WO2021103234A1 - 一种d-阿洛酮糖3-差向异构酶突变体及其应用 - Google Patents

一种d-阿洛酮糖3-差向异构酶突变体及其应用 Download PDF

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WO2021103234A1
WO2021103234A1 PCT/CN2019/128050 CN2019128050W WO2021103234A1 WO 2021103234 A1 WO2021103234 A1 WO 2021103234A1 CN 2019128050 W CN2019128050 W CN 2019128050W WO 2021103234 A1 WO2021103234 A1 WO 2021103234A1
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mutant
psicose
ntdpe
site
mutation
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柳志强
贾东旭
孙晨奕
彭晨
金利群
郑裕国
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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    • 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.)
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    • 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
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    • 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
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)

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  • the invention relates to a D-psicose 3-epimerase mutant and its application in preparing D-psicose by catalyzing the isomerization of D-fructose by microorganisms.
  • D-psicose is the C-3 epimer of D-fructose. It belongs to the rare sugar family. Because of its high sweetness and low energy, it is an ideal sucrose substitute. D-psicose can inhibit the absorption of D-fructose and D-glucose by competing for absorption and excretion of transport glycoproteins, thereby reducing body fat accumulation and reducing the risk of diabetes. Long-term and short-term D-psicose dietary supplementation has obvious benefits for the cells of type II diabetes patients and patients with pancreatic ⁇ -cell function defects. D-psicose is scarce in nature and is only found in some plants (such as wheat and rat spines). Direct extraction leads to waste of resources and environmental damage. Using chemical synthesis, the catalytic reaction is complicated, the purification process is cumbersome, and the chemical pollution is serious. Therefore, a more efficient synthesis method is needed to prepare D-psicose.
  • Biotransformation is a process that uses one or several special extracellular or intracellular enzymes produced by microorganisms as biocatalysts to convert substrates into products. Biotransformation has the characteristics of mild reaction conditions and high raw material utilization. At the same time, the transformation process has excellent chemical selectivity, regioselectivity and stereoselectivity, which can ensure the efficient synthesis of target compounds. At present, the use of isomerase or cells containing the enzyme as a biocatalyst for isomerization reactions to prepare a variety of sugar compounds has become an important economic growth point for the sugar industry.
  • D-psicose 3-epimerase belongs to the ketose 3-epimerase family and is used to catalyze the isomerization of D-fructose to D-psicose.
  • the current D-psicose production technology is mainly concentrated in Japan Rare Sugar Research Center, Seoul National University and other institutions.
  • the DPE used is mainly derived from Agrobacterium tumefaciens ATCC33970, Clostridium cellulolyticum H10 and Rhodobacter sphaeroides SK011 (Kim T et al., PLoS) ONE,2016,11(7):e0160044.).
  • the above-mentioned DPEs are not heat-resistant enzymes, and can only be catalyzed at an isomerization temperature of 50-60°C, and the D-fructose conversion rate is between 25-35%.
  • the purpose of the present invention is to provide a D-psicose 3-epimerase mutant that still has high catalytic activity above 70°C, and the preparation of D-psicose 3-epimerase mutants that can catalyze the isomerization of D-fructose by microorganisms. Application of ketose.
  • a D-psicose 3-epimerase mutant obtained by site-directed mutation of the amino acid shown in SEQ ID NO.5, and the site of the point mutation is one or more of the following: (1 ) 242nd, (2) 105th, (3) 210th, (4) 147th, (5) 184th.
  • the point mutation is one or more of the following: (1) The mutation of Valine V at position 242 to Lysine K, Leucine L, Tyrosine Y or Asparagine N; (2) ) Mutation of glycine G at position 105 to asparagine N, aspartic acid D or glutamate E; (3) Mutation of isoleucine I at position 210 to threonine T, phenylalanine F, glutamine Amide Q or Serine S; (4) Mutation of Leucine L at position 147 to Lysine K, Asparagine N or Cysteine C, (5) Mutation of Threonine T at position 184 to Tyrosine Y Or phenylalanine F.
