WO2021128432A1 - 一种l-阿拉伯糖异构酶异构体及其应用 - Google Patents

一种l-阿拉伯糖异构酶异构体及其应用 Download PDF

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WO2021128432A1
WO2021128432A1 PCT/CN2019/130811 CN2019130811W WO2021128432A1 WO 2021128432 A1 WO2021128432 A1 WO 2021128432A1 CN 2019130811 W CN2019130811 W CN 2019130811W WO 2021128432 A1 WO2021128432 A1 WO 2021128432A1
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mutant
arabinose
epimerase
mutation
seq
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French (fr)
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柳志强
贾东旭
孙晨奕
郑裕国
金利群
彭晨
陈德水
廖承军
程新平
李勉
毛宝兴
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浙江工业大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
    • C12Y503/01004—L-Arabinose isomerase (5.3.1.4)

Definitions

  • the invention relates to an L-arabinose isomerase isomer mutant and its application in preparing D-tagatose by catalyzing the isomerization of D-galactose by microorganisms.
  • D-tagatose is a rare ketulose found in nature and an isomer of D-galactose. It was first discovered in the gum of a tropical evergreen plant, and later in yogurt and cheese. D-tagatose has the properties and effects of high sweetness, low calories, low absorption rate, effective reduction of blood sugar, anti-caries, and improvement of 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. Chemical methods have problems such as high cost, high acid and alkali dosage, serious pollution, complex product components, and difficult separation and purification. In contrast, the biological method of preparing D-tagatose has more advantages and has gradually become a research hotspot in recent years.
  • the process of preparing D-tagatose by biological method uses D-galactose as a raw material, and uses L-arabinose isomerase (L-arabinose isomerase, LAI for short) to perform a catalytic reaction to generate D-tagatose in one step.
  • L-arabinose isomerase L-arabinose isomerase, LAI for short
  • the raw material D-galactose is cheap and easy to obtain
  • the overall reaction process is mild and easy to control
  • the reaction process meets the requirements of environmental protection, which is an optimal sugar production method instead of chemical methods.
  • Research reports point out that high temperature and weakly acidic reaction conditions are more conducive to the biotransformation of D-tagatose.
  • the substrate concentration when preparing D-tagatose from D-galactose catalyzed by biological methods is low, all below 100g/L, which is not conducive to the application in industrial production.
  • Bioinformatics takes nucleic acid, protein and other biological macromolecule databases as the main object, assisting computers to compare and analyze biological information, and obtain rational information such as gene coding, protein structure and function, and their relationships.
  • biotechnology methods such as bioinformatics and genetic engineering to screen and transform to obtain new high-temperature and acid-resistant LAI enzyme preparations is of great significance to meet the people's growing sugar demand.
  • the purpose of the present invention is to provide a high-temperature and acid-resistant L-arabinose isomerase isomer mutant and its application in preparing D-tagatose by catalyzing the isomerization of D-galactose by microorganisms.
  • a mutant of L-arabinose epimerase which is derived from the amino acid sequence shown in SEQ ID NO. 3 through site-directed mutation, and the mutation site is one or more of the following: (1) 304th, (2) 75th, (3) 274th, (4) 167th.
  • the mutant is derived from the amino acid sequence shown in SEQ ID NO. 3 through one or more of the following mutations: (1) Mutation of glycine G at position 304 to histidine H, isoleucine I. Tyrosine Y or Tryptophan W, (2) Mutation of glycine G at position 75 to lysine K, methionine M, phenylalanine F or proline P, (3) Alanine at position 274 G mutation to serine S, threonine T, tyrosine Y, valine V or cysteine C, (4) 167 alanine A mutation to asparagine N, aspartic acid D or arginine Amino acid R.
  • mutations (1) Mutation of glycine G at position 304 to histidine H, isoleucine I. Tyrosine Y or Tryptophan W, (2) Mutation of glycine G at position 75 to lysine K, methionine M, phenylalanine F or proline P, (3)
  • the high temperature resistant TIM barrel protease mutant is obtained by mutating the amino acid shown in SEQ ID NO. 3 through one or more of the following sites: (1) Mutation of glycine G at position 304 to tyrosine Y, (2) Glycine G at position 75 was mutated to phenylalanine F, (3) Alanine G at position 274 was changed to cysteine C, and (4) Alanine A at position 167 was mutated to arginine R.
  • the sequence of the L-arabinose epimerase mutant is shown in SEQ ID NO. 7 (the coding gene is shown in SEQ ID NO. 8).
  • the invention also relates to the application of the L-arabinose epimerase mutant in catalyzing the isomerization of D-galactose to prepare D-tagatose.
  • the application is: wet bacteria obtained by fermentation culture of recombinant genetically engineered bacteria containing L-arabinose epimerase encoding gene, or supernatants or bacteria obtained by ultrasonic disintegration of wet bacteria
  • the immobilized enzyme preparation obtained by cell immobilization is used as a catalyst, using D-galactose as a substrate, in the presence of Mn 2+ and/or Co 2+ , in a 6.0-7.0 KH 2 PO 4 -NaOH buffer, The reaction is conducted 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 the bacterial cells, suspend them in Na 2 HPO 4 -NaH 2 PO 4 buffer (pH 6.5), add Celite 545, and stir appropriately. Polyethyleneimine aqueous solution was added to flocculate under the conditions of 25°C and 100r/min, and then trimethylol phosphate (THP) aqueous solution was added, and the cross-linking reaction was carried out at 25°C and 100r/min for 2h.
  • THP trimethylol phosphate
  • the filter cake is washed with distilled water and then extruded into a long strip with an axial extruder, and then air-dried at room temperature, then crushed into granules (preferably with a particle size of 0.5-2 mm) to obtain immobilized enzyme granules.
  • the coding gene sequence of the L-arabinose epimerase mutant is shown in SEQ ID NO.8.
  • the present invention provides a brand-new L-arabinose epimerase and its mutant.
  • the mutant has a higher optimal reaction temperature of 75°C, and its enzyme activity is comparable to that of the original enzyme. The ratio is increased by 280%.
  • the product yield can reach up to 73.3%.
