A method for catalyzing the synthesis of tagatase at high temperature and its application

Abstract

The present invention relates to a biofermentation method of tagatose, and in particular to a tagatase enzyme for catalytic synthesis at high temperature and its application. The enzyme comprises tagatose six phosphate synthase and tagatose six phosphate phosphatase, wherein the amino acid sequence of the tagatose six phosphate synthase is shown in SEQ ID NO.1, and the amino acid sequence of the tagatose six phosphate phosphatase is shown in SEQ ID NO.5. The tagatase enzyme provided by the present invention has higher temperature stability and is more conducive to improving the efficiency of converting starch into tagatose.

Description

Technical Field

[0001] The invention relates to a biological fermentation method of tagatose, and in particular to a tagatase enzyme catalyzed and synthesized at high temperature and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] According to the inventor's research, starch is converted into tagatose in one step through the joint action of glycogen phosphorylase, phosphoglucomutase, glucose-6-phosphate isomerase, tagatose-6-phosphate synthase, and phosphatase. The specific process is as follows: starch and phosphate form glucose-1-phosphate (glucose monophosphate) under the catalytic action of glycogen phosphorylase, glucose-6-phosphate (glucose-6-phosphate) can be converted into glucose-6-phosphate (glucose-6-phosphate) by phosphoglucomutase, glucose-6-phosphate can be isomerized into fructose-6-phosphate (fructose-6-phosphate) under the catalytic action of glucose-6-phosphate isomerase, fructose-6-phosphate isomerized into tagatose-6-phosphate (tagatose-6-phosphate) by tagatose-6-phosphate synthase, and tagatose-6-phosphate is dephosphorylated to form tagatose under the action of phosphatase. However, the above method currently used to convert starch into tagatose in one step has many byproducts and low yield, thereby affecting its application in the production of tagatose. Summary of the invention

[0004] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a tagatase that catalyzes the synthesis of tagatase at high temperature and its application. The tagatase provided by the present invention has higher temperature stability and is more conducive to improving the efficiency of converting starch into tagatose.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In a first aspect, a tagatase that catalyzes the synthesis of tagatose at high temperature comprises tagatose six-phosphate synthase and tagatose six-phosphate phosphatase, wherein the amino acid sequence of the tagatose six-phosphate synthase is shown in SEQ ID NO.1, and the amino acid sequence of the tagatose six-phosphate phosphatase is shown in SEQ ID NO.5.

[0007] The present invention has found that tagatose 6-phosphate synthase has low catalytic efficiency and high affinity for fructose 6-phosphate substrate, resulting in low efficiency of starch conversion to tagatose 6-phosphate; at the same time, phosphatase has poor specific selectivity for tagatose 6-phosphate during the dephosphorylation process, resulting in a large amount of glucose monophosphate, glucose 6-phosphate and fructose 6-phosphate being dephosphorylated in the reaction system, ultimately producing more glucose and fructose as by-products; therefore, the yield of tagatose prepared by the one-step starch method is affected.

[0008] Therefore, the present invention first screened and cloned a tagatose 6-phosphate synthase (T6PE-T) derived from Thermoflexus sp. from the metagenome of high-temperature hot springs. Compared with the existing tagatose 6-phosphate synthase, T6PE-T has higher high-temperature catalytic stability and extremely low fructose 6-phosphate Km, and can continuously and more specifically catalyze the production of more tagatose 6-phosphate by high temperature (50-70°C) under the condition of adding less enzyme; secondly, the present invention screened and cloned a tagatose 6-phosphate phosphatase derived from Thermoanaerobacterales bacterium from the metagenome of high-temperature hot springs. Compared with the existing tagatose 6-phosphate phosphatase, the tagatose 6-phosphate phosphatase provided by the present invention can highly selectively dephosphorylate tagatose 6-phosphate under higher temperature (70°C) conditions, greatly reducing the generation of by-products such as glucose and fructose, thereby improving the yield of tagatose.

[0009] In a second aspect, a nucleic acid molecule comprises a nucleotide encoding a tagatose six-phosphate synthase and a nucleotide encoding a tagatose six-phosphate phosphatase, wherein the amino acid sequence of the tagatose six-phosphate synthase is shown in SEQ ID NO.1, and the amino acid sequence of the tagatose six-phosphate phosphatase is shown in SEQ ID NO.5.

