N-acetyl glucosamine isomerase mutant, recombinant expression vector, recombinant strain and application of N-acetyl glucosamine isomerase mutant

Abstract

The invention belongs to the field of gene engineering, and particularly relates to an N-acetyl glucosamine isomerase mutant, a recombinant expression vector, a recombinant strain and application thereof. According to the invention, modification is carried out based on amino acid of wild type N-acetyl glucosamine isomerase from Anabaena sp.CH1, the modified mutant comprises two mutation sites, namely K84D and F349Y, and the activity is improved by 1.84 times. The invention further constructs a double-enzyme co-expression strain of N-acetylglucosamine isomerase and N-acetylneuraminic acid aldolase, and the strain is used for producing N-acetylneuraminic acid, so that the dosage of thalli is reduced, the conversion efficiency is improved, a more economical enzyme catalysis scheme is provided for industrial catalytic preparation of N-acetylneuraminic acid, and the method has a wide application prospect. The method has an important industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular to a N-acetylglucosamine isomerase mutant, a recombinant expression vector, a recombinant strain and application thereof. BACKGROUND

[0002] N-acetylneuraminic acid, a form of sialic acid, has many effects on human health. The main effects are: promoting brain development and improving memory, improving immunity, promoting liver metabolism, having antioxidant and anti-inflammatory effects, protecting neurons, etc.

[0003] In the prior art, N-acetylneuraminic acid can be synthesized by a double-enzyme catalytic system of N-acetylglucosamine isomerase (AGE) and N-acetylneuraminic acid aldolase (NAL). For example, Chinese patent CN104878035A discloses the synthesis of N-acetylneuraminic acid using two recombinant E. coli expressing N-acetylglucosamine-2-isomerase gene and N-acetylneuraminic acid aldolase gene as whole-cell catalyst. However, in the prior art, there are still disadvantages such as high enzyme dosage and low substrate conversion efficiency. For example, in previous studies, the company has carried out site-directed mutagenesis on wild-type N-acetylneuraminic acid aldolase from Corynebacterium propinquum in Chinese patent CN119685298A, and the enzyme activity and N-acetylneuraminic acid yield have been significantly improved, but there is still a problem of unsatisfactory conversion efficiency. Therefore, the present application further provides a N-acetylglucosamine isomerase mutant to improve the conversion efficiency and reduce the substrate dosage, thereby saving production costs. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a N-acetylglucosamine isomerase mutant with higher activity to overcome the shortcomings of the prior art.

[0005] The technical problem to be solved by the present application is to provide a N-acetylglucosamine isomerase mutant with higher activity to overcome the shortcomings of the prior art.

[0006] The technical problem to be solved by the present application is to provide a N-acetylglucosamine isomerase mutant with higher activity to overcome the shortcomings of the prior art.

[0007] The technical problem to be solved by the present application is to provide a N-acetylglucosamine isomerase mutant with higher activity to overcome the shortcomings of the prior art.

[0008] The technical problem to be solved by the present application is to provide a N-acetylglucosamine isomerase mutant with higher activity to overcome the shortcomings of the prior art.

[0009] To solve the above technical problems, the technical solutions adopted by the present application are as follows:

[0010] In a first aspect, the present application provides an N-acetylglucosamine isomerase mutant, whose amino acid sequence is shown in SEQ ID NO. 3.

[0011] The N-acetylglucosamine isomerase mutant is obtained by mutating the wild-type N-acetylglucosamine isomerase at position 84 from lysine to aspartic acid and at position 349 from phenylalanine to tyrosine.

[0012] Specifically, the wild-type N-acetylglucosamine isomerase is derived from Anabaena sp. CH1, whose amino acid sequence is shown in SEQ ID NO. 1, and the nucleotide sequence of the gene encoding the wild-type N-acetylglucosamine isomerase is shown in SEQ ID NO. 2.

[0013] The gene sequence of the wild-type N-acetylglucosamine isomerase is entrusted to General Bio (Anhui) Co., Ltd. for full gene synthesis.

