技术领域Technical Field
本发明涉及塔格糖的生物发酵方法，具体涉及一种高温下高效催化合成塔格糖酶及其应用。The invention relates to a biological fermentation method of tagatose, and in particular to an enzyme for efficiently catalyzing and synthesizing tagatose at high temperature and an application thereof.
背景技术Background technique
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。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.
据发明人研究了解，通过糖原磷酸化酶、葡糖磷酸变位酶、葡萄糖六磷酸异构酶、塔格糖六磷酸合成酶、磷酸酶的共同作用将淀粉一步转化形成塔格糖，其具体过程为：淀粉与磷酸在糖原磷酸化酶的催化作用下形成萄糖-1-磷酸(葡萄糖一磷酸)，通过葡糖磷酸变位酶可将葡萄糖一磷酸变构形成葡萄糖-6-磷酸(葡萄糖六磷酸)，在葡萄糖六磷酸异构酶的催化作用下可以将葡萄糖六磷酸异构形成果糖-6-磷酸(果糖六磷酸)，果糖六磷酸经过塔格糖六磷酸合成酶的催化异构形成塔格糖-6-磷酸(塔格糖六磷酸)，塔格糖六磷酸在磷酸酶的作用下脱磷形成塔格糖。然而，目前采用上述方法将淀粉一步转化形成塔格糖的副产物较多，产率较低，从而影响其在生产塔格糖中的应用。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, which affects its application in the production of tagatose.
发明内容Summary of the invention
为了解决现有技术的不足，本发明的目的是提供一种高温下高效催化合成塔格糖酶及其应用，本发明提供的塔格糖酶具有更高的温度稳定性，更有利于提高淀粉转化为塔格糖的效率。In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a tagatase that can efficiently catalyze 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.
为了实现上述目的，本发明的技术方案为：In order to achieve the above object, the technical solution of the present invention is:
第一方面，一种高温下高效催化合成塔格糖酶，包括塔格糖六磷酸合成酶和塔格糖六磷酸磷酸酶，所述塔格糖六磷酸合成酶的氨基酸序列如SEQ ID NO.1所示，所述塔格糖六磷酸磷酸酶的氨基酸序列如SEQ ID NO.5所示。In the first aspect, a tagatase that efficiently 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.
本发明研究发现，塔格糖六磷酸合成酶的催化效率较低以及对果糖六磷酸底物的亲和度较高，导致淀粉转化形成塔格糖六磷酸效率低；同时，磷酸酶在脱磷酸过程中塔格糖六磷酸的特意选择性较差，导致反应体系中会有大量的葡萄糖一磷酸、葡萄糖六磷酸以及果糖六磷酸被脱磷最终产生较多的葡萄糖与果糖为副产物；因此，影响淀粉一步法制备塔格糖的产率。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.
因此，本发明首先从高温热泉的宏基因组中筛选并克隆出了一个来源于Thermoflexus sp.的塔格糖六磷酸合成酶(T6PE-T)，相比现有的塔格糖六磷酸合成酶，T6PE-T具有更高高温催化稳定性以及极低的果糖六磷酸Km，能够在在添加更少的酶的条件下通过高温(50～70℃)持续更专一地催化产生更多的塔格糖六磷酸；其次，本发明从高温热泉宏基因组中筛选并克隆出了一个来源于Thermoanaerobacterales bacterium的塔格糖六磷酸磷酸酶，与现有塔格糖六磷酸磷酸酶相比，本发明提供的塔格糖六磷酸磷酸酶可以在更高的温度(70℃)条件下能够高选择性地将塔格糖六磷酸脱磷形成，极大程度上降低葡萄糖和果糖等副产物的生成，从而提高塔格糖的产率。Therefore, the present invention first screened and cloned a tagatose six-phosphate synthase (T6PE-T) derived from Thermoflexus sp. from the metagenome of high-temperature hot springs. Compared with the existing tagatose six-phosphate synthase, T6PE-T has higher high-temperature catalytic stability and extremely low fructose six-phosphate Km, and can continuously and more specifically catalyze the production of more tagatose six-phosphate by high temperature (50-70°C) under the condition of adding less enzyme; secondly, the present invention screened and cloned a tagatose six-phosphate phosphatase derived from Thermoanaerobacterales bacterium from the metagenome of high-temperature hot springs. 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 under higher temperature (70°C) conditions, greatly reducing the generation of by-products such as glucose and fructose, thereby improving the yield of tagatose.
第二方面，一种核酸分子，包括编码塔格糖六磷酸合成酶的核苷酸和编码塔格糖六磷酸磷酸酶的核苷酸，所述塔格糖六磷酸合成酶的氨基酸序列如SEQ ID NO.1所示，所述塔格糖六磷酸磷酸酶的氨基酸序列如SEQ ID NO.5所示。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.
进一步地，编码塔格糖六磷酸合成酶的核苷酸的核酸序列如SEQ ID NO.2所示。Furthermore, the nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase is shown in SEQ ID NO.2.
进一步地，编码塔格糖六磷酸磷酸酶的核苷酸的核酸序列如SEQ ID NO.6所示。Furthermore, the nucleic acid sequence of the nucleotide encoding tagatose six-phosphate phosphatase is shown in SEQ ID NO.6.
第三方面，一种微生物，能够表达塔格糖六磷酸合成酶和塔格糖六磷酸磷酸酶，所述塔格糖六磷酸合成酶的氨基酸序列如SEQ ID NO.1所示，所述塔格糖六磷酸磷酸酶的氨基酸序列如SEQ ID NO.5所示。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.
本发明所述的微生物可以为一组分别能够表达塔格糖六磷酸合成酶和塔格糖六磷酸磷酸酶的工程菌组，也可以为一株能够同时能够表达塔格糖六磷酸合成酶和塔格糖六磷酸磷酸酶的工程菌。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.
