Efficient biosynthesis of β-alanine with a tandem reaction strategy to eliminate amide by-product in the nitrilase-catalyzed hydrolysis

Article abstract of DOI:10.1016/j.molcatb.2016.11.018

An efficient biosynthesis of β-alanine from 3-aminopropionitrile at high concentration has been developed using a one-pot bienzymatic cascade of a nitrilase and an amidase. The nitrilase BjNIT3397 from Bradyrhizobium japonicum strain USDA110 catalyzes the hydrolysis of 3-aminopropionitrile to β-alanine at the concentration up to 3.0 mol/L with the formation 23% of 3-aminopropanamide. In order to eliminate the by-product 3-aminopropanamide, we cloned and characterized a new amidase from Pseudomonas nitroreducens through gene mining. Under the optimal conditions (50 mmol/L Na₂HPO₄-NaH₂PO₄ buffer, pH 6.0, 40 °C), 2.0 mol/L (176 g/L) of 3-aminopropanamide was completely hydrolyzed within 12 h. A tandem reaction system was then established to eliminate the by-product 3-aminopropanamide and increase the production of β-alanine to 90% isolated yield with 15.02 g/(L.h) space-time-yield. These results demonstrated that the tandem reaction strategy was an effective method of eliminating the amide by-products in the nitrilase-catalyzed hydrolysis at high substrate concentration.

Full text of DOI:10.1016/j.molcatb.2016.11.018

Accepted Manuscript
Title: Efficient biosynthesis of ␤-alanine with a tandem
reaction strategy to eliminate amide by-product in the
nitrilase-catalyzed hydrolysis
Author: Yanyang Tao Peiyuan Yao Jing Yuan Chao Han
Jinhui Feng Min Wang Qiaqing Wu Dunming Zhu
PII:
S1381-1177(16)30231-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.11.018
MOLCAB 3483
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Journal of Molecular Catalysis B: Enzymatic
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5-10-2016
16-11-2016
17-11-2016
Please cite this article as: Yanyang Tao, Peiyuan Yao, Jing Yuan, Chao Han,
Jinhui Feng, Min Wang, Qiaqing Wu, Dunming Zhu, Efficient biosynthesis of
␤-alanine with a tandem reaction strategy to eliminate amide by-product in
the nitrilase-catalyzed hydrolysis, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2016.11.018
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Efficient biosynthesis of β-alanine with a tandem reaction strategy to eliminate
amide by-product in the nitrilase-catalyzed hydrolysis
Yanyang Tao a, b, Peiyuan Yao b, Jing Yuanb, Chao Han a, b, Jinhui Fengb, Min Wanga,
Qiaqing Wu*, b, Dunming Zhu*,b
a
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education,
College of Biotechnology, Tianjin University of Science & Technology, Tianjin
300457, China
b National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering
Research Center of Biocatalytic Technology, Tianjin Institute of Industrial
Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport
Economic Area, Tianjin, 300308, China
*Corresponding authors: Dunming Zhu, Qiaqing Wu
Telephone: 86-22-84861963
Fax: 86-22-84861996
E-mail: zhu_dm@tib.cas.cn, wu_qq@tib.cas.cn
1

Graphical abstract
2

Research Highlights
 An amidase from Pseudomonas nitroreducens (AMI-pn) was characterized.
 It catalyzed the hydrolysis of 3-aminopropanamide (2.0 mol/L, 176 g/L) to
β-alanine.
 An effective bienzymatic synthesis of β-alanine from 3-aminopropionitrile (3.0
mol/L) was achieved.
 Amidase effectively eliminated the by-product in the nitrilase-catalyzed
hydrolysis of nitriles.
Abstract An efficient biosynthesis of β-alanine from 3-aminopropionitrile at high
concentration has been developed using a one-pot bienzymatic cascade of a nitrilase
and an amidase. The nitrilase BjNIT3397 from Bradyrhizobium japonicum strain
USDA110 catalyzes the hydrolysis of 3-aminopropionitrile to β-alanine at the
concentration up to 3.0 mol/L with the formation 23% of 3-aminopropanamide. In
order to eliminate the by-product 3-aminopropanamide, we cloned and characterized
a new amidase from Pseudomonas nitroreducens through gene mining. Under the
optimal conditions (50 mmol/L Na2HPO4-NaH2PO4 buffer, pH 6.0, 40°C), 2.0 mol/L
(176 g/L) of 3-aminopropanamide was completely hydrolyzed within 12 h. A tandem
reaction system was then established to eliminate the by-product
3-aminopropanamide and increase the production of β-alanine to 90% isolated yield
3

