CN117327684A - Enzyme for preparing tagatose, composition and application thereof - Google Patents

Enzyme for preparing tagatose, composition and application thereof Download PDF

Info

Publication number
CN117327684A
CN117327684A CN202210730019.6A CN202210730019A CN117327684A CN 117327684 A CN117327684 A CN 117327684A CN 202210730019 A CN202210730019 A CN 202210730019A CN 117327684 A CN117327684 A CN 117327684A
Authority
CN
China
Prior art keywords
tagatose
phosphate
leu
ala
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210730019.6A
Other languages
Chinese (zh)
Inventor
马延和
石婷
李运杰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin Yihe Biotechnology Co ltd
Original Assignee
Tianjin Yihe Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Yihe Biotechnology Co ltd filed Critical Tianjin Yihe Biotechnology Co ltd
Priority to CN202210730019.6A priority Critical patent/CN117327684A/en
Priority to PCT/CN2023/073198 priority patent/WO2023246099A1/en
Publication of CN117327684A publication Critical patent/CN117327684A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/265—Micrococcus
    • C12R2001/28—Micrococcus glutamicus ; Corynebacterium glutamicum

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The present disclosure relates to an enzyme for the preparation of tagatose, a composition thereof and uses thereof. In particular, the disclosure relates to the use of polypeptides as tagatose 6-phosphate epimerase, as tagatose 6-phosphate phosphatase; and the use of an enzyme composition comprising the aforementioned enzyme in the preparation of tagatose, and a method for preparing tagatose using the enzyme composition of the aforementioned enzyme. The method for preparing tagatose has the advantages of simplicity, economy and improved yield.

Description

Enzyme for preparing tagatose, composition and application thereof
Technical Field
The present disclosure relates to the fields of genetic engineering and biocatalysis, in particular to the biotechnology field related to tagatose production. More particularly, the present disclosure relates to a composition comprising a novel tagatose 6-phosphate epimerase and/or tagatose 6-phosphate phosphatase and a method of preparing tagatose using the same.
Background
D-Tagatose (hereinafter referred to as Tagatose) is a naturally occurring rare monosaccharide, which is a ketose form of galactose, an epimer of fructose. Natural tagatose is mainly found in dairy products such as yogurt and milk powder. The sweetness characteristics are similar to those of sucrose, and the calories generated are only one third of those of sucrose, so they are called low calorie sweeteners. Tagatose has excellent nutritional characteristics of low calorific value, zero glycemic index, blood glucose inactivation, no caries, prebiotic action, antioxidant activity, etc., and is formally approved by the U.S. food and drug administration (US FDA) as a generally recognized safe food (GRAS) in 2001 and approved by the european union for market 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 the fields of foods, beverages, tooth care products, and the like.
The current industrial production processes of tagatose mainly include chemical processes (basic catalytic reactions) and biological processes (isomerase reactions) using galactose as a main raw material (CN 201080067326.6 and CN 201810018301.5). However, in the current production method, lactose as a base material of galactose is unstable in price, which depends on the yield, supply amount, demand amount, etc. of raw milk and lactose on the global market, which makes stable supply of tagatose production materials limited. Meanwhile, due to the nature of the reaction, the conversion rate of tagatose in the preparation methods is lower, and a complex separation and purification process is also required, so that the cost of tagatose production is increased undoubtedly. There are also patents reporting methods for producing tagatose from fructose (CN 105431541B, CN111344405A, etc.), but the enzyme activities reported in the patents are very low, which undoubtedly increases the amount of enzyme used in the catalytic process, thus increasing the cost of tagatose production.
Chinese patent document CN 201610937656.5 discloses a method for efficiently converting a substrate into tagatose by using inexpensive starch, cellulose or derivatives thereof or sucrose as a substrate through an in vitro multi-enzyme molecular machine. The method involves sequentially preparing glucose 1-phosphate (G1P), glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) from starch, cellulose or derivatives thereof or sucrose as a substrate, then converting the fructose 6-phosphate into tagatose 6-phosphate (T6P) by using tagatose 6-phosphate epimerase (TPE), and preparing tagatose by catalyzing the tagatose 6-phosphate by using tagatose 6-phosphate phosphatase (T6 PP) through the irreversible reaction of the last step. The method can obviously improve the conversion rate of tagatose, has low production cost and is beneficial to large-scale production. However, this in vitro multi-enzyme molecular machine method of producing tagatose is significantly improved, but there remains a desire and need to provide further improved methods of producing tagatose, for example, using more efficient enzymes or combinations of enzymes than used in previous approaches to reduce the amount of enzymes used or to increase the yield of tagatose, thereby further reducing the cost of tagatose production and facilitating commercial conversion of tagatose.
Disclosure of Invention
Problems to be solved by the invention
The inventors have conducted intensive studies on methods for producing tagatose using in vitro multi-enzyme molecular machines, and found that some unreported novel enzymes have the activity of converting fructose 6-phosphate into tagatose 6-phosphate, and some of the novel enzymes have more excellent activity of converting fructose 6-phosphate into tagatose 6-phosphate; other enzymes were found to have tagatose 6-phosphate dephosphorylation to produce tagatose, some of which were found to have more excellent tagatose 6-phosphate dephosphorylation to produce tagatose, and these enzymes were found to give better results when used for tagatose production, thereby completing the present disclosure.
Solution for solving the problem
The present disclosure provides the following technical solutions.
(1) Use of a polypeptide selected from any one of the following groups (i) - (iv) as tagatose 6-phosphate epimerase, wherein the polypeptide:
(i) Has the sequence shown in SEQ ID NO: 1-4;
(ii) Has at least 70% sequence identity to the sequence set forth in (i) and excludes the sequence set forth in SEQ ID NO: 1-4;
(iii) A polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with a polynucleotide as set forth in (a) or (b):
(a) A polynucleotide encoding a polypeptide of the amino acid sequence shown in (i);
(b) The full-length complementary polynucleotide of (a);
(iv) Fragments of the polypeptides shown by (i), (ii), (iii) and which still have tagatose 6-phosphate epimerase activity.
(2) The use according to (1), wherein the tagatose 6-phosphate epimerase is derived from a thermostable microorganism; preferably, the thermostable microorganism is selected from Rhodothermus, anaerorinea, ignosphaera or thermostremia; 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 following groups (v) - (viii) as tagatose 6-phosphate phosphatase, wherein the polypeptide:
(v) Has the sequence shown in SEQ ID NO: 5-7;
(vi) Has at least 70% sequence identity to the sequence set forth in (v) and excludes the sequence set forth in SEQ ID NO: 5-7;
(vii) A polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with a polynucleotide as set forth in (a) or (b):
(a) A polynucleotide encoding a polypeptide of the amino acid sequence shown in (v);
(b) The full-length complementary polynucleotide of (a);
(viii) Fragments of the polypeptides shown by (v), (vi), (vii), and which still have tagatose 6-phosphate phosphatase activity.
(4) The use according to (3), wherein the tagatose 6-phosphate phosphatase is derived from a thermostable microorganism; preferably, the heat-resistant microorganism is selected from the group consisting of thermospora, spiraeta or huntateiclotriostrichum; 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 comprises 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 any one of the uses (1) to (2), and the tagatose 6-phosphate phosphatase is the polypeptide in any one of the uses (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 group consisting of: starch branching enzymes (including isoamylase and pullulanase), alpha-glucan phosphorylase, glucose phosphomutase, glucose phosphoisomerase, maltose phosphorylase, beta-glucose phosphomutase, polyphosphate glucokinase, cellodextrin phosphorylase, cellobiose phosphorylase, sucrose phosphorylase, glucose isomerase, 4-glucan transferase, alpha-amylase, beta-amylase.
(8) A strain or strain composition expressing the enzyme composition according to any one of (5) to (7).
(9) The strain or strain composition according to (8), wherein the host cell of the strain or strain composition is derived from corynebacterium, brevibacterium, arthrobacter, microbacterium or Escherichia; preferably, the host cell is Bacillus subtilis, corynebacterium glutamicum or Escherichia coli.
(10) The strain or strain composition according to any one of (8) - (9), wherein the strain or strain composition converts an expression vector as follows:
an expression vector comprising a nucleic acid encoding tagatose 6-phosphate epimerase for use according to any one of (1) to (2); and/or
An expression vector comprising a nucleic acid encoding tagatose 6-phosphate phosphatase for use according to any one of (3) to (4).
(11) The strain or strain composition of (10), wherein the strain or strain composition further comprises transformed an expression vector comprising a nucleic acid encoding a starch branching enzyme (including isoamylase and pullulanase), an alpha-glucan phosphorylase, a glucose phosphomutase, a glucose phosphoisomerase, a maltose phosphorylase, a beta-glucose phosphomutase, a polyphosphate glucokinase, a cellodextrin phosphorylase, a cellobiose phosphorylase, a sucrose phosphorylase, a glucose isomerase, a 4-glucan transferase, an alpha-amylase, or a beta-amylase.
(12) Use of the enzyme composition of any one of (5) to (7) or the strain or strain composition of any one of (8) to (11) for producing tagatose.
(13) A process for producing tagatose, wherein the process comprises: a step of adding the enzyme composition of any one of (5) to (7) or inoculating the strain or strain composition of any one of (8) to (11) to convert a substrate into tagatose;
optionally, the method further comprises the step of pre-treating the substrate; or (b)
And purifying or separating the tagatose.
(14) The method according to (13), wherein the method comprises a step of further adding a metal ion or a metal salt 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 of the group consisting of magnesium, nickel, manganese, zinc, cobalt, iron, copper, calcium, molybdenum, selenium.
(15) The method according to any one of (13) to (14), wherein the substrate is selected from saccharides or derivatives thereof; preferably, the fermentation substrate is selected from one or more of the group consisting of: starch or its derivative, cellulose or its derivative, fructose, glucose, sucrose, maltose.
(16) The method according to any one of (13) - (15), wherein the method is selected from one or more of the group consisting of: multienzyme catalysis, whole cell catalysis, ferment catalysis containing enzymes/whole cells, immobilized multienzyme catalysis, immobilized whole cell catalysis.