进一步地，所述微生物的出发菌株为大肠杆菌。具体为大肠杆菌BL21。Furthermore, the starting strain of the microorganism is Escherichia coli, specifically Escherichia coli BL21.
更进一步地，能够表达塔格糖六磷酸合成酶的工程菌的构建方法为：将第一引物对编码塔格糖六磷酸合成酶的核苷酸的核酸序列进行扩增，将扩增产物与载体制成重组载体，将重组载体转入至出发菌中；所述第一引物对的碱基序列分别如SEQ ID NO.3和SEQ IDNO.4所示。具体地，所述载体为pet41载体。Furthermore, 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 the first primer pair, preparing a recombinant vector with the amplified product and the vector, and transferring the recombinant vector into the starting bacterium; 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.
进一步地，能够表达塔格糖六磷酸磷酸酶的工程菌的构建方法为：将第二引物对编码塔格糖六磷酸磷酸酶的核苷酸的核酸序列进行扩增，将扩增产物与载体制成重组载体，将重组载体转入至出发菌中；所述第二引物对的碱基序列分别如SEQ ID NO.7和SEQ IDNO.8所示。具体地，所述载体为pet41载体。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.
第四方面，一种上述高温下高效催化合成塔格糖酶、核酸分子或微生物在淀粉一步制备塔格糖中的应用。In a fourth aspect, a use of the above-mentioned high-efficiency catalytic synthesis of tagatase, nucleic acid molecule or microorganism in the one-step preparation of tagatose from starch.
进一步地，将所述高温下高效催化合成塔格糖酶、核酸分子或微生物，以及淀粉磷酸化酶、葡萄糖6磷酸异构酶、果糖六磷酸异构酶加入至含有淀粉的反应体系中，加热至65～70℃进行催化反应60～84h。Furthermore, the tagatase, nucleic acid molecule or microorganism that efficiently catalyzes the synthesis of tagatase 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 heated to 65-70° C. for catalytic reaction for 60-84 hours.
更进一步地，反应体系中淀粉的起始浓度为250～350g/L。Furthermore, the initial concentration of starch in the reaction system is 250-350 g/L.
更进一步地，反应体系中含有磷酸氢二钾和硫酸镁。具体地，磷酸氢二钾和硫酸镁的摩尔比为9～11:1。具体地，反应体系中含有磷酸氢二钾的浓度为45～55mM。本发明中单位“M”是指mol/L，单位“mM”是指mmol/L。具体地，反应体系中的pH为6.9～7.2。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.
第五方面，一种产品1在催化果糖六磷酸制备塔格糖六磷酸或催化淀粉一步制备塔格糖中的应用，所述产品1为塔格糖六磷酸合成酶、编码所述塔格糖六磷酸合成酶的核苷酸或表达所述塔格糖六磷酸合成酶的微生物，所述塔格糖六磷酸合成酶的氨基酸序列如SEQ ID NO.1所示。In a fifth aspect, a product 1 is used in 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.
本发明从高温热泉的宏基因组中筛选并克隆出了一个来源于Thermoflexus sp.的塔格糖六磷酸合成酶(T6PE-T)，相比现有的塔格糖六磷酸合成酶，T6PE-T具有更高高温催化稳定性以及极低的果糖六磷酸Km，能够在在添加更少的酶的条件下通过高温(50～70℃)持续更专一地催化产生更多的塔格糖六磷酸，从而提高塔格糖六磷酸的收率。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.
进一步地，编码塔格糖六磷酸合成酶的核苷酸的核酸序列如SEQ ID NO.2所示。Furthermore, the nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase is shown in SEQ ID NO.2.
进一步地，所述微生物的出发菌株为大肠杆菌。具体为大肠杆菌BL21。Furthermore, the starting strain of the microorganism is Escherichia coli, specifically Escherichia coli BL21.
更进一步地，能够表达塔格糖六磷酸合成酶的微生物的构建方法为：将第一引物对编码塔格糖六磷酸合成酶的核苷酸的核酸序列进行扩增，将扩增产物与载体制成重组载体，将重组载体转入至出发菌中；所述第一引物对的碱基序列分别如SEQ ID NO.3和SEQ IDNO.4所示。具体地，所述载体为pet41载体。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.
进一步地，催化果糖六磷酸制备塔格糖六磷酸中，向含有果糖六磷酸的反应液中添加产品1，在65～70℃的条件下进行催化反应。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.
更进一步地，所述反应液中果糖六磷酸的浓度为40～60mM。Furthermore, the concentration of fructose hexaphosphate in the reaction solution is 40-60 mM.
更进一步地，所述反应液中含有磷酸二氢钾和硫酸镁。具体地，磷酸氢二钾和硫酸镁的摩尔比为9～11:1。具体地，反应体系中含有磷酸氢二钾的浓度为45～55mM。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.
进一步地，催化淀粉一步制备塔格糖中，向含有淀粉的反应体系中添加所述产品1，以及淀粉磷酸化酶、葡萄糖6磷酸异构酶、果糖六磷酸异构酶和塔格糖六磷酸磷酸酶，加热至65～70℃催化反应60～84h。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.
更进一步地，所述反应液中含有磷酸二氢钾和硫酸镁。具体地，磷酸氢二钾和硫酸镁的摩尔比为9～11:1。具体地，反应体系中含有磷酸氢二钾的浓度为45～55mM。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.
第六方面，一种产品2在催化塔格糖六磷酸脱磷形成塔格糖中的应用，所述产品2为塔格糖六磷酸磷酸酶、编码所述塔格糖六磷酸磷酸酶的核苷酸或表达所述塔格糖六磷酸磷酸酶的微生物，所述塔格糖六磷酸合成酶的氨基酸序列如SEQ ID NO.5所示。In a sixth aspect, a product 2 is used for catalyzing the dephosphorylation of tagatose hexaphosphate to form tagatose, wherein the product 2 is a 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.
