TW200904458A - Method of producing N-acetyl-d-neuraminic acid and application thereof - Google Patents

Abstract

Genes of Anabaena sp. CH1 N-acetyl-d-glucosamine 2-epimerase (bage) and E. coli NeuAc lyase were cloned and, wholly or individually, expressed in E. coli. The bage gene has a length of 1167bp. The molecular mass of the transformed protein was determined to be 43 kDa. A stirred glass vessel containing transformed E. coli cells expressing bage gene from Anabaena sp. CH1 and NeuAc lyase gene separately is used for the conversion of GlcNAc and pyruvate to NeuAc. A maximal productivity of 10.2 g NeuAc l/L h with 33.3% conversion yield from GlcNAc could be obtained when the concentrations of GlcNAc and pyruvate are both 1.2M. The recombinant E. coli cells can be reused for more than eight cycles with a productivity higher than 80%. This new process of using recombinant whole cells to produce NeuAc has advantages of high productivity and high recycling use of enzyme, and the necessity of using ATP can be omitted.

Description

200904458 IX. INSTRUCTIONS: TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for producing N-acety^D-neuraminic acid and an application thereof. [Prior Art] N-acetamidine-D-neuraminidase (NeuAc) is abundantly present on the cell surface glycoprotein and glycolipid (g丨ycoh_pid), and is recognized by cell-cell recognition. ), bio-message (transduction) and pathogen infection play a very important role. Many NeuAc-based drugs have been used to treat a variety of diseases such as epidemic, diabetes, and cancer. They are very important and expensive APIs. At present, NeuAc is produced in the following four ways: 1 · From natural sources Extract

NeuAc can be produced by natural extraction method, from defatted egg yolk (egg_y〇丨k), by acid or protease (p "otease" hydrolysis and desalting, and then purified by ion exchange tree. The other raw material is whey (mj |k whey), the same method is used to produce NeuAc. In addition, NeuAc is also produced from polysialic acid (c〇|〇rnjnjc acid) by hydrolysis and chromatographic purification. Due to the abundant source of raw materials, natural matter is extracted as the current The main method of industrial production of NeuAc. 2. Enzymatic Process Enzymatic Process The synthesis of NeuAc by enzyme method is a two-step biochemical method. The required biocatalyst is W-acetam-D-glucosamine ( G丨cNAc) is converted to N-ethyl-D-mannosamine (ManNAc) N-acetamidine-D-glucosamine 2-epoxidase (A/-acety D-glucosamine 2-epimerase; AGE), and will be 200904458

ManNAc and pyruvate condensed into NeuAc N-acetyl-D-neuraminic acid lyase (A/-acetyl-D-neuraminic acid lyase; NeuAc lyase). Ghosh et al. first purified AGE (pAGE) from pig kidney cortex in 1965, which proved that it can catalyze the reversible epimerization reaction between ManNAc and GIcNAc. It is also documented that this enzyme is in the presence of ATP. The reverse reaction rate can be increased by about 2 times. E. co// NeuAc dissociation enzyme was first used by Kim et al. (1988) to catalyze the synthesis of NeuAc from ManNAc and pyruvate. Based on the understanding of pAGE and NeuAc dissociation enzymes, the literature proposes a two-step enzyme method of epi-asease/lyase (epjmerase/lyase) to synthesize NeuAc. The gene expression method was used to express pAGE and NeuAc dissociation enzyme in Ε· co//· in large quantities. Then the enzyme was obtained by separation and purification technology. The pAGE and NeuAc dissociation enzymes were simultaneously immobilized on the filter membrane.