  • the recombinase was immobilized and continuously catalyzed for 30 batches, and the conversion rate was all greater than 73%.
  • the invention solves the problem of low efficiency of the existing enzyme-catalyzed preparation of D-tagatose, and the obtained continuous conversion effect that is better than that reported in the literature is of great significance for improving the industrialization level of D-tagatose.
  • Figure 1 is a schematic diagram of the optimum temperature of the recombinase
  • Figure 2 is a schematic diagram of the influence of metal ions on the activity of recombinase
  • Example 1 Screening and vitality determination of a new type of LAI
  • New LAIs were obtained from the NCBI database, which were derived from Novibacillus thermophilus (GenBank No. WP_077718551.1), Lactobacillus Oris (GenBank No. WP_003716014.1), Pseudothermotoga Thermorum (GenBank No. WP_013932424.1), and named NtLAI, LoLAI and PtLAI.
  • the codon optimization was carried out, and three selected nucleotide sequences were synthesized by the conventional operation of genetic engineering by a fully synthetic method, such as SEQ ID NO.2 and SEQ ID NO.4, respectively.
  • SEQ ID NO. SEQ ID NO.2 and SEQ ID NO.4
  • 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 to LB liquid medium; 121°C high pressure Sterilize for 20 minutes; add kanamycin at a final concentration of 100 ⁇ g/mL before use.
  • the genetically engineered bacteria were inoculated into LB liquid medium containing kanamycin at a final concentration of 100 ⁇ g/mL, and cultured at 37°C and 150r/min to an OD 600 of about 0.6-0.8 to obtain seed liquid; the volume concentration of the seed liquid was 2% (v/v) Inoculation amount to fresh LB medium containing 100 ⁇ g/mL kanamycin at a final concentration of 100 ⁇ g/mL, culture at 37°C, 150r/min OD 600 to 0.4 ⁇ 0.6, and then add the final concentration to the culture solution 0.5mM IPTG was induced and expressed at 28°C for 12h, centrifuged at 6000r/min at 4°C for 10min, the supernatant was discarded, the wet cells were washed twice with 0.85% normal saline, and the wet cells were collected for use.
  • Ultrasonic breaking is used to ultrasonically break the wet bacteria. Take 1 g of the prepared wet bacterial cells, suspend them in 50 mL of 50 mM KH 2 PO 4 -NaOH buffer (pH 6.5), sonicate the suspension for 15 min under 39W conditions, prepare a suspension after sonication, centrifuge, and collect the supernatant.
  • Enzyme activity reaction system 50 g/L D-galactose, 1 mM MnCl 2 , 50 ⁇ L 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 boiling water for 10 minutes to terminate the reaction, after diluting 10 times, use a 0.22 ⁇ m filter membrane to pass through the membrane; use HPLC to detect the concentration of D-tagatose.
  • the analytical column is an Aminex HPX-87H column (300 ⁇ 7.8mm, 9 ⁇ m, Bo Le Life Medical Products Co., Ltd.). Waters 2414 refractive index detector, Waters 1525 pump, Waters 717 sampler.
  • Enzyme activity definition At 40°C and pH 6.5, the amount of enzyme required to isomerize D-galactose to 1 ⁇ mol D-tagatose per minute is defined as one enzyme activity unit (U).
  • the primers are:
  • PCR reaction system 2 ⁇ Phanta Max Buffer (containing Mg 2+ ) 25 ⁇ L, dNTPs 10mM, forward primer 304G 2 ⁇ L (5pmol/ ⁇ L, the same below), reverse primer 304G 2 ⁇ L (5pmol/ ⁇ L, the same below), template DNA 1 ⁇ L (20ng/ ⁇ L, the same below), Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 O to 50 ⁇ L.
  • PCR amplification conditions were 95°C for 3min; (95°C for 15s, 64°C for 15s, 72°C for 6.5min) 30 cycles; 72°C for 5min.
  • the composition of the reaction mixture 5 g/L of D-galactose, 1 mM MnCl 2 , and then adding 50 mM KH 2 PO 4 -NaOH buffer (pH 6.5) to 1L of the total reaction system for use.
  • the reaction system includes 2 ⁇ L of the reaction solution, 5 ⁇ L of 1.5% (w/v) cysteine hydrochloride, and 150 ⁇ L of 70% (w/ w) Concentrated sulfuric acid, 5 ⁇ L of 0.12% (w/v) carbazole ethanol, and observe the color change after incubating at 60°C for 10 min.
  • the result of this example is: 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 remained unchanged or decreased due to the mutation of glycine (G) at position 304 to amino acids other than H, I, Y, and W.
  • G glycine
  • LoLAI-1 The LoLAI-G304Y mutant with the most increased enzyme activity was recorded as LoLAI-1, and the recombinant strain E. coli BL21(DE3)/pET28b/LoLAI-1 was obtained.
  • the mutation primers for site-directed mutation were designed, using rapid PCR technology, using the recombinant vector pET28b/LoLAI-1 as the template, a single mutation was introduced at position 75, and the primers were:
  • Forward primer 75G CGACAAAGTTGCA NNN GTGATGACCT (mutated bases are underlined)
  • Reverse primer 75G AGGTCATCAC NNN TGCAACTTTGTCG (mutated bases are underlined)
  • PCR reaction system 2 ⁇ Phanta Max Buffer (containing Mg 2+ ) 25 ⁇ L, dNTPs 10mM, forward primer 75G 2 ⁇ L, reverse primer 75G 2 ⁇ L, template DNA 1 ⁇ L, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 O to 50 ⁇ L.
  • PCR amplification conditions were 95°C for 3min; (95°C for 15s, 60.5°C for 15s, 72°C for 6.5min) 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 ⁇ g/mL kanamycin and cultured overnight at 37°C.
  • the mutants were initially screened using the cysteine carbazole method (the operation is the same as the "high-throughput screening of positive transformants" in Example 2), and the positive clones were subjected to the accurate determination of enzyme activity (the operation was the same as that in Example 1. Enzyme activity determination of positive transformants").
  • the result of this example is: 399 strains of recombinant transformed bacteria were initially screened, and 4 mutant strains with increased enzyme activity were screened out, and then the enzyme activity was accurately measured. The specific results are shown in Table 3. After analysis, it was determined that the enzyme activity of the remaining 395 strains of recombinant bacteria remained unchanged or decreased due to the mutation of glycine (G) at position 75 to amino acids other than K, M, F, and P.
