L-arabinose isomerase mutant with improved enzyme activity

Abstract

The application discloses an L-arabinose isomerase mutant with improved enzyme activity, and belongs to the field of genetic engineering and modern enzyme technology engineering. The application uses a site-directed mutagenesis technology to perform molecular modification on L-arabinose isomerase (LbAI), and constructs a series of combined mutants. Compared with a wild type, the specific enzyme activity of the mutant is significantly improved, the highest specific enzyme activity of four-point and five-point mutants can reach 4.01-6.34 U / mg, which is 2.35-3.72 times of the wild type, and the specific enzyme activity of the five-point mutant K220A / N141K / Q258A / K339H / I297Q reaches 6.34 U / mg, which is 372.28% of the wild type. 2+ Co is the best metal ion activator, and can further greatly improve the enzyme activity. The application provides an enzyme catalyst with excellent performance for efficient and green biosynthesis of D-tagatose, and has important industrial application prospect.

Description

Technical Field

[0001] This invention relates to an L-arabinose isomerase mutant with enhanced enzyme activity, belonging to the fields of genetic engineering and modern enzyme technology engineering. Background Technology

[0002] L-arabinose isomerase (EC 5.3.1.4, L-AI) catalyzes the reversible isomerization of L-arabinose and the epimerization of D-galactose, producing a high-value rare sugar—D-tagatose. D-tagatose has a sweetness comparable to sucrose but only one-third the calories. It possesses important physiological functions such as a low glycemic index, prebiotic properties, and antioxidant effects, making it in high demand in the food and pharmaceutical industries. Synthesizing tagatose using inexpensive galactose as a substrate with L-AI offers advantages such as mild reaction conditions, high specificity, and environmental friendliness, making it a research hotspot in recent years.

[0003] Based on its reversible catalytic biochemical properties, L-AI shows great application potential in the field of in vitro biocatalysis. However, the core bottleneck for the industrialization of this technology lies precisely in the insufficient performance of its key catalytic enzyme, L-AI. Currently, most reported wild-type L-AI still face numerous limitations in practical applications. On the one hand, wild-type enzymes have limited overall catalytic efficiency, insufficient affinity for the non-natural substrate D-galactose, and low catalytic conversion rates. On the other hand, these enzymes generally exhibit poor thermal stability; most L-AI derived from mesophilic bacteria rapidly inactivate above 60 °C, while suitable high-temperature conditions can improve substrate solubility, accelerate the reaction process, and reduce the risk of contamination. For example, Levilactobacillus brevis Although the L-AI (LbAI) from this source possesses certain catalytic activity, its enzyme activity decreases by more than half after incubation at 70 °C for 1 hour. Furthermore, the optimal pH of some L-AI is alkaline, which is difficult to perfectly match the pH conditions required for the optimal dissolution / conversion of the substrate galactose, further limiting the stability and applicability of this enzyme in industrial continuous production.

[0004] Therefore, constructing an L-arabinose isomerase mutant with high catalytic activity, high thermal stability, and excellent pH adaptability is the key to breaking through the bottleneck of the in vitro biosynthesis of tagatose. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention addresses the deficiencies arising from... Levilactobacillus brevisL-arabinose isomerase (LbAI, GenBank accession number: WP_015474061.1) was modified by iterative combination mutations of key amino acid sites (such as positions 297, 258, 339, 141, and 220) near the substrate binding pocket and catalytic active site. This successfully yielded a mutant with significantly increased specific enzyme activity and enhanced thermostability, providing a novel solution for the efficient and green production of tagatose.

[0006] The first technical solution provided by this invention is an L-arabinose isomerase mutant, which... Levilactobacillus brevis The L-arabinose isomerase from which this was derived is the parent and has mutations at one or more of the following sites: I297Q, Q258A, K339H, N141K, and K220A.

[0007] In one embodiment, the amino acid sequence of the parent is shown in SEQ ID NO.1.

[0008] In one implementation, the mutation is as shown in any of (1) to (7): (1) The lysine at position 339 is mutated to histidine; (2) Asparagine at position 141 is mutated to lysine; (3) The lysine at position 220 is mutated to alanine; (4) Lysine at position 339 was mutated to histidine, isoleucine at position 297 was mutated to glutamine, and glutamine at position 258 was mutated to alanine; (5) Asparagine at position 141 was mutated to lysine, isoleucine at position 297 was mutated to glutamine, and glutamine at position 258 was mutated to alanine; (6) Lysine at position 220 was mutated to alanine, glutamine at position 258 was mutated to alanine, isoleucine at position 297 was mutated to glutamine, and lysine at position 339 was mutated to histidine; (7) Lysine at position 220 is mutated to alanine, asparagine at position 141 is mutated to lysine, glutamine at position 258 is mutated to alanine, lysine at position 339 is mutated to histidine and isoleucine at position 297 is mutated to glutamine.

