Aspartase Research Articles

Fine-Regulating the Carbon Flux of l-Isoleucine Producing Corynebacterium glutamicum WM001 for Efficient l-Threonine Production.

l-Threonine, an essential amino acid, is widely used in various industries, with an annually growing demand. However, the present Corynebacterium glutamicum strains are difficult to achieve industrialization of l-threonine due to low yield and purity. In this study, we engineered an l-isoleucine-producing C. glutamicum WM001 to efficiently produce l-threonine by finely regulating the carbon flux. First, the threonine dehydratase in WM001 was mutated to lower the level of l-isoleucine production, then the homoserine dehydrogenase and aspartate kinase were mutated to release the feedback inhibition of l-threonine, and the resulting strain TWZ006 produced 14.2 g/L l-threonine. Subsequently, aspartate ammonia-lyase and aspartate transaminase were overexpressed to accumulate the precursor l-aspartate. Next, phosphoenolpyruvate carboxylase, pyruvate carboxylase and pyruvate kinase were overexpressed, and phosphoenolpyruvate carboxykinase, oxaloacetate decarboxylase were inactivated to fine-regulate the carbon flux among oxaloacetate, pyruvate and phosphoenolpyruvate. The resulting strain TWZ017 produced 21.5 g/L l-threonine. Finally, dihydrodipicolinate synthase was mutated with strong allosteric inhibition from l-lysine to significantly decrease byproducts accumulation, l-threonine export was optimized, and the final engineered strain TWZ024/pXTuf-thrE produced 78.3 g/L of l-threonine with the yield of 0.33 g/g glucose and the productivity of 0.82 g/L/h in a 7 L bioreactor. To the best of our knowledge, this represents the highest l-threonine production in C. glutamicum, providing possibilities for industrial-scale production.

Salmonella Typhimurium expansion in the inflamed murine gut is dependent on aspartate derived from ROS-mediated microbiota lysis

Inflammation boosts the availability of electron acceptors in the intestinal lumen, creating a favorable niche for pathogenic Enterobacteriaceae. However, the mechanisms linking intestinal inflammation-mediated changes in luminal metabolites and pathogen expansion remain unclear. Here, we show that mucosal inflammation induced by Salmonella enterica serovar Typhimurium (S. Tm) infection increases intestinal levels of the amino acid aspartate. S. Tm used aspartate-ammonia lyase (aspA)-dependent fumarate respiration for growth in the murine gut only during inflammation. AspA-dependent growth advantage was abolished in the gut of germ-free mice and restored in gnotobiotic mice colonized with members of the classes Bacteroidia and Clostridia. Reactive oxygen species (ROS) produced during the host response caused lysis of commensal microbes, resulting in the release of microbiota-derived aspartate that was used by S. Tm, in concert with nitrate-dependent anaerobic respiration, to outcompete commensal Enterobacteriaceae. Our findings demonstrate the role of microbiota-derived amino acids in driving respiration-dependent S. Tm expansion during colitis.

Insight into the Substrate Specificity of Lactobacillus paracasei Aspartate Ammonia-Lyase

Aspartate ammonia-lyase (AAL) catalyzes the reversible conversion reactions of aspartate to fumaric acid and ammonia. In this work, Lactobacillus paracasei LpAAL gene was heterologously expressed in Escherichia coli. As well as a recombinant His-tagged LpAAL protein, a maltose-binding protein (MBP) fused LpAAL protein was used to enhance its protein solubility and expression level. Both recombinant proteins showed broad substrate specificity, catalyzing aspartic acid, fumaric acid, phenylalanine, and tyrosine to produce fumaric acid, aspartic acid, trans-cinnamic acid, and p-coumaric acid, respectively. The optimum reaction pH and temperature of LpAAL protein for four substrates were measured at 8.0 and 40 °C, respectively. The Km values of LpAAL protein for aspartic acid, fumaric acid, phenylalanine, and tyrosine as substrates were 5.7, 8.5, 4.4, and 1.2 mM, respectively. The kcat values of LpAAL protein for aspartic acid, fumaric acid, phenylalanine, and tyrosine as substrates were 6.7, 0.45, 4.96, and 0.02 s−1, respectively. Therefore, aspartic acid, fumaric acid, phenylalanine, and tyrosine are bona fide substrates for LpAAL enzyme.