It is an object of the present disclosure to provide a composition for preparing tagatose 6-phosphate, comprising tagatose 6-phosphate epimerase, a microorganism expressing the tagatose 6-phosphate epimerase, or a culture of the microorganism.
Tagatose 6-phosphate epimerase of the present disclosure can be a polypeptide consisting of SEQ ID NO:1 (Uniprot ID: A0A7V2B2J 0), SEQ ID NO:2 (Uniprot ID: A0A3M1DFN 1), SEQ ID NO:3 (Uniprot ID: A0A7C4PIG 5) or SEQ ID NO:4 (Uniprot ID: A0A7J2U4S 4), or a polypeptide comprising an amino acid sequence identical to 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 99% sequence identity. Has said homology and exhibits NO: 1. SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4 (i.e., the activity of epimerising the 4-carbon position of fructose in fructose 6-phosphate to convert fructose 6-phosphate to fructose 6-phosphate C4-epimerisation of tagatose 6-phosphate), even with a portion of the amino acid sequence deleted, modified, substituted or added. In addition, probes prepared from known nucleotide sequences, for example, polypeptides encoded by polynucleotides that hybridize under stringent conditions to the complement of all or part of the nucleotide sequence encoding the polypeptide, may be included without limitation as long as they have fructose 6-phosphate C4-epimerization activity. In addition, the composition may comprise one or more amino acid sequences consisting of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, and tagatose 6-phosphate epimerase consisting of the amino acid sequence of seq id no.
Wherein, SEQ ID NO:1 (Uniprot ID: A0A7V2B2J0, rhodothermus marinus):
MGEQVPHGEALVRALRARHRSFVDWVVELLRGPLAYAHTLLAVCPNSVAVVEAALEAAAEANAPLLFAATLNQVDLDGGYTGWTPHTLAQFVAQKRQHLALDLPVVLGLDHGGPWKKDVHARDRLSFQETFRSVLRTIEACLDAGYGLLHLDPTVDLEASPGSPIPIDRIVERTVALLQHAESYRQARKLPPVAYEVGTEEVGGGLEAETRMAAFLDQLWKALDREGLPHPVFVVGDIGTQLDTSDFDFKRAQRLDALVRRYGALIKGHYTDGVTRLELYPKAGIGGANVGPGLAAVEFEALETLIHEARHRGLPVTLDIALKQAVVESGRWKKWLQPEEKDLPFEALSPERQRWLVATGSRYVWTHPAVQAARRQLYAMLAPWIDAQAYVRDRLKAYLQKYFHAFNLIGFNERLRALWPG
SEQ ID NO:2 (Uniprot ID: A0A3M1DFN1, thermoflexia bacterium):
MYMRGNYLDFVVAAHKFGVPFGIASICSAHPLVLEAALRHGLVHGMPVLIEATCNQVNQFGGYTGMTPMDFARQVMEQAERVGFPQERLILGGDHLGPLPWAHEPAEEAMQKASDLVRAFVQAGCTKIHLDCSMPLGGETVLPVEVIAQRVARLAQVAEEAAGERRGALRYVIGSEVPPAGGAKAGEGPPSVTRPEDAAEAIEATHRAFRALGLEEAWERVIALVVQPGVEFGDETIHEYDRAAAAPLVRYIEGVPGLVYEAHSTDYQPLRALRAMVEDHFAILKVGPALTFALREAVFALADIEAAMGLEPPSGIREAFEAAMLSNPVHWQRYYRGDPMSQKLARQYSLSDRIRYYWTAPEVQAAFSRLMRNLGDRPIPPGLLSQYMPEEFRKVRAGELKNRPDDLLLGRVMGVLEAYRLATQGVPG
SEQ ID NO:3 is as follows: (Uniprot ID: A0A7C4PIG5, anaerolinea thermolimosa)
MKPLKEVVRRLIELRKQGRKMTLLAVCPNSSAVLEAAVQSAALHRSVMLFAATLNQVDRDGGYTGWTPESFVQEMQRRAARINWNGPLYPCLDHGGPWLKDNQAQFPYAKTEAEVKESLFACLHAGYALLHIDTTVDRSLPPGMAPAIEVVVDRAVSLIGAVEKERIEHQLPEIAYEVGSDEVHGGLVEFDRFREFLILLKERLDRAGLGEVWPAFLVTQVGTDLHTTRFDGEVAKRLFDLVSPYGSLIKGHYTDWVENPEMYPETGMGGANVGPEFTTVEYLALKELCARESELLAGNPGKASEFLWHLEHAVLDSGRWKKWLFPEERGLPFEELSKERREWLTQTGARYVWSQPVVRQARIRLYENLRGVISDPHAWVVRKIQQAIDRYIEAFHLTDSASLFE
SEQ ID NO:4 (Uniprot ID: A0A7J2U4S4, ignisphaera aggregans):
MIGVRSTLDKACKLPGKATLLCLSPISRHVVQAYIKIAKEFNTPICFATSLNQVDRGGGYTGWTPIDFKNYVMDMAREYSISTPIILQLDHGGPWLKDEHIAKKYSYEEALNDFLKSLELFIKAGFDVIHIDTTIDLDSRDGYADVEVASKRTADLIMYSEEIASRYGVKLEYEIGSDRWGYKPLEIVENFVSKAISMLRDRGFDINRLVFGVADVGTKVCPGNRVDPVIVREFSSLMRRHGLYLKIHSGDYLENPGELPKNSVGGVNIGPMLAHIMYSTFKEILYEKLDKDRALELLEELNNFIASSDKLAKYVGKGLGEAEEYKLGLASRYIWSTTKAKEFIDRISKIIGIDIEKLFIEKLAQTVKRYVIELNIYKLYETNKKHATLKQY
in some embodiments, tagatose 6-phosphate epimerase of the present disclosure can be an enzyme derived from a thermostable microorganism, for example, an enzyme derived from Rhodothermus or a variant thereof, in particular, an enzyme derived from Rhodothermus marinus or a variant thereof; an enzyme derived from anaerorinea or a variant thereof, in particular an enzyme derived from Anaerolinea thermolimosa or a variant thereof; an enzyme derived from Ignisphaera or a variant thereof, in particular an enzyme derived from Ignisphaera aggregans or a variant thereof; an enzyme derived from thermoslexia or a variant thereof, in particular an enzyme derived from Thermoflexia bacterium or a variant thereof; but is not limited thereto.
Tagatose 6-phosphate epimerases in the present disclosure are specific for fructose 6-phosphate and tagatose 6-phosphate, i.e., fructose 6-phosphate/tagatose 6-phosphate is more active than other phosphorylated monosaccharides and monosaccharides present in the reaction. For example, tagatose 6-phosphate epimerase has higher epimerization activity on fructose 6-phosphate/tagatose 6-phosphate than on glucose 6-phosphate, glucose 1-phosphate, fructose or tagatose.
The tagatose 6-phosphate epimerase of the present disclosure has a comparable level of activity but better substrate specificity than the previously disclosed Thermoanaerobacter indiensis-derived tagatose 6-phosphate epimerase (CN 109750024A). Specifically, the tagatose 6-phosphate epimerase in the present disclosure has much lower C4-epimerization activity on fructose or tagatose monosaccharides than that of the tagatose 6-phosphate epimerase of Thermoanaerobacter indiensis, thereby enabling higher yields of products to be ensured. Certain tagatose 6-phosphate epimerases of the present disclosure have better thermostability.
In the present disclosure, the tagatose 6-phosphate epimerase may be used directly, or immobilized to maintain stability and be recycled; the microorganism and/or culture of microorganisms containing the tagatose 6-phosphate epimerase may be used as such, or immobilized to maintain stability and be recycled, or whole cells may be subjected to permeabilization to obtain a rapid reaction rate.
It is another object of the present disclosure to provide a composition for preparing tagatose, comprising tagatose 6-phosphatase, a microorganism expressing the tagatose 6-phosphatase, or a culture of the microorganism.
Tagatose 6-phosphate phosphatase may be a nucleotide sequence consisting of SEQ ID NO:5 (Uniprot ID: D1A2R 1), SEQ ID NO:6 (Uniprot ID: G0GB 57), SEQ ID NO:7 (Uniprot ID: A3DJZ 0) or a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 5. SEQ ID NO:6 or SEQ ID NO:7 has an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Has said homology and exhibits NO: 5. SEQ ID NO:6 or SEQ ID NO:7 (i.e., the activity of dephosphorylating tagatose 6-phosphate to produce tagatose), even an amino acid sequence having a deletion, modification, substitution, or addition of a portion of the sequence is included within the scope of the present disclosure. In addition, a probe prepared from a known nucleotide sequence, for example, a polypeptide encoded by a polynucleotide which hybridizes under stringent conditions to the complement of all or part of the nucleotide sequence encoding the polypeptide may be included without limitation as long as it has an activity of dephosphorylating tagatose 6-phosphate to tagatose. In addition, the composition may comprise one or more amino acid sequences consisting of SEQ ID NO: 5. SEQ ID NO:6 or SEQ ID NO:7, and tagatose 6-phosphate phosphatase consisting of the amino acid sequence of tagatose.