  • G glycine
  • LoLAI-2 The LoLAI-G304Y-G75F mutant with the most increased enzyme activity was recorded as LoLAI-2, and the recombinant strain E. coli BL21(DE3)/pET28b/LoLAI-2 was obtained.
  • the mutation primers for site-directed mutation were designed. Using rapid PCR technology, using the recombinant vector pET28b/LoLAI-2 as the template, a single mutation was introduced at position 274.
  • the primers were:
  • Forward primer 274G GGTTATGAT NNN TTCACCACCAACTTCCAGG (mutated bases are underlined)
  • Reverse primer 274G CCTGGAAGTTGGTGGTGAA NNN ATCATAACC (mutated bases are underlined)
  • PCR reaction system 2 ⁇ Phanta Max Buffer (containing Mg 2+ ) 25 ⁇ L, dNTPs 10mM, forward primer 274G 2 ⁇ L, reverse primer 274G 2 ⁇ L, template DNA 1 ⁇ L, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 O to 50 ⁇ L.
  • PCR amplification conditions were 95°C for 3min; (95°C for 15s, 59.5°C for 15s, 72°C for 6.5min) 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 ⁇ g/mL kanamycin and cultured overnight at 37°C.
  • the mutants were initially screened using the cysteine carbazole method (the operation is the same as the "high-throughput screening of positive transformants" in Example 2), and the positive clones were subjected to the accurate determination of enzyme activity (the operation was the same as that in Example 1. Enzyme activity determination of positive transformants").
  • the result of this example is: 735 strains of recombinant transformed bacteria were initially screened, and 5 mutant strains with increased enzyme activity were screened out, and then the enzyme activity was measured on them.
  • the specific results are shown in Table 4. After analysis, it was determined that the enzyme activity of the remaining 730 strains of recombinant bacteria remained unchanged or decreased due to the mutation of alanine (G) at position 274 to amino acids other than S, T, Y, V, and C.
  • LoLAI-3 The LoLAI-G304Y-G75F-G274C mutant with the most increased enzyme activity was recorded as LoLAI-3, and the recombinant strain E. coli BL21(DE3)/pET28b/LoLAI-3 was obtained.
  • the mutation primers for site-directed mutation were designed. Using rapid PCR technology, using the recombinant vector pET28b/LoLAI-3 as the template, a single mutation was introduced at position 167.
  • the primers were:
  • Forward primer 167A GGCAGAAAGTG NNN ATTGCATACGATATGAGC (mutated bases are underlined)
  • Reverse primer 167A GCTCATATCGTATGCAAT NNN CACTTTCTGCC (mutated bases are underlined)
  • PCR reaction system 2 ⁇ Phanta Max Buffer (containing Mg 2+ ) 25 ⁇ L, dNTPs 10mM, forward primer 167A 2 ⁇ L, reverse primer 167A 2 ⁇ L, template DNA 1 ⁇ L, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 O to 50 ⁇ L.
  • the PCR amplification conditions were 95°C for 3min; (95°C for 15s, 59°C for 15s, 72°C for 6.5min) 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 ⁇ g/mL kanamycin and cultured overnight at 37°C.
  • the mutants were initially screened by the cysteine carbazole method (the operation is the same as the "high-throughput screening of positive transformants” in Example 2), and the enzyme activity is accurately determined (the operation is the same as the "positive transformation” in Example 1 Sub-enzyme activity determination").
  • the result of this example is: 444 strains of recombinant transformed bacteria were initially screened, and 3 mutant strains with increased enzyme activity were screened out, and then the enzyme activity was measured on them. The specific results are shown in Table 5. After analysis, it was determined that the enzyme activity of the remaining 441 strains remained unchanged or decreased due to the mutation of alanine (A) at position 167 to other amino acids other than N, D, and R.
  • A alanine
  • LoLAI-4 The LoLAI-G304Y-G75F-G274C-A167R mutant with the most increased enzyme activity was recorded as LoLAI-4, and the recombinant strain E. coli BL21(DE3)/pET28b/LoLAI-4 was obtained.
  • E.coli BL21(DE3)/pET28b/LoLAI 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 ⁇ g/mL kanamycin, and cultured at 37°C, 150r/min, OD 600 approximately 0.6 ⁇ 0.8 to obtain seed liquid; inoculate the seed liquid with 2% (v/v) inoculum into fresh LB liquid medium containing 100 ⁇ g/mL kanamycin, and cultivate at 37°C, 150r/min OD 600 to 0.4 ⁇ 0.6, then add IPTG with a final concentration of
  • the wet bacterial cells were ultrasonically broken by the ultrasonic breaking method, and the supernatant was collected.
  • nickel-NTA sepharose column for purification, equilibrate the column with equilibration buffer (20mM phosphate buffer, 300mM NaCl, 20mM imidazole, pH 8.0), and then use eluent (50mM phosphate buffer, 300mM NaCl, 500mM imidazole, pH 8.0) was used for elution. According to the signal response of the UV detector, the corresponding eluate was collected, which is the respective pure enzyme solution.
  • equilibration buffer 20mM phosphate buffer, 300mM NaCl, 20mM imidazole, pH 8.0
  • eluent 50mM phosphate buffer, 300mM NaCl, 500mM imidazole, pH 8.0
  • the pure enzyme solution in Example 6 was used as the enzyme for conversion, and the optimal reaction temperature of the enzyme was determined.
  • the reaction system is: 50 g/L D-galactose, 1 mM MnCl 2 , 50 ⁇ L of the pure enzyme solution obtained in the above example, and then add 50 mM KH 2 PO 4 -NaOH buffer (pH 6.5) to the total system 1 mL.
  • the activity of recombinant LAI was measured at different transformation temperatures: 60, 65, 70, 75, 80, 85, 90°C (the operation method is the same as the "determination of enzyme activity of recombinant bacteria" in Example 1). It can be seen from Figure 1 that the optimal reaction temperature of LoLAI-4 is 75°C, which is 10°C higher than that of the original enzyme LoLAI.