Enzyme membrane reactor (EMR), in the presence of ATP, uses EMR as a catalyst to catalyze the synthesis of NeuAc from Mannac and pyruvate. This is a technique for membrane-enclosed enzymatic catalysis (MEEC) applied to the enzyme production of NeuAc. In addition, there are also reports that directly using a large amount of purified pAGE and NeuAc dissociation enzyme as a catalyst, the reaction is directed to the synthesis of NeuAc by continuously adding a large amount of pyruvic acid, and the conversion rate of G|cNAc to NeuAc is as high as 77%. This method is considered to have the advantages of simple process, high yield and low pollution compared with chemical synthesis. 3. Chemistry and Enzyme Synthesis The disadvantage of synthesizing NeUAc by enzyme catalysis is that expensive ATP is required to activate PAGE, and NeuAc dissociation enzyme recombination 200904458 protein is relatively easy to obtain in large quantities relative to pAGE. Therefore, Dawson et al. of the Drug Research and Development Center of Glaxo Pharmaceuticals in the United Kingdom proposed a process for synthesizing NeuAc by chemical and chemoenzymatic process. First, the GIcNAc is surface-isomerized into ManNAc by alkali, and then ec〇J/ NeuAc dissociation enzyme catalyzes the condensation of ManN Ac with pyruvate to NeuAc. They found the optimum reaction conditions for NeuAc dissociation enzyme between ManNAc's bath resolution and concentration, pyruvate concentration, NeUAc concentration and pΗ value, which determined the reaction mode of NeuAc production by NeuAc dissociation enzyme. However, the equilibrium of the NeuAc dissociation enzyme reaction is in the direction of decomposition of NeuAc. To make the NeuAc dissociation enzyme direction toward the synthesis of NeuAc, a high concentration of ManNAc ′ means that a high concentration of G|cNAc is required. However, high concentrations of GIcNAc inhibit the activity of NeuAc dissociation enzymes, causing a bottleneck in the labor process. Therefore, Dawson et al. proposed to use ManNAc and GlcNAc to have different solubility properties in different solutions, reduce the concentration of GIcNAc and increase the concentration of ManNAc. Reuse of E. co//_ NeuAc dissociation enzyme to synthesize NeuAc. Dawson et al. filed a PCT patent for this "test treatment and NeuAc dissociation enzyme catalysis" process, the application number of which is yy〇 Patent: 94/29476. In addition, T本 Tsukada et al. also developed a similar chemical and enzyme method to synthesize

NeuAc' was converted to ManNAc under the conditions of PH 10-12, and then NeuAc was catalyzed by NeuAc dissociation enzyme, and the process was also obtained from U.S. Patent No. 5,472,860. 4. Whole cell conversion synthesis method Different from the above three methods, Tabata et al. proposed a NeuAc synthesis method for whole cell enzymes. The large amount of SyA?ec/?ocysi/s sp. 200904458 PCC6803 AGE gene was used as the first biocatalyst to catalyze the isomerization of GIcNAc to ManNAc. The C〇"NeuAc synthetase gene ((10)...) is the first biocatalyst, catalyzing ManN Ac and phosphoenolpyruvate (Ph〇sph〇_iPy "uvate; PEPU NeuAc, and pEp is provided by glucose or fructose by the third microbial c〇r coffee " continued ammo/7/agenes fermentation. This whole cell synthesis method does not need to add ATP or chemical waste in the above three methods. The shortcomings of the object to be treated. The process of synthesizing NeuAc by the enzyme method is a two-step biochemical method. The required biocatalyst is to convert G|cNAc into ManNAc AGE; and add ManNAc to pyruvate. The NeuAc dissociation enzyme of NeuAc was synthesized. In the enzyme-membrane reactor (EMR) process of Krag丨 et al., 5 mM ATP was added to the reaction solution, and the NeuAc yield was 4 5 g/Lh (1〇gg/L). -Day) 'The conversion rate of GIcNAc and pyruvate is 28% and 35%. EMR is reusable and free By-products have the greatest advantage for this process. In the free enzyme process of Tsukada et al., the strategy of continuous addition of pyruvate was used to avoid the inhibition of PAGE, thus obtaining a GIcNAc conversion rate of up to 77%, while the concentration of NeuAc was 153. g/L. However, this process requires the addition of 9 mM ATP, and the reaction time of 250 hours results in extremely low yield (0.64 g NeuAc/Lh). The biggest disadvantage of the above two processes for synthesizing NeuAc by enzyme method In order to increase the activity of pAGE by adding ATp, the dependence property greatly enhances the production cost of NeuAc. The chemical and enzyme synthesis method is used to form the isomerization of GIcNAc into ManNAc with alkali, and then use £·co// NeuAc The dissociation enzyme catalyzes the synthesis of ManNAc and C-200904458 keto acid to synthesize P11 Δ-AC' in the preparation of Daws〇n et al., the spread of ~150 g/L and the G| of peaches However, compared with the two y biochemical methods, the chemical and enzyme synthesis and Dinghua pretreatment steps seem quite complicated. In order to make the NeuAc dissociation enzyme toward the synthesis side, ', ΚΙΛ stone removal reaction' must be improved.