  • amino acid sequence of the mutant is shown in SEQ ID NO.7.
  • the present invention excavates and screens new DPE and conducts site-directed mutations, and constructs high-expressing genetically engineered bacteria through genetic engineering technology to produce D-psicose at high temperature, which improves the synthesis level of existing sugar isomer compounds and is important
  • the theoretical significance and application development potential are of great significance to fill the gap in the market lacking high-temperature enzymes.
  • the invention also relates to the application of the mutants in the preparation of D-psicose by the isomerization of D-fructose catalyzed by microorganisms.
  • the catalysis is carried out at 70-85°C.
  • the application is: using wet bacteria obtained by fermentation culture of engineered bacteria containing the D-psicose-3-epimerase mutant gene as the enzyme source, using D-fructose as the substrate, Cobalt salt is used as auxiliary agent, Na 2 HPO 4 /NaH 2 PO 4 buffer is used as reaction medium, and the reaction is performed at 75-85° C. and 100-300 r/min to prepare D-psicose.
  • the initial concentration of the substrate is 300-700 g/L
  • the amount of wet bacteria is 10-50 g/L
  • the initial concentration of the cobalt salt is 0.5-5 mM.
  • the beneficial effects of the present invention are mainly embodied in that the present invention provides a brand-new DPE and its mutant.
  • the mutant has a high optimal reaction temperature of 85° C. and solves the technical problem that the existing enzyme cannot produce D-psicose at high temperature. Use this mutant to produce D-psicose.
  • the obtained NtDPE mutant produces D-psicose conversion rate as high as 50.12%, which is higher than the transformation effect of the original enzyme and other mutant enzymes, and has good application prospects. .
  • Figure 1 is a schematic diagram of the optimum temperature of the recombinase
  • Figure 2 is a schematic diagram of the effect of metal ions on the activity of recombinase.
  • Example 1 Screening and viability determination of a new type of DPE
  • New DPEs were obtained from the NCBI database, which were derived from Rhizobiales bacterium (GenBank code WP_112533378.1), Martelella sp. (GenBank code MAU19484.1), Novibacillus thermophilus (GenBank code WP_077721022.1), and named RbDPE, MsDPE and NtDPE .
  • 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. 6 the amino acid sequence encoding the enzyme is shown in SEQ ID NO.
  • 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 to 0.8 to obtain seed liquid; the volume concentration of the seed liquid was 2% (v/v)
  • the inoculum volume is inoculated into fresh LB medium containing 100 ⁇ g/mL kanamycin at a final concentration, cultured at 37°C and 150r/min with an OD 600 to 0.4-0.6, and then the final concentration is added to the culture solution 1mM IPTG was induced to express at 25°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 method is used to ultrasonically break the wet bacteria. Take 1g of the prepared wet bacteria, suspend it in 50mL Na 2 HPO 4 /NaH 2 PO 4 (pH 7.5) buffer, sonicate it at 39W for 15 min, prepare the sonicated suspension, centrifuge, and collect the supernatant liquid.
  • Enzyme activity reaction system 50g/L D-fructose, 1mM CoCl 2 .6H 2 O 100 ⁇ L of the above supernatant (enzyme solution), then add an appropriate amount of 50mM Na 2 HPO 4 /NaH 2 PO 4 (pH 7.5) buffer to the total The volume is 1mL.
  • Reaction conditions react at 60°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-psicose.
  • 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 60°C and pH 7.5, the amount of enzyme required to isomerize D-fructose to 1 ⁇ mol D-psicose per minute is defined as one enzyme activity unit (U).
  • PCR reaction system 2 ⁇ Phanta Max Buffer (containing Mg 2+ ) 25 ⁇ L, dNTPs 10mM, forward primer 2 ⁇ L, reverse primer 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, 55°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-fructose, 1 mM CoCl 2 ⁇ 6H 2 O, and then adding 50 mM Na 2 HPO 4 /NaH 2 PO 4 (pH 7.5) buffer to 1L of the total reaction system for use.
  • the reaction system includes 2.5 ⁇ 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 incubation at 60°C for 10 min.
