CN110951717B - A kind of L-arabinose isomerase isomer and its application - Google Patents

Abstract

The invention relates to an L-arabinose epimerase mutant and its application in the preparation of D-tagatose by microorganism-catalyzed D-galactose isomerization. The L-arabinose epimerase mutant is obtained by site-directed mutation of the indicated amino acids, and the point mutation site is one or more of the following: (1) 304th, (2) 75th, (3) 274th, (4) 167th. The beneficial effects of the present invention are mainly reflected in: the present invention provides a brand-new L-arabinose epimerase and its mutant, which has a high optimum reaction temperature of 75°C, and its enzyme activity is compared with that of the original enzyme 280% improvement. Using this mutant to produce D-tagatose, the product yield can reach up to 73.3%. The recombinant enzyme was immobilized and continuously catalyzed for 30 batches, and the conversion rates were all greater than 73%. The transformation level of the mutant shows excellent industrial application prospects.

Description

A kind of L-arabinose isomerase isomer and its application

(1) Technical field

The invention relates to an L-arabinose isomerase isomer mutant and its application in the preparation of D-tagatose by microorganism-catalyzed isomerization of D-galactose.

(2) Background technology

D-tagatose is a rare ketohexose found in nature and is an isomer of D-galactose. It was first found in the gum of a tropical evergreen plant, and later found in yogurt and cheese. D-tagatose has the properties and effects of high sweetness, low calorie, low absorption rate, effectively lowering blood sugar, anti-caries, and improving intestinal flora. The US Food and Drug Administration has passed the safety certification of D-tagatose, allowing it to be used in the food field. The production methods of D-tagatose mainly include chemical method and biological method. The chemical method has problems such as high cost, high acid and alkali consumption, serious pollution, complex product components, and difficult separation and purification. In contrast, the biological preparation of D-tagatose has more advantages, and has gradually become a research hotspot in recent years.

The process of preparing D-tagatose by biological method is to use D-galactose as raw material, utilize L-arabinose isomerase (L-arabinose isomerase, LAI for short) to carry out catalytic reaction, and generate D-tagatose in one step. Among them, the raw material D-galactose is cheap and easy to obtain, the overall reaction process is mild and easy to control, and the reaction process meets environmental protection requirements. It is an optimal sugar production method that replaces chemical methods. Research reports pointed out that the reaction conditions of high temperature and weak acidity are more conducive to the biotransformation of D-tagatose. At present, the substrate concentration of D-tagatose prepared from D-galactose by biological method is low, all below 100g/L, which is not conducive to the application in industrial production. In addition, there are few enzyme sources that can be developed, which restricts the production level of D-tagatose.

Bioinformatics takes nucleic acid, protein and other biomacromolecular databases as the main object, and assists computers to compare and analyze biological information, and obtain rational information such as gene codes, protein structure and function, and their relationship. In this context, using modern biotechnology methods such as bioinformatics and genetic engineering to screen and transform new high-temperature and acid-resistant LAI enzyme preparations is of great significance for meeting the growing sugar intake needs of the people.

(3) Contents of the invention

The purpose of the present invention is to provide a high-temperature and acid-resistant L-arabinose isomerase isomer mutant and its application in the preparation of D-tagatose by microorganism-catalyzed isomerization of D-galactose.

The technical scheme adopted in the present invention is:

An L-arabinose epimerase mutant, which is obtained by site-directed mutation of the amino acid sequence shown in SEQ ID NO.3, and the mutation site is one or more of the following: (1) 304th, (2) 75th, (3) 274th, (4) 167th.

Preferably, the mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.3 through one or more of the following sites: (1) Glycine G at position 304 is mutated to histidine H, isoleucine I. Tyrosine Y or tryptophan W, (2) 75th glycine G is mutated to lysine K, methionine M, phenylalanine F or proline P, (3) 274th alanine G is mutated to serine S, threonine T, tyrosine Y, valine V or cysteine C, (4) alanine A at position 167 is mutated to asparagine N, aspartic acid D or arginine amino acid R.

Further, the high-temperature-resistant TIM barrel protease mutant is obtained by mutating the amino acids shown in SEQ ID NO.3 through one or more of the following sites: (1) Glycine G at position 304 is mutated into tyrosine Y, (2) Glycine G at position 75 is mutated to phenylalanine F, (3) Alanine G at position 274 is mutated to cysteine C, and (4) Alanine A at position 167 is mutated to arginine R.

More preferably, the sequence of the L-arabinose epimerase mutant is shown in SEQ ID NO.7 (its coding gene is shown in SEQ ID NO.8).

The present invention also relates to the application of the L-arabinose epimerase mutant in catalyzing the isomerization of D-galactose to prepare D-tagatose.

Specifically, the application is: the supernatant or the thalline obtained by fermenting and culturing the recombinant genetically engineered bacteria containing the gene encoding L-arabinose epimerase or the wet thalline after ultrasonic crushing The immobilized enzyme preparation obtained by immobilizing cells is used as a catalyst, with D-galactose as a substrate, in the presence of Mn ²⁺ and/or Co ²⁺ , in 6.0-7.0 KH ₂ PO ₄ -NaOH buffer, The reaction is carried out at a temperature of 65-80° C. After the reaction is completed, the reaction liquid is separated and purified to obtain D-tagatose.

The preparation method of the immobilized enzyme preparation is as follows: weigh bacterial cells, suspend them with Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer solution (pH6.5), add Celite 545, and stir properly. Add polyethyleneimine aqueous solution to flocculate at 25°C and 100r/min, then add trihydroxymethylphosphine (THP) aqueous solution, and conduct crosslinking reaction at 25°C and 100r/min for 2 hours. Then suction filtration, the filter cake is washed with distilled water, extruded into long strips with an axial extruder, air-dried at room temperature, and crushed into granules (preferably 0.5-2 mm in diameter) to obtain enzyme immobilized granules.

Preferably, the coding gene sequence of the L-arabinose epimerase mutant is shown in SEQ ID NO.8.

The beneficial effects of the present invention are mainly reflected in: the present invention provides a brand-new L-arabinose epimerase and its mutant, which has a higher optimum reaction temperature of 75°C, and its enzyme activity is comparable to that of the original enzyme. Ratio increased by 280%. Using the mutant to produce D-tagatose, the highest product yield can reach 73.3%. The recombinant enzyme was immobilized and continuously catalyzed for 30 batches, and the conversion rates were all greater than 73%. The invention solves the problem of low efficiency of the existing enzyme-catalyzed preparation of D-tagatose, and obtains a continuous conversion effect better than that reported in literature, which is of great significance for improving the industrialization level of D-tagatose.