The concentration of ManNAc' must therefore use a 兀/谷 、 and a larger amount of the process. The synthesis of NeuAc by the material method requires the addition of hydrazine to activate AGE, which is an unfavorable factor in the cost test. Therefore, if the Dependence can be reduced or even eliminated, the enzyme synthesis of NeuAc will be more industrially valuable. Therefore, there are still many problems in the existing production methods, which need to be further resolved. SUMMARY OF THE INVENTION In view of the many problems still existing in the production of NeuAc, the present invention mainly provides a method for efficiently producing NeuAc to overcome the difficulties of existing production methods. In order to achieve the aforementioned object, the present invention mainly provides an isolated nucleic acid molecule comprising SEQ丨D Ν Ο · 1, and encoding a N-acety-D-glucosamine. An enzyme (bAGE) that is converted to N-acety-D-manosamine. Preferably, the nucleic acid molecule is a nucleic acid molecule deposited under the accession number BCRC940532 of the Institute for Food Industry Development. The present invention is further related to a recombinant vector which is a recombinant vector deposited under the registration number BCRC940532 of the Institute of Food Industry Development. Preferably, the vector comprises the 200904458 nucleic acid molecule, a promoter and a regulatory sequence as described in claim 1 of the scope of the patent application. Preferably, the vector is translated into a BAGE which converts N_acetyl-Dg|UC0samine into N_acetyi-D-manosamine. More preferably, the enzyme transcribed from the vector has a molecular weight of 44.7 kDa; preferably, the enzyme transcribed from the vector comprises the sequence of SEQ ID NO. The invention further relates to a microorganism which is produced by converting N-acety Dg|UC0samine into ... acetyl-D-manosamine enzyme, wherein The microorganisms comprise the nucleic acid sequences as claimed in item i of the patent application. Preferably, the host cell is prokaryotic ^ ^ = 1 . y pole , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Microorganisms of the registration number BCRC940532 of the Food Industry Development Institute. The invention further relates to a method for producing N-acetyl-D-glucosamine 2_epoxidase (/V-acetyND-glucosaminp · 9mine 2-epimerase; bAGE) enzyme in a large amount of 咮M ^ ^ Including the following steps: Charge the ^ before ^ please the microbial culture #于培# liquid; and take the N-acetamidine-D-glucosamine 2_epoxidase enzyme from the culture medium. Preferably, N is the time method (4) column chromatography to perform '·more preferably' when the acidity of bismuth acid 〇 (4) glycosamine 2-epoxidase enzyme is half-reduced, 2-different The enzyme enzyme has no inhibition on the two h, Co2+ and Ni2+, and the N-acetylglucosamine metal ion Mg2+, guanidine 2+, Zn2+, Ca or enhance the activity. 10 200904458 The invention further relates to a method for producing N-acetyl-D-neuraminic acid, which comprises: the N-acetyl-D-glucosamine 2 produced as described above - Epimerase is co-reacted with N-acetamidine-D-glucosamine and pyruvic acid, wherein N-acetamidine-D-glucosamine 2-isomerase and neu A c dissociation enzyme are 1 ·· 4~1 : The ratio of 2 4 works together. Preferably, the ratio of N-acetamidine-D-glucosamine 2-epoxidase to NeuAc dissociation enzyme is 1:16; more preferably, the concentration of GIcNAc is 0.1-1.5M, and the concentration of pyruvate is 〇 · ί_ι.5Μ; optimally, the GIcNAc concentration is 0.8M and the pyruvic acid concentration is 1.2M. Thus, the present invention can utilize the reusability of bAGE and NeuAc dissociation enzyme whole cell catalyst, and greatly improve the yield and efficiency of production of NeuAc. [Embodiment] In the whole-cell biocatalytic system established by the present invention, the whole cell catalyst exhibiting bAG E and NeuAc dissociating enzyme can directly convert the matrix gicnAc and acetone to synthesize NeuAc. Since extracellular (/n W 〖ro) analysis proves that bAGE can act as an activator for ATP, dATP and ADp' and ATP does not involve the energy consumption of ATP in the activation of bAGE for surface isomerization. Hydrolysis. Therefore, bAGE can continuously utilize these ATp in E. C〇// bacteria as an activator. This solves the shortcomings of adding ATp in the process of synthesizing NeuAc by the enzyme method, and greatly reduces the production cost of NeuAc. Second, the non-hydrolyzable activation of bAGE by Ατρ makes the whole-cell biocatalyst reusable. In the production process established by the present invention, the whole cell biocatalyst was maintained at 80% or more when the product was reused for 8 times. In addition, activity analysis with purified enzyme showed that bAGE catalyzed the conversion of GIcNAc to ManNAc with a specific activity of 124 U/mg, which is higher than the prior patent of pig kidney N_acetamidine_D_glucosamine isomerase (A/-acetyl- Dg|UC0samine 2-ePime "ase" is 4 times higher, and the bAGE/ccat value is as high as 7·2χ1〇3 minM, indicating its high catalytic efficiency. These characteristics prove that bAGE is competitive in the industrial production of NeuAc.