Magnetically agitated continuous-flow tube reactors with aspartate ammonia-lyase immobilized on magnetic nanoparticles

Continuous-flow tube reactors agitating the aspartate ammonia-lyase on magnetic nanoparticles by two external permanent magnets within the reaction stream showed AAL-MNPs being the most efficient in the rotating magnets system.

Re-designing Escherichia coli for high-yield production of β-alanine by metabolic engineering

β-Alanine is the only natural β-amino acid and an important precursor for pantothenic acid (vitamin B5) synthesis. In this work, to improve the β-alanine fermentative production, collaborative modifications mainly focused on aspartate ammonia-lyase (aspA), phosphoenolpyruvate (PEP) – pyruvate (PYR) – oxaloacetate (OAA) – L-aspartate nodes, glucose uptake system, and β-alanine uptake system were performed, including knock out of aspA, reducing the consumption of central metabolic nodes PEP and PYR, enforcement of the non-PEP-dependent transferase system (non-PTS), inactivation of gluconeogenesis through deleting pck gene, and disruption of β-alanine uptake system. As a result, a significantly enhanced β-alanine accumulation was achieved. The β-alanine titer was increased to 6.67 g/L (final strain B15) from 2.46 g/L (original strain B1) in shake flask fermentation, and a titer of 41.12 g/L was achieved in 5-L fermentor fermentation. With the presence of succinic acid (0.25 g/L), the β-alanine titer eventually reached 52.61 g/L at 72 h, and a high productivity (0.73 g/L/h) and yield (0.268 g/g glucose) were also obtained. The yield was relatively higher compared with the reported levels and the B15 exhibited a remarkable fermentation stability over a long period. This result laid foundation for industrialization of β-alanine fermentative production.

Metabolic engineering of Bacillus subtilis for high-level production of uridine from glucose.

As an intermediate in drug synthesis, uridine has practical applications in the pharmaceutical field. Bacillus subtilis is used as a host to boost uridine yield by manipulating its uridine biosynthesis pathway. In this study, we engineered a high-uridine-producing strain of B. subtilis by modifying its metabolic pathways invivo. Overexpression of the aspartate ammonia-lyase (ansB) gene increased the relative transcriptional level of ansB in B. subtilis TD320 by 13·18 times and improved uridine production to 15·13 g l-1 after 72-h fermentation. Overexpression of the putative 6-phosphogluconolactonase (ykgB) gene increased uridine production by the derivative strain TD325 to 15·43 g l-1 . Reducing the translation of the amido phosphoribosyl transferase (purF) gene and inducing expression of the subtilisin E (aprE) gene resulted in a 1·99-fold increase in uridine production after 24 h shaking. Finally, uridine production in the optimal strain B. subtilis TD335, which exhibited reduced urease expression, reached 17·9 g l-1 with a yield of 314 mg of uridine g-1 glucose. To our knowledge, this is the first study to obtain high-yield uridine-producing B. subtilis in a medium containing only three components (80 g l-1 glucose, 20 g l-1 yeast powder, and 20 g l-1 urea).

The VarA-CsrA regulatory pathway influences cell shape in Vibrio cholerae.

Despite extensive studies on the curve-shaped bacterium Vibrio cholerae, the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA-deficient V. cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V. cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment.

Engineering precursor and co-factor supply to enhance D-pantothenic acid production in Bacillus megaterium.

High-yielding chemical and chemo-enzymatic methods of D-pantothenic acid (DPA) synthesis are limited by using poisonous chemicals and DL-pantolactone racemic mixture formation. Alternatively, the safe microbial fermentative route of DPA production was found promising but suffered from low productivity and precursor supplementation. In this study,Bacillus megateriumwas metabolically engineered to produce DPA without precursor supplementation. In order to provide a higher supply of precursor D-pantoic acid, key genes involved in its synthesis are overexpressed, resulting strain was produced 0.53 ± 0.08g/L DPA was attained in shake flasks. Cofactor CH2-THF was found to be vital for DPA biosynthesis and was regenerated through the serine-glycine degradation pathway. Enhanced supply of another precursor, β-alanine was achieved by codon optimization and dosing of the limiting L-asparate-1-decarboxylase (ADC). Co-expression of Pantoate-β-alanine ligase, ADC, phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate ammonia-lyase enhanced DPA concentration to 2.56 ± 0.05g/L at shake flasks level. Fed-batch fermentation in a bioreactor with and without the supplementation of β-alanine increased DPA concentration to 19.52 ± 0.26 and 4.78 ± 0.53g/L, respectively. This present study successfully demonstrated a rational approach combining precursor supply engineering with cofactor regeneration for the enhancement of DPA titer in recombinantB. megaterium.