(4) Description of drawings

Fig. 1 is the optimal temperature schematic diagram of recombinase;

Figure 2 is a schematic diagram of the impact of metal ions on the activity of recombinases;

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1: Screening and activity determination of novel LAI

1. Source of enzyme and construction of recombinant bacteria

The new LAIs were obtained from the NCBI database, respectively from Novibacillus thermophilus (GenBank No. WP_077718551.1), Lactobacillus oris (GenBank No. WP_003716014.1), Pseudothermotoga thermarum (GenBank No. WP_013932424.1), and named NtLAI, LoLAI and PtLAI. According to the amino acid sequence, the codon was optimized according to the codon preference of Escherichia coli, and three selected nucleotide sequences were synthesized by the method of total synthesis through the conventional operation of genetic engineering, such as SEQ ID NO.2, SEQ ID NO.4 and shown in SEQ ID NO.6; the amino acid sequences encoding the enzyme are shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 respectively. Add 6×His-tag tags at the end of the nucleic acid sequence, add restriction sites Xba I and Xho I at both ends, clone the gene into the Xba I and Xho I sites corresponding to pET28b(+), and obtain the recombinant expression plasmid pET28b/ NtLAI, pET28b/LoLAI and pET28b/PtLAI.

2. Transformation and induced expression of recombinant bacteria

The obtained recombinant expression plasmids pET28b/NtLAI, pET28b/LoLAI and pET28b/PtLAI were transformed into Escherichia coli BL21 (DE3) recipient bacteria, spread on LB agar plates containing a final concentration of 100 μg/mL kanamycin, Cultivate overnight at 37°C, and on the second day, randomly pick clones from the colonies grown on the plate and extract the plasmids for identification by agarose gel electrophoresis to obtain genetically engineered bacteria containing the LAI gene.

LB liquid medium composition: tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L, solvent is water, pH value is natural; LB solid medium is added 15g/L agar in LB liquid medium; 121℃ high pressure Sterilize for 20 minutes; add kanamycin at a final concentration of 100 μg/mL before use.

Inoculate genetically engineered bacteria into LB liquid medium containing kanamycin at a final concentration of 100 μg/mL, and culture them at 37°C and 150 r/min until the OD ₆₀₀ is about 0.6-0.8 to obtain seed liquid; (v/v) Inoculum amount was inoculated into fresh LB medium containing kanamycin with a final concentration of 100 μg/mL, cultured at 37°C and 150 r/min to OD ₆₀₀ to 0.4-0.6, and then added to the culture medium with the final concentration 0.5mM IPTG, induced expression at 28°C for 12h, centrifuged at 4°C, 6000r/min for 10min, discarded the supernatant, washed the wet cells twice with 0.85% normal saline, collected the wet cells, and set aside.

3. Determination of enzyme activity of recombinant bacteria

Ultrasonic disruption was performed on the wet bacteria. Take 1 g of the prepared wet bacteria, suspend it with 50 mL of 50 mM KH ₂ PO ₄ -NaOH buffer (pH 6.5), and sonicate it at 39W for 15 minutes to prepare a sonicated suspension, centrifuge, and collect the supernatant.

Enzyme activity reaction system: 50 g/L D-galactose, 1 mM MnCl ₂ , 50 μL of the above supernatant (enzyme solution), and then add an appropriate amount of 50 mM KH ₂ PO ₄ -NaOH buffer (pH 6.5) to a total volume of 1 mL. Reaction conditions: react at 40°C for 10 minutes, boil in boiling water for 10 minutes to terminate the reaction, dilute 10 times, and pass through a 0.22 μm filter membrane; use HPLC to detect the concentration of D-tagatose. The analytical column is an Aminex HPX-87H column (300×7.8mm, 9 μm, Bio-Rad Life Medical Products Co., Ltd.). Waters 2414 differential refractive index detector, Waters 1525 pump, Waters 717 injector.

Definition of enzyme activity: at 40°C and pH 6.5, the amount of enzyme required to isomerize D-galactose into 1 μmol D-tagatose per minute is defined as one enzyme activity unit (U).

Table 1: Enzyme Activity Assays for Recombinases

Example 2: Construction and screening of LoLAI single point mutants

1. Mutant construction

Design the mutation primers for site-directed mutation according to the LoLAI parental sequence, and use the rapid PCR technology to introduce a single mutation at position 304 with the recombinant vector pET28b/LoLAI as a template. The primers are:

Forward primer 304G:

ATGGCTATGGCTTC NNN GCAGAAGGTGACTTCAAAA (the underline is the mutated base)

Reverse primer 304G:

TTTTGAAGTCACCTTCTGC NNN GAAGCCATAGCCAT (the underline is the mutated base)

PCR reaction system: 2×Phanta Max Buffer (containing Mg ²⁺ ) 25 μL, dNTPs 10 mM, forward primer 304G 2 μL (5 pmol/μL, the same below), reverse primer 304G 2 μL (5 pmol/μL, the same below), template DNA 1 μL (20ng/μL, the same below), Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH ₂ O to 50 μL.

The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 64°C for 15s, 72°C for 6.5min) for 30 cycles; 72°C for 5min.

2. Transformation and expression of mutants

Take 5 μL of the PCR product, add it to 100 μL of E.coli BL21(DE3) competent cell suspension in an ice bath, let it stand on ice for 30 minutes, heat-shock the transformed product at 42°C for 90 seconds, and quickly place it on ice to cool for 5 minutes. Add 600 μL of LB liquid medium to the tube, incubate at 37°C for 60 minutes at 150 r/min, take 100 μL of the above bacterial solution to smear the plate, and after the bacterial solution is completely absorbed by the medium, incubate at 37°C for 12 hours.

3. High-throughput screening of positive transformants

The composition of the reaction mixture: 5 g/L D-galactose, 1 mM MnCl ₂ , and 50 mM KH ₂ PO ₄ -NaOH buffer solution (pH 6.5) were added to make the total reaction system 1 L, and set aside.

Add 100 μL of LB culture solution containing a final concentration of 100 μg/mL kanamycin to each well of a 96-well polystyrene microwell culture plate, inoculate different transformed colonies, and culture at 37°C and 150 r/min to OD ₆₀₀ to 0.4-0.6 , and then add IPTG with a final concentration of 0.5mM to the culture medium, induce expression at 28°C for 10h, centrifuge at 6000r/min at 4°C for 10min, and discard the supernatant. Take 100 μL of the above reaction mixture and add it to a 96-well plate containing bacteria, shake and mix with an oscillator, react at 40°C, 600 r/min for 10 minutes, and stop the reaction in an ice bath for 10 minutes. Take 2 μL of reaction solution to screen mutants by cysteine-carbazole chromogenic method, and the reaction system includes 2 μL of reaction solution, 5 μL of 1.5% (w/v) cysteine hydrochloride, 150 μL of 70% (w/v) w) Concentrated sulfuric acid, 5 μL of 0.12% (w/v) carbazole ethanol, and observe the color change after incubation at 60° C. for 10 minutes. Taking the reaction of recombinant bacteria E.coli BL21(DE3)/pET28b/LoLAI as a control, the mutant strains with a darker color than the reaction of E.coli BL21(DE3)/pET28b/LoLAI were used for accurate determination of enzyme activity.

4. Accurate determination of enzyme activity of positive transformants

The operation is the same as that of "Determination of Enzyme Activity of Recombinant Bacteria" in Example 1.

The results of this example are: 665 strains of recombinant transformed bacteria were initially screened, and 4 mutant strains with improved enzyme activity were screened out, and then the enzyme activity was accurately measured. The specific results are shown in Table 2. After analysis, it was determined that the enzyme activity of the remaining 661 strains of recombinant bacteria remained unchanged or decreased because the 304th glycine (G) was mutated to other amino acids other than H, I, Y and W.