本发明本发明从高温热泉宏基因组中筛选并克隆出了一个来源于Thermoanaerobacterales bacterium的塔格糖六磷酸磷酸酶，与现有塔格糖六磷酸磷酸酶相比，本发明提供的塔格糖六磷酸磷酸酶可以在更高的温度(70℃)条件下能够高选择性地将塔格糖六磷酸脱磷形成，极大程度上降低葡萄糖和果糖等副产物的生成。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.
进一步地，编码塔格糖六磷酸合成酶的核苷酸的核酸序列如SEQ ID NO.6所示。Furthermore, the nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase is shown in SEQ ID NO.6.
进一步地，所述微生物的出发菌株为大肠杆菌。具体为大肠杆菌BL21。Furthermore, the starting strain of the microorganism is Escherichia coli, specifically Escherichia coli BL21.
更进一步地，能够表达塔格糖六磷酸磷酸酶的微生物的构建方法为：将第二引物对编码塔格糖六磷酸磷酸酶的核苷酸的核酸序列进行扩增，将扩增产物与载体制成重组载体，将重组载体转入至出发菌中；所述第二引物对的碱基序列分别如SEQ ID NO.7和SEQ IDNO.8所示。具体地，所述载体为pet41载体。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.
进一步地，向含有塔格糖六磷酸的反应液中添加产品2，在65～70℃的条件下进行催化反应。Furthermore, product 2 is added to the reaction solution containing tagatose hexaphosphate, and the catalytic reaction is carried out at 65-70°C.
更进一步地，所述反应液中含有磷酸二氢钾和硫酸镁。具体地，磷酸氢二钾和硫酸镁的摩尔比为9～11:1。具体地，反应体系中含有磷酸氢二钾的浓度为45～55mM。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.
本发明的有益效果为：The beneficial effects of the present invention are:
本发明通过对高温热泉宏基因组分别筛选了新的塔格糖六磷酸合成酶和塔格糖六磷酸磷酸酶，通过该塔格糖六磷酸合成酶能够在50～70℃的高温条件下持续、专一地催化产生更多的塔格糖六磷酸；并在此基础上，通过筛选的新的塔格糖六磷酸磷酸酶可以在70℃下更加高效的选择塔格糖六磷酸为底物进行脱磷，从而大大提高淀粉一步法制备塔格糖的效率。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.
具体实施方式Detailed ways
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例与对比例详细说明本发明的技术方案。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.
实施例1Example 1
塔格糖六磷酸合成酶(T6PE-T)及其制备：Tagatose six phosphate synthase (T6PE-T) and its preparation:
从高温热泉的宏基因组中筛选并克隆出了一个来源于Thermoflexus sp.的塔格糖六磷酸合成酶(T6PE-T)。现在已知可以合成塔格糖六磷酸的合成酶是来源于Caldilineaaerophila的gatZ(accession number：WP_014433578)。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).
塔格糖六磷酸合成酶T6PE-T的氨基酸序列为：The amino acid sequence of tagatose hexaphosphate synthase T6PE-T is:
MKGYDVVAAGGGSCSAHVAARHGRASGKVATSNVNGGYTGMTAAGAAKMARGRRGGDHGWSHAMRKADVRAVAGTKHDCSMGGVARARARVA AAGRRDRYVVGSVAGGAAAGVTRDAAAAARRARRDTAWRVAVVGGDHYDRAAAAVRWGVGAHSTDYAARAVDHAKVGATARAVAADAAGSRAAAMARVHWKYYRGDARVRARRYSSDRRYYWAAVTARMRNGDRVGSYMYRRVRRGRNHRDGRVMAVGYAATVNGCR，如SEQ ID NO.1所示。MKGYDVVAAGGGSCSAHVAARHGRASGKVATSNVNGGYTGMTAAGAAKMARGRRGGDHGWSHAMRKADVRAVAGTKHDCSMGGVARARARVA AAGRRDRYVVGSVAGGAAAGVTRDAAAAARRARRDTAWRVAVVGGDHYDRAAAAVRWGVGAHSTDYAARAVDHAKVGATARAVAADAAGSRAAAMARVHWKYYRGDARVRARRYSSDRRY YWAAVTARMRNGDRVGSYMYRRVRRGRNHRDGRVMAVGYAATVNGCR, as shown in SEQ ID NO.1.
