WO2023246099A1

WO2023246099A1 - Enzyme for preparing tagatose, composition and use thereof

Info

Publication number: WO2023246099A1
Application number: PCT/CN2023/073198
Authority: WO
Inventors: 马延和; 石婷; 李运杰
Original assignee: 中国科学院天津工业生物技术研究所
Priority date: 2022-06-24
Filing date: 2023-01-19
Publication date: 2023-12-28
Also published as: CN117327684A

Abstract

Provided are polypeptides with tagatose 6-phosphate epimerase activity, polypeptides with tagatose 6-phosphate phosphatase activity, a composition comprising the polypeptides with tagatose 6-phosphate epimerase activity and the polypeptides with tagatose 6-phosphate phosphatase activity, a method for preparing tagatose from the composition, and use of the composition in the preparation of tagatose.

Description

Enzymes for the preparation of tagatose, compositions thereof and uses thereof

Priority and related applications

This disclosure claims priority to Chinese patent application 202210730019.6 titled "Enzymes for preparing tagatose, compositions thereof and applications thereof" submitted on June 24, 2022. The entire content of this application, including the appendix, is used as a reference. incorporated into this disclosure.

Technical field

The present disclosure relates to the fields of genetic engineering and biocatalysis, and specifically to the field of biotechnology related to tagatose production. More specifically, the present disclosure relates to a composition comprising a novel tagatose 6-phosphate epimerase and/or tagatose 6-phosphate phosphatase and a method for preparing tagatose using the same.

Background technique

D-Tagatose (hereinafter referred to as tagatose) is a rare naturally occurring monosaccharide. It is the ketose form of galactose and the epimer of fructose. Natural tagatose mainly exists in dairy products such as yogurt and milk powder. Its sweetness characteristics are similar to sucrose, but the calories generated are only one-third of sucrose, so it is called a low-calorie sweetener. Tagatose has excellent nutritional properties such as low caloric value, zero glycemic index, blood sugar inactivation, no caries, prebiotic effect and antioxidant activity. It was officially approved by the United States Food and Drug Administration (US FDA) in 2001 It is a generally recognized as safe food (GRAS) and was approved by the European Union for marketing in Europe in 2005. Tagatose has four major functions: low energy, lowering blood sugar, improving intestinal flora and anti-caries (Oh D-K: Tagatose: properties, applications, and biotechnological processes. App. Microbiol. Biotechnol. 2007, 76: 1-8) . Therefore, tagatose has been widely used in food, beverages, dental care products and other fields.

The current industrial preparation methods of tagatose mainly include chemical methods (alkaline catalytic reaction) and biological methods (isomerase reaction) using galactose as the main raw material (CN 201080067326.6 and CN 201810018301.5). However, in the current preparation method, the price of lactose, which is the basic raw material of galactose, is unstable and depends on the production, supply and demand of raw milk and lactose in the global market, which makes the stable supply of tagatose production raw materials restricted. At the same time, due to the nature of the reaction itself, these preparation methods have relatively low tagatose conversion rates and require complex separation and purification processes, which will undoubtedly increase the cost of tagatose production. There are also patent reports on the production method of tagatose from fructose (CN105431541B, CN111344405A, etc.), but the enzyme activity reported in the patent is very low, which undoubtedly increases the amount of enzyme used in the catalytic process, thereby increasing the cost of tagatose production.

Chinese patent document CN 201610937656.5 discloses a method for efficiently converting the substrate into tagatose through an in vitro multi-enzyme molecular machine using cheap starch, cellulose or their derivatives or sucrose as the substrate. The method involves sequentially preparing glucose 1-phosphate (G1P), glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) using starch, cellulose or their derivatives or sucrose as a substrate, and then utilizing tagatose 6-phosphate epimerase (TPE) converts fructose 6-phosphate into tagatose 6-phosphate (T6P), and uses tagatose 6-phosphate phosphatase (T6PP) to catalyze tagatose 6-phosphate through the final irreversible reaction. Tagatose is prepared from the sugar 6-phosphate. This method can significantly improve the conversion rate of tagatose, has low production cost and is conducive to large-scale production. However, this method of in vitro multi-enzyme molecular machines for the production of tagatose represents a significant improvement, but there is still a desire and need to provide further improved methods for the production of tagatose, e.g., using more efficient enzymes than those used in previous pathways. Or a combination of enzymes to reduce the usage of enzymes or increase the yield of tagatose, thereby further reducing the production cost of tagatose and promoting the commercial conversion of tagatose.

Contents of the invention

Invent the problem to be solved

The inventor conducted in-depth research on the method of producing tagatose using in vitro multi-enzyme molecular machines, and discovered some unreported new enzymes with the activity of converting fructose 6-phosphate into tagatose 6-phosphate, some of which Has better conversion of fructose 6-phosphate into tagatose 6-phosphate activity; some other enzymes were found to have the activity of dephosphorylating tagatose 6-phosphate to generate tagatose, and some of the new enzymes have better activity of dephosphorylating tagatose 6-phosphate to generate tagatose. , and found that these enzymes gave better results when used for tagatose production, thus completing the present disclosure.

solutions to problems

This disclosure provides the following technical solutions.

(1) Use of a polypeptide selected from any one of the group consisting of (i) to (iv) as tagatose 6-phosphate epimerase, wherein the polypeptide:

(i) A polypeptide having a sequence shown in any one of SEQ ID NO: 1-4;

(ii) Having at least 70% sequence identity with the sequence shown in (i), and excluding the polypeptide of the sequence shown in any one of SEQ ID NO: 1-4;

(iii) A polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions to a polynucleotide represented by (a) or (b):

(a) A polynucleotide encoding a polypeptide having the amino acid sequence shown in (i);

(b) the full-length complementary polynucleotide of (a);

(iv) A fragment of the polypeptide represented by (i), (ii), (iii), and the fragment still has tagatose 6-phosphate epimerase activity.

(2) The use according to (1), wherein the tagatose 6-phosphate epimerase is derived from a heat-resistant microorganism; preferably, the heat-resistant microorganism is selected from Rhodothermus, Anaerolinea, Ignisphaera or Thermoflexia ; More preferably, the heat-resistant microorganism is selected from Rhodothermus marinus, Anaerolinea thermolimosa, Ignisphaera aggregans or Thermoflexia bacterium.

(3) Use of a polypeptide selected from any one of the group consisting of (v) to (viii) as tagatose 6-phosphate phosphatase, wherein the polypeptide:

(v) A polypeptide having a sequence shown in any one of SEQ ID NO: 5-7;

(vi) has at least 70% sequence identity with the sequence shown in (v), and does not include the polypeptide of the sequence shown in any one of SEQ ID NO: 5-7;

(vii) A polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions to a polynucleotide represented by (a) or (b):

(a) A polynucleotide encoding a polypeptide having the amino acid sequence shown in (v);

(b) the full-length complementary polynucleotide of (a);

(viii) A fragment of the polypeptide represented by (v), (vi), (vii), and the fragment still has tagatose 6-phosphate phosphatase activity.

(4) The use according to (3), wherein the tagatose 6-phosphate phosphatase is derived from a heat-resistant microorganism; preferably, the heat-resistant microorganism is selected from Thermomonospora, Spirochaeta or Hungateiclostridium; more preferably, The heat-resistant microorganism is selected from Thermomonospora curvata, Spirochaeta thermophila or Hungateiclostridium thermocellum.

(5) An enzyme composition for producing tagatose, wherein the enzyme composition contains tagatose 6-phosphate epimerase and/or tagatose 6-phosphate phosphatase.

(6) The enzyme composition according to (5), wherein the tagatose 6-phosphate epimerase is the polypeptide in the use according to any one of (1) to (2), The tagatose 6-phosphate phosphatase is the polypeptide in the use described in any one of (3) to (4).

(7) The enzyme composition according to any one of (5) to (6), wherein the enzyme composition further contains one or more of the following enzymes: starch branching enzymes (including isostarch enzyme and pullulanase), alpha-glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, maltose phosphorylase, beta-glucose phosphomutase, polyphosphate glucokinase, fiber paste Sperm phosphorylase, Cellobiose phosphorylase, sucrose phosphorylase, glucose isomerase, 4-glucan transferase, α-amylase, β-amylase.

(8) A strain or strain composition expressing the enzyme composition described in any one of (5) to (7).

(9) The strain or strain composition according to (8), characterized in that the host cell of the strain or strain composition is derived from Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia coli Bacillus; preferably, the host cell is Bacillus subtilis, Corynebacterium glutamicum or Escherichia coli.

(10) The strain or strain composition according to any one of (8) to (9), wherein the strain or strain composition is transformed with the following expression vector:

An expression vector containing a nucleic acid encoding tagatose 6-phosphate epimerase in any of the uses described in (1) to (2); and/or

An expression vector containing a nucleic acid encoding tagatose 6-phosphate phosphatase for use in any one of (3) to (4).

(11) The strain or strain composition according to (10), wherein the strain or strain composition is also transformed with genes encoding starch branching enzymes (including isoamylase and pullulanase), α-glucan Phosphorylase, glucose phosphomutase, glucose phosphate isomerase, maltose phosphorylase, β-glucose phosphomutase, polyphosphate glucokinase, cellodextrin phosphorylase, cellobiose phosphorylase, sucrose Expression vector for nucleic acid of phosphorylase, glucose isomerase, 4-glucan transferase, α-amylase, or β-amylase.

(12) Use of the enzyme composition described in any one of (5)-(7) or the strain or strain composition described in any one of (8)-(11) in the production of tagatose.

(13) A method for producing tagatose, wherein the method includes: adding the enzyme composition described in any one of (5)-(7) or inoculating the strain described in any one of (8)-(11) or a strain composition, a step of converting a substrate to tagatose;

Optionally, the method further includes the step of pretreating the substrate; or

The step of purifying or isolating said tagatose.

