L-arabinose isomerase mutant and use thereof in preparing d-tagatose

Abstract

This invention discloses an L-arabinose isomerase mutant and its application in the preparation of D-tagatose. The invention screened and obtained mutants with improved enzyme activity, thermostability, and substrate affinity. Compared to the wild-type enzyme, which requires 25 hours to reach reaction equilibrium, the mutant TsAI / F276A reaches equilibrium in only 6 hours at 50°C. This mutant not only improves enzyme activity but also maintains its thermostability (Tm value) without a significant decrease compared to the wild-type enzyme, and enhances its affinity for D-galactose. It is suitable for production applications with high substrate concentrations. This invention addresses the problem of low wild-type enzyme activity requiring a long time to reach reaction equilibrium through mutation, expands the relatively limited enzyme library, and demonstrates the technological advantages of D-galactose in the synthesis of D-tagatose invertase: it is environmentally friendly, has low toxicity, and produces few byproducts. It overcomes the waste problems associated with chemical synthesis methods and has significant industrial application prospects.

Description

(I) Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a novel L-arabinose isomerase mutant and its application in the preparation of D-tagatose. (II) Background Technology

[0002] D-Tagsulfate is a natural hexose, an epimer of fructose at the C-4 position and D-sorbose at the C-3 position, and also an isomer of D-galactose. In 2000, D-Tagsulfate was recognized as a Generally Recognized As Safe (GRAS) low-calorie sweetener by the U.S. Food and Drug Administration (FDA) and is widely used in non-chronic medicines, toothpaste, mouthwash, cosmetics, and veterinary drugs. D-Tagsulfate has a taste similar to sucrose, with 92% of the sweetness of sucrose and only one-third the calories, making it a hot topic in natural sweetener research in recent years. In addition to its sweetness, D-Tagsulfate possesses several unique physiological functions, such as preventing obesity, combating oxidative cell damage, lowering blood sugar, improving gut microbiota, preventing tooth decay, and alleviating gingivitis. Therefore, it has attracted significant attention not only in the food industry but also in the field of health management.

[0003] Currently, the biological method for preparing D-tagatose using L-arabinose isomerase (L-AI) is considered the most efficient. L-arabinose isomerase catalyzes the isomerization of L-arabinose to L-ribulose, and due to the structural similarity between L-arabinose and D-galactose, it also exhibits some activity in catalyzing the conversion of D-galactose to D-tagatose. However, existing L-arabinose isomerases suffer from low catalytic activity and poor temperature stability when catalyzing the conversion of D-galactose to D-tagatose, making them unsuitable for industrial production of D-tagatose.

[0004] With the increasing market demand for D-tagatose, the development of efficient biocatalysts has become crucial for its industrial applications. To meet the needs of industrial production, many studies have used protein engineering techniques to modify the molecular activity of different types of L-arabinose isomerases. Meanwhile, computer-aided methods for designing enzyme active molecules are also constantly evolving, which is of great significance for meeting the growing demand for carbohydrates among the population.

[0005] Therefore, developing L-arabinose isomerases with high catalytic activity is of great significance for improving the production efficiency and industrial application of D-tagatose. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a novel L-arabinose isomerase mutant and its application in the preparation of D-tagatose, in order to solve the technical problem that the activity of L-arabinose isomerase is too low in industrial production and is not suitable for industrial production.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides an L-arabinose isomerase mutant, which is obtained by single or multiple mutations of amino acids at positions 19, 186, and 276 of the amino acid sequence shown in SEQ ID NO.6.

[0009] Furthermore, the L-arabinose isomerase mutant is a mutant that mutates the amino acid sequence shown in SEQ ID NO.6 to one of the following: (1) L19A, i.e., leucine at position 19 is mutated to alanine; (2) M186A, i.e. methionine at position 186 is mutated to alanine; (3) F276A, i.e. phenylalanine at position 276 is mutated to alanine, with the nucleotide sequence being SEQ ID NO.7 and the amino acid sequence being SEQ ID NO.8; (4) a combination of the above three single mutations.

[0010] Due to the specificity of amino acid sequences, any variant of the aforementioned mutant amino acid sequence, such as its conserved variants, bioactive fragments, or derivatives, is within the scope of protection of this invention, provided that the fragment or variant of the polypeptide shares more than 95% homology with the aforementioned amino acid sequence. The alterations may include the deletion, insertion, or substitution of amino acids in the amino acid sequence; for conserved alterations in variants, the substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine; variants may also have non-conserved alterations, such as replacing glycine with tryptophan.