NeuAc promotes the contact of drugs with enzymes in the body, enhances the efficacy, and blocks the contact of antigens on the cell surface, so that the drug is not destroyed by the immune system and causes rejection. NeuAc is also a receptor for pathogens, viral infections or their toxins, which can interfere with infections of viruses and viruses. Therefore, it has been used in cold medicines, anticancer drugs, cardiovascular drugs and synthetic ganglioside drugs. Wait. A number of new drugs based on NeuAc have been approved by the US FDA, especially for the suppression of influenza viruses such as 'Glaxo We丨丨come and Biota'.

Holding) Zanamivir (trade name) used to inhibit neuraminidases of influenza A and B viruses

NeuAc is synthesized as a raw material and is currently the least resistant clinical drug for all anti-influenza drugs. Therefore, NeuAc is a very important and expensive raw material drug today. The present invention is a gene (6age) selected from the blue-green algae Ana£)aeA7a sp. CH1, which combines hge with the E. co// NeuAc dissociation enzyme gene. Or a large number of performances in E. c 0 / /, for the first time with the bAGE and NeuAc dissociation enzyme Ε · co / / cells as a biocatalyst catalyzed GIcNAc and acetone 12 200904458 acid synthesis NeuAc. Compared with the existing industrial production, this whole-cell biocatalytic system has the advantages of high productivity, high catalyst recycling, and no need to add ATP, and has the potential for industrial utilization.

NeuAc is the most important basic structure in sialic acid. It exists on the end of many secretory or cell surface glycoproteins and glycolipids, and cell-cell recognition, cell attachment (ce 丨丨adhesj〇n ), there is a considerable correlation between signal transduction, leucocyte diapedesis, and activation of b cells, which is a very important sugar in organisms. In addition, NeuAc is also associated with pathogenic bacteria infections. ' Neu Ac is a component of the surface polysaccharides of pathogenic bacteria' is a virulence factor for pathogens, providing a camouflage effect when infected with bacteria without being affected by the innate immune system ( Innate immunity) is eliminated. The relative NeuAc can exhibit receptor (recept〇r) characteristics on the cell surface, and is recognized by microorganisms, viruses, toxins, hormones, and immune systems, including certain pathogenic bacteria to identify the surface of the cell. It is able to bind to specific host cells and invade cells. For example, the pathogenic microorganism He//co6acier Py/on· ‘Specially attached to NeuAc' on gastric mucosal cells causes gastric ulcer in the host. Therefore, NeuAc plays a very important role in the biochemical function of the human body or microorganisms. So far, the age genes of humans, pigs, mice, and rabbits have been cloned in mammals, expressed in co//· and analyzed for biochemical properties of enzymes. However, among the published microbial gene sequences, only blue-green vines with the age gene were found, including Anabaerja \/ar/ajb/7/s ATCC 29314 (accession: NZ_AAEAOOOOOOO〇), 13 200904458

Nostoc sp. PCC 7120 (serial number: NC_003276), Nostoc punctiforme PCC 73102 (serial number: NZ_AAAY0000000〇), and Synechocystis sp. PCC6803 (serial number: NC_005232). The AGE of Synec/7〇cysi/s sp. PCC6803 has been used as a biocatalyst for the synthesis of NeuAc, except that the yield is only 0.6 g NeuAc/L-h. In order to obtain AG E with high catalytic activity, 俾 is used to establish a whole cell biotransformation method to synthesize NeuAc, and the present invention selects its gene from /A/7a/?ae"a sp. CH1 and is in E. co// In the performance, the activity analysis and biochemical properties of the enzyme were discussed. E. co//_ cells, which express bage and E. co//· NeuAc dissociation enzymes, act as biocatalysts, catalyzing GIcNAc and pyruvate as important raw materials for NeuAc. The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Any person skilled in the art can make any modifications and changes without departing from the spirit and scope of the invention, and should be included in the scope of the invention. Example 1. Colonization, expression, purification and analysis of the enzyme gene A. Gene selection According to the published A/osioc sp. PCC 7120 (sequence number: NC_003276) age gene sequence, two different restriction enzymes were designed. The specific primer of the position, the primer sequences are Ep5: 5, -CCATATGGGGAAAAACTTACAAGC-3, {Nde I cleavage) (SEQ ID NO. 2); and Ep3: 5, -CClCOAG.ACTCAAGGCCTCGAATTGTTG-3, (X/7〇 I cut 14 200904458 bit) (SEQ ID Ν〇·3). Using the polymerase chain reaction (PCR), the ibaye gene was largely replicated from the Anahaena sp. CH1 chromosome, and after restriction enzyme cleavage, it was cloned into the pET-32a expression vector and became pET-bage. In addition, two specific primers were designed based on the published sequence of the £1; NeuAc lyase gene, and the sequence of the primers was Ald5:

