Abstract:
It discloses a use of N-acetylneuraminic acid aldolase with an amino acid sequence as shown in SEQ ID NO: 2 in catalytic synthesis of N-acetylneuraminic acid. The preparation of N-acetylneuraminic acid is to use the N-acetylneuraminic acid aldolase with the amino acid sequence as shown in SEQ ID NO: 2 as a catalyst, and N-acetylmannosamine and pyruvic acid as substrates.

Description:
[0001]    This application is the U.S. national phase of International Application No. PCT/CN32013/087987 filed on 27 Nov. 2013 which designated the U.S. and claims priority to Chinese Application Nos. 201310482695.7 filed on 16 Oct. 2013, the entire contents of each of which are hereby incorporated by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention belongs to the field of biotechnology, and it relates to the use of N-acetylneuraminic acid aldolase (Nal), particularly relates to the use of Nal from  Corynebacterium glutamicum  ATCC 13032 in producing N-acetylneuraminic acid (Neu5Ac) by using N-acetylmannosamine (ManNAc) and pyruvic acid as the substrates. 
       BACKGROUND OF THE INVENTION 
       [0003]    N-acetylneuraminic acid (N-acetyl-D-neuraminic acid, Neu5Ac) is an important milk powder additive, which can improve immunity of infants [1], meanwhile it can be used as a precursor for synthesizing anti-influenza A/B type virus drugs [2]. Synthesizing Neu5Ac by using ManNAc and pyruvic acid as the substrates under catalysis by Nal is the currently most primary synthesis route for Neu5Ac. Nal has been used in industrial synthesis of Neu5Ac [3-5], and synthesis of Neu5Ac from ManNAc and pyruvic acid under catalysis by Nal is a reversible reaction. Nal is widely distributed in the nature, and it is found in bacteria and mammals [6]. Many pathogenic bacteria, after invasion into human body, decompose human Neu5Ac by Nal as their carbon source and nitrogen source [6], thus currently there are a great number of reports on Nal from pathogenic bacteria. Besides Nal derived from pathogenic bacteria, there is also Nal found from food safe (generally regarded as safe, GRAS) strains, such as  Lactobacillus plantarum  WCFS1 [7] and  Taphylococcus carnosus  TM300 [8]. Because the substrate pyruvic acid is cheap [9] and Nals have relatively high temperature stability [10], it has been widely applied in the synthesis of Neu5Ac. However, currently all of Nal have a common defect: in a reversible catalytic synthesis reaction of Neu5Ac, the Nal is more prone to decompose Neu5Ac [7, 8, 11-14]. 
         [0004]    To obtain Nal of high activity and to resolve the issue of the chemical equilibrium of Nal being prone to decomposing Neu5Ac, this patent cloned and expressed N-acetylneuraminic acid aldolase (CgNal) from food safe strain  Corynebacterium glutamicum  ATCC 13032 [15]. It belongs to one of Nal family, comprising 312 amino acids, and its accession number in Genbank is NP — 601846, its amino acid sequence is shown in SEQ ID NO: 2. The gene encoding this protein comprises 939 bp bases, its accession number in the Genbank is NC — 003450.3, and its gene sequence is shown in SEQ ID NO:1. Reports on using CgNal in Neu5Ac synthesis has not been found until now. 
       SUMMARY OF THE INVENTION 
       [0005]    The technical issue to be resolved by the present invention is to provide use of N-acetylneuraminic acid aldolase (Nal) in catalytic synthesis of N-acetylneuraminic acid (Neu5Ac) from N-acetylmannosamine (ManNAc) and pyruvic acid. 
         [0006]    To resolve the above-described technical issue, a technical solution adopted by the present invention is as follow: 
         [0007]    Use of N-acetylneuraminic acid aldolase (Nal) with an amino acid sequence as shown in SEQ ID NO: 2 in catalytic synthesis of N-acetylneuraminic acid (Neu5Ac) from N-acetylmannosamine (ManNAc) and pyruvic acid. 
         [0008]    A specific method is synthesizing N-acetylneuraminic acid by using N-acetylneuraminic acid aldolase with the amino acid sequence as shown in SEQ ID NO: 2 as a catalyst, and using N-acetylmantosamine and pyruvic acid as substrates. 
         [0009]    A more specific method is to express a recombinant strain comprising a gene sequence as shown in SEQ ID NO: 1, and a crude N-acetylneuraminic acid aldolase after lysis or a pure N-acetylneuraminic acid aldolase obtained by further nickel column purification is reacted with N-acetylmannosamine and pyruvic acid in a buffer, to obtain N-acetylneuraminic acid. 