[0009] In one embodiment, the mutant is obtained by mutating lysine at position 339 to histidine, isoleucine at position 297 to glutamine, and glutamine at position 258 to alanine, resulting in the three-point mutant K339H / I297Q / Q258A.

[0010] In one embodiment, the mutant is obtained by mutating asparagine at position 141 to lysine, isoleucine at position 297 to glutamine, and glutamine at position 258 to alanine, resulting in the three-point mutant N141K / I297Q / Q258A.

[0011] In one embodiment, the mutant is obtained by mutating lysine at position 220 to alanine, glutamine at position 258 to alanine, isoleucine at position 297 to glutamine, and lysine at position 339 to histidine, resulting in a four-point mutant K220A / Q258A / I297Q / K339H.

[0012] In one embodiment, the mutant is obtained by mutating lysine at position 220 to alanine, asparagine at position 141 to lysine, glutamine at position 258 to alanine, lysine at position 339 to histidine, and isoleucine at position 297 to glutamine, resulting in a five-point mutant K220A / N141K / Q258A / K339H / I297Q.

[0013] In one embodiment, the amino acid sequences of the mutants K339H / I297Q / Q258A, N141K / I297Q / Q258A, K220A / Q258A / I297Q / K339H and K220A / N141K / Q258A / K339H / I297Q are shown in SEQ ID NO.3 to SEQ ID NO.6, respectively.

[0014] In one embodiment, the nucleotide sequences of the mutants K339H / I297Q / Q258A, N141K / I297Q / Q258A, K220A / Q258A / I297Q / K339H and K220A / N141K / Q258A / K339H / I297Q are shown in SEQ ID NO.7 to SEQ ID NO.10, respectively.

[0015] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0016] The third technical solution provided by the present invention is a recombinant plasmid carrying the gene described in the second technical solution.

[0017] In one embodiment, the expression vector of the recombinant plasmid includes, but is not limited to, the pET-22b plasmid.

[0018] The fourth technical solution provided by the present invention is a recombinant microorganism expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant plasmid described in the third technical solution.

[0019] In one embodiment, the host cell of the recombinant microorganism is Escherichia coli or Bacillus subtilis.

[0020] Preferably, the recombinant microorganism uses Escherichia coli BL21(DE3) as the host cell and pET-22b(+) as the vector.

[0021] The fifth technical solution provided by the present invention is a catalyst for synthesizing tagatose, wherein the catalyst contains the mutant described in the first technical solution or the recombinant microorganism described in the fourth technical solution.

[0022] The present invention also provides a method for preparing the L-arabinose isomerase mutant, which involves culturing recombinant Escherichia coli expressing the mutant in a culture medium for a period of time and collecting the L-arabinose isomerase mutant.

[0023] In one embodiment, the method involves culturing the recombinant Escherichia coli in a culture medium until the OD600 reaches 0.5-0.8, and then inducing it with IPTG.

[0024] In one embodiment, the induction is performed at 16-20 °C.

[0025] In one embodiment, the method involves culturing the recombinant Escherichia coli in LB medium at 35-40°C until OD reaches zero. 600 The concentration of IPTG was 0.5-0.8, and IPTG was added to a final concentration of 0.4-0.6 mmol / L. The mixture was induced at 16-20 °C for 22-26 h.

[0026] The sixth technical solution provided by this invention is a method for improving the activity and / or thermostability of L-arabinose isomerase, wherein the method involves performing any one of the following mutations on the L-arabinose isomerase parent with the amino acid sequence shown in SEQ ID NO.1: (1) The lysine at position 339 is mutated to histidine; (2) Asparagine at position 141 is mutated to lysine; (3) The lysine at position 220 is mutated to alanine; (4) Lysine at position 339 was mutated to histidine, isoleucine at position 297 was mutated to glutamine, and glutamine at position 258 was mutated to alanine; (5) Asparagine at position 141 was mutated to lysine, isoleucine at position 297 was mutated to glutamine, and glutamine at position 258 was mutated to alanine; (6) Lysine at position 220 was mutated to alanine, glutamine at position 258 was mutated to alanine, isoleucine at position 297 was mutated to glutamine, and lysine at position 339 was mutated to histidine; (7) Lysine at position 220 is mutated to alanine, asparagine at position 141 is mutated to lysine, glutamine at position 258 is mutated to alanine, lysine at position 339 is mutated to histidine and isoleucine at position 297 is mutated to glutamine.