[0011] The present invention also provides a coding gene for the L-arabinose isomerase mutant, preferably the nucleotide sequence of the coding gene is shown in SEQ ID NO.7.

[0012] Due to the specific nature of nucleotide sequences, any polynucleotide variant encoding the aforementioned gene, provided it shares more than 95% homology with the polynucleotide, falls within the scope of protection of this invention. The polynucleotide variant refers to a polynucleotide sequence with one or more nucleotide alterations. This polynucleotide variant can be a live or non-live allelic variant, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may involve the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the encoded amino acid.

[0013] This invention also relates to a recombinant vector containing the L-arabinose isomerase mutant encoding gene, and recombinant genetically engineered bacteria constructed from the recombinant vector. The recombinant vector is based on pET-28a(+), and the recombinant genetically engineered bacteria are based on... E. coli BL21(DE3) is the host bacterium. A recombinant vector containing the L-arabinose isomerase gene was constructed, and the recombinant vector was transformed into Escherichia coli. The resulting recombinant genetically engineered bacteria were induced and cultured, and bacterial cells containing L-arabinose isomerase were isolated from the culture medium. These cells can then be used as an enzyme source for the microbial catalysis of D-galactose isomerization to prepare D-tagatose.

[0014] This invention also relates to the application of the L-arabinose isomerase mutant in the microbial catalytic isomerization of D-galactose to prepare D-tagatose. The application is as follows: using wet bacterial cells obtained by fermentation culture of recombinant bacteria containing the L-arabinose isomerase mutant encoding gene or pure enzyme solution obtained by ultrasonic disruption and purification of wet bacteria as the enzyme source, D-galactose as the substrate, metal ions as the co-catalyst, and a buffer solution with pH 4.0-10.0 (preferably pH 7.0, 50mM KH2PO4-NaOH buffer) as the reaction medium to form a reaction system, the reaction is carried out at 35-65℃ (preferably 50-60℃) and 1000r / min. After the reaction is complete, a mixture of D-galactose and D-tagatose is obtained.

[0015] Furthermore, the metal ions include Mn 2+ Co 2+ Zn 2+ Ni 2+ Preferred Mn 2+ The manganese ions are added in the form of MnCl2.

[0016] Furthermore, in the reaction system, the final concentration of wet bacterial cells is 10-50 g / L (preferably 10 g / L), the final concentration of pure enzyme solution (based on protein content) is 0.01-0.5 mg / mL (preferably 0.3 mg / mL), the final concentration of metal ions is 0.1-10 mM (preferably 1 mM), and the initial final concentration of substrate is 20-180 g / L (preferably 100 g / L).

[0017] Furthermore, the wet bacterial cells were prepared as follows: recombinant bacteria containing the L-arabinose isomerase mutant encoding gene were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 200 r / min until OD200. 600 The seed culture was obtained by setting the inoculum size to 0.8–1.0. The seed culture was then transferred at a volume concentration of 1–5% into LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37°C and 180–200 rpm until the OD value reached 0.8–1.0. 600Add IPTG to a final concentration of 0.1 mM at a concentration of 0.4-0.6, and induce culture at 28℃ and 200 r / min for 12-20 h. Centrifuge (4℃, 8000 r / min for 10 min), wash the wet bacterial cells twice with 0.85% physiological saline, and collect the wet bacterial cells.

[0018] Further, the pure enzyme solution was prepared as follows: Wet bacterial cells were suspended in 50 mM KH₂PO₄-NaOH (pH 7.0) buffer (preferably 10 mL / g), sonicated at 120 W for 20 min, sonicated for 1 s, paused for 2 s, and centrifuged at 4 °C and 8000 r / min for 10 min. The supernatant was collected to obtain the crude enzyme solution. The crude enzyme solution was purified using a Nickel-NTA affinity chromatography column (Bio-Scale Mini Profinity IMAC pre-packed column, 40 mm long × 12.6 mm inner diameter). The column was first equilibrated with equilibration buffer (50 mM KH₂PO₄-NaOH buffer, 300 mM NaCl, 20 mM imidazole, pH 8.0). The crude enzyme solution was loaded at a rate of 1 mL / min for 4 column volumes, and then eluted with elution buffer (50 mM KH₂PO₄-NaOH buffer, 300 mM NaCl, 500 mM imidazole, pH 8.0). 8.0) Elution was performed at a rate of 2 mL / min, monitored by a UV detector and a conductivity detector. When the signals of the UV detector and the conductivity detector both increased simultaneously, the corresponding eluent was collected. When the conductivity detector signal remained unchanged and the UV detector signal decreased, the collection was stopped. The collected eluent was dialyzed overnight in deionized water (pH 7.0) at 4°C. The molecular weight cutoff of the dialysis bag was 10 kDa. The cutoff fluid was collected to obtain the pure enzyme solution.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0020] (1) This invention first screened wild-type arabinose isomerases and identified a novel L-arabinose isomerase that achieved a 50% conversion efficiency for D-galactose at reaction equilibrium. Then, based on the wild-type, further screening was conducted through mutation to obtain mutants with improved enzyme activity, thermostability, and substrate affinity. Compared to the wild-type enzyme which required 25 hours to reach reaction equilibrium, the mutant TsAI / F276A reached equilibrium in just 6 hours at 50°C. This mutant not only improved enzyme activity but also showed no significant decrease in thermostability (Tm value) compared to the wild-type enzyme.