  • Example 8 The influence of metal ions on the enzyme activity of the optimal mutant of LAI
  • Example 6 Using the pure enzyme solution in Example 6 as the enzyme for transformation, the effect of metal ions on the enzyme activity of the recombinase was measured.
  • 1mL reaction system comprising: 50mM KH 2 PO 4 -NaOH buffer (pH 6.5), 50g / L D- galactose, 50 ⁇ L 1mM pure enzyme solution and different metal ions (anions as Cl -).
  • 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 measured at 40°C.
  • Example 9 Preparation of D-tagatose from whole cells of mutant recombinant bacteria with original enzyme and mutant enzyme
  • 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.
  • D-tagatose was prepared by biotransformation.
  • 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 (pH 6.5) to the total system 100mL.
  • the reaction system was reacted at 65°C and 150r/min for 8h. Samples were taken every 1h, centrifuged, filtered with 0.22 ⁇ m membrane, and then subjected to HPLC to detect the concentration of D-tagatose.
  • E.coli BL21(DE3)/pET28b/LoLAI-4 cells suspend them in 50 mL of Na 2 HPO 4 -NaH 2 PO 4 buffer (pH 6.5), add 0.3 g of Celite 545, and stir appropriately.
  • the filter cake is washed with distilled water and then extruded into long strips with an axial extruder. After air drying at room temperature, it is pulverized into granules (preferably with a particle size of 0.5-2 mm) to obtain immobilized granules of LoLAI-5 mutant.
  • D-tagatose is prepared by biotransformation.
  • the 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. The reaction was carried out at 65°C, 200r/min, and isomerization for 2h.
  • the reaction solution was centrifuged at 4°C, a small amount of supernatant was filtered through a 0.22 ⁇ m membrane and 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 for 30 batches of continuous catalysis, the product yields are all greater than 73%.

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Abstract

提供了一种L-阿拉伯糖差向异构酶突变体，及其在微生物催化D-半乳糖异构化制备D-塔格糖中的应用。该L-阿拉伯糖差向异构酶突变体，由SEQ ID NO：3所示氨基酸经第304位、第75位、第274位和第167位中一个或多个氨基酸定点突变而得。该突变体具有高最适反应温度75℃，其酶活与原始酶相比提高280％，D-塔格糖得率最高可达73.3％。将重组酶进行固定化并连续催化30批次，转化率均大于73％。

Description

一种L-阿拉伯糖异构酶异构体及其应用 (一)技术领域
本发明涉及一种L-阿拉伯糖异构酶异构体突变体，及其在微生物催化D-半乳糖异构化制备D-塔格糖中的应用。
(二)背景技术
D-塔格糖(D-tagatose)是自然界中发现的稀有己酮糖，是D-半乳糖的异构体。它最早被发现存在于一种热带长青植物的树胶中，后来在酸奶、奶酪中也有发现。D-tagatose具有甜度高、热量低、吸收率低、有效降低血糖及抗龋齿、改善肠道菌群等性质与功效。美国食品及药物管理局已通过对D-tagatose的安全认证，准许其用于食品领域。D-tagatose的生产方法主要有化学法和生物法。化学法存在成本较高、酸碱用量高、污染严重、产物成分复杂、分离纯化难等问题。相比之下，生物法制备D-tagatose更具有优势，近年来逐渐成为研究热点。
生物法制备D-tagatose的工艺是以D-半乳糖为原料，利用L-阿拉伯糖异构酶(L-arabinose isomerase，简称LAI)进行催化反应，一步法生成D-tagatose。其中，原料D-半乳糖廉价易得、整体反应过程温和易控、反应过程符合环保要求，是一种替代化学法的最优制糖方法。研究报道指出，高温和弱酸性的反应条件更有利于D-tagatose的生物转化。当前，生物法催化D-半乳糖制备D-tagatose时底物浓度偏低，均处于100g/L以下，不利于应用到工业化生产中。除此之外，可开发的酶源较少，该弊端制约着D-tagatose的生产水平。