SEQ ID NO:5 (Uniprot ID: D1A2R1, thermomonospora curvata):
MAGGGLQAVLFDMDGLLIDSEPMWLEVETEVMAWLGGEWGPQHQQKLLGGSVTYAAHYMLSLVEATVAPQEVERRLVDGMAERLAGSVPLMPGAKELLAEVRAAGVATALVSSSERRLVEAALAGIGREHFDVTVAGDEVARRKPDPEPYLTAMARLGVSPGRCVVLEDSPTGLAAAEAAGCVTVAVPGVVPVPPAPGRTVVESLRNVDLQMLNGLLP
SEQ ID NO:6 (Uniprot ID: G0GB57, spirochaeta thermophila):
MRMRRECAPPGIRAAIFDMDGTLVNSEDVYWDADCAFLDRYGIPHDDALREYMIGRGTKGFIEWMRTQKEIPRSDEELAREKIGLFLEHARGRVQVFPEMRRLLGLFEEAGMSCALASGSPRRVIEVLLEETGLVGFFRVVVSADEVARPKPAPDVFLEAAGRLGVEPGGCVVFEDSEPGVQAALDAGMVCVAIPTLVKDRYPEVFYQADVLFEGGMGEFCAERVWEWLGCGVGVRR
SEQ ID NO:7 as follows (Uniprot ID: A3DJZ0, acetivibrio thermocellus):
MKKVKAVIFDMDGLMIDTERLYFEVERIMARKFGKEVKDETLWKMMGRKPLEAITVFAEDLELDISPKKLLEIRDELFVKKLVNEVEPMPGLFDILNILKGKVKMAIATGSPQKFLKIVLDKLKIESYFDVFVTSDEVEKGKPDPEVYNTAVKRLKVAPFECVVLEDSSNGALAAVRAGCYTIAVPTVYTNKQDFSFVNYVAKDLKDAAEKINEFLCSQEIEF
in some embodiments, tagatose 6-phosphate phosphatase of the present disclosure can be an enzyme derived from a thermostable microorganism, e.g., an enzyme derived from thermo monospora or a variant thereof, in particular an enzyme derived from Thermomonospora curvata or a variant thereof; an enzyme derived from spiraeta or a variant thereof, in particular an enzyme derived from Spirochaeta thermophila or a variant thereof; an enzyme derived from huntateiclotrichum or a variant thereof, in particular an enzyme derived from Hungateiclostridium thermocellum or a variant thereof; but is not limited thereto.
The tagatose 6-phosphatase of the present disclosure has a higher activity than the previously disclosed Archaeoglobus fulgidus-derived tagatose 6-phosphatase (Uniprot code O29805) (CN 106399427B). Preferably, tagatose 6-phosphate phosphatase in the present disclosure has at least 10%, at least 30%, at least 1-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 150-fold improved enzymatic activity compared to the previously disclosed Archaeoglobus fulgidus-derived tagatose 6-phosphate phosphatase.
Tagatose 6-phosphate phosphatase used in the methods of the present disclosure is specific for tagatose 6-phosphate. I.e. the dephosphorylation activity for tagatose 6-phosphate is higher than for other phosphorylated monosaccharides. For example, tagatose 6-phosphate phosphatase has higher dephosphorylation activity on tagatose 6-phosphate than on, for example, glucose 1-phosphate, glucose 6-phosphate or fructose 6-phosphate. In the present disclosure, certain tagatose 6-phosphatase enzymes have better specificity and/or activity than the previously disclosed Archaeoglobus fulgidus-derived tagatose 6-phosphatase (Uniprot code O29805).
In the present disclosure, the tagatose 6-phosphate phosphatase may be directly used, or immobilized to maintain stability and be recycled; the microorganism and/or culture of microorganisms containing the tagatose 6-phosphatase may be used directly, or immobilized to maintain stability and be recycled, or whole cells may be subjected to a permeabilization treatment to obtain a rapid reaction rate.
It is a further object of the present disclosure to provide a composition for producing tagatose comprising the tagatose 6-phosphate epimerase described in the present disclosure, a microorganism expressing the tagatose 6-phosphate epimerase, and/or a culture of the microorganism; and tagatose 6-phosphatase, a microorganism expressing tagatose 6-phosphatase, and/or a culture of a microorganism expressing tagatose 6-phosphatase as described in the present disclosure. The above description of tagatose 6-phosphate epimerase, a microorganism expressing said tagatose 6-phosphate epimerase and/or a culture of said microorganism applies here as well to tagatose 6-phosphate phosphatase, a microorganism expressing tagatose 6-phosphate phosphatase and/or a culture of a microorganism expressing tagatose 6-phosphate phosphatase.
In some embodiments, the compositions of the present disclosure for producing tagatose may further comprise an enzyme that participates in the tagatose production pathway of the present disclosure (fig. 1), a microorganism that expresses an enzyme that participates in the tagatose production pathway of the present disclosure, and/or a culture of a microorganism that expresses an enzyme that participates in the tagatose production pathway of the present disclosure. This is merely as some examples, i.e., there is no limitation in the present disclosure on the enzyme contained in the composition of the present disclosure for producing tagatose and the substrate for producing tagatose, as long as tagatose can be produced by using the tagatose 6-phosphate epimerase and/or tagatose 6-phosphate phosphatase of the present disclosure.
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 comprising a divalent cation. In particular, 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, and more specifically, the metal salt may be magnesium chloride, magnesium sulfate, nickel chloride, manganese sulfate, cobalt chloride, ferric chloride, ferrous chloride, zinc sulfate, cupric chloride, calcium chloride, sodium molybdate, sodium selenate, and the like. The concentration of the metal salt may be in the range of 0.001mM to 100 mM. Preferably, the concentration of the metal salt may be in the range of 0.01mM to 50 mM.
It is yet another object of the present disclosure to provide a more superior process for preparing tagatose, which comprises preparing tagatose by reacting the composition with saccharides and derivatives such as polysaccharides, oligosaccharides, disaccharides, monosaccharides or phosphate compounds of saccharides (fig. 2).
In a preferred embodiment, the more superior method of making tagatose comprises the step of converting fructose 6-phosphate to tagatose 6-phosphate using a tagatose-6-phosphate epimerase, wherein the tagatose 6-phosphate epimerase comprises a nucleotide sequence that hybridizes with SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4 has an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; more preferably, tagatose 6-phosphate epimerase comprises a nucleotide sequence identical to SEQ ID NO:4 has an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; most preferably, tagatose 6-phosphate epimerase has the amino acid sequence as set forth in SEQ ID NO:4, and a sequence of amino acids listed in 4.
In another preferred embodiment, the more superior method of making tagatose comprises the step of converting tagatose 6-phosphate to tagatose using tagatose 6-phosphate phosphatase, wherein the tagatose 6-phosphate phosphatase comprises a nucleotide sequence that matches SEQ ID NO: 5. SEQ ID NO:6 or SEQ ID NO:7 has an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity; more preferably, tagatose 6-phosphate phosphatase comprises a nucleotide sequence identical to SEQ id no:7 has an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity; most preferably, tagatose 6-phosphate phosphatase has the amino acid sequence as set forth in SEQ ID NO: 7.
In yet another preferred embodiment, the more superior method of making tagatose comprises the steps of converting fructose 6-phosphate to tagatose 6-phosphate using a tagatose 6-phosphate epimerase and converting tagatose 6-phosphate to tagatose using a tagatose 6-phosphate phosphatase, wherein the tagatose 6-phosphate epimerase comprises an amino acid sequence that hybridizes with SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5. SEQ ID NO: 6. or SEQ ID NO:7 has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity amino acid sequence. More preferably, tagatose 6-phosphate epimerase comprises a nucleotide sequence identical to SEQ id no:4, having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:7 has an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Most preferably, tagatose 6-phosphate epimerase has the amino acid sequence as set forth in SEQ ID NO:4, the tagatose 6-phosphate phosphatase has the amino acid sequence as set forth in SEQ ID NO: 7.
In some embodiments, the more superior methods of making tagatose further comprise the step of converting glucose 6-phosphate to fructose 6-phosphate by utilizing glucose 6-phosphate isomerase (PGI), a microorganism expressing glucose 6-phosphate isomerase, and/or a culture of a microorganism expressing glucose 6-phosphate isomerase, prior to the step of converting fructose 6-phosphate to tagatose 6-phosphate of the present disclosure. Sources of the glucose 6-phosphate isomerase include, but are not limited to Hungateiclostridium thermocellum, thermus thermophilus, pyreococcus sp., pyrococcus horikoshii, pyrococcus furiosus, thermococcus barophilus, thermococcus kodakarensis, thermotoga maritima, and the like. In some examples, glucose phosphate isomerase includes, but is not limited to, one derived from Hungateiclostridium thermocellum, uniprot database number A3DBX9; can also be derived from Thermus thermophilus, and Uniprot database number Q5SLL6.
In some embodiments, the more superior methods of making tagatose further comprise the step of converting glucose-1-phosphate to glucose 6-phosphate by utilizing glucose Phosphomutase (PGM), a microorganism expressing glucose phosphomutase, and/or a culture of a microorganism expressing glucose phosphomutase prior to the step of converting glucose 6-phosphate to fructose 6-phosphate of the present disclosure. Sources of the glucose phosphomutases include, but are not limited to Hungateiclostridium thermocellum, thermus thermophilus, pyrococcus sp., pyrococcus horikoshii, pyrococcus furiosus, thermococcus barophilus, thermococcus kodakarensis, thermotoga maritima, and the like. In some examples, the glucose phosphomutase includes, but is not limited to, those derived from Thermococcus kodakarensis, uniprot database number Q68BJ6; can also be derived from Pyrococcus furiosus, and the Uniprot database is numbered as Q8U383.
In addition, the more superior method of preparing tagatose of the present disclosure may further include a step of converting a saccharide such as polysaccharide, oligosaccharide, disaccharide, monosaccharide) into glucose-1-phosphate, wherein the step is catalyzed by at least one enzyme, a microorganism expressing the enzyme, and/or a culture of a microorganism expressing the enzyme, and the saccharide may be selected from the group consisting of, but not limited to, starch or derivatives thereof, cellulose or derivatives thereof, fructose, glucose, and/or sucrose (fig. 2). The one or more enzymes used in the step of converting the saccharide to glucose-1-phosphate according to the methods of the present disclosure may be an alpha-glucan phosphorylase, a maltose phosphorylase, a sucrose phosphorylase, a cellodextrin phosphorylase, a cellobiose phosphorylase, and/or a cellulose phosphorylase, and mixtures thereof, and/or a microorganism expressing the one or more enzymes, and mixtures thereof, and/or a culture of the microorganism expressing the one or more enzymes, and mixtures thereof.