核酸序列：Nucleic acid sequence:
ATGAAGGGTTATGATGTGGTTGCAGCAGGTGGTGGTAGCTGTAGCGCCCATGTGGCCGCACGTCATGGCCGTGCCAGTGGCAAAGTTGCAACCAGTAATGTGAATGGTGGTTATACCGGCATGACCGCAGCCGGCGCCGCCAAAATGGCACGCGGTCGTCGCGGCGGCGATCATGGTTGGAGTCATGCCATGCGTAAAGCCGATGTTCGTGCAGTGGCCGGTACCAAACATGATTGCAGCATGGGTGGTGTGGCCCGCGCACGTGCACGCGTTGCTGCTGCAGGCCGTCGTGATCGCTATGTGGTTGGCAGTGTGGCAGGCGGCGCAGCAGCCGGTGTGACACGTGATGCAGCCGCCGCCGCCCGTCGTGCAAGAAGAGATACCGCATGGCGCGTTGCAGTTGTTGGTGGTGACCATTATGATCGTGCAGCAGCCGCAGTGCGCTGGGGTGTGGGTGCACATAGCACCGATTATGCAGCCCGCGCAGTGGATCATGCCAAAGTGGGTGCAACCGCCCGTGCAGTGGCGGCTGATGCCGCAGGTAGTCGTGCCGCAGCCATGGCACGTGTTCATTGGAAATATTATCGCGGTGACGCCCGTGTTCGTGCACGTCGTTATAGTAGCGATCGTCGCTATTATTGGGCAGCAGTTACCGCCCGTATGCGCAATGGCGATCGCGTGGGTAGTTATATGTATCGTCGCGTGCGTCGCGGCCGTAATCATCGTGATGGTCGTGTTATGGCCGTGGGCTATGCAGCCACCGTGAATGGTTGTCGC，如SEQ ID NO.2所示。ATGAAGGGTTATGATGTGGTTGCAGCAGGTGGTGGTAGCTGTAGCGCCCATGTGGCCGCACGTCATGGCCGTGCCAGTGGCAAAGTTGCAACCAGTAATGTGAATGGTGGTTATACCGGCATGACCGCAGCCGGCGCCGCCAAAATGGCACGCGGTCGTCGCGGCGGCGATCATGGTTGGAGTCATGCCATGCGTAAAGCCGATGTTCGTGCAGTGGCCGGTACCAAACATGATTGCAGCATGGGTGGTGGTGGCCC GCGCACGTGCACGCGTTGCTGCTGCAGGCCGTCGTGATCGCTATGTGGTTGGCAGTGTGGCAGGCGGCGCAGCAGCCGGTGTGACACGTGATGCAGCCGCCGCCGCCCGTCGTGCAAGAAGAGATACCGCATGGC GCGTTGCAGTTGTTGGTGGTGACCATTATGATCGTGCAGCAGCCGCAGTGCTGGGGTGTGGGTGCACATAGCACCGATTATGCAGCCCGCGCAGTGGATCATGCCAAAGTGGGTGCAACCGCCCGTGCAGTGGCGGCTGATGCCGCAGGTAGTCGTGCCGCAGCCATGGCACGTGTTCATTGGAAATATTATCGCGGTGACGCCCGTGTTCGTGCACGTCGTTATAGTAGCGATCGTCGCTATTATTGGGCAG CAGTTACCGCCCGTATGCGCAATGGCGATCGCGTGGGTAGTTATATGTATCGTCGCGTGCGTCGCGGCCGTAATCATCGTGATGGTCGTGTTATGGCCGTGGGCTATGCAGCCACCGTGAATGGTTGTCGC, as shown in SEQ ID NO.2.
设计引物P1：aactttaagaaggagatatacatATGAAGGGTTATGATGTGG(如SEQ ID NO.3所示)；P2：agtggtggtggtggtggtggtggtgctcgagGCGACAACCATTCACG(如SEQ ID NO.4所示)以T6PE-T序列为模板扩增获得扩增产物，利用琼脂糖凝胶电泳对产物进行分离及回收浓缩。利用NdeI+XhoI对pet41载体进行酶切，利用琼脂糖凝胶电泳对产物进行分离及回收浓缩。利用DNA重组试剂盒将酶切好的pet41载体与扩增片段进行重组，转化至大肠杆菌DH5a感受态中，培养筛选出阳性克隆，测序验证。将阳性克隆转化入大肠杆菌BL21中，构建出可重组表达T6PE-T的工程菌株。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.
利用TB培养基对上述重组菌株进行培养，用IPTG诱导表达。离心收集诱导表达好的菌体，用含有20mM咪唑、0.5M氯化钠的PBS缓冲液重悬菌体，按照1g湿菌体加入10ml缓冲液比例重悬，超声破碎，离心收集上清液。用Ni柱对上清液中的重组蛋白进行吸附，用含有300mM咪唑、0.5M氯化钠的PBS缓冲液对重组蛋白进行洗脱，利用脱盐柱对洗脱的蛋白进行脱盐，即得T6PE-T。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 10ml buffer according to the ratio of 1g wet bacteria, ultrasonically broken, and the supernatant was collected by centrifugation. The recombinant protein in the supernatant was adsorbed by Ni column, and the recombinant protein was eluted with PBS buffer containing 300mM imidazole and 0.5M sodium chloride. The eluted protein was desalted using a desalting column to obtain T6PE-T.
利用His标签纯化出T6PE-T，对其脱盐后，利用考马斯亮蓝G250法对其蛋白含量分别进行标定。T6PE-T was purified using the His tag, and after desalting, its protein content was calibrated using the Coomassie Brilliant Blue G250 method.
实施例2Example 2
T6PE-T催化果糖六磷酸合成塔格糖六磷酸：T6PE-T catalyzes the synthesis of tagatose hexaphosphate from fructose hexaphosphate:
将20mg T6PE-T添加至100ml含有50mM磷酸二氢钾(pH 7.2)、5mM硫酸镁以及50mM果糖六磷酸的反应液，在70℃条件下催化反应1小时，获得塔格糖六磷酸，产量8.5mM，收率17％。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%.
对比例1Comparative Example 1
gatZ催化果糖六磷酸合成塔格糖六磷酸：gatZ catalyzes the synthesis of tagatose hexaphosphate from fructose hexaphosphate:
gatZ催化果糖六磷酸合成塔格糖六磷酸：gatZ catalyzes the synthesis of tagatose hexaphosphate from fructose hexaphosphate:
将20mg gatZ添加至100ml含有50mM磷酸二氢钾(pH 7.2)、5mM硫酸镁以及50mM果糖六磷酸的反应液，在70℃条件下催化反应1小时，获得塔格糖六磷酸，产量4.9mM，收率9.8％。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%.
实施例3Example 3
T6PE-T的催化的效率检测：Catalytic efficiency test of T6PE-T:
将T6PE-T放置在不同温度下(例如55℃-70℃)热处理不同时间(例如1500分钟-5000分钟)，将20mg热处理的T6PE-T与未处理的T6PE-T分别添加至100ml含有50mM磷酸二氢钾(pH 7.2)、5mM硫酸镁以及50mM果糖六磷酸的反应液中在70℃催化1小时，通过液相色谱检测塔格糖六磷酸的生成量来判定其催化的效率，生成量如表1所示。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.