生物信息学以核酸、蛋白质等生物大分子数据库为主要对象，辅助计算机对生物信息进行比较分析，从中获取基因编码、蛋白质结构功能及其相互关系等理性信息。在此背景下，利用生物信息学和基因工程等现代生物技术手段，筛选改造获得新型耐高温耐酸的LAI酶制剂，对于满足人民群众日益增长的摄糖需求具有重要意义。
(三)发明内容
本发明目的是提供一种耐高温耐酸的L-阿拉伯糖异构酶异构体突变体，及其在微生物催化D-半乳糖异构化制备D-塔格糖中的应用。
本发明采用的技术方案是：
一种L-阿拉伯糖差向异构酶突变体，由序列如SEQ ID NO.3所示氨基酸经定点突变而来，所述突变的位点为下列中的一个或多个：(1)第304位、(2)第75位、(3)第274位、(4)第167位。
优选的，所述突变体由序列如SEQ ID NO.3所示氨基酸经下列之一或多个位点突变而得：(1)第304位甘氨酸G突变为组氨酸H、异亮氨酸I、酪氨酸Y或色氨酸W，(2)第75位甘氨酸G突变为赖氨酸K、蛋氨酸M、苯丙氨酸F或脯氨酸P，(3)第274位丙氨酸G突变为丝氨酸S、苏氨酸T、酪氨酸Y、缬氨酸V或半胱氨酸C，(4)167位丙氨酸A突变为天冬酰胺N、天冬氨酸D或精氨酸R。
进一步，所述耐高温TIM barrel蛋白酶突变体由序列如SEQ ID NO.3所示氨基酸经下列之一或多个位点突变而得：(1)第304位甘氨酸G突变为酪氨酸Y、(2)第75位甘氨酸G突变为苯丙氨酸F、(3)第274位丙氨酸G突半胱氨酸变C、(4)167位丙氨酸A突变为精氨酸R。
更为优选的，所述L-阿拉伯糖差向异构酶突变体序列如SEQ ID NO.7所示(其编码基因如SEQ ID NO.8所示)。
本发明还涉及所述L-阿拉伯糖差向异构酶突变体在催化D-半乳糖异构化制备D-塔格糖中的应用。
具体的，所述应用为：以含L-阿拉伯糖差向异构酶编码基因的重组基因工程菌经发酵培养获得的湿菌体或湿菌体经超声破碎后获得的上清液或菌体细胞经固定化获得的固定化酶制剂作为催化剂，以D-半乳糖为底物，在Mn 2+和/或Co 2+存在下，在6.0～7.0的KH 2PO 4-NaOH缓冲液中，65～80℃温度下反应，反应结束后，反应液分离纯化，获得D-塔格糖。
所述固定化酶制剂制备方法如下：称取菌体细胞，用Na 2HPO 4-NaH 2PO 4缓冲液(pH 6.5)悬浮，加入Celite 545，适当搅拌。加入聚乙烯亚胺水溶液在25℃、100r/min条件下絮凝，再加入三羟甲基磷(THP)水溶液，在25℃、100r/min条件下交联反应2h。然后抽滤，滤饼用蒸馏水洗涤后用轴向挤压机挤压成长条状，室温风干后，粉碎成粒(优选粒径为0.5～2mm)，获得酶的固定化颗粒。
优选的，所述L-阿拉伯糖差向异构酶突变体编码基因序列如SEQ ID NO.8所示。
本发明有益效果主要体现在：本发明提供了一种全新的L-阿拉伯糖差向异构酶及其突变体，该突变体具有较高最适反应温度75℃，其酶活与原始酶相比提高280％。使用本突变体生产D-tagatose，产物得率最高可达73.3％。将重组酶进行固定化并连续催化30批次，转化率均大于73％。本发明解决了现有酶催化制备D-tagatose效率低下的难题，获得的优于文献报道的连续转化效果对提升D-tagatose工业化水平具有重要意义。
(四)附图说明
图1为重组酶的最适温度示意图；
图2为金属离子对重组酶活力的影响示意图；
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1：新型LAI的筛选与活力测定
1、酶的来源与重组菌的构建
从NCBI数据库中获得新型LAI，分别来源于Novibacillus thermophilus(GenBank编号WP_077718551.1)、Lactobacillus oris(GenBank编号WP_003716014.1)、Pseudothermotoga thermarum(GenBank编号WP_013932424.1)，并命名为NtLAI、LoLAI和PtLAI。根据氨基酸序列，依据大肠杆菌密码子偏好性进行密码子优化，通过基因工程的常规操作以全合成的方法合成了三条选择的核苷酸序列，分别如SEQ ID NO.2、SEQ ID NO.4和SEQ ID NO.6所示；编码酶的氨基酸序列分别如SEQ ID NO.1、SEQ ID NO.3和SEQ ID NO.5所示。在核酸序列末端加入6×His-tag标签，两端加入酶切位点Xba I和Xho I，将该基因克隆至pET28b(+)对应的Xba I和Xho I位点，获得重组表达质粒pET28b/NtLAI、pET28b/LoLAI和pET28b/PtLAI。
2、重组菌的转化与诱导表达
将获得的重组表达质粒pET28b/NtLAI、pET28b/LoLAI和pET28b/PtLAI转化至 Escherichia coli BL21(DE3)受体菌中，涂布于含终浓度为100μg/mL卡那霉素的LB琼脂平板上，37℃下培养过夜，第2天于平板上长出的菌落中随机挑取克隆并抽提质粒进行琼脂糖凝胶电泳鉴定，获得含LAI基因的基因工程菌。
LB液体培养基组成：胰蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，溶剂为水，pH值自然；LB固体培养基在LB液体培养基中添加15g/L琼脂；121℃高压灭菌20min；使用前添加终浓度100μg/mL卡那霉素。
将基因工程菌接种至含终浓度100μg/mL卡那霉素的LB液体培养基，在37℃、150r/min培养至OD 600约0.6～0.8，获得种子液；将种子液以体积浓度2％(v/v)接种量接种至新鲜的含有终浓度100μg/mL卡那霉素的LB培养基中，于37℃、150r/min培养OD 600至0.4～0.6，再向培养液中加入终浓度0.5mM的IPTG，于28℃下诱导表达12h后，4℃、6000r/min离心10min，弃去上清液，用0.85％的生理盐水清洗两遍湿菌体，并收集湿菌体，备用。
3、重组菌的酶活测定
采用超声破碎方法对湿菌体进行超声破碎。取1g制备的湿菌体，用50mL 50mM KH 2PO 4-NaOH缓冲液(pH 6.5)悬浮，在39W条件下超声破碎15min，制备获得超声破碎后的混悬液，离心，收集上清液。
酶活反应体系：50g/L的D-半乳糖、1mM MnCl 2、50μL上述上清液(酶液)，再加入适量50mM KH 2PO 4-NaOH缓冲液(pH 6.5)至总体积1mL。反应条件：于40℃条件下反应10min，沸水煮沸10min终止反应，稀释10倍后，使用0.22μm滤膜过膜；采用HPLC检测D-tagatose浓度。分析柱为Aminex HPX-87H柱(300×7.8mm，9μm，伯乐生命医学产品有限公司)。Waters 2414示差折光检测器，Waters 1525泵，Waters 717进样器。
酶活定义：40℃和pH 6.5下，每分钟将D-半乳糖异构化生成1μmol D-tagatose所需酶量定义为一个酶活单位(U)。