When the saccharide is starch or a derivative thereof, the starch or derivative thereof may be selected from the group consisting of, but not limited to, amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltooligosaccharide, maltose, glucose, and mixtures thereof. In certain embodiments of the present disclosure, when the saccharide is starch or a derivative thereof, including, but not limited to, e.g., amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltooligosaccharide, enzymes for converting the saccharide to glucose 1-phosphate comprise an alpha-glucan phosphorylase, a microorganism expressing the alpha-glucan phosphorylase, and/or a culture of a microorganism expressing the alpha-glucan phosphorylase. Sources of the α -glucan phosphorylase include, but are not limited to Hungateiclostridium thermocellum, thermus thermophilus, pyreococcus sp., pyrococcus horikoshii, pyrococcus furiosus, thermococcus barophilus, thermococcus kodakarensis, thermotoga maritima, and the like. In some examples, the α -glucan phosphorylase includes, but is not limited to, a gene numbered TM1168 on KEGG derived from Thermotoga maritima; can also be derived from Thermococcus kodakarensis, the enzyme is numbered Q5JH18 in the Uniprot database.
Some methods of the present disclosure may further comprise the step of converting the starch into a starch derivative, wherein the starch derivative is prepared by enzymatic hydrolysis of the starch and/or acid hydrolysis of the starch. In certain embodiments of the present disclosure, to increase the yield of tagatose, the more superior methods of making tagatose may further comprise converting the starch to dextrins using starch branching enzymes such as Isoamylase (IA), an isoamylase-expressing microorganism, and/or a culture of an isoamylase-expressing microorganism. Sources of the isoamylase include, but are not limited to Hungateiclostridium thermocellum, thermus thermophilus, pyreococcus sp., pyrococcus horikoshii, pyrococcus furiosus, thermococcus barophilus, thermococcus kodakarensis, thermotoga maritima, sulfolobus tokodaii, and the like. In some embodiments, the isoamylase is derived from Sulfolobus tokodaii and the enzyme is numbered Q973H3 in the Uniprot database. In certain embodiments of the present disclosure, to increase the yield of tagatose, the more superior methods of making tagatose may further comprise converting the starch to dextrins using starch branching enzymes such as isoamylase, pullulanase (PA), pullulanase-expressing microorganisms, and/or cultures of pullulanase-expressing microorganisms. 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, and the like. In some embodiments, the pullulanase is derived from Thermotoga maritima and the enzyme is numbered O33840 in the Uniprot database.
In further embodiments of the present disclosure, to increase the yield of tagatose, the more superior method of preparing tagatose may further comprise converting degradation products of starch or derivatives thereof, glucose, maltose and maltotriose, into longer maltooligosaccharides using 4-glucanotransferase (4 GT), a microorganism expressing 4-glucanotransferase and/or a culture of a microorganism expressing 4-glucanotransferase, wherein the longer maltooligosaccharides may be converted into glucose 1-phosphate using alpha-glucanotransferase, a microorganism expressing alpha-glucanotransferase and/or a culture of a microorganism expressing alpha-glucanotransferase. In some examples, the 4-glucanotransferase is derived from Thermococcus litoralis and the enzyme is numbered O32462 in the Uniprot database. Sources of the 4-glucanotransferases 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, and the like. In still further embodiments of the present disclosure, in order to increase the yield of tagatose, the more superior method of preparing tagatose may further include converting glucose, which is a degradation product of starch or a derivative thereof, into glucose 6-phosphate by adding polyphosphate using polyphosphate glucokinase (PPGK), a microorganism expressing polyphosphate glucokinase, and/or a culture of a microorganism expressing polyphosphate glucokinase. 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, and the like. In some examples, the polyphosphate glucokinase is derived from Thermobifida fusca and the enzyme is numbered Q47NX5 in the Uniprot database.
When the saccharide is maltose and/or maltooligosaccharide, the enzyme for converting the saccharide to beta-glucose 1-phosphate comprises a maltose phosphorylase, a microorganism expressing the maltose phosphorylase, and/or a culture of a microorganism expressing the maltose phosphorylase. The beta-glucose 1-phosphate is further converted to glucose 6-phosphate by beta-glucose phosphomutase (beta-PGM), a beta-glucose phosphomutase-expressing microorganism, and/or a culture of a beta-glucose phosphomutase-expressing microorganism. In some examples, the maltose phosphorylase is derived from Bacillus sp.RK-1, the gene of which is numbered AB084460.1 on Genebank. Sources of the beta-glucose phosphomutases 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, and the like. Preferably a beta-glucose phosphomutase derived from Pyrococcus horikoshii OT3, enzyme number O58510 in Uniprot database.
When the saccharide is sucrose, the enzyme for converting the saccharide to glucose 1-phosphate comprises a sucrose phosphorylase, a microorganism expressing the sucrose phosphorylase and/or a culture of a microorganism expressing the sucrose phosphorylase. In still further embodiments of the present disclosure, to increase the yield of tagatose, the more superior method of preparing tagatose may further comprise converting fructose, a degradation product of sucrose, into glucose using Glucose Isomerase (GI), a microorganism expressing glucose isomerase, and/or a culture of a microorganism expressing glucose isomerase. Glucose and polyphosphate are in turn catalyzed by polyphosphate glucokinase to glucose 1-phosphate. Sources of the sucrose phosphorylase, glucose isomerase, polyphosphate glucokinase include, but are not limited to Hungateiclostridium thermocellum, thermus thermophilus, pyreococcus sp., pyrococcus horikoshii, pyrococcus furiosus, thermococcus barophilus, thermococcus kodakarensis, thermotoga maritima, thermococcus litoralis, thermobifida fusca, sulfolobus tokodaii, streptomyces murinus, bifidobacterium adolescentis, and the like. In some examples, the glucose isomerase is derived from Streptomyces murinus and the enzyme is numbered P37031 in the Uniprot database. In some examples, the polyphosphate glucokinase is derived from Thermobifida fusca and the enzyme is numbered Q47NX5 in the Uniprot database. In some examples, the sucrose phosphorylase is derived from Bifidobacterium adolescentis and the enzyme is numbered A0ZZH6 in the Uniprot database.
Tagatose can also be produced from glucose when the saccharide is glucose (fig. 2). The method 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 the use of glucose and polyphosphate to produce glucose 6-phosphate catalyzed by polyphosphate glucokinase (PPGK). Glucose can be produced by enzymatic conversion of sucrose (fig. 2). Sources of the glucokinase, polyphosphate glucokinase include, but are not limited to Hungateiclostridium thermocellum, thermus thermophilus, pyreococcus sp., pyrococcus horikoshii, pyrococcus furiosus, thermococcus barophilus, thermococcus kodakarensis, thermotoga maritima, thermococcus litoralis, thermobifida fusca, sulfolobus tokodaii, streptomyces murinus, bifidobacterium adolescentis, and the like. In some examples, the polyphosphate glucokinase is derived from Thermobifida fusca and the enzyme is numbered Q47NX5 in the Uniprot database.
Tagatose can also be produced from fructose when the saccharide is fructose (fig. 2). The process according to the present disclosure may further include, but is not limited to, a step of converting fructose to glucose or fructose 6-phosphate, wherein the step is catalyzed by at least one enzyme, and, optionally, a step of converting sucrose to fructose, wherein the step is catalyzed by at least one enzyme. For example, the method involves the catalytic production of glucose from fructose via glucose isomerase. For example, the method involves the catalytic production of fructose 6-phosphate from fructose via a fructokinase. Sources of the glucose isomerase, 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 tokodaii, streptomyces murinus, bifidobacterium adolescentis, and the like. In some examples, the glucose isomerase is derived from Streptomyces murinus and the enzyme is numbered P37031 in the Uniprot database. The conversion of glucose to tagatose is as described above. Fructose may be produced by enzymatic conversion of sucrose. The phosphate ions generated when tagatose 6-phosphate is converted to tagatose can be recycled in the step of converting sucrose to glucose 1-phosphate.
When the saccharide 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 (e.g., cellulose, hemicellulose and/or lignin and other minor ingredients), pure cellulose (Avicel (microcrystalline cellulose), regenerated amorphous cellulose, bacterial cellulose, filter paper, etc.), partially hydrolyzed cellulosic substrates (including water insoluble cellodextrins having a degree of polymerization greater than 7, water soluble cellodextrins having a degree of polymerization of 3-6, cellobiose, glucose and fructose). In certain embodiments of the present disclosure, when the saccharide is cellulose and derivatives thereof, the enzyme for converting the saccharide to glucose 1-phosphate comprises cellodextrin phosphorylase (CDP), a culture of a microorganism expressing cellodextrin phosphorylase and/or a microorganism expressing cellodextrin phosphorylase, and cellobiose phosphorylase (CBP), a microorganism expressing cellobiose phosphorylase and/or a culture of a microorganism expressing cellobiose phosphorylase. Sources of the cellodextrin phosphorylase, 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, and the like. In some examples, the cellodextrin phosphorylase source Hungateiclostridium thermocellum, enzyme number A3DJQ6 in the Uniprot database; cellobiose phosphorylase was derived from Hungateiclostridium thermocellum and the enzyme was numbered A3DC35 in Uniprot database.
Some methods of the present disclosure may further comprise a step of converting cellulose to a cellulose derivative, for example hydrolyzing solid cellulose to water-soluble cellodextrin and cellobiose using an endoglucanase, a microorganism expressing the endoglucanase, and/or a culture of a microorganism expressing the endoglucanase and/or a cellobiohydrolase, a microorganism expressing the cellobiohydrolase, and/or a culture of a microorganism expressing the cellobiohydrolase. In some embodiments, the more superior methods of making tagatose may further include pre-treating the cellulose to increase their reactivity and reduce the degree of polymerization of the cellulose chains prior to hydrolysis of the cellulose and formation of glucose 1-phosphate. The pretreatment methods of cellulose include, but are not limited to, dilute acid pretreatment, lignocellulose fractionation based on cellulose solvents, ammonia fiber swelling, ammonia water soaking, ionic liquid treatment, and partial hydrolysis by use of concentrated acids (including hydrochloric acid, sulfuric acid, phosphoric acid, and combinations thereof).