(14) The method according to (13), wherein the method includes the step of further adding metal ions or metal salts in the reaction; preferably, the metal is selected from metals capable of forming divalent cations; more Preferably, the metal is selected from one or more selected from the group consisting of magnesium, nickel, manganese, zinc, cobalt, iron, copper, calcium, molybdenum, and selenium.

(15) The method according to any one of (13)-(14), wherein the substrate is selected from sugars or derivatives thereof; preferably, the fermentation substrate is selected from the group consisting of the following components One or more of the group: starch or its derivatives, cellulose or its derivatives, fructose, glucose, sucrose, maltose.

(16) The method according to any one of (13) to (15), wherein the method is selected from one or more of the group consisting of: multi-enzyme catalysis, whole cell catalysis, containing Enzyme/whole cell fermentation catalysis, immobilized multi-enzyme catalysis, and immobilized whole cell catalysis.

One object of the present disclosure is to provide a composition for preparing tagatose 6-phosphate, which comprises tagatose 6-phosphate epimerase, expresses the tagatose 6-phosphate epimerase of a microorganism or a culture of said microorganism.

The tagatose 6-phosphate epimerase of the present disclosure may be SEQ ID NO: 1 (Uniprot ID: A0A7V2B2J0), SEQ ID NO: 2 (Uniprot ID: A0A3M1DFN1), SEQ ID NO: 3 (Uniprot ID: A0A7C4PIG5) or a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 (Uniprot ID: A0A7J2U4S4), or comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 Amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity . Having said homology and exhibiting an effect corresponding to a protein consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 (i.e., converting fructose 6-phosphate A polypeptide with an amino acid sequence that epimerizes the 4th carbon position of fructose and converts fructose 6-phosphate into tagatose 6-phosphate (fructose 6-phosphate C4-epimerization activity) Amino acid sequences in which part of the sequence is deleted, modified, substituted or added are included in the scope of the present disclosure. In addition, a probe prepared based on a known nucleotide sequence, for example, a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to the complement of all or part of the nucleotide sequence encoding the polypeptide, may or may not be present. This is included with limitations as long as it has fructose 6-phosphate C4-epimerization activity. Additionally, the composition may comprise one or more compounds consisting of SEQ. Tagatose 6-phosphate epimerase consisting of the amino acid sequence of ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

Among them, the amino acid sequence of SEQ ID NO: 1 is as follows (Uniprot ID: A0A7V2B2J0, Rhodothermus marinus):

The amino acid sequence of SEQ ID NO: 2 is as follows (Uniprot ID: A0A3M1DFN1, Thermoflexia bacterium):

The amino acid sequence of SEQ ID NO: 3 is as follows: (Uniprot ID: A0A7C4PIG5, Anaerolinea thermolimosa)

The amino acid sequence of SEQ ID NO: 4 is as follows (Uniprot ID: A0A7J2U4S4, Ignisphaera aggregans):

In some embodiments, the tagatose 6-phosphate epimerase of the present disclosure may be an enzyme derived from a thermotolerant microorganism, such as, for example, an enzyme derived from Rhodothermus or a variant thereof, specifically, derived from Rhodothermus marinus An enzyme or a variant thereof; an enzyme derived from Anaerolinea or a variant thereof, specifically an enzyme derived from Anaerolinea thermolimosa or a variant thereof; an enzyme derived from Ignisphaera or a variant thereof, specifically an enzyme derived from Ignisphaera aggregans or a variant thereof body; an enzyme derived from Thermoflexia or a variant thereof, specifically, an enzyme derived from Thermoflexia bacterium or a variant thereof; but is not limited thereto.

The tagatose 6-phosphate epimerase in the present disclosure is specific for fructose 6-phosphate and tagatose 6-phosphate, that is, the activity of fructose 6-phosphate/tagatose 6-phosphate is higher than that present in the reaction. of other phosphorylated monosaccharides and monosaccharides. For example, tagatose 6-phosphate epimerase has a higher epimerization for fructose 6-phosphate/tagatose 6-phosphate than for glucose 6-phosphate, glucose 1-phosphate, fructose, or tagatose chemical activity.

Compared with the previously disclosed tagatose 6-phosphate epimerase (CN 109750024A) derived from Thermoanaerobacter indiensis, the tagatose 6-phosphate epimerase of the present disclosure has a comparable level of activity, but has better substrate specificity. Specifically, the C4-epimerase activity of the tagatose 6-phosphate epimerases of the present disclosure toward fructose or tagatose monosaccharides is much lower than the tagatose 6-phosphate epimerase of Thermoanaerobacter indiensis structure enzyme, thereby ensuring a higher yield of product. Certain tagatose 6-phosphate epimerases in the present disclosure have better thermal stability.

In the present disclosure, the tagatose 6-phosphate epimerase can be used directly, or immobilized to maintain stability and be recyclable; microorganisms containing the tagatose 6-phosphate epimerase Cultures of microorganisms and/or microorganisms can be used directly, or immobilized for stability and recyclability, or whole cells can be permeabilized to obtain fast reaction rates.

Another object of the present disclosure is to provide a composition for preparing tagatose, comprising tagatose 6-phosphate phosphatase, a microorganism expressing the tagatose 6-phosphate phosphatase, or a culture of the microorganism things.

Tagatose 6-phosphate phosphatase can be composed of the amino acid sequences of SEQ ID NO: 5 (Uniprot ID: D1A2R1), SEQ ID NO: 6 (Uniprot ID: G0GB57), and SEQ ID NO: 7 (Uniprot ID: A3DJZ0) A polypeptide, or containing an amino acid sequence corresponding to SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 Amino acid sequences with %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Having said homology and exhibiting an effect corresponding to a protein consisting of the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 (i.e., dephosphorylating tagatose 6-phosphate to generate Tagatose activity), even if a part of the amino acid sequence is deleted, modified, substituted or added, is included in the scope of the present disclosure. In addition, a probe prepared based on a known nucleotide sequence, for example, a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to the complement of all or part of the nucleotide sequence encoding the polypeptide, may or may not be present. It is included with limitations as long as it has the activity of dephosphorylating tagatose 6-phosphate to form tagatose. Additionally, the composition may comprise one or more tagatose 6-phosphate phosphatases consisting of the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

The amino acid sequence of SEQ ID NO: 5 is as follows (Uniprot ID: D1A2R1, Thermomonospora curvata):

The amino acid sequence of SEQ ID NO: 6 is as follows (Uniprot ID: G0GB57, Spirochaeta thermophila):

The amino acid sequence of SEQ ID NO: 7 is as follows (Uniprot ID: A3DJZ0, Acetivibrio thermocellus):

In some embodiments, the tagatose 6-phosphate phosphatase of the present disclosure may be an enzyme derived from a thermotolerant microorganism, for example, an enzyme derived from Thermomonospora or a variant thereof, specifically, an enzyme derived from Thermomonospora curvata or a variant thereof ; An enzyme derived from Spirochaeta or a variant thereof, specifically, an enzyme derived from Spirochaeta thermophila or a variant thereof; An enzyme derived from Hungateiclostridium or a variant thereof, specifically, an enzyme derived from Hungateiclostridium thermocellum or a variant thereof; but not limited to this.

Compared with the previously disclosed tagatose 6-phosphate phosphatase derived from Archaeoglobus fulgidus (Uniprot code O29805) (CN 106399427 B), the tagatose 6-phosphate phosphatase of the present disclosure has higher activity. Preferably, compared with the previously disclosed tagatose 6-phosphate phosphatase derived from Archaeoglobus fulgidus, the tagatose 6-phosphate phosphatase in the present disclosure has an improvement of at least 10%, at least 30%, at least 1 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 150 times the enzyme activity.

Tagatose 6-phosphate phosphatase used in the methods of the present disclosure is specific for tagatose 6-phosphate. That is, the dephosphorylation activity of tagatose 6-phosphate is higher than that of other phosphorylated monosaccharides. For example, the dephosphorylating activity of tagatose 6-phosphate phosphatase on tagatose 6-phosphate is higher than that on, for example, glucose 1-phosphate, glucose 6-phosphate or fructose 6-phosphate. In the present disclosure, certain tagatose 6-phosphate phosphatases have better specificity and/or activity than the previously disclosed tagatose 6-phosphate phosphatase derived from Archaeoglobus fulgidus (Uniprot code O29805).

In the present disclosure, the tagatose 6-phosphate phosphatase can be used directly, or immobilized to maintain stability and recyclability; microorganisms and/or culture of microorganisms containing the tagatose 6-phosphate phosphatase Materials can be used directly, immobilized for stability and recyclability, or whole cells can be permeabilized to obtain fast reaction rates.

Yet another object of the present disclosure is to provide a composition for producing tagatose, which includes the tagatose 6-phosphate epimerase of the present disclosure, expresses the tagatose 6-phosphate epimerase enzyme microorganisms and/or cultures of said microorganisms; and tagatose 6-phosphate phosphatase, tagatose 6-phosphate phosphatase-expressing microorganisms and/or tagatose 6-phosphate-expressing phosphatase of the present disclosure Enzymatic microbial cultures. In the above aspect, tagatose 6-phosphate epimerase, a microorganism expressing the tagatose 6-phosphate epimerase and/or a culture of the microorganism, and tagatose 6-phosphate phosphatase The description of microorganisms expressing tagatose 6-phosphate phosphatase and/or cultures of microorganisms expressing tagatose 6-phosphate phosphatase also applies here.

In some embodiments, the composition of the present disclosure for producing tagatose may further comprise an enzyme participating in the tagatose production pathway of the present disclosure (FIG. 1), a microorganism expressing an enzyme participating in the tagatose production pathway of the present disclosure. , and/or a culture of a microorganism expressing an enzyme involved in the tagatose production pathway of the present disclosure. This is only as some examples, that is, the enzymes contained in the compositions of the present disclosure for producing tagatose and the substrates for producing tagatose are not limited in the present disclosure, as long as they can be produced by using the present disclosure. Tagatose 6-phosphate epimerase and/or tagatose 6-phosphate phosphatase may be used to produce tagatose.