[0021] (2) Compared with the wild type, the mutant of the present invention has significantly improved tolerance to the substrate D-galactose. The conversion rate did not decrease significantly with the increase of substrate concentration within the range of 20-100 g / L, which improved the affinity for D-galactose and is suitable for production applications with high substrate concentration.

[0022] (3) This invention improves the problem of low wild enzyme activity requiring a long time to reach reaction equilibrium by mutation, expands the relatively scarce enzyme library, and demonstrates the technical advantages of D-galactose in the synthesis of D-tagatose invertase, which is green, environmentally friendly, low in toxicity and has few by-products. It overcomes the problem of easy generation of three wastes by chemical synthesis method and has important industrial application prospects. (iv) Description of the attached drawings

[0023] Figure 1 This is a bar graph showing the relative enzyme activity of wild-type enzyme TsAI under the action of different metal ions.

[0024] Figure 2 The graph shows the relative enzyme activity curves of wild-type TsAI and mutants TsAI / L19A, TsAI / M186A and TsAI / F276A at different reaction times.

[0025] Figure 3 The image shows the SDS-PAGE electrophoresis result of the pure enzyme solution in Example 7; Lane M: Marker, Lane 1: TsAI pure enzyme solution, Lane 2: TsAI / L19A pure enzyme solution, Lane 3: TsAI / M186A pure enzyme solution, Lane 4: TsAI / F276A pure enzyme solution.

[0026] Figure 4 The Tm detection diagram shows the pure enzymes of wild-type TsAI and mutants TsAI / L19A, TsAI / M186A and TsAI / F276A.

[0027] Figure 5 The bar chart shows the relative enzyme activity of the mutant TsAI / F276A at different temperatures.

[0028] Figure 6 The bar chart shows the relative enzyme activities of wild-type TsAI and mutant TsAI / F276A at different substrate concentrations.

[0029] Figure 7 The graph shows the relative enzyme activity curves of the mutant TsAI / F276A at different pH values. (V) Detailed Implementation Methods

[0030] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0031] Example 1: Screening and activity determination of a novel L-arabinose isomerase L-AI

[0032] 1. Screening of wild-type enzymes and construction of recombinant expression plasmids

[0033] Novel L-AIs were screened from the NCBI database, respectively from... Capillibacterium thermochitinicola (GenBank ID WP_181339886.1) Marispirochaeta aestuarii (GenBank ID WP_083051604.1) Paenibacillus terrae (GenBank ID WP_149094981.1) Terribacillus saccharophilus (GenBank ID WP_095235122.1) Gracilinema caldarium (GenBank accession number WP_304240302.1), and named CtAI, MaAI, PtAI, TsAI, and GcAI. Based on the amino acid sequence, codon optimization was performed according to the codon preference of *E. coli*. Five nucleotide sequences were synthesized using conventional genetic engineering methods, as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. A 6×His-tag was added to the end of the nucleic acid sequence, and the gene was cloned into the multiple cloning site (MCS) corresponding to pET28a(+), obtaining the recombinant expression plasmids pET28a / CtAI, pET28a / MaAI, pET28a / PtAI, pET28a / TsAI, and pET28a / GcAI.

[0034] 2. Transformation and Induction Expression of Recombinant Bacteria

[0035] The obtained recombinant expression plasmids pET28a / CtAI, pET28a / MaAI, pET28a / PtAI, pET28a / TsAI, and pET28a / GcAI were transformed into [a specific gene / particle]. Escherichia coli BL21(DE3) recipient bacteria were plated on LB solid medium plates containing a final concentration of 50 μg / mL kanamycin and cultured overnight at 37°C. Then, clones were randomly selected from the colonies that grew on the plates, and plasmids were extracted for identification by agarose gel electrophoresis to obtain recombinant genetically engineered bacteria containing the target gene.