In certain embodiments of the present disclosure, to increase the yield of tagatose, the more superior method of preparing tagatose may further comprise converting the degradation product maltose of cellulose or a derivative thereof into glucose-1-phosphate and glucose using cellobiose phosphorylase, a microorganism expressing cellobiose phosphorylase, and/or a culture of a microorganism expressing cellobiose phosphorylase. Sources of the cellobiose phosphorylase include, but are not limited to Hungateiclostridium thermocellum, thermus thermophilus, pyreococcus sp., pyrococcus horikoshii, pyrococcus furiosus, thermococcus barophilus, thermococcus kodakarensis, thermotoga maritima, thermococcus litoralis, thermobifida fusca, sulfolobus tokodaii, streptomyces murinus, bifidobacterium adolescentis, and the like. In some examples, the cellobiose phosphorylase is derived from Hungateiclostridium thermocellum and the enzyme is numbered A3DC35 in the Uniprot database. In still further embodiments of the present disclosure, in order to increase the yield of tagatose, the more superior method of preparing tagatose may further include converting glucose, which is a degradation product of cellulose or a derivative thereof, into glucose 6-phosphate by adding polyphosphate using polyphosphate glucokinase (PPGK), a microorganism expressing polyphosphate glucokinase, and/or a culture of a microorganism expressing polyphosphate glucokinase. 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, and the like. In some examples, the polyphosphate glucokinase is derived from Thermobifida fusca and the enzyme is numbered Q47NX5 in the Uniprot database.
In the present disclosure, the enzymes used may be used directly, and/or immobilized to maintain stability and be recycled; the microorganisms and/or cultures of microorganisms containing the enzymes described above may be used directly and/or immobilized to maintain stability and be recycled or whole cells may be permeabilized to achieve rapid reaction rates.
ADVANTAGEOUS EFFECTS OF INVENTION
Because the tagatose 6-phosphate epimerase and the tagatose 6-phosphate phosphatase are both thermostable and have higher activity and specificity, the use of the enzyme can be reduced or the yield of the tagatose can be improved when the tagatose is used for industrial production, so that the production cost of the tagatose is further reduced, and the industrial production of the tagatose is facilitated.
Drawings
FIG. 1 shows the tagatose pathway prepared with fructose 6-phosphate. The abbreviations used in fig. 1 have the following meanings: TPE: tagatose 6-phosphate epimerase; TPP: tagatose 6-phosphate phosphatase; pi: inorganic phosphorus.
Figure 2 shows the catalytic route for the preparation of tagatose from starch and its derivatives, cellulose and its derivatives, maltose, sucrose, fructose. The abbreviations used in fig. 2 have the following meanings: IA: an isoamylase; αgp: alpha-glucan phosphorylase; PGM: glucose phosphate mutase; PGI: glucose phosphate isomerase; TPE: tagatose 6-phosphate epimerase; TPP: tagatose 6-phosphate phosphatase; MP: maltose phosphorylase; beta-PGM: beta-glucose phosphomutase; PPGK: polyphosphate glucokinase; CDP: a cellodextrin phosphorylase; CBP, cellobiose phosphorylase; SP: sucrose phosphorylase; GI: glucose isomerase; pi: inorganic phosphorus; (Pi) n Or (Pi) n-1 : polyphosphoric acid.
Detailed Description
Definition of the definition
The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or specification may refer to "one" but may also refer to "one or more", "at least one" and "one or more".
As used in the claims and specification, the words "comprise," "have," "include" or "contain" mean including or open-ended, and do not exclude additional, unrecited elements or method steps.
Throughout this application, the term "about" means: one 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 being inclusive of alternatives and "and/or", the term "or" in the claims means "and/or" unless expressly indicated otherwise as being exclusive of each other, as defined by the alternatives or alternatives.
When used in the claims or specification, the term "numerical range" is intended to include both the numerical endpoints of the range and all natural numbers covered in the middle of the numerical endpoints relative to the numerical endpoints.
As used in this disclosure, the term "conversion" refers to the chemical conversion from one molecule to another, primarily catalyzed by one or more polypeptides (enzymes), although other organic or inorganic catalysts may be used; it may also refer to the ratio (in%) between the molar amount of the desired product and the molar amount of the limiting substrate
As used in the present disclosure, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and are polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).
As used in the present disclosure, the term "fragment" means a polypeptide or a catalytic or carbohydrate binding module that lacks one or more (e.g., several) amino acids from the amino and/or carboxy terminus of a mature polypeptide or domain.
In some specific embodiments, the fragment has tagatose 6-phosphate epimerase (i.e., conversion to "tagatose 6-phosphate" with "fructose 6-phosphate" as a substrate).
In other specific embodiments, the fragment has tagatose 6-phosphate phosphatase (i.e., dephosphorylation to produce "tagatose" using "tagatose 6-phosphate" as a substrate).
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 is present in an organism, can be isolated from a source in nature, and is not intentionally modified by man in the laboratory is naturally occurring. As used in this disclosure, "naturally occurring" and "wild-type" are synonymous.
As used in this disclosure, the term "mutant" refers to a polynucleotide or polypeptide comprising an alteration (i.e., substitution, insertion, and/or deletion) at one or more (e.g., several) positions relative to a "wild-type" or "comparable" polynucleotide or polypeptide, wherein a substitution refers to a substitution of a nucleotide or amino acid occupying one position with a different nucleotide or amino acid. Deletions refer to the removal of a nucleotide or amino acid occupying a position. Insertion refers to the addition of a nucleotide or amino acid following the nucleotide or amino acid that abuts and immediately occupies 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, repetition, deletion, or addition of one or more amino acids or nucleotides. In the present disclosure, the term "mutation" refers to a change in nucleotide sequence or amino acid sequence. In a specific embodiment, the term "mutation" refers to a "substitution".
In some embodiments, a "mutation" of the present disclosure may be selected from "conservative mutations". In the present disclosure, the term "conservative mutation" refers to a mutation that can normally maintain the function of a protein. Representative examples of conservative mutations are conservative substitutions.
As used in this disclosure, the term "conservative substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Amino acid residue families having similar side chains have been defined in the art and include those having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branches (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).
As used in this disclosure, "conservative substitutions" typically exchange one amino acid at one or more sites of a protein. Such substitutions may be conservative. As substitutions considered as conservative substitutions, there may be exemplified Ala to Ser or Thr substitutions, arg to Gln, his or Lys substitutions, asn to Glu, gln, lys, his or Asp substitutions, asp to Asn, glu or Gln substitutions, cys to Ser or Ala substitutions, gln to Asn, glu, lys, his, asp or Arg substitutions, glu to Gly, asn, gln, lys or Asp substitutions, gly to Pro substitutions, his to Asn, lys, gln, arg or Tyr substitutions, ile to Leu, met, val or Phe substitutions, leu to Ile, met, val or Phe substitutions, lys to Asn, glu, gln, his or Arg substitutions, met to Ile, leu, val or Phe substitutions, phe to Trp, tyr, met, ile or Leu substitutions, ser to Thr or Ala substitutions, thr to Ser or Ala substitutions, trp to Phe or Tyr substitutions, tyr to His, phe or Trp substitutions, and Val to Met, ile or Leu substitutions. In addition, conservative mutations include naturally occurring mutations resulting from individual differences, strains, species differences, and the like from which the gene is derived.
As used in this disclosure, the term "sequence identity" or "percent identity" in the comparison of two nucleic acids or polypeptides refers to that they are identical or have a specified percentage of identical sequences when compared and aligned for maximum correspondence using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection. That is, the identity of nucleotide or amino acid sequences can be defined by a ratio of the number of identical nucleotides or amino acids in the aligned part to the total number of nucleotides or amino acids in such a manner that two or more nucleotide or amino acid sequences are maximized and gaps are added as needed to align the identical numbers of nucleotides or amino acids.
Methods of determining "sequence identity" or "percent identity" to which the present disclosure relates include, but are not limited to: computer molecular biology (Computational Molecular Biology), lesk, a.m. editions, oxford university press, new york, 1988; biological calculation: informatics and genome project (Biocomputing: informatics and Genome Projects), smith, d.w. editions, academic press, new york, 1993; computer analysis of sequence data (Computer Analysis of Sequence Data), first part, griffin, a.m. and Griffin, h.g. editions, humana Press, new jersey, 1994; sequence analysis in molecular biology (Sequence Analysis in Molecular Biology), von Heinje, g., academic Press, 1987 and sequence analysis primer (Sequence Analysis Primer), gribskov, m. and deveverux, j. Code M Stockton Press, new york, 1991 and carllo, 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: GCG package (Devereux, J. Et al, 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. Et al, 1990). BLASTX programs are available to the public from NCBI and other sources (BLAST handbook, 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 judgment/calculation of "sequence identity" or "percent identity" may be based on any suitable region of the sequence. For example, a region of at least about 50 residues in length, 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 over the entire length of either or both of the compared biopolymers (i.e., nucleic acids or polypeptides).
In some embodiments, a polypeptide of the disclosure having tagatose 6-phosphate epimerase activity comprises a sequence that is identical to SEQ ID NO:1-4 has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid residues.
In still other embodiments, a polynucleotide of the present disclosure encoding a polypeptide having tagatose 6-phosphate phosphatase activity comprises a nucleotide sequence that hybridizes to a nucleotide sequence encoding SEQ ID NO:5-7 has a "sequence identity" or "percent identity" of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide.
As used in this disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. Polynucleotides may be in the form of individual fragments or may be an integral part of a larger nucleotide sequence structure, derived from nucleotide sequences that are separated at least once in number or concentration, and capable of identifying, manipulating and recovering sequences and their constituent nucleotide sequences by standard molecular biological methods (e.g., using cloning vectors). 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, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (individual fragments or whole fragments), or may be a component or constituent of a larger nucleotide structure, such as an 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 exist 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, which is at least partially removed from one or more or all of the naturally occurring components with which it is essentially associated; (3) Any substance that is artificially modified with respect to a naturally occurring substance; or (4) any agent modified by increasing the amount of the agent relative to other components naturally associated therewith (e.g., recombinant production in a host cell; multiple copies of a gene encoding the agent; and use of a stronger promoter than the promoter naturally associated with the gene encoding the agent). The isolated material may be present in a fermentation broth sample. For example, a host cell may be genetically modified to express a polypeptide of the disclosure. The fermentation broth from the host cell will comprise the isolated polypeptide. The "recombinant polynucleotide" belongs to one of the "polynucleotides".
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 may be included in a suitable vector, and the vector may be used for transformation into a suitable host cell. Host cells containing recombinant polynucleotides are referred to as "recombinant host cells". 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 involving 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 comprises a polynucleotide encoding a polypeptide and the polynucleotide is operably linked to control sequences for its expression.