         [0010]    Wherein, said recombinant strain comprising the gene sequence as shown in SEQ ID NO: 1 is established by the following method: the gene of N-acetylneuraminic acid aldolase Nal is amplified by using  Corynebacterium glutamicum  ATCC13032 genome as a template and ligated to a pET-28a vector, then the recombinant plasmid is transformed into  E. coli  Rosetta (DE3). Wherein, the method to express the recombinant strain comprising the gene sequence as showing in SEQ ID NO: 1 is: when the recombinant strain is incubated to OD 600 =0.4 to 0.8, IPTG of final concentration 0.2 to 1.0 mmol·L −1  is added at 15 to 37° C., and induced at 150 to 220 rpm for 4 to 12 hours. The preferred method is: when the recombinant strain is incubated to OD 600  of 0.6, IPTG of final concentration 0.2 mmol·L −1  is added at 30° C., and induced at 220 rpm for 10 hours. Wherein, the condition of the nickel column purification is: a mixed protein is eluted with a 20 mmol·L −1  imidazole solution, and the pure enzyme is eluted with a 500 mmol·L −1  imidazole solution. 
         [0011]    Wherein, the reaction ratio of N-acetylneuraminic acid aldolase with N-acetylglucosamine and pyruvic acid is: 0.36 to 300 U·mL −1  crude enzyme or pure enzyme is reacted with 100 to 1000 mmol·L −1 N-acetylmannosamine and 100 to 2000 mmol·L −1  pyruvic acid. 
         [0012]    Wherein, said buffer is 20 to 200 mmol·L −1  Tris-HCl buffer of pH7 to 8.8 or 20 to 200 mmol·L −1  glycine-NaOH buffer of pH 9.0 to 9.5, preferably Tris-HCl buffer of pH 7 to 8.5, most preferably Tris-HCl buffer of pH 7.5 or Tris-HCl buffer of pH 8.5. 
         [0013]    Wherein, the reaction condition in the buffer is: the temperature being 25 to 60° C., and reaction time being 0.1 to 12 hours; preferably, the temperature being 35 to 45° C., and reaction time being 0.15 to 0.5 hours; most preferably, the temperature being 40° C., and reaction time being 0.15 to 0.5 hours. 
         [0014]    The inventors, based on modern bioinformatics principle and in combination with molecular biotechnology, cloned the gene of N-acetylneuraminic acid aldolase from  Corynebacterium glutamicum  ATCC13032 by the method of genetic engineering and expressed it in  Escherichia coli , and it was found be able to catalyze and synthesize Neu5Ac from ManNAc and pyruvate. Beneficial effects: the present invention firstly used the N-acetylneuraminic acid aldolase with the amino acid sequence as shown in SEQ ID NO: 2 in catalytic synthesis of Neu5Ac from ManNAc and pyruvate, and obtained very good effects, its enzyme activity was up to 12 U/mg. Because this reaction is a reversible reaction, compared with other Nals, the chemical equilibrium of this aldolase is more prone to a direction of N-acetylneuraminic acid synthesis i.e., sialic acid synthesis, meanwhile the expression effect of the enzyme is very good, no inclusion body is formed, and the expression amount of N-acetylneuraminic acid aldolase is large, being 5 folds of expression amount of aldolase gene derived from  Escherichia coli , meanwhile  Corynebacterium glutamicum  is a food safe bacteria. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
         [0015]      FIG. 1  is a graph of establishing the N-acetylneuraminic acid aldolase gene. 
           [0016]      FIG. 2  is a graph of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after expression of CgNal (from  Corynebacterium glutamicum  ATCC13032) and EcNal (from  Escherichia coli ). Wherein, 1 is CgNal crude enzyme, 2 is CgNal pure enzyme, 3 is EcNal crude enzyme, 4 is EcNal pure enzyme, and 5 is Marker. 
           [0017]      FIG. 3  is effect of pH on CgNal enzyme activity. 
           [0018]      FIG. 4  is effect of temperature on CgNal enzyme activity. 
           [0019]      FIG. 5  is change of the enzyme activity of CgNal during the warm water bath. 
           [0020]      FIG. 6  is effects of metal ion and surfactant at pH 7.5 on CgNal enzyme activity 
           [0021]      FIG. 7  is effects of metal ion and surfactant at pH 8.5 on CgNal enzyme activity. 
           [0022]      FIG. 8  is change of product concentration during synthesis of Neu5Ac using CgNal as the catalyst. 
       