[0010] Furthermore, the nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase is shown in SEQ ID NO.2.

[0011] Furthermore, the nucleic acid sequence of the nucleotide encoding tagatose six-phosphate phosphatase is shown in SEQ ID NO.6.

[0012] In a third aspect, a microorganism is provided, which can express tagatose six-phosphate synthase and tagatose six-phosphate phosphatase, wherein the amino acid sequence of the tagatose six-phosphate synthase is shown in SEQ ID NO.1, and the amino acid sequence of the tagatose six-phosphate phosphatase is shown in SEQ ID NO.5.

[0013] The microorganism of the present invention can be a group of engineered bacteria that can express tagatose 6-phosphate synthase and tagatose 6-phosphate phosphatase, or can be an engineered bacteria that can express tagatose 6-phosphate synthase and tagatose 6-phosphate phosphatase at the same time.

[0014] Furthermore, the starting strain of the microorganism is Escherichia coli, specifically Escherichia coli BL21.

[0015] Furthermore, the construction method of the engineered bacteria capable of expressing tagatose hexaphosphate synthase is as follows: amplifying the nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase with the first primer pair, preparing a recombinant vector with the amplified product and the vector, and transferring the recombinant vector into the starting bacteria; the base sequences of the first primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. Specifically, the vector is a pet41 vector.

[0016] Furthermore, the construction method of the engineered bacteria capable of expressing tagatose six-phosphate phosphatase is as follows: amplifying the nucleic acid sequence of the nucleotide encoding tagatose six-phosphate phosphatase with the second primer pair, preparing a recombinant vector with the amplified product and the vector, and transferring the recombinant vector into the starting bacteria; the base sequences of the second primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively. Specifically, the vector is a pet41 vector.

[0017] In a fourth aspect, a use of the above-mentioned tagatase, nucleic acid molecule or microorganism catalyzing the synthesis of tagatose at high temperature in one-step preparation of tagatose from starch.

[0018] Furthermore, the tagatase, nucleic acid molecule or microorganism catalyzing synthesis at high temperature, as well as starch phosphorylase, glucose 6-phosphate isomerase and fructose 6-phosphate isomerase are added to the reaction system containing starch, and heated to 65-70° C. for catalytic reaction for 60-84 hours.

[0019] Furthermore, the initial concentration of starch in the reaction system is 250-350 g / L.

[0020] Furthermore, the reaction system contains dipotassium hydrogen phosphate and magnesium sulfate. Specifically, the molar ratio of dipotassium hydrogen phosphate to magnesium sulfate is 9 to 11:1. Specifically, the concentration of dipotassium hydrogen phosphate in the reaction system is 45 to 55 mM. In the present invention, the unit "M" refers to mol / L, and the unit "mM" refers to mmol / L. Specifically, the pH in the reaction system is 6.9 to 7.2.

[0021] In a fifth aspect, a product 1 is used for catalyzing fructose hexaphosphate to prepare tagatose hexaphosphate or catalyzing starch to prepare tagatose in one step, wherein the product 1 is tagatose hexaphosphate synthase, a nucleotide encoding the tagatose hexaphosphate synthase, or a microorganism expressing the tagatose hexaphosphate synthase, and the amino acid sequence of the tagatose hexaphosphate synthase is shown in SEQ ID NO.1.

[0022] The present invention screened and cloned a tagatose 6-phosphate synthase (T6PE-T) derived from Thermoflexus sp. from the metagenome of high-temperature hot springs. Compared with the existing tagatose 6-phosphate synthase, T6PE-T has higher high-temperature catalytic stability and extremely low fructose 6-phosphate Km, and can continuously and more specifically catalyze the production of more tagatose 6-phosphate at high temperature (50-70°C) under the condition of adding less enzyme, thereby improving the yield of tagatose 6-phosphate.

[0023] Furthermore, the nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase is shown in SEQ ID NO.2.

[0024] Furthermore, the starting strain of the microorganism is Escherichia coli, specifically Escherichia coli BL21.

[0025] Furthermore, the method for constructing a microorganism capable of expressing tagatose hexaphosphate synthase is as follows: amplifying the nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase with the first primer pair, preparing a recombinant vector with the amplified product and the vector, and transferring the recombinant vector into the starting bacteria; the base sequences of the first primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. Specifically, the vector is a pet41 vector.

[0026] Furthermore, in the preparation of tagatose hexaphosphate by catalyzing fructose hexaphosphate, product 1 is added to a reaction solution containing fructose hexaphosphate, and the catalytic reaction is carried out at 65-70°C.

[0027] Furthermore, the concentration of fructose hexaphosphate in the reaction solution is 40-60 mM.

[0028] Furthermore, the reaction solution contains potassium dihydrogen phosphate and magnesium sulfate. Specifically, the molar ratio of potassium dihydrogen phosphate to magnesium sulfate is 9 to 11: 1. Specifically, the concentration of potassium dihydrogen phosphate in the reaction system is 45 to 55 mM.

[0029] Furthermore, in the one-step preparation of tagatose from starch, the product 1, as well as starch phosphorylase, glucose 6-phosphate isomerase, fructose 6-phosphate isomerase and tagatose 6-phosphate phosphatase are added to a reaction system containing starch, and the system is heated to 65-70° C. to catalyze the reaction for 60-84 hours.

[0030] Furthermore, the reaction solution contains potassium dihydrogen phosphate and magnesium sulfate. Specifically, the molar ratio of potassium dihydrogen phosphate to magnesium sulfate is 9 to 11: 1. Specifically, the concentration of potassium dihydrogen phosphate in the reaction system is 45 to 55 mM.

[0031] In a sixth aspect, a product 2 is used for catalyzing the dephosphorylation of tagatose hexaphosphate to form tagatose, wherein the product 2 is tagatose hexaphosphate phosphatase, a nucleotide encoding the tagatose hexaphosphate phosphatase, or a microorganism expressing the tagatose hexaphosphate phosphatase, and the amino acid sequence of the tagatose hexaphosphate synthase is shown in SEQ ID NO.5.

[0032] The present invention screened and cloned a tagatose six-phosphate phosphatase derived from Thermoanaerobacterales bacterium from the high-temperature hot spring metagenome. Compared with the existing tagatose six-phosphate phosphatase, the tagatose six-phosphate phosphatase provided by the present invention can highly selectively dephosphorylate tagatose six-phosphate at a higher temperature (70°C), greatly reducing the generation of by-products such as glucose and fructose.

[0033] Furthermore, the nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase is shown in SEQ ID NO.6.

[0034] Furthermore, the starting strain of the microorganism is Escherichia coli, specifically Escherichia coli BL21.

[0035] Furthermore, the method for constructing a microorganism capable of expressing tagatose six-phosphate phosphatase is as follows: amplifying the nucleic acid sequence of the nucleotide encoding tagatose six-phosphate phosphatase with the second primer pair, preparing a recombinant vector with the amplified product and the vector, and transferring the recombinant vector into the starting bacteria; the base sequences of the second primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively. Specifically, the vector is a pet41 vector.

[0036] Furthermore, product 2 is added to the reaction solution containing tagatose hexaphosphate, and the catalytic reaction is carried out at 65-70°C.

[0037] Furthermore, the reaction solution contains potassium dihydrogen phosphate and magnesium sulfate. Specifically, the molar ratio of potassium dihydrogen phosphate to magnesium sulfate is 9 to 11: 1. Specifically, the concentration of potassium dihydrogen phosphate in the reaction system is 45 to 55 mM.

[0038] The beneficial effects of the present invention are:

[0039] The present invention screens new tagatose six phosphate synthase and tagatose six phosphate phosphatase respectively through the high-temperature hot spring metagenome, and the tagatose six phosphate synthase can continuously and specifically catalyze the production of more tagatose six phosphate under high temperature conditions of 50-70°C; and on this basis, the screened new tagatose six phosphate phosphatase can more efficiently select tagatose six phosphate as a substrate for dephosphorylation at 70°C, thereby greatly improving the efficiency of preparing tagatose by a one-step starch method. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0041] Example 1

[0042] Tagatose six phosphate synthase (T6PE-T) and its preparation:

[0043] A tagatose 6-phosphate synthase (T6PE-T) from Thermoflexus sp. was screened and cloned from the metagenome of a high-temperature hot spring. Currently, the synthase that is known to synthesize tagatose 6-phosphate is gatZ from Caldilinea aerophila (accession number: WP_014433578).

[0044] The amino acid sequence of tagatose hexaphosphate synthase T6PE-T is:

[0045] MKGYDVVAAGGGSCSAHVAARHGRASGKVATSNVNGGYTGMTAAGAAKMARGRRGGDHGWSHAMRKADVRAVAGTKHDCSMGGVARARARVAAAGRRDRYVVGSVAGGAAAGVTRDAAAAARRARRDTAWRV AVVGGDHYDRAAAAVRWGVGAHSTDYAARAVDHAKVGATARAVAADAAGSRAAAMARVHWKYYRGDARVRARRYSSDRRYYWAAVTARMRNGDRVGSYMYRRVRRGRNHRDGRVMAVGYAATVNGCR, as in SEQ Shown as ID NO.1.

[0046] Nucleic acid sequence:

[0047] ATGAAGGGTTATGATGTGGTTGCAGCAGGTGGTGGTAGCTGTAGCGCCCATGTGGCCGCACGTCATGGCCGTGCCAGTGGCAAAGTTGCAACCAGTAATGTGAATGGTGGTTATACCGGCATGACCGCAGCCGGCGCCGCCAAAATGGCACGCGGTCGTCGCGGCGGCGATCATGGTTGGAGTCATGCCATGCGTAAAGCCGATGTTCGTGCAGTGGCCGGTACCAAACATGATTGCAGCATGGGTGGTGTGGCCCGCGCACGTGCACGCGTTGCTGCTGCAGGCCGTCGTGATCGCTATGTGGTTGGCAGTGTGGCAGGCGGCGCAGCAGCCGGTGTGACACGTGATGCAGCCGCCGCCGCCCGTCGTGCAAGAAGAGATACCGCATGGCGCGTTGCAGTTGTTGGTGGTGACCATTATGATCGTGCAGCAGCCGCAGTGCGCTGGGGTGTGGGTGCACATAGCACCGATTATGCAGCCCGCGCAGTGGATCATGCCAAAGTGGGTGCAACCGCCCGTGCAGTGGCGGCTGATGCCGCAGGTAGTCGTGCCGCAGCCATGGCACGTGTTCATTGGAAATATTATCGCGGTGACGCCCGTGTTCGTGCACGTCGTTATAGTAGCGATCGTCGCTATTATTGGGCAGCAGTTACCGCCCGTATGCGCAATGGCGATCGCGTGGGTAGTTATATGTATCGTCGCGTGCGTCGCGGCCGTAATCATCGTGATGGTCGTGTTATGGCCGTGGGCTATGCAGCCACCGTGAATGGTTGTCGC, as shown in SEQ ID NO.2.

[0048] Primers P1: aactttaagaaggagatatacatATGAAGGGTTATGATGTGG (as shown in SEQ ID NO.3); P2: agtggtggtggtggtggtggtggtggtgctcgagGCGACAACCATTCACG (as shown in SEQ ID NO.4) were designed to amplify the T6PE-T sequence as a template to obtain the amplified product, and the product was separated, recovered and concentrated by agarose gel electrophoresis. The pet41 vector was digested with NdeI+XhoI, and the product was separated, recovered and concentrated by agarose gel electrophoresis. The digested pet41 vector and the amplified fragment were recombined using a DNA recombination kit, transformed into Escherichia coli DH5a competent cells, and positive clones were cultured and screened for sequencing verification. The positive clones were transformed into Escherichia coli BL21 to construct an engineering strain that can recombinantly express T6PE-T.

[0049] The above recombinant strain was cultured using TB medium and induced to express using IPTG. The induced expression bacteria were collected by centrifugation, and the bacteria were resuspended in PBS buffer containing 20mM imidazole and 0.5M sodium chloride. The bacteria were resuspended in a ratio of 10ml buffer to 1g wet bacteria, ultrasonically broken, and the supernatant was collected by centrifugation. The recombinant protein in the supernatant was adsorbed by a Ni column, and the recombinant protein was eluted with a PBS buffer containing 300mM imidazole and 0.5M sodium chloride. The eluted protein was desalted using a desalting column to obtain T6PE-T.

[0050] T6PE-T was purified using the His tag, and after desalting, its protein content was calibrated using the Coomassie Brilliant Blue G250 method.

[0051] Example 2

[0052] T6PE-T catalyzes the synthesis of tagatose hexaphosphate from fructose hexaphosphate:

[0053] 20 mg of T6PE-T was added to 100 ml of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate and 50 mM fructose hexaphosphate, and the reaction was catalyzed at 70°C for 1 hour to obtain tagatose hexaphosphate with a yield of 8.5 mM and a yield of 17%.

[0054] Comparative Example 1

[0055] gatZ catalyzes the synthesis of tagatose hexaphosphate from fructose hexaphosphate:

[0056] gatZ catalyzes the synthesis of tagatose hexaphosphate from fructose hexaphosphate:

[0057] 20 mg of gatZ was added to 100 ml of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate and 50 mM fructose hexaphosphate, and the reaction was catalyzed at 70° C. for 1 hour to obtain tagatose hexaphosphate with a yield of 4.9 mM and a yield of 9.8%.

[0058] Example 3

[0059] Catalytic efficiency test of T6PE-T:

[0060] T6PE-T was placed at different temperatures (e.g., 55°C-70°C) and heat-treated for different times (e.g., 1500 minutes-5000 minutes). 20 mg of heat-treated T6PE-T and untreated T6PE-T were added to 100 ml of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate and 50 mM fructose hexaphosphate, respectively, and catalyzed at 70°C for 1 hour. The amount of tagatose hexaphosphate produced was detected by liquid chromatography to determine its catalytic efficiency. The amount produced is shown in Table 1.

[0061] Table 1 The amount of tagatose hexaphosphate generated by heat-treated T6PE-T and untreated T6PE-T

[0062]

[0063] Comparative Example 2

[0064] Detection of the catalytic efficiency of gatZ:

[0065] GatZ was placed at different temperatures (e.g., 55°C-70°C) and heat-treated for different times (e.g., 1500 minutes-5000 minutes). 20 mg of heat-treated gatZ and untreated gatZ were added to 100 ml of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate and 50 mM fructose hexaphosphate, respectively, and catalyzed at 70°C for 1 hour. The amount of tagatose hexaphosphate produced was detected by liquid chromatography to determine its catalytic efficiency. The amount produced is shown in Table 2.

[0066] Table 2 The amount of tagatose hexaphosphate generated by heat-treated gatZ and untreated gatZ

[0067]

[0068] By comparing the catalytic efficiency of Example 3 and Comparative Example 2, the half-lives of different tagatose hexaphosphate synthases under different heat treatments can be known, thereby knowing their thermal stability. The results are shown in Table 3.

[0069] Table 3 Thermal stability of T6PE-T and gatZ

[0070]

[0071]

[0072] Table 3 shows that compared with gatZ, T6PE-T has stronger catalytic stability under high temperature conditions of 55-70°C, and can produce more products through continuous catalysis at high temperature with less enzyme added.

[0073] Example 4

[0074] Detection of T6PE-T and gatZ's affinity for substrates:

[0075] Determination of 1. Km value of fructose hexaphosphate: prepare a catalytic reaction solution of 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, 20 mg of catalytic enzyme, and fructose hexaphosphate (0.5 mM-100 mM) at 70°C for 1 hour, detect the amount of tagatose hexaphosphate produced by liquid chromatography, and use Linewaver-Burk plotting to measure the Km values ​​of T6PE-T and gatZ for fructose hexaphosphate.

[0076] Determination 2. Fructose-1,6-bisphosphate (fructose-1,6-bisphosphate) Km value: prepare a catalytic reaction solution of 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, 20 mg of catalytic enzyme, and fructose-1,6-bisphosphate (0.5 mM-100 mM) at 70°C for 1 hour, detect the amount of tagatose-1,6-bisphosphate (tagatose-1,6-bisphosphate) produced by liquid chromatography, and use the Linewaver-Burk plot method to measure the Km values ​​of T6PE-T and gatZ for fructose-1,6-bisphosphate.

[0077] Determination 3. Km value of tagatose 1,6-diphosphate: prepare a catalytic reaction solution of 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, 20 mg of catalytic enzyme, and tagatose 1,6-diphosphate (0.5 mM-100 mM) at 70°C for 1 hour, detect the amount of fructose 1,6-diphosphate produced by liquid chromatography, and use Linewaver-Burk plotting to measure the Km values ​​of T6PE-T and gatZ for tagatose 1,6-diphosphate.

[0078] Determination 4. Km value of tagatose hexaphosphate: prepare a catalytic reaction solution of 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, 20 mg of catalytic enzyme, and tagatose hexaphosphate (0.5 mM-100 mM) at 70°C for 1 hour, detect the amount of fructose hexaphosphate generated by liquid chromatography, and use Linewaver-Burk plotting to determine the Km values ​​of T6PE-T and gatZ for tagatose hexaphosphate.

[0079] The test results are shown in Table 4.

[0080] Table 4 Affinity of T6PE-T and gatZ for substrates

[0081] T6PE-T gatZ Fructose 6-phosphate Km value (mmol / L) 1.84 36 Fructose 1,6-diphosphate Km value (mmol / L) 0.51 0.68 Tagatose 1,6-diphosphate Km value (mmol / L) 0.92 1.5 Tagatose hexaphosphate Km value (mmol / L) 189 124

[0082] Table 4 shows that T6PE-T has a lower Km value of fructose 6-phosphate during the catalytic process, so the enzyme can catalyze the formation of tagatose 6-phosphate more specifically and will not catalyze tagatose 6-phosphate to form other substances.

[0083] Example 5

[0084] T6PE-T catalyzes the synthesis of tagatose from starch:

[0085] Prepare a catalyst solution (pH 7.0) of 50 mM potassium dihydrogen phosphate, 5 mM magnesium sulfate, and 300 g / L starch. Add 10 U starch phosphorylase, 10 U glucose 6-phosphate isomerase, 10 U fructose 6-phosphate isomerase, 10 U T6PE-T, and 10 U T6PP to a 1 L reaction system and catalyze at 70°C for 72 h. The content of tagatose detected by liquid chromatography is 168 g, and the yield is 56%.

[0086] Comparative Example 3

[0087] gatZ catalyzes the synthesis of tagatose from starch:

[0088] Prepare a catalyst solution (pH 7.0) of 50 mM potassium dihydrogen phosphate, 5 mM magnesium sulfate, and 300 g / L starch. Add 10 U starch phosphorylase, 10 U glucose 6-phosphate isomerase, 10 U fructose 6-phosphate isomerase, 10 U gatZ, and 10 U T6PP to a 1 L reaction system and catalyze at 70°C for 72 h. The content of tagatose detected by liquid chromatography is 103 g, and the yield is 34.3%.

[0089] Example 6

[0090] Tagatose six phosphate phosphatase (T6PP-T) and its preparation:

[0091] A new type of tagatose six-phosphate phosphatase (T6PP-T) was screened from the high-temperature hot spring metagenome, and the species source of the enzyme is: Thermoanaerobacterales bacterium. The species source of the commonly used tagatose six-phosphate phosphatase (T6PP) is: Sphaerobacter thermophilus, sequence accession number: WP_012872856.1.

[0092] The amino acid sequence of the tagatose six-phosphate phosphatase is:

[0093] MVFDLDGVLLDSEQAWDEARRALVRERGGRWRPEAQRAMMGMSSPEWSRYLRDELGVDMAPEDISRAVVERLVAGYRRHLPLLPGAVEAVRALAARWPLGLASSSNRPVIDLVLELAGLAGAFAATVSSEEVARGKPAPDVYLEAARRLGVAPEACVAVEDSTNGLRSAAAAGMRVVALPNRDYPPAADALALAAVRLGSLDELRPELIERLDRPGA, as shown in SEQ ID NO.5.

[0094] Nucleic acid sequence:

[0095] ATGGTTTTCGATCTGGATGGTGTTCTGCTGGATAGTGAACAGGCCTGGGATGAAGCACGCCGTGCACTGGTGCGCGAACGCGGTGGTCGTTGGCGTCCGGAAGCACAGCGCGCAATGATGGGTATGAGTAGCCCGGAATGGAGCCGCTATCTGCGTGATGAACTGGGCGTTGATATGGCACCGGAAGATATTAGTCGTGCAGTGGTGGAACGTCTGGTGGCCGGTTATCGTCGTCATCTGCCGCTGCTGCCGGGCGCAGTGGAAGCTGTGCGCGCTCTGGCCGCACGCTGGCCTTTAGGTCTGGCCAGCAGTAGTAATCGTCCGGTTATTGATCTGGTTCTGGAACTGGCCGGCCTGGCCGGCGCATTTGCAGCAACCGTTAGCAGTGAAGAAGTGGCACGTGGTAAACCGGCCCCGGATGTTTATCTGGAAGCAGCACGTCGTCTGGGTGTTGCCCCGGAAGCCTGCGTTGCAGTGGAAGATAGTACCAATGGTCTGCGTAGCGCAGCAGCCGCAGGCATGCGCGTGGTTGCCCTGCCTAATCGTGATTATCCGCCGGCCGCAGATGCACTGGCCCTGGCAGCAGTTCGCCTGGGTAGCCTGGATGAACTGCGTCCGGAACTGATTGAACGCCTGGATCGTCCGGGCGCC, as shown in SEQ ID NO.6.