with 15.02 g/(L.h) space-time-yield. These results demonstrated that the tandem
reaction strategy was an effective method of eliminating the amide by-products in
the nitrilase-catalyzed hydrolysis at high substrate concentration.
Keywords: Amidase; Nitrilase; β-Alanine; Hydrolysis; Tandem reaction
1. Introduction
Nitrilases are becoming important and powerful biocatalysts with potential
applications in the synthesis of high value-added carboxylic acids under mild
conditions [1-8]. However, the undesirable amide by-products, which are generated
from nitriles due to the nitrile hydratase activity contributed by nitrilase, have adverse
effect on the yield and purity of the products [9-11]. For example, a recombinant
nitrilase from Arabidopsis thaliana (AtNIT4) catalyzed the hydrolysis of
3-cyano-L-alanine and >60% amide was formed [12]. Similarly, a recombinant
nitrilase originating from Pseudomonas fluorescens EBC191 is also prone to amide
formation [13]. The nitrilase from Gibberella intermedia CA3-1 displays prominent
catalytic efficiency towards 3- and 4-cyanopyridine to produce the corresponding
nicotinic and isonicotinic acids, but with about 3% of amide in the products [14].
Therefore, development of practical methods to eliminate the amide by-products in
nitrilase-catalyzed hydrolysis of nitriles is highly in demand.
Recently, Gong et al employed sequence analysis and saturation mutagenesis to
obtain new mutant enzymes with the aim of reducing the by-product formation in the
hydrolysis of 3-and 4-cyanopyridine catalyzed by a fungal nitrilase from Gibberella
4

intermedia. Their best mutant I128L-N161Q reduced the amount of amide to one
third of that in the hydrolysis with wild-type enzyme [11]. As such, modification of
nitrilases offers an approach to reduce the formation of amide by-product. However,
only two protein crystal structures of nitrilases from the archaeon Pyrococcus abyssi
[15] and Synechocystis sp. PCC6803 [16] have been reported up to now. The lack of
the structural information hindered the rational modification of nitrilases to obtain the
effective mutants without the formation of amide by-product.
On the other hand, the control of hydrolysis process offers an alternative strategy.
Vejvoda et al reported that immobilization of fungal nitrilase and bacterial amidase
resulted in the decrease of isonicotinamide in the products from 25% to 5% [17].
Chmura et al reported that the cascade reaction by a triple cross-linked enzyme
aggregate of a (S)-selective oxynitrilase, a non-selective nitrilase and an amidase
produced (S)-mandelic acid as the sole product at 10 mM substrate concentration [18].
In order to prevent the formation of 3-aminopropanamide, we constructed a tandem
reaction strategy by using aspartate ammonia-lyase and fumaric acid to consume the
by-product ammonia, resulting in the decrease of 3-aminopropanamide from 33% to 3%
[19]. However, the mixture of L-aspartic acid, 3-aminopropanamide and β-alanine
increases the downstream process cost because of additional purification steps.
In this study, we cloned and screened a series of amidases, and a new amidase
from Pseudomonas nitroreducens was found to catalyze the hydrolysis of
3-aminopropanamide to β-alanine at high concentration. Furthermore, a tandem
5

reaction strategy was established to eliminate the 3-aminopropanamide by-product in
the nitrilase-catalyzed hydrolysis by using a nitrilase and an amidase as the
biocatalysts.
2 Materials and methods
2.1 Materials
3-Aminopropionitrile, 3-aminopropanamide hydrochloride, β-alanine and
1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (FDAA) were purchased from TCI (Tokyo
Chemical Industry Co. Ltd.), Bide Pharmatech Ltd, Sinopharm Chemical Reagent Co.,
Ltd. and Innochem (Beijing) Technology Co., Ltd., respectively. All chemical reagents
were of analytical grade. The genes AMI-pa from Pseudomonas aeruginosa (GenBank:
CEI05647.1), AMI-pp from Pseudomonas putida KT2440 (NCBI Reference Sequence:
NP_745976.1), AMI-pc1 from Pseudomonas chlororaphis (NCBI Reference Sequence:
WP_025806143.1) and AMI-pc2 from Pseudomonas chlororaphis (NCBI Reference
Sequence: WP_023969146.1) were synthesized by Shanghai Xuguan Biotechnological
13
Development Co., Ltd (Shanghai, China). 1H and C NMR spectra were recorded on a
Bruker AVANCE-Ⅲ 400 MHz NMR spectrometer. High-resolution MS was recorded
on a Bruker microTOF-QII.
2.2 Gene cloning, functional expression and preparation of whole cells
The full-length amidase gene (NCBI Reference Sequence: WP_024765375.1)
was amplified by PCR from Pseudomonas nitroreducens using the following primer
pair: pn-F (5’- GGGAATTCCATATGATGAAAGTAGAACTCGTCCAACT-3’) and
6