表1：重组酶的酶活测定
Figure PCTCN2019130811-appb-000001
Figure PCTCN2019130811-appb-000002
实施例2：LoLAI单位点突变体的构建与筛选
1、突变体构建
根据LoLAI亲本序列设计定点突变的突变引物，利用快速PCR技术，以重组载体pET28b/LoLAI为模板，对第304位引入单突变，引物为：
正向引物304G：
ATGGCTATGGCTTC NNNGCAGAAGGTGACTTCAAAA(下划线为突变碱基)
反向引物304G：
TTTTGAAGTCACCTTCTGC NNNGAAGCCATAGCCAT(下划线为突变碱基)
PCR反应体系：2×Phanta Max Buffer(含Mg 2+)25μL，dNTPs 10mM，正向引物304G 2μL(5pmol/μL，下同)，反向引物304G 2μL(5pmol/μL，下同)，模板DNA 1μL(20ng/μL，下同)，Phanta Max Super-Fidelity DNA Polymerase 50U，加入ddH 2O至50μL。
PCR扩增条件为95℃3min；(95℃15s，64℃15s，72℃6.5min)30循环；72℃5min。
2、突变体转化表达
取5μL的PCR产物，加入100μL冰浴的E.coli BL21(DE3)感受态细胞悬液中，冰上静置30min，将转化产物于42℃热击90s，迅速置于冰上冷却5min，向管中加入600μL的LB液体培养基，37℃，150r/min培养60min，取100μL上述菌液涂板，待菌液完全被培养基吸收后，37℃倒置培养12h。
3、高通量筛选阳性转化子
反应混合液组成：5g/L的D-半乳糖、1mM MnCl 2，再加入50mM KH 2PO 4-NaOH缓冲液(pH 6.5)至总反应体系1L，备用。
在96孔聚苯乙烯微孔培养板中每孔加入100μL含有终浓度100μg/mL卡那霉素的LB培养液，接种不同的转化菌落，于37℃、150r/min培养OD 600至0.4～0.6，再向培养液中加入终浓度为0.5mM的IPTG，于28℃下诱导表达10h后，4℃、6000r/min离心10min，弃去上清液。取100μL上述反应混合液加入含有菌体的96孔板中，振荡器振荡混匀后在40℃、600r/min反应10min，冰浴10min停止反应。取2μL反应液以半胱氨酸-咔唑显色法筛选突变体，反应体系包括2μL反应液、5μL的1.5％(w/v)半胱氨酸盐酸盐、150μL的70％(w/w)浓硫酸、5μL的0.12％(w/v)咔唑乙醇，60℃下保温10min后观察颜色变化。以重组菌E.coli BL21(DE3)/pET28b/LoLAI的反应为对照，取颜色比E.coli BL21(DE3)/pET28b/LoLAI的反应深的突变株进行酶活精准测定。
4、阳性转化子酶活的精准测定
操作同实施例1的“重组菌的酶活测定”。
该实施例的结果为：对665株重组转化菌初筛，筛选出4株酶活提高的突变株，再对其进行酶活精准测定，具体结果见表2。经分析确定，其余661株重组菌酶活保持不变或下降的原因是第304位甘氨酸(G)突变为H、I、Y和W外的其他氨基酸。
表2：单点突变重组菌的酶活测定
Figure PCTCN2019130811-appb-000003
将酶活提高最多的LoLAI-G304Y突变体记为LoLAI-1，获得重组菌E.coli BL21(DE3)/pET28b/LoLAI-1。
实施例3：LoLAI双位点突变体的构建与筛选
根据实施例2构建的单突变体LoLAI-1序列设计定点突变的突变引物，利用快速PCR技术，以重组载体pET28b/LoLAI-1为模板，对第75位引入单突变，引物为：
正向引物75G：CGACAAAGTTGCA NNNGTGATGACCT(下划线为突变碱基)
反向引物75G：AGGTCATCAC NNNTGCAACTTTGTCG(下划线为突变碱基)
PCR反应体系：2×Phanta Max Buffer(含Mg 2+)25μL，dNTPs 10mM，正向引物75G 2μL，反向引物75G 2μL，模板DNA 1μL，Phanta Max Super-Fidelity DNA Polymerase 50U，加入ddH 2O至50μL。
PCR扩增条件为95℃3min；(95℃15s，60.5℃15s，72℃6.5min)30循环；72℃5min。
PCR产物转化E.coli BL21(DE3)感受态细胞，挑单克隆于含100μg/mL卡那霉素的LB液体培养基中，37℃培养过夜。利用半胱氨酸咔唑法显色法对突变体进行初筛(操作同实施例2的“高通量筛选阳性转化子”)，阳性克隆进行酶活精准测定(操作同实施例1的“阳性转化子酶活测定”)。
该实施例的结果为：对399株重组转化菌初筛，筛选出4株酶活提高的突变株，再对其进行酶活精准测定，具体结果见表3。经分析确定，其余395株重组菌酶活保持不变或下降的原因是第75位甘氨酸(G)突变为K、M、F和P外的其他氨基酸。
表3：双点突变重组菌的酶活测定
Figure PCTCN2019130811-appb-000004
将酶活提高最多的LoLAI-G304Y-G75F突变体记为LoLAI-2，获得重组菌E.coli  BL21(DE3)/pET28b/LoLAI-2。
实施例4：LoLAI三位点突变体的构建与筛选
根据实施例3构建的突变体LoLAI-2序列设计定点突变的突变引物，利用快速PCR技术，以重组载体pET28b/LoLAI-2为模板，对第274位引入单突变，引物为：
正向引物274G：GGTTATGAT NNNTTCACCACCAACTTCCAGG(下划线为突变碱基)
反向引物274G：CCTGGAAGTTGGTGGTGAA NNNATCATAACC(下划线为突变碱基)
PCR反应体系：2×Phanta Max Buffer(含Mg 2+)25μL，dNTPs 10mM，正向引物274G 2μL，反向引物274G 2μL，模板DNA 1μL，Phanta Max Super-Fidelity DNA Polymerase 50U，加入ddH 2O至50μL。
PCR扩增条件为95℃3min；(95℃15s，59.5℃15s，72℃6.5min)30循环；72℃5min。
PCR产物转化E.coli BL21(DE3)感受态细胞，挑单克隆于含100μg/mL卡那霉素的LB液体培养基中，37℃培养过夜。利用半胱氨酸咔唑法显色法对突变体进行初筛(操作同实施例2的“高通量筛选阳性转化子”)，阳性克隆进行酶活精准测定(操作同实施例1的“阳性转化子酶活测定”)。
该实施例的结果为：对735株重组转化菌初筛，筛选出5株酶活提高的突变株，再对其进行酶活测定，具体结果见表4。经分析确定，其余730株重组菌酶活保持不变或下降的原因是第274位丙氨酸(G)突变为S、T、Y、V和C外的其他氨基酸。