The composition for preparing tagatose of the present disclosure may further comprise a metal. In one embodiment, the metal of the present disclosure may be a metal containing divalent cations. Specifically, the metals of the present disclosure may be magnesium, nickel, manganese, zinc, cobalt, iron, copper, calcium, molybdenum, selenium. More specifically, the metal of the present disclosure may be a metal ion or a metal salt. More specifically, the metal salt may be magnesium chloride, magnesium sulfate, nickel sulfate, nickel chloride, manganese chloride, manganese sulfate, cobalt chloride, chlorine Ferric chloride, ferrous chloride, zinc chloride, zinc sulfate, copper chloride, calcium chloride, sodium molybdate, sodium selenate, etc. The concentration of the metal salt may range from 0.001mM to 100mM. Preferably, the concentration of the metal salt may range from 0.01mM to 50mM.

Yet another object of the present disclosure is to provide a more superior method for preparing tagatose, which method comprises combining the composition with carbohydrates and derivatives (such as polysaccharides, oligosaccharides, disaccharides, monosaccharides or sugars). phosphate compound) reaction to prepare tagatose (Figure 2).

In a preferred embodiment, the more excellent method for preparing tagatose includes the step of converting fructose 6-phosphate into tagatose 6-phosphate using tagatose 6-phosphate epimerase, wherein tagatose 6-phosphate is converted into tagatose 6-phosphate. Gritose 6-phosphate epimerase comprising at least 70%, 75%, 80%, 85%, 90 An amino acid sequence with %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; more preferably, the tagatose 6-phosphate epimer Enzyme containing SEQ ID NO: 4 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % sequence identity of the amino acid sequence; most preferably, the tagatose 6-phosphate epimerase has an amino acid sequence as listed in SEQ ID NO: 4.

In another preferred embodiment, the more excellent method for preparing tagatose includes the step of converting tagatose 6-phosphate into tagatose using tagatose 6-phosphate phosphatase, wherein tagatose 6 - Phosphophosphatase contains SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:7 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% An amino acid sequence with sequence identity; more preferably, the tagatose 6-phosphate phosphatase comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% with SEQ ID NO: 7 , an amino acid sequence with 94%, 95%, 96%, 97%, 98% or 99% sequence identity; most preferably, the tagatose 6-phosphate phosphatase has an amino acid sequence as listed in SEQ ID NO: 7 .

In yet another preferred embodiment, the more excellent method for preparing tagatose includes the steps of converting fructose 6-phosphate into tagatose 6-phosphate using tagatose 6-phosphate epimerase and utilizing Tagatose 6-phosphate phosphatase converts tagatose 6-phosphate into tagatose, wherein tagatose 6-phosphate epimerase contains SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3 or SEQ ID NO:4 has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or an amino acid sequence that has 99% sequence identity, tagatose 6-phosphate phosphatase comprising at least 70%, 75%, 80%, 85% with SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence. More preferably, the tagatose 6-phosphate epimerase comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, An amino acid sequence having 95%, 96%, 97%, 98% or 99% sequence identity, tagatose 6-phosphate phosphatase comprising at least 70%, 75%, 80%, 85% with SEQ ID NO:7 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Most preferably, the tagatose 6-phosphate epimerase has an amino acid sequence as listed in SEQ ID NO: 4, and the tagatose 6-phosphate phosphatase has an amino acid sequence as listed in SEQ ID NO: 7.

In some embodiments, the more superior method of preparing tagatose further includes, before the step of converting fructose 6-phosphate of the present disclosure into tagatose 6-phosphate, by utilizing glucose 6-phosphate isomerase (PGI). ), a step in which a microorganism expressing glucose 6-phosphate isomerase and/or a culture of a microorganism expressing glucose 6-phosphate isomerase converts glucose 6-phosphate into fructose 6-phosphate. The sources of the glucose 6-phosphate isomerase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcusfuriosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, etc. In some examples, glucose phosphate isomerase includes but is not limited to derived from Hungateiclostridium thermocellum, Uniprot database number A3DBX9; it can also be derived from Thermus thermophilus, Uniprot database number Q5SLL6.

In some embodiments, the more superior method of preparing tagatose further includes, before the step of converting glucose 6-phosphate of the present disclosure into fructose 6-phosphate, by utilizing phosphoglucomutase (PGM), expressing glucose A step in which a phosphomutase microorganism and/or a culture of a microorganism expressing glucose phosphomutase converts glucose-1-phosphate into glucose-6-phosphate. The sources of the glucose phosphomutase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, etc. In some examples, the phosphoglucomutase includes, but is not limited to, derived from Thermococcus kodakarensis, Uniprot database number Q68BJ6; it can also be derived from Pyrococcus furiosus, Uniprot database number Q8U383.

In addition, the more excellent method of preparing tagatose described in the present disclosure may further include the step of converting sugars (such as polysaccharides, oligosaccharides, disaccharides, monosaccharides) into glucose-1-phosphate, wherein this step is performed by at least one catalyzed by an enzyme, a microorganism expressing the enzyme and/or a culture of a microorganism expressing the enzyme, and the sugars may be selected from the group consisting of but not limited to starch or its derivatives, cellulose or its derivatives, fructose, glucose and /or sucrose (Figure 2). According to the method of the present disclosure, the one or more enzymes used in the step of converting sugars into glucose-1-phosphate may be alpha-glucan phosphorylase, maltose phosphorylase, sucrose phosphorylase, fiber Dextrin phosphorylase, cellobiose phosphorylase and/or cellulose phosphorylase, and mixtures thereof, and/or microorganisms expressing one or more of the above-mentioned enzymes and mixtures thereof, and/or expressing one or more of the above-mentioned enzymes Cultures of multiple enzymatic microorganisms and their mixtures.

When the sugar is starch or its derivatives, the starch or its derivatives may be selected from the group consisting of, but not limited to, amylose, amylopectin, Soluble starch, starch dextrin, maltodextrin, malto-oligosaccharides, maltose, glucose and mixtures thereof. In certain embodiments of the present disclosure, when the carbohydrate is starch or a derivative thereof, including but not limited to amylose, amylopectin, soluble starch, starch dextrin, maltodextrin, malto-oligosaccharides, The enzyme for converting sugars into glucose 1-phosphate includes α-glucan phosphorylase, a microorganism expressing α-glucan phosphorylase, and/or a culture of a microorganism expressing α-glucan phosphorylase things. The sources of the α-glucan phosphorylase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, etc. In some examples, α-glucan phosphorylase includes, but is not limited to, derived from Thermotoga maritima, and the gene number in KEGG is TM1168; it can also be derived from Thermococcus kodakarensis, and the enzyme number in the Uniprot database is Q5JH18.

Some methods of the present disclosure may further include the step of converting starch into a starch derivative, wherein the starch derivative is prepared by enzymatic hydrolysis of starch and/or acid hydrolysis of starch. In certain embodiments of the present disclosure, in order to increase the production of tagatose, the more excellent method for preparing tagatose may further include utilizing a starch branching enzyme such as isoamylase (IA), expressing isoamylase A culture of a microorganism and/or an isoamylase-expressing microorganism converts starch into dextrin. The sources of the isoamylase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, Sulfolobus tokodaii, etc. In some embodiments, the isoamylase is derived from Sulfolobus tokodaii, and the enzyme number in the Uniprot database is Q973H3. In certain embodiments of the present disclosure, in order to increase the production of tagatose, the more excellent method for preparing tagatose may further include utilizing starch branching enzymes such as isoamylase, pullulanase (PA) , a pullulanase-expressing microorganism and/or a culture of a pullulanase-expressing microorganism converts starch into dextrin. The sources of the pullulanase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, Sulfolobus tokodaii, etc. In some embodiments, pullulanase is derived from Thermotoga maritima, and the enzyme number in the Uniprot database is O33840.

In other embodiments of the present disclosure, in order to increase the production of tagatose, the more excellent method for preparing tagatose may further include utilizing 4-glucan transferase (4GT), expressing 4-glucan A culture of a glycotransferase microorganism and/or a 4-glucan transferase-expressing microorganism converts the degradation products of starch or its derivatives, glucose, maltose and maltotriose, into longer malto-oligosaccharides, wherein said Longer malto-oligosaccharides can be converted to glucose using alpha-glucan phosphorylase, microorganisms expressing alpha-glucan phosphorylase, and/or cultures of microorganisms expressing alpha-glucan phosphorylase1 -Phosphoric acid. In some examples, the 4-glucan transferase is derived from Thermococcus litoralis and the enzyme number is O32462 in the Uniprot database. The sources of the 4-glucan transferase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, Thermococcus litoralis, Sulfolobus tokodaii, etc. In some embodiments of the present disclosure, in order to increase the production of tagatose, the more excellent method for preparing tagatose may further include utilizing polyphosphate glucokinase (PPGK), expressing polyphosphate glucose A culture of a kinase microorganism and/or a microorganism expressing polyphosphate glucokinase converts glucose, the degradation product of starch or its derivatives, into glucose 6-phosphate by the addition of polyphosphate. The source of polymerogenic glucosin kinase includes, but not limited to the HUNGATEICLOSTRIDIDIDIDIUMTRMOCELLUM, Thermus Thermophilus, Pyrococcus SP., Pyrocococococcus Furiosus, TH. Ermococcus Barophilus, Thermococcus Kodakarensis, Thermotoga Maritima, Thermococcus Litoralis, ThermobiFida Fusca, Sulfolobus Tokodaii, etc. In some examples, the polyphosphate glucokinase is derived from Thermobifidafusca and the enzyme has the Uniprot database number Q47NX5.