pn-R (5’- CCGCTCGAGTCAGGCGGGAATGATCAGCTCGCGC-3’). NdeI and
XhoI restriction sites (underlined) were used to clone the amidase gene into the same
sites on pET32a (+), and the resulting plasmid was transformed into E. coli BL21
(DE3) strain. The constructed plasmid was confirmed by restriction analysis and DNA
sequencing.
The E. coli BL21 (DE3) strain harboring the amidase gene was grown in LB
medium containing 100 μg/mL ampicillin at 37°C and 200 rpm. When the optical
density at 600 nm (OD600) reached 0.6  0.8, 0.1 mmol/L isopropyl
β-D-1-thiogalactopyranoside (IPTG) was added to induce gene expression at 25°C for
12 h. The cells were harvested by centrifugation, washed once with deionized water
and stored at -20°C for further use.
2.3 Purification of amidase and determination of the molecular mass
All purifications steps were performed at 4°C using 50 mmol/L pH7.0
Na2HPO4/NaH2PO4 buffer (A). Cells were suspended in buffer A and disrupted by a
high pressure slurry disruptor (APV 2000). Cell debris were removed by
centrifugation at 6000 g, 4°C for 30 min. Poly(ethylene imine) (PEI, the final
concentration of 5%) was added to the resulting cell free extract and stirred for 1 h.
After centrifugation, the cell-free supernatant was subjected to precipitation with 60%
ammonium sulfate overnight. The resulting precipitate was collected and dissolved in
buffer A and dialyzed against the same buffer overnight. The dialysate was then
applied to a DEAE-Sephadex column equilibrated with buffer A. The column was
7

washed with the same buffer, the absorbed protein was eluted by the buffer A
supplementing with 0  1.0 mol/L NaCl, and the eluent was collected in 2.0 mL
fractions. The resulting target protein solution was dialyzed in buffer A overnight. The
purity of the protein was determined by SDS-PAGE analysis.
The enzyme sample was subjected to a Sephadex TM 200 Increase 10/300GL
column to determine the native molecular mass of the purified enzyme. The column
was equilibrated with buffer A and eluted at a flow rate of 0.5 ml/min and the
absorbance at 280 nm was monitored. The relative molecular mass of the enzyme was
calculated by retention volume compared with those of the standard proteins,
ovalbumin (44kDa), conalbumin (75kDa), aldolase (158kDa), ferritin (440kDa), and
thyroglobulin (669kDa).
2.4 Amidase activity assay
Amidase activity was determined by measuring the release of ammonia using the
phenol-hypochlorite ammonia assay method [20]. A standard reaction mixture (100
μL) contained Na2HPO4-NaH2PO4 (50 mmol/L, pH 7.0), 10 mmol/L substrate, and 2
μL of the purified enzyme or cell-free extract of E. coli BL21 (DE3) strain harboring
the amidase gene. The reaction mixture was shaken at 40°C in an orbital shaker at 200
rpm for 10 min, then 350 μL of reagent A (0.59 mol/L phenol and 1 mmol/L sodium
nitroprusside) was added. Color was developed by the addition of 100 μL reagent B
(2.0 mol/L sodium hydroxide and 0.11 mol/L sodium hypochlorite) after 5 min of
incubation at 60°C and the absorbance at 600 nm was measured. Control reactions
8

were performed without enzyme. Standard curve was prepared using NH4Cl. One unit
of enzyme activity was defined as the amount of enzyme that catalyzed the release of
1 μmol of NH3 per min under standard assay conditions. All assays were performed in
triplicate.
2.5 Effects of pH and temperature on the hydrolysis of 3-aminopropanamide
Similar to the procedure in Section 2.4, citrate buffer (50 mmol/L, pH 4.0  6.0)
and sodium phosphate buffer (50 mmol/L, pH 6.0  8.0) were used to determine the
optimum pH. The optimum temperature was established at pH 6.0 within the temperature
range of 20  60°C. Stability of the enzyme at different pHs was studied by incubating
the enzyme at pH 6.0  8.0 for an appropriate period of time (6 h) followed by
measurement of the residual enzyme activity. The thermal stability of the enzyme was
studied by incubation of the enzyme at 30  50°C for an appropriate period of time (12 h)
followed by the measurement of the residual enzyme activity.
2.6 Substrate specificity
All reactions were carried out at 40°C under standard conditions. Fifty μL of
purified amidase (2.75 U) was added to 40 μL of sodium phosphate buffer (50
mmol/L, pH 6.0), and the mixture was pre-incubated for 5 min. Ten μL of amide (100
mmol/L in water) was added to the reaction mixture and incubated for additional 20
min. The reaction mixtures were treated and analyzed as described in Section 2.4. The
activity of AMI-pn for 3-aminopropanamide was regarded as 100%.
2.7 Determination of kinetic constants
9

