.8–
1.3 µm (Fig. 2). The microorganism was a Gram neg-
ative and non-sporulating bacterium able to produce
indole, hydrolyze starch and reduce nitrate. Further
physiological characterization of the strain was car-

J. M. Xu et al./Chemical Papers 67 (10) 1262–1270 (2013)
1265
ried out using the ATB Expression system (ID32 GN,
bioMérieux, France); the results (Table 1) indicated
that there was a 99.9 % probability that the mi-
croorganism belonged to the species Flavobacterium
aquatile.
To confirm such an identification, a 16S rDNA frag-
ment from the strain was amplified by PCR and its
nucleotide sequence (1587 bp) was determined. Then,
the 16S rDNA sequence obtained from ZJB-09211 was
compared with similar sequences from other bacteria,
and an unrooted phylogenetic tree was constructed
by the neighbor-joining method (Fig. 3). This anal-
Fig. 2. Transmission electron micrograph of strain ZJB-09211
grown at 30◦C for 24 h.
Fig. 3. Phylogenetic tree based on 16S rDNA sequences, constructed by the neighbor-joining method, showing the relationship
between strain ZJB-09211 and representatives of some related taxa. Numbers in parentheses are accession numbers of
published sequences.
Table 1. Characteristics of strain ZJB-09211
Characteristics
ZJB-09211
Characteristics
ZJB-09211
Gram-staining
Oxidase
Citrate
–
+
–
D-Ribose
2-Ketogluconate
Inositol
–
–
–
Itaconic acid
Histidine
D-Maltose
D-Mannitol
Arabinose
Sodium acetate
3-Hydroxybutyric acid
Propionic acid
D-Sorbitol
Salicin
Lactic acid
–
Glucogen
L-Fucose
Capric acid
Alanine
5-Ketogluconate
D-Glucose
Suberic acid
Serine
Sucrose
L-Rhamnose
Proline
N-Acetylglucosamine
Sodium malonate
Nitrate reduction
Indole
–
–
+
+
–
–
+
–
+
–
+
+
+
–
+
+
–
+
–
–
+
–
+
+
+
–
Valeric acid
4-Hydroxybenzoic acid
3-Hydroxybenzoic acid
D-Melibiose
–
+
–
+ – Positive, – – negative.

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J. M. Xu et al./Chemical Papers 67 (10) 1262–1270 (2013)
Fig. 4. Effects of different carbon sources (a) and combinations of two carbon sources (b) on amidase activity and biomass. Carbon
sources (Fig. 5a); 1 – glucose, 2 – lactose, 3 – sodium citrate, 4 – sorbitol, 5 – glycerin, 6 – starch, 7 – maltose, 8 – sucrose,
9 – mannitol, 10 – dextrin, and 11 – gelatin. Combinations of two carbon sources (Fig. 5b); 1 – glycerin and gelatin, 2 –
lactose and gelatin, 3 – starch and gelatin, 4 – sucrose and gelatin, and 5 – maltose and gelatin; – activity units,
biomass.
–
ysis confirmed a strong relationship between strain
ZJB-09211 and the members of the genus Flavobac-
terium. In particular, ZJB-09211 was closely clustered
in the phylogenetic tree with Flavobacterium sp. WB
(GenBank accession No. AM167560.1), and their 16S
rDNA sequences were identical to 99 %. Results of the
phylogenetic analysis were consistent with the phys-
iological tests, the microorganism was therefore des-
ignated as Flavobacterium aquatile ZJB-09211. The
bacterium was deposited in the China Center for Type
Culture Collection (Wuhan, China) as strain CCTCC
M 2011354.
Effect of different carbon sources on growth
and amidase production of strain ZJB-09211
Carbon source has a crucial role in the determi-
nation of the growth and metabolic rates of microor-
ganisms. Therefore, as a first step in the optimization
of the fermentation medium, eleven carbon sources
were tested for their effect on the growth and amidase
activity of the ZJB-09211 strain (Fig. 4a). High en-
zyme activities were obtained with sucrose, sorbitol, or
glycerin, whereas maximum cell growth was achieved
with gelatin. The results suggest that a combination
of different carbon sources leads to even higher yields
of both cell growth and amidase production. There-
fore, the effects of various combinations of gelatin
with other carbon sources were investigated (Fig. 4b).
The maximum enzyme activity (238.21 U L−1) was
achieved with sucrose and gelatin. To determine the
optimum ratio of sucrose and gelatin for amidase pro-
duction, the effects of different mole ratio of carbon
atoms of the two components were tested. As shown
in Fig. 5a, the highest amidase production (396.20
U L−1) was obtained when the mole ratio of gelatin
carbon atoms was 50 %. To determine the optimum
concentration of the gelatin–sucrose combination for
amidase production, different carbon concentrations
Fig. 5. Effect of different ratios of gelatin (a) and gelatin–
sucrose carbon source concentrations (b) on amidase
activity and biomass; – activity units, – biomass.
(from 0.15 M to 0.60 M) were tested while keeping the
50 % mole ratio of gelatin carbon atoms. Maximum
amidase production (418.66 U L−1) was observed for
carbon concentrations of 0.40 M (Fig. 5b), which cor-
responds to 5.48 g L−1 of sucrose and 6.25 g L−1 of
gelatin. Therefore, these concentrations of sucrose and
gelatin were used as carbon source in the following
tests.