One-Pot Biosynthesis of 3-Aminopropionic Acid from Fumaric Acid Using Recombinant Bacillus megaterium Containing a Linear Dual-Enzyme Cascade.

3-Aminopropionic acid (3-APA) has wide applications in food, cosmetics, pharmaceuticals, chemical, and polymer industries. This present study aimed to develop an eco-friendly whole-cell biocatalytic process for the bio-production of 3-APA from fumaric acid (FA) using Bacillus megaterium. A dual-enzyme cascade route with aspartate-1-decarboxylases (ADC) from Bacillus subtilis and native aspartate ammonia-lyase (AspA) was developed. Divergent catalytic efficiencies between these two enzymes led to an imbalance between both enzyme reactions. In order to coordinate AspA and ADC expression levels, gene mining, optimization, and duplication strategies were employed. Additionally, culture cultivation conditions and biocatalysis process parameters were optimized. A maximum 3-APA titer was obtained (11.68 ± 0.26g/L) with a yield of 0.78g/g under the following optimal conditions: 45°C, pH 6.0, and 15g/L FA. This study established a biocatalysis process for the production of 3-APA from FA using the whole cells of the recombinant B. megaterium.

Central Carbon Metabolism, Sodium-Motive Electron Transfer, and Ammonium Formation by the Vaginal Pathogen Prevotella bivia.

Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.

Magnetically Agitated Nanoparticle-Based Batch Reactors for Biocatalysis with Immobilized Aspartate Ammonia-Lyase

In this study, we investigated the influence of different modes of magnetic mixing on effective enzyme activity of aspartate ammonia-lyase from Pseudomonas fluorescens immobilized onto epoxy-functionalized magnetic nanoparticles by covalent binding (AAL-MNP). The effective specific enzyme activity of AAL-MNPs in traditional shake vial method was compared to the specific activity of the MNP-based biocatalyst in two devices designed for magnetic agitation. The first device agitated the AAL-MNPs by moving two permanent magnets at two opposite sides of a vial in x-axis direction (being perpendicular to the y-axis of the vial); the second device unsettled the MNP biocatalyst by rotating the two permanent magnets around the y-axis of the vial. In a traditional shake vial, the substrate and biocatalyst move in the same direction with the same pattern. In magnetic agitation modes, the MNPs responded differently to the external magnetic field of two permanent magnets. In the axial agitation mode, MNPs formed a moving cloud inside the vial, whereas in the rotating agitation mode, they formed a ring. Especially, the rotating agitation of the MNPs generated small fluid flow inside the vial enabling the mixing of the reaction mixture, leading to enhanced effective activity of AAL-MNPs compared to shake vial agitation.

Role of aspartate ammonia-lyase in Pasteurella multocida

BackgroundPasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. However, the pathogenesis of this disease is poorly understood. We recently identified an aspartate ammonia-lyase (aspA) in P. multocida that was significantly upregulated under iron-restricted conditions, the protein of which could effectively protect chicken flocks against P. multocida. However, the functions of this gene remain unclear. In the present study, we constructed aspA mutant strain △aspA::kan and complementary strain C△aspA::kan to investigate the function of aspA in detail.ResultDeletion of the aspA gene in P. multocida resulted in a significant reduction in bacterial growth in LB (Luria-Bertani) and MH (Mueller-Hinton) media, which was rescued by supplementation with 20 mM fumarate. The mutant strain △aspA::kan showed significantly growth defects in anaerobic conditions and acid medium, compared with the wild-type strain. Moreover, growth of △aspA::kan was more seriously impaired than that of the wild-type strain under iron-restricted conditions, and this growth recovered after supplementation with iron ions. AspA transcription was negatively regulated by iron conditions, as demonstrated by quantitative reverse transcription-polymerase chain reaction. Although competitive index assay showed the wild-type strain outcompetes the aspA mutant strain and △aspA::kan was significantly more efficient at producing biofilms than the wild-type strain, there was no significant difference in virulence between the mutant and the wild-type strains.ConclusionThese results demonstrate that aspA is required for bacterial growth in complex medium, and under anaerobic, acid, and iron-limited conditions.