Apparent Km value for 3-aminopropanamide was measured with various
substrate concentrations (10 mmol/L  500 mmol/L) in Na2HPO4-NaH2PO4 buffer (50
mmol/L, pH6.0). The reaction was carried out at 40°C for 3 min, quenched by equal
volume of 10% sodium carbonate and derivatized with 1-fluoro-2-4-dinitrophenyl-5-L
-alanine amide (FDAA). The reaction rate was determined by HPLC analysis, which
was performed on an Agilent 1200 series HPLC system with an Eclipse XDB-C18
column and a UV detector at wavelength of 340 nm as previously reported [19].
Initial reaction velocities measured at different substrate concentrations were analyzed
by non-linear regression, which was performed using SigmaPlot program.
2.8 Substrate and product inhibition
The wet E. coli whole cells producing AMI-pn enzyme (50 mg, 41 U) were
added to the phosphate buffer (1.0 mL, 50 mmol/L, pH 6.0) with appropriate amounts
of 3-aminopropanamide, and the reaction mixture was shaken for 12 h at 200 rpm and
40°C on an orbital shaker. For substrate inhibition, the concentrations of
3-aminopropanamide were 0.5  3.0 mol/L. For product inhibition, the reaction was
performed with 0.5 mol/L 3-aminopropanamide by adding 0.5  3.0 mol/L β-alanine.
The effect of 3-aminopropionitrile was investigated at 0.5 mol/L 3-aminopropanamide
by adding 0.5  3.0 mol/L 3-aminopropionitrile. The reactions in the presence of
3-aminopropionitrile and β-alanine were carried out for 30 min. Aliquots (50 μL)
were taken and analyzed by HPLC analysis as described in Section 2.7.
2.9 Biosynthesis of β-alanine through bienzymatic one-pot two-step cascade
10

To a solution of 3-aminopropionitrile (4.20 g, 60 mmol) in water, which was
adjusted to pH 7.0 with concentrated hydrochloric acid (12 mol/L), 1.6 g of whole cells
of E. coli Rosetta2(DE3) strain expressing nitrilase BjNIT3397 (800 U) was added
[19]. The initial volume of the reaction mixture was 20 mL. The suspension was stirred at
40°C for 8 hours. At the indicated time intervals, aliquots (50 μL) were taken and
analyzed by HPLC analysis. After 3-aminopropionitrile was completely hydrolyzed to
3-aminopropanamide and β-alanine, the whole cells were removed by centrifugation at
6000 g, 4°C for 15 min. The supernatant was adjusted to pH 6.0 with concentrated
hydrochloric acid (12 mol/L). The wet E. coli whole cells producing AMI-pn enzyme
(1.6 g, 1312 U) was added to the mixture and the suspension was stirred at 40°C for
additional 8 hours and monitored by HPLC analysis. Upon completion, the reaction was
acidified to pH 1 with concentrated hydrochloric acid (12 mol/L). The supernatant was
obtained by centrifugation at 6000 g, 4°C for 15 min, and run through a column with
strong acid cation exchange resin. The product was eluted with an ammonia solution
(about 7%, w/w) and detected by thin-layer chromatography (TLC), which was
developed with a mixture of 1-butanol, water and glacial acetic acid (4/1/1, v/v), and
visualized by 2, 2-dihdroxyindane-1, 3-dione. The desired product was obtained as pale
yellow solid (4.81 g, 90% yield) by removal of the solvent. 1H NMR (400 MHz, D2O):
δ 2.90 (t, J=6.8 Hz, 2 H), 2.34 (t, J=6.8 Hz, 2 H). 13C NMR (100 MHz, D2O): δ
-
179.88, 37.15, 36.69. High-resolution MS (ESI): m/z: calcd for C3H6NO2 : 88.0399
[M-1]-, found: 88.0369.
11

3 Results and discussion
3.1 Cloning and functional expression of AMI-pn
NCBI BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) was carried out to
identify a series of candidate amidase genes using an amidase from Pseudomonas sp.
MCI3434 (AMI-ps, GenBank: BAE02667.1) as a template, because it has been
reported to hydrolyze 3-aminopropanamide [21]. Five putative carbon-nitrogen
hydrolases with the homology between 30% and 80% were selected, cloned into
pET32a (+) vector and transformed into E. coli BL21 (DE3) strain for further detailed
studies. The amidases AMI-ta from Thermoplasma acidophilum DSM 1728 (NCBI
Reference Sequence: WP_010901615.1), AMI-gp from Geobacillus pallidus RAPc8
(GenBank: AAO23013.1) [22], AMI-rs from Rhodococcus sp. N-771 (GenBank:
BAA36596.1) [23] and AMI-ms from Microbacterium sp. AJ115 (GenBank:
CAG29798.1) [24] were available at our laboratory. After screening 10 amidases with
3-aminopropanamide at a substrate concentration of 500 mmol/L at pH 7.0 for 12h,
we chose a carbon-nitrogen hydrolase (AMI-pn) from Pseudomonas nitroreducens
(61.2% identity to AMI-ps) for further research (Table 1).
3.2 Expression, Purification of amidase and determination of the molecular mass
The AMI-pn gene was expressed in E. coli BL21 (DE3) strain as a soluble
protein, and the SDS-PAGE analysis indicates that the subunit molecular weight of
the enzyme is about 30 kDa (Figure 1). The recombinant enzyme was purified by PEI
precipitation, ammonium sulfate fractionation and DEAE-Sephadex chromatography
12