[0036] LB liquid medium composition: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, water as solvent, natural pH; LB solid medium is LB liquid medium with 15 g / L agar added; autoclave at 121℃ for 20 min; add kanamycin to a final concentration of 50 μg / mL before use.

[0037] The recombinant genetically engineered bacteria were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 200 r / min until OD500 was reached. 600 Seed culture was obtained by setting the concentration to 0.8–1.0. The seed culture was then inoculated at a volume concentration of 1% (v / v) into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm until the OD reached a certain level. 600 The concentration was set at 0.4–0.6. IPTG was then added to the culture medium to a final concentration of 0.1 mM. After inducing expression at 28 °C and 200 r / min for 12 h, the cells were centrifuged at 4 °C and 8000 r / min for 10 min. The supernatant was discarded, and the wet cells were washed twice with 0.85% physiological saline and collected for later use.

[0038] 3. Enzyme activity assay of recombinant bacteria

[0039] The final concentration composition of the enzyme activity assay reaction system was: 20 g / L D-galactose, 1 mM MnCl2, 10 g / L wet bacterial cells, and then an appropriate amount of 50 mM KH2PO4-NaOH buffer (pH 7.0) was added to a total volume of 1 mL. Reaction conditions: The reaction was carried out at 50℃ in a metal bath at 1000 rpm for 5 h, and then terminated by boiling in water for 10 min. After diluting 10 times with deionized water, the solution was filtered through a 0.22 μm filter membrane. The concentrations of D-galactose and D-tagatose in the filtrate were determined by HPLC.

[0040] HPLC detection conditions: Agilent 1260 HPLC system, Agilent autosampler, Sugar-Park column, Agilent differential detector, ultrapure water as mobile phase, column temperature set at 80℃, flow rate at 0.5 mL / min, external standard method, the yield of D-tagatose was determined based on peak retention time and peak area. The retention time of D-galactose was 9.9 min, and the retention time of D-tagatose was 12.7 min.

[0041] Enzyme activity unit definition: The amount of enzyme required to isomerize D-galactose to produce 1 μmol of D-tagatose per minute at 50℃ and pH 7.0 is defined as one enzyme activity unit (U). The specific enzyme activity of L-arabinose isomerase is expressed as enzyme activity units per gram of wet cell weight (U / g·wet cell weight).

[0042] The specific enzyme activities of five recombinant bacteria from different sources catalyzing the synthesis of D-tagatose from D-galactose are shown in Table 1. One strain with higher enzyme activity was selected. Terribacillus saccharophilus The TsAI source exhibited the highest enzyme activity, reaching 124.8 U / (g·wet cell weight). The amino acid sequence of TsAI is shown in SEQ ID NO.6.

[0043] Table 1. Enzyme activity assay of wild-type L-AI recombinase

[0044]

[0045] SEQ ID NO.6

[0046] PMLQVKPYVFWFVTGSQHLYGEETLNQVQGNSEDLVNKLNEQGTLPPFIAFKEVLTNAADIQRVSLEANADPECAGLITWMHTFSPAKMWIGGLKTLQKPLLHLHTQYNRDVPWDTIDMDFM NLNQSAHGDREYGFMGTRLNKARKVIVGYWGNKEIQNRVADWMTTAVGFNESQHIKVARFGDNMRTVAVTDGDKVEAQIKFGWTVDYYGIGDLVAEMKEVSESDVDELVSTYKEAYDLPTEED QLASVREQARIGVALKRFLDRGGYQAFSTNFEDLHGMKQLPGLAVQHLMAQGYGFAGEGDWRTAALVRLLKAMANNDRTSFMEDYTYHLEEGNELILGSHMLEVCPTVAANQPSIQVHPLGIG GKEDPARIVFDGIAGEAVNVSIIELGGRFRMIINKVDAVESEKETPNLPVAKVLWKPQPSLSEATEAWIYAGGAHHTALSFALTAEQLEDFAELVGIECVTIDNNTVLKQFRKELQWNQVVWK

[0047] Example 2: Effect of metal ions on TsAI enzyme activity

[0048] The TsAI wet cells from Example 1 were used as the transformation catalyst to determine the effect of metal ions on the recombinase activity. The final composition of the 1 mL reaction system was: 50 mM KH₂PO₄-NaOH buffer (pH 7.0), 20 g / L D-galactose, 10 g / L wet cells, and 1 mM metal ions. The selected metal ion was Mn. 2+ Co 2+ Mg 2+Cu 2+ Zn 2+ Fe 2+ Fe 3+ Ni 2+ and Ca 2+ The activity of TsAI was determined at 50°C using the method described in Example 1. A control without added metal ions was used (100% enzyme activity). Figure 1 It can be seen that Mn 2+ With Co 2+ Mn has a significant promoting effect on the enzyme activity of TsAI, and is chosen for cost considerations. 2+ Auxiliary ions as reaction conditions.