Protection of chickens against fowl cholera by supernatant proteins of Pasteurella multocida cultured in an iron-restricted medium

ABSTRACTPasteurella multocida (P. multocida), a causative agent of fowl cholera, is an important pathogen in the poultry industry. In the present study, we found that the inactivated vaccine of P. multocida grown in an iron-restricted medium provided better protection than that grown in normal medium. Thus, we adopted a comparative proteomics approach, by using two-dimensional gel electrophoresis (2-DE), coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS), to profile the supernatant proteins associated with P. multocida under both conditions. Eleven upregulated proteins were identified, including aspartate ammonia-lyase (AspA), diacylglycerol kinase (DgK), 30S ribosomal protein S6 (RpsF), and eight outer membrane proteins (OMPs). To further characterize the three novel supernatant proteins identified under iron-restricted conditions, the AspA, DgK and RpsF proteins were expressed and purified, and used as immunogens to vaccinate chickens. The results showed that AspA, DgK and RpsF proteins induced 80.0%, 66.7%, and 80.0% immunity, respectively. These data indicate that the three novel proteins identified in the supernatant of the culture media might play important roles in the survival of bacteria under iron-restricted conditions, and thus protect chickens against P. multocida. These findings also suggest that the proteins identified can be used as subunit vaccines.

Immunoproteomic identification of antigenic candidate Campylobacter jejuni and human peripheral nerve proteins involved in Guillain-Barré syndrome

Immunoproteomics is become a potent methodology used for identifying immunoreactive proteins. In this study, an immunoproteomic approach based on 2-dimensional gel electrophoresis (2D-PAGE) and immunoblotting combined with high resolution mass spectrometry (MS) was used to identify immunoreactive proteins that might be involved in mechanisms of Guillain-Barré syndrome (GBS) development, regardless of their potential reciprocal molecular mimicry. Proteins isolated from C. jejuni and human peripheral nerve tissue (HPN) were separated with 2D SDS-PAGE and subjected to western blotting using serum samples from GBS patients. The peptides generated after proteolysis of the immunoreactive proteins were submitted to nanoflow-high performance liquid chromatography-nano electrospray ionization coupled to high resolution mass spectrometry (nHPLC-nESI-MS and MS/MS) followed by SEQUESTdata analysis for proteins identification. In C. jejuni, immunoreactivity was found for GroEL and DnaK, structural proteins (MOMP), key enzymatic proteins necessary for the microbial proliferation (adenylate kinase, enolase, inorganic pyrophosphatase and aspartate ammonia-lyase), and antioxidant enzymes (alkyl hydroperoxide reductase–AhpC and DNA protection during starvation protein - DNA protection factor against Fe2+-mediated oxidative stress).HPN immunoreactive proteins identified were heat shock proteins (HSP), intermediate filaments (vimentin and desmin), and other proteins and enzymes such as troponin/tropomyosin complex and ATP synthase subunit beta and the keratan sulfate proteoglycan lumican. The targeting of vimentin and desmin, suggested that the neuronal autoimmune damage is specifically directed to intermediate neuronal (vimentin) and neuromuscular IF, probably localized nearby cell surface, affording increased accessibility to autoantibodies. These findings suggest that the post-infectious development of GBS may be also associated to additional concomitant immune factors that lead to nerve damage generated by auto-immune trigger(s) different from molecular mimicry.

Development of an Amperometric Biosensor Platform for the Combined Determination of L-Malic, Fumaric, and L-Aspartic Acid.

Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD+) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of +0.3V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7μAmM-1 (L-malate biosensor) and 0.4μAmM-1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0-10.0mM with a sensitivity of 0.09μAmM-1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.