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
       [0023]    The present invention can be better understood based on the following examples. However, one skilled in the field will easily understand that the specific material ratio described in the examples, process conditions and their results are used to illustrate the present invention only and should not be used to limit the invention described in detail in the claims. 
       Example 1 
     Establishing the Recombinant  E. coli  Rosseta (pET28a-CgNal) 
     1. Obtaining N-Acetylneuraminic Acid Aldolase Gene: 
       [0024]    The genome of  Corynebacterium glutamicum  ATCC 13032 was extracted, then PCR was carried out by using the extracted genome as the template. 
         [0025]    The primer adding enzyme digestion site used in expression vector was established, the sequence of the primer was as follow: 
         [0000]    
       
         
               
             
           
               
                 An upstream primer (CgNal-sense comprising BamH I)  
               
               
                 is: 
               
               
                 (SEQ ID NO: 3) 
               
               
                 5′-GACAGCAAATGGGTCGCGGATCCATGGCTTCCGCAACTTTCACC 
               
               
                 G-3′ 
               
               
                   
               
               
                 A downstream primer (CgNal-anti comprising Hind 
               
               
                 III) is: 
               
               
                 (SEQ ID NO: 4) 
               
               
                 5′-TGCTCGAGTGCGGCCGCAAGCTTTTAAGCGGTGTACAGGAATTCAT 
               
               
                 C-3′ 
               
             
          
         
       