表1热处理的T6PE-T与未处理的T6PE-T催化生成塔格糖六磷酸的量Table 1 The amount of tagatose hexaphosphate generated by heat-treated T6PE-T and untreated T6PE-T
对比例2Comparative Example 2
gatZ的催化的效率检测：Detection of the catalytic efficiency of gatZ:
将gatZ放置在不同温度下(例如55℃-70℃)热处理不同时间(例如1500分钟-5000分钟)，将20mg热处理的gatZ与未处理的gatZ分别添加至100ml含有50mM磷酸二氢钾(pH7.2)、5mM硫酸镁以及50mM果糖六磷酸的反应液中在70℃催化1小时，通过液相色谱检测塔格糖六磷酸的生成量来判定其催化的效率，生成量如表2所示。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.
表2热处理的gatZ与未处理的gatZ催化生成塔格糖六磷酸的量Table 2 The amount of tagatose hexaphosphate generated by heat-treated gatZ and untreated gatZ
通过对比实施例3和对比例2的催化效率，可以获知不同塔格糖六磷酸合成酶不同热处理的半衰期，从而获知其热稳定性，结果如表3所示。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.
表3T6PE-T与gatZ的热稳定性Table 3 Thermal stability of T6PE-T and gatZ
表3表明，与gatZ相比，T6PE-T在55～70℃的高温条件下催化的稳定性更强，可以在添加更少的酶的条件下通过高温持续催化产生更多的产物。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.
实施例4Example 4
T6PE-T、gatZ对底物亲和能力的检测：Detection of T6PE-T and gatZ's affinity for substrates:
测定1、果糖六磷酸Km值:配制催化反应液50mM磷酸二氢钾(pH 7.2)、5mM硫酸镁、催化酶20mg、果糖六磷酸(0.5mM-100mM)在70℃催化1小时，通过液相色谱检测塔格糖六磷酸的生成量，利用Linewaver-Burk作图法测出T6PE-T与gatZ对果糖六磷酸Km值。Determination 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.
测定2、果糖-1,6-二磷酸(果糖一六二磷酸)Km值：配制催化反应液50mM磷酸二氢钾(pH 7.2)、5mM硫酸镁、催化酶20mg、果糖一六二磷酸(0.5mM-100mM)在70℃催化1小时，通过液相色谱检测塔格糖-1,6-二磷酸(塔格糖一六二磷酸)的生成量，利用Linewaver-Burk作图法测出T6PE-T与gatZ对果糖一六二磷酸Km值。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.
测定3、塔格糖一六二磷酸Km值：配制催化反应液50mM磷酸二氢钾(pH7.2)、5mM硫酸镁、催化酶20mg、塔格糖一六二磷酸(0.5mM-100mM)在70℃催化1小时，通过液相色谱检测果糖一六二磷酸的生成量，利用Linewaver-Burk作图法测出T6PE-T与gatZ对塔格糖一六二磷酸Km值。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 determine the Km values of T6PE-T and gatZ for tagatose 1,6-diphosphate.
测定4、塔格糖六磷酸Km值：配制催化反应液50mM磷酸二氢钾(pH 7.2)、5mM硫酸镁、催化酶20mg、塔格糖六磷酸(0.5mM-100mM)在70℃催化1小时，通过液相色谱检测果糖六磷酸的生成量，利用Linewaver-Burk作图法测出T6PE-T与gatZ对塔格糖六磷酸Km值。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.
检测结果如表4所示。The test results are shown in Table 4.
表4T6PE-T与gatZ的对底物亲和能力Table 4 Affinity of T6PE-T and gatZ for substrates
表4表明，T6PE-T在催化过程中具有更低的果糖六磷酸Km值，因此该酶可以更加专一的催化形成塔格糖六磷酸，且不会将塔格糖六磷酸催化形成其它物质。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.
实施例5Example 5
T6PE-T催化淀粉合成塔格糖：T6PE-T catalyzes the synthesis of tagatose from starch:
配制50mM磷酸氢二钾、5mM硫酸镁、300g/L淀粉的催化液(pH7.0)。向1L反应体系中加入10U淀粉磷酸化酶、10U葡萄糖6磷酸异构酶、10U果糖六磷酸异构酶、10U T6PE-T、10UT6PP在70℃催化72h，液相色谱检测塔格糖的含量为168g，收率为56％。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%.
对比例3Comparative Example 3
gatZ催化淀粉合成塔格糖：gatZ catalyzes the synthesis of tagatose from starch:
配制50mM磷酸氢二钾、5mM硫酸镁、300g/L淀粉的催化液(pH7.0)。向1L反应体系中加入10U淀粉磷酸化酶、10U葡萄糖6磷酸异构酶、10U果糖六磷酸异构酶、10U gatZ、10UT6PP在70℃催化72h，液相色谱检测塔格糖的含量为103g，收率为34.3％。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%.
实施例6Example 6
塔格糖六磷酸磷酸酶(T6PP-T)及其制备：Tagatose six phosphate phosphatase (T6PP-T) and its preparation:
从高温热泉宏基因组中筛选获得了一个新型的塔格糖六磷酸磷酸酶(T6PP-T)，该酶的物种来源是：Thermoanaerobacterales bacterium。现在常用的塔格糖六磷酸磷酸酶(T6PP)的物种来源是：Sphaerobacter thermophilus，序列登录号：WP_012872856.1。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.
该塔格糖六磷酸磷酸酶的氨基酸序列为：The amino acid sequence of the tagatose six-phosphate phosphatase is:
MVFDLDGVLLDSEQAWDEARRALVRERGGRWRPEAQRAMMGMSSPEWSRYLRDELGVDMAPEDISRAVVERLVAGYRRHLPLLPGAVEAVRALAARWPLGLASSSNRPVIDLVLELAGLAGAFAATVSSEEVARGKPAPDVYLEAARRLGVAPEACVAVEDSTNGLRSAAAAGMRVVALPNRDYPPAADALALAAVRLGSLDELRPELIERLDRPGA，如SEQ ID NO.5所示。MVFDLDGVLLDSEQAWDEARRALVRERGGRWRPEAQRAMMGMSPEWSRYLRDELGVDMAPEDISRAVVERLVAGYRRHLPLLPGAVEAVRALAARWPLGLASSSNRPVIDLVLELAGLAGAFAATVSSEEVARGKPAPDVYLEAARRLGVAPEACVAVEDSTNGLRSAAAAGMRVVALPNRDYPPAADALALAAVRLGSLDELRPELIERLDRPGA, as shown in SEQ ID NO.5.