表4：三点突变重组菌的酶活测定
Figure PCTCN2019130811-appb-000005
Figure PCTCN2019130811-appb-000006
将酶活提高最多的LoLAI-G304Y-G75F-G274C突变体记为LoLAI-3，获得重组菌E.coli BL21(DE3)/pET28b/LoLAI-3。
实施例5：LoLAI四位点突变体的构建与筛选
根据实施例4构建的突变体LoLAI-3序列设计定点突变的突变引物，利用快速PCR技术，以重组载体pET28b/LoLAI-3为模板，对第167位引入单突变，引物为：
正向引物167A：GGCAGAAAGTG NNNATTGCATACGATATGAGC(下划线为突变碱基)
反向引物167A：GCTCATATCGTATGCAAT NNNCACTTTCTGCC(下划线为突变碱基)
PCR反应体系：2×Phanta Max Buffer(含Mg 2+)25μL，dNTPs 10mM，正向引物167A 2μL，反向引物167A 2μL，模板DNA 1μL，Phanta Max Super-Fidelity DNA Polymerase 50U，加入ddH 2O至50μL。
PCR扩增条件为95℃3min；(95℃15s，59℃15s，72℃6.5min)30循环；72℃5min。
PCR产物转化E.coli BL21(DE3)感受态细胞，挑单克隆于含100μg/mL卡那霉素的LB液体培养基中，37℃培养过夜。利用半胱氨酸咔唑法显色法对突变体进行初筛(操作同实施例2的“高通量筛选阳性转化子”)，进行酶活精准测定(操作同实施例1的“阳性转化子酶活测定”)。
该实施例的结果为：对444株重组转化菌初筛，筛选出3株酶活提高的突变株，再对其进行酶活测定，具体结果见表5。经分析确定，其余441株重组菌酶活保持不变或下降的原因是第167位丙氨酸(A)突变为N、D和R外的其他氨基酸。
表5：四位点突变重组菌的酶活测定
Figure PCTCN2019130811-appb-000007
将酶活提高最多的LoLAI-G304Y-G75F-G274C-A167R突变体记为LoLAI-4，获得重组菌E.coli BL21(DE3)/pET28b/LoLAI-4。
实施例6：重组大肠杆菌发酵产酶与纯化
分别将重组菌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接种至含终浓度100μg/mL卡那霉素的LB液体培养基，在37℃、150r/min培养OD 600约0.6～0.8，获得种子液；将种子液以2％(v/v)接种量接种至新鲜的含有终浓度100μg/mL卡那霉素的LB液体培养基中，于37℃、150r/min培养OD 600至0.4～0.6，再向培养液中加入终浓度为0.5mM的IPTG，于28℃下诱导表达12h后，4℃、6000r/min离心10min，弃去上清液，用0.85％的生理盐水清洗两遍湿菌体，并收集湿菌体。
采用超声破碎方法对湿菌体进行超声破碎，收集上清液。
使用nickel-NTA琼脂糖凝胶柱进行纯化，用平衡缓冲液(20mM磷酸盐缓冲液，300mM NaCl，20mM咪唑，pH 8.0)平衡层析柱，再使用洗脱液(50mM磷酸盐缓冲液，300mM NaCl，500mM咪唑，pH 8.0)进行洗脱，根据紫外检测器的信号响应，收集相应的洗脱液，即为各自纯酶液。
实施例7：纯化LoLAI及其突变体的最适反应温度
将实施例6中的纯酶液作为转化用酶，测定酶的最适反应温度。反应体系为：50 g/L的D-半乳糖、1mM MnCl 2、50μL上述实施例获得的纯酶液，再加入50mM KH 2PO 4-NaOH缓冲液(pH 6.5)至总体系1mL。分别于不同转化温度：60、65、70、75、80、85、90℃测定重组LAI的活力(操作方法同实施例1的“重组菌的酶活测定”)。由图1中可知，LoLAI-4的最适反应温度为75℃，比原始酶LoLAI提高10℃。
实施例8：金属离子对LAI最优突变体酶活的影响
将实施例6中的纯酶液作为转化用酶，测定金属离子对重组酶酶活的影响。1mL反应体系包括：50mM KH 2PO 4-NaOH缓冲液(pH 6.5)、50g/L D-半乳糖、50μL纯酶液和1mM不同金属离子(阴离子为Cl -)。其中，金属离子的选择如下：(1)选用单金属离子：Mn 2+、Co 2+、Mg 2+、Cu 2+、Zn 2+、Ba 2+、Fe 2+、和Ca 2+。于40℃测定LAI的活力。(2)设置组合金属离子，分别为1mM Mn 2+和0.5mM Co 2+、1mM Mn 2+和0.5mM Zn 2+、1mM Mn 2+和0.5mM Mg 2+进行酶活测定。以不加金属离子作为对照。由图2可知，Mn 2+对LAI的酶活有极大的促进作用，并且比组合金属的效果更为明显。
实施例9：原始酶与突变酶突变重组菌全细胞制备D-tagatose
按实施例6的发酵方法，大规模发酵获得重组菌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。分别以上述湿菌体作为生物催化剂，以D-半乳糖为底物，生物转化制备D-tagatose。催化体系包括：不同浓度的D-半乳糖、1mM MnCl 2、15g/L湿菌体，再加入适量50mM KH 2PO 4-NaOH缓冲液(pH 6.5)至总体系100mL。反应体系于65℃、150r/min条件下反应8h。每隔1h取样、离心，用0.22μm膜过滤后进行HPLC检测D-tagatose浓度。由表6可知，E.coli BL21(DE3)/pET28b/LoLAI-4的产物得率最终达到73.3％，高于原始酶E.coli BL21(DE3)/pET28b/LoLAI和其他突变酶的得率。
表6：各重组菌得率的比较
Figure PCTCN2019130811-appb-000008
实施例10：E.coli BL21(DE3)/pET28b/LoLAI-4的固定化与连续转化
制备30％(v/v)THP水溶液：取31.8g的四羟甲基硫酸磷溶于80mL去离子水，3.4g的氢氧化钾溶于10mL去离子水，室温25℃、100r/min条件下将两者缓慢混合，制得30％(v/v)THP水溶液，现用现配，四羟甲基硫酸磷和氢氧化钾按摩尔比1:0.995。
称取5g重组E.coli BL21(DE3)/pET28b/LoLAI-4细胞，分别用50mL的Na 2HPO 4-NaH 2PO 4缓冲液(pH 6.5)悬浮，加入0.3g Celite 545，适当搅拌。加入2mL5％(v/v)聚乙烯亚胺水溶液在25℃、100r/min条件下絮凝，再加入0.25mL体积浓度30％THP水溶液，在25℃、100r/min条件下交联反应2h。然后抽滤，滤饼用蒸馏水洗涤后用轴向挤压机挤压成长条状，室温风干后，粉碎成粒(优选粒径为0.5～2mm)，获得LoLAI-5突变体的固定化颗粒。