[0014] Specifically, the N-acetylglucosamine isomerase mutant has an enzyme activity of 1.84 times that of the wild-type N-acetylglucosamine isomerase.

[0015] In a second aspect, the present application provides a coding gene encoding the N-acetylglucosamine isomerase mutant.

[0016] Specifically, the nucleotide sequence of the coding gene is shown in SEQ ID NO. 4.

[0017] In a third aspect, the present application provides a recombinant expression vector.

[0018] Specifically, the recombinant expression vector contains the coding gene of the N-acetylglucosamine isomerase mutant.

[0019] Further, the recombinant expression vector contains a coding gene of N-acetylneuraminic acid aldolase.

[0020] Further, the N-acetylneuraminic acid aldolase is the N-acetylneuraminic acid aldolase mutant V149K-R233Q-K235D-V241R disclosed in Chinese patent CN119685298A.

[0021] Further, the coding gene of the N-acetylneuraminic acid aldolase mutant V149K-R233Q-K235D-V241R is also disclosed in Chinese patent CN119685298A.

[0022] Figure 1 The recombinant expression vector plasmid map containing the N-acetylglucosamine isomerase mutant coding gene and the N-acetylneuraminic acid aldolase coding gene is shown.

[0023] In a fourth aspect, the present application provides a recombinant strain.

[0024] Specifically, the recombinant strain is obtained by introducing the recombinant expression vector into a host strain.

[0025] In the application, the host strain is Escherichia coli BL21 (DE3).

[0026] Further, the recombinant strain is a N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase double-enzyme co-expression strain.

[0027] In a fifth aspect, the present application provides the use of the N-acetylglucosamine isomerase mutant in catalyzing the preparation of N-acetylneuraminic acid.

[0028] In the application, the catalysis is catalyzing the synthesis of N-acetylneuraminic acid by using the N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase double-enzyme co-expression strain with N-acetylglucosamine and sodium pyruvate as substrates.

[0029] In the application, the catalysis is catalyzing the synthesis of N-acetylneuraminic acid by using the N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase double-enzyme co-expression strain with N-acetylglucosamine and sodium pyruvate as substrates.

[0030] In some embodiments of the present application, the catalysis is catalyzing the synthesis of N-acetylneuraminic acid by using the N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase double-enzyme co-expression strain with N-acetylglucosamine and sodium pyruvate as substrates.

[0031] Preferably, the concentration of the crude enzyme solution of the N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase double-enzyme co-expression strain is 60 g / L.

[0032] In the application, the catalysis is catalyzing the synthesis of N-acetylneuraminic acid by using the N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase double-enzyme co-expression strain with N-acetylglucosamine and sodium pyruvate as substrates.

[0033] In some embodiments of the present application, the catalysis is catalyzing the synthesis of N-acetylneuraminic acid by using the N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase double-enzyme co-expression strain with N-acetylglucosamine and sodium pyruvate as substrates.

[0034] Beneficial effects:

[0035] (1) The present application obtains a mutant of N-acetylglucosamine isomerase with significantly improved activity by site-directed modification of wild-type N-acetylglucosamine isomerase from Anabaena sp. CH1, and the enzyme activity of the mutant is 1.84 times that of the wild-type N-acetylglucosamine isomerase.

[0036] (2) The present application catalytically produces N-acetylneuraminic acid by constructing a double-enzyme co-expression strain of N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase, and optimizing the addition amount of the crude enzyme solution of the co-expression strain, thereby significantly improving the concentration and conversion efficiency of N-acetylneuraminic acid. Thus, the N-acetylglucosamine isomerase mutant obtained by site-directed modification has extremely high catalytic application potential and broad application prospects in industrial applications. Compared with the crude enzyme solution of the strain using N-acetylglucosamine isomerase and N-acetylneuraminic acid aldolase alone, the co-expression strategy significantly reduces the enzyme consumption by 33%, effectively reducing the catalytic cost. The co-expression strain breaks through the enzyme efficiency bottleneck of traditional single-expression strain through synergistic catalysis, and at the same time of reaching the peak of yield and conversion rate, the enzyme consumption is also minimized, thereby providing a more economical enzyme catalysis scheme for industrial catalytic preparation of N-acetylneuraminic acid. BRIEF DESCRIPTION OF DRAWINGS