(Table 2, Figure 2). Size exclusion chromatography reveals that the molecular weight
of the native enzyme is 62 kDa, suggesting that the native enzyme is composed of two
subunits (dimer). According to sequence alignment, we may conclude that AMI-pn
belongs to nitrilase superfamily with the characteristic catalytic triad residues
Glu-Lys-Cys(E-K-C) [25-29] (Figure 3). Some amidases belonging to the nitrilase
superfamily also form dimers, such as the amidases from Pyrococcus yayanosii CH1
(Gene ID: 10837614) [30] and Nesterenkonia strain AN1 (GenBank: ACS35546)
[31].
3.3 Effects of pH and temperature on AMI-pn activity and stability
Effects of pH and temperature on the enzyme activity of AMI-pn were
investigated at 10 mmol/L of substrate concentration. As shown in Figure 4, the
optimal pH for AMI-pn was 6.0. Different buffering systems of pH6 gave quite
different AMI-pn activities with deviation approaching 20%. We tested the assay
method with ammonia standard solution in these two buffer systems, and the results
showed no significant difference in the measured amount of ammonia. This suggests
that the observed difference in AMI-pn activity should not be resulted from the effect
of the buffers on the assay method, but these buffers do affect the AMI-pn activity,
although it is not clear why these buffering systems affect the enzyme activity. The
purified AMI-pn exhibited the maximum relative activities at 40°C (Figure 5).
The pH and thermal stability was investigated by measuring the remaining
activity of the enzyme after incubation at different pH and temperatures, and the
13

initial activity without incubation was defined as 100%. As shown in Figure 6, the
purified AMI-pn retained more than 85% of activity in the pH range of 6.0  8.0 for 6
h, suggesting that the enzyme is stable in this pH range. Although AMI-pn was not
stable at 50°C, it retained 94% of activity after incubation at 40°C for 12 h (Figure 7).
As such, AMI-pn shows relatively high thermal stability.
3.4 Substrate spectrum and kinetic constants
A number of amides were tested to investigate the substrate spectrum of AMI-pn
(Table 3). AMI-pn showed activity toward the majority of aliphatic amides, such as
butyrylamide, 3-hydroxypropionamide, 3-pyrrolidine formamide hydrochloride,
piperidine-3-carboxamide and piperidine-4-carboxamide, but no or little activity
towards aromatic amides and 2-amino amides.
The purified amidase showed apparent Km for 3-aminopropanamide of 42.5±5.9
mmol/L and kcat was 17.3±0.9 s-1.
3.5 Substrate and product inhibition
The hydrolysis of 3-aminopropanamide of different initial concentrations was
performed with 50 mg/mL of wet E. coli whole cells producing AMI-pn enzyme in
phosphate buffer (50 mmol/L, pH 6.0) at 40°C. Up to 2.0 mol/L (176 g/L) of
3-aminopropanamide was completely hydrolyzed within 12 hours. When further
increasing the initial concentration of the substrate to 2.5 mol/L and 3.0 mol/L, the
conversions of the reaction were about 98% and 91%, respectively.
14

The results showed that low concentration of 3-aminopropionitrile could
facilitate the hydrolysis of 3-aminopropanamide, while high concentration of
3-aminopropionitrile inhibited the activity of AMI-pn and the activity was 79% at 3.0
mol/L concentration relative to the control experiment (Figure 8). β-Alanine showed a
little inhibitory effect on the activity of AMI-pn, and the activity at 3.0 mol/L of
β-alanine was 75% relative to the control experiment (Figure 9).
3.6 Biosynthesis of β-alanine through bienzymatic one-pot two-step cascade
In the coupled reaction shown in Figure 10, the wet E. coli whole cells producing
nitrilase BjNIT3397 (80 mg/ml) completely hydrolyzed 3-aminopropionitrile (3.0 mol/L)
in 8 hours, producing 77% β-alanine and 23% 3-aminopropanamide. In the following 8
hours, the wet E. coli whole cells producing AMI-pn enzyme (80 mg/ml) completely
hydrolyzed the by-product 3-aminopropanamide to β-alanine. After purification by
strong acid cation exchange resin, the isolated yield of β-alanine was 90% and the
space-time-yield was 15.02 g/(L.h). Thus, the one pot two-step cascade reaction
strategy provided an efficient method to eliminate the by-product amide in the
nitrilase-catalyzed hydrolysis.
4. Conclusion
A new amidase from Pseudomonas nitroreducens (AMI-pn) was found to
catalyze the hydrolysis of 3-aminopropanamide (2.0 mol/L, 176 g/L) to β-alanine at
pH 6.0 and 40°C within 12 h. Furthermore, the amidase was applied to eliminate the
amide by-product of nitrilase, and an effective bienzymatic synthesis of β-alanine
15