Table 2: Enzyme Activity Determination of Single Point Mutation Recombinant Bacteria

The LoLAI-G304Y mutant with the most improved enzyme activity was designated as LoLAI-1, and the recombinant strain E.coli BL21(DE3)/pET28b/LoLAI-1 was obtained.

Example 3: Construction and screening of LoLAI double-site mutants

According to the single mutant LoLAI-1 sequence constructed in Example 2, the mutation primers for site-directed mutation were designed, and the rapid PCR technology was used to introduce a single mutation at the 75th position with the recombinant vector pET28b/LoLAI-1 as a template. The primers were:

Forward primer 75G: CGACAAAGTTGCA NNN GTGATGACCT (the underline is the mutated base)

Reverse primer 75G: AGGTCATCAC NNN TGCAACTTTGTCG (the underline is the mutated base)

PCR reaction system: 2×Phanta Max Buffer (containing Mg ²⁺ ) 25 μL, dNTPs 10 mM, forward primer 75G 2 μL, reverse primer 75G 2 μL, template DNA 1 μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH ₂ O to 50 μL .

The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 60.5°C for 15s, 72°C for 6.5min) for 30 cycles; 72°C for 5min.

The PCR product was transformed into E.coli BL21(DE3) competent cells, and a single clone was picked in LB liquid medium containing 100 μg/mL kanamycin, and cultured overnight at 37°C. The mutants were initially screened by the cysteine carbazole chromogenic method (the operation is the same as in the "high-throughput screening of positive transformants" in Example 2), and the positive clones were subjected to precise enzyme activity determination (the operation was the same as in the "Positive Transformants" in Example 1). Enzyme activity assay of positive transformants").

The results of this example are: 399 strains of recombinant transformed bacteria were initially screened, and 4 mutant strains with improved enzyme activity were screened out, and then the enzyme activity was accurately measured. The specific results are shown in Table 3. It was determined by analysis that the enzyme activity of the remaining 395 recombinant strains remained unchanged or decreased because the 75th glycine (G) was mutated to other amino acids other than K, M, F and P.

Table 3: Enzyme Activity Determination of Double Point Mutation Recombinant Bacteria

The LoLAI-G304Y-G75F mutant with the most improved enzyme activity was designated as LoLAI-2, and the recombinant strain E.coliBL21(DE3)/pET28b/LoLAI-2 was obtained.

Example 4: Construction and screening of LoLAI three-site mutants

According to the mutant LoLAI-2 sequence constructed in Example 3, the mutation primers for site-directed mutation were designed, using the rapid PCR technology, using the recombinant vector pET28b/LoLAI-2 as a template, and introducing a single mutation at position 274, the primers were:

Forward primer 274G: GGTTATGAT NNN TTCACCACCAACTTCCAGG (the underline is the mutated base)

Reverse primer 274G: CCTGGAAGTTGGTGGTGAA NNN ATCATAACC (the underline is the mutated base)

PCR reaction system: 2×Phanta Max Buffer (containing Mg ²⁺ ) 25 μL, dNTPs 10 mM, forward primer 274G 2 μL, reverse primer 274G 2 μL, template DNA 1 μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH ₂ O to 50 μL.

The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 59.5°C for 15s, 72°C for 6.5min) for 30 cycles; 72°C for 5min.

The PCR product was transformed into E.coli BL21(DE3) competent cells, and a single clone was picked in LB liquid medium containing 100 μg/mL kanamycin, and cultured overnight at 37°C. The mutants were initially screened by the cysteine carbazole chromogenic method (the operation is the same as in the "high-throughput screening of positive transformants" in Example 2), and the positive clones were subjected to precise enzyme activity determination (the operation was the same as in the "Positive Transformants" in Example 1). Enzyme activity assay of positive transformants").

The results of this example are: 735 strains of recombinant transformed bacteria were initially screened, and 5 mutants with improved enzyme activity were screened out, and then their enzyme activity was determined. The specific results are shown in Table 4. It was determined by analysis that the enzyme activity of the remaining 730 recombinant strains remained unchanged or decreased because the 274th alanine (G) was mutated to other amino acids other than S, T, Y, V and C.

Table 4: Enzyme Activity Determination of Three-point Mutation Recombinant Bacteria

The LoLAI-G304Y-G75F-G274C mutant with the most improved enzyme activity was designated as LoLAI-3, and the recombinant strain E.coli BL21(DE3)/pET28b/LoLAI-3 was obtained.

Example 5: Construction and screening of LoLAI four-site mutants

According to the mutant LoLAI-3 sequence constructed in Example 4, the mutation primers for site-directed mutagenesis were designed, and the recombinant vector pET28b/LoLAI-3 was used as a template to introduce a single mutation at position 167 using rapid PCR technology. The primers were:

Forward primer 167A: GGCAGAAAGTG NNN ATTGCATACGATATGAGC (the underline is the mutated base)

Reverse primer 167A: GCTCATATCGTATGCAAT NNN CACTTTCTGCC (the underline is the mutated base)

PCR reaction system: 2×Phanta Max Buffer (containing Mg ²⁺ ) 25 μL, dNTPs 10 mM, forward primer 167A 2 μL, reverse primer 167A 2 μL, template DNA 1 μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH ₂ O to 50 μL.

The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 59°C for 15s, 72°C for 6.5min) for 30 cycles; 72°C for 5min.

The PCR product was transformed into E.coli BL21(DE3) competent cells, and a single clone was picked in LB liquid medium containing 100 μg/mL kanamycin, and cultured overnight at 37°C. Utilize the cysteine carbazole color method to carry out primary screening on the mutant (operation is the same as the "high-throughput screening positive transformant" in Example 2), and carry out accurate determination of enzyme activity (operation is the same as the "positive transformation" in Example 1 Subenzyme activity assay").

The results of this example are: 444 strains of recombinant transformed bacteria were initially screened, and 3 mutant strains with improved enzyme activity were screened out, and then their enzyme activity was determined. The specific results are shown in Table 5. It was determined by analysis that the enzyme activity of the remaining 441 recombinant strains remained unchanged or decreased because the 167th alanine (A) was mutated to amino acids other than N, D and R.

Table 5: Enzyme Activity Determination of Four-site Mutation Recombinant Bacteria

The LoLAI-G304Y-G75F-G274C-A167R mutant with the most improved enzyme activity was designated as LoLAI-4, and the recombinant strain E.coli BL21(DE3)/pET28b/LoLAI-4 was obtained.

Embodiment 6: Recombinant Escherichia coli fermentation enzyme production and purification

The recombinant bacteria E.coli BL21(DE3)/pET28b/LoLAI, E.coli BL21(DE3)/pET28b/LoLAI-1, E.coli BL21(DE3)/pET28b/LoLAI-2, E.coli BL21(DE3 )/pET28b/LoLAI-3, E.coli BL21(DE3)/pET28b/LoLAI-4 were inoculated into LB liquid medium containing a final concentration of 100 μg/mL kanamycin, and cultured at 37°C and 150 r/min at an OD ₆₀₀ of about 0.6 to 0.8 to obtain seed liquid; inoculate the seed liquid with 2% (v/v) inoculum into fresh LB liquid medium containing kanamycin with a final concentration of 100 μg/mL, and cultivate at 37°C and 150r/min OD ₆₀₀ to 0.4-0.6, then add IPTG with a final concentration of 0.5mM to the culture medium, induce expression at 28°C for 12h, centrifuge at 6000r/min at 4°C for 10min, discard the supernatant, and replace with 0.85% physiological Wash the wet cells twice with salt water, and collect the wet cells.