[0049] Example 3: Construction of TsAI unit point mutant

[0050] 1. Design of mutation sites and primers

[0051] A three-dimensional structural model of TsAI was constructed using the AlphaFold3 online website, and molecular docking with the substrate D-galactose was performed using AutoDock software to predict the binding mode and affinity between the substrate and the enzyme's active site. Three key sites on the active pocket were identified: leucine at position 19, methionine at position 186, and phenylalanine at position 276. Activity analysis of these three key sites revealed that replacing each site with alanine significantly increased the rate at which the substrate entered the active pocket, thereby enhancing enzyme activity.

[0052] Primers for site-directed mutagenesis were designed based on the parental TsAI sequence and mutation sites. Using rapid PCR technology and the recombinant vector pET28a / TsAI as a template, single mutations were introduced at positions 19, 186, and 276. The primers are as follows:

[0053] Forward primer L19A: CTCAGCAT GCG TATGGTGAAGAAACCCTGAACC (underlined bases are mutant bases)

[0054] Reverse primer L19A: CACCATA CGC ATGCTGAGAGCCGGTTAC (Underlined bases are mutant bases)

[0055] Forward primer M186A: GACAAC GCG CGTACCGTTGCTGTTACC (underlined bases are mutant bases)

[0056] Reverse primer M186A:CGGTA CGC GCGTTGTCGCCGAAACGAG (underlined bases are mutant bases).

[0057] Forward primer F276A: CTACTAAC GCC GAGGATCTGCATGGTATGAAAC (underlined bases are mutant bases)

[0058] Reverse primer F276A:GATCCTC GGC GTTAGTAGAGAACGCCTGGTAACC (underlined bases are mutant bases).

[0059] 2. The PCR reaction system (50 μL) is as follows: 2×Phanta Max Buffer 25 μL, dNTPs 1 μL, forward primer 2 μL (5 pmol / μL), reverse primer 2 μL (5 pmol / μL), template DNA 1 μL (20 ng / μL), Phanta Max Super-Fidelity DNA Polymerase 1 μL, and ddH2O added to bring the total volume to 50 μL.

[0060] 3. The PCR reaction program is as follows: 95℃ pre-denaturation for 5 min; 30 cycles (95℃ denaturation for 10 s, 60-70℃ annealing for 15 s, 72℃ extension for 3.3 min); 72℃ extension for 10 min, and finally incubation at 16℃. The PCR products are verified by 0.9% agarose gel electrophoresis. When the amplified fragment matches the size of the target vector, the next step of the experiment can be carried out.

[0061] 4. After digesting the template with DpnI at 37℃ for 3 hours, take 3 μL of the PCR product and add it to 100 μL of water incubated on ice. E. coli In a suspension of BL21(DE3) competent cells, the cells were incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then rapidly cooled on ice for 5 min. 600 μL of LB liquid medium was added, and the cells were incubated at 37°C and 200 rpm for 50 min, followed by centrifugation at 12000 rpm for 1 min. 400 μL of supernatant was discarded, and the bacterial pellet was resuspended. 100 μL of the resuspended cell pellet was plated onto LB agar plates containing 50 μg / mL kanamycin-resistant material and incubated upside down at 37°C for 12 h. A single colony was picked and inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin-resistant material and incubated at 37°C for 12 h to obtain the bacterial culture.

[0062] 5. The bacterial culture from step 4 was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were correctly matched, yielding a unit point mutant recombinant bacterium. E. coli BL21(DE3) / pET28a / TsAI / L19A, E. coli BL21(DE3) / pET28a / TsAI / M186A and E. coliBL21(DE3) / pET28a / TsAI / F276A, the corresponding enzymes are denoted as mutant enzymes TsAI / L19A, TsAI / M186A and TsAI / F276A (nucleotide sequences are shown in SEQ ID NO.7, amino acid sequences are shown in SEQ ID NO.8).

[0063] Wet bacterial cells were prepared using the method in Example 1 and enzyme activity was measured. The results are shown in Table 2.

[0064] Example 4: Construction of TsAI two-site mutants

[0065] Using the recombinant vector pET28a / TsAI / L19A from Example 3 as a template, a single mutation was introduced at position 186 using primers from Example 3 (forward and reverse M186A) and at position 276 using primers from Example 3 (forward and reverse F276A). Using the recombinant vector pET28a / TsAI / M186A from Example 3 as a template, a single mutation was introduced at position 276 using primers from Example 3 (forward and reverse F276A). Other operations were the same as in Example 3, resulting in a two-site mutant recombinant bacterium. E. coli BL21(DE3) / pET28a / TsAI / L19A / M186A, E. coli BL21(DE3) / pET28a / TsAI / L19A / F276A and E. coli BL21(DE3) / pET28a / TsAI / M186A / F276A, the corresponding enzymes are denoted as mutant enzymes TsAI / L19A / M186A, TsAI / L19A / F276A and TsAI / M186A / F276A.

[0066] Wet bacterial cells were prepared using the method in Example 1 and enzyme activity was measured. The results are shown in Table 2.

[0067] Example 5: Construction of TsAI three-point mutant

[0068] Using the recombinant vector pET28a / TsAI / L19A / M186A from Example 4 as a template, a single mutation was introduced at position 276 using primers from Example 3 (forward and reverse F276A). Other procedures were the same as in Example 3, resulting in a three-point mutant recombinant bacterium. E. coli BL21(DE3) / pET28a / TsAI / L19A / M186 / F276A, the corresponding enzyme is denoted as the mutant enzyme TsAI / L19A / M186A / F276A.

[0069] Wet bacterial cells were prepared using the method in Example 1 and enzyme activity was measured. The results are shown in Table 2.

[0070] Table 2. Enzyme activity assay of TsAI mutant recombinant bacteria

[0071]

[0072] Example 6: Reaction process of wild-type L-arabinoside TsAI and its mutants TsAI / L19A, TsAI / M186A and TsAI / F276A

[0073] The wild-type enzyme TsAI constructed in Examples 1 and 3, and the mutants TsAI / L19A, TsAI / M186A, and TsAI / F276A, were used as catalysts for transformation by preparing wet cells according to the method in Example 1.

[0074] The final concentration composition of the 1 mL reaction system was: 50 mM KH2PO4-NaOH buffer (pH 7.0), 100 g / L D-galactose, 50 g / L wet bacterial cells, and 1 mM MnCl2, totaling 1 mL. Reaction conditions: 50℃ metal bath, 1000 rpm for 25 h. Samples were taken at 10 min, 1 h, 3 h, 6 h, 9 h, 12 h, and 25 h. The reaction was terminated by boiling in a water bath for 10 min, centrifuged at 12000 rpm, and the supernatant was diluted 10-fold with deionized water and filtered through a 0.22 μm water membrane. The concentrations of D-galactose and D-tagatose in the filtrate were determined using the method described in Example 1, and the residual enzyme activity was calculated. The results are shown in [Figure 1]. Figure 2 TsAI, TsAI / L19A, TsAI / M186A, and TsAI / F276A were compared with TsAI at its highest conversion rate of 100%. The results showed that, unlike the wild-type enzyme which required 25 hours to reach equilibrium, the mutant TsAI / F276A reached equilibrium in only 6 hours at 50℃. While TsAI / L9A and TsAI / M186A took less time to reach equilibrium than the wild-type, they still required 13 hours. This indicates that the enzyme activity of the mutant TsAI / F276A was significantly enhanced.

[0075] Conversion rate is defined as the ratio of the product concentration to the residual substrate concentration in the system after the reaction is complete and terminated. The formula is as follows:

[0076] Conversion rate = C 产物 / (C 产物 +C 底物 )

[0077] Example 7: Determination of Tm values ​​for TsAI and its mutants TsAI / L19A, TsAI / M186A, and TsAI / F276A

[0078] 1. Preparation of pure enzyme solution

[0079] (1) The wild-type enzymes TsAI, TsAI / L19A, TsAI / M186A and TsAI / F276A constructed in Examples 1 and 3 were prepared into wet cells according to the method in Example 1.

[0080] (2) 1g of wet bacterial cells were suspended in 10mL of 50mM KH2PO4-NaOH buffer (pH 7.0), and the cells were sonicated at 120W for 20min, sonicated for 1s, paused for 2s, centrifuged at 4℃ and 8000r / min for 10min, and the supernatant was collected to obtain crude enzyme solution.