[0027] The seventh technical solution provided by this invention is the application of the mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant plasmid described in the third technical solution, or the recombinant microorganism described in the fourth technical solution, or the catalyst described in the fifth technical solution in the catalytic synthesis of tagatose from D-galactose.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention is based on Levilactobacillus brevis LbAI was used as the parent enzyme for molecular modification. Through co-evolutionary analysis and computer-aided design, single-point and iterative combination mutations were performed on key amino acid residues such as positions 297, 258, 339, 141 and 220 to obtain a series of mutants with enhanced enzyme activity.

[0029] (2) The enzyme activities of the series of L-arabinose isomerase mutants provided by the present invention are significantly improved compared with wild-type enzymes: the specific enzyme activity of wild-type is 1.71 U / mg, and the highest enzyme activities of K339H / I297Q / Q258A, N141K / I297Q / Q258A, K220A / Q258A / I297Q / K339H, K220A / N141K / Q258A / K339H / I297Q can reach 4.33 U / mg, 4.62 U / mg, 4.01 U / mg, and 6.34 U / mg, respectively, which are 253.80%, 270.80%, 235.24%, and 372.28% compared with wild-type enzyme activities. Attached Figure Description

[0030] Figure 1 The relative enzyme activities of wild-type enzyme LbAI and its mutants at their optimal temperature and optimal pH are given.

[0031] Figure 2 The effects of different temperatures and pH on the activity of L-arabinose isomerase mutants K339H / I297Q / Q258A were investigated.

[0032] Figure 3 The effects of different temperatures and pH on the activity of L-arabinose isomerase mutants N141K / I297Q / Q258A were investigated.

[0033] Figure 4The effects of different temperatures and pH on the activity of L-arabinose isomerase mutants K220A / Q258A / I297Q / K339H were investigated.

[0034] Figure 5 The effects of different temperatures and pH on the activity of L-arabinose isomerase mutants K220A / N141K / Q258A / K339H / I297Q were investigated.

[0035] Figure 6 The effect of metal ion type and concentration on the enzyme activity of the five-point mutant.

[0036] Figure 7 The thermostability of the L-arabinose isomerase mutant K220A / N141K / Q258A / K339H / I297Q.

[0037] Figure 8 This is a map of the wild-type LbAI recombinant plasmid. Detailed Implementation

[0038] Reference Appendix Figures 1-8 The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0039] Test method: 1. Protein concentration was determined using the BCA protein kit. The specific steps are as follows: (1) Preparation of BCA working solution. Based on the number of samples, BCA working solution was prepared by mixing BCA Reagent A and B at a volume ratio of 50:1 and then thoroughly mixed.

[0040] (2) Draw the standard curve.

[0041] (3) Sample preparation: Dilute the protein sample to be tested with deionized water to an appropriate concentration, and add 200 μL of LBCA working solution to 20 μL of sample.

[0042] (4) After shaking and mixing, place at 37 ℃ for 20 min to 30 min.

[0043] (5) Use the absorbance value without BSA as a blank control, and use an ELISA reader to detect the absorbance value of the sample at A562 nm.

[0044] (6) Plot a standard curve with protein content (μg) as the x-axis and absorbance as the y-axis.

[0045] (7) The protein content of the sample can be calculated by using a linear equation in two variables based on the measured absorbance value.

[0046] (8) Calculation of protein concentration: Divide the obtained protein content by the sample volume (20 μL) and multiply by the corresponding dilution factor to determine the true concentration of the sample.

[0047] 2. Recombinant enzyme activity assay: The activity of L-arabinose isomerase was determined by high performance liquid chromatography (HPLC). The standard reaction system consisted of 100 μL of purified enzyme solution mixed with 100 mM D-galactose, 50 mM KH₂PO₄-NaOH (pH 6.0), and 1 mM Co. 2+ The solution was prepared in a volume of 1 mL. After reacting at 60 °C for 30 min, it was immediately inactivated by boiling in a water bath for 10 min, centrifuged at 12,000 rpm for 5 min, and the supernatant was passed through a 0.22 μm aqueous membrane for HPLC detection.

[0048] Chromatographic conditions: A Waters Sugar-Pak™ I column (6.5 mm inner diameter, 300 mm length, product number WAT085188) was used, with a column temperature of 85 ℃; the mobile phase was 50 mg / L EDTA calcium salt aqueous solution, with a flow rate of 0.4 mL / min; a differential refractive index detector was used; and the injection volume was 10 μL.

[0049] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of D-tagatose per minute under the conditions described above. Specific enzyme activity (U / mg) is calculated based on the pure enzyme protein content, and the protein concentration is determined using the BCA method.