[0037] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0038] Figure 1 A recombinant expression vector containing a gene encoding the N-acetylglucosamine isomerase mutant and a gene encoding N-acetylneuraminic acid aldolase. DETAILED DESCRIPTION

[0039] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0040] In the following examples, the experimental methods described are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0041] Example 1: Construction and expression of wild-type N-acetylglucosamine isomerase

[0042] The amino acid sequence of wild-type N-acetylglucosamine isomerase AGE (denoted as WT) from Anabaena sp. CH1 is shown in SEQ ID NO. 1, and the nucleotide sequence of the gene encoding the wild-type N-acetylglucosamine isomerase is shown in SEQ ID NO. 2.

[0043] The gene of wild type N-acetylglucosamine isomerase WT (SEQ ID NO. 2) was entrusted to General Biosystems (Anhui) Co., Ltd. for full gene synthesis, and was ligated to pET28a vector to obtain plasmid pET28a-AGE, and then the plasmid pET28a-AGE was transformed into E. coli BL21 (DE3) competent cells to obtain a recombinant E. coli strain of wild type N-acetylglucosamine isomerase.

[0044] The recombinant E. coli strain of wild type N-acetylglucosamine isomerase was inoculated into 20 mL of LB medium (containing kanamycin at a final concentration of 50 mg / L) and cultured at 37°C and 200 rpm for 12 hours. Then, 1% v / v of the culture was inoculated into 1 L of TB medium (containing kanamycin at a final concentration of 50 mg / L), and cultured at 37°C and 200 rpm until the OD 600 When the OD was 0.6-0.8, IPTG at a final concentration of 0.2 mM was added, and the culture was induced at 28°C and 200 rpm for 12-16 hours. The culture was centrifuged at 8000 rpm for 10 minutes, and the N-acetylglucosamine isomerase wild type bacteria were collected and stored in a -80°C refrigerator.

[0045] Example 2: Construction and expression of N-acetylglucosamine isomerase single point mutant

[0046] The construction of N-acetylglucosamine isomerase single point mutant was carried out by full plasmid PCR method. The mutant primers are shown in Table 1. The plasmid pET28a-AGE constructed in Example 1 was used as a template, and the corresponding primers were used for PCR amplification. The PCR reaction system included 5 μL of 10×PCR Buffer, 5 μL of 2 mM dNTPs, 3 μL of 25 mM magnesium sulfate, 1.5 μL of 10 pmol / μL primer F and primer R, 1 μL of plasmid template, 1 μL of KOD-Plus-Neo polymerase (purchased from Toyobo Co., Ltd.), and finally distilled water was added to 50 μL. The PCR reaction parameters were as follows: 94°C for 2 minutes, 98°C for 10 seconds, 60°C for 30 seconds, 68°C for 3 minutes and 30 seconds, 30 cycles, and 68°C for 5 minutes, and then 4°C for storage.

[0047] Table 1: Primer sequences corresponding to each single point mutation site

[0048]