from 3-aminopropionitrile at 3.0 mol/L of substrate concentration was achieved by
using the wet E. coli whole cells producing AMI-pn and a nitrilase BjNIT3397 from
Bradyrhizobium japonicum strain USDA110. This one-pot bienzymatic
transformation provides a practical method to eliminate the by-product in the
nitrilase-catalyzed hydrolysis of nitriles.
Acknowledgement
This work was financially supported by the National Natural Science Foundation
of China (No. 21572261), Key Project of Tianjin Municipal Science and Technology
Support Program (No.15ZCZDSY00490), and Youth Innovation Promotion
Association of the Chinese Academy of Sciences (Grant No. 2016166). We greatly
appreciate Professor Peter Lau (Tianjin Institute of Industrial Biotechnology) for
proofreading the manuscript and suggestions.
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18

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(2011) 3696-3702.
19

Figure 1 The protein expression level of AMI-pn. Lane M: protein marker, Lane 1:
the cell-free lysate, Lane 2: the supernatant of lysate, Lane 3: the precipitation of
lysate.
Figure 2 The purification of AMI-pn. Lane M: protein marker, Lane 1: the column
flow-through of the protein solution obtained from ammonium sulfate fractionation,
Lane 2: 60% ammonium sulfate fractionation protein, Lane 3: impurity protein after
60% ammonium sulfate fractionation, Lane 4-5: purified AMI-pn after column.
Figure 3 Amino acid sequence alignment of amidases from different origins. The
alignment was generated with DNAMAN. Identical amino acid residues are marked
in black, similar residues are marked in pink. The blue arrows above the sequence
indicate the residues of the characteristic Glu-Lys-Cys (E-K-C) catalytic triad
residues.
Figure 4 Effects of pH on the activity of 3-aminopropanamide to β-alanine using
purified enzyme. The buffers were citrate buffer (50 mmol/L, pH 4.0-6.0), sodium
phosphate buffer (50 mmol/L, pH 6.0-8.0). The reaction activity in sodium phosphate
buffer (50 mmol/L, pH 6.0) at 37 °C for 10 min was defined as 100%.
Figure 5 Effects of temperature on the activity of 3-aminopropanamide to β-alanine
using purified enzyme in the temperature range of 20 - 60 °C. The reaction activity at
40 °C for 1 h in sodium phosphate buffer (50 mmol/L, pH 6.0) was defined as 100%.
20

Figure 6 The stability of purified enzyme at different pHs in the sodium phosphate
buffer (50 mmol/L, pH 6.0-8.0) for 6h. The reaction activity without incubation in
sodium phosphate buffer (50 mmol/L, pH 6.0-8.0) at 40 °C was defined as 100%.
Figure 7 The thermal stability of purified enzyme in the temperature range of 30 -
50 °C for 12 h. The reaction activity without incubation at 30 - 50 °C was defined as
100%.
Figure 8 Effects of 3-aminopropionitrile on the activity of AMI-pn. The
transformation was carried out with 0.5 mol/L of 3-aminopropanamide and 50 mg/ml
of E. coli whole cells producing AMI-pn enzyme in the presence of different
concentrations of 3-aminopropionitrile. The activity without 3-aminopropionitrile was
defined as 100%.
Figure 9 Effects of β-alanine on the activity of AMI-pn. The transformation was
carried out with 0.5 mol/L of 3-aminopropanamide and 50 mg/ml of E. coli whole
cells producing AMI-pn enzyme in the presence of different concentrations of
β-alanine. The activity without β-alanine was defined as 100%.
Figure 10 Time course for the biotransformation of 3-aminopropionitrile to β-alanine
by using the E. coli whole cells producing BjNIT3397 and AMI-pn. Reaction
conditions: A: 3.0 mol/L 3-aminopropionitrile and 80 mg/ml E. coli whole cells
producing BjNIT3397 at pH 7.0; B: then 80 mg/ml E. coli whole cells producing
AMI-pn at pH 6.0.
21