[0050] Materials used in the examples: (I) Materials and Reagents Primers used were purchased from Ansonda Biotechnology Co., Ltd.; plasmid extraction kit, genome extraction kit, agarose purification kit, E. coil DH5α, E. coil BL21(DE3) strain, yeast extract, tryptone, agar, sodium chloride, glucose, electrophoresis buffer (TAE), nucleic acid dye (4S Green Plus), kanamycin (Kan), isopropyl-β-D-thiogalactoside (IPTG), agarose, imidazole (C3H4N2), rapid competent cell preparation kit (one-step method), BCA protein concentration assay kit for recombinant protein concentration determination, and nickel ion chelate affinity chromatography packing material were purchased from Sangon Biotech (Shanghai) Co., Ltd. Novozymes Biotechnology Co., Ltd. (Nanjing, China) provided chemical reagents and supplies for protein purification. Agarose gel DNA recovery kit and plasmid extraction kit were purchased from Novozymes (Nanjing) Biotechnology Co., Ltd. All other reagents were purchased from Maclean's Biotech (Shanghai, China) and were all analytical or chromatographic grade.

[0051] (ii) Culture medium All culture media were prepared using ddH2O and sterilized at 121 °C for 15-20 min after preparation.

[0052] LB liquid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0053] LB solid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L.

[0054] (iii) Buffer solution Binding Buffer: 50 mmol / L Tris, 500 mmol / L NaCl, pH 8.0; Washing Buffer: 50 mmol / L Tris, 500 mmol / L NaCl, pH 8.0, 50 mmol / L imidazole; Elution Buffer: 50 mmol / L Tris, 500 mmol / L NaCl, pH 8.0, 500 mmol / L imidazole; Dialysis solution A: 10 mM EDTA·2Na, 50 mM Tris, pH=8.0; Dialysis solution B: 50 mM Tris, pH=8.0.

[0055] Example 1: Design of a single-point mutation site for L-arabinose isomerase Co-evolutionary analysis of the homology sequence of L-arabinose isomerase (LbAI, amino acid sequence shown in SEQ ID NO.1) was performed using the EVcouplings server, identifying a network of key amino acid sites that have been cooperatively conserved over long-term evolution. These sites are crucial for maintaining the enzyme's structure and function. Subsequently, based on the three-dimensional structural model obtained through homology modeling, structural hotspot analysis was performed using the HotSpot tool. By calculating the free energy changes and residue interactions caused by residue mutations, mutation hotspots with potential optimization effects on structural stability and the catalytic microenvironment were further screened. Combined with conformational observations of the substrate-binding pocket, subunit interface, and flexible loop region, the amino acid sites to be mutated were finally determined to be: glutamine at position 258 (Q258), isoleucine at position 297 (I297), lysine at position 339 (K339), asparagine at position 141 (N141), and lysine at position 220 (K220). The predicted mutations at these sites can effectively enhance the enzyme's affinity for substrates / cofactors and improve the thermal stability of the catalytic center.

[0056] Example 2: Single-point site-directed mutagenesis of L-arabinose isomerase and construction of recombinant plasmids and recombinant Escherichia coli. According to SEQ ID NO.2 Levilactobacillus brevis Using the L-arabinose isomerase gene from the source, primers were designed, and site-directed mutagenesis was performed using the parent plasmid pET-22b-LbAI as a template to gradually construct a combined mutant plasmid.

[0057] The designed site-directed mutagenesis primers are as follows (mutation sites are marked with lowercase letters and underscores): Q258A-F: 5'-TTCAGCTG gcg CAGTATCTGGGCATCAAGCGCT-3'; Q258A-R: 5'-GATACTG cgc CAGCTGAACACGCACGCTGTGTT-3'; I297Q-F:5'-CTG cag CGTGACGGTTACGGCTTCGGCGCAGA-3'; I297Q-R: 5'-TAACCGTCACG ctg CAGCAGCTGAGCTGCCAGA-3'; K339H-F: 5'-TGCGT cat GGTCACGAAGCAATCCTGGGCTCT-3'; K339H-R: 5'-TTCGTGACC atgACGCAGGTCCAGAGTGTAATCTTC-3'; N141K-F: 5'-CCCGTCTG aaa GTGAATAACAAAATCGTTTACGGTTAC-3'; N141K-R: 5'-ATTCAC ttt CAGACGGGCGTTAATGTAAGCGT-3'; K220A-F: 5'-AGATTAAC gcg GTGTCTGATGCGGATGTTGACA-3'; K220A-R: 5'-CAGACAC cgc GTTAATCTCTTCGACCAGGTCACC-3'.

[0058] The PCR reaction conditions are shown in Tables 1 and 2.

[0059] Table 1 Reaction System

[0060] Table 2 Reaction conditions

[0061] After PCR amplification, 2 μL of Dpn I restriction endonuclease (10 U / μL) was added to the reaction solution, and the mixture was incubated at 37°C for 2 h to eliminate the template. The PCR product was then transformed into... E. coil In DH5α cells, LB plates were used to spread the cells, and single colonies were picked and transferred to LB liquid medium. Plasmids were extracted and sequenced to obtain the correct single-point mutant plasmids pET-22b(+)-LbAI-Q258A, pET-22b(+)-LbAI-I297Q, pET-22b(+)-LbAI-K339H, pET-22b(+)-LbAI-N141K, and pET-22b(+)-LbAI-K220A.

[0062] Using single-point mutation plasmids as templates, the desired sites were gradually superimposed using sequential site-directed mutagenesis or overlap extension PCR to obtain the following combined mutant plasmids: pET-22b(+)-LbAI-Q258A / I297Q / K339H, pET-22b(+)-LbAI-N141K / I297Q / Q258A, pET-22b(+)-LbAI-K220A / Q258A / I297Q / K339H, and pET-22b(+)-LbAI-K220A / N141K / Q258A / K339H / I297Q.

[0063] All combined mutant plasmids were sequenced to confirm the correct mutation sites and the absence of additional mutations. The above combined mutant plasmids were transformed into E. coli BL21(DE3) competent cells to obtain the following recombinant engineered strains: BL21(DE3) / pET-22b(+)-LbAI-Q258A / I297Q / K339H, BL21(DE3) / pET-22b(+)-LbAI-N141K / I297Q / Q258A, BL21(DE3) / pET-22b-LbAI-K220A / Q258A / I297Q / K339H, and BL21(DE3) / pET-22b(+)-LbAI-K220A / N141K / Q258A / K339H / I297Q.

[0064] Example 3: Induced Expression of Mutants (1) The recombinant engineered bacteria obtained in Example 2 were streaked from glycerol tubes to LB solid medium plates (containing 100 μg / mL Amp) and incubated upside down at 37 °C for 12 h.

[0065] (2) Single colonies were picked from LB solid medium and cultured in 5 mL LB liquid medium containing 100 μg / mL Amp at 37 ℃ and 200 r / min for 12 h to prepare seed liquids.

[0066] (3) The prepared seed culture was transferred to 50 mL LB liquid medium containing 100 μg / mL Amp at an inoculation rate of 2% (v / v). The culture was incubated at 37 °C and 200 r / min until the OD600 value reached 0.6. IPTG was added to bring the final concentration of IPTG in the medium to 0.5 mmol / L. The culture was induced at 18 °C and 200 r / min for 24 h to obtain the fermentation broth. The enzyme activity of the mutant cells was measured by the fermentation broth. The results are shown in Table 3.

[0067] Table 3 Whole-cell enzyme activity of mutants

[0068] (4) Centrifuge the obtained fermentation broth at 4 ℃ and 8000 rpm for 10 min, resuspend the cells in buffer solution, and then sonicate for 15 min (1 s over, 2 s intermittent). After sonication, centrifuge at 4 ℃ and 8000 rpm for 10 min and take the supernatant as crude enzyme solution.

[0069] (5) Protein purification The fermentation broth prepared in step (4) was centrifuged at 4 ℃ and 6000 rpm for 10 min, the supernatant was removed, and the broth was resuspended in an equal volume of 50 mM phosphate buffer (pH=7.0). Then, it was sonicated for 20 min (2 s for sonication, 3 s for rest), and the supernatant was collected by centrifugation. Impurities were removed by passing the supernatant through a 0.45 μm aqueous membrane. The supernatant was then connected to the constant flow pump and Ni in the protein purification system. 2+ For the tubing connecting the affinity chromatography column, UV detector, and other components, set the constant flow pump to 1 mL / min and check for leaks using deionized water. After the protein purification system is ready, equilibrate Ni with two column volumes of binding buffer (50 mM Tris, 500 mM NaCl, pH=7.0). 2+ Affinity chromatography column was used, and the crude protein solution was pumped into the column at a flow rate of 0.5 mL / min. After all the crude protein solution had entered the column, unadsorbed protein and other impurities were washed away with binding buffer. Once the detector reading stabilized, wash buffer (50 mM imidazole, 50 mM Tris, 500 mM NaCl, pH=7.0) was pumped in to wash away weakly binding proteins. Once the detector reading stabilized again, elution buffer (500 mM imidazole, 50 mM Tris, 500 mM NaCl, pH=7.0) was pumped in to elute the adsorbed recombinant protein. The eluent was collected based on the UV detector signal value; this was the target recombinant protein. The target recombinant protein solution obtained above was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa. After clamping with dialysis clamps, the bag was placed in dialysis solution A (10 mM EDTA·2Na, 50 mM Tris, pH=7.0) and in a chromatography cabinet at 4 ℃ for dialysis for 18 h. Fresh dialysis solution was replaced every 6 h to remove imidazole and other metal ions from the target recombinant protein solution. The dialysis bag was then transferred to dialysis solution B (50 mM Tris, pH=7.0) and dialyzed for another 18 h, with fresh dialysis solution replaced every 6 h. After dialysis, the target recombinant protein solution was collected into a tube, which is the pure enzyme solution.

[0070] Example 4: Effects of different temperatures and pH on mutant enzyme activity To investigate the adaptability of the three-point, four-point, and five-point mutants to temperature and pH, the enzyme activity changes under different combinations of reaction temperature and pH were systematically studied using the purified enzyme solutions obtained in Example 3 as materials and an orthogonal experimental design.

[0071] (1) Preparation of buffer solution Prepare buffer solutions at pH 4.0, pH 5.0, and pH 5.5 using: Acetate-Sodium Acetate (HAc-NaAc) buffer (50mM); Phosphate (KH₂PO₄-NaOH) buffer (50mM); Tris-HCl buffer (50mM); and Tris-HCl buffer (50mM); and Tris-HCl buffer (50mM).

[0072] (2) Temperature-pH orthogonal experiment Take an appropriate amount of pure enzyme solution (100 μL), add 100 mM D-galactose and 1 mM Co² + The reaction mixture was added to a 1 mL pH buffer solution. Each reaction tube was placed in a constant temperature water bath at 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, and 80 °C. After reacting for 30 min, the mixture was immediately inactivated by boiling in a water bath for 10 min, centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm aqueous membrane. The amount of D-tagatose produced was determined by HPLC. The highest enzyme activities of wild-type LbAI and mutants Q258A / I297Q / K339H, N141K / I297Q / Q258A, K220A / Q258A / I297Q / K339H, and K220A / N141K / Q258A / K339H / I297Q at different temperatures and pH values ​​were 1.71 U / mg, 4.33 U / mg, 4.62 U / mg, 4.01 U / mg, and 6.34 U / mg, respectively. Taking this highest enzyme activity as 100%, the relative enzyme activity at each condition point was calculated. The optimal reaction temperature and optimal pH for each mutant were extracted from the orthogonal experimental data, and optimal temperature and optimal pH curves were plotted. Figures 2-5 ).

[0073] The results showed that, compared with wild-type LbAI (Topt=60 ℃, optimum pH=6.0), the obtained series of combined mutants exhibited beneficial shifts in both temperature and pH adaptability. Mutant Q258A / I297Q / K339H: Optimal temperature is 60 ℃, and optimal pH is 5.5 (KH2PO4-NaOH). Mutant N141K / I297Q / Q258A: Optimal temperature is 65 ℃, and optimal pH is 5.5 (KH2PO4-NaOH). Mutant K220A / Q258A / I297Q / K339H: Optimal temperature is 60 ℃, and optimal pH is 5.5 (KH2PO4-NaOH). Mutant K220A / N141K / Q258A / K339H / I297Q: Optimal temperature is 60℃, and optimal pH is 6.0 (KH2PO4-NaOH).

[0074] Example 5: Effects of metal ion type and concentration on enzyme activity of L-arabinose isomerase mutant To clarify the regulatory effect of different metal ions on the catalytic activity of the mutants of this invention, and to determine the optimal cofactors and their concentrations, the five-point mutant K220A / N141K / Q258A / K339H / I297Q, which had the highest enzyme activity obtained from the purification of Example 3, was used as a representative. The enzyme activity changes under various metal ion and cobalt ion concentration gradients were systematically evaluated with reference to the optimal reaction temperature and pH determined in Example 4.

[0075] (1) Preparation of mother liquor containing metal ions: Weigh out the contents of K and K respectively. + Na + Mg 2+ Fe 3+ Fe 2+ Ni 2+ Co 2+ Ca 2+ Zn 2+ Cu 2+ NH4 + Mn 2+ The inorganic salts were dissolved and diluted to volume with a pH 8.5 buffer solution to prepare a stock solution with a concentration of 1 mol / L for each metal ion, which was then stored at 4°C for later use.

[0076] (2) Effect of metal ion type on mutant enzyme activity: In a standard reaction system with a total volume of 1 mL (containing 100 mM M-galactose, 50 mM optimal pH buffer, and 100 μL pure enzyme solution), 5 μL of the above-mentioned different metal ion stock solutions were added to make the final concentration of metal ions uniform at 5 mM, so as to compare the activation effect of different metal ions at the same concentration. The reaction group without any metal ions was used as a blank control. The reaction was carried out at the optimal temperature and optimal pH conditions for each mutant for 30 min, and then inactivated by boiling water bath. The D-tagatose yield was determined by HPLC, and the relative enzyme activity was calculated.

[0077] The results showed that different metal ions had significantly different activating or inhibiting effects on the mutant enzyme activity. Among the 12 metal ions tested, the enzyme activity in the control group (without any metal ions) was 1.73 U / mg, and Co... 2+ It exhibited the strongest activation effect, increasing enzyme activity to 2.45 times that of the control group; Fe 2+ Secondly, the relative enzyme activity is 215%; Mn 2+ Ni 2+ Moderate activation was also observed in Ca.2+ Fe 3+ Zn 2+ Co²⁻ significantly inhibits enzyme activity. Therefore, Co²⁻ was chosen. + As the best metal ion cofactor for subsequent applications.

[0078] (3) Effect of cobalt ion concentration on mutant enzyme activity: To further optimize Co 2+ The concentration of added Co was adjusted in the above reaction system by adjusting the concentration of 1 mol / L Co. 2+ The amount of mother liquor added makes Co 2+ Final concentrations were 0, 1, 1.5, 2, 3, 5, 8, 10, and 20 mM. Enzyme activity was measured at the optimum temperature and pH, with the highest enzyme activity defined as 100%. Relative enzyme activities at each concentration were calculated and trend curves were plotted. Figure 6 ).

[0079] The results showed that the mutant enzyme activity increased with Co. 2+ The enzyme activity exhibits a typical activation-inhibition curve, initially increasing and then decreasing with increasing concentration. Within the range of 0–1.5 mM, enzyme activity increases with increasing concentration; when Co… 2+ At a concentration of 1.5 mM, the enzyme activity reached its peak; with further increases in concentration, the enzyme activity gradually decreased due to ion poisoning or steric hindrance. This optimal Co... 2+ At the concentration, the specific enzyme activity of the five-point mutant was lower than that of the unadded Co. 2+ The control group showed a 2.1-fold increase. This example determined Co 2+ The optimal cofactor and concentration for L-arabinose isomerase mutants lay the necessary foundation for the high-activity conversion of D-tagatose.

[0080] Example 6: Thermostability of L-arabinose isomerase mutant To evaluate the thermal stability of the obtained combined mutants under practical application conditions, the purified wild-type LbAI obtained in Example 3 and the pure enzyme solutions of each mutant (Q258A / I297Q / K339H, N141K / I297Q / Q258A, K220A / Q258A / I297Q / K339H, K220A / N141K / Q258A / K339H / I297Q) were placed in constant temperature water baths at 40 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, and 70 ℃ for a total incubation time of 8 h. Samples were taken at regular intervals (0 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h), and immediately cooled to 4 ℃ in an ice bath after sampling to terminate the thermal denaturation process.

[0081] Subsequently, referring to the optimal reaction temperature and pH conditions for each enzyme determined in Example 4, and the metal ion concentration determined in Example 5, in a standard reaction system (100 mM D-galactose, 1 mM Co²⁻¹), + 100 μL of the incubated enzyme solution was added to a 50 mM optimal pH buffer (total volume 1 mL), and reacted at the optimal temperature for 0.5 h. The enzyme was then inactivated by boiling in a water bath for 10 min, centrifuged at 12000 rpm for 5 min, and the supernatant was passed through a 0.22 μm aqueous membrane for HPLC analysis to calculate residual enzyme activity. The enzyme activity measured under the same conditions after 0 h of incubation (i.e., no incubation) was taken as 100%. The relative residual enzyme activity (%) at each time point was calculated using the following formula: Residual enzyme activity (%) = (Enzyme activity after 0 h of incubation / Enzyme activity after 0 h of incubation) × 100%.

[0082] Based on the measurement results, a thermal deactivation curve was plotted. Figure 7 The results showed that the series of combined mutants obtained in this invention exhibited superior thermal stability within the tested temperature range: at temperatures below or equal to 50 °C, the relative enzyme activity remained above 95% after 8 hours of incubation, demonstrating excellent thermal stability at low temperatures; at 55 °C, the enzyme activity decreased slowly with prolonged incubation time, with approximately 71% residual enzyme activity after 8 hours; at 60 °C, the relative enzyme activity of the five-point mutant was 53% of the initial enzyme activity after 0.5 hours of incubation. At 65 °C and 70 °C, the enzyme inactivation rate accelerated significantly, and the enzyme activity was essentially lost after 1 hour of incubation.