When the sugar is maltose and/or malto-oligosaccharide, the enzyme for converting the sugar into β-glucose 1-phosphate includes maltose phosphorylase, a microorganism expressing maltose phosphorylase, and/or a maltose phosphorylase-expressing microorganism. cultures of microorganisms. The β-glucose 1-phosphate The acid is further converted into glucose 6-phosphate by β-glucophosphomutase (β-PGM), a microorganism expressing β-glucophosphomutase, and/or a culture of a microorganism expressing β-glucophosphomutase. In some examples, maltose phosphorylase is derived from Bacillus sp.RK-1, and its gene number on Genebank is AB084460.1. The sources of the β-glucose phosphomutase include, but are not limited to, Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, Thermococcus litoralis, Thermobifida fusca, Sulfolobus tokodaii, etc. Preferably, it is β-glucose phosphomutase derived from Pyrococcus horikoshiiOT3, and the enzyme number in the Uniprot database is O58510.

When the sugar is sucrose, the enzyme for converting the sugar into glucose 1-phosphate includes sucrose phosphorylase, a microorganism expressing sucrose phosphorylase, and/or a culture of a microorganism expressing sucrose phosphorylase. In some embodiments of the present disclosure, in order to increase the production of tagatose, the more excellent method for preparing tagatose may further include utilizing glucose isomerase (GI), a microorganism expressing glucose isomerase, and /or a culture of a microorganism expressing glucose isomerase converts fructose, a degradation product of sucrose, into glucose. Glucose and polyphosphate are in turn catalyzed by polyphosphate glucokinase to generate glucose 1-phosphate. The sources of the sucrose phosphorylase, glucose isomerase, and polyphosphate glucokinase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, Thermococcus litoralis, Thermobifida fusca, Sulfolobus tokodaii, Streptomyces murinus, Bifidobacterium adolescentis, etc. In some examples, glucose isomerase is derived from Streptomyces murinus and the enzyme number in the Uniprot database is P37031. In some instances, the polyphosphate glucokinase is derived from Thermobifida fusca and the enzyme has the Uniprot database number Q47NX5. In some examples, sucrose phosphorylase is derived from Bifidobacterium adolescentis, and the enzyme number in the Uniprot database is A0ZZH6.

When the sugar is glucose, tagatose can also be produced from glucose (Figure 2). Methods according to the present disclosure may further comprise a step of converting glucose to glucose 6-phosphate, catalyzed by at least one enzyme, and, optionally, a step of converting sucrose to fructose, catalyzed by at least one enzyme. For example, the method involves using glucose and polyphosphate to produce glucose 6-phosphate catalyzed by polyphosphate glucokinase (PPGK). Glucose can be produced by the enzymatic conversion of sucrose (Figure 2). The sources of the glucokinase and polyphosphate glucokinase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, Thermococcus litoralis, Thermobifida fusca, Sulfolobus toko daii , Streptomyces murinus, Bifidobacterium adolescentis, etc. In some instances, the polyphosphate glucokinase is derived from Thermobifida fusca and the enzyme has the Uniprot database number Q47NX5.

When the sugar is fructose, tagatose can also be produced from fructose (Figure 2). Methods according to the present disclosure may also include, but are not limited to, a step of converting fructose to glucose or fructose 6-phosphate, wherein this step is catalyzed by at least one enzyme, and, optionally, a step of converting sucrose to fructose, wherein the step The step is catalyzed by at least one enzyme. For example, the method involves the generation of glucose from fructose catalyzed by glucose isomerase. For example, the method involves the production of fructose 6-phosphate from fructose via fructokinase catalysis. The sources of the glucose isomerase and fructokinase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, Thermococcus litoralis, Thermobifida fusca, Sulfolobus tokodai i. Streptomyces murinus, Bifidobacterium adolescentis, etc. In some examples, glucose isomerase is derived from Streptomyces murinus and the enzyme number in the Uniprot database is P37031. Conversion of glucose to tagatose is as described above. Fructose can be produced by the enzymatic conversion of sucrose. The phosphate ions generated when tagatose 6-phosphate is converted into tagatose can be recycled in the step of converting sucrose into glucose 1-phosphate.

When the sugar is cellulose or a derivative thereof, the cellulose or derivative thereof may be selected from the group consisting of, but not limited to, non-edible lignocellulosic materials (such as cellulose, hemicellulose and/or lignin and other minor Ingredients), pure cellulose (Avicel (microcrystalline cellulose), regenerated amorphous cellulose, bacterial cellulose, filter paper, etc.), partially hydrolyzed cellulose substrate (including water-insoluble fiber with a degree of polymerization greater than 7 Vidextrin, water-soluble cellodextrin with a degree of polymerization of 3-6, cellobiose, glucose and fructose). In certain embodiments of the present disclosure, when the carbohydrate is cellulose and its derivatives, the enzyme used to convert the carbohydrate into glucose 1-phosphate includes cellodextrin phosphorylase (CDP), expressing cellodextrin Cultures of microorganisms that phosphorylase and/or microorganisms that express cellodextrin phosphorylase and cellobiose phosphorylase (CBP), microorganisms that express cellobiose phosphorylase and/or that express cellobiose phosphorylase cultures of microorganisms. The cellodextrin phosphorylase and sources of cellodextrin phosphorylase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, Thermococcus litoralis, Thermobifida fusca, Sulfolobus tokodaii, Streptomyces murinus, Bifidobacterium adolescentis, etc. In some examples, cellodextrin phosphorylase is derived from Hungateiclostridium thermocellum, and the enzyme number in the Uniprot database is A3DJQ6; cellobiose phosphorylase is derived from Hungateiclostridium thermocellum, and the enzyme number in the Uniprot database is A3DC35.

Some methods of the present disclosure may further include the step of converting cellulose into a cellulose derivative, such as using an endoglucanase, an endoglucanase-expressing microorganism, and/or an endoglucanase-expressing microorganism A culture of and/or a cellobiohydrolase, a cellobiohydrolase-expressing microorganism and/or a culture of a cellobiohydrolase-expressing microorganism hydrolyzes solid cellulose into water-soluble cellodextrin and cellobiose. In some embodiments, the more excellent method for preparing tagatose may further include pretreating cellulose before hydrolyzing the cellulose and generating glucose 1-phosphate to increase their reactivity and reduce cellulose The degree of polymerization of the chain. The pretreatment methods of cellulose include, but are not limited to, dilute acid pretreatment, lignocellulose fractionation based on cellulose solvents, ammonia fiber expansion, ammonia soaking, ionic liquid treatment and by using concentrated acids (including hydrochloric acid, sulfuric acid, phosphoric acid and combination) for partial hydrolysis.

In certain embodiments of the present disclosure, in order to increase the production of tagatose, the more excellent method for preparing tagatose may further include utilizing cellobiose phosphorylase and expressing cellobiose phosphorylase. Cultures of microorganisms and/or microorganisms expressing cellobiose phosphorylase convert maltose, a degradation product of cellulose or its derivatives, into glucose-1-phosphate and glucose. Sources of the cellobiose phosphorylase include but are not limited to Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga mantima, Therrnococcus litoralis, Thermobifida fusca, Sulfolobus tokodai i. Streptomyces murinus , Bifidobacterium adolescentis, etc. In some examples, cellobiose phosphorylase is derived from Hungateiclostridium thermocellum, and the enzyme number in the Uniprot database is A3DC35. In some embodiments of the present disclosure, in order to increase the production of tagatose, the more excellent method for preparing tagatose may further include utilizing polyphosphate glucokinase (PPGK), expressing polyphosphate glucose A culture of a kinase microorganism and/or a microorganism expressing polyphosphate glucokinase converts glucose, the degradation product of cellulose or its derivatives, into glucose 6-phosphate by the addition of polyphosphate. The sources of the polyphosphate glucokinase include, but are not limited to, Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, Thermococcus litoralis, Thermobifida fusca, Sulfolobus tokodaii, Streptomyces murinus , Bifidobacterium adolescentis, etc. In some instances, the polyphosphate glucokinase is derived from Thermobifida fusca and the enzyme has the Uniprot database number Q47NX5.

In the present disclosure, the enzymes used can be used directly, and/or immobilized to maintain stability and recyclable; the microorganisms and/or cultures of microorganisms containing the above-mentioned enzymes can be used directly, and/or immobilized ized for stability and recyclability, or whole cells can be permeabilized for fast reaction rates.

Effect of the invention

Since the tagatose 6-phosphate epimerase and tagatose 6-phosphate phosphatase disclosed in the present disclosure are both thermostable and have high activity and specificity, they can be used to produce tagatose in industry. The amount of enzyme used may increase the yield of tagatose, thereby further reducing the production cost of tagatose and being more conducive to the industrial production of tagatose.

Description of the drawings

Figure 1 shows the route to tagatose from fructose 6-phosphate. Among them, the meanings of the abbreviations used in Figure 1 are as follows: TPE: tagatose 6-phosphate epimerase; TPP: tagatose 6-phosphate phosphatase; Pi: inorganic phosphorus.

Figure 2 shows the catalytic pathway for preparing tagatose from starch and its derivatives, cellulose and its derivatives, maltose, sucrose, and fructose. Among them, the meanings of the abbreviations used in Figure 2 are as follows: IA: isoamylase; aGP: α-glucan phosphorylase; PGM: phosphoglucose mutase; PGI: phosphoglucose isomerase; TPE: tagg Sugar 6-phosphate epimerase; TPP: tagatose 6-phosphate phosphatase; MP: maltose phosphorylase; β-PGM: β-glucose phosphomutase; PPGK: polyphosphate glucokinase; CDP: Cellodextrin phosphorylase; CBP: cellobiose phosphorylase; SP: sucrose phosphorylase; GI: glucose isomerase; Pi: inorganic phosphorus; (Pi) _n or (Pi) _n-1 : polyphosphate .

Detailed ways

definition

When used in conjunction with the term "comprising" in the claims and/or description, the word "a" or "an" may refer to "one", but may also refer to "one or more", "at least one” and “one or more than one”.

As used in the claims and description, the words "comprises," "having," "including," or "containing" mean inclusive or open-ended and do not exclude additional, unrecited elements or methods. step.

Throughout this application, the term "about" means that a value includes the standard deviation of the error of the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as an alternative only and "and/or", unless it is expressly stated as an alternative only or the alternatives are mutually exclusive, the term "or" in the claims means "and / or".