Figure 1
Figure 2
22

Figure 3
pn.txt ....................MKVELVQLAGRDGDVAWNLA
Methylophilus_methylotrophus.txt .MIHGDISSSQDTVGVAVVNYKMPRLHTKAEILANCRNIA
20
39
39
39
40
39
Helicobacter_pylori_B8.txt
Pseudomonas_aeruginosa.txt
Bacillus_cereus.txt
.MRHGDISSSPDTVGVAVVNYKMPRLHTKNEVLENCRNIA
.MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKIA
MGSSGSMVKPISGFLTALIQYPVPVVESRADIDKQIQQII
.MRHGDISSSHDTVGIAVVNYKMPRLHTKAEVIENAKKIA
Geobacillus_Pallidus_Rapc8.txt
Consensus
pn.txt RTLEAIHACADDTRLVVFPETQLTGFPTAQNIA.....AI
Methylophilus_methylotrophus.txt DMVVNMKAGLPGMDLVIFPEYSTHGIMYDSQEMYDNATTV
55
79
79
79
80
79
Helicobacter_pylori_B8.txt
Pseudomonas_aeruginosa.txt
Bacillus_cereus.txt
Geobacillus_Pallidus_Rapc8.txt
Consensus
KVIGGVKQGLPGLDLIIFPEYSTHGIMYDRQEMFDTAASV
DMIVGMKQGLPGMDLVVFPEYSLQGIMYDPAEMMETAVAI
KTIHSTKSGYPGLELIVFPEYSTQGLNTKKWTTEEFLCTV
DMVVGMKQGLPGMDLVVFPEYSTMGIMYDQDEMFATAASI
l fpe
g
pn.txt AEPVDGPSVQAVQRAAQEKNISIAVGFAEAADDGRFYNTT
Methylophilus_methylotrophus.txt PGPETDIFAEACRKAKVWGVFSITGEQHEEHPKKAPYNTL
95
119
118
119
117
119
Helicobacter_pylori_B8.txt
Pseudomonas_aeruginosa.txt
Bacillus_cereus.txt
Geobacillus_Pallidus_Rapc8.txt
Consensus
PGEETAILAEACKKNRVWGVFSLTGEKHEQAK.KNPYNTL
PGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPYNTL
PGPETDLFAEACKESKVYGVFSIMEKNPDGGE...PYNTA
PGEETAIFAEACKKADTWGVFSLTGEKHEDHPNKAPYNTL
a
s
ynt
pn.txt LLITPEGIAM.KYRKTHLWASDRGIFTPGDRYATC.LWNG
Methylophilus_methylotrophus.txt ILMNDQGEIVQKYRKIMPWVPIEGWYPGKETFISE.GPKG
133
158
157
158
157
158
Helicobacter_pylori_B8.txt
Pseudomonas_aeruginosa.txt
Bacillus_cereus.txt
Geobacillus_Pallidus_Rapc8.txt
Consensus
ILVNDKGEIVQKYRKILPWCPIECWYPGDKTYVVD.GPKG
VLIDNNGEIVQKYRKIIPWCPIEGWYPGGQTYVSE.GPKG
VIIDPQGEMILKYRKLNPWVPVEPWKAGDLGLPVCDGPGG
VLINNKGEIVQKYRKIIPWCPIEGWYPGDTTYVTE.GPKG
g
kyrk w
g
pn.txt IRVGIVICFDIEFPESARALGQLGAELIIVTNGNMDPYGP
Methylophilus_methylotrophus.txt LKVSLIICDDGNYPEIWRDCAMKGAELIVRCQGYMYPAKE
173
198
197
198
197
198
Helicobacter_pylori_B8.txt
Pseudomonas_aeruginosa.txt
Bacillus_cereus.txt
Geobacillus_Pallidus_Rapc8.txt
Consensus
LKVSLIICDDGNYPEIWRDCAMRGAELIVRCQGYMYPAKE
MKISLIICDDGNYPEIWRDCAMKGAELIVRCQGYMYPAKD
SKLAVCICHDGMFPEVAREAAYKGANVLIRISGYSTQVSE
LKISLIVCDDGNYPEIWRDCAMKGAELIVRCQGYMYPAKE
c d pe r
ga
g
pn.txt THRTAIMGRAMENQAYAVMVNRVGEGDDGLVFAGGSAVVD
Methylophilus_methylotrophus.txt QQILISKAMAFANNTY.VAVSNAAGFDGVYSYFGHSAIIG
213
237
236
237
236
237
Helicobacter_pylori_B8.txt
Pseudomonas_aeruginosa.txt
Bacillus_cereus.txt
Geobacillus_Pallidus_Rapc8.txt
Consensus
QQIAIVKAMAWANQCY.VAVANATGFDGVYSYFGHSSIIG
QQVMMAKAMAWANNCY.VAVANAAGFDGVYSYFGHSAIIG
QWMLTNRSNAWQNLMY.TLSVNLAGYDGVFYYFGEGQVCN
QQIMMAKAMAWANNTY.VAVANATGFDGVYSYFGHSAIIG
a n y
d
g
pn.txt
PYGQLIVEAGREEC...............RQIVELDFERL
238
277
276
277
276
277
Methylophilus_methylotrophus.txt FDGRTLGECGEEENGVQYAALSKHLIRDFRKHGQSENHLF
Helicobacter_pylori_B8.txt
Pseudomonas_aeruginosa.txt
Bacillus_cereus.txt
Geobacillus_Pallidus_Rapc8.txt
Consensus
FDGHTLGECGEEENGLQYAQLSVQQIRDARKYDQSQNQLF
FDGRTLGECGEEEMGIQYAQLSLSQIRDARANDQSQNHLF
FDGTTLVQGHRNPWEIVTAEVYPELADQARLGWGLENNIY
FDGRTLGECGTEENGIQYAEVSISQIRDFRKNAQSQNHLF
g
r
pn.txt AQSRRDYSYLAERRFVLPGELREHDGGLRELIIPA.....
Methylophilus_methylotrophus.txt KLLHRGYTGMLNSGDGDQGVATCPYSFYSKWVQDPAAARD
273
317
316
317
304
317
Helicobacter_pylori_B8.txt
Pseudomonas_aeruginosa.txt
Bacillus_cereus.txt
KLLHRGYSGVFASGDGDKGVAECPFEFYKTWVNDPKKAQE
KILHRGYSGLQASGDGDRGLAECPFEFYRTWVTDAEKARE
NLGSRGY............VATPGGVKENPYTFVKDLAEG
KLLHRGYTGLINSGEGDRGVAECPFDFYRTWVLDAEKARE
r y
Geobacillus_Pallidus_Rapc8.txt
Consensus
pn.txt
..............................
273
340
339
346
332
347
Methylophilus_methylotrophus.txt MVESFTRTTLGTKEAPIAGLPNE.......
Helicobacter_pylori_B8.txt
Pseudomonas_aeruginosa.txt
Bacillus_cereus.txt
Geobacillus_Pallidus_Rapc8.txt
Consensus
NVEKFTRPSVGVAACPVGDLPTK.......
NVERLTRSTTGVAQCPVGRLPYEGLEKEA.
KYKVPWEDEIKVKDGSIYGYPVKKTIHS..
NVEKITRSTVGTAECPIQGIPNEGKTKEIG
23