[0096] Design primer P3: aactttaagaaggagatatacatATGGTTTTCGATCTGGATGG (as shown in SEQ ID NO.7); P4:

[0097] agtggtggtggtggtggtggtggtggtgctcgagGGCGCCCGGACGATCCAGGCG (as shown in SEQ ID NO.8) was amplified using the T6PP-T sequence as a template to obtain an amplified product, which was separated and recovered and concentrated by agarose gel electrophoresis. The pet41 vector was digested with NdeI+XhoI, and the product was separated and recovered and concentrated by agarose gel electrophoresis. The digested pet41 vector and the amplified fragment were recombined using a DNA recombination kit, transformed into the competent E. coli DH5a, and positive clones were cultured and screened for sequencing verification. The positive clones were transformed into E. coli BL21 to construct an engineered strain that can recombinantly express T6PP-T.

[0098] The above recombinant strain was cultured using TB medium and induced to express using IPTG. The induced expression bacteria were collected by centrifugation, and the bacteria were resuspended in PBS buffer containing 20mM imidazole and 0.5M sodium chloride. The bacteria were resuspended in a ratio of 10ml buffer to 1g wet bacteria, ultrasonically broken, and the supernatant was collected by centrifugation. The recombinant protein in the supernatant was adsorbed by a Ni column, and the recombinant protein was eluted with a PBS buffer containing 300mM imidazole and 0.5M sodium chloride. The eluted protein was desalted using a desalting column to obtain T6PP-T.

[0099] T6PP-T was purified using the His tag, and after desalting, its protein content was calibrated using the Coomassie Brilliant Blue G250 method.

[0100] Example 7

[0101] T6PP-T catalyzes the dephosphorylation of tagatose hexaphosphate to synthesize tagatose:

[0102] 10 mg of T6PP-T was added to 100 mL of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate and 50 mM tagatose hexaphosphate, and the reaction was catalyzed at 70° C. for 1 hour to obtain tagatose with a yield of 48 mM and a yield of 96%.

[0103] Comparative Example 4

[0104] T6PP catalyzes the dephosphorylation of tagatose hexaphosphate to synthesize tagatose:

[0105] 10 mg of T6PP was added to 100 mL of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate and 50 mM tagatose hexaphosphate, and the reaction was catalytically reacted at 70° C. for 1 hour to obtain tagatose hexaphosphate with a yield of 33 mM and a yield of 66%.

[0106] Example 8

[0107] Detection of T6PP-T's substrate affinity:

[0108] The catalytic reaction solution was prepared with 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, 1 μmol / L catalytic enzyme, and substrates (tagatose hexaphosphate, fructose hexaphosphate, glucose hexaphosphate, glucose monophosphate, 0.1 mM-100 mM) and catalyzed at 70°C for 1 hour. The amount of dephosphorylation product generated was detected by liquid chromatography, and the Km value of T6PP-T for the substrate was measured by Linewaver-Burk plotting. The results are shown in Table 5.

[0109] Table 5 Affinity of tagatose six phosphate phosphatase (T6PP-T) for different sugar phosphate substrates

[0110]

[0111] Table 5 shows that the tagatose 6-phosphate phosphatase can more efficiently select tagatose 6-phosphate as a substrate for dephosphorylation at 70°C, while fructose 6-phosphate, glucose 6-phosphate and glucose monophosphate in the same system cannot be dephosphorylated in this case. Therefore, the use of the tagatose 6-phosphate phosphatase in the reaction can further improve the conversion rate of tagatose catalysis and greatly reduce the yield of byproducts (glucose and fructose).

[0112] Example 9

[0113] Prepare a catalytic solution (pH 7.0) of 50 mM potassium hydrogen phosphate, 5 mM magnesium sulfate, and 300 g / L starch. Add 10 U starch phosphorylase, 10 U glucose 6-phosphate isomerase, 10 U fructose 6-phosphate isomerase, 10 U gatZ or 10 U T6PE-T, 10 U T6PP or T6PP-T to 1 L reaction system respectively. Catalyze at 70 ° C for 72 hours. After the catalysis is completed, the content of tagatose and sorbose is detected by liquid chromatography, as shown in Table 6 below.

[0114] Table 6 Liquid chromatography detection of tagatose and sorbose content

[0115] Tagetang (g / L) Sorbitol(g / L) T6PP-T 198 3.82 T6PP 161 12.48

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An enzyme combination for catalyzing the synthesis of tagatose at high temperature, characterized in that: The enzyme combination comprises tagatose six phosphate synthase and tagatose six phosphate phosphatase, the amino acid sequence of the tagatose six phosphate synthase is shown in SEQ ID NO.1, and the amino acid sequence of the tagatose six phosphate phosphatase is shown in SEQ ID NO.

5.

2. A nucleic acid molecule, characterized in that: The invention comprises nucleotides encoding tagatose six phosphate synthase and nucleotides encoding tagatose six phosphate phosphatase, wherein the amino acid sequence of the tagatose six phosphate synthase is shown in SEQ ID NO.1, and the amino acid sequence of the tagatose six phosphate phosphatase is shown in SEQ ID NO.

5.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase is shown in SEQ ID NO.

2.

4. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid sequence of the nucleotide encoding tagatose six-phosphate phosphatase is shown in SEQ ID NO.

6.

5. A microorganism characterized by: The invention can express tagatose six phosphate synthase and tagatose six phosphate phosphatase, wherein the amino acid sequence of the tagatose six phosphate synthase is shown in SEQ ID NO.1, and the amino acid sequence of the tagatose six phosphate phosphatase is shown in SEQ ID NO.

5.

6. The microorganism according to claim 5, characterized in that The starting strain of the microorganism is Escherichia coli.

7. The microorganism according to claim 6, characterized in that The starting strain of the microorganism is Escherichia coli BL21.

8. The microorganism according to any one of claims 5 to 7, characterized in that The method for constructing an engineered bacterium capable of expressing tagatose hexaphosphate synthase is as follows: amplifying the nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase with a first primer pair, preparing a recombinant vector with the amplified product and a vector, and transferring the recombinant vector into a starting bacterium; the base sequences of the first primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

9. The microorganism according to claim 8, characterized in that The vector is a pet41 vector.

10. The microorganism according to any one of claims 5 to 7, characterized in that: The method for constructing an engineered bacterium capable of expressing tagatose six-phosphate phosphatase is as follows: amplifying the nucleic acid sequence of the nucleotide encoding tagatose six-phosphate phosphatase with a second primer pair, preparing a recombinant vector with the amplified product and a vector, and transferring the recombinant vector into the starting bacterium; the base sequences of the second primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively.

11. The microorganism according to claim 10, characterized in that The vector is a pet41 vector.

12. Use of the enzyme combination for catalyzing the synthesis of tagatose at high temperature as claimed in claim 1 in preparing tagatose from starch in one step.

13. The use according to claim 12, characterized in that: The enzyme combination for catalyzing the synthesis of tagatose at high temperature, as well as starch phosphorylase, glucose 6-phosphate isomerase and fructose 6-phosphate isomerase are added to a reaction system containing starch, and the mixture is heated to 65-70° C. for catalytic reaction for 60-84 h.

14. The use according to claim 13, characterized in that: The initial concentration of starch in the reaction system was 250~350 g / L.

15. The use according to claim 13, characterized in that: The reaction system contains dipotassium hydrogen phosphate and magnesium sulfate.

16. The use according to claim 15, characterized in that: The molar ratio of dipotassium hydrogen phosphate to magnesium sulfate is 9-11:

1.

17. The use according to claim 15, characterized in that: The concentration of potassium dihydrogen phosphate in the reaction system is 45-55 mM.

18. The use according to claim 13, characterized in that: The pH of the reaction system is 6.9~7.2.

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