[0049] The PCR product was digested with Dpn I (purchased from TaKaRa Company) in the following system: 8.5 μL PCR product, 1 μL 10 x QuickCut Buffer, 0.5 μL Dpn I, and the reaction was carried out at 37 °C for 0.5 h, and then the product was transformed into E. coli DH5α competent cells, which were incubated on LB solid plate (containing 50 mg / L kanamycin) at 37 °C for 12 h to obtain clones. Three clones were picked and incubated in LB medium (containing 50 mg / L kanamycin) at 37 °C and 200 rpm for 12 h, and then the clones were sequenced. After sequencing, the plasmid of each single-point mutant was extracted using a plasmid extraction kit (pET28a-AGE S31P , pET28a-AGE K84D , pET28a-AGE Q88K , pET28a-AGE N272E , pET28a-AGE N277K , pET28a-AGE A289E , pET28a-AGE S309A , pET28a-AGE A324E , pET28a-AGE A327E , pET28a-AGE F349Y , pET28a-AGE N361P ), and then the single-point mutant plasmid verified by sequencing was transformed into E. coli BL21 (DE3) competent cells to obtain each mutant strain. The mutant strain was induced and expressed in the same manner as the wild-type N-acetylglucosamine isomerase WT strain.

[0050] Example 3: Enzyme activity detection of N-acetylglucosamine isomerase single-point mutant

[0051] The enzyme activity detection system was as follows: 20 mM N-acetylglucosamine and 20 mM sodium pyruvate, pH 8.0, 10 g / L N-acetylglucosamine isomerase, 10 g / L N-acetylneuraminic acid aldehyde lyase, and 0.2% Triton X-100 were added, and the reaction was carried out at 37 °C and 200 rpm, and the sample was taken every 30 min, and then boiled in water bath for quenching. The enzyme activity of N-acetylglucosamine isomerase was defined as follows: the amount of enzyme required to produce 1 μmol N-acetylneuraminic acid per minute, and the enzyme activity unit was calculated based on the wet bacterial weight. Relative specific enzyme activity = mutant enzyme activity / wild-type N-acetylglucosamine isomerase enzyme activity.

[0052] Table 2: Comparison of relative specific enzyme activity of wild-type N-acetylglucosamine isomerase and single-point mutant

[0053] Single point mutation sites Relative specific enzyme activity (%) WT 100 S31P 124 K84D 136 Q88K 56 N272E 68 N277K 15 A289E 36 S309A 48 A324E 56 A327E 64 F349Y 143 N361P 15

[0054] The experimental results are shown in Table 2. The results showed that not all single-point mutants exhibited increased relative enzyme activity after mutation; only mutants with mutation sites S31P, K84D, and F349Y showed higher relative enzyme activity than wild-type N-acetylglucosamine isomerase. Further combined mutations will be performed using any two or more of these sites.

[0055] Example 4: Construction, expression, and enzyme activity detection of N-acetylglucosamine isomerase mutants (combined mutants)

[0056] Combinatorial mutations are obtained by using plasmids containing single- or double-point mutants as templates and then performing full-plasmid PCR construction. The specific process is as follows:

[0057] (1) Construction and expression of mutant S31P-K84D

[0058] The plasmid pET28a-AGE of the single-point mutant S31P S31P Using 84-F and 84-R primers as templates, PCR amplification was performed. The PCR amplification process, PCR product digestion and transformation, and plasmid pET28a-AGE were also described. S31P-K84D The extraction, transformation, and induction expression of mutant S31P-K84D cells were performed in the same manner as in Example 2.

[0059] (2) Construction and expression of mutant S31P-F349Y

[0060] The plasmid pET28a-AGE of the single-point mutant S31P S31P Using 349-F and 349-R primers as templates, PCR amplification was performed. The PCR amplification process, PCR product digestion and transformation, and plasmid pET28a-AGE were also described. S31P-F349Y The extraction, transformation, and induction expression of mutant S31P-F349Y cells were performed in the same manner as in Example 2.

[0061] (3) Construction and expression of mutant K84D-F349Y

[0062] Using the single-point mutant K84D plasmid pET28a-AGE K84D Using 349-F and 349-R primers as templates, PCR amplification was performed. The PCR amplification process, PCR product digestion and transformation, and plasmid pET28a-AGE were also described. K84D-F349Y The extraction, transformation, and induction expression of mutant K84D-F349Y cells were performed in the same manner as in Example 2.

