CN117925576A - N-acetylglucosaminidase mutant De10A and application thereof - Google Patents

Abstract

The invention discloses an N-acetylglucosaminidase mutant De10A and application thereof, belonging to the technical field of genetic engineering, wherein the amino acid sequence of the mutant De10A is shown as SEQ ID NO.1, and the nucleotide sequence of a coding gene of the mutant De10A is shown as SEQ ID NO. 2. The invention solves the problem of poor catalytic efficiency of the existing N-acetylglucosaminidase. The K _m value of the mutant De10A is 0.2mM, the catalytic efficiency is 2035.7s ^‑1mM^‑1, and compared with the wild enzyme opt-HJ5NagNC, the mutant De10A has the advantages that the substrate affinity is improved by 250%, the catalytic efficiency is improved by 95%, the application cost is reduced, and the mutant De10A has a wide application prospect in the field of functional food production and processing.

Description

A N-acetylglucosaminidase mutant De10A and its application

Technical Field

The invention belongs to the technical field of genetic engineering, and specifically relates to an N-acetylglucosaminidase mutant De10A and an application thereof.

Background Art

N-acetylhexosaminidase (EC 3.2.1.52) is a glycoside hydrolase that catalyzes the hydrolysis of N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-galactosamine (GalNAc) at the non-reducing ends of oligosaccharides, glycoproteins, glycolipids and other glycoconjugates. Among them, a class of enzymes with a strong affinity for GlcNAc is called acetylglucosaminidase (GlcNAcases). Existing literature 1( et al. The FEBS Journal, 2018, 285(3): 580–598) disclosed that GlcNAcases are widely present in organisms ranging from humans to bacteria, and play important physiological functions, including the catabolism of ganglioside storage in the human body, the regulation of fruit ripening-related softening, the renewal of chitin exoskeletons in insects, chitin utilization in bacteria, cell wall recycling and flagella formation, etc.

In the CAZY database, GlcNAcases are mainly distributed in three glycoside hydrolase families: GH3, GH20 and GH84. Among them, reference 2 (Zhang et al. Appl Microbiol Biot, 2018, 102 (1): 93–103) discloses that the hydrolysis activity of N-acetylglucosaminidase of GH20 family on chitosan oligosaccharides is generally higher than that of enzymes of other families. It is the enzyme with the most functional verification and structural analysis among all families, and it is also the enzyme with the greatest potential for industrial application, but the catalytic efficiency is still poor. Therefore, it is necessary to screen new enzymes or design and modify existing N-acetylglucosaminidase to improve its catalytic efficiency.

Summary of the invention

The purpose of the present invention is to provide an N-acetylglucosaminidase mutant De10A and application thereof, so as to solve the problem of poor catalytic efficiency of existing N-acetylglucosaminidase.

In order to achieve the above object, the present invention provides an N-acetylglucosaminidase mutant De10A, the amino acid sequence of the mutant is shown in SEQ ID NO.1.

The present invention provides a gene encoding the N-acetylglucosaminidase mutant De10A, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.2.

The present invention provides a recombinant expression vector comprising the N-acetylglucosaminidase mutant De10A encoding gene as described above.

Preferably, the recombinant expression vector includes pET series plasmids.

The present invention provides a recombinant expression bacterium comprising the above-mentioned N-acetylglucosaminidase mutant De10A encoding gene.

Preferably, the recombinant expression bacteria is selected from Escherichia coli BL21 (DE3).

The invention provides a method for improving the catalytic efficiency of N-acetylglucosaminidase, which comprises deleting the 10th alanine in the amino acid sequence of N-acetylglucosaminidase as shown in SEQ ID NO.3.

The present invention provides an application of the N-acetylglucosaminidase mutant De10A or a gene encoding the N-acetylglucosaminidase mutant De10A in the field of functional food production and processing.

The present invention provides an application of the above-mentioned group expression vector in the production and processing of functional foods.

The present invention provides an application of the recombinant expression bacteria in the production and processing of functional foods.

The N-acetylglucosaminidase mutant De10A of the present invention and its application solve the problem of poor catalytic efficiency of existing N-acetylglucosaminidase and have the following advantages:

The present invention utilizes genetic engineering technology to provide an N-acetylglucosaminidase mutant De10A, wherein the mutant has a K _m value of 0.2 mM and a catalytic efficiency of 2035.7 s ^-1 mM ^-1 . Compared with the wild enzyme opt-HJ5NagNC, the mutant De10A has a substrate affinity increased by 250%, a catalytic efficiency increased by 95%, and an application cost reduced. The mutant has broad application prospects in the field of functional food production and processing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the SDS-PAGE analysis results of the wild enzyme opt-HJ5NagNC and the mutant De10A provided by the present invention.

FIG. 2 is a graph showing the pH activity measurement results of the wild enzyme opt-HJ5NagNC and the mutant De10A provided by the present invention.

FIG3 is a graph showing the results of thermal activity determination of the wild enzyme opt-HJ5NagNC and the mutant De10A provided by the present invention.

FIG. 4 is a graph showing the results of determination of kinetic parameters of the wild enzyme opt-HJ5NagNC and the mutant De10A provided by the present invention.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Some experimental materials and reagents used in the following examples:

1. Strains and vectors: Escherichia coli BL21 (DE3) and expression vector pET-22b (+) were purchased from Novagen.

2. Enzymes and other biochemical reagents: Nickel-NTA Agarose was purchased from QIAGEN, QuickMutation ^TM gene site-directed mutagenesis kit was purchased from Shanghai Bio-Tech, p-nitrophenol-β-N-acetylglucosamine (pNPGlcNAc) was purchased from Shanghai Yuanye, and the others were domestic reagents (all available from ordinary biochemical reagent companies).

3. Culture medium

LB medium: 10 g peptone, 5 g yeast extract, 10 g sodium chloride, add distilled water to 1000 mL, pH natural (about 7). Solid medium is based on this and 2.0% (w/v) agar is added.

The recombinant N-acetylglucosaminidase in the embodiment of the present invention is the expression product of wild-type N-acetylglucosaminidase HJ5Nag (GenBank accession number ARJ33352) inserted into the recombination region of the pEasy-E2 expression vector, and its amino acid sequence is shown in SEQ ID NO. 3. The molecular biology experimental methods not specifically described in detail in this embodiment are all carried out with reference to the specific methods listed in the book "Molecular Cloning Experiment Guide" (3rd Edition) by J. Sambrook, or according to the kit and product instructions.

Example 1 Construction and transformation of mutant De10A expression vector

The nucleotide sequence of the N-acetylglucosaminidase gene hj5nag, whose GenBank accession number is KX400857, is deleted from the signal peptide NRRRGRAIAAATVLAASLA

(AATCGTCGCCGAGGACGGGCCATCGCCGCCGCCACGGTGCTCGCCGCGTC GCTGGCG), since its GC content is as high as 72% and there are more hairpin structures, codon optimization is performed, and coding sequence 7 is introduced after its start codon, and its nucleotide sequence is shown in SEQ ID NO.7, and coding sequence 8 is introduced before its stop codon, and its nucleotide sequence is shown in SEQ ID NO.8, as shown in detail as follows:

Because the N-acetylglucosaminidase gene opt-hj5nagNC is constructed between the NdeI and XhoI restriction sites in the vector pET-22b(+), and the ATG on NdeI is used as the start codon, the introduced coding sequences 7 and 8 correspond to the nucleotide fragments of the amino acid fragments ‘ELAL and KGQF’, respectively, to eliminate the changes in the recombinant N-acetylglucosaminidase expression sequence caused by replacing the original vector pEasy-E2 with the current vector pET-22b(+). After optimization, the N-acetylglucosaminidase optimized gene opt-hj5nagNC with a nucleotide sequence as shown in SEQ ID NO.4 was obtained. The optimized gene opt-hj5nagNC was synthesized by Suzhou Hongxun Biotechnology Co., Ltd., and a recombinant expression plasmid pET22b-opt-hj5nagNC containing the optimized gene opt-hj5nagNC was constructed, and the recombinant expression plasmid pET22b-opt-hj5nagNC was transferred into Escherichia coli BL21 (DE3) to obtain a recombinant expression bacterium containing the optimized gene. The protein expressed by the optimized gene is the optimized wild enzyme opt-HJ5NagNC, which is the same as the amino acid HJ5Nag sequence of the recombinant N-acetylglucosaminidase in the pEasy-E2 vector, and its amino acid sequence is shown in SEQ ID NO.3.

The introduced coding sequence is as follows (5'→3'):

Coding sequence 7 (SEQ ID NO. 7): GAATTGGCACTT.

Coding sequence 8 (SEQ ID NO. 8): AAGGGACAATTC.

1. According to the sequence of the wild enzyme optimized gene opt-hj5nagNC shown in SEQ ID NO.4 and the plasmid pET-22b-opt-hj5nagNC, the recombination primers F and R were designed using CE Design software. The specific sequences are as follows:

The recombination primer sequence is as follows (5'→3'):

F (SEQ ID NO.5):

TGTAGTCCGGCACAAAACGGCGATAACCGTTCCG.

R (SEQ ID NO.6):

GTTTTGTGCCGGACTACAACCAAGTGCCAATTC.

According to the manufacturer's instructions of the QuickMutation ^TM gene site-directed mutagenesis kit, PCR amplification was performed using the plasmid pET-22b-opt-hj5nagNC as a template to obtain the PCR product of pET-22b-de10A. The PCR amplification reaction procedure was as follows: pre-denaturation at 95°C for 30 min; then denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 68°C for 7 min, for a total of 20 cycles; extension and completion at 68°C for 15 min; and temporary storage at 4°C for 30 min.

2. After the PCR reaction, add 1 μL DpnI directly to the PCR reaction system, mix well and incubate in a 37°C water bath for 5 minutes. After DpnI digestion, it can be used directly for transformation or stored at -20°C for later use.

3. Transformation and identification: The PCR digestion product was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method. After picking a single colony for culture and preservation, a recombinant expression bacterium containing the expression vector pET-22b-de10A was obtained. It was further confirmed by sequencing by Beijing Qingke Biotechnology Co., Ltd. that the nucleotide sequence of the mutant De10A encoding gene is shown in SEQ ID NO.2. At the same time, it can be seen that the amino acid sequence of the mutant De10A is shown in SEQ ID NO.1. Compared with the amino acid sequence of the optimized wild enzyme opt-HJ5NagNC (SEQ ID NO.3), De10A deletes the 10th amino acid residue alanine in the N-terminal loop region.

Example 2 Preparation of wild enzyme opt-HJ5NagNC and mutant De10A

The recombinant expression strain containing the coding genes of the wild enzyme opt-HJ5NagNC and the mutant De10A was inoculated into LB (containing 100 μg mL ^-1 ampicillin) culture medium at an inoculation amount of 0.1% (v/v), and cultured overnight at 37° C. and 180 rpm to obtain an activated bacterial solution.

The activated bacterial solution was inoculated into fresh LB (containing 100 μg mL ^-1 ampicillin) culture medium at a 1% (v/v) inoculation volume, and cultured with rapid shaking at 37°C and 180 rpm constant temperature shaker for about 2 to 3 hours. When the OD ₆₀₀ of the culture reached between 0.6 and 1.0, IPTG was added at a final concentration of 0.7 mM for induction, and cultured at 20°C and 160 rpm constant temperature shaker for about 20 hours. The induced bacterial solution was centrifuged in a low-temperature centrifuge at 4°C and 6000 rpm for 10 minutes to collect the bacteria. After suspending the bacteria with an appropriate amount of pH = 7.0 McIlvaine buffer, the bacteria were ultrasonically broken in a low-temperature water bath. After the above intracellular concentrated crude enzyme solution was centrifuged at 12000 rpm for 10 minutes, the supernatant was aspirated and the target protein was affinity and eluted with Nickel-NTAAgarose and 0 to 500 mM imidazole, respectively.

As shown in Figure 1, the SDS-PAGE analysis result diagram of the wild enzyme opt-HJ5NagNC and the mutant De10A provided by the present invention, wherein M is a protein marker; the crude enzyme solution of opt-HJ5NagNC is the unpurified wild enzyme opt-HJ5NagNC; opt-HJ5NagNC is the purified wild enzyme opt-HJ5NagNC; and De10A is the purified mutant De10A. As can be seen from Figure 1, both the wild enzyme opt-HJ5NagNC and the mutant De10A have been purified, and the product is a single band.

Example 3 Determination of properties of wild enzyme opt-HJ5NagNC and mutant De10A

1. Activity analysis of purified wild-type enzyme opt-HJ5NagNC and mutant De10A

The activity determination method adopts the p-nitrophenol (pNP) method: the substrate pNPGlcNAc is dissolved in a buffer solution to a final concentration of 2 mM; the reaction system contains 50 μL of an appropriate amount of enzyme solution and 450 μL of substrate; the substrate is preheated at the reaction temperature for 5 minutes, and then the enzyme solution is added to react for 10 minutes, and then 2 mL of 1M Na ₂ CO ₃ is added to terminate the reaction. After cooling to room temperature, the OD value is measured at a wavelength of 405 nm; 1 enzyme activity unit (U) is defined as the amount of enzyme required to decompose the substrate to produce 1 μmol pNP per minute under given conditions.

2. Determination of the optimal pH of the purified wild-type enzyme opt-HJ5NagNC and mutant De10A

The optimal pH of the wild enzyme and mutants was determined by a two-step method. The activity was measured at every pH unit point, and the second time at the pH unit point with the highest activity and the points 0.5 pH units to the left and right. The results can be accurately and verified when the sample volume is basically unchanged. At 30°C, the buffer was 0.1M MClvaine buffer (pH = 5.0-8.0) and 0.1M glycine-NaOH (pH = 9.0). The enzyme activity of the purified N-acetylglucosaminidase was determined by taking pNPGlcNAc as the substrate and reacting for 10 minutes. First, the enzyme solution was placed in a buffer solution of pH = 5.0, 6.0, 7.0, 8.0, and 9.0 for the above enzymatic reaction. It was found that the wild enzyme opt-HJ5NagNC and the mutant De10A both had the highest enzyme activity at pH 6.0. Then the enzyme activity of the wild enzyme and the mutant was determined in buffer solutions of pH = 5.5, 6.0, and 6.5, respectively.

As shown in Figure 2, the pH activity measurement results of the wild enzyme opt-HJ5NagNC and the mutant De10A provided by the present invention are shown in Figure 2. As can be seen from Figure 2, the optimum pH of the wild enzyme opt-HJ5NagNC and the mutant De10A are both 6.0, the wild enzyme opt-HJ5NagNC and the mutant De10A both have the highest enzyme activity at pH 6.0, and the mutant De10A can maintain more than 60% enzyme activity at pH 5.0-8.0.

3. Thermal activity assay of purified wild-type enzyme opt-HJ5NagNC and mutant De10A

The optimal temperature of the wild enzyme and mutant was determined by a two-step method. The activity was measured at each temperature interval of 10°C for the first time, and the activity was measured at the temperature point with the highest activity and the temperature points 5°C to the left and right for the second time. The measurement results can be accurately verified when the sample volume is basically unchanged. In a buffer solution of pH = 6.0, the enzymatic reaction was first carried out at 0-60°C, and the enzyme activity was measured every 10°C from 0°C. It was found that the wild enzyme opt-HJ5NagNC and the mutant De10A had the highest enzyme activity at 40°C. Then, the enzyme activity of the wild enzyme and mutant was measured at 35°C, 40°C and 45°C in a buffer solution of pH = 6.0.

As shown in Figure 3, the thermal activity test results of the wild enzyme opt-HJ5NagNC and the mutant De10A provided by the present invention are shown in Figure 3. As shown in Figure 3, the optimum temperatures of the wild enzyme opt-HJ5NagNC and the mutant De10A are 45°C and 40°C, respectively.

4. Determination of kinetic parameters of wild-type enzyme opt-HJ5NagNC and mutant De10A

At 30°C and pH=6.0, pNPGlcNAc was used as a substrate to determine the kinetic parameters of the wild enzyme and the mutant, with a substrate concentration between 0.1mM and 2mM and a reaction time of 6min. The substrate concentration was used as the horizontal axis, and the enzyme activity units per milligram of enzyme were calculated according to the reaction determination results as the vertical axis to draw a nonlinear fitting curve, as shown in Figure 4. The kinetic parameters were calculated using the Michaelis-Menten equation in the nonlinear fitting calculation method of GraphPadPrism 5.0 software. The results are shown in Table 1. Compared with the wild enzyme, the mutant De10A has a K _m reduced by 250%, that is, the substrate affinity is improved, and the catalytic efficiency is increased by about 95%.

Table 1 Kinetic parameters of wild-type enzymes opt-HJ5NagNC and De10A

In summary, the present invention uses genetic engineering technology to delete the 10th amino acid alanine in the N-terminal loop region of the wild enzyme opt-HJ5NagNC to obtain the mutant De10A. The catalytic efficiency of the mutant is 2035.7s ^-1 mM ^-1 , which is 95% higher than that of the wild N-acetylglucosaminidase. The present invention provides an N-acetylglucosaminidase mutant De10A with improved catalytic efficiency, reduces application costs, and provides effective protection for its use in the field of functional food production and processing.

Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims (10)

1. An N-acetylglucosaminidase mutant De10A, characterized in that the amino acid sequence of the mutant is shown in SEQ ID NO.1. 2. A gene encoding the N-acetylglucosaminidase mutant De10A as claimed in claim 1, characterized in that the nucleotide sequence of the gene encoding is shown in SEQ ID NO.2. 3 . A recombinant expression vector comprising the gene encoding the N-acetylglucosaminidase mutant De10A as claimed in claim 2 . 4. The recombinant expression vector according to claim 3, characterized in that the recombinant expression vector comprises a pET series plasmid. 5 . A recombinant expression bacterium comprising the gene encoding the N-acetylglucosaminidase mutant De10A as claimed in claim 2 . 6. The recombinant expression bacteria according to claim 5 is characterized in that the recombinant expression bacteria is selected from Escherichia coli BL21 (DE3). 7. A method for improving the catalytic efficiency of N-acetylglucosaminidase, characterized in that the method is to delete the 10th alanine in the amino acid sequence of N-acetylglucosaminidase as shown in SEQ ID NO.3. 8. Use of the N-acetylglucosaminidase mutant De10A according to claim 1 or the gene encoding the N-acetylglucosaminidase mutant De10A according to claim 2 in the field of functional food production and processing. 9. Use of the group expression vector as claimed in claim 3 in the field of functional food production and processing. 10. Use of the recombinant expression bacteria as claimed in claim 5 in the field of functional food production and processing.