5,-CCATATGGCAACGAATTTACGTG-3, (Λ/c/e I cleavage) and Ald3 5'-C.C_I^GAGCCCGCGCTCTTGCATCAAC-3, (Xho I cleavage). The target gene was largely replicated from the e. co// chromosome by PCR and cloned into the pET-32a expression vector by restriction enzyme digestion to become pET-/7a/7/\. pET-hage and pET-nan/\ were transformed into co//BL21 (DE3), and a single colony of co//· BL21 (DE3) carrying the expression vector of the target gene was selected and inoculated into 1〇m| LB medium (containing 100/vg/ml ampicillin). After being cultured for 12 to 16 hours in a 37 〇c incubator, the nucleus dnA was extracted for 5 m each for gene sequencing analysis. The result showed that the protein sequence of the bage gene of 1,167 bp (SEQ ID NO. 1)' was SEQ ID NO. 4 and the molecular weight was calculated to be 44.7 kDa. In addition, 1 ml of the culture solution was taken for 12 to 16 hours, inoculated into 1 ml of LB medium (containing 100 amps of ampicillin), and cultured for about 4 to 6 hours until 〇D6.00 was approximately equal to 1, followed by the addition of 丨PTG ( The final concentration was 〇_5 mM) and placed in a 28 c strange temperature incubator to induce protein f expression. The recombinant protein was purified from the crude bacterial extract by Nj_NTA resin (Qiagen) affinity chromatography, and the performance and purity of the protein were analyzed by U SDS-PAGE. ^ 15 200904458 b A G E has a molecular weight of about 4 5 k D a ', and its solubility accounts for about 20% of the total soluble protein. B. Analysis of enzyme activity 1. Activity analysis of bAGE and NeuAc dissociation enzyme The activity of b A G E was determined by G丨c N A c as the substrate, and the activity of conversion to ManNAc was analyzed. In the reaction mixture (0.2 ml), containing 〇〇 Mm

Tris-HCI, pH 8_0, 50 mM GIcNAc, 1 mM ATP and an appropriate amount of enzyme solution were reacted at 37 ° C for 30 minutes. After the reaction, add 2 丨 of '0% SDS, and boil at 1 〇〇 ° C. Minute to stop the reaction. After cooling on ice, the cells were centrifuged at 12,000 rpm for 5 minutes at 4 ° C. The supernatant was filtered through a 〇 45 # m filter membrane and analyzed by ΗPLC. The activity of NeuAc dissociation enzyme was determined by ManNAc and pyruvate as the substrate, and the activity of synthesizing NeuAc was analyzed. The reaction mixture (0.2 ml) contained 100 mM Tris-HC Bu pH 8.0, 50 mM ManNAc, 0.5 Μ pyruvic acid and an appropriate amount of the enzyme solution, and the reaction conditions and analytical methods were the same as those of AGΕ. ΗPLC analysis conditions: Aminex Η-87Η Ion Exclusion column 300 mm x 7.8 mm 'Bio-Rad' was used at 65 ° C under heating to 〇·〇55〇/. h2S04 is the mobile phase, and the concentrations of ManNAc, G mountain NAc, pyruvic acid and NeuAc are analyzed. The specific activity of the purified enzyme was analyzed, and the specific activity of bAGE to GIcNAc to ManNAc was measured in the presence of 1 mM ATP. The specific activity of ManNAc and the pyruvate synthesis of NeuAc was also analyzed by NeuAc dissociation enzyme. The enzyme activity of 1 unit (u) is defined as the amount of enzyme required to catalyze the production of 1 mole of ManNAc per minute. The results showed that the specific activity of bAGE was 124 u/mg; it was 7.2 xi〇3 (min-1). In the absence of ATP in 16 200904458, the specific activity of bAGE was approximately 8 u/mg. The specific activity of NeuAc dissociated enzyme-catalyzed synthesis of NeuAc was 132 u/mg. The activation of bAGE by different concentrations of ATP was analyzed, and it was shown that bAGE reached the highest activity when the concentration of ATp was 2〇 μΜ. 2 · bAG E optimum pH and temperature and metal ion dependence analysis of bAGE activity at pH 3~10, showing that bAGE has maximum activity at pH 8.0 and maintains 9 PH between pH 7 and 9.5. Relative activity above % (see the first figure). In addition, it is 25~6〇 respectively. At the reaction temperature of 〇, the activity of bAGE was analyzed by GIcNAc as the substrate, and the results showed that the maximum activity was obtained at 45 °C, while the relative activity was maintained at 80% or more between 35 and 5 CTC (see the second figure). ). Thermal stability analysis showed that the active half-life of bAGE at 45 ° C was approximately 48 hours. The effects of Mg, Mn2, Zn2, Ca2+, Co2+ and Ni2+ (1 Mm) on the activity of bAGE were analyzed. It was found that all metal ions had no effect on the activity of bAGE. 3. Activation of bAGE by nucleotides The method of analyzing the activation of bAGE by various nucleotides was carried out by substituting ATP for various nucleotides in the above activity analysis. From the results of Table 1, it was found that AMPPNP and dATP have enzymatic activation similar to ATP for bAGE' while ADP has about 90% activation ability, and other nucleotides do not have activation. Since AMPPNP is an ATp analog that is not hydrolyzed and has the same activation as ATP, and ADp still has similar activation ability, it shows that bAGE binds to ATP and promotes the process of enzyme reaction, and does not involve ATP hydrolysis. Energy consumption. 17 200904458 Table 1. Nucleotide vs. bAGE^ Relative effects Nucleotide (1 mM) __ii Pair activity (%) None 5.4 ATP 1 〇〇GTP 8.5 CTP 3.7 UTP 5.3 AMPPNP 101 ADP 90.5 AMP 9.3 dATP 95 dGTP 4.6 dCTP 6.7 dTTP 5.4 c. Activity analysis of whole-cell biocatalysts The cells collected by IPTG-induced bAGE and NeuAc dissociation enzyme were collected by centrifugation in '0.1 M Tris-HCI (pH 8.0) buffer three times, and weighed to buffer. Suspension into a concentration of 〇 2 g / m ,, formulated into a whole cell enzyme solution. ', κ 0 reaction mixture (10 ml) containing 0.1 M Tris-HCI (pH 8.0), 〇 5 μ GlcNAc, and 2% bAGE or pAGE whole cell enzyme solution at 37.