When used in the claims or description, the selected/optional/preferred "numeric range" includes both the numerical endpoints at both ends of the range, and also includes all natural numbers covered by the numerical endpoints relative to the aforementioned numerical endpoints.

As used in this disclosure, the term "converting" refers to a chemical transformation from one molecule to another catalyzed primarily by one or more polypeptides (enzymes), although other organic or inorganic catalysts may be used; It can also refer to the ratio (in %) between the moles of desired product and the moles of limiting substrate

As used in this disclosure, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

As used in this disclosure, the term "fragment" means a polypeptide or a catalytic or carbohydrate-binding module in which one or more (e.g., several) amino acids are deleted from the amino and/or carboxyl termini of a mature polypeptide or domain. .

In some specific embodiments, the fragment has the activity of tagatose 6-phosphate epimerase (that is, using "fructose 6-phosphate" as a substrate to convert "tagatose 6-phosphate").

In other specific embodiments, the fragment has the activity of tagatose 6-phosphate phosphatase (that is, using "tagatose 6-phosphate" as a substrate to dephosphorylate "tagatose").

As used in this disclosure, the term "wild-type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a source in nature, and has not been intentionally modified by humans in the laboratory is naturally occurring. As used in this disclosure, "naturally occurring" and "wild-type" are synonyms.

As used in this disclosure, the term "mutant" refers to a polynucleotide or polypeptide that contains an alteration at one or more (e.g., several) positions (i.e., several) relative to a "wild-type", or "comparison" , substitution, insertion and/or deletion) polynucleotide or polypeptide, Substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to the removal of a nucleotide or amino acid occupying a certain position. Insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position. Illustratively, a "mutant" in the present disclosure is a polypeptide having increased tagatose 6-phosphate epimerase or tagatose 6-phosphate phosphatase activity.

As used in this disclosure, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion, or addition of one or more amino acids or nucleotides." In this disclosure, the term "mutation" refers to a change in a nucleotide sequence or an amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution."

In some embodiments, "mutations" of the present disclosure may be selected from "conservative mutations." In this disclosure, the term "conservative mutation" refers to a mutation that normally maintains the function of a protein. Representative examples of conservative mutations are conservative substitutions.

As used in this disclosure, the term "conservative substitution" involves replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid) ), non-polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), non-polar side chains (such as alanine, valine acid, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), β-branched chains (e.g. threonine, valine and isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan and histidine).

As used in this disclosure, a "conservative substitution" typically exchanges one amino acid at one or more sites in a protein. This substitution can be conservative. Examples of substitutions considered as conservative substitutions include substitution of Ala to Ser or Thr, substitution of Arg to Gln, His or Lys, substitution of Asn to Glu, Gln, Lys, His or Asp, substitution of Asp to Substitution of Asn, Glu or Gln, substitution of Cys to Ser or Ala, substitution of Gln to Asn, Glu, Lys, His, Asp or Arg, substitution of Glu to Gly, Asn, Gln, Lys or Asp, substitution of Gly to Pro Substitution, substitution of His to Asn, Lys, Gln, Arg or Tyr, substitution of Ile to Leu, Met, Val or Phe, substitution of Leu to Ile, Met, Val or Phe, substitution of Lys to Asn, Glu, Gln, His or Substitution of Arg, substitution of Met to I1e, Leu, Val or Phe, substitution of Phe to Trp, Tyr, Met, Ile or Leu, substitution of Ser to Thr or Ala, substitution of Thr to Ser or Ala, substitution of Trp to Phe or Substitution of Tyr, substitution of Tyr to His, Phe or Trp, and substitution of Val to Met, Ile or Leu. In addition, conservative mutations also include naturally occurring mutations resulting from individual differences, strain, and species differences in where the gene comes from.

As used in this disclosure, the term "sequence identity" or "percent identity" in the comparison of two nucleic acids or polypeptides means that when measured using a nucleotide or amino acid residue sequence comparison algorithm or by visual inspection, When compared and aligned for maximum correspondence, they are identical or have a specific percentage number of the same sequence. That is to say, the identity of nucleotide or amino acid sequences can be defined by the ratio that maximizes the number of identical nucleotides or amino acids between two or more nucleotide or amino acid sequences, Gaps are added as necessary to achieve a consistent ratio of the number of nucleotides or amino acids in the alignment to the total number of nucleotides or amino acids in the alignment.

Methods for determining "sequence identity" or "percent identity" involved in the present disclosure include, but are not limited to: Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG Editor, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds. M Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

The determination/calculation of "sequence identity" or "percent identity" can be based on any suitable region of the sequence. For example, a region of at least about 50 residues, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In certain embodiments, the sequences are substantially identical throughout the entire length of either or both compared biopolymers (that is, nucleic acids or polypeptides).

In some embodiments, the polypeptide having tagatose 6-phosphate epimerase activity of the present disclosure comprises a mutant having at least 70%, 75 %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% “sequence identity” or “identity” of amino acid residues percentage".

In other embodiments, a polynucleotide encoding a polypeptide having tagatose 6-phosphate phosphatase activity of the present disclosure comprises a polynucleotide encoding a mutant of a polypeptide encoding the sequence set forth in any one of SEQ ID NOs: 5-7. The nucleotide has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotides" Sequence identity” or “percent identity”.

As used in this disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide may be in the form of an individual fragment or may be a component of a larger nucleotide sequence structure derived from a nucleotide sequence that has been isolated at least once in quantity or concentration and is capable of passing standards Molecular biology methods (eg, using cloning vectors) identify, manipulate, and recover sequences and their component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), where "U" replaces "T". In other words, "polynucleotide" refers to a polymer of nucleotides that has been removed from other nucleotides (individual fragments or entire fragments), or may be a component or component of a larger nucleotide structure, such as the expression Vector or polycistronic sequence. Polynucleotides include DNA, RNA and cDNA sequences.

As used in this disclosure, the term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, mutant, nucleic acid, protein, peptide or cofactor derived, at least in part, from Any substance that is removed from one or more or all of the naturally occurring components with which it is intrinsically related; (3) by artificial modification relative to a substance found in nature; or (4) by addition of a substance relative to other components with which it is naturally associated Any substance modified in quantity (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). Isolated substances can be present in fermentation broth samples. For example, host cells can be genetically modified to express polypeptides of the present disclosure. The fermentation broth from the host cells will contain the isolated polypeptide. "Recombinant polynucleotide" is one type of "polynucleotide".

As used in this disclosure, the term "recombinant polynucleotide" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used for transformation into a suitable host cell. A host cell containing a recombinant polynucleotide is called a "recombinant host cell." The polynucleotide is then expressed in a recombinant host cell to produce, for example, a "recombinant polypeptide."

As used in this disclosure, the term "expression" includes any step involved in the production of a polypeptide, including, but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

As used in this disclosure, the term "expression vector" refers to a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and that is operably linked to control sequences for expression thereof.

As used in this disclosure, the term "recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired polypeptide. Recombinant expression vectors may include, for example, i) a collection of genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; ii) structural or coding sequences that are transcribed into mRNA and translated into proteins; and iii) appropriate transcription and transcriptional subunits of translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not critical and any vector may be used, including plasmids, viruses, phages and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal and synthetic DNA sequences such as bacterial plasmids, phage DNA, yeast plasmids and vectors derived from combinations of plasmid and phage DNA derived from e.g. vaccinia, adenovirus, chicken DNA from viruses such as pox, baculovirus, SV40 and pseudorabies.

As used in this disclosure, the term "recombinant gene" is a gene that does not occur naturally. Recombinant genes are man-made. Recombinant genes include A protein-coding sequence operably linked to expression control sequences. Embodiments include, but are not limited to, exogenous genes introduced into a microorganism, endogenous protein-coding sequences operably linked to heterologous promoters, and genes with modified protein-coding sequences. Recombinant genes are stored in the genome of microorganisms, plasmids in microorganisms, or phages in microorganisms.

The term "host cell" in the present disclosure means any cell type that is susceptible to transformation, transfection, transduction, etc., with a mutant polypeptide, a polynucleotide encoding a mutant polypeptide, or a recombinant expression vector of the present disclosure. The term "recombinant host cell" covers a host cell that is different from the parent cell after the introduction of a polynucleotide encoding a mutant polypeptide or a recombinant expression vector. The recombinant host cell is specifically achieved by transformation. The host cell of the present disclosure may be a prokaryotic cell or a eukaryotic cell, as long as it can introduce the polypeptide or recombinant polypeptide of the present disclosure having encoding tagatose 6-phosphate epimerase or tagatose 6-phosphate phosphatase activity. Polynucleotide cells can be.

The terms "transformation, transfection, and transduction" in this disclosure have meanings generally understood by those skilled in the art, that is, the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method of introducing nucleic acid into cells. These methods include but are not limited to electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl2) precipitation, and microinjection. method, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.

The culture of the host cells of the present disclosure can be carried out according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and can be appropriately adjusted according to the actual situation. Various culture conditions such as temperature, time and pH value of the culture medium.

As used in this disclosure, the term "high stringency conditions" means, for probes of at least 100 nucleotides in length, following standard Southern blotting procedures, 5X SSPE (saline sodium phosphate EDTA) at 42°C. Prehybridize and hybridize for 12 to 24 hours in , 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide. Finally, wash the carrier material three times using 2X SSC, 0.2% SDS at 65°C for 15 minutes each time.

As used in this disclosure, the term "very high stringency conditions" means, for probes of at least 100 nucleotides in length, following standard Southern blotting procedures, 5X SSPE (saline sodium phosphate EDTA) at 42°C. ), 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide to prehybridize and hybridize for 12 to 24 hours. Finally, wash the carrier material three times using 2X SSC, 0.2% SDS at 70°C for 15 minutes each time.

Unless defined otherwise or clearly indicated by context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Example

The present disclosure will be further described below in conjunction with specific embodiments, and the advantages and features of the present disclosure will become clearer with the description. However, it should be understood that the embodiments are only exemplary and do not constitute any limitation on the scope of the present disclosure. Those skilled in the art should understand that the details and forms of the technical solution of the present disclosure can be modified or replaced without departing from the spirit and scope of the present disclosure, but these modifications or substitutions all fall within the protection scope of the present disclosure.

Example 1: Tagatose 6-phosphate epimerase (TPE) with better specificity and activity

A series of genes that may have tagatose 6-phosphate epimerase activity were synthesized into the pET20b vector by gene synthesis. The expression plasmid containing the target gene was transformed into E. coli BL21(DE3), and the cells were obtained by shake flask fermentation. The bacterial cells were collected by centrifugation, and after high-pressure homogenization and crushing, nickel filler affinity chromatography was used to obtain pure enzyme. SDS-PAGE electrophoresis detects the purity of the enzyme. Protein concentration was determined by Bradford method.

First, it was tested whether TPEs had tagatose 6-phosphate epimeric activity. The activity of different TPEs was measured at 50°C. The reaction system contained 10mM fructose 6-phosphate (F6P), 100mM HEPES buffer, 5mM MgSO4, 0.2g/L tagatose 6-phosphate phosphatase (derived from Archaeoglobus fulgidus , Uniprot ID: O29805) and appropriate amount of TPE. The reaction was terminated by ice bath. The activity of TPEs is characterized by measuring the production of free phosphorus (Anal. Chem. 1956, 28, 1756-1759).

The test results are shown in Table 1. The TPEs all have tagatose 6-phosphate epimeric activity. Among them, the enzyme activities of Uniprot IDs A0A7V2B2J0, A0A3M1DFN1, A0A7C4PIG5 and A0A7J2U4S4 were higher than those in the control group.

Secondly, verify the monosaccharide epimerization activity of TPEs. The activity of different TPEs was measured at 50°C. The reaction system contained 50g/L tagatose, 100mM HEPES buffer, 5mM MgSO4, and 1g/L TPE. Stop the reaction in a boiling water bath for 5 minutes. HPLC was used to detect fructose production to characterize enzyme activity. The liquid chromatography column is waters sugar-Pak1, the column temperature is 80°C, the flow rate is 0.5mL/min, and the detector is a differential refractive index detector.

The test results are shown in Table 1, the UniProt ID is A0A3B0UCF1, A0A7V2B2J0, A0A3M1DFN1, A0A7C4PIG5, A0A497GDS3, A0A7J2U4S4, A0A7J3I828, A0A7C5XKK1 and A0A77C2V2881. There are no activity of the transformation of Tagose and fructose.

Table 1. Verification of TPEs enzyme activity

Example 2: Tagatose 6-phosphate phosphatases (TPPs) with higher activity and specificity

A series of genes that may have tagatose 6-phosphate phosphatase activity were synthesized into the pET20b vector by gene synthesis. The expression plasmid containing the target gene was transformed into E. coli BL21(DE3), and the cells were obtained by shake flask fermentation. The bacterial cells were collected by centrifugation, and after high-pressure homogenization and crushing, nickel filler affinity chromatography was used to obtain pure enzyme. SDS-PAGE electrophoresis detects the purity of the enzyme. Protein concentration was determined by Bradford method.

The activity of TPPs towards tagatose 6-phosphate (T6P) was determined. The activity of different TPPs was measured at 50°C. The reaction system contained 10mM fructose 6-phosphate, 100mM HEPES buffer, 5mM MgSO4, 0.5g/L TPE (Uniprot number: A0A7J2U4S4) and an appropriate amount of TPP. The reaction was terminated by ice bath. The activity of TPPs was characterized by measuring the production of free phosphorus (Anal. Chem. 1956, 28, 1756-1759).

The test results are shown in Table 2. The TPPs all have tagatose 6-phosphate phosphatase activity. The activity of enzymes with Uniprot IDs D1A2R1, G0GB57 and A3DJZ0 on T6P increased to 14.26, 35.44 and 159.41 times respectively.

The activity of TPPs on glucose 1-phosphate (G1P), glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) was determined. The reaction is carried out at 50°C, and the reaction system contains 10mM G1P or G6P or F6P, 100mM HEPES buffer, 5mM MgSO4, and an appropriate amount of TPP. The reaction was terminated by ice bath. The activity of TPPs was characterized by measuring the production of free phosphorus (Anal. Chem. 1956, 28, 1756-1759).

It has been determined that the enzymes with Uniprot IDs O29805, D1A2R1, G0GB57 and A3DJZ0 have no activity on G1P, but have weak activity on G6P and F6P. The specificity of TPPs was characterized by the ratio of the activity of TPPs on T6P to the sum of the activities of TPPs on F6P and G6P (i.e., specificity = SP _T6P/ (SP _G6P +SP _F6P )). As shown in Table 2, A3DJZ0 not only increased the enzyme activity by 159 times, but its specificity was also significantly better than that of the control group.

Table 2. TPPs enzyme activity verification

Example 3: Production of tagatose from fructose 6-phosphate

F6P is converted into tagatose through an in vitro multi-enzyme catalytic system (Figure 1). These key enzymes include: (1) tagatose 6-phosphate epimerase (TPE), which catalyzes the dephosphorylation of F6P to T6P; (2) tagatose 6-phosphate phosphatase (TPP), which catalyzes the dephosphorylation of T6P to generate tagatose. Sugar and inorganic phosphorus.

In this example, A0A7J2U4S4 derived from Ignisphaera aggregans was selected as the tagatose 6-phosphate epimerase, and A3DJZ0 derived from Hungateiclostridium thermocellum as the tagatose 6-phosphate phosphatase was selected. These plasmids were transformed into E. coli expression strain BL21(DE3) (Invitrogen, Carlsbad, CA), and protein expression and purification were performed.

A 3 ml reaction system contains 100mM HEPES buffer (pH 7.0), 5mM divalent magnesium ions, 50mM F6P, and the dosage of tagatose 6-phosphate epimerase is 2U/mL, so The dosage of tagatose 6-phosphate phosphatase is 1 U/mL, the catalytic reaction is carried out at 60°C, and the reaction time is 1 hour. After the reaction, HPLC was used to determine the yield of tagatose. HPLC detection conditions were the same as Example 1. As a result, the yield of tagatose was 8.8g/L, and the yield of tagatose was 97.8%.

It is worth noting that the TPEs described in the present disclosure include enzymes with Uniprot IDs A0A3B0UCF1, A0A7V2B2J0, A0A3M1DFN1, A0A7C4PIG5, A0A497GDS3, A0A7J2U4S4, A0A7J3I828, A0A7C5XKK1 and A0A7C2V281, and enzymes derived from Hungateiclostridium ther tagatose 6-phosphate phosphatase A3DJZ0 from mocellum The above reactions were carried out, and the yields of tagatose exceeded 95%.

The TPE described in the present disclosure includes enzymes with Uniprot IDs of A0A3B0UCF1, A0A7V2B2J0, A0A3M1DFN1, A0A7C4PIG5, A0A497GDS3, A0A7J2U4S4, A0A7J3I828, A0A7C5XKK1 and A0A7C2V281 and Tagatose 6 derived from Spirochaeta thermophila - Phosphophosphatase G0GB57 is used together to perform the above reaction. The yield of tagatose exceeds 90%.

Example 4: Production of tagatose using starch as substrate

Starch is converted into tagatose through an in vitro multi-enzyme catalytic system (Figure 2). These key enzymes include: (1) α-glucan phosphorylase, which adds 1 phosphate to the non-reducing end of starch to release glucose 1-phosphate; (2) glucose phosphomutase, which catalyzes glucose 1-phosphate to glucose 6-phosphate; (3) glucose phosphate isomerase, which converts glucose 6-phosphate into fructose 6-phosphate; (4) tagatose 6-phosphate epimerase, which converts fructose 6-phosphate into tagatose 6-phosphate. Tagatose 6-phosphate; (5) Tagatose 6-phosphate phosphatase dephosphorylates tagatose 6-phosphate and converts it into tagatose and phosphate.

In this example, α-glucan phosphorylase is derived from Thermotoga maritima, and the gene number in KEGG is TM1168; Glucose phosphomutase is derived from Thermococcus kodakarensis, and the enzyme number in the Uniprot database is Q68BJ6; Glucose phosphate The isomerase is derived from Thermus thermophilus, and the enzyme number in the Uniprot database is Q5SLL6; Tagatose 6-phosphate epimerase comes from Ignisphaera aggregans, and the enzyme number in the Uniprot database is A0A7J2U4S4; Tagatose 6 -Phosphophosphatase is derived from Hungateiclostridium thermocellum, and the enzyme number in the Uniprot database is A3DJZ0. The genes corresponding to these enzymes were cloned into the pET20b vector, transformed into E. coli expression strain BL21 (DE3) (Invitrogen, Carlsbad, CA), and the proteins were expressed and purified.

In a 3 ml reaction system containing 30mM phosphate buffer (pH 7.0), 5mM divalent magnesium ions, the α- The dosage of glucan phosphorylase is 1 U/mL, the dosage of glucose phosphomutase is 1 U/mL, the dosage of glucose phosphate isomerase is 1 U/mL, and the tagatose 6-phosphate difference The dosage of isomerase is 1 U/mL, the dosage of tagatose 6-phosphate phosphatase is 1 U/mL, and 10 g/L soluble starch is used to catalyze the reaction at 55°C for 24 hours. After the reaction, HPLC was used to determine the final concentration of tagatose to be 4.4g/L, and the yield of tagatose to starch was 44%.

Since starch has branched chains, α-glucan phosphorylase alone cannot completely hydrolyze starch, because α-glucan phosphorylase only acts on α-1,4 glycosidic bonds, and branched chains are α-1,6 glycosidic bond connected to the main chain. This requires the addition of isoamylase to hydrolyze α-1,6 glycosidic bonds. Finally, the final products of starch hydrolyzed by these two enzymes are maltose and glucose. In order to convert these final products into tagatose, 4-glucan transferase and polyphosphate glucokinase need to be added. 4-glucan transfer The enzyme condenses short-chain maltopolysaccharides into long-chain maltopolysaccharides and releases a molecule of glucose. Glucokinase polyphosphate catalyzes polyphosphate and glucose to generate glucose 6-phosphate. In this example, the starch debranching enzyme is derived from Sulfolobus tokodaii, and the enzyme number in the Uniprot database is Q973H3; the polyphosphate glucokinase is derived from Thermobifida fusca, and the enzyme number in the Uniprot database is Q47NX5; 4-glucan Glycotransferase is derived from Thermococcus litoralis, and the enzyme number in the Uniprot database is O32462. The genes corresponding to these enzymes were cloned into the pET20b vector, transformed into E. coli expression strain BL21 (DE3) (Invitrogen, Carlsbad, CA), and the proteins were expressed and purified.

A 3 ml reaction system contains 30mM phosphate buffer (pH 7.0), 5mM divalent magnesium ions, the dosage of the α-glucan phosphorylase is 10U/mL, and the glucose phosphomutase The dosage of the glucose phosphate isomerase is 10 U/mL, the dosage of the tagatose 6-phosphate epimerase is 10 U/mL, and the dosage of the tagatose 6-phosphate epimerase is 10 U/mL. The dosage of enzyme is 10U/mL, 100g/L IA pre-treated soluble starch (40mM sodium acetate buffer (pH 5.5), 5mM divalent magnesium ion, S/E=3000:1, incubated at 80°C for 6 hours), After performing the catalytic reaction at 55°C for 12 hours, 5 U/mL glucokinase polyphosphate, 5 U/ml glucan transferase, and 50 mM sodium polyphosphate were added. After the reaction, HPLC was used to determine the final concentration of tagatose to be 85g/L, and the yield of tagatose to starch was 85%.

Example 5: Production of tagatose using cellulose as substrate

The schematic diagram of the conversion of cellulose into tagatose through an in vitro multi-enzyme catalytic system is shown in Figure 2.

In this example, cellulase is a product from Sigma Company, product number is C2730. Cellodextrin phosphorylase is derived from Hungateiclostridium thermocellum, and the enzyme number in the Uniprot database is A3DJQ6; cellobiose phosphorylase is derived from Hungateiclostridium thermocellum, and the enzyme number in the Uniprot database is A3DC35; glucose phosphomutase is derived from Pyrococcus furiosus , the enzyme number in the Uniprot database is Q8U383; glucose phosphate isomerase comes from Hungateiclostridium thermocellum, and the number in the Uniprot database is A3DBX9; tagatose 6-phosphate epimerase comes from Ignisphaera aggregans, the enzyme is in the Uniprot database The number is A0A7J2U4S4; tagatose 6-phosphate phosphatase is derived from Hungateiclostridium thermocellum, and the enzyme number in the Uniprot database is A3DJZ0; the polyphosphate glucokinase is derived from Thermobifida fusca, and the enzyme number in the Uniprot database is Q47NX5. The genes corresponding to these enzymes were cloned into the pET20b vector, transformed into E. coli expression strain BL21 (DE3) (Invitrogen, Carlsbad, CA), and the proteins were expressed and purified.

This experiment used microcrystalline cellulose (Avicel) as the substrate. First, mix commercial cellulase (10U/ml) and cellulose (100g/L) on an ice-water bath, place in the ice-water bath for 5 minutes, centrifuge at 4°C, and remove the supernatant. The precipitate is a mixture of cellulose and cellulase that can bind to cellulose. This treatment can remove almost all glucosidases in commercial cellulases, thus preventing glucosidases from hydrolyzing cellobiose to produce large amounts of glucose, so that the main hydrolysis products are cellobiose and cellopolysaccharide.

A 3 ml reaction system contains 30mM phosphate buffer (pH 7.2), 5mM divalent magnesium ions, 10U/mL cellulan phosphorylase, 50U/mL cellobiose phosphorylase, 10U/mL Glucose phosphomutase, 10 U/mL glucose phosphate isomerase, 10 U/mL tagatose 6-phosphate epimerase, 10 U/mL tagatose 6-phosphate phosphatase, 100 g/L The mixture of cellulose and cellulase as described above, 10 U/mL polyphosphate glucokinase, and 50 mM polyphosphate was used to catalyze the reaction at 50°C for 72 hours. After the reaction, HPLC was used to determine the final concentration of tagatose to be 20 g/L, and the yield of tagatose to cellulose was 20%.

Example 6: Production of tagatose using maltose as substrate

The schematic diagram of converting maltose into tagatose through an in vitro multi-enzyme catalytic system is shown in Figure 2.

Maltose phosphorylase is derived from Bacillus sp.RK-1, and its gene number on Genebank is AB084460.1. β-glucose phosphomutase is derived from Pyrococcus horikoshii OT3, and the enzyme number in the Uniprot database is O58510. Glucose phosphate isomerase comes from Hungateiclostridium thermocellum, and the number in the Uniprot database is A3DBX9; Tagatose 6-phosphate epimerase comes from Ignisphaera aggregans, and the enzyme number in the Uniprot database is A0A7J2U4S4; Tagatose 6-phosphate The phosphatase is derived from Hungateiclostridium thermocellum, and the enzyme number in the Uniprot database is A3DJZ0; the polyphosphate glucokinase is derived from Thermobifida fusca, and the enzyme number in the Uniprot database is Q47NX5. The genes corresponding to these enzymes were cloned into the pET20b vector, transformed into E. coli expression strain BL21 (DE3) (Invitrogen, Carlsbad, CA), and the proteins were expressed and purified.

A 3 ml reaction system contains 10mM phosphate buffer (pH 7.2), 10mM divalent magnesium ions, 1U/mL maltose phosphorylase, 1U/mL β-glucose phosphomutase, 1U/mL Glucose phosphate isomerase, 1U/mL tagatose 6-phosphate epimerase, 1U/mL tagatose 6-phosphate phosphatase, 10g/L maltose, 1U/mL polyphosphate glucokinase , 10mM polyphosphate, react at 37°C for 24 hours. After the reaction, HPLC was used to determine the final concentration of tagatose to be 6g/L, and the yield of tagatose to starch was 60%.

Example 7: Production of tagatose using sucrose as substrate

The schematic diagram of the conversion of sucrose into tagatose through an in vitro multi-enzyme catalytic system is shown in Figure 2.

Sucrose phosphorylase is derived from Bifidobacterium adolescentis, and the enzyme number in the Uniprot database is A0ZZH6. Glucose isomerase comes from Streptomyces murinus, and the enzyme number in the Uniprot database is P37031. The sources of other enzymes are the same as in Example 5.

A 3 ml reaction system contains 10mM phosphate buffer (pH 7.2), 10mM divalent magnesium ions, 1U/mL sucrose phosphorylase, 1U/mL glucose phosphomutase, and 1U/mL glucose. Phosphoisomerase, 1 U/mL tagatose 6-phosphate epimerase, 1 U/mL tagatose 6-phosphate phosphatase, 10 g/L sucrose, 1 U/mL polyphosphate glucokinase, 10mM Polyphosphate, 10U/mL glucose isomerase, react at 37°C for 24 hours. After the reaction, HPLC was used to determine the final concentration of tagatose to be 5.5g/L, and the yield of tagatose to starch was 55%.

Example 8: Production of tagatose using fructose as substrate

The source of the enzyme is the same as in Example 7.

A 3 ml reaction system contains 10mM phosphate buffer (pH 7.2), 10mM divalent magnesium ion, 10U/mL glucose isomerase, 1U/mL glucose phosphate isomerase, 1U/mL Taggar Sugar 6-phosphate epimerase, 1U/mL tagatose 6-phosphate phosphatase, 10g/L fructose, 1U/mL polyphosphate glucokinase, 10mM polyphosphate, react at 50°C for 24 hours. After the reaction, HPLC was used to determine the final concentration of tagatose to be 6g/L, and the yield of tagatose to starch was 60%.

Example 9: Expression of enzyme using Bacillus subtilis

As the most commonly used heterologous protein expression host, Escherichia coli has many good characteristics, but it also has immunogen, endogenous Unavoidable problems such as toxins and low secretion expression efficiency limit the cost reduction of downstream processes and the convenience of operation. Bacillus subtilis, as a model strain of Gram-positive bacteria, has many excellent properties such as secretory expression, high heterologous expression levels, and safety. It is considered an ideal protein-producing strain and is widely used in various Enzyme protein production. Therefore, Bacillus subtilis is also used as the protein expression host in the present invention.

The nucleotide sequences of tagatose 6-phosphate epimerase (TPE) and tagatose 6-phosphate phosphatase (TPPs) described in Example 1 and Example 2 were cloned into pWB980 vector (Development of improved pUB110 -Based Vectors for Expression and Secretion Studies in Bacillus Subtilis.Journal of BioteChnology, 1999.72 (3); High Copy Number and Highly Stable Escheric Hia Coli-Bacillus Subtilis Shuttle Plasmids Based on PWB980, 2020, 19 (1)) -A0A3B0UCF1, pWB980-A0A7V2B2J0, pWB980-A0A3M1DFN1, pWB980-A0A7C4PIG5, pWB980-A0A497GDS3, pWB980-A0A7J2U4S4, pWB980-A0A7J3I828, pWB980-A0A7C5XKK 1. pWB980-A0A7C2V281, pWB980-D1A2R1, pWB980-G0GB57, pWB980-A3DJZ0. The above expression plasmid was transformed into Bacillus subtilis SCK23, and SR medium was used for amplification and culture. Collect the cells by centrifugation, wash once with physiological saline, and resuspend in phosphate buffer to obtain cells expressing the corresponding enzymes.

Similarly, isoamylase, α-glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, maltose phosphorylase, β-glucose phosphomutase, polyphosphate glucokinase, fiber paste The corresponding nucleotide sequences of refined phosphorylase, cellobiose phosphorylase, sucrose phosphorylase, glucose isomerase, 4-glucan transferase, α-amylase, and β-amylase were cloned into the vector pWB980, And transformed into Bacillus subtilis SCK23 to obtain the corresponding Bacillus subtilis cells.