  • the result of this example is: 415 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 accurately measured. The specific results are shown in Table 2. After analysis, it was determined that the enzyme activity of the remaining 410 strains remained unchanged or decreased due to the mutation of valine (V) at position 242 to amino acids other than Y, T, K, N, and L.
  • V valine
  • NtDPE-V242K mutant with the most increased enzyme activity was recorded as NtDPE-1, and the recombinant strain E. coli BL21(DE3)/pET28b/NtDPE-1 was obtained.
  • a mutation primer for site-directed mutation was designed, using rapid PCR technology, using the recombinant vector pET28b/NtDPE-1 as a template, a single mutation was introduced to position 105, and the primers were:
  • PCR reaction system 2 ⁇ Phanta Max Buffer (containing Mg 2+ ) 25 ⁇ L, dNTPs 10mM, forward primer 2 ⁇ L, reverse primer 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, 58°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: 587 strains of recombinant transformed bacteria were initially screened, and 3 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. After analysis, it was determined that the enzyme activity of the remaining 584 strains remained unchanged or decreased due to the mutation of glycine (G) at position 105 to amino acids other than N, D, and E.
  • G glycine
  • NtDPE-2 The NtDPE-V242K-G105N mutant with the most increased enzyme activity was marked as NtDPE-2, and the recombinant strain E. coli BL21(DE3)/pET28b/NtDPE-2 was obtained.
  • a mutation primer for site-directed mutation was designed. Using rapid PCR technology, using the recombinant vector pET28b/NtDPE-2 as a template, a single mutation was introduced at position 210.
  • the primers were:
  • PCR reaction system 2 ⁇ Phanta Max Buffer (containing Mg 2+ ) 25 ⁇ L, dNTPs 10mM, forward primer 2 ⁇ L, reverse primer 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, 55°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: 610 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 measured on them. The specific results are shown in Table 4. After analysis, it was determined that the enzyme activity of the remaining 606 strains remained unchanged or decreased due to the mutation of isoleucine (I) at position 210 to amino acids other than Q, S, F and T.
  • NtDPE-V242K-G105N-I210T mutant with the most increased enzyme activity was recorded as NtDPE-3, and the recombinant strain E. coli BL21(DE3)/pET28b/NtDPE-3 was obtained.
  • mutant NtDPE-3 constructed in Example 4, a mutation primer for site-directed mutation was designed. Using rapid PCR technology, using the recombinant vector pET28b/NtDPE-3 as a template, a single mutation was introduced at position 147.
  • the primers were:
  • PCR reaction system 2 ⁇ Phanta Max Buffer (containing Mg 2+ ) 25 ⁇ L, dNTPs 10mM, forward primer 2 ⁇ L, reverse primer 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, 54°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: 576 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 measured on them. The specific results are shown in Table 5. After analysis, it was determined that the enzyme activity of the remaining 572 strains of recombinant bacteria remained unchanged or decreased due to the mutation of leucine (L) at position 147 to amino acids other than R, N, C, and K.
  • NtDPE-4 The NtDPE-V242K-G105N-I210T-L147K mutant with the most increased enzyme activity was recorded as NtDPE-4, and the recombinant strain E. coli BL21(DE3)/pET28b/NtDPE-4 was obtained.
  • a mutation primer for site-directed mutation was designed. Using rapid PCR technology, using the recombinant vector pET28b/NtDPE-4 as a template, a single mutation was introduced at position 184.
  • the primers were:
  • PCR reaction system 2 ⁇ Phanta Max Buffer (containing Mg 2+ ) 25 ⁇ L, dNTPs 10mM, forward primer 2 ⁇ L, reverse primer 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, 56°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 up 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: 587 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 6. After analysis, it was determined that the enzyme activity of the remaining 584 strains remained unchanged or decreased due to the mutation of threonine (T) at position 184 to amino acids other than Y, G, and F.
  • T threonine
  • NtDPE-5 ie SEQ ID NO. 7
  • E. coli BL21(DE3)/pET28b/NtDPE-5 was obtained.