The reaction was oscillated for 6 hours in C. After the reaction, 0.5 ml of the reaction solution was centrifuged in a small test (eppendoff) at 12,0 rpm for 1 minute, and the supernatant was diluted 1× times with 0.1 M Tris-HCI (pH 8_0) buffer. After filtration through a filtration membrane of 〇45 18 200904458, HPLC analysis was carried out. The analysis of the whole cell enzyme activity of the NeuAc dissociating enzyme was carried out in the same manner as the analysis of bAGE whole cell enzyme activity except that GI.5 M ManNAc and 1 Μpyruvate were substituted for GIcNAc. The IPTG-induced E. co// BL21 (DE3) (PET-0age) transformed strain was transformed into GIcNAc to become ManNAc. The results showed that the whole cell catalyst specific activity of bAGE was obtained without the addition of ATP. 32 U/g cel. In addition, the activity of E· co// BL21 (DE3) (ρΕΤ α?3α7Λ) for catalyzing the synthesis of neu A c from pyranic acid was determined. The specific activity of the whole cell catalyst was determined to be 1 32 U/g cells. It is indicated that the recombinant cells constructed by the present invention can be used as a biocatalyst, and can be used in the process of establishing a whole cell biocatalyst to synthesize NeuAc. D. Analysis of whole cell biocatalyst synthesis NeuAc The £.co// BL21 (DE3) transformation strain with plastid pET-bage and pET-na/7/\ was selected and cultured in 500 ml of LB medium (containing 1〇0 amperecil/ml) ' Incubate at 37 °C until 〇D6〇0 is approximately equal to 1. IPTG (final concentration q.5 mM) was then added to a constant temperature incubator at 17 ° C, and cultured for 10 hours with shaking to induce gene expression. The induced bacterial liquid was centrifuged at 8,000 rpm for 15 minutes at 4 ° C to recover the cells, and the cells were washed vigorously by adding 〇M Tns-HCI (pH 8.0) buffer 50 rrM', and the washing was repeated twice. . The wet weight of the cells was weighed and then suspended in a buffer of 〇 1 M Tr is - H C丨 (p Η 8 · 0) to a concentration of 2 g/ml, and stored at 4 °C until use. When the whole cell catalyst is used to synthesize NeuAc, 100 m of the reaction solution containing the prepared substrate solution and the whole cell biocatalyst is placed in a 25 〇m glass tube 19 200904458 (glass vessel) at 37 art. The water bath was subjected to a synthesis reaction by magnet stirring] and the pH during the reaction was controlled at 8_0 using a pH-regulated reactor (pH/〇Rp Contro丨丨er PC310, Suntex'Taiwan) with 1N HCI and 1N Na〇H. Samples were taken at intervals (7) after centrifugation, sputum and dilution, and the results of the conversion were analyzed by ΗPLC. Results: 1. The optimal ratio of whole-cell catalysts When two-cell catalysts were used to synthesize NeuAc with GICNAC and C-acid as the substrate, the optimal amount of whole-cell catalyst was first analyzed. Table 2 shows the results of synthesizing NeuAc in a 5-hour reaction at different ratios of two whole cell catalysts. The immobilized bAGE whole cell catalyst was added at 6 25 χ 102 U/L, and the NeuAc dissociation enzyme whole cell catalyst with 4 to 24 times of bAGE was synthesized with 0.8 Μ of GincNAc and 1.2 Μpyruvate as the substrate. NeuAc. It was found that with the increase of the concentration of whole cell catalyst of NeuAc dissociation enzyme, the conversion rate of GIcNAc (conversi〇ri yjeld) and the yield of NeuAc also increased, when the ratio of whole cell catalyst activity of 仏^ and NeuAc dissociation enzyme was 1 At 16 o'clock, the conversion rate of GlcNAc is 彳5 % and the yield of NeuAc reaches the highest value of 7.4 g/Lh (concentration is 〇12..., the activity ratio of 1:16 corresponds to the weight concentration (West/... is 2%: 8 %. When the dissociation enzyme whole cell catalyst exceeds 6 times, there is no significant increase in conversion rate and yield. In addition, under various ratios, the cumulative amount of intermediate ManNAc is about 0.13 Μ to 〇·18 M. As a result, in the process of synthesizing NeuAc in the whole cell biocatalyst established in this study, the activity amount of the two whole cell biocatalysts was set as i: 16 (6 25x1〇2 U/L: 1χ1〇4... 