核酸序列：Nucleic acid sequence:
ATGGTTTTCGATCTGGATGGTGTTCTGCTGGATAGTGAACAGGCCTGGGATGAAGCACGCCGTGCACTGGTGCGCGAACGCGGTGGTCGTTGGCGTCCGGAAGCACAGCGCGCAATGATGGGTATGAGTAGCCCGGAATGGAGCCGCTATCTGCGTGATGAACTGGGCGTTGATATGGCACCGGAAGATATTAGTCGTGCAGTGGTGGAACGTCTGGTGGCCGGTTATCGTCGTCATCTGCCGCTGCTGCCGGGCGCAGTGGAAGCTGTGCGCGCTCTGGCCGCACGCTGGCCTTTAGGTCTGGCCAGCAGTAGTAATCGTCCGGTTATTGATCTGGTTCTGGAACTGGCCGGCCTGGCCGGCGCATTTGCAGCAACCGTTAGCAGTGAAGAAGTGGCACGTGGTAAACCGGCCCCGGATGTTTATCTGGAAGCAGCACGTCGTCTGGGTGTTGCCCCGGAAGCCTGCGTTGCAGTGGAAGATAGTACCAA TGGTCTGCGTAGCGCAGCAGCCGCAGGCATGCGCGTGGTTGCCCTGCCTAATCGTGATTATCCGCCGGCCGCAGATGCACTGGCCCTGGCAGCAGTTCGCCTGGGTAGCCTGGATGAACTGCGTCCGGAACTGATTGAACGCCTGGATCGTCCGGGCGCC，如SEQ ID NO.6所示。ATGGTTTTTCGATCTGGATGGTGTTCTGCTGGATAGTGAACAGGCCTGGGATGAAGCACGCCGTGCACTGGTGCGCGAACGCGGTGGTCGTTGGCGTCCGGAAGCACAGCGCGCAATGATGGGTATGAGTAGCCCGGAATGGAGCCGCTATCTGCGTGATGAACTGGGCGTTGATATGGCACCGGAAGATATTAGTCGTGCAGTGGTGGAACGTCTGGTGGCCGGTTATCGTCGTCATCTGCCGCT GCTGCCGGGCGCAGTGGAAGCTGTGCGCGCTCTGGCCGCACGCTGGCCTTTAGGTCTGGCCAGCAGTAGTAATCGTCCGGTTATTGATCTGGTTCTGGAACTGGCCGGCCTGGCCGGCGCATTTGCAGCAACCGTTAGCAGTGAAGAAGTGGCACGTGGTAAACCGGCCCCGGATGTTTATCTGGAAGCAGCACGTCGTCTGGGTGTTGCCCCGGAAGCCTGCGTTGCAGTGGAAGATAGTACCAA TGG TCTGCGTAGCGCAGCAGCCGCAGGCATGCGCGTGGTTGCCCTGCCTAATCGTGATTATCCGCCGGCCGCAGATGCACTGGCCCTGGCAGCAGTTCGCCTGGGTAGCCTGGATGAACTGCGTCCGGAACTGATTGAACGCCTGGATCGTCCGGGCGCC, as shown in SEQ ID NO.6.
设计引物P3：aactttaagaaggagatatacatATGGTTTTCGATCTGGATGG(如SEQ ID NO.7所示)；P4：Design primer P3: aactttaagaaggagatatacatATGGTTTTCGATCTGGATGG (as shown in SEQ ID NO.7); P4:
agtggtggtggtggtggtggtggtgctcgagGGCGCCCGGACGATCCAGGCG(如SEQ ID NO.8所示)以T6PP-T序列为模板扩增获得扩增产物，利用琼脂糖凝胶电泳对产物进行分离及回收浓缩。利用NdeI+XhoI对pet41载体进行酶切，利用琼脂糖凝胶电泳对产物进行分离及回收浓缩。利用DNA重组试剂盒将酶切好的pet41载体与扩增片段进行重组，转化至大肠杆菌DH5a感受态中，培养筛选出阳性克隆，测序验证。将阳性克隆转化入大肠杆菌BL21中，构建出可重组表达T6PP-T的工程菌株。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, 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 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.
利用TB培养基对上述重组菌株进行培养，用IPTG诱导表达。离心收集诱导表达好的菌体，用含有20mM咪唑、0.5M氯化钠的PBS缓冲液重悬菌体，按照1g湿菌体加入10ml缓冲液比例重悬，超声破碎，离心收集上清液。用Ni柱对上清液中的重组蛋白进行吸附，用含有300mM咪唑、0.5M氯化钠的PBS缓冲液对重组蛋白进行洗脱，利用脱盐柱对洗脱的蛋白进行脱盐，即得T6PP-T。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.
利用His标签纯化出T6PP-T，对其脱盐后，利用考马斯亮蓝G250法对其蛋白含量分别进行标定。T6PP-T was purified using the His tag, and after desalting, its protein content was calibrated using the Coomassie Brilliant Blue G250 method.
实施例7Example 7
T6PP-T催化塔格糖六磷酸脱磷酸合成塔格糖：T6PP-T catalyzes the dephosphorylation of tagatose hexaphosphate to synthesize tagatose:
将10mg T6PP-T添加至100mL含有50mM磷酸二氢钾(pH7.2)、5mM硫酸镁以及50mM塔格糖六磷酸的反应液，在70℃条件下催化反应1小时，获得塔格糖，产量48mM，收率96％。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%.