Ultrasonic disruption was performed on the wet bacteria, and the supernatant was collected.

Purify using a nickel-NTA agarose gel column, equilibrate the chromatography column with an equilibration buffer (20mM phosphate buffer, 300mM NaCl, 20mM imidazole, pH 8.0), and then use an eluent (50mM phosphate buffer, 300mM NaCl , 500mM imidazole, pH 8.0) for elution, according to the signal response of the ultraviolet detector, collect the corresponding eluate, which is the respective pure enzyme solution.

Embodiment 7: the optimal reaction temperature of purifying LoLAI and mutants thereof

The pure enzyme solution in Example 6 was used as the enzyme for transformation, and the optimum reaction temperature of the enzyme was determined. The reaction system was: 50 g/L D-galactose, 1 mM MnCl ₂ , 50 μL of the pure enzyme solution obtained in the above examples, and then 50 mM KH ₂ PO ₄ -NaOH buffer solution (pH 6.5) was added to make the total system 1 mL. The activity of the recombinant LAI was measured at different transformation temperatures: 60, 65, 70, 75, 80, 85, and 90° C. (the operation method was the same as that of “Determination of Enzyme Activity of Recombinant Bacteria” in Example 1). It can be seen from Figure 1 that the optimum reaction temperature of LoLAI-4 is 75°C, which is 10°C higher than that of the original enzyme LoLAI.

Embodiment 8: Effect of metal ions on LAI optimal mutant enzyme activity

The pure enzyme solution in Example 6 was used as the enzyme for transformation, and the influence of metal ions on the enzyme activity of the recombinase was determined. The 1 mL reaction system includes: 50 mM KH ₂ PO ₄ -NaOH buffer solution (pH 6.5), 50 g/L D-galactose, 50 μL pure enzyme solution and 1 mM different metal ions (the anion is Cl ⁻ ). Among them, ^the selection of metal ions is as follows: (1) Single metal ions are selected: Mn ²⁺ , Co ²⁺ , Mg ²⁺ , Cu ²⁺ , Zn ²⁺ , Ba ²⁺ , Fe ²⁺ , and Ca ²⁺ . The activity of LAI was determined at 40°C. (2) Set combined metal ions, respectively 1mM Mn ²⁺ and 0.5mM Co ²⁺ , 1mM Mn ²⁺ and 0.5mM Zn ²⁺ , 1mM Mn ²⁺ and 0.5mM Mg ²⁺ for enzyme activity determination. No metal ions were used as a control. It can be seen from Figure 2 that Mn ²⁺ greatly promotes the enzymatic activity of LAI, and the effect is more obvious than that of combined metals.

Example 9: Preparation of D-tagatose from whole cells of the original enzyme and mutant enzyme mutant recombinant bacteria

According to the fermentation method of Example 6, large-scale fermentation obtained recombinant bacteria E.coli BL21(DE3)/pET28b/LoLAI, E.coli BL21(DE3)/pET28b/LoLAI-1, E.coli BL21(DE3)/pET28b/ LoLAI-2, E.coli BL21(DE3)/pET28b/LoLAI-3, E.coli BL21(DE3)/pET28b/LoLAI-4. D-tagatose was prepared by biotransformation with the above-mentioned wet bacteria as a biocatalyst and D-galactose as a substrate. The catalytic system includes: different concentrations of D-galactose, 1mM MnCl ₂ , 15g/L wet bacteria, and then add an appropriate amount of 50mM KH ₂ PO ₄ -NaOH buffer solution (pH 6.5) to make the total system 100mL. The reaction system was reacted at 65°C and 150r/min for 8h. Samples were taken every 1 hour, centrifuged, filtered through a 0.22 μm membrane, and the concentration of D-tagatose was detected by HPLC. It can be seen from Table 6 that the product yield of E.coli BL21(DE3)/pET28b/LoLAI-4 finally reached 73.3%, which was higher than that of the original enzyme E.coli BL21(DE3)/pET28b/LoLAI and other mutant enzymes.

Table 6: Comparison of the yield of each recombinant bacteria

Example 10: Immobilization and continuous transformation of E.coli BL21(DE3)/pET28b/LoLAI-4

Preparation of 30% (v/v) THP aqueous solution: Take 31.8g of tetrakis hydroxymethyl phosphorus sulfate dissolved in 80mL of deionized water, 3.4g of potassium hydroxide dissolved in 10mL of deionized water, room temperature 25 ℃, 100r/min conditions The two are slowly mixed to obtain a 30% (v/v) THP aqueous solution, which is ready-to-use, and the molar ratio of four hydroxymethyl phosphorus sulfate and potassium hydroxide is 1:0.995.

Weigh 5 g of recombinant E.coli BL21(DE3)/pET28b/LoLAI-4 cells, suspend them with 50 mL of Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer solution (pH 6.5), add 0.3 g of Celite 545, and stir properly. Add 2mL of 5% (v/v) polyethyleneimine aqueous solution to flocculate at 25°C and 100r/min, then add 0.25mL of 30% THP aqueous solution, and conduct crosslinking reaction at 25°C and 100r/min for 2h. Then suction filtration, the filter cake was washed with distilled water and then extruded into long strips with an axial extruder. After air-drying at room temperature, it was pulverized into granules (preferably with a particle size of 0.5-2 mm) to obtain immobilized granules of the LoLAI-5 mutant.

The above-mentioned immobilized particles are used as a biocatalyst and D-galactose is used as a substrate to prepare D-tagatose through biotransformation. 100mL catalytic system includes: 50mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer (pH 6.5), 500g/L D-galactose, 1mM MnCl ₂ , 6g/L immobilized particles. Reaction at 65°C, 200r/min, isomerization for 2h. The reaction solution was centrifuged at 4°C, and a small amount of the supernatant was filtered through a 0.22 μm membrane, and then the concentration of D-tagatose was detected by HPLC; the immobilized particles were collected and washed with buffer for the next batch of transformation. The experimental results show that the product yields are all greater than 73% after continuous catalysis for 30 batches.