Figure 4
Figure 5
Figure 6
24

Figure 7
Figure 8
Figure 9
25

Figure 10
26

Table 1 Information of amidases from gene mining and the results of screening
Identity
Amidase
Source
GenBank
Activityb)
(%)
100
AMI-ps
AMI-pna)
AMI-pa a)
AMI-ppa)
AMI-pc1a)
AMI-pc2a)
Pseudomonas sp. MCI3434
Pseudomonas nitroreducens
Pseudomonas aeruginosa
Pseudomonas putida KT2440
Pseudomonas chlororaphis
Pseudomonas chlororaphis
Thermoplasma acidophilum DSM
1728
BAE02667.1
WP_024765375.1
CEI05647.1
+
+++
-
61.2
63.5
71.5
75.2
34.8
NP_745976.1
++
++
+
WP_025806143.1
WP_023969146.1
27.7
AMI-ta
WP_010901615.1
+
17.2
8.6
AMI-gp
AMI-rs
AMI-ms
Geobacillus pallidus RAPc8
Rhodococcus sp. N-771
AAO23013.1
BAA36596.1
CAG29798.1
+
+
+
Microbacterium sp. AJ115
9.8
a) Previously unreported amidases. b) The activity of amidase towards
3-aminopropanamide (500 mmol/L) at pH 7.0 for 12 h. “-” no activity, “+” a little
activity, “++” higher activity but 3-aminopropanamide was not converted completely;
“+++” 3-aminopropanamide was converted completely.
Table 2 Summary of the purification of AMI-pn
Purification step
Total protein Total activity Specific activity Purification
Yield
(%)
(mg)
457.9
95.8
(U)
(U/mg)
7.5
(fold)
1
Crude AMI-pn
3451.2
2301.1
100
Purified AMI-pn
24.0
3.2
66.7%
27

Table 3 Substrate spectrum of purified enzyme AMI-pn
Relative
substrate
Relative
substrate
activity (%)
activity (%)
3-aminopropanamide
100.0±5.0
513.0±6.5
97.0±1.2
5.3±0.9
D-valinamide hydrochloride
4.8±1.6
1.9±0.3
7.1±0.5
4.8±0.7
6.1±0.4
8.3±1.9
4.8±0.7
hydrochloride
(S)-2-amino butyramide
hydrochloride
butyramide
(R)-2-amino butyramide
hydrochloride
3-hydroxypropionamide
piperidine-2-carboxamid
piperidine-3-carboxamide
piperidine-4-carboxamide
(R)-2-amino-4-methyl amyl
amide
(2R)-2-amino-3-phenyl
acrylic amide
42.5±2.4
57.9±6.4
48.0±4.7
L-leucine amide
hydrochloride
pyrrolidine-3-carboxamide
hydrochloride
acrylamide
L-phenylalaninamide
L-phenylglycinamide
urea
11.9±1.1
10.4±2.3
9.8±2.5
6.8±1.4
7.8±2.4
5.5±1.0
D-(-)-phenylglycinamid
glycinamide hydrochloride
L-alaninamide hydrochloride
nicotinamide
1.9±0.4
0.5±0.2
0
0
0
D-prolinamide
L-prolinamide
benzamide
L-valinamide hydrochloride
28