[0081] (3) The crude enzyme solution was purified using a Nickel-NTA affinity chromatography column (Bio-Scale MiniProfinity IMAC pre-packed column, 40 mm long × 12.6 mm inner diameter). The column was first equilibrated with equilibration buffer (50 mM KH2PO4-NaOH buffer, 300 mM NaCl, 20 mM imidazole, pH 8.0). The crude enzyme solution was loaded at a rate of 1 mL / min for 4 column volumes. Elution was then performed with elution buffer (50 mM KH2PO4-NaOH buffer, 300 mM NaCl, 500 mM imidazole, pH 8.0) at a rate of 2 mL / min. Ultraviolet (UV) and conductivity (C) detectors were used for monitoring. When the signals of both UV and C rise simultaneously, the corresponding eluent was collected. When the C signal remained unchanged and the UV signal decreased, collection was stopped. The collected eluent was then rinsed in deionized water (pH 7.0). The enzyme was dialyzed overnight at 4°C. The molecular weight cutoff of the dialysis bag was 10 kDa. The retentate was used as the pure enzyme solution. The protein concentration of each pure enzyme solution was tested using a protein quantification kit (BCA method).

[0082] Take 15 μL of the purified enzyme solution after dialysis, add 5 μL of 4×SDS buffer, mix, heat in a boiling water bath for 15 min, and take 8 μL for SDS-PAGE electrophoresis analysis. Figure 3 The expression of the target protein is considered successful if a protein band with a molecular weight of approximately 59 kDa is obtained.

[0083] 2. Tm value determination

[0084] The obtained pure enzyme solution was diluted with 10mM KH2PO4-NaOH buffer (pH 7.0) to a protein concentration of 0.1-0.2 g / L. The protein denaturation temperature (Tm) was then determined using circular dichroism spectroscopy. The results are as follows: Figure 4As shown in Table 2, the Tm value of wild-type TsAI is 52.3℃, while the Tm values ​​of mutants TsAI / L19A, TsAI / M186A, and TsAI / F276A are 52.5℃, 47.9℃, and 52.4℃, respectively. According to Table 2, although the enzyme activities of mutants TsAI / L19A, TsAI / M186A, and TsAI / F276A are all higher than those of wild-type TsAI, the mutant TsAI / F276A shows the greatest increase in enzyme activity without a decrease in its Tm value.

[0085] Example 8: Optimal temperature of mutant TsAI / F276A

[0086] The wet bacterial cells prepared by the mutant enzyme TsAI / F276A constructed in Example 3 according to the method in Example 1 were used as recombinant bacteria for transformation.

[0087] The final concentration composition of the 1 mL reaction system was: 50 mM KH2PO4-NaOH buffer (pH 7.0), 10 g / L D-galactose, 10 g / L wet bacterial cells, and 1 mM MnCl2, totaling 1 mL. Reaction conditions: Reacted at 1000 rpm for 5 h in a metal bath at 35-65℃ (35, 40, 45, 50℃, 55, 60, 65℃), the reaction was terminated by boiling in a water bath for 10 min, centrifuged at 12000 rpm, the supernatant was diluted 4 times with deionized water, and filtered through a 0.22 μm water membrane. The concentrations of D-galactose and D-tagatose in the filtrate were determined using the method described in Example 1. The relative enzyme activity at other temperatures was calculated with the highest enzyme activity as 100%. The results are shown in [Figure 1]. Figure 5 The optimal reaction temperature for the mutant TsAI / F276A is 50℃-60℃, indicating that the mutant TsAI / F276A is suitable for industrial production applications.

[0088] Example 9: Tolerance of mutant TsAI / F276A to different substrate concentrations

[0089] The wet bacterial cells prepared by recombinant bacteria TsAI and mutant TsAI / F276A according to the method in Example 1 were used as recombinant bacteria for transformation.

[0090] The final concentration composition of the 1 mL reaction system was: 50 mM KH2PO4-NaOH buffer (pH 7.0), 10 g / L wet bacterial cells, and a final concentration of 1 mM MnCl2. Substrate D-galactose was prepared with final concentrations of 20 g / L, 40 g / L, 60 g / L, 80 g / L, 100 g / L, 140 g / L, and 180 g / L, each in a 1 mL system. Reaction conditions: The reaction was carried out at 50℃ in a metal bath at 1000 rpm for 5 h, terminated by boiling in a water bath for 10 min, centrifuged at 12000 rpm, and the supernatant was diluted 4-fold with deionized water and filtered through a 0.22 μm water membrane. The concentrations of D-galactose and D-tagatose in the filtrate were determined using the method described in Example 1. The relative enzyme activities at other substrate concentrations were calculated with their respective highest enzyme activities as 100%. The results are shown in […]. Figure 6 The results showed that with increasing substrate concentration, the relative enzyme activity (i.e., D-tagatose yield) of TsAI decreased significantly at a substrate concentration of 40 g / L. However, the enzyme activity of the mutant TsAI / F276A did not decrease significantly with increasing substrate concentration within the range of 20-100 g / L, indicating that the beneficial mutant TsAI / F276A improved the affinity of wild-type TsAI for D-galactose. It is suitable for production applications with high substrate concentrations.