[0063] (4) Construction and expression of mutant S31P-K84D-F349Y

[0064] Plasmid pET28a-AGE of double point mutant S31P-K84D S31P-K84D PCR amplification was performed with 349-F and 349-R primers using the plasmid pET28a-AGE of double point mutant S31P-K84D as a template. S31P-K84D-F349Y The extraction and induced expression process of the mutant S31P-K84D-F349Y strain were the same as in Example 2.

[0065] The relative specific enzyme activity of the combined mutant was detected, and the detection process was the same as in Example 3.

[0066] Table 3 Comparison of relative specific enzyme activity of wild-type N-acetylglucosamine isomerase and combined mutants

[0067] Combination mutation sites Relative specific enzyme activity (%) WT 100 S31P-K84D 146 S31P-F349Y 152 K84D-F349Y 184 S31P-K84D-F349Y 175

[0068] The results are shown in Table 3. After combined mutation, the relative specific enzyme activity of the mutant K84D-F349Y was the highest, which was 1.84 times that of the wild type. The amino acid sequence of the N-acetylglucosamine isomerase mutant K84D-F349Y is shown in SEQ ID NO. 3, and the corresponding nucleotide sequence is shown in SEQ ID NO. 4.

[0069] Example 5: Catalytic preparation of N-acetylneuraminic acid by N-acetylglucosamine isomerase (AGE) and N-acetylneuraminic acid aldolase (NAL) single expression strain

[0070] In a 50 mL reaction system, 800 mM N-acetylglucosamine and 1200 mM sodium pyruvate were added, respectively, and the pH was adjusted to 8.0. Then, the crude enzyme solution of the N-acetylglucosamine isomerase strain and the N-acetylneuraminic acid aldolase strain was added at 37°C, and the reaction was carried out for 16 h to prepare N-acetylneuraminic acid.

[0071] Strain disruption process: 2 g of bacterial cells were weighed into a 50 mL centrifuge tube, washed twice with 0.1 mol / L PBS (pH 7.5) solution, centrifuged to collect the bacterial cells, resuspended with 20 mL of 0.1 mol / L PBS (pH 7.5) solution to obtain a 100 g / L bacterial solution, and the cells were disrupted by ultrasonic disrupter in an ice water bath, with the instrument parameters set as power 200 W, ultrasonic 3 s and pause 7 s, for about 40 min until the bacterial solution was clear. After completion, centrifugation was performed at 4°C and 8000 rpm for 10 min to obtain the supernatant, which was the crude enzyme solution of the strain.

[0072] The crude enzyme solution of N-acetylglucosamine isomerase strain and N-acetylneuraminic acid aldolase strain in the reaction system was added and optimized, and 5 groups of reactions were set as shown in Table 4. From the optimization results, it can be seen that the catalytic effect of reaction group 3 is optimal, at this time the crude enzyme solution of N-acetylglucosamine isomerase strain is added in an amount of 60 g / L, the crude enzyme solution of N-acetylneuraminic acid aldolase strain is added in an amount of 30 g / L, the yield of N-acetylneuraminic acid is 174.1 g / L, and the conversion rate is 70.3%.

[0073] Table 4 Optimization of the addition amount of AGE and NAL crude enzyme solution

[0074]

[0075] Example 6: Construction of N-acetylglucosamine isomerase (AGE) and N-acetylneuraminic acid aldolase (NAL) co-expressed strain