对比例4Comparative Example 4
T6PP催化塔格糖六磷酸脱磷酸合成塔格糖：T6PP catalyzes the dephosphorylation of tagatose hexaphosphate to synthesize tagatose:
将10mg T6PP添加至100mL含有50mM磷酸二氢钾(pH7.2)、5mM硫酸镁以及50mM塔格糖六磷酸的反应液，在70℃条件下催化反应1小时，获得塔格糖六磷酸，产量33mM，收率66％。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 catalyzed at 70° C. for 1 hour to obtain tagatose hexaphosphate with a yield of 33 mM and a yield of 66%.
实施例8Example 8
T6PP-T对底物亲和能力的检测：Detection of T6PP-T's substrate affinity:
配制催化反应液50mM磷酸二氢钾(pH7.2)、5mM硫酸镁、催化酶1μmol/L、底物(塔格糖六磷酸、果糖六磷酸、葡萄糖六磷酸、葡萄糖一磷酸，0.1mM-100mM)在70℃催化1小时，通过液相色谱检测脱磷产物的生成量，利用Linewaver-Burk作图法测出T6PP-T对底物Km值，结果如表5所示。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 dephosphorylated 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.
表5塔格糖六磷酸磷酸酶(T6PP-T)不同糖磷酸底物的亲和能力Table 5 Affinity of tagatose six phosphate phosphatase (T6PP-T) for different sugar phosphate substrates
表5表明，该塔格糖六磷酸磷酸酶可以在70℃下更加高效的选择塔格糖六磷酸为底物进行脱磷，而在该情况下同一体系的果糖六磷酸、葡萄糖六磷酸和葡萄糖一磷酸无法被脱磷。因此利用该塔格糖六磷酸磷酸酶参与反应可以更高的提升塔格糖催化的转化率且在极大程度上降低副产物(葡萄糖和果糖)的产率。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).
实施例9Embodiment 9
配制50mM磷酸氢二钾、5mM硫酸镁、300g/L淀粉的催化液(pH7.0)。分别向1L反应体系中加入10U淀粉磷酸化酶、10U葡萄糖6磷酸异构酶、10U果糖六磷酸异构酶、10U gatZ或者10U T6PE-T、10U T6PP或者T6PP-T。在70℃催化72h。催化完毕后通过液相色谱检测塔格糖与山梨糖的含量，如下表6所示。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 h. After the catalysis is completed, the content of tagatose and sorbose is detected by liquid chromatography, as shown in Table 6 below.
表6液相色谱检测塔格糖与山梨糖的含量Table 6 Liquid chromatography detection of tagatose and sorbose content
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。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.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411385472.3ACN119432804B (en) | 2024-05-29 | 2024-05-29 | Application of product in catalyzing tagatose hexaphosphoric acid dephosphorization to form tagatose |
| CN202410681025.6ACN118406675B (en) | 2024-05-29 | 2024-05-29 | A method for catalyzing the synthesis of tagatase at high temperature and its application |
| CN202411385471.9ACN119265171B (en) | 2024-05-29 | 2024-05-29 | Application of a product in catalyzing fructose hexaphosphate to prepare tagatose hexaphosphate or catalyzing starch to prepare tagatose in one step |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410681025.6ACN118406675B (en) | 2024-05-29 | 2024-05-29 | A method for catalyzing the synthesis of tagatase at high temperature and its application |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411385472.3ADivisionCN119432804B (en) | 2024-05-29 | 2024-05-29 | Application of product in catalyzing tagatose hexaphosphoric acid dephosphorization to form tagatose |
| CN202411385471.9ADivisionCN119265171B (en) | 2024-05-29 | 2024-05-29 | Application of a product in catalyzing fructose hexaphosphate to prepare tagatose hexaphosphate or catalyzing starch to prepare tagatose in one step |
| Publication Number | Publication Date |
|---|---|
| CN118406675Atrue CN118406675A (en) | 2024-07-30 |
| CN118406675B CN118406675B (en) | 2025-02-14 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410681025.6AActiveCN118406675B (en) | 2024-05-29 | 2024-05-29 | A method for catalyzing the synthesis of tagatase at high temperature and its application |
| CN202411385471.9AActiveCN119265171B (en) | 2024-05-29 | 2024-05-29 | Application of a product in catalyzing fructose hexaphosphate to prepare tagatose hexaphosphate or catalyzing starch to prepare tagatose in one step |
| CN202411385472.3AActiveCN119432804B (en) | 2024-05-29 | 2024-05-29 | Application of product in catalyzing tagatose hexaphosphoric acid dephosphorization to form tagatose |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411385471.9AActiveCN119265171B (en) | 2024-05-29 | 2024-05-29 | Application of a product in catalyzing fructose hexaphosphate to prepare tagatose hexaphosphate or catalyzing starch to prepare tagatose in one step |
| CN202411385472.3AActiveCN119432804B (en) | 2024-05-29 | 2024-05-29 | Application of product in catalyzing tagatose hexaphosphoric acid dephosphorization to form tagatose |
| Country | Link |
|---|---|
| CN (3) | CN118406675B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118530980A (en)* | 2024-04-30 | 2024-08-23 | 山东三元生物科技股份有限公司 | One-step production method for producing tagatose from starch with extremely low side reaction products |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3480304A1 (en)* | 2016-06-30 | 2019-05-08 | Cj Cheiljedang Corporation | Tagatose 6-phosphoric acid-specific novel heat-resistant phosphatase, and method for preparing tagatose by using same |
| CN117512033A (en)* | 2023-12-21 | 2024-02-06 | 山东三元生物科技股份有限公司 | Method for simultaneously producing D-tagatose and D-psicose from glucose |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| UA126434C2 (en)* | 2015-10-02 | 2022-10-05 | Бонамоуз Інк. | Enzymatic synthesis of d-tagatose |
| CN107988286B (en)* | 2016-11-02 | 2021-03-19 | 中国科学院天津工业生物技术研究所 | A kind of method for whole cell catalytic preparation of tagatose |
| KR102074957B1 (en)* | 2017-03-31 | 2020-02-10 | 씨제이제일제당 주식회사 | A composition for preparing tagatose and Methods for producing tagatose using The Same |