20 200904458 Table 1. Effect of the ratio of whole cell catalyst on the yield and conversion rate of synthetic NeuAc _ _ (x 1〇Ul ) NeuAc GIcNAc ManNac NeuAc Yield conversion rate of cell dissociation enzyme (%) (M) (M) (g丨. Enzyme activity and bAGE, ----- h'1) bAGE NeuAc ratio dissociation enzyme 6.25 6.25 6 .25 6.25 6.25 6.25 25 50 75 100 125 150 4 8 12 16 20 24 6·3±〇_7 7·5±1.1 10.0±1.5 15.0±2.8 16·0±3.0 15.0±2.5 0.18 0.18 0.16 0.13 0.14 0.14 0.05 0.06 0.08 0.12 0.13 12 3.1 3.7 4.9 7.4 8.0 7.4 2· The optimal substrate concentration is the maximum GIcNAc conversion rate and NeuAc yield. The activity ratio is 1:16. Two whole cell organisms are used as catalysts. GlcNAc and pyruvate concentration were used to analyze the conversion rate of G丨cNAc and the yield of NeuAc. From the values in Table 3, it was found that in the 12-hour reaction, when G|cNAc was 〇4M, with the increase of pyruvate, GIcNAc The conversion rate and the yield of NeuAc also increased. When the pyruvate was 1.6 ,, the conversion rate of GIcNAc reached the highest value of 5〇0/〇, and the yield of NeuAc was 51 g/L_h. At this time, the concentration of NeuAc was 〇·2 M (61.4 g/L). Please refer to the third figure. The third figure is the concentration change of 1丨 Μ G丨cNAc and ^ M pyruvate. 21 200904458 Figure, in 6 hours Both GlcNAc and propionic acid are rapidly consumed, and the consumption rate is slowed after 6 hours. NeuAc started to build and continued to accumulate after 2 hours' to reach the highest value in 10 hours, and did not continue to increase in 12 hours, indicating that the reaction had reached equilibrium. As for ManNAc, it is produced in the first hour and remains between 〇彳2 and 〇彳4 M after about 4 hours. E. Reuse analysis of whole cell biocatalysts Analysis of the reusability of bAGE|% NeuAc dissociation enzyme whole cell catalyst. After the first 12 hours of reaction reached equilibrium, the cell catalyst was centrifuged and washed three times (M M Tris_HC|buffer (10) 8 Q), followed by a second cycle. The fourth panel shows that in 8 cycles of reaction, the conversion rate of GICNAC can be maintained above 80% of the first cycle, and the yield of NeUAC is greater than 8 g/Lh, indicating that the whole cell of levine bAGE and NeuAc dissociation enzyme Catalyst synthesis NeuAc is reusable. The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any one of ordinary skill in the art, without departing from the technical features of the present invention. Equivalent embodiments that make partial changes or modifications made by the technical content disclosed in the present disclosure, and 'without departing from the technical features of the present invention, are still within the scope of the technical features of the present invention. [Simple description of the diagram] The first figure is to analyze the activity of bAGE at different pH values. The first plot is to analyze the activity of bAGE at different temperatures. The second graph is a graph showing the change in concentration of 12 M GIcNAc with 1.2 Μ pyruvate. 22 200904458 The fourth figure is a representation of the reusability of NeuAc using bAGE and NeuAc dissociation enzyme whole cell catalyst. [Main component symbol description] None 23