[0076] The genes of N-acetylglucosamine isomerase and N-acetylneuraminic acid aldolase were constructed into the same expression vector pET28a. According to the nucleotide sequence corresponding to N-acetylneuraminic acid aldolase V149K-R233Q-K235D-V241R, upstream primer 1F (5'-CATGCCATGGGCATGTCAGAAACCATCGCGCC-3') and downstream primer 1R (5'-TCTCCTTCTTAAAGTTAAACAAACTACACCCCGTGGCCGAAT-3') were designed and synthesized, and the plasmid pET28a-NAL V149K-R233Q-K235D-V241R was used as the template for PCR amplification. According to the nucleotide sequence corresponding to the N-acetylglucosamine isomerase mutant constructed in Example 4, upstream primer 2F (5'-TTTGTTTAACTTTAAGAAGGAGAATGGGGAAAAACTTACAAGCACTG-3') and downstream primer 2R (5'-CCGCTCGAGTCAACTCAACGCCTCGAACTGT-3') were designed and synthesized, and pET28a-AGE K84D-F349YThe PCR amplification was performed using the template. The PCR amplification product was recovered by 1% agarose gel electrophoresis using a DNA gel recovery kit (purchased from Takara Company) to obtain the DNA fragments of NAL and AGE mutants, respectively. The PCR reaction system included 5 μL of 10x PCR Buffer, 5 μL of 2 mM dNTPs, 3 μL of 25 mM magnesium sulfate, 1.5 μL of 10 pmol / μL primer F and primer R, 1 μL of template, 1 μL of KOD-Plus-Neo polymerase, and finally distilled sterilized water was added to 50 μL. The PCR reaction parameters were 2 minutes of 94°C, 10 seconds of 98°C, 30 seconds of 55°C, 45 seconds of 68°C, 30 cycles, 5 minutes of 68°C, and then 4°C.

[0077] Further, the upstream primer 1F and the downstream primer 2R were used for the overlapping PCR amplification using the DNA fragments of NAL and AGE mutants as the template, and the DNA fragment of NAL-AGE mutant was obtained by gel recovery. The PCR reaction system included 5 μL of 10x PCR Buffer, 5 μL of 2 mM dNTPs, 3 μL of 25 mM magnesium sulfate, 1.5 μL of 10 pmol / μL primer F and primer R, 1 μL of template, 1 μL of KOD-Plus-Neo polymerase, and finally distilled sterilized water was added to 50 μL. The PCR reaction parameters were 2 minutes of 94°C, 10 seconds of 98°C, 30 seconds of 55°C, 1 minute and 15 seconds of 68°C, 30 cycles, 5 minutes of 68°C, and then 4°C.

[0078] The NAL-AGE mutant gene fragment and the pET-28a expression vector (purchased from Novagen Company) after amplification were respectively subjected to enzyme cutting using NcoI and XhoI restriction endonucleases (purchased from Takara Company). The DNA fragment purification product was connected with the linearized pET28a expression vector after enzyme cutting, and the connection system was as follows: 4 μL of DNA fragment purification product, 6 μL of linearized pET28a expression vector, 10 μL of Solution I, reaction at 16°C for 2 h, to obtain the recombinant expression vector pET28a-NAL-AGE Figure 1 ), and then the recombinant expression vector was transformed into the competent cells of Escherichia coli BL21 (DE3) to obtain the recombinant Escherichia coli. After the single colony was picked on the LB plate (containing a final concentration of 50 mg / L Kan) and cultured at 37°C for 12 hours, the culture was sent for sequencing to determine the sequence after being cultured at 37°C and 200 rpm (containing a final concentration of 50 mg / L Kan) for 12 hours in the LB medium, and the NAL and AGE co-expression strain was obtained.

[0079] The induced expression process of the co-expression strain with correct sequencing is the same as that in Example 1. The process of obtaining the crude enzyme solution by breaking the co-expression strain is the same as that in Example 5.

[0080] Example 7: Application of N-acetylglucosamine isomerase and N-acetylneuraminic acid aldolase co-expression strain in catalytic preparation of N-acetylneuraminic acid

[0081] In a 50 mL reaction system, 800 mM N-acetylglucosamine and 1200 mM sodium pyruvate were added respectively, the pH was adjusted to 8.0, then the crude enzyme solution of the co-expression strain was added at 37℃, and N-acetylneuraminic acid was prepared by reacting for 16 h.