sequence listing

<110> Zhejiang University of Technology

<120> A kind of L-arabinose isomerase isomer and its application

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 500

<212> PRT

<213> Novibacillus thermophilus

<400> 1

Met Ile Ala Leu Lys Pro Tyr Glu Phe Trp Phe Val Thr Gly Ser Gln

1 5 10 15

His Leu Tyr Gly Glu Glu Thr Leu Gln Glu Val Glu Ala His Ala Arg

20 25 30

Gln Val Ile Asp Gly Leu Asn Ala Asp Ser Ser Ile Pro Phe Pro Ile

35 40 45

Ala Phe Lys Pro Val Leu Thr Glu Pro Asp Ala Ile Leu Arg Leu Cys

50 55 60

Leu Asp Ala Asn Arg Asp Glu Lys Cys Ala Gly Ile Val Thr Trp Met

65 70 75 80

His Thr Phe Ser Pro Ala Lys Met Trp Ile Ala Gly Leu Ser Ala Leu

85 90 95

Gln Lys Pro Leu Leu His Leu His Thr Gln Phe Asn Arg Asp Ile Pro

100 105 110

Trp Ser Ser Ile Asp Met Asp Phe Met Asn Leu Asn Gln Ser Ala His

115 120 125

Gly Asp Arg Glu Tyr Gly Phe Ile Gly Ser Arg Met Asn Val Arg Arg

130 135 140

Lys Val Val Val Gly His Trp Lys Ser Lys Ala Val Arg Glu Arg Ile

145 150 155 160

Gly Gly Trp Met Arg Val Ala Val Ala Tyr Val Glu Gly Lys Gln Leu

165 170 175

Lys Val Ala Arg Phe Gly Asp Asn Met Arg Glu Val Ala Val Thr Glu

180 185 190

Gly Asp Lys Val Glu Ala Gln Ile Gln Phe Gly Trp Ser Ile Asn Gly

195 200 205

Tyr Gly Val Gly Asp Leu Val Gln Arg Ile Asn Asp Ile Gln Glu Ala

210 215 220

Glu Val Asn Ala Leu Leu Asp Glu Tyr Ala Glu Gln Tyr Asp Ile Ala

225 230 235 240

Pro Glu Gly Leu Lys Glu Gly Ala Val Arg Asp Ser Val Arg Glu Gln

245 250 255

Ala Arg Ile Glu Leu Gly Leu Lys Ala Phe Leu Gln Glu Gly Gly Phe

260 265 270

Thr Ala Phe Thr Thr Thr Thr Phe Glu Asp Leu His Gly Met Lys Gln Leu

275 280 285

Pro Gly Leu Ala Val Gln Arg Leu Met Ala Glu Gly Tyr Gly Phe Gly

290 295 300

Gly Glu Gly Asp Trp Lys Thr Ala Ala Leu Val Arg Met Met Lys Ile

305 310 315 320

Leu Ala Asp Asn Glu Gly Thr Ser Phe Met Glu Asp Tyr Thr Tyr His

325 330 335

Phe Glu Pro Asp Asn Glu Leu Val Leu Gly Ser His Met Leu Glu Val

340 345 350

Cys Pro Thr Val Ala Ala Thr Lys Pro Arg Val Glu Val His Pro Leu

355 360 365

Ser Ile Gly Gly Lys Glu Asp Pro Ala Arg Leu Val Phe Asp Gly Lys

370 375 380

Ser Gly Ala Ala Leu Asn Ala Ser Leu Val Asp Met Gly Asn Arg Phe

385 390 395 400

Arg Leu Leu Val Asn Glu Val Asp Ala Val Gln Pro Glu Cys Ala Met

405 410 415

Pro Lys Leu Pro Val Ala Arg Val Leu Trp Lys Pro Ala Pro Ser Leu

420 425 430

Ser Glu Ala Ala Glu Cys Trp Ile Val Ala Gly Gly Ala His His Thr

435 440 445

Cys Phe Ser Tyr Arg Val Thr Thr Glu Gln Leu Ile Asp Trp Ala Asp

450 455 460

Met Ala Gly Ile Glu Cys Val Val Ile Asp Lys Asp Thr Arg Arg His

465 470 475 480

Ala Phe Arg Asn Glu Leu Lys Trp Asn Glu Val Val Tyr Gly His His

485 490 495

His His His His

500

<210> 2

<211> 1500

<212>DNA

<213> Novibacillus thermophilus

<400> 2

atgatcgcac tgaaaccgta tgagttctgg ttcgttaccg gcagccagca tctgtatggc 60

gaagaaacct tacaggaagt ggaagcacat gcacgccagg tgattgatgg cctgaatgcc 120

gatagcagta ttccgttccc gattgcattc aaaccggtgc tgaccgaacc ggatgcaatt 180

ctgcgcctgt gcctggatgc aaatcgtgat gaaaaatgcg ccggcattgt tacctggatg 240

cataccttca gtccggcaaa aatgtggatt gcaggcctga gcgcactgca gaaaccgctg 300

ctgcatctgc atacccagtt caatcgtgat attccgtgga gtagtattga tatggacttc 360

atgaatctga atcagagtgc acatggtgat cgtgaatatg gcttcattgg cagtcgtatg 420

aatgtgcgcc gcaaagttgt tgttggtcat tggaaaagca aagcagttcg cgaacgcatt 480

ggcggctgga tgcgcgtggc agtggcatac gttgaaggca aacagctgaa agtggcacgc 540

ttcggtgata atatgcgcga agtggcagtg accgaaggtg ataaagttga agcccagatt 600

cagttcggtt ggagcattaa tggttatggc gttggcgatc tggttcagcg tattaatgat 660

attcaggaag ccgaagtgaa tgcactgctg gatgaatatg ccgaacagta tgatattgca 720

ccggaaggtc tgaaagaagg tgcagttcgt gatagcgtgc gtgaacaggc ccgtattgaa 780

ctgggtctga aagccttcct gcaggaaggt ggcttcaccg cattcaccac caccttcgaa 840

gatctgcatg gcatgaaaca gctgccgggc ctggccgtgc agcgtcttat ggcagaaggc 900

tatggcttcg gcggcgaagg tgattggaaa accgccgccc tggttcgtat gatgaaaatt 960

ctggccgata atgaaggtac cagcttcatg gaagattata cctatcactt cgaaccggat 1020

aatgaactgg ttctgggtag ccacatgctg gaagtgtgtc cgaccgtggc cgccaccaaa 1080

ccgcgtgtgg aagtgcatcc gctgagcatt ggcggtaaag aagatccggc ccgtctggtg 1140

ttcgatggta aaagcggcgc cgcactgaat gccagcctgg ttgatatggg caatcgcttc 1200

cgtctgctgg tgaatgaagt tgatgccgtt cagccggaat gcgccatgcc gaaactgccg 1260

gtggcacgcg tgctgtggaa accggcaccg agcctgagtg aagccgccga atgttggatt 1320

gttgccggtg gcgcccatca tacctgcttc agctatcgcg ttaccaccga acagctgatt 1380