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, vectors comprising i) a collection of genetic elements, such as promoters and enhancers, that have a regulatory effect on gene expression; ii) a structural or coding sequence transcribed into mRNA and translated into protein; and iii) transcriptional subunits of appropriate transcription and translation initiation and termination sequences. The recombinant expression vector is 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, nonchromosomal and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, chicken pox, baculovirus, SV40, and pseudorabies.
As used in this disclosure, the term "recombinant gene" is a gene that does not occur in nature. The recombinant gene is artificial. The recombinant gene includes a protein coding sequence operably linked to an expression control sequence. Embodiments include, but are not limited to, exogenous genes introduced into a microorganism, endogenous protein coding sequences operably linked to a heterologous promoter, and genes having modified protein coding sequences. The recombinant gene is stored on the genome of the microorganism, a plasmid in the microorganism or a phage in the microorganism.
The term "host cell" in the present disclosure means any cell type that is readily transformed, transfected, transduced, or the like with a polynucleotide or recombinant expression vector comprising a mutant polypeptide, encoding a mutant polypeptide of the present disclosure. The term "recombinant host cell" encompasses host cells which differ from the parent cell upon introduction of a polynucleotide encoding a mutant polypeptide or recombinant expression vector, in particular by transformation. The host cell of the present disclosure may be a prokaryotic cell or eukaryotic cell, as long as it is a cell into which a polynucleotide encoding a tagatose 6-phosphate epimerase or tagatose 6-phosphate phosphatase activity of the present disclosure, a recombinant polypeptide, can be introduced.
The term "transformation, transfection, transduction" in the present disclosure has the meaning commonly understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The transformation, transfection, transduction methods include any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate (CaPO) 4 ) Precipitation method, calcium chloride (CaCl) 2 ) Precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposome, and lithium acetate-DMSO.
The cultivation of the host cells of the present disclosure may be performed according to conventional methods in the art, including, but not limited to, well plate cultivation, shake flask cultivation, batch cultivation, continuous cultivation, fed-batch cultivation, and the like, and various cultivation conditions such as temperature, time, and pH value of the medium, and the like, may be appropriately adjusted according to the actual situation.
As used in this disclosure, the term "high stringency conditions" refers to prehybridization and hybridization in 5X SSPE (saline sodium phosphate EDTA), 0.3% sds, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42 ℃ for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. Finally, the carrier material was washed three times, 15 minutes each, with 2 XSSC, 0.2% SDS at 65 ℃.
As used in this disclosure, the term "very high stringency conditions" refers to prehybridization and hybridization in 5X SSPE (saline sodium phosphate EDTA), 0.3% sds, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42 ℃ for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. Finally, the carrier material was washed three times, 15 minutes each, with 2 XSSC, 0.2% SDS at 70 ℃.
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.
Examples
The present disclosure is further described below in conjunction with specific embodiments, and advantages and features of the present disclosure will become apparent as the description proceeds. It should be understood that the embodiments described are exemplary only and are not intended to limit the scope of the present disclosure in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the disclosed technical solution without departing from the spirit and scope of the disclosure, but these changes and substitutions fall within the scope of the disclosure.
Example 1: tagatose 6-phosphate epimerase (TPE) with better specificity and activity
A series of genes possibly having tagatose 6-phosphate epimerase activity were synthesized into pET20b vector by means of gene synthesis. The expression plasmid containing the target gene is transformed into escherichia coli BL21 (DE 3), and the bacterial cells are obtained by shake flask fermentation. And centrifugally collecting thalli, homogenizing and crushing at high pressure, and carrying out affinity chromatography by using nickel filler to obtain pure enzyme. SDS-PAGE electrophoresis detects the purity of the enzyme. The Bradford method determines protein concentration.
First, it was examined whether TPEs had tagatose 6-phosphate epimeric activity. The activity of various TPEs was measured at 50℃and the reaction system contained 10mM fructose 6-phosphate (F6P), 100mM HEPES buffer, 5mM MgSO4,0.2g/L tagatose 6-phosphatase (derived from Archaeoglobus fulgidus, uniprot ID: O29805) and an appropriate amount of TPE. The reaction was stopped in an ice bath. The activity of TPEs was characterized by measuring the amount of free phosphorus produced (anal. Chem.1956,28, 1756-1759).
The results of the assay are shown in Table 1, and the TPEs all have tagatose 6-phosphate epimeric activity. Enzyme activities of Uniprot ID A0A7V2B2J0, A0A3M1DFN1, A0A7C4PIG5 and A0A7J2U4S4 were higher than those of the control group.
Second, TPEs monosaccharide epimerization activity was verified. The activity of the various TPEs was measured at 50℃and the reaction system contained 50g/L tagatose, 100mM HEPES buffer, 5mM MgSO4,1g/L TPE. The reaction was terminated in a boiling water bath for 5 minutes. The formation of fructose was detected by HPLC to characterize the enzyme activity. The liquid chromatographic column is waters sugam-Pak 1, the column temperature is 80 ℃, the flow rate is 0.5mL/min, and the detector is a differential refraction detector.
As shown in Table 1, the Uniprot ID was A0A3B0UCF1, A0A7V2B2J0, A0A3M1DFN1, A0A7C4PIG5, A0A497GDS3, A0A7J2U4S4, A0A7J3I828, A0A7C5XKK1 and A0A7C2V281 enzymes were not active in catalyzing interconversion of tagatose and fructose.
TABLE 1 verification of TPEs enzymatic Activity
Example 2: tagatose 6-phosphate phosphatase (TPPs) with higher activity and specificity
A series of genes possibly having tagatose 6-phosphatase activity were synthesized into pET20b vector by means of gene synthesis. The expression plasmid containing the target gene is transformed into escherichia coli BL21 (DE 3), and the bacterial cells are obtained by shake flask fermentation. And centrifugally collecting thalli, homogenizing and crushing at high pressure, and carrying out affinity chromatography by using nickel filler to obtain pure enzyme. SDS-PAGE electrophoresis detects the purity of the enzyme. The Bradford method determines protein concentration.
TPPs were assayed for tagatose 6-phosphate (T6P) activity. The activity of the various TPPs was measured at 50℃and the reaction system contained 10mM fructose 6-phosphate, 100mM HEPES buffer, 5mM MgSO4,0.5g/L TPE (Uniprot number: A0A7J2U4S 4) and an appropriate amount of TPP. The reaction was stopped in an ice bath. The activity of TPPs was characterized by measuring the amount of free phosphorus produced (Anal. Chem.1956,28, 1756-1759).
The results of the detection are shown in Table 2, and the TPPs have tagatose 6-phosphatase activity. The activities of enzymes with Uniprot ID of D1A2R1, G0GB57 and A3DJZ0 on T6P were 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 was carried out at 50℃and the reaction system contained 10mM G1P or G6P or F6P,100mM HEPES buffer, 5mM MgSO4, and an appropriate amount of TPP. The reaction was stopped in an ice bath. The activity of TPPs was characterized by measuring the amount of free phosphorus produced (Anal. Chem.1956,28, 1756-1759).
As a result, the enzymes with Uniprot ID of O29805, D1A2R1, G0GB57 and A3DJZ0 were all inactive against G1P and were only weakly active against G6P and F6P. Characterization of TPPs specificity by ratio of TPPs activity on T6P to TPPs activity on the sum of F6P and G6P activities (i.e., specificity = SP T6P/ (SP G6P +SP F6P )). As shown in Table 2, the A3DJZ0 has 159 times higher enzyme activity and obviously better specificity than the control group.
TABLE 2 enzyme Activity validation of TPPs
TPEs(Uniprot ID) Relative Activity against T6P% Specificity (specificity)
Control group (O29805) 100 9.7
D1A2R1 1426 1.4
G0GB57 3544 8.3
A3DJZ0 15941 12.6
Example 3: production of tagatose from fructose 6-phosphate
F6P was converted to tagatose by an in vitro multienzyme catalytic system (FIG. 1). These key enzymes include: (1) Tagatose 6-phosphate epimerase (TPE), which catalyzes the production of T6P from F6P; (2) Tagatose 6-phosphate phosphatase (TPP), which catalyzes the dephosphorization of T6P to produce tagatose and inorganic phosphorus.
In this example, tagatose 6-phosphate epimerase was selected from A0A7J2U4S4 derived from Ignisphaera aggregans, and tagatose 6-phosphate phosphatase was selected from A3DJZ0 derived from Hungateiclostridium thermocellum. These plasmids were all transformed into E.coli expression strain BL21 (DE 3) (Invitrogen, carlsbad, calif.) and protein expression and purification were performed.
A3-mL reaction system contains 100mM HEPES buffer (pH 7.0), 5mM divalent magnesium ion, 50mM F6P, the amount of tagatose 6-phosphate epimerase is 2U/mL, the amount of tagatose 6-phosphate phosphatase is 1U/mL, and the reaction is catalyzed at 60 ℃ for 1 hour. After the reaction was completed, the yield of tagatose was measured by HPLC. HPLC detection conditions were the same as in example 1. As a result, the yield of tagatose was 8.8g/L, and the yield of tagatose was 97.8%.
Notably, the TPEs described in this disclosure, including enzymes Uniprot ID A0A3B0UCF1, A0A7V2B2J0, A0A3M1DFN1, A0A7C4PIG5, A0a497GDS3, A0A7J2U4S4, A0A7J3I828, A0A7C5XKK1, and A0A7C2V281, were used in combination with tagatose 6-phosphate phosphatase A3DJZ0 derived from Hungateiclostridium thermocellum to perform the above reactions, with yields of tagatose all exceeding 95%.
The TPE disclosed in the disclosure comprises enzymes with Uniprot ID of A0A3B0UCF1, A0A7V2B2J0, A0A3M1DFN1, A0A7C4PIG5, A0A497GDS3, A0A7J2U4S4, A0A7J3I828, A0A7C5XKK1 and A0A7C2V281 and tagatose 6-phosphatase G0GB57 from Spirochaeta thermophila which are matched for the above reaction, and the yield of tagatose is more than 90%.
Example 4: tagatose production by using starch as substrate
Starch was converted to tagatose by an in vitro multienzyme catalytic system (fig. 2). These key enzymes include: (1) Alpha-glucan phosphorylase, which releases 1-phosphoglucose from the non-reducing end of starch by adding 1 phosphate; (2) Glucose phosphomutase catalyzes the conversion of 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 converts fructose 6-phosphate into tagatose 6-phosphate; (5) Tagatose 6-phosphate phosphatase dephosphorizes tagatose 6-phosphate to tagatose and phosphoric acid.
In this example, the α -glucan phosphorylase is derived from Thermotoga maritima and the gene is numbered TM1168 on KEGG; the glucose phosphate mutase is derived from Thermococcus kodakarensis, and the enzyme is numbered as Q68BJ6 in the Uniprot database; glucose phosphate isomerase is derived from Thermus thermophilus, and the enzyme is numbered Q5SLL6 in Uniprot database; tagatose 6-phosphate epimerase was from Ignisphaera aggregans, enzyme number A0A7J2U4S4 in Uniprot database; tagatose 6-phosphate phosphatase was derived from Hungateiclostridium thermocellum, enzyme number A3DJZ0 in Uniprot database. The genes corresponding to these enzymes were cloned into pET20b vector, transformed into E.coli expression strain BL21 (DE 3) (Invitrogen, carlsbad, calif.), and protein expressed and purified.
A3 mL reaction system contains 30mM phosphate buffer (pH 7.0), 5mM divalent magnesium ion, the dosage of the alpha-glucan phosphorylase is 1U/mL, the dosage of the glucose phosphomutase is 1U/mL, the dosage of the glucose phosphoisomerase is 1U/mL, the dosage of the tagatose 6-phosphate epimerase is 1U/mL, the dosage of the tagatose 6-phosphate phosphatase is 1U/mL,10g/L of soluble starch is subjected to catalytic reaction at 55 ℃ for 24 hours. After the reaction, the final concentration of tagatose was determined to be 4.4g/L by HPLC, and the yield of tagatose to starch was 44%.
Because starch is branched, the mere use of α -glucan phosphorylase does not completely hydrolyze starch, because α -glucan phosphorylase acts only on α -1,4 glycosidic linkages, and the branched chains are linked to the main chain by α -1,6 glycosidic linkages. This requires the addition of an isoamylase to hydrolyze the alpha-1, 6 glycosidic bond. Finally, the final products of starch hydrolysis by these two enzymes are maltose and glucose, and in order to convert these final products into tagatose, it is also necessary to add 4-glucanotransferase and polyglucose kinase, 4-glucanotransferase being able to condense short-chain maltopolysaccharides to long-chain maltopolysaccharides and release one molecule of glucose. The polyphosphate glucokinase catalyzes the production of glucose 6-phosphate from polyphosphate and glucose. In this example, the starch debranching enzyme is derived from Sulfolobus tokodaii, the enzyme number in Uniprot database is Q973H3; polyphosphate glucokinase is derived from Thermobifida fusca, and the enzyme is numbered Q47NX5 in Uniprot database; the 4-glucanotransferase is derived from Thermococcus litoralis and the enzyme is numbered O32462 in the Uniprot database. The genes corresponding to these enzymes were cloned into pET20b vector, transformed into E.coli expression strain BL21 (DE 3) (Invitrogen, carlsbad, calif.), and protein expressed and purified.
A3 mL reaction system contains 30mM phosphate buffer (pH 7.0), 5mM divalent magnesium ion, 10U/mL of alpha-glucan phosphorylase, 10U/mL of glucose phosphomutase, 10U/mL of glucose phosphoisomerase, 10U/mL of tagatose 6-phosphate epimerase, 10U/mL of tagatose 6-phosphate phosphatase, 100g/L of IA pretreated soluble starch (40 mM sodium acetate buffer (pH 5.5), 5mM divalent magnesium ion, S/E=3000:1, incubation at 80 ℃ for 6 hours) and 5U/mL of polyglucose kinase, 5U/mL of glucan transferase, 50mM sodium polyphosphate after a catalytic reaction at 55 ℃ for 12 hours. After the reaction is finished, the final concentration of tagatose is 85g/L by adopting HPLC, and the yield of tagatose to starch is 85%.
Example 5: tagatose production using cellulose as substrate
A schematic representation of the conversion of cellulose to tagatose by an in vitro multienzyme catalytic system is shown in FIG. 2.
In this example, the cellulase is a product from Sigma company under the product number C2730. Cellodextrin phosphorylase source Hungateiclostridium thermocellum, enzyme number A3DJQ in Uniprot database; cellobiose phosphorylase was derived from Hungateiclostridium thermocellum, enzyme number A3DC35 in Uniprot database; the glucose phosphate mutase is derived from Pyrococcus furiosus, and the enzyme is numbered as Q8U383 in the Uniprot database; glucose phosphate isomerase is derived from Hungateiclostridium thermocellum and is numbered A3DBX9 in Uniprot database; tagatose 6-phosphate epimerase was from Ignisphaera aggregans, enzyme number A0A7J2U4S4 in Uniprot database; tagatose 6-phosphate phosphatase is derived from Hungateiclostridium thermocellum, and the enzyme is numbered A3DJZ0 in the Uniprot database; polyphosphate glucokinase is derived from Thermobifida fusca and the enzyme is numbered Q47NX5 in Uniprot database. The genes corresponding to these enzymes were cloned into pET20b vector, transformed into E.coli expression strain BL21 (DE 3) (Invitrogen, carlsbad, calif.), and protein expressed and purified.
This experiment uses microcrystalline cellulose (Avicel) as a substrate. Commercial cellulase (10U/ml) and cellulose (100 g/L) were first mixed on an ice-water bath, placed in the ice-water bath for 5 minutes, centrifuged at 4℃and the supernatant removed. The precipitate is a mixture of cellulose and cellulase capable of binding to cellulose. This treatment allows removal of almost all the glucosidase in commercial cellulases, thus avoiding the hydrolysis of cellobiose by glucosidase to produce large amounts of glucose, thus making the major hydrolysis products cellobiose and cellopolysaccharide.
A3 mL reaction system contains 30mM phosphate buffer (pH 7.2), 5mM divalent magnesium ion, 10U/mL cellopolysaccharide phosphorylase, 50U/mL cellobiose phosphorylase, 10U/mL glucose phosphomutase, 10U/mL glucose phosphoisomerase, 10U/mL tagatose 6-phosphate epimerase, 10U/mL tagatose 6-phosphate phosphatase, 100g/L of a mixture of cellulose and cellulase as described above, 10U/mL polyphosphate glucokinase, 50mM polyphosphate, and the reaction is catalyzed at 50℃for 72 hours. After the reaction, the final concentration of tagatose is 20g/L by HPLC, and the yield of tagatose to cellulose is 20%.
Example 6: production of tagatose using maltose as substrate
A schematic representation of the conversion of maltose to tagatose by an in vitro multienzyme catalytic system is shown in FIG. 2.
The maltose phosphorylase is derived from Bacillus sp.RK-1, and the gene thereof is numbered AB084460.1 on Genebank. Beta-glucose phosphomutase is derived from Pyrococcus horikoshii OT3 and the enzyme is numbered O58510 in the Uniprot database. Glucose phosphate isomerase is derived from Hungateiclostridium thermocellum and is numbered A3DBX9 in Uniprot database; tagatose 6-phosphate epimerase was from Ignisphaera aggregans, enzyme number A0A7J2U4S4 in Uniprot database; tagatose 6-phosphate phosphatase is derived from Hungateiclostridium thermocellum, and the enzyme is numbered A3DJZ0 in the Uniprot database; polyphosphate glucokinase is derived from Thermobifida fusca and the enzyme is numbered Q47NX5 in Uniprot database. The genes corresponding to these enzymes were cloned into pET20b vector, transformed into E.coli expression strain BL21 (DE 3) (Invitrogen, carlsbad, calif.), and protein expressed and purified.
A3 mL reaction system contained 10mM phosphate buffer (pH 7.2), 10mM magnesium, 1U/mL maltose phosphorylase, 1U/mL beta-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, and reacted at 37℃for 24 hours. After the reaction is finished, the final concentration of tagatose is 6g/L by adopting HPLC, and the yield of tagatose to starch is 60%.
Example 7: tagatose production by using sucrose as substrate
A schematic representation of the conversion of sucrose to tagatose by an in vitro multienzyme catalytic system is shown in FIG. 2.
Sucrose phosphorylase was derived from Bifidobacterium adolescentis and the enzyme was numbered A0 zh6 in Uniprot database. Glucose isomerase was derived from Streptomyces murinus and the enzyme was numbered P37031 in Uniprot database. The sources of other enzymes were the same as in example 5.
A3 mL reaction system contained 10mM phosphate buffer (pH 7.2), 10mM magnesium ions, 1U/mL sucrose phosphorylase, 1U/mL glucose phosphomutase, 1U/mL glucose phosphoisomerase, 1U/mL tagatose 6-phosphate epimerase, 1U/mL tagatose 6-phosphate phosphatase, 10g/L sucrose, 1U/mL polyphosphate glucokinase, 10mM polyphosphate, 10U/mL glucose isomerase, and reacted at 37℃for 24 hours. After the reaction is finished, the final concentration of tagatose is 5.5g/L by adopting HPLC, and the yield of tagatose to starch is 55%.
Example 8: tagatose production by using fructose as substrate
A schematic representation of the conversion of sucrose to tagatose by an in vitro multienzyme catalytic system is shown in FIG. 2.
The enzyme source was the same as in example 7.
A3 mL reaction system contained 10mM phosphate buffer (pH 7.2), 10mM magnesium ions, 10U/mL glucose isomerase, 1U/mL glucose phosphate isomerase, 1U/mL tagatose 6-phosphate epimerase, 1U/mL tagatose 6-phosphate phosphatase, 10g/L fructose, 1U/mL polyphosphate glucokinase, 10mM polyphosphate, and reacted at 50℃for 24 hours. After the reaction is finished, the final concentration of tagatose is 6g/L by adopting HPLC, and the yield of tagatose to starch is 60%.