[0082] The amount of the crude enzyme solution of the co-expression strain added to the reaction system was optimized, and four groups of reactions were set as shown in Table 5. It can be seen from the optimization results that the catalytic effect of reaction group 2 is optimal, that is, when the amount of the crude enzyme solution of the co-expression strain added is 60 g / L, the yield of N-acetylneuraminic acid is 174.5 g / L, and the conversion rate is 70.5%. Further increasing the amount of the crude enzyme solution added, the yield and conversion rate of N-acetylneuraminic acid do not increase significantly, indicating that the contribution of enzyme amount increase to reaction efficiency is limited when the amount of the crude enzyme solution added is more than 60 g / L. In Example 5, at least 60 g / L of N-acetylglucosamine isomerase crude enzyme solution and 30 g / L of N-acetylneuraminic acid aldolase crude enzyme solution are required to achieve the same conversion rate of about 70%, that is, a total of 90 g / L of crude enzyme solution needs to be added. Compared with this, the co-expression strategy significantly reduces the enzyme consumption by 33%, effectively reducing the catalytic cost. It is shown that the co-expression strain breaks through the enzyme efficiency bottleneck of the traditional single expression strain through synergistic catalysis, and at the same time of reaching the peak of yield and conversion rate, the enzyme consumption is also the lowest, which provides a more economical enzyme catalysis scheme for industrial catalytic preparation of N-acetylneuraminic acid.

[0083] Table 5 Optimization of the amount of crude enzyme solution of the co-expression strain

[0084] Group Amount of crude enzyme solution added (g / L) Production of N-acetylneuraminic acid (g / L) Conversion yield (%) 1 40 122.2 49.4 2 60 174.5 70.5 3 80 173.7 70.2 4 100 175.2 70.8

[0085] The present application provides a kind of N-acetylglucosamine isomerase mutant, recombination expression vector, recombination strain and its application mentality and method, the method and approach for specifically realizing this technical scheme are many, above-mentioned only preferred embodiment of the present application, it should be pointed out, for the ordinary skilled person in the art, without departing from the principle of the present application, can make a number of improvements and refinements, these improvements and refinements also should be considered as the protection scope of the present application. The components not explicitly described in the example can be realized by existing technology.

Claims

1. A mutant of N-acetylglucosamine isomerase, characterized in that, The N-acetylglucosamine isomerase mutant has an amino acid sequence as shown in SEQ ID NO.

3.

2. The mutant N-acetylglucosamine isomerase according to claim 1, wherein The N-acetylglucosamine isomerase mutant is obtained by mutating the lysine at position 84 of wild-type N-acetylglucosamine isomerase with aspartic acid and mutating the phenylalanine at position 349 with tyrosine, wherein the amino acid sequence of the wild-type N-acetylglucosamine isomerase is shown in SEQ ID NO.

1.

3. A gene encoding the N-acetylglucosamine isomerase mutant according to any one of claims 1 or 2, characterized in that, The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the coding gene of claim 3.

5. The recombinant expression vector of claim 4, wherein, The recombinant expression vector contains the coding gene of N-acetylneuraminic acid aldolase.

6. A recombinant bacterial strain, characterized in that, The recombinant strain is obtained by introducing the recombinant expression vector of claim 5 into a host strain.

7. The recombinant strain of claim 6, wherein, The recombinant strain is a N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase double-enzyme co-expression strain.

8. The N-acetylglucosamine isomerase mutant of any one of claims 1 or 2 for use in catalyzing the preparation of N-acetylneuraminic acid.

9. Use according to claim 8, characterized in that, The catalysis is performed by using the N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase double-enzyme co-expression strain to catalyze the synthesis of N-acetylneuraminic acid with N-acetylglucosamine and sodium pyruvate as substrates.

10. Use according to claim 8 or 9, characterized in that, The catalysis is performed in a reaction system containing 400-1000 mM N-acetylglucosamine, 600-1500 mM sodium pyruvate, 60-100 g / L crude enzyme solution of the N-acetylglucosamine isomerase mutant and N-acetylneuraminic acid aldolase double-enzyme co-expression strain, and the catalysis is performed under the reaction conditions of 35-40℃, pH 6.0-9.0 for 10-20 h.

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