Example 10: Using whole cell catalytic starch to produce tagatose

It will express α-glucan phosphorylase, thermostable glucose phosphomutase, thermostable glucose phosphate isomerase, thermostable 6-phosphate tagatose epimerase, and thermostable 6-phosphate tagatose phosphate. subtilis cells or E. coli cells using 50mM sodium phosphate buffer (pH 7.5), and resuspend the cells to OD 600 = 200. The resuspended bacterial cells were heat-treated at 55°C for 90 min to obtain heat-treated whole cells. The source of the enzyme is the same as in Example 4.

In a 3 ml reaction system containing 30mM phosphate buffer (pH 7.0), 5mM divalent magnesium ions, 100g/L soluble starch, the above-mentioned heat-treated α-glucan phosphorylase, thermostable glucose phosphate Bacillus subtilis cells or Escherichia coli mutase, thermostable glucose phosphate isomerase, thermostable tagatose 6-phosphate epimerase, thermostable tagatose 6-phosphate phosphatase and thermostable isoamylase Cells were added to the reaction system in amounts of 10U/mL, 10U/mL, 10U/mL, 10U/mL, 10U/mL, and 2U/mL, and the catalytic reaction was carried out at 55°C for 24 hours. After the reaction, HPLC was used to determine the final concentration of tagatose to be 70 g/L, and the yield of tagatose to starch was 70%.

Example 11: Production of tagatose by immobilized enzyme

Fermentation to obtain whole cells expressing thermostable α-glucan phosphorylase, whole cells expressing thermostable glucose phosphomutase, whole cells expressing thermostable glucose phosphate isomerase, and expressing thermostable tagatose 6-phosphate. Whole cells expressing epimerase, expressing thermostable tagatose 6-phosphate phosphatase, and whole cells expressing thermostable isoamylase. Add 50mM sodium phosphate buffer (pH 7.5) to the above cells respectively, and resuspend the cells to OD 600 = 200. The resuspended bacterial cells were heat treated at 55°C for 90 min. Use pH 7.0 sodium phosphate buffer to mix the above-mentioned permeable whole cells according to the enzyme activity ratio 1:1:1:1:1, so that OD600=100, add 5% w/v diatomaceous earth to the bacterial suspension, and stir Evenly. Subsequently, 0.5% w/v polyethyleneimine aqueous solution with a molecular weight of 70,000 was added to flocculate at room temperature. Then, 0.5% v/v glutaraldehyde aqueous solution was added for cross-linking at room temperature for 2 h. After vacuum filtration, a filter cake is obtained, which is then extruded and granulated; the obtained immobilized cell particles are dried at 30°C to obtain immobilized cells. The source of the enzyme is the same as in Example 4, and the expression host of the enzyme is Bacillus subtilis or Escherichia coli.

In a 3 ml reaction system containing 30mM phosphate buffer (pH 7.0), 5mM divalent magnesium ions, the α- The dosage of glucan phosphorylase immobilized enzyme granules is 10 U/mL, the dosage of the glucose phosphomutase immobilized enzyme granules is 10 U/mL, and the dosage of the glucose phosphate isomerase immobilized enzyme granules is 10 U /mL, the dosage of the tagatose 6-phosphate epimerase immobilized enzyme particles is 10 U/mL, the dosage of the tagatose 6-phosphate phosphatase immobilized enzyme particles is 10 U/mL, the The dosage of isoamylase immobilized enzyme granules is 2U/mL, 100g/L soluble starch, and the catalytic reaction is carried out at 55°C for 24 hours. After the reaction, HPLC was used to determine the final concentration of tagatose to be 70 g/L, and the yield of tagatose to starch was 70%.

The supernatant was removed, and fresh reaction solution was added to the enzyme particles for batch reaction. 60 batches were continuously catalyzed, and the product yields were all greater than 50%.

All technical features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification may also be replaced by other features having the same, equivalent or similar effect. Therefore, unless expressly stated otherwise, each feature disclosed is only an example of a series of equivalent or similar features.

In addition, from the above description, those skilled in the art can easily understand the key features of the present disclosure, and without departing from the spirit and scope of the present disclosure, many modifications can be made to the invention to adapt to various applications. purposes and conditions of use, and therefore such modifications are intended to fall within the scope of the appended claims.

Claims

Use of a polypeptide selected from any one of the group consisting of (i) to (iv) as tagatose 6-phosphate epimerase, wherein the polypeptide:

(i) A polypeptide having a sequence shown in any one of SEQ ID NO: 1-4;

(ii) Having at least 70% sequence identity with the sequence shown in (i), and excluding the polypeptide of the sequence shown in any one of SEQ ID NO: 1-4;

(iii) A polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions to a polynucleotide represented by (a) or (b):

(a) A polynucleotide encoding a polypeptide having the amino acid sequence shown in (i);

(b) the full-length complementary polynucleotide of (a);

(iv) A fragment of the polypeptide represented by (i), (ii), (iii), and the fragment still has tagatose 6-phosphate epimerase activity.
The use according to claim 1, wherein the tagatose 6-phosphate epimerase is derived from a heat-resistant microorganism; preferably, the heat-resistant microorganism is selected from Rhodothermus, Anaerolinea, Ignisphaera or Thermoflexia; more preferably , the heat-resistant microorganism is selected from Rhodothermus marinus, Anaerolinea thermolimosa, Ignisphaera aggregans or Thermoflexia bacterium.
Use of a polypeptide selected from any one of the group consisting of (v) to (viii) as tagatose 6-phosphate phosphatase, wherein the polypeptide:

(v) A polypeptide having a sequence shown in any one of SEQ ID NO: 5-7;

(vi) It has at least 70% sequence identity with the sequence shown in (v), and does not include the polypeptide of the sequence shown in any one of SEQ ID NO: 5-7;

(vii) A polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions to a polynucleotide represented by (a) or (b):

(a) A polynucleotide encoding a polypeptide having the amino acid sequence shown in (v);

(b) the full-length complementary polynucleotide of (a);

(viii) A fragment of the polypeptide represented by (v), (vi), (vii), and the fragment still has tagatose 6-phosphate phosphatase activity.
The use according to claim 3, wherein the tagatose 6-phosphate phosphatase is derived from a heat-resistant microorganism; preferably, the heat-resistant microorganism is selected from Thermomonospora, Spirochaeta or Hungateiclostridium; more preferably, the heat-resistant microorganism The thermomicroorganism is selected from Thermomonospora curvata, Spirochaeta thermophila or Hungateiclostridium thermocellum.
An enzyme composition for producing tagatose, wherein the enzyme composition comprises tagatose 6-phosphate epimerase and/or tagatose 6-phosphate phosphatase.
The enzyme composition according to claim 5, wherein the tagatose 6-phosphate epimerase is the polypeptide in the use of any one of claims 1-2, and the tagatose 6-phosphate epimerase is -Phosphophosphatase is the polypeptide in the use according to any one of claims 3-4.
The enzyme composition according to any one of claims 5-6, wherein the enzyme composition further contains one or more of the following enzymes: starch branching enzymes (including isoamylase and pullulan enzyme), α-glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, maltose phosphorylase, β-glucose phosphomutase, polyphosphate glucokinase, cellodextrin phosphorylase, Cellobiose phosphorylase, sucrose phosphorylase, glucose isomerase, 4-glucan transferase, α-amylase, β-amylase.
A strain or strain composition expressing the enzyme composition according to any one of claims 5-7.
The strain or strain composition according to claim 8, characterized in that the host cell of the strain or strain composition is derived from the genus Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia. ; Preferably, the host cell is Bacillus subtilis, Corynebacterium glutamicum or Escherichia coli.
The strain or strain composition according to any one of claims 8-9, wherein the strain or strain composition is transformed into the following expression vector:

An expression vector containing a nucleic acid encoding tagatose 6-phosphate epimerase for use according to any one of claims 1-2; and/or

An expression vector containing a nucleic acid encoding tagatose 6-phosphate phosphatase for use according to any one of claims 3-4.
The strain or strain composition according to claim 10, wherein the strain or strain composition is also transformed to contain encoding starch branching enzymes (including isoamylase and pullulanase), α-glucan phosphorylase , glucose phosphomutase, glucose phosphate isomerase, maltose phosphorylase, β-glucose phosphomutase, polyphosphate glucokinase, cellodextrin phosphorylase, cellobiose phosphorylase, sucrose phosphorylase , expression vector of nucleic acid of glucose isomerase, 4-glucan transferase, α-amylase, or β-amylase.
Application of the enzyme composition according to any one of claims 5 to 7 or the strain or strain composition according to any one of claims 8 to 11 in the production of tagatose.
A method for producing tagatose, wherein the method includes: adding the enzyme composition described in any one of claims 5-7 or inoculating the strain or strain composition described in any one of claims 8-11, and The step of converting the substance into tagatose;

Optionally, the method further includes the step of pretreating the substrate; or

The step of purifying or isolating said tagatose.
The method according to claim 13, wherein the method includes the step of further adding metal ions or metal salts in the reaction; preferably, the metal is selected from metals capable of forming divalent cations; more preferably, The metal is selected from one or more selected from the group consisting of magnesium, nickel, manganese, zinc, cobalt, iron, copper, calcium, molybdenum, and selenium.
The method according to any one of claims 13-14, wherein the substrate is selected from sugars or derivatives thereof; preferably, the fermentation substrate is selected from one of the group consisting of the following components Or more: starch or its derivatives, cellulose or its derivatives, fructose, glucose, sucrose, maltose.
The method according to any one of claims 13 to 15, wherein the method is selected from one or more of the group consisting of: multi-enzyme catalysis, whole cell catalysis, enzyme/whole cell containing Fermentation catalysis, immobilized multi-enzyme catalysis, and immobilized whole cell catalysis.