J. M. Xu et al./Chemical Papers 67 (10) 1262–1270 (2013)
1267
Fig. 6. Effects of different nitrogen sources (a) and beef extract
concentrations (b) on amidase activity and biomass.
Nitrogen source; 1 – peptone, 2 – corn steep, 3 – yeast
extract, 4 – beef extract, 5 – NH4Cl, 6 – (NH4)2SO4, 7 –
NH4NO3, 8 – glutamate, 9 – beef extract and peptone,
and 10 – yeast extract and peptone; – activity units,
– biomass.
Fig. 7. Effect of different inducers (a) and caprolactam concen-
trations (b) on amidase activity and biomass. Inducer;
1 – control (no inducer), 2 – caprolactam, 3 – acry-
lamide, 4 – acetamide, 5 – benzamide, 6 – cyclopropane
carboxamide, 7 – methionine amide, 8 – nicotinamide,
and 9 – diamino-benzamide;
biomass.
– activity units,
–
Effect of different nitrogen sources on cell
growth and amidase production
Because organic and inorganic nitrogen sources
play an essential role in microbial production of en-
zymes, the effects of ten different nitrogen sources on
the amidase activity were evaluated (Fig. 6a). The
maximum enzyme activity (454.77 U L−1) was ob-
tained with beef extract, whereas both amidase ac-
tivity and biomass were very low when corn steep or
glutamate was used as the nitrogen source. In addi-
tion, the use of NH4Cl, (NH4)2SO4, or NH4NO3 as
the nitrogen source led to low enzyme activity (below
210.98 U L−1) and biomass (below 2.11 U L−1), which
indicates that F. aquatile ZJB-09211 cannot efficiently
utilize inorganic nitrogen. The optimum concentration
of beef extract for amidase production was found to be
8 U L−1, which yielded the enzyme activity of 488.35
U L−1 (Fig. 6b). Therefore, 8 g L−1 of beef extract
were used as the nitrogen source in the following ex-
periments.
ture media induce or enhance microbial amidase pro-
duction. For this reason, the effects of eight differ-
ent amides on the growth and amidase production of
F. aquatile ZJB-09211 were examined. All the tested
amides improved the enzyme production, and the
maximum amidase activity was achieved with capro-
lactam (Fig. 7a). It was found that the optimum con-
centration of caprolactam for both cell growth and
amidase production was 1.0 g L−1, which resulted in
the enzyme activity of 478.11 U L−1 (Fig. 7b). There-
fore, 1.0 g L−1 of caprolactam was used as the inducer
in the experiments.
Effect of various ions on cell growth and ami-
dase production
Microorganisms require certain metal ions for cell
growth and metabolite biosynthesis. It was found that
the presence of Mg2+, Cu2+, Co2+, Mn2+, or Fe3+
leads to low amidase activity, whereas the presence
of Ag+, Ni2+, Ca2+, Ba2+, and Fe2+ resulted in high
enzyme activity (Table 2). The maximum enzyme ac-
tivity was achieved with Ag+ (which was supplied in
Effect of different inducers on cell growth and
amidase production
It is known that some amides when added to cul-