gattgggcag atatggccgg tattgaatgc gtggttatg ataaagatac ccgtcgccat 1440

gcattccgta atgaactgaa atggaatgaa gttgtgtatg gtcatcatca tcatcaccat 1500

<210> 3

<211> 480

<212> PRT

<213> Lactobacillus oris

<400> 3

Met Leu Lys Thr Pro Asp Tyr Glu Phe Trp Phe Ala Val Gly Ser Gln

1 5 10 15

Pro Leu Tyr Gly Pro Glu Ala Leu Glu Gln Val Glu Lys Asp Gly Arg

20 25 30

Lys Leu Val Asp Gly Leu Asn Ala Ser Gly Lys Leu Pro Tyr Lys Val

35 40 45

Val Phe Lys Leu Val Ala Thr Thr Thr Ala Asp Ser Ile Thr Lys Phe Met

50 55 60

Lys Asp Ala Asn Tyr Asn Asp Lys Val Ala Gly Val Met Thr Trp Met

65 70 75 80

His Thr Phe Ser Pro Ala Lys Asn Trp Ile Arg Gly Thr Gln Leu Leu

85 90 95

Gln Lys Pro Leu Leu His Leu Ala Thr Gln Met Leu Asp His Ile Pro

100 105 110

Phe Asp Thr Ile Asp Phe Asp Tyr Met Asn Leu Asn Gln Ser Ala His

115 120 125

Gly Asp Arg Glu Tyr Asp Tyr Ile Asn Ala Arg Leu Gly Val Pro Glu

130 135 140

Lys Ile Val Tyr Gly Trp Trp Asn Asp Pro Glu Val Gln Glu Glu Ile

145 150 155 160

Ala Asp Trp Gln Lys Val Ala Ile Ala Tyr Asp Met Ser Phe Lys Ile

165 170 175

Lys Ile Ala Arg Phe Gly Asp Thr Met Arg Asp Val Ala Val Thr Glu

180 185 190

Gly Asp Lys Val Ala Ala Gln Ile Lys Leu Gly Trp Thr Val Asp Tyr

195 200 205

Trp Pro Val Ala Glu Leu Val Ala Ala Val Asn Ala Val Ser Glu Ala

210 215 220

Glu Ile Asp Glu Gln Tyr Lys Arg Leu Glu Glu Asn Tyr Asp Met Val

225 230 235 240

Glu Gly Asp Asn Asp His Asp Lys Tyr Val His Ser Val Arg Tyr Gln

245 250 255

Leu Arg Glu Tyr Leu Gly Ile Lys His Phe Leu Asp Glu Lys Gly Tyr

260 265 270

Asp Ala Phe Thr Thr Asn Phe Gln Asp Leu Glu Gly Leu Glu Gln Leu

275 280 285

Pro Gly Leu Ala Val Gln Leu Leu Met Ile Asp Gly Tyr Gly Phe Gly

290 295 300

Ala Glu Gly Asp Phe Lys Ser Ala Gly Leu Ser Arg Leu Leu Lys Ile

305 310 315 320

Ala Ala Glu Asn Lys Glu Thr Ala Phe Met Glu Asp Tyr Thr Leu Asp

325 330 335

Leu Arg Ser Gly His Gln Ala Ile Met Gly Ser His Met Leu Glu Val

340 345 350

Asp Pro Thr Leu Ala Ser Asp Lys Pro Arg Val Glu Val His Pro Leu

355 360 365

Asp Ile Gly Asp Lys Ala Asp Pro Ala Arg Leu Val Phe Thr Gly Ser

370 375 380

Glu Gly Glu Gly Ile Asp Val Thr Leu Ser Tyr Phe Asp Asp Gly Tyr

385 390 395 400

Lys Phe Ile Ala Tyr Asp Val Asp Cys Ala Lys Pro Glu Lys Glu Met

405 410 415

Pro Asn Leu Pro Val Ala Lys Gln Met Trp Thr Pro Lys Cys Gly Leu

420 425 430

Lys Lys Gly Ala Thr Ala Trp Met His Asn Gly Gly Gly His His Thr

435 440 445

Val Leu Ser Met Asn Leu Ser Met Asp Gln Met Ala Thr Leu Ala Asn

450 455 460

Leu Phe Gly Ile Glu Leu Ile Asp Ile Lys His His His His His His His His

465 470 475 480

<210> 4

<211> 1440

<212>DNA

<213> Lactobacillus oris

<400> 4

atgctgaaaa cacctgatta tgagttctgg ttcgccgttg gtagccagcc gctgtatggt 60

ccggaagcac tggaacaggt ggaaaaagat ggccgcaaac tggtggatgg cctgaatgca 120

agcggcaaac tgccgtataa agttgtgttc aaactggttg ccaccaccgc cgatagcatt 180

accaaattca tgaaagatgc aaattacaac gacaaagttg caggcgtgat gacctggatg 240

cataccttca gtccggccaa aaattggatt cgtggcaccc agctgctgca gaaaccgctg 300

ctgcatctgg caacccagat gctggatcat attccgttcg ataccattga cttcgattat 360

atgaatctga atcagagcgc acatggtgat cgcgaatatg attatattaa tgcacgcctg 420

ggcgttccgg aaaaaattgt gtatggttgg tggaatgatc cggaagttca ggaagaaatt 480

gcagattggc agaaagtggc aattgcatac gatatgagct tcaaaattaa gattgcccgc 540

ttcggcgata ccatgcgtga tgttgcagtg accgaaggcg ataaagtggc cgcacagatt 600

aaactgggtt ggaccgttga ttatggccg gtggccgaac tggtggcagc cgtgaatgca 660

gttagcgaag ccgaaattga tgaacagtat aaacgtctgg aagaaaatta tgatatggtt 720

gaaggcgata atgatcatga taaatatgtt catagcgtgc gctatcagct gcgcgaatat 780

ctgggtatta aacacttcct ggatgaaaaa ggttatgatg cattcaccac caacttccag 840

gatctggaag gtctggaaca gctgccgggc ctggccgtgc agctgttaat gattgatggc 900

tatggcttcg gcgcagaagg tgacttcaaa agcgcaggcc tgagccgtct gctgaaaatt 960

gcagccgaaa ataaagaaac cgcattcatg gaagattata ccctggatct gcgcagcggt 1020

catcaggcaa ttatgggtag tcacatgctg gaagttgatc cgaccctggc aagcgataaa 1080

ccgcgtgttg aagttcatcc gctggatatt ggtgataaag cagatccggc ccgtctggtg 1140

ttcaccggca gcgaaggtga aggcattgat gttaccctga gctacttcga tgatggttat 1200

aaattcattg cctatgatgt tgattgcgcc aaaccggaaa aagaaatgcc gaatctgccg 1260

gttgccaaac agatgtggac cccgaaatgc ggtctgaaaa aaggtgcaac cgcctggatg 1320

cataatggcg gcggccatca taccgtgctg agtatgaatc tgagtatgga tcagatggcc 1380

accctggcca atctgttcgg cattgaactg attgatatta aacatcatca ccatcatcac 1440

<210> 5

<211> 500

<212> PRT

<213> Pseudothermotoga thermarum