[0091] Example 10: Optimal pH of mutant TsAI / F276A

[0092] The wet bacterial cells prepared by the mutant enzyme TsAI / F276A according to the method in Example 1 were used as recombinant bacteria for transformation.

[0093] Buffer preparation: Prepare 50mM sodium citrate buffer by adjusting the pH of the sodium citrate solution to 4.0, 5.0, and 6.0 using low concentrations of HCl and NaOH; prepare 50mM sodium phosphate buffer by adjusting the pH of the buffer to 6.0, 6.5, 7.0, 7.5, and 8.0 using phosphate and NaOH; prepare 50mM Tris buffer by adjusting the pH of the buffer to 8.0, 9.0, and 10.0 using HCl; set aside.

[0094] The final concentration composition of the 1 mL reaction system was: 10 g / L wet bacterial cells, 1 mM MnCl2, and 20 g / L D-galactose substrate, totaling 1 mL. Different pH buffers prepared above were used for the reaction. Reaction conditions: 50℃ metal bath, 1000 rpm for 5 h; boiling water bath for 10 min to terminate the reaction; centrifugation at 12000 rpm; supernatant diluted 4-fold with deionized water; filtration through a 0.22 μm water membrane; concentrations of D-galactose and D-tagatose in the filtrate were determined using the method described in Example 1. Relative enzyme activities at other pH values ​​were calculated with the highest enzyme activity as 100%. Results are shown in [Figure 1]. Figure 7The mutant TsAI / F276A can react stably in an environment of pH 7.0-10.0 and is suitable for industrial production applications.

Claims

1. An L-arabinose isomerase mutant, characterized in that, The mutant was obtained by single or multiple mutations of amino acids at positions 19, 186, and 276 of the amino acid sequence shown in SEQ ID NO.

6.

2. The L-arabinose isomerase mutant as described in claim 1, characterized in that, The L-arabinose isomerase mutant is a mutation of the amino acid sequence shown in SEQ ID NO.6 to one of the following: (1) L19A; (2) M186A; (3) F276A; (4) a combination of the above three single mutations.

3. A recombinant genetically engineered bacterium containing the L-arabinose isomerase mutant encoding gene as described in claim 1.

4. The application of the L-arabinose isomerase mutant of claim 1 in the microbial catalytic D-galactose isomerization to prepare D-tagatose.

5. The application as described in claim 4, characterized in that, The application is as follows: using wet bacterial cells obtained by fermentation culture of recombinant bacteria containing the L-arabinose isomerase mutant encoding gene or pure enzyme solution obtained by ultrasonic disruption and purification of wet bacteria as the enzyme source, D-galactose as the substrate, metal ions as the catalyst, and a buffer solution with pH 4.0-10.0 as the reaction medium to form a reaction system, the reaction is carried out at 35-65℃ and 1000 r / min. After the reaction is complete, a mixture of D-galactose and D-tagatose is obtained.

6. The application as described in claim 5, characterized in that, The metal ions include Mn 2+ Co 2+ Zn 2+ Ni 2+ .

7. The application as described in claim 5, characterized in that, The reaction medium is a pH 7.0, 50mM KH2PO4-NaOH buffer solution.

8. The application as described in claim 5, characterized in that, In the reaction system, the final concentration of wet bacterial cells added is 10–50 g / L; the final concentration of metal ions added is 0.1–10 mM; and the initial final concentration of substrate added is 20–180 g / L.

9. The application as described in claim 5, characterized in that, The wet bacterial cells were prepared as follows: recombinant bacteria containing the L-arabinose isomerase mutant encoding gene were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 200 r / min until OD200. 600 The seed culture was obtained by setting the inoculum size to 0.8–1.

0. The seed culture was then transferred at a volume concentration of 1–5% into LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37°C and 180–200 rpm until the OD value reached 0.8–1.

0. 600 Add IPTG to a final concentration of 0.1 mM at a concentration of 0.4-0.6, induce culture at 28℃ and 200 r / min for 12-20 h, centrifuge, and collect wet cells.

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