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J. M. Xu et al./Chemical Papers 67 (10) 1262–1270 (2013)
Table 2. Effect of different metal ions on cell biomass and amidase activity
Metal ion
Biomass/(g L−1
)
Activity units/(U L−1
)
Relative activity/%
Blank
Mg2+
Cu+
3.58
4.00
3.45
3.43
3.25
3.48
2.68
3.23
3.28
3.20
2.88
3.28
3.18
314.56
228.32
249.01
95.63
248.24
382.26
373.94
464.85
259.97
401.71
361.47
478.17
438.79
100.00
72.58
79.16
30.40
78.92
121.52
118.87
147.78
82.64
127.70
114.91
152.01
139.49
Co2+
Mn2+
Fe2+
Zn2+
Ni2+
Fe3+
Ba2+
Al3+
Ag+
Ca2+
Table 3. Effect of different anions on cell biomass and amidase activity
Anionic
Biomass/(g L−1
)
Activity units/(U L−1
)
Relative activity/%
Blank
3.56
3.27
3.48
3.18
3.10
3.40
3.35
3.20
3.28
284.13
468.23
486.95
298.10
370.43
284.14
282.42
328.82
300.72
100.00
164.80
171.38
104.92
130.37
100.00
99.39
AgNO3
NaNO3
NaCl
Na2SO4
Na2HPO4
NaH2PO4
Na2CO3
Na2SO3
115.73
105.84
the form of AgNO3). However, Ag+ has not been pre-
viously described as an activator of amidase produc-
tion in other microorganisms. Moreover, it inhibited
cell growth in our experiments. Therefore, it is pos-
sible that the NO−3 anions (and not the metal ion
Ag+) in AgNO3 are promoting the amidase produc-
tion. The effects of different anions (NO−3 , Cl−, SO42−
,
HPO24−, H2PO−4 , CO32−, and SO23−) on the enzyme
activity were therefore examined and the results show
that amidase production was significantly improved by
NaNO3 (Table 3). Optimum enzyme activity (501.50
U L−1) was obtained with 20.0 mM NaNO3 (Fig. 8),
and this concentration was used in further experi-
ments.
Fig. 8. Effect of different NaNO3 concentrations on amidase
production and biomass; – activity units, – biomass.
Substrate specificity
Amidase produced by strain ZJB-09211 was found
to be able to catalyze the hydrolysis of a variety of
amide substrates (Table 4). High activity against aro-
matic, heterocyclic, and aliphatic amides was exhib-
ited. For substrates in which the α-carbon is chiral,
amidase displayed enantioselectivity. In addition, the
amidase activity and enantioselectivity increased with
the size of the substituent at the α-position, as indi-
cated by the conversion rates and enantiomeric excess
(e.e.) values obtained for 2,2-dimethylcyclopropane
carboxamide (12.05 % conversion, e.e. = 60.01 %), me-
thioninamide (28.02 % conversion, e.e. = 99.80%), and
tryptophanamide (50 % conversion, e.e. > 99.90 %).
The enantiomeric ratio (E), calculated according to
the e.e. of the acidic fraction for tryptophanamide,
was higher than 200 for tryptophanamide, methioni-
namide and mandelic amide, whereas it was only 4.3
for 2,2-dimethylcyclopropane carboxamide.
Conclusions
It has been shown here that F. aquatile ZJB-

J. M. Xu et al./Chemical Papers 67 (10) 1262–1270 (2013)
Table 4. Substrate specificity of amidase produced by F. aquatile ZJB-09211
1269
Substrates
Relative activitya/%
Enantioselective
e.e. value/%
E-value
Tryptophan amide
Butyl amide
Isobutyramide
Acetamide
Methionin amide
Caprolactam
100.00
15.28
261.17
17.40
49.35
16.36
–
S
–
–
–
S
–
–
R
–
–
R
–
>99.9
–
–
–
99.8
–
–
60.01
–
–
>99.9
–
>200
–
–
–
>200
–
–
4.334
–
Levetiracetam
2,2-Dimethylcyclopro-panecarboxamide
2-Chloro-nicotinamide
Nicotinamide
Mandelic amide
Benzamide
12.11
–
–
–
298.25
277.66
>200
–
a) Activity for tryptophanamide corresponding to 147.86 U g−1 was taken as 100 %.
09211 can selectively hydrolyze the L-enantiomer to
produce optically active L-Trp in high yields when
fed D,L-tryptophanamide. To the best of our knowl-
edge, this is the first report on one strain of the
genus Flavobacterium producing stereospecific ami-
dase. The F. aquatile ZJB-09211 amidase exhibits
strict S-enantioselectivity (e.e. >99.9 %, E >200) for
tryptophan amides. Under optimized culture condi-
tions, enzyme production reaches 501.5 U L−1, an
activity that is by about six times higher than that
achieved before optimization. Our results indicate that
this strain has great potential for L-Trp production in
scientific and industrial applications.
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