<400> 5

Met Ile Asp Leu Lys Lys Tyr Glu Phe Trp Leu Leu Val Gly Ser Gln

1 5 10 15

His Leu Tyr Gly Ser Glu Thr Leu Lys Lys Val Glu Gln Gln Ala Arg

20 25 30

Lys Ile Val Glu Glu Leu Ser Lys Asp Leu Pro Ser Pro Leu Leu Phe

35 40 45

Lys Gly Val Leu Thr Thr Pro Glu Glu Ile Leu Arg Thr Phe Glu Gln

50 55 60

Ala Asn Ala Gln Thr Asn Cys Ala Gly Val Ile Thr Trp Met His Thr

65 70 75 80

Phe Ser Pro Ser Lys Met Trp Ile Lys Gly Leu Leu Ala Asn Lys Lys

85 90 95

Pro Leu Leu His Leu His Thr Gln Phe Asn Arg Glu Ile Pro Trp Asp

100 105 110

Thr Ile Asp Met Asp Tyr Met Asn Leu Asn Gln Ser Ala His Gly Asp

115 120 125

Arg Glu His Gly Tyr Ile His Ala Arg Leu Arg Leu Pro Arg Lys Val

130 135 140

Val Val Gly His Trp Gln Asp Ser Glu Val Lys Arg Glu Ile Ser Lys

145 150 155 160

Trp Met Arg Val Ala Cys Ala Ile Ala Asp Gly Arg Ser Gly Gln Ile

165 170 175

Val Arg Phe Gly Asp Asn Met Arg Glu Val Ala Ser Thr Glu Ala Asp

180 185 190

Lys Val Glu Ala Gln Ile Lys Ile Gly Trp Ser Ile Asn Thr Trp Gly

195 200 205

Val Gly Glu Leu Ala Glu Arg Val Lys Ser Val Ser Glu Asn Leu Val

210 215 220

Glu Asp Leu Ile Lys His Tyr Ala Glu Lys Tyr Val Leu Pro Ser Gly

225 230 235 240

Glu Tyr Glu Leu Lys Ala Ile Lys Glu Gln Ala Arg Ile Glu Ile Ala

245 250 255

Leu Arg Glu Phe Leu Lys Glu Lys Asn Ala Ile Ala Phe Thr Thr Thr Thr

260 265 270

Phe Glu Asp Leu His Asp Leu Pro Gln Leu Pro Gly Leu Ala Val Gln

275 280 285

Arg Leu Met Glu Glu Gly Tyr Gly Phe Gly Ala Glu Gly Asp Trp Lys

290 295 300

Val Ala Gly Leu Val Arg Ala Leu Lys Val Met Gly Val Gly Leu Lys

305 310 315 320

Gly Gly Thr Ser Phe Met Glu Asp Tyr Thr Tyr His Leu Pro Glu Gly

325 330 335

Asn Glu Leu Val Leu Gly Ala His Met Leu Glu Ile Cys Pro Ser Ile

340 345 350

Ala Lys Glu Lys Pro Arg Ile Glu Val His Pro Leu Ser Ile Gly Gly

355 360 365

Lys Ala Asp Pro Ala Arg Leu Val Phe Glu Ala Gln Val Gly Glu Ala

370 375 380

Leu Asn Ala Ser Ile Val Asp Leu Gly Asn Arg Phe Arg Leu Val Val

385 390 395 400

Asn Lys Val Ile Ser Val Pro Leu Val Lys Pro Met Pro Lys Leu Pro

405 410 415

Val Ala Arg Val Leu Trp Lys Pro Leu Pro Asn Phe Lys Ala Ala Ala

420 425 430

Thr Ala Trp Ile Leu Ala Gly Gly Ser His His Thr Ala Phe Ser Thr

435 440 445

Ala Val Asp Pro Ser Tyr Leu Ile Asp Trp Ala Glu Met Leu Asp Ile

450 455 460

Glu Cys Val Val Ile Asp Glu Lys Leu Asp Leu Glu Arg Phe Lys Ala

465 470 475 480

Glu Leu Arg Ala Asn Glu Val Tyr Trp Gly Phe Phe Lys Lys His His His

485 490 495

His His His His

500

<210> 6

<211> 1500

<212>DNA

<213> Pseudothermotoga thermarum

<400> 6

atgatcgatc tgaaaaagta cgagttctgg ctgctggttg gcagtcagca tctgtatggt 60

agcgaaacct taaaaaaagt tgaacagcag gcccgcaaaa ttgttgaaga actgagcaaa 120

gatctgccga gcccgctgct gttcaaaggt gttctgacca ccccggaaga aattctgcgt 180

accttcgaac aggccaatgc ccagaccaat tgcgcaggcg ttattacctg gatgcatacc 240

ttcagcccga gtaaaatgtggattaaaggc ctgctggcca ataaaaaacc gctgctgcat 300

ctgcataccc agttcaatcg tgaaattccg tgggatacca ttgatatgga ttatatgaat 360

ctgaaccaga gtgcccacgg tgatcgcgaa catggttata ttcatgcccg tctgcgtctg 420

ccgcgtaaag tggtggtggg ccattggcag gatagtgaag tgaaacgcga aattagtaaa 480

tggatgcgcg tggcatgtgc cattgcagat ggccgtagtg gccagattgt gcgcttcggt 540

gataatatgc gtgaagttgc aagtaccgaa gcagataaag tggaagccca gattaaaatt 600

ggttggagta ttaatacctg gggtgttggc gaactggcag aacgcgtgaa aagtgtgagt 660

gaaaatctgg tggaagatct gattaaacat tatgccgaaa aatacgttct gccgagtggt 720

gaatatgaac tgaaagccat taaagaacag gcacgcattg aaattgcact gcgcgagttc 780

ctgaaagaaa aaaatgccat tgcattcacc accaccttcg aagatctgca tgatctgccg 840

cagctgccgg gcctggccgt tcaacgtctg atggaagaag gttatggctt cggtgccgaa 900

ggtgattgga aagtggcagg cctggtgcgc gcactgaaag ttatgggcgt tggcctgaaa 960

ggcggcacca gcttcatgga agattatacc tatcatctgc cggaaggtaa tgaactggtg 1020

ctgggtgcac acatgctgga aatctgtccg agcattgcaa aagaaaaacc gcgtattgaa 1080

gttcatccgc tgagtattgg tggtaaagca gatccggccc gcctggtgtt cgaagcacag 1140

gtgggtgaag ccctgaatgc cagtattgtt gatctgggca atcgcttccg tctggtggtg 1200

aataaagtga ttagcgttcc gctggttaaa ccgatgccga aactgccggt tgcacgcgtt 1260

ctgtggaaac cgctgccgaa cttcaaagcc gccgccaccg catggattct ggccggtggt 1320

agtcatcata ccgcattcag taccgccgtt gatccgagtt atctgattga ttgggccgaa 1380

atgctggata ttgaatgcgt tgtgattgat gaaaaactgg atctggaacg cttcaaagcc 1440

gaactgcgtg caaatgaagt gtattggggc ttcttcaaaa aacatcatca tcatcaccat 1500

<210> 7

<211> 480

<212> PRT

<213> Artificial sequence (Unknown)