Example 9: expression of enzymes using Bacillus subtilis
Coli has many good characteristics as the most commonly used heterologous protein expression host, but has the problems of unavoidable immunogen, endotoxin, low secretion expression efficiency and the like, and limits the cost reduction and the convenience of operation of downstream processes. Bacillus subtilis (Bacillus subtilis) is used as a model strain of gram-positive bacteria, has many excellent characteristics of secretion expression, high heterologous expression level, safety and the like, is considered to be an ideal protein production strain, and is widely applied to the production of various enzyme proteins. Thus, bacillus subtilis is also used as an expression host for the protein in the invention.
Nucleotide sequences of tagatose 6-phosphate epimerase (TPE) and tagatose 6-phosphate phosphatase (TPPs) described in example 1 and example 2 were cloned into a 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 Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980,2020,19 (1)), and expression plasmids pWB980-A0A3B0UCF1, pWB980-A0A7V2B2J0, pWB980-A0A3M1DFN1, pWB980-A0A7C4PIG5, pWB980-A0A497GDS3, pWB980-A0A7J2U4S4, pWB980-A0A7J3I828, pWB980-A0A7C5XKK1, pWB980-A0A7C2V281, pWB980-D1A2R1, pWB 980-A0B 7G 5, pWB 980-A0B 3D 3. The expression plasmid is transformed into bacillus subtilis SCK23 and amplified and cultured by adopting an SR culture medium. And centrifugally collecting thalli, washing the thalli once by using normal saline, and re-suspending the thalli by using phosphate buffer solution to obtain cells expressing corresponding enzymes.
Similarly, the nucleotide sequences corresponding to isoamylase, alpha-glucan phosphorylase, glucose phosphomutase, glucose phosphoisomerase, maltose phosphorylase, beta-glucose phosphomutase, polyphosphoric glucokinase, cellodextrin phosphorylase, cellobiose phosphorylase, sucrose phosphorylase, glucose isomerase, 4-glucan transferase, alpha-amylase and beta-amylase are cloned into a vector pWB980 and transformed into bacillus subtilis SCK23 to obtain corresponding bacillus subtilis cells.
Example 10: tagatose production by whole cell catalytic starch
Will expressAlpha-glucan phosphorylase, thermostable glucose phosphomutase, thermostable glucose phosphoisomerase, thermostable 6-phosphate tagatose epimerase, thermostable 6-phosphate tagatose phosphatase and thermostable isoamylase bacillus subtilis cells or escherichia coli cells were resuspended to od600=200 in 50mM sodium phosphate buffer (pH 7.5). And (3) carrying out heat treatment on the resuspended thalli for 90min at 55 ℃ to obtain heat-treated whole cells. The enzyme source was the same as in example 4.
A3-mL reaction system containing 30mM phosphate buffer (pH 7.0), 5mM divalent magnesium ion, 100g/L soluble starch, and the above heat-treated alpha-glucan phosphorylase, thermostable glucose phosphomutase, thermostable glucose phosphoisomerase, thermostable 6-phosphate tagatose epimerase, thermostable 6-phosphate tagatose phosphatase and thermostable isoamylase were added to the reaction system in amounts of 10U/mL, 2U/mL, respectively, and catalytic reaction was carried out at 55℃for 24 hours. After the reaction is finished, the final concentration of tagatose is 70g/L by adopting HPLC, and the yield of tagatose to starch is 70%.
Example 11: tagatose produced by immobilized enzyme
Fermenting to obtain whole cells expressing heat-resistant alpha-glucan phosphorylase, whole cells expressing heat-resistant glucose phosphomutase, whole cells expressing heat-resistant glucose phosphoisomerase, whole cells expressing heat-resistant 6-phosphate tagatose epimerase, whole cells expressing heat-resistant 6-phosphate tagatose phosphatase and whole cells expressing heat-resistant isoamylase. 50mM sodium phosphate buffer (pH 7.5) was added to each of the above cells, and the cells were resuspended to OD600 = 200. The resuspended cells were heat treated at 55℃for 90min. The permeable whole cells were mixed with pH 7.0 sodium phosphate buffer at an enzyme activity ratio of 1:1:1:1 so that OD600 = 100, 5% w/v celite was added to the bacterial suspension and stirred well. Subsequently, an aqueous solution of polyethylenimine of molecular weight 70000 was added at 0.5% w/v and flocculated at room temperature. Then, 0.5% v/v glutaraldehyde aqueous solution was added to crosslink for 2 hours at room temperature. Vacuum filtering to obtain filter cake, and extruding and granulating; the obtained immobilized cell particles are dried at 30 ℃ 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.
A3 mL reaction system contains 30mM phosphate buffer (pH 7.0), 5mM divalent magnesium ion, the amount of alpha-glucan phosphorylase immobilized enzyme particles is 10U/mL, the amount of glucose phosphomutase immobilized enzyme particles is 10U/mL, the amount of glucose phosphoisomerase immobilized enzyme particles is 10U/mL, the amount of tagatose 6-phosphate epimerase immobilized enzyme particles is 10U/mL, the amount of tagatose 6-phosphate phosphatase immobilized enzyme particles is 10U/mL, the amount of isoamylase immobilized enzyme particles is 2U/mL, and the catalytic reaction is performed at 55 ℃ for 24 hours. After the reaction is finished, the final concentration of tagatose is 70g/L by adopting HPLC, and the yield of tagatose to starch is 70%.
And removing the supernatant, adding fresh reaction liquid into enzyme particles to carry out batch reaction, and continuously catalyzing 60 batches, wherein the product yield is more than 50%.
All technical features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
Furthermore, from the foregoing description, it will be apparent to those skilled in the art from this disclosure that many modifications may be made to the invention without departing from the spirit or scope of the disclosure, and it is therefore intended that such modifications be within the scope of the appended claims.
SEQUENCE LISTING
<110> institute of Tianjin Industrial biotechnology, national academy of sciences
<120> enzyme for producing tagatose, composition thereof and use thereof
<130> 6A17-2243109IP
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 421
<212> PRT
<213> Rhodothermus marinus
<400> 1
Met Gly Glu Gln Val Pro His Gly Glu Ala Leu Val Arg Ala Leu Arg
1 5 10 15
Ala Arg His Arg Ser Phe Val Asp Trp Val Val Glu Leu Leu Arg Gly
20 25 30
Pro Leu Ala Tyr Ala His Thr Leu Leu Ala Val Cys Pro Asn Ser Val
35 40 45
Ala Val Val Glu Ala Ala Leu Glu Ala Ala Ala Glu Ala Asn Ala Pro
50 55 60
Leu Leu Phe Ala Ala Thr Leu Asn Gln Val Asp Leu Asp Gly Gly Tyr
65 70 75 80
Thr Gly Trp Thr Pro His Thr Leu Ala Gln Phe Val Ala Gln Lys Arg
85 90 95
Gln His Leu Ala Leu Asp Leu Pro Val Val Leu Gly Leu Asp His Gly
100 105 110
Gly Pro Trp Lys Lys Asp Val His Ala Arg Asp Arg Leu Ser Phe Gln
115 120 125
Glu Thr Phe Arg Ser Val Leu Arg Thr Ile Glu Ala Cys Leu Asp Ala
130 135 140
Gly Tyr Gly Leu Leu His Leu Asp Pro Thr Val Asp Leu Glu Ala Ser
145 150 155 160
Pro Gly Ser Pro Ile Pro Ile Asp Arg Ile Val Glu Arg Thr Val Ala
165 170 175
Leu Leu Gln His Ala Glu Ser Tyr Arg Gln Ala Arg Lys Leu Pro Pro
180 185 190
Val Ala Tyr Glu Val Gly Thr Glu Glu Val Gly Gly Gly Leu Glu Ala
195 200 205
Glu Thr Arg Met Ala Ala Phe Leu Asp Gln Leu Trp Lys Ala Leu Asp
210 215 220
Arg Glu Gly Leu Pro His Pro Val Phe Val Val Gly Asp Ile Gly Thr
225 230 235 240
Gln Leu Asp Thr Ser Asp Phe Asp Phe Lys Arg Ala Gln Arg Leu Asp
245 250 255
Ala Leu Val Arg Arg Tyr Gly Ala Leu Ile Lys Gly His Tyr Thr Asp
260 265 270
Gly Val Thr Arg Leu Glu Leu Tyr Pro Lys Ala Gly Ile Gly Gly Ala
275 280 285
Asn Val Gly Pro Gly Leu Ala Ala Val Glu Phe Glu Ala Leu Glu Thr
290 295 300
Leu Ile His Glu Ala Arg His Arg Gly Leu Pro Val Thr Leu Asp Ile
305 310 315 320
Ala Leu Lys Gln Ala Val Val Glu Ser Gly Arg Trp Lys Lys Trp Leu
325 330 335
Gln Pro Glu Glu Lys Asp Leu Pro Phe Glu Ala Leu Ser Pro Glu Arg
340 345 350
Gln Arg Trp Leu Val Ala Thr Gly Ser Arg Tyr Val Trp Thr His Pro
355 360 365
Ala Val Gln Ala Ala Arg Arg Gln Leu Tyr Ala Met Leu Ala Pro Trp
370 375 380
Ile Asp Ala Gln Ala Tyr Val Arg Asp Arg Leu Lys Ala Tyr Leu Gln
385 390 395 400
Lys Tyr Phe His Ala Phe Asn Leu Ile Gly Phe Asn Glu Arg Leu Arg
405 410 415
Ala Leu Trp Pro Gly
420
<210> 2
<211> 428
<212> PRT
<213> Thermoflexia bacterium
<400> 2
Met Tyr Met Arg Gly Asn Tyr Leu Asp Phe Val Val Ala Ala His Lys
1 5 10 15
Phe Gly Val Pro Phe Gly Ile Ala Ser Ile Cys Ser Ala His Pro Leu
20 25 30
Val Leu Glu Ala Ala Leu Arg His Gly Leu Val His Gly Met Pro Val
35 40 45
Leu Ile Glu Ala Thr Cys Asn Gln Val Asn Gln Phe Gly Gly Tyr Thr
50 55 60
Gly Met Thr Pro Met Asp Phe Ala Arg Gln Val Met Glu Gln Ala Glu
65 70 75 80
Arg