| CN109750024B (en)* | 2017-11-02 | 2021-12-14 | 中国科学院天津工业生物技术研究所 | A kind of 4-position epimerase of tagatose 6-phosphate and its application |
| CN109666620B (en)* | 2018-05-29 | 2022-05-10 | 中国科学院天津工业生物技术研究所 | Engineering strain for producing tagatose, construction method and application thereof |
| AU2019297508A1 (en)* | 2018-07-03 | 2021-02-25 | Arzeda Corp. | Methods and compositions for preparing tagatose from fructose |
| EP3867393A4 (en)* | 2018-10-19 | 2022-08-24 | Bonumose, Inc. | ENZYMATIC PRODUCTION OF TAGATOSE |
| KR102791960B1 (en)* | 2020-12-30 | 2025-04-08 | 주식회사 삼양사 | Tagatose 6-phosphatase with high substrate specificity and use of the same |
| CN112342179B (en)* | 2021-01-05 | 2021-04-06 | 中国科学院天津工业生物技术研究所 | Tagatose-producing Bacillus subtilis genetically engineered bacteria and method for preparing tagatose |
| CN117305204A (en)* | 2023-08-29 | 2023-12-29 | 杭州唯铂莱生物科技有限公司 | Method for producing D-tagatose by whole cell catalysis and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3480304A1 (en)* | 2016-06-30 | 2019-05-08 | Cj Cheiljedang Corporation | Tagatose 6-phosphoric acid-specific novel heat-resistant phosphatase, and method for preparing tagatose by using same |
| CN109790524A (en)* | 2016-06-30 | 2019-05-21 | Cj第一制糖株式会社 | Tagatose -6- phospho-specif iotac heat resistance acid phosphatase and the method for preparing Tagatose using it |
| US20190203189A1 (en)* | 2016-06-30 | 2019-07-04 | Cj Cheiljedang Corporation | Novel thermostable tagatose-6-phosphate phosphatase and a method for producing tagatose using the same |
| CN117512033A (en)* | 2023-12-21 | 2024-02-06 | 山东三元生物科技股份有限公司 | Method for simultaneously producing D-tagatose and D-psicose from glucose |
| Title |
|---|
| GENBANK: "MBO2501773.1", NCBI, 24 March 2021 (2021-03-24)* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118530980A (en)* | 2024-04-30 | 2024-08-23 | 山东三元生物科技股份有限公司 | One-step production method for producing tagatose from starch with extremely low side reaction products |
| CN118530980B (en)* | 2024-04-30 | 2024-12-10 | 山东三元生物科技股份有限公司 | Method for producing tagatose by starch with extremely low side reaction product through one-step method |
| Publication number | Publication date |
|---|---|
| CN119432804A (en) | 2025-02-14 |
| CN119265171B (en) | 2025-04-15 |
| CN119432804B (en) | 2025-06-24 |
| CN119265171A (en) | 2025-01-07 |
| CN118406675B (en) | 2025-02-14 |
| Publication | Publication Date | Title |
|---|---|---|
| CN113046403B (en) | Method for efficiently catalyzing and synthesizing PAPS (PAPS) based on construction of ATP (adenosine triphosphate) regeneration system | |
| AU2017287766A1 (en) | A Novel Thermostable Tagatose-6-Phosphate Phosphatase and A Method for Producing Tagatose Using the Same | |
| JP7266594B2 (en) | Novel tagatose 6-phosphate 4-epimerase and its application | |
| CN118406675A (en) | Efficient catalytic synthesis tagatose at high temperature and application thereof | |
| CN113583997B (en) | Phosphatase mutant and application thereof in preparation of mannose by catalyzing maltodextrin | |
| CN109988778A (en) | Sucrose phosphorylase gene and application thereof | |
| CN107603994A (en) | A kind of κ carrageenases and its gene and application | |
| CN113604454A (en) | Phosphatase mutant and application thereof in preparing fructose by catalyzing maltodextrin | |
| CN113913481B (en) | The biological preparation method of mannose | |
| CN112921022B (en) | Method for producing 1, 4-butanediamine by using recombinant escherichia coli | |
| CN111172215B (en) | A kind of preparation method of D-type rare ketohexose | |
| CN116790550B (en) | Nicotinamide ribokinase mutant and design method and application thereof | |
| KR102063908B1 (en) | A novel thermostable fructose-6-phosphate 3-epimerase and methods for producing allulose using the same | |
| KR20220096207A (en) | Tagatose 6-phosphatase with high substrate specificity and use of the same | |
| CN107475274B (en) | Gene of coding phosphoglucomutase in kelp, protein and application thereof | |
| CN113215125B (en) | Inulin sucrase mutant with improved thermal stability and enzyme activity | |
| CN116240195A (en) | Mannose 6-phosphodephosphorization enzyme mutant and application thereof | |
| KR20210052317A (en) | Fructose 6-phosphate 4-epimerase and use thereof | |
| KR102055875B1 (en) | A novel thermostable fructose-6-phosphate 3-epimerase and methods for producing allulose using the same | |
| CN114729345A (en) | Fructose 6-phosphate 3-epimerase and use thereof | |
| CN112481245A (en) | Recombinant D-tagatose 3 epimerase DTE-CM and construction method and application thereof | |
| CN119120415B (en) | A L-rhamnose sugar kinase mutant and preparation method thereof | |
| KR101228974B1 (en) | A thermostable H2O forming NADH oxidase from Lactobacillus rhamnosus and a preparation method thereof | |
| CN111944784A (en) | Maltogenic oligosaccharide based seaweed hydrolase mutant with improved heat stability and application thereof | |
| CN115786320B (en) | Method for synthesizing PAPS (sulfonic acid group donor) by using immobilized enzyme based on self-assembly strategy |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |