Thermophilic glucosamine-6-phosphate deaminase mutant, encoding gene and application thereof

Abstract

The present invention relates to a thermophilic glucosamine 6-phosphate deaminase mutant and its encoding gene, recombinant expression vector and engineered bacteria, as well as its application in enzymatic catalysis of the reversible conversion of fructose 6-phosphate and ammonium ions into glucosamine 6-phosphate. The present invention obtains a thermophilic glucosamine 6-phosphate deaminase mutant with improved enzyme activity through site-directed saturation mutagenesis technology. The mutant is 2.15 times more effective than the wild type in catalyzing the reversible conversion of fructose 6-phosphate and ammonium ions into glucosamine 6-phosphate, with an activity of 3.16U / mg. The mutant can still play a catalytic role at high temperatures, can be protein purified by heat treatment, simplifies the purification step, and has good thermal stability under normal temperature conditions. By using the deaminase mutant, fructose 6-phosphate and ammonium ions can be used as substrates to more efficiently synthesize glucosamine 6-phosphate, thereby improving the efficiency of thermodynamically driven glucosamine biosynthesis in vitro and having good application prospects.

Description

(1) Technical field

[0001] The present invention relates to a thermophilic glucosamine-6-phosphate deaminase mutant and its encoding gene, recombinant expression vector and engineered bacteria, as well as application of the mutant in enzymatically catalyzing the reversible conversion of fructose-6-phosphate and ammonium ions into glucosamine-6-phosphate. (2) Background technology

[0002] Glucosamine, also known as glucosamine (GlcN), is a natural amino monosaccharide formed by replacing one of the hydroxyl groups of glucose with an amino group. Glucosamine, commonly found in polysaccharides and conjugated sugars derived from microorganisms and animals in the form of N-acetyl derivatives (such as chitin and chitosan), is a crucial nutrient for the formation of chondrocytes in the human body. It stimulates chondrocyte growth, promotes cartilage repair, and has the potential to prevent the progression of osteoarthritis. In recent years, with the increasing aging of the population, the market share of glucosamine has grown annually.

[0003] Due to the broad market prospects of glucosamine, its synthesis has become a research hotspot. Currently, the main method for industrial glucosamine production is acid hydrolysis, which involves hydrolyzing chitin and chitosan from crab and shrimp shells into glucosamine using high-concentration hydrochloric acid at high temperatures (approximately 100°C). Although this method is simple to operate, the use of large amounts of acid leads to serious environmental concerns (J. Agric. Food Chem. 2007, 55, 2246). Consequently, researchers are working to develop new glucosamine synthesis processes. For example, chitinase, exochitinase, β-N-acetylglucosaminidase, and N-acetyl-glucosamine deacetylase are used to biodegrade chitin from crab and shrimp shells into glucosamine. However, due to the complex structure of chitin from crab and shrimp shells, chitin cannot be completely hydrolyzed, resulting in low glucosamine yields (J. Agric. Food Chem. 2018, 66, 8061). Microbial fermentation, which has emerged in recent years, has attracted much attention from researchers. However, its products are mostly N-acetylglucosamine, which requires further hydrolysis and deacetylation to synthesize glucosamine. This has problems such as low yield and heavy environmental burden (Bioresour. Technol. 2018, 250, 642-649).

[0004] You Chun et al. reported a thermodynamically driven in vitro biosynthesis method for glucosamine, which uses cheap starch or starch derivatives and inorganic ammonium salts as substrates, and uses five cascade enzymes including α-glucan phosphorylase, glucose phosphomutase, glucose phosphoisomerase, glucosamine-6-phosphate deaminase (GlmD) and phosphatase as catalysts to synthesize glucosamine through steps such as phosphorylation, isomerization, amination and dephosphorylation (ACS Catal.2020,10,13809-13819). Since the last step of the reaction system is irreversible dephosphorylation catalyzed by phosphatase, the theoretical yield of this method can reach 100%, which is an ideal way to synthesize glucosamine. Among them, glucosamine-6-phosphate deaminase (EC 3.5.99.6) is a type of allosteric enzyme that catalyzes the reversible conversion of glucosamine-6-phosphate into fructose-6-phosphate and ammonium ions. It is one of the key enzymes for achieving thermodynamically driven efficient biosynthesis of glucosamine. However, currently reported glucosamine-6-phosphate deaminases suffer from low activity and poor stability, making them difficult to use in the industrial synthesis of glucosamine. Therefore, it is of great significance to develop a thermostable, thermophilic deaminase that can efficiently catalyze the conversion of fructose-6-phosphate and ammonium ions into glucosamine-6-phosphate. (3) Summary of the invention

[0005] To solve the above problems, the present invention provides a thermophilic glucosamine-6-phosphate deaminase mutant, an encoding gene, and use thereof in efficiently catalyzing the conversion of fructose-6-phosphate and ammonium ions into glucosamine-6-phosphate.

[0006] The technical solution adopted in the present invention is:

[0007] A thermophilic glucosamine-6-phosphate deaminase mutant is obtained by subjecting the glucosamine-6-phosphate deaminase derived from Pyrococcus abyssi to single-point mutation or multiple-point mutation, wherein the mutation sites of the single-point mutation or multiple-point mutation are one or more of the following: (1) cysteine ​​at position 40, (2) arginine at position 88, and (3) threonine at position 92.

[0008] The present invention screened and mined glucosamine-6-phosphate deaminase from the deep-sea fireball bacteria (Pyrococcus abyssi) from a natural enzyme library, and molecularly modified it through protein engineering technology, thereby improving its activity in catalyzing the conversion of fructose-6-phosphate and ammonium ions into glucosamine-6-phosphate, thereby further improving the efficiency of glucosamine biosynthesis in vitro and laying the foundation for the large-scale preparation of glucosamine.

[0009] Specifically, the point mutation is one or more of the following: (1) cysteine ​​at position 40 mutates to alanine, (2) arginine at position 88 mutates to aspartic acid, and (3) threonine at position 92 mutates to phenylalanine.

[0010] Preferably, the amino acid sequence of the thermophilic glucosamine-6-phosphate deaminase mutant is shown as SEQ ID NO.3 (i.e. mutant C40A), SEQ ID NO.5 (i.e. mutant R88N) or SEQ ID NO.7 (i.e. mutant T92F).

[0011] Any deletion, insertion or substitution of one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1 that has the activity of catalyzing the reversible conversion of fructose-6-phosphate and ammonium ions into glucosamine-6-phosphate still falls within the scope of protection of the present invention.

[0012] The present invention also relates to recombinant expression vectors containing the encoding gene. These recombinant vectors can be constructed by connecting the nucleotide sequence of the sucrose phosphorylase mutant of the present invention to various vectors using conventional methods in the art. The vectors can be various conventional vectors in the art, such as various plasmids, phages, or viral vectors, preferably pET-28a.

[0013] Preferably, the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 4 (encoding mutant C40A), SEQ ID NO. 6 (encoding mutant R88N) or SEQ ID NO. 8 (encoding mutant T92F).

[0014] The present invention also relates to engineered bacteria containing the encoding gene. As a recombinant expression vector, genetically engineered bacteria can be obtained by transforming the recombinant expression vector of the present invention into a host microorganism. The host microorganism can be any of a variety of conventional host microorganisms in the art, provided that the recombinant expression vector can stably replicate and effectively express the sucrose phosphorylase mutant gene carried by the present invention. Escherichia coli is preferred, and E. coli BL21 (DE3) is more preferred.

[0015] The present invention also relates to the use of the thermophilic glucosamine-6-phosphate deaminase mutant in enzymatically catalyzing the reversible conversion of fructose-6-phosphate and ammonium ions into glucosamine-6-phosphate.

[0016] The thermophilic glucosamine-6-phosphate deaminase mutant of the present invention can be used in the form of whole cells, in the form of unpurified crude enzymes, or in the form of partially purified or completely purified enzyme proteins. If desired, the thermophilic glucosamine-6-phosphate deaminase mutant of the present invention can also be prepared into an immobilized enzyme or immobilized cell form using immobilization techniques known in the art.

[0017] The beneficial effects of the present invention are primarily reflected in the following: A thermophilic glucosamine-6-phosphate deaminase mutant with enhanced enzyme activity is obtained through site-directed saturation mutagenesis. This mutant catalyzes the reversible conversion of fructose-6-phosphate and ammonium ions into glucosamine-6-phosphate 2.15 times more efficiently than the wild-type, achieving an activity of 3.16 U / mg. Furthermore, this mutant can still function catalytically at high temperatures and can be purified by heat treatment, simplifying the purification process. Furthermore, this deaminase mutant exhibits excellent thermal stability at room temperature. Using this deaminase mutant, glucosamine-6-phosphate can be synthesized more efficiently using fructose-6-phosphate and ammonium ions as substrates, thereby improving the efficiency of thermodynamically driven glucosamine biosynthesis in vitro and demonstrating promising application prospects. (IV) Specific implementation methods

[0018] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0019] Example 1: Acquisition of Glucosamine-6-Phosphate Deaminase Gene Sequence and Vector Construction

[0020] The amino acid sequence from Pyrococcus abyssi (GenBank: WP_048147071.1) was obtained by mining databases such as NCBI. Without changing the amino acid sequence of the polypeptide, the codons of the gene were replaced with codons preferred by Escherichia coli. The nucleotide sequence of the codon-optimized glucosamine-6-phosphate deaminase gene is shown in SEQ ID NO. 2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 1.

[0021] The gene sequence shown in SEQ ID NO.2 was connected to the pET-28b(+) vector, and the recombinant plasmid was named pET-28b(+)-GlmD.

[0022] Example 2: Inducible expression and purification of wild-type glucosamine-6-phosphate deaminase

[0023] 1. Construction of recombinant bacteria

[0024] The recombinant plasmid pET-28b(+)-GlmD in Example 1 was transformed into E. coli BL21(DE3) competent cells to obtain wild-type recombinant bacteria.

[0025] 2. Cultivation of Recombinant Bacteria

[0026] After streaking overnight from a stored glycerol tube, a single colony was inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C, 180 rpm. A 2% (v / v) inoculum was inoculated into 100 mL of LB liquid medium and cultured at 37°C to an OD of 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, the culture temperature was adjusted to 16°C, and expression was induced for 16 hours. Centrifuge and discard the supernatant to obtain wet cells.

[0027] 3. Thermal Purification of Crude Enzyme Solution

[0028] The wet cells collected above were resuspended with 100mM HEPES buffer (pH 7.5), and the bacterial suspension was placed on ice for an ice bath. Ultrasonic disruption (2 seconds for disruption, 4 seconds for rest, a total of 15 minutes, and a power of 60W) was performed to obtain a cell disruption solution. The solution was then centrifuged at 8000rpm for 10 minutes, and the supernatant was heat-treated in a water bath at 80°C for 20 minutes. The solution was centrifuged at 8000rpm for 10 minutes, and the supernatant was the preliminarily purified enzyme solution. The purity and molecular weight of the target protein were estimated by SDS-PAGE gel electrophoresis, and its protein concentration was detected using a BCA protein kit.

[0029] Example 3: Construction of a Glucosamine-6-Phosphate Deaminase Mutant Library

[0030] 1. Selection of deaminase mutation sites

[0031] To improve the catalytic activity of glucosamine-6-phosphate deaminase (GlmD), site-directed mutagenesis sites were selected using computer-aided design. The crystal structures of Pyrococcus horikoshii OT3 deaminase (PDB ID: 2DEC) and Pyrococcus furiosu deaminase (PDB ID: 2CB0), both of which share 61.92% identity with GlmD, were used as templates for homology modeling using MODELLER software. The model with the highest DOPE score was selected, and its reliability was verified using Procheck. Based on the catalytic mechanism of the deaminase and the molecular docking model of GlmD and fructose-6-phosphate, key amino acid residues in the catalytically active region were identified. Ten amino acid residues that may affect enzymatic activity were further screened: Cys40, Ser42, Ser43, Ser87, Arg88, Thr92, Val133, Met135, Glu211, and Lys319.

[0032] 2. Site-directed saturation mutagenesis

[0033] According to the amino acid sequence shown in SEQ ID NO. 1, site-directed saturation mutagenesis primers were designed, as shown in Table 1. (Note: N = A / G / C / T, K = G / T, M = A / C).

[0034] Table 1: Site-directed saturation mutagenesis primer sequence list

[0035]

[0036] The entire plasmid was amplified by PCR using the vector pET28b as a template. The reaction system is shown in Table 2 and the reaction conditions are shown in Table 3 to obtain the mutant sequence.

[0037] Table 2: PCR reaction system

[0038]

[0039] Table 3: PCR reaction conditions

[0040]

[0041] Example 4: Initial screening of a library of site-saturated mutants of glucosamine-6-phosphate deaminase

[0042] 1. Construction and expression of deaminase mutant recombinant bacteria

[0043] Amplified bands of the correct size were detected by 0.8% agarose gel electrophoresis. The PCR product was digested with the restriction endonuclease DpnI at 37°C for 2 hours to digest the methylated plasmid template. 10 μL of the digested PCR product was transformed into Escherichia coli BL21(DE3) cells and plated with LB solid medium containing 50 μg / mL kanamycin. The cells were incubated at 37°C for 12 hours to obtain single colonies.

[0044] The obtained single colony was inoculated into a 96-well plate containing LB liquid medium containing 50μg / mL kanamycin and cultured at 37°C for 12h to obtain a seed solution. 200μL of the seed solution was transferred to a new sterile 96-deep-well plate, and each well contained 400μL of LB liquid medium with a final concentration of 50μg / mL kanamycin and 0.1mM IPTG. After culture at 28°C and 180rpm for 12h, the plate was centrifuged at 4000rpm for 20min, and the supernatant was discarded to collect the wet bacteria. 400μL of HEPES buffer (pH 7.5, 100mM) was added to resuspend the bacteria in each well, and the cells were disrupted by repeated freezing and thawing (freezing at -80°C for 40min, thawing at 37°C for 30min, repeated 4 times), heat treated at 80°C for 20min, and then centrifuged at 8000rpm for 20min. The supernatant was taken to obtain the mutant enzyme solution.

[0045] 2. High-throughput screening of deaminases

[0046] A high-throughput screening method for GlmD was established using the Elson-Morgan colorimetric assay. The reaction principle is as follows: under alkaline conditions, hexosamine can react with acetylacetone to form a pyrrole derivative. The resulting pyrrole derivative reacts with an acidic alcohol solution of p-dimethylaminobenzaldehyde to form a red condensate. Within a certain concentration range, the absorbance of the red condensate at 530 nm is proportional to the hexosamine concentration, thus reflecting the catalytic activity of the deaminase mutant.

[0047] Activity assay steps: add 200 μL of reaction solution (containing 155 μL mutant enzyme solution, 25 μL (NH4)2SO4 (final concentration in the reaction solution is 25 mM), and 20 μL fructose-6-phosphate (final concentration in the reaction solution is 5 mM)) to a 96-well plate reaction plate, react at 40°C for 10 minutes, terminate in an ice bath, and quickly pipette 100 μL of the reaction solution into a 96-deep-well plate containing 200 μL acetylacetone reagent and incubate in a water bath at 90°C for 1 hour; after cooling to room temperature, pipette 30 μL into a 96-well ELISA plate, add 100 μL of 96% ethanol, and then add 120 μL of DMAB reagent, mix well, react at 37°C for 30 minutes, perform colorimetry at 530 nm, calculate the amount of glucosamine-6-phosphate in the reaction sample according to the standard curve, and select mutants with improved activity for the next step of rescreening.

[0048] Example 5: Rescreening of the Glucosamine-6-Phosphate Deaminase Site-Saturation Mutant Library

[0049] The mutants selected from the primary screening of Example 4 were inoculated into 10 mL LB liquid medium containing 50 μg / mL of kanamycin and cultured at 37°C, 180 rpm for 10 h. 2% by volume of the inoculum was inoculated into 100 mL LB liquid medium containing 50 μg / mL of kanamycin and cultured at 37°C, 180 rpm until OD was 0.6-0.8. IPTG was then added at a final concentration of 0.1 mM and induced for 16 h at 16°C. The culture solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the wet cells obtained were resuspended in HEPES buffer (pH 7.5, 100 mM). The cell suspension was ultrasonically disrupted (60 W for 2 s, 4 s between intervals, for a total of 15 min) to obtain a cell lysis solution, which was then centrifuged at 8000 rpm for 10 min. The supernatant was heat-treated in a water bath at 80° C. for 20 minutes and centrifuged to obtain the GlmD mutant enzyme solution. The target protein content was detected using a BCA protein content detection kit.

[0050] The enzyme activity assay reaction system (1 mL) consists of 100 mM HEPES (pH 7.5) buffer, 10 mM fructose-6-phosphate, 50 mM (NH₄)₂SO₄, and an appropriate amount of enzyme solution. The reaction is incubated at 40°C for 10 min to determine the deaminase catalytic activity. The reaction is terminated with perchloric acid, neutralized with sodium hydroxide, and centrifuged at 12,000 rpm for 1 min. The supernatant is then analyzed by HPLC for glucosamine-6-phosphate content. Activity Definition: One unit of activity is defined as the amount of enzyme required to convert 1 μmol of product per minute at 40°C in 100 mM HEPES buffer (pH 7.5).

[0051] The HPLC detection method is as follows: chromatographic conditions: mobile phase A: 20 mM sodium acetate (pH 7.2±0.5), mobile phase B: 10% 20 mM sodium acetate + 20% acetonitrile + 20% methanol (pH adjusted to 7.2±0.5); column temperature, 35°C; chromatographic column model: Welchrom-C18 (4.6 x 250 mm, 5 μm); flow rate, 0.8 mL / min; injection volume: 5 μL of sample + 6 μL of 0.4 mol / L boric acid buffer (pH 9.5) + 2 μL of OPA derivatization reagent.

[0052] As shown in Table 4, GlmD-C40A, GlmD-R80N, and GlmD-T92F showed significant improvements in activity. Mutation at the Cys40 site was the most effective, with mutant C40A achieving an enzyme activity of 3.16 U / mg for the reversible conversion of fructose-6-phosphate and ammonium ions to glucosamine-6-phosphate, 2.15 times that of wild-type glucosamine-6-phosphate deaminase.

[0053] Table 4: Enzyme activities of wild-type GlmD and its mutants

[0054]

[0055] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of protection of the present invention.

Claims

1. A thermophilic glucosamine-6-phosphate deaminase mutant, having an amino acid sequence as shown in SEQ ID NO.1 from Pyrococcus pyrococcus ( Pyrococcus abyssi ) was obtained by single-point mutation of glucosamine-6-phosphate deaminase derived from β-lactamase, wherein the single-point mutation was the mutation of cysteine ​​at position 40 to alanine.

2. The thermophilic glucosamine-6-phosphate deaminase mutant according to claim 1, characterized in that The amino acid sequence of the thermophilic glucosamine-6-phosphate deaminase mutant is shown in SEQ ID NO.

3.

3. A gene encoding the thermophilic glucosamine-6-phosphate deaminase mutant according to claim 1.

4. The coding gene according to claim 3, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

5. A recombinant expression vector containing the coding gene according to claim 3.

6. An engineered bacterium containing the coding gene according to claim 3.

7. Use of the thermophilic glucosamine-6-phosphate deaminase mutant according to claim 1 in enzymatically catalyzing the reversible conversion of fructose-6-phosphate and ammonium ions into glucosamine-6-phosphate.

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