Discovery of novel highly active and stable aspartate dehydrogenases

Aspartate family amino acids (AFAAs) have important commercial values due to their wide spectrum of applications. Most if not all AFAAs are produced under aerobic conditions which is energy-intensive. To establish a cost-effective anaerobic process for production of AFAAs, it holds great promise to develop a new pathway enabling the conversion of oxoloacetate into aspartate through direct amination which is catalyzed by aspartate dehydrogenase (AspDH). Compared with the well studied aspartate aminotransferase and aspartate ammonia-lyase, only a few AspDHs are characterized till date, and failure to reproduce the high activity of AspDH from Rastonia eutropha documented in the literature encouraged us to screen and characterize novel AspDHs from different origins. Interestingly, the AspDHs from Klebsiella pneumoniae 34618 (KpnAspDH) and Delftia sp. Cs1–4 (DelAspDH) showed successful soluble expression. KpnAspDH and DelAspDH containing C-terminal hexa-histidine tags were purified and characterized for their catalytic properties. Notably, in addition to its high reductive amination activity, DelAspDH exhibited considerable stability as compared to the other source of AspDHs. This work thus provides novel enzyme resource for engineering strains capable of producing AFAAs under anaerobic conditions.

Bioproduction of L-Aspartic Acid and Cinnamic Acid by L-Aspartate Ammonia Lyase from Pseudomonas aeruginosa PAO1.

Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) catalyses the reversible amination and deamination of L-aspartic acid to fumaric acid which can be used to produce important biochemical. In this study, we have explored the characteristics of aspartase from Pseudomonas aeruginosa PAO1 (PA-AspA). To overproduce PA-AspA, the 1425-bp gene was introduced in Escherichia coli BL21 and purified. A 51.0-kDa protein was observed as a homogenous purified protein on SDS-PAGE. The enzyme was optimally active at pH8.0 and 35°C. PA-AspA has retained 56% activity after 7days of incubation at 35°C, which displays the hyperthermostablility characteristics of the enzyme. PA-AspA is activated in the presence of metal ions and Mg2+ is found to be most effective. Among the substrates tested for specificity of PA-AspA, L-phenylalanine (38.35±2.68) showed the highest specific activity followed by L-aspartic acid (31.21±3.31) and fumarate (5.42±2.94). K m values for L-phenylalanine, L-aspartic acid and fumarate were 1.71mM, 0.346μM and 2M, respectively. The catalytic efficiency (k cat/K m) for L-aspartic acid (14.18s-1mM-1) was higher than that for L-phenylalanine (4.65s-1mM-1). For bioconversion, from an initial concentration of 1000mM of fumarate and 30mM of L-phenylalanine, PA-AspA was found to convert 395.31μM L-aspartic acid and 3.47mM cinnamic acid, respectively.

The highly efficient expression of the aspartase gene (L-aspartate ammonia-lyase) in Escherichia coli cells

Cloning of aspartase gene (L-aspartate ammonia-lyase) from the natural isolate Escherichia coli VKPM V-7188 in the pLATE31 plasmid composition and its expression under the control of the T7 phage promoter in Escherichia coli BLR (DE3) cells was performed. It was shown that the aspartase content in recombinant cells under optimal conditions is up to 60% of the sum of all dissolved proteins, while the maximal specific activity of cells increases to 700 µmole/mg S/min. Mutant variants carrying an aspartase gene with end sequence deletions and coding proteins with an increased level of aspartase activity were obtained. The developed system for a highly efficient expression of aspartase gene can be used for the selection and quick estimation of catalytic properties of enzymatic mutant forms generated in vitro, as well as for the creation of new, more efficient biocatalists of L-aspartic acid synthesis.

Efficient aspartic acid production by a psychrophile-based simple biocatalyst.

We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds.

Nitrilase-catalyzed hydrolysis of 3-aminopropionitrile at high concentration with a tandem reaction strategy for shifting the reaction to β-alanine formation

Given the importance of β-alanine, the nitrilase BjNIT3397 from Bradyrhizobium japonicum strain USDA110 was examined toward the hydrolysis of 3-aminopropionitrile. It has been found that nitrilase BjNIT3397 effectively hydrolyzed 3-aminopropionitrile with substrate concentration up to 3M (210g/L) at the pH 7.3 and temperature 30°C. With the increase of substrate concentration from 0.6 to 3M, 3-aminopropanamide was formed and its percentage in the products was increased up to 33%. In order to reduce the formation of 3-aminopropanamide, aspartate ammonia-lyase and fumaric acid were added into the reaction system to consume the byproduct ammonia. As expected, the reaction was shifted toward the formation of β-alanine, resulting in the decrease of 3-aminopropanamide from 33% to 3%. Therefore, a tandem reaction strategy was developed to effectively prevent the formation of 3-aminopropanamide. This might also offer a possibility of producing β-alanine and l-aspartic acid in one process.