  • Example 7 Fermentation and purification of recombinant E. coli
  • the wet bacterial cells were ultrasonically broken by the ultrasonic breaking method, and the supernatant was collected.
  • the supernatant was heat-treated at 75° C. for 15 min, and then centrifuged at 4° C., 8000 r/min for 10 min, the precipitate was discarded, and the heat-treated supernatant was collected. Then use 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) for elution. According to the signal response of the UV detector, the corresponding eluate is 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 7 was used as the enzyme for conversion, and the optimal reaction temperature of the enzyme was measured.
  • the reaction system is: 50 g/L D-fructose, 1 mM CoCl 2 ⁇ 6H 2 O, 100 ⁇ L of the pure enzyme solution obtained in the above example, and then add 50 mM Na 2 HPO 4 /NaH 2 PO 4 (pH 7.5) buffer to the total System 1mL.
  • the activity of the recombinant DPE was determined at different transformation temperatures: 50, 55, 60, 65, 70, 75, 80, 85, 90°C (the operation method is the same as the "enzyme activity determination of recombinant bacteria" in Example 1). It can be seen from Figure 1 that the optimal reaction temperature of NtDPE-5 is 85°C, which is 15°C higher than that of the original enzyme NtDPE.
  • Example 9 The effect of metal ions on the enzyme activity of DPE optimal mutants
  • the 1mL reaction system includes: 50mM Na 2 HPO 4 /NaH 2 PO 4 buffer (pH 7.5), 50g/L D-fructose, 100 ⁇ L pure enzyme solution and 1mM different metal ions.
  • the selection of metal ions is as follows: (1) Use single metal ions: Co 2+ , Mg 2+ , Mn 2+ , Cu 2+ , Zn 2+ , Ba 2+ , Fe 2+ , Ni 2+ , Pb 2 + And Ca 2+ .
  • the activity of DPE was measured at 60°C.
  • Example 10 Preparation of D-psicose from whole cells of mutant recombinant bacteria with original enzyme and mutant enzyme
  • the recombinant bacteria E.coli BL21(DE3)/pET28b/NtDPE, E.coli BL21(DE3)/pET28b/NtDPE-1, E.coli BL21(DE3)/pET28b/ NtDPE-2, E.coli BL21(DE3)/pET28b/NtDPE-3, E.coli BL21(DE3)/pET28b/NtDPE-4, E.coliBL21(DE3)/pET28b/NtDPE-5.
  • D-psicose was prepared by biotransformation.
  • the catalytic system includes: 500g/L D-fructose, 1mM CoCl 2 ⁇ 6H 2 O, 20g/L wet bacteria, and then add an appropriate amount of 50mM Na 2 HPO 4 /NaH 2 PO 4 (pH 7.5) buffer to the total system 100mL .
  • the reaction system was reacted for 8h at 70°C and 150r/min. Samples were taken every 1h, centrifuged, filtered with a 0.22 ⁇ m membrane, and then subjected to HPLC to detect the D-psicose concentration.