[0083] Comparative example: Following the same strategy as in Examples 1-6, conserved sites K269, E58, and E438 were screened, and mutants K269R, E58D, and E438A were constructed, along with plasmids pET-22b(+)-LbAI-K269R, pET-22b(+)-LbAI-E58D, and pET-22b(+)-LbAI-E438A. Recombinant bacteria BL21(DE3) / pET-22b(+)-LbAI-K269R, BL21(DE3) / pET-22b(+)-LbAI-E58D, and BL21(DE3) / pET-22b(+)-LbAI-E438A were also constructed. The recombinant bacteria were cultured using the same method as in Example 3, and whole-cell enzyme activities were detected. The results showed that the whole-cell enzyme activities of K269R, E58D, and E438A were 0.08, respectively. The levels of K269, E58, and E438 decreased to 54%, 53%, and 7% of the wild type, respectively, indicating that K269, E58, and E438 are important residues that enable the enzyme to catalyze. After mutation, the enzyme activity was not improved but was lost.

[0084] The primers involved in this site-directed mutagenesis example are: K269R-F: 5'-TTCTGGAA cga GGTGGTTATACCGCCTTCACTA-3'; K269R-R: 5'-AACCACC tcg TTCCAGAAAGCGCTTGATGCCCA-3'; E58D -F: 5'-C gac TCCATCACCAACTTCATGAAGGAAGTGA-3'; E58D -R: 5'-AGTTGGTGATGGA GTC GGCGGTGGTCATCACA-3'; E438A -F: 5'-CCCTG gca TGGATGAAAGCCGGTGGTGGTCAC-3'; E438A-R: 5'-TTTCATCCA tgc CAGGGCACCTTTTTTCAGAC-3'.

[0085] The lowercase part represents the codons corresponding to lysine at position 269, glutamic acid at position 58, and glutamic acid at position 437 encoded by the mutant gene.

[0086] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An L-arabinose isomerase mutant, characterized in that, Using L-arabinose isomerase with the amino acid sequence shown in SEQ ID NO.1 as the parent, perform any of the following mutations: (1) mutate lysine at position 339 to histidine; (2) mutate asparagine at position 141 to lysine; (3) mutate lysine at position 220 to alanine; (4) mutate lysine at position 339 to histidine, isoleucine at position 297 to glutamine, and glutamine at position 258 to alanine; (5) mutate asparagine at position 141 to lysine and isoleucine at position 297 to glutamine. (6) Lysine at position 220 is mutated to alanine, glutamine at position 258 is mutated to alanine, isoleucine at position 297 is mutated to glutamine, and lysine at position 339 is mutated to histidine; (7) Lysine at position 220 is mutated to alanine, asparagine at position 141 is mutated to lysine, glutamine at position 258 is mutated to alanine, lysine at position 339 is mutated to histidine, and isoleucine at position 297 is mutated to glutamine.

2. The gene encoding the mutant of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The recombinant plasmid was expressed using pET-22b plasmid as the expression vector.

5. A recombinant microorganism expressing the mutant of claim 1, or containing the gene of claim 2, or transformed with the recombinant plasmid of any one of claims 3 to 4.

6. The recombinant microorganism according to claim 5, characterized in that, The recombinant microorganisms use Escherichia coli or Bacillus subtilis as host cells.

7. The recombinant microorganism according to claim 5, characterized in that, The recombinant microorganism uses Escherichia coli BL21(DE3) as the host cell and pET-22b(+) as the vector.

8. A catalyst for the synthesis of tagatose, characterized in that, The catalyst contains the mutant as described in claim 1 or the recombinant microorganism as described in any one of claims 5 to 7.

9. A method for improving the activity and / or thermal stability of L-arabinose isomerase, characterized in that, The method involves performing one of the following mutations on the L-arabinose isomerase parent with the amino acid sequence shown in SEQ ID NO.1: (1) The lysine at position 339 is mutated to histidine; (2) Asparagine at position 141 is mutated to lysine; (3) The lysine at position 220 is mutated to alanine; (4) Lysine at position 339 was mutated to histidine, isoleucine at position 297 was mutated to glutamine, and glutamine at position 258 was mutated to alanine; (5) Asparagine at position 141 was mutated to lysine, isoleucine at position 297 was mutated to glutamine, and glutamine at position 258 was mutated to alanine; (6) Lysine at position 220 was mutated to alanine, glutamine at position 258 was mutated to alanine, isoleucine at position 297 was mutated to glutamine, and lysine at position 339 was mutated to histidine; (7) Lysine at position 220 was mutated to alanine, asparagine at position 141 was mutated to lysine, glutamine at position 258 was mutated to alanine, lysine at position 339 was mutated to histidine, and isoleucine at position 297 was mutated to glutamine.

10. The use of the mutant of claim 1, or the gene of claim 2, or the recombinant plasmid of any one of claims 3 to 4, or the recombinant microorganism of any one of claims 5 to 7, or the catalyst of claim 8 in the catalytic synthesis of tagatose from D-galactose.

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