以上述固定化颗粒为生物催化剂，以D-半乳糖为底物，生物转化制备D-tagatose。100mL催化体系包括：50mM Na 2HPO 4/NaH 2PO 4缓冲液(pH 6.5)、500g/L D-半乳糖、1mM MnCl 2、6g/L固定化颗粒。反应于65℃、200r/min、异构化2h。反应液于4℃离心，少量上清液经0.22μm膜过滤后用HPLC检测D-tagatose浓度；收集固定化颗粒，经缓冲液洗涤进行下一批转化。实验结果表明，连续催化30批次，产物得率均大于73％。

Claims (7)

  1. 一种L-阿拉伯糖差向异构酶突变体，由序列如SEQ ID NO.3所示氨基酸经定点突变而来，所述突变的位点为下列中的一个或多个：(1)第304位、(2)第75位、(3)第274位、(4)第167位。
  2. 如权利要求1所述的L-阿拉伯糖差向异构酶突变体，其特征在于所述突变体由序列如SEQ ID NO.3所示氨基酸经下列之一或多个位点突变而得：(1)第304位甘氨酸G突变为组氨酸H、异亮氨酸I、酪氨酸Y或色氨酸W，(2)第75位甘氨酸G突变为赖氨酸K、蛋氨酸M、苯丙氨酸F或脯氨酸P，(3)第274位丙氨酸G突变为丝氨酸S、苏氨酸T、酪氨酸Y、缬氨酸V或半胱氨酸C，(4)167位丙氨酸A突变为天冬酰胺N、天冬氨酸D或精氨酸R。
  3. 如权利要求1所述的L-阿拉伯糖差向异构酶突变体，其特征在于所述突变体由序列如SEQ ID NO.3所示氨基酸经下列之一或多个位点突变而得：(1)第304位甘氨酸G突变为酪氨酸Y、(2)第75位甘氨酸G突变为苯丙氨酸F、(3)第274位丙氨酸G突半胱氨酸变C、(4)167位丙氨酸A突变为精氨酸R。
  4. 如权利要求3所述的L-阿拉伯糖差向异构酶突变体，其特征在于所述L-阿拉伯糖差向异构酶突变体序列如SEQ ID NO.7所示。
  5. 权利要求1～4之一所述L-阿拉伯糖差向异构酶突变体在催化D-半乳糖异构化制备D-塔格糖中的应用。
  6. 如权利要求5所述的应用，其特征在于所述应用为：以含L-阿拉伯糖差向异构酶编码基因的重组基因工程菌经发酵培养获得的湿菌体或湿菌体经超声破碎后获得的上清液或菌体细胞经固定化获得的固定化酶制剂作为催化剂，以D-半乳糖为底物，在Mn 2+和/或Co 2+存在下，在6.0～7.0的KH 2PO 4-NaOH缓冲液中，65～80℃温度下反应，反应结束后，反应液分离纯化，获得D-塔格糖。
  7. 如权利要求6所述的应用，其特征在于所述L-阿拉伯糖差向异构酶突变体编码基因序列如SEQ ID NO.8所示。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101845429A (zh) * 2010-04-21 2010-09-29 南京工业大学 一种耐高温l-阿拉伯糖异构酶及其应用
CN106062188A (zh) * 2014-03-05 2016-10-26 Cj第制糖株式会社 具有提高的转化活性的l‑阿拉伯糖异构酶变异体及利用该变异体生产d‑塔格糖的方法
CN106480006A (zh) * 2016-10-13 2017-03-08 南京林业大学 一种l‑阿拉伯糖异构酶及其应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ531074A (en) * 2001-07-16 2005-10-28 Bioneer As A thermostable isomerase and use thereof, in particular for producing tagatose
EP1765978A2 (en) * 2004-05-19 2007-03-28 Biotechnology Research And Development Corporation Methods for production of xylitol in microorganisms
EP2211643B1 (en) * 2007-11-23 2015-04-15 Institut National De La Recherche Agronomique (INRA) L-arabinose isomerase for converting d-galactose into d-tagatose in a dairy product which contains d-galactose
CN101215554A (zh) * 2008-01-08 2008-07-09 江南大学 一种耐高温l-阿拉伯糖异构酶及其基因序列
KR101015343B1 (ko) * 2008-08-05 2011-02-16 씨제이제일제당 (주) 효소 활성이 향상된 아라비노스 이성화효소 변이체
CN101768581B (zh) * 2010-02-20 2012-05-23 江南大学 具有高产d-塔格糖能力的l-阿拉伯糖异构酶的突变体酶l20a及其突变方法
CN104073481B (zh) * 2014-07-10 2016-08-17 江南大学 一种耐酸性提高的l-阿拉伯糖异构酶突变体
CN104152430B (zh) * 2014-09-01 2016-08-17 江南大学 一种催化活性提高且最适pH降低的L-阿拉伯糖异构酶的突变体酶D478N

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101845429A (zh) * 2010-04-21 2010-09-29 南京工业大学 一种耐高温l-阿拉伯糖异构酶及其应用
CN106062188A (zh) * 2014-03-05 2016-10-26 Cj第制糖株式会社 具有提高的转化活性的l‑阿拉伯糖异构酶变异体及利用该变异体生产d‑塔格糖的方法
CN106480006A (zh) * 2016-10-13 2017-03-08 南京林业大学 一种l‑阿拉伯糖异构酶及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 1 June 2019 (2019-06-01), ANONYMOUS: "L-arabinose isomerase [Limosilactobacillus oris]", XP055823694, retrieved from NCBI Database accession no. WP_003716014 *

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