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Abstract

提供了一种D-阿洛酮糖3-差向异构酶突变体，及其在微生物催化D-果糖异构化制备D-阿洛酮糖中的应用。该D-阿洛酮糖3-差向异构酶突变体，由SEQ ID NO：5所示氨基酸经第242位、第105位、第210位、第147位和第184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85℃，产物得率最高可达40.1％，优于原始酶和其他突变酶的转化效果，具有较好工业应用前景。

Description

一种D-阿洛酮糖3-差向异构酶突变体及其应用 (一)技术领域
本发明涉及一种D-阿洛酮糖3-差向异构酶突变体，及其在微生物催化D-果糖异构化制备D-阿洛酮糖中的应用。
(二)背景技术
D-阿洛酮糖(D-psicose)是D-果糖的C-3差向异构体，属于稀有糖家族，因其高甜度低能量，是一种理想的蔗糖替代物。D-psicose通过竞争吸收和排泄转运糖蛋白，可以抑制D-果糖和D-葡萄糖的吸收，进而减少体内脂肪累积，降低糖尿病风险。长期和短期的D-psicose膳食补充，对II型糖尿病患者和胰岛β-细胞功能缺陷患者细胞均有明显的益处。D-psicose在自然界中含量稀少，仅在部分植物(如小麦和鼠刺属植物)中有发现，直接提取导致资源浪费和环境破坏。采用化学合成法，催化反应复杂，纯化过程繁琐，化学污染严重。因此，需要采用更加高效的合成方式制备D-psicose。
生物转化是一种利用微生物产生的一种或几种特殊的胞外或胞内酶作为生物催化剂，将底物转化为产物的过程。生物转化具有反应条件温和、原料利用率高的特点，同时转化过程具有优良的化学选择性、区域选择性和立体选择性，能够保证目标化合物的高效合成。目前，利用异构酶或含有该酶的细胞为生物催化剂进行异构化反应制备多种糖类化合物，已成为制糖工业重要的经济增长点。
D-阿洛酮糖3-差向异构酶(D-psicose3-epimerase，简称DPE)属于酮糖3-差向异构酶家族，用于催化D-果糖异构化生成D-psicose。当前的D-psicose生产技术主要集中在日本稀有糖研究中心、首尔国立大学等机构，所用的DPE主要来源于Agrobacterium tumefaciens ATCC33970、 Clostridium cellulolyticum H10和Rhodobacter sphaeroides SK011等野生菌(Kim T et al.，PLoS ONE,2016,11(7):e0160044.)。上述DPE均不属于耐热性酶，只能在50～60℃的异构化温度下进行催化反应，D-果糖转化率介于25～35％之间。
有报道指出，DPE介入的D-果糖异构化过程是一个热力学平衡反应，随着异构化温度的升高，会促进异构化反应向D-psicose方向进行。如果能在高温如70℃或更高的异构化温度下催化，促使平衡正向进行，提高D-果糖转化率，获得高浓度产物，将大幅度减小生产、提取等成本。目前，在用于合成的D-psicose的DPE酶源较少，能够满足高温催化制备高浓度D-psicose的酶更是罕见。在耐高温酶制剂没有成功投放于市场的背景下，研发新型耐高温DPE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在70℃以上仍具有较高催化活性的D-阿洛酮糖3-差向异构酶突变体，及其在微生物催化D-果糖异构化制备D-阿洛酮糖中的应用。
本发明采用的技术方案是：
一种D-阿洛酮糖3-差向异构酶突变体，由SEQ ID NO.5所示氨基酸经定点突变而得，所述点突变位点为下列中的一个或多个：(1)第242位、(2)第105位、(3)第210位、(4)第147位、(5)第184位。
具体的，所述点突变为下列中的一个或多个：(1)第242位缬氨酸V突变为赖氨酸K、亮氨酸L、酪氨酸Y或天冬酰胺N；(2)第105位甘氨酸G突变为天冬酰胺N、天冬氨酸D或谷氨酸E；(3)第210位异亮 氨酸I突变为苏氨酸T、苯丙氨酸F、谷氨酰胺Q或丝氨酸S；(4)第147位亮氨酸L突变为赖氨酸K、天冬酰胺N或半胱氨酸C、(5)第184位苏氨酸T突变为酪氨酸Y或苯丙氨酸F。
优选的，所述突变体氨基酸序列如SEQ ID NO.7所示。
本发明挖掘筛选到新型DPE并进行定点突变，通过基因工程技术构建高表达的基因工程菌用于在高温下生产D-psicose，提升了现有的糖异构体化合物的合成水平，具有重要的理论意义和应用开发潜力，对于填补缺乏耐高温酶的市场空白具有重大意义。
本发明还涉及所述的突变体在微生物催化D-果糖异构化制备D-阿洛酮糖中的应用。
优选的，所述催化在70～85℃下进行。
具体的，所述应用为：以含D-阿洛酮糖-3-差向异构酶突变体基因的工程菌经发酵培养获得的湿菌体为酶源，以D-果糖为底物，以钴盐为助剂，以Na 2HPO 4/NaH 2PO 4缓冲液为反应介质，在75～85℃，100～300r/min条件下反应，制得D-阿洛酮糖。
优选的，所述反应体系中，底物初始浓度为300～700g/L，湿菌体的用量为10～50g/L，钴盐初始浓度为0.5～5mM。
本发明有益效果主要体现在：本发明提供了一种全新的DPE及其突变体，该突变体具有高最适反应温度85℃，解决了现有酶无法在高温生产D-psicose的技术难题。使用本突变体生产D-psicose，在80℃转化条件下，获得的NtDPE突变体所生产的D-psicose转化率高达50.12％，高于原始酶和其他突变酶的转化效果，具有较好应用前景。