Claims (1)

200904458 X. Patent application scope: 1 · An isolated nucleic acid molecule comprising SEQ丨D Ν〇.Ί, and encoding a N-acety-D-glucosamine (N-acety|-Dg|UCOSamine ) An enzyme that is converted to N-acety D-manosamine. 2. The nucleic acid molecule according to the above-mentioned patent application, which is a nucleic acid molecule deposited in the registration number BCRC 940532 of the Food Industry Development Research Institute. 3. A recombinant vector which is a recombinant vector deposited under the registration number BCRC94〇532 of the Food Industry Development Research Institute. 4. The recombinant vector of claim 3, wherein the vector comprises the nucleic acid molecule of claim i, a promoter, and a regulatory sequence. ^ # 印 寻 第 第 第 第 第 第 一 一 一 一 一 一 一 一 一 一 一 一 一 一 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第Acety d-manosamine). 6. The recombinant vector of claim 5, wherein the enzyme transcribed by the vector has a molecular weight of 44 7 kDa. 7. The recombinant vector of claim 5, wherein the enzyme transcribed by the vector comprises the sequence of SEQ丨D N〇 4. Eight kinds of productions are directly catalyzed by N-acetylglucosamine (GIcNAc) and pyruvate (Pyruvate)... The microorganisms of the enzymes of 0-neuroglycine (NeuAc), the pots in the pots _ The Pharmacy VIII μ ^ Biology Department contains the nucleic acid sequence as requested in the patent application No. 1 24 200904458. 9. The method of claim 8, wherein the host cell is a prokaryotic cell. 1 0. The method of claim 9, wherein the prokaryotic cell comprises a co., a human, or a microbe, as claimed in the scope of claim ii. For the microorganisms registered in the registration number BCRC940532 of the Food and Drug Research Institute. 1 2 . - Mass production of N_acetamidine _D_glucosamine 2_epoxidase (/V-aCetyl-Dg|UC0samine 2_epjmerase; method of (^) enzyme, including the following steps: i the microorganisms to be cultured in the culture solution; and the cells having the activity of N_acetamidine_D_glucosamine 2_epoxidase enzyme are collected from the culture solution. 13. As described in the scope of claim i2 The method wherein the method for concentrating the cells is by centrifugation or filtration. 14 methods for producing N-acet-D-neuramim'c acid, the system comprising: The n-acetamidine-D-glucosamine 2_epimerase produced by the patent application range of items 12 to 13 is co-reacted with N_acetamidine-7-glucosamine and propionic acid, and the towel N thereof - acetamidine glucosamine 2_epim isomerase and accompanying. The dissociating enzyme can act in various ratios; and the N-acetyl-D-neuraminic acid produced by the above-mentioned action is collected. The method described in 2, 4, wherein n_ 25 200904458 acetaminophen (IV) 2_the isomerase gene and the dissociation enzyme gene coexist - a cell in or There are two ways of expressing the bacteria. 16. The method according to claim 14 of the patent application, wherein the concentration of G丨cNAc is CM·', and the degree of pyruvate is ο". The method of claim 15 is wherein the concentration of GlcNAc is 〇·1-1, and the concentration of pyruvic acid is om 1 8 . The method according to claim 17 of the patent application, wherein (4) the hidden concentration is Q. 8M, the concentration of C (IV) is 1.2 Μ. 9. The method described in claim 18, Lizhong N-acetam-D-glucosamine 2_epoxidase and a dissociation enzyme system The ratio of 10:24 is synergistic. The method of 9th, wherein the N-NeuAc dissociation enzyme system is 1:16 2 0. As claimed in the patent range 1 醯-D-glucosamine 2_epim isomerase The ratio of the GIcNAc is 〇^ 5M is 忐勹υ.丨, the concentration of acrylic acid is m 5M, 2 2 is the scope of the patent. The method has a concentration of GIcNAc of 〇8M, and the concentration of zhongzhong M-propionic acid is 1.2M. XI. Schema: as shown in the next page 26