     
         [0026]    All the primers were synthesized by Suzhou GENEWIZ Corporation. 
       PCR Conditions for Gene: 
       [0027]    Cycle 30 times according to the following parameters: denaturation at 98° C. for 10 seconds, annealing and extension at 68° C. for 1 minute, finally extension at 72° C. for 10 minutes. 
         [0000]    2. Transforming the Recombinant  E. coli  Rosseta (DE3): 
         [0028]    The pET-28a vector (pET-28a, purchased from Novagen (Merck China)) was digested by BamH I and Hind III respectively, after conforming that the vectors were completely linearized, the target fragment of PCR and the linearized expression vector were extracted respectively, then with one-step clone kit (ClonExpress), 10 μL of linking product pET-28a-CgNal was added into 100 μL of Rosetta (DE3) competent cells, and placed on ice for 30 minutes, heat shocked at 42° C. for 90 seconds, placed on ice for 5 minutes. A pre-heated 0.9 mL of LB medium was added. Centrifuged at 200 rpm at 37° C. for 1 hour. A 200 μL of bacteria solution was added onto a LB plate containing 100 μg/mL kanamycin and chloramphenicol respectively, incubated at 37° C. overnight for 12 to 16 hours. The graph of establishment is seen in  FIG. 1 . 
       Example 2 
     Obtaining the Aldolase CgNal 
     1. Expression of N-Acetylneuraminic Acid Aldolase CgNal. 
       [0029]    The recombinant strain  E. coli  Rosseta (pET-28a-CgNal) was picked up into a LB liquid medium containing antibiotics, incubated under vibration at 37° C. overnight. Then, inoculated to a fresh culture solution in a 1 (v/v) % inoculation amount, when incubated to OD 600  of about 0.6 at 37° C., IPTG was added to a final concentration of 0.2 mmol·L −1 , centrifuged at 200 rpm at 30° C., induced expression for 10 hours, then centrifuged (4° C., 10000 rpm, 10 minutes). 
       2. Purifying N-Acetylneuraminic Acid Aldolase CgNal. 
       [0030]    The collected bacterial sludge was re-suspended in a 100 mmol·L −1  Tris-HCl (PH 7.5) buffer, and the cells were ultrasonically lysed (power 300W, sonicated for 3 seconds, interrupted for 5 seconds, totally 5 minutes), centrifuged (4° C., 12000 rpm, 15 minutes), and supernatant was removed. 
         [0031]    The collected enzyme supernatant was added to a Ni-NTA column (Ni-NTA His Bind Resin, Novagen), and incubated on ice for 30 minutes. After the supernatant flowed through the column, the mixed protein was washed away with a 100 mmol·L −1  Tris-HCl (pH 7.5) containing 20 mmol·L −1  imidazole. Then, the target protein CgNal was eluted down with a 100 mmol·L −1  Tris-HCl (pH 7.5) containing 500 mmol·L −1  imidazole. Use aldolase EcNal from  Escherichia coli  as a control, and the purity and expression level of CgNal were detected by SDS-PAGE, which was shown in  FIG. 2 . The protein concentration of the purified CgNal was determined by Bradford method. 
       Example 3 
     Study on the Enzymatic Properties of the Aldolase CgNal 
     1. Detecting Method for CgNal Enzyme Activities 
       [0032]    The enzyme activities of CgNal were divided into the enzyme activity of Neu5Ac synthesis reaction and the enzyme activity of Neu5Ac decomposition reaction. The enzyme activity on Neu5Ac synthesis reaction was defined as the enzyme amount required for synthesizing 1 μmol Neu5Ac per minute, and the enzyme activity on Neu5Ac decomposition reaction was defined as the enzyme amount required for decomposing 1 μmol Neu5Ac per minute, the enzyme activity detection solution for Neu5Ac decomposition reaction was 0.1 M pyruvic acid, 0.1 mol·L −1 ManNAc and 0.1 mol·L −1  Tris-HCl (pH 7.5 or 8.5); the enzyme activity detection solution for Neu5Ac decomposition reaction was 100 mmol·L −1  Neu5Ac and 0.1 mol. L −1  Tris-HCl (pH 7.5 or 8.5). The purified Nal was added into 1 ml of enzyme activity detection solution to the final concentration of 30 μg/ml (about 0.36 U/ml). After reaction at 37 V for 20 minutes, the tube was heated in a boiling water for 5 minutes to stop the reaction, centrifuged at 12000 g for 10 minutes, and the sample was filtered with a 0.22 μm filter. 
         [0033]    The substrate and the product were detected with Bio-Rad Aminex 87-H column by using Agilent 1200 HPLC, (5 mmol·L −1  H 2 SO 4  as a mobile phase, flow rate 0.6 ml/min, differential refractive index detector). 
       2. The Enzyme Activities of CgNal at Different pH Values. 
       [0034]    The following buffer was used in effect of pH on CgNal: 0.1 M Tris-HCl (pH 7 to 8.8) and 0.1 M glycine-NaOH buffer (pH 9.0 to 9.5). The enzyme activity was detected in the enzyme activity detection solutions of different pHs, in order to detect the effect of pH on the enzyme activity. The detection results showed that the activity of CgNal on Neu5Ac decomposition reaction between pH 7.5 and 8.4 was much higher than the activity on Neu5Ac synthesis reaction; when the pH was between 8.6 and 8.8, the decomposition activity of Neu5Ac was close to Neu5Ac synthesis activity ( FIG. 3 ). 
       3. Effect of the Temperature on the CgNal  
       [0035]    The enzyme activities of both directions of the aldolase CgNal were detected by using a temperature gradient at pH 7.5 (25° C. to 60° C.), in order to find the optimum reaction temperature. At pH 7.5, the optimum temperature for CgNal was 40° C., the optimum temperature for decomposition and synthesis reaction of Neu5Ac were identical. At pH 8.5, the optimum temperature of Neu5Ac synthesis direction was 40° C., and the optimum temperature of Neu5Ac decomposition direction was 45° C. ( FIG. 4 ). The CgNal was suspended in a 0.1 M Tris-HCl (pH 8.5) buffer, then placed in a warm water bath at 37° C. for 48 hours, and the change of enzyme activities was detected during the warm water bath, in order to determine the stability of CgNal. Within 10 hours prior to the warm water bath, Neu5Ac synthesis activity of CgNal showed a rising trend, after 36 hours of warm water bath it can still maintain about 80% of starting activity ( FIG. 5 ). 
       4. Effects of Metal Ions and Surfactant on the CgNal Activity 
       [0036]    To an enzyme activity detection solution of CgNal, 5 mM of CaCl 2 , NaCl, BaCl 2 , FeCl 3 , KCl, ZnCl 2 , CoCl 2 , MgCl 2 , NH 4 Cl, NiSO 4 , EDTA, CTAB and SDS were added, and a sample without adding any metal ion and surfactant was used as a control. The enzyme activities of CgNal at both pH 7.5 and pH 8.5 were detected. The enzyme activity of CgNal at pH 8.5 was much higher than the enzyme activity of CgNal at pH 7.5, and the effects of metal ions on CgNal under different pH conditions were quite different. At pH 7.5, ZnCl 2 , CoCl 2 , NiSO 4 , CTAB and SDS all promoted reaction of CgNal in the synthesizing Neu5Ac direction, meanwhile they also inhibited the reaction in the decomposing Neu5Ac direction ( FIG. 6 ). Whereas the metal ions at pH 8.5 had no significant activation effect on CgNal. ZnCl 2 , CTAB and SDS promoted the superiority of Neu5Ac synthesis over Neu5Ac decomposition, but overall decreased the enzyme activity ( FIG. 7 ). Therefore, at suitable pH value and under effect of metal ions and surfactant, the rate of CgNal in Neu5Ac synthesis direction was greater than the rate in Neu5Ac decomposition direction. 
       5. Determining the Enzyme Reaction Kinetic Constants for CgNal 
       [0037]    The enzymatic reaction kinetic constants of CgNal at pH 7.5 and pH 8.5 on the substrates ManNAc, Neu5Ac and pyruvic acid were determined at different concentration of substrates. When the kinetic constant on pyruvic acid was determined, the fixed ManNAc concentration was 100 mM, and the pyruvic acid concentration varied between 1 and 100 mM. When the kinetic constant on ManNAc was determined, the fixed pyruvic acid concentration was 100 mM, and the concentration of ManNAc varied between 1 and 400 mM. When the kinetic constant on Neu5Ac was determined, the concentration of Neu5Ac varied between 1 and 200 mM. The kinetic constants of CgNal on Neu5Ac, ManNAc and pyruvic acid at pH 7.5 and pH 8.5 were as shown in Table 1. When the pH value was increased from 7.5 to 8.5, the Km and Vmax values of the substrate were greatly increased. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Kinetic constants of CgNal 
               