(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DPE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DPE，分别来源于Rhizobiales bacterium(GenBank编号WP_112533378.1)、Martelella sp.(GenBank编号MAU19484.1)、Novibacillus thermophilus(GenBank编号WP_077721022.1)，并命名为RbDPE、MsDPE和NtDPE。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/RbDPE、pET28b/MsDPE和pET28b/NtDPE。
SEQIDNO.1：
Figure PCTCN2019128050-appb-000001
Figure PCTCN2019128050-appb-000002
SEQIDNO.3：
Figure PCTCN2019128050-appb-000003
SEQIDNO.5：
Figure PCTCN2019128050-appb-000004
2、重组菌的转化与诱导表达
将获得的重组表达质粒pET28b/RbDPE、pET28b/MsDPE和pET28b/NtDPE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DPE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1mM的IPTG，于25℃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 Na 2HPO 4/NaH 2PO 4(pH 7.5)缓冲液悬浮，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-果糖、1mM CoCl 2.6H 2O100μL上述上清液(酶液)，再加入适量50mM Na 2HPO 4/NaH 2PO 4(pH 7.5)缓冲液至总体积1mL。反应条件：于60℃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-psicose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：60℃和pH 7.5下，每分钟将D-果糖异构化生成1μmol D-阿洛酮糖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 PCTCN2019128050-appb-000005
实施例2：NtDPE单位点突变体的构建与筛选
1、突变体构建
根据NtDPE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/NtDPE为模板，对第242位引入单突变，引物为：
正向引物GATGGTTATGTG NNNATGGAACCG(下划线为突变碱基)
反向引物CGGTTCCATCAC NNNATAACCATC(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2μL，反向引物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，55℃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-果糖、1mM CoCl 2·6H 2O，再加入50mM Na 2HPO 4/NaH 2PO 4(pH 7.5)缓冲液至总反应体系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1mM的IPTG，于25℃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60℃、600r/min反应10min，冰浴10min停止反应。取2.5μL反应液以半胱氨酸-咔唑显色法筛选突变体，反应体系包括2.5μL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。以重组菌菌E.coli BL21(DE3)/pET28b/NtDPE的反应为对照，取颜色比E.coli BL21(DE3)/pET28b/NtDPE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415株重组转化菌初筛，筛选出5株酶活提高的突变株，再对其进行酶活精准测定，具体结果见表2。经分析确定，其余410株重组菌酶活保持不变或下降的原因是第242位缬氨酸(V)突变为Y、T、K、N和L外的其他氨基酸。
表2：单点突变重组菌的酶活测定
Figure PCTCN2019128050-appb-000006
将酶活提高最多的NtDPE-V242K突变体记为NtDPE-1，获得重组菌E.coli BL21(DE3)/pET28b/NtDPE-1。
实施例3：NtDPE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NtDPE-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/NtDPE-1为模板，对第105 位引入单突变，引物为：
正向引物CATTGATCGTGTG NNNGGTACCGTGTAT(下划线为突变碱基)
反向引物ATACACGGTACCA NNNACACGATCAATG(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2μL，反向引物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，58℃15s，72℃6.5min)30循环；72℃5min。
PCR产物转化E.coliBL21(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587株重组转化菌初筛，筛选出3株酶活提高的突变株，再对其进行酶活精准测定，具体结果见表3。经分析确定，其余584株重组菌酶活保持不变或下降的原因是第105位甘氨酸(G)突变为N、D和E外的其他氨基酸。
表3：双点突变重组菌的酶活测定
Figure PCTCN2019128050-appb-000007
将酶活提高最多的NtDPE-V242K-G105N突变体记为NtDPE-2，获得 重组菌E.coli BL21(DE3)/pET28b/NtDPE-2。
实施例4：NtDPE三位点突变体的构建与筛选
根据实施例3构建的突变体NtDPE-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/NtDPE-2为模板，对第210位引入单突变，引物为：
正向引物GGGTCATCTGCAT NNNGGTGAAGC(下划线为突变碱基)
反向引物GCTTCACCAATAT NNNGATGACCC(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2μL，反向引物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，55℃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610株重组转化菌初筛，筛选出4株酶活提高的突变株，再对其进行酶活测定，具体结果见表4。经分析确定，其余606株重组菌酶活保持不变或下降的原因是第210位异亮氨酸(I)突变为Q、S、F和T外的其他氨基酸。
表4：三点突变重组菌的酶活测定
Figure PCTCN2019128050-appb-000008
Figure PCTCN2019128050-appb-000009
将酶活提高最多的NtDPE-V242K-G105N-I210T突变体记为NtDPE-3，获得重组菌E.coli BL21(DE3)/pET28b/NtDPE-3。
实施例5：NtDPE四位点突变体的构建与筛选
根据实施例4构建的突变体NtDPE-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/NtDPE-3为模板，对第147位引入单突变，引物为：
正向引物CACGTCAGTATGATATTACC NNNCTG(下划线为突变碱基)
反向引物CAGCAGGGTAATATCATACT NNNGTG(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2μL，反向引物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，54℃15s，72℃6.5min)30循环；72℃5min。
PCR产物转化E.coliBL21(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576株重组转化菌初筛，筛选出4株酶活提高的突变株，再对其进行酶活测定，具体结果见表5。经分析确定，其余572 株重组菌酶活保持不变或下降的原因是第147位亮氨酸(L)突变为R、N、C和K外的其他氨基酸。
表5：四位点突变重组菌的酶活测定
Figure PCTCN2019128050-appb-000010
将酶活提高最多的NtDPE-V242K-G105N-I210T-L147K突变体记为NtDPE-4，获得重组菌E.coli BL21(DE3)/pET28b/NtDPE-4。
实施例6：NtDPE五位点突变体的构建与筛选
根据实施例5构建的突变体NtDPE-4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/NtDPE-4为模板，对第184位引入单突变，引物为：
正向引物AGTGATGCTGGAT NNNTTCCACATG(下划线为突变碱基)
反向引物CATGTGGAAGGTA NNNAGCATCACT(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2μL，反向引物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，56℃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100 μg/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587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6。经分析确定，其余584株重组菌酶活保持不变或下降的原因是第184位苏氨酸(T)突变为Y、G和F外的其他氨基酸。
表6：五点突变重组菌的酶活测定
Figure PCTCN2019128050-appb-000011
将酶活提高最多的NtDPE-V242K-G105N-I210T-L147K-T184Y突变体记为NtDPE-5(即SEQIDNO.7)，获得重组菌E.coli BL21(DE3)/pET28b/NtDPE-5。
SEQIDNO.7：
Figure PCTCN2019128050-appb-000012
实施例7：重组大肠杆菌发酵产酶与纯化
分别将重组菌E.coli BL21(DE3)/pET28b/NtDPE、E.coli BL21(DE3)/pET28b/NtDPE-1、E.coli BL21(DE3)/pET28b/NtDPE-2、E.coli BL21(DE3)/pET28b/NtDPE-3、E.coli BL21(DE3)/pET28b/NtDPE-4、E.coli BL21(DE3)/pET28b/NtDPE-5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1mM的IPTG，于25℃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。
上清液在75℃热处理15min，然后在4℃、8000r/min离心10min，弃去沉淀，收集热处理后的上清液。接着使用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8：纯化NtDPE及其突变体的最适反应温度
将实施例7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：50g/L的D-果糖、1mM CoCl 2·6H 2O、100μL上述实施例获得的纯酶液，再加入50mM Na 2HPO 4/NaH 2PO 4(pH 7.5)缓冲液至总体系1mL。分别于不同转化温度：50、55、60、65、70、75、80、85、90℃测 定重组DPE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，NtDPE-5的最适反应温度为85℃，比原始酶NtDPE提高15℃。
实施例9：金属离子对DPE最优突变体酶活的影响
将实施例7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 Na 2HPO 4/NaH 2PO 4缓冲液(pH 7.5)、50g/L D-果糖、100μL纯酶液和1mM不同金属离子。其中，金属离子的选择如下：(1)选用单金属离子：Co 2+、Mg 2+、Mn 2+、Cu 2+、Zn 2+、Ba 2+、Fe 2+、Ni 2+、Pb 2+和Ca 2+。于60℃测定DPE的活力。(2)设置组合金属离子，分别为1mM Co 2+和0.5mM Ca 2+、1mM Co 2+和0.5mM Zn 2+、1mM Co 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，Co 2+对DPE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10：原始酶与突变酶突变重组菌全细胞制备D-psicose
按实施例7的发酵方法，大规模发酵获得重组菌E.coli BL21(DE3)/pET28b/NtDPE、E.coli BL21(DE3)/pET28b/NtDPE-1、E.coli BL21(DE3)/pET28b/NtDPE-2、E.coli BL21(DE3)/pET28b/NtDPE-3、E.coli BL21(DE3)/pET28b/NtDPE-4、E.coliBL21(DE3)/pET28b/NtDPE-5。分别以上述湿菌体作为生物催化剂，以D-果糖为底物，生物转化制备D-psicose。催化体系包括：500g/L的D-果糖、1mM CoCl 2·6H 2O、20g/L湿菌体，再加入适量50mM Na 2HPO 4/NaH 2PO 4(pH 7.5)缓冲液至总体系100mL。反应体系于70℃、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-psicose浓度。由表7可知，E.coli BL21(DE3)/pET28b/NtDPE-5的产物得率最终达到40.1％，高于原 始酶E.coli BL21(DE3)/pET28b/NtDPE和其他突变酶的得率。
表7：各重组菌得率的比较
Figure PCTCN2019128050-appb-000013

Claims (7)

  1. 一种D-阿洛酮糖3-差向异构酶突变体，由SEQ ID NO.5所示氨基酸经定点突变而得，所述点突变位点为下列中的一个或多个：(1)第242位、(2)第105位、(3)第210位、(4)第147位、(5)第184位。
  2. 如权利要求1所述的突变体，其特征在于所述点突变为下列中的一个或多个：(1)第242位缬氨酸突变为赖氨酸、亮氨酸、酪氨酸、苏氨酸或天冬酰胺；(2)第105位甘氨酸突变为天冬酰胺、天冬氨酸或谷氨酸；(3)第210位异亮氨酸突变为苏氨酸、苯丙氨酸、谷氨酰胺或丝氨酸；(4)第147位亮氨酸突变为赖氨酸、天冬酰胺、精氨酸或半胱氨酸；(5)第184位苏氨酸突变为酪氨酸或苯丙氨酸。
  3. 如权利要求1所述的突变体，其特征在于所述突变体氨基酸序列如SEQ ID NO.7所示。
  4. 权利要求1～3之一所述的突变体在微生物催化D-果糖异构化制备D-阿洛酮糖中的应用。
  5. 如权利要求4所述的应用，其特征在于所述催化在60～85℃下进行。
  6. 如权利要求5所述的应用，其特征在于所述应用为：以含D-阿洛酮糖3-差向异构酶突变体基因的工程菌经发酵培养获得的湿菌体为酶源，以D-果糖为底物，以钴盐为助剂，以Na 2HPO 4/NaH 2PO 4缓冲液为反应介质，在65～85℃，100～300r/min条件下反应，制得D-阿洛酮糖。
  7. 如权利要求6所述的应用，其特征在于：所述反应体系中，底物初始浓度为300～700g/L，湿菌体的用量为10～50g/L，钴盐初始浓度为0.5～5mM。
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