<400> 7

Met Leu Lys Thr Pro Asp Tyr Glu Phe Trp Phe Ala Val Gly Ser Gln

1 5 10 15

Pro Leu Tyr Gly Pro Glu Ala Leu Glu Gln Val Glu Lys Asp Gly Arg

20 25 30

Lys Leu Val Asp Gly Leu Asn Ala Ser Gly Lys Leu Pro Tyr Lys Val

35 40 45

Val Phe Lys Leu Val Ala Thr Thr Thr Ala Asp Ser Ile Thr Lys Phe Met

50 55 60

Lys Asp Ala Asn Tyr Asn Asp Lys Val Ala Phe Val Met Thr Trp Met

65 70 75 80

His Thr Phe Ser Pro Ala Lys Asn Trp Ile Arg Gly Thr Gln Leu Leu

85 90 95

Gln Lys Pro Leu Leu His Leu Ala Thr Gln Met Leu Asp His Ile Pro

100 105 110

Phe Asp Thr Ile Asp Phe Asp Tyr Met Asn Leu Asn Gln Ser Ala His

115 120 125

Gly Asp Arg Glu Tyr Asp Tyr Ile Asn Ala Arg Leu Gly Val Pro Glu

130 135 140

Lys Ile Val Tyr Gly Trp Trp Asn Asp Pro Glu Val Gln Glu Glu Ile

145 150 155 160

Ala Asp Trp Gln Lys Val Asn Ile Ala Tyr Asp Met Ser Phe Lys Ile

165 170 175

Lys Ile Ala Arg Phe Gly Asp Thr Met Arg Asp Val Ala Val Thr Glu

180 185 190

Gly Asp Lys Val Ala Ala Gln Ile Lys Leu Gly Trp Thr Val Asp Tyr

195 200 205

Trp Pro Val Ala Glu Leu Val Ala Ala Val Asn Ala Val Ser Glu Ala

210 215 220

Glu Ile Asp Glu Gln Tyr Lys Arg Leu Glu Glu Asn Tyr Asp Met Val

225 230 235 240

Glu Gly Asp Asn Asp His Asp Lys Tyr Val His Ser Val Arg Tyr Gln

245 250 255

Leu Arg Glu Tyr Leu Gly Ile Lys His Phe Leu Asp Glu Lys Gly Tyr

260 265 270

Asp Cys Phe Thr Thr Asn Phe Gln Asp Leu Glu Gly Leu Glu Gln Leu

275 280 285

Pro Gly Leu Ala Val Gln Leu Leu Met Ile Asp Gly Tyr Gly Phe Tyr

290 295 300

Ala Glu Gly Asp Phe Lys Ser Ala Gly Leu Ser Arg Leu Leu Lys Ile

305 310 315 320

Ala Ala Glu Asn Lys Glu Thr Ala Phe Met Glu Asp Tyr Thr Leu Asp

325 330 335

Leu Arg Ser Gly His Gln Ala Ile Met Gly Ser His Met Leu Glu Val

340 345 350

Asp Pro Thr Leu Ala Ser Asp Lys Pro Arg Val Glu Val His Pro Leu

355 360 365

Asp Ile Gly Asp Lys Ala Asp Pro Ala Arg Leu Val Phe Thr Gly Ser

370 375 380

Glu Gly Glu Gly Ile Asp Val Thr Leu Ser Tyr Phe Asp Asp Gly Tyr

385 390 395 400

Lys Phe Ile Ala Tyr Asp Val Asp Cys Ala Lys Pro Glu Lys Glu Met

405 410 415

Pro Asn Leu Pro Val Ala Lys Gln Met Trp Thr Pro Lys Cys Gly Leu

420 425 430

Lys Lys Gly Ala Thr Ala Trp Met His Asn Gly Gly Gly His His Thr

435 440 445

Val Leu Ser Met Asn Leu Ser Met Asp Gln Met Ala Thr Leu Ala Asn

450 455 460

Leu Phe Gly Ile Glu Leu Ile Asp Ile Lys His His His His His His His His

465 470 475 480

<210> 8

<211> 1440

<212>DNA

<213> Artificial sequence (Unknown)

<400> 8

atgctgaaaa cacctgatta tgagttctgg ttcgccgttg gtagccagcc gctgtatggt 60

ccggaagcac tggaacaggt ggaaaaagat ggccgcaaac tggtggatgg cctgaatgca 120

agcggcaaac tgccgtataa agttgtgttc aaactggttg ccaccaccgc cgatagcatt 180

accaaattca tgaaagatgc aaattacaac gacaaagttg catttgtgat gacctggatg 240

cataccttca gtccggccaa aaattggatt cgtggcaccc agctgctgca gaaaccgctg 300

ctgcatctgg caacccagat gctggatcat attccgttcg ataccattga cttcgattat 360

atgaatctga atcagagcgc acatggtgat cgcgaatatg attatattaa tgcacgcctg 420

ggcgttccgg aaaaaattgt gtatggttgg tggaatgatc cggaagttca ggaagaaatt 480

gcagattggc agaaagtgaa tattgcatac gatatgagct tcaaaattaa gattgcccgc 540

ttcggcgata ccatgcgtga tgttgcagtg accgaaggcg ataaagtggc cgcacagatt 600

aaactgggtt ggaccgttga ttatggccg gtggccgaac tggtggcagc cgtgaatgca 660

gttagcgaag ccgaaattga tgaacagtat aaacgtctgg aagaaaatta tgatatggtt 720

gaaggcgata atgatcatga taaatatgtt catagcgtgc gctatcagct gcgcgaatat 780

ctgggtatta aacacttcct ggatgaaaaa ggttatgatt gtttcaccac caacttccag 840

gatctggaag gtctggaaca gctgccgggc ctggccgtgc agctgttaat gattgatggc 900

tatggcttct atgcagaagg tgacttcaaa agcgcaggcc tgagccgtct gctgaaaatt 960

gcagccgaaa ataaagaaac cgcattcatg gaagattata ccctggatct gcgcagcggt 1020

catcaggcaa ttatgggtag tcacatgctg gaagttgatc cgaccctggc aagcgataaa 1080

ccgcgtgttg aagttcatcc gctggatatt ggtgataaag cagatccggc ccgtctggtg 1140

ttcaccggca gcgaaggtga aggcattgat gttaccctga gctacttcga tgatggttat 1200

aaattcattg cctatgatgt tgattgcgcc aaaccggaaa aagaaatgcc gaatctgccg 1260

gttgccaaac agatgtggac cccgaaatgc ggtctgaaaa aaggtgcaac cgcctggatg 1320

cataatggcg gcggccatca taccgtgctg agtatgaatc tgagtatgga tcagatggcc 1380

accctggcca atctgttcgg cattgaactg attgatatta aacatcatca ccatcatcac 1440

Claims (3)

1. An L-arabinose epimerase mutant, which is obtained by site-directed mutagenesis of an amino acid with a sequence shown in SEQ ID NO.3, wherein the mutation is one of the following: (1) glycine G at position 304 is mutated to tyrosine Y; (2) Glycine G at position 304 is mutated to tyrosine Y and glycine G at position 75 is mutated to phenylalanine F; (3) Glycine G at position 304 to tyrosine Y, glycine G at position 75 to phenylalanine F, and alanine a at position 274 to cysteine C; (4) Glycine G at position 304 was mutated to tyrosine Y, glycine G at position 75 was mutated to phenylalanine F, alanine a at position 274 was mutated to cysteine C, and alanine a at position 167 was mutated to arginine R.

2. Use of the L-arabinose epimerase mutant according to claim 1 for catalyzing isomerization of D-galactose to prepare D-tagatose.

3. The application according to claim 2, characterized in that it is: wet thalli or wet bacteria obtained by fermenting and culturing recombinant genetic engineering bacteria containing L-arabinose epimerase mutant coding genesImmobilized enzyme preparation obtained by immobilizing supernatant or somatic cells obtained by ultrasonic disruption of the body is used as a catalyst, D-galactose is used as a substrate, and Mn is used as a catalyst ²⁺ And/or Co ²⁺ In the presence of KH of 6.0-7.0 ₂ PO ₄ And (3) reacting in NaOH buffer solution at 65-80 ℃, and separating and purifying the reaction solution after the reaction is finished to obtain the D-tagatose.