             
          
           
               
                   
                 Neu5Ac 
                   
                 ManNAc 
                   
                 pyruvic acid 
                   
               
             
          
           
               
                   
                 Km 
                 Vmax 
                 Km 
                 Vmax 
                 Km 
                 Vmax 
               
               
                 pH 
                 (mM) 
                 (U/mg) 
                 (mM) 
                 (U/mg) 
                 (mM) 
                 (U/mg) 
               
               
                   
               
             
          
           
               
                 7.5 
                 33.5 
                 16.74 
                 53.3 
                 10.2 
                 14.7 
                 10.98 
               
               
                 8.5 
                 87.7 
                 79.6 
                 92.1 
                 73.2 
                 72.4 
                 76.64 
               
               
                   
               
             
          
         
       
     
       Example 4 
     Catalytic Synthesis of Neu5Ac by Using CgNal as the Catalyst 
       [0038]    In a 20 ml of reaction system, the reaction solution was 50 mM Tris-HCl (pH 7.5 and pH 8.5) comprising 0.8 mol·L −1  ManNAc and 2 mol·L −1  pyruvic acid. Under the same conditions, 1 mL of inducer purified CgNal (180 U/mL) and EcNal (64 U/mL) were respectively added into the reaction solution. The catalytic condition was 37° C., 200 rpm for 12 hours. pH was respectively maintain to 7.5 and 8.5, During the reaction, the solution was sampled and the contents of ManNAc, pyruvic acid and Neu5Ac were detected. The catalysis results showed that the yield of CgNal was much higher than the yield of ECNal, and CgNal synthesized the highest amount of 185 g/L Neu5Ac within 12 hours. Within 6 hours prior to catalysis, the yield at pH 8.5 were significantly higher than the yield at pH 7.5, then in terms of catalysis results of EcNal and CgNal, the yield at pH 8.5 was very close to the yield at pH 7.5 ( FIG. 8 ). 
       REFERENCES 
       [0000]    
       
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         13. Schauer, R. and M. Wember,  Isolation and characterization of sialate lyase from pig kidney . Biol Chem Hoppe Seyler, 1996. 377(5): p. 293-9. 
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         15. Zahoor ul Hassan, A., S. Lindner, and V. F. Wendisch,  Metabolic engineering of Corynebacterium glutamicum aimed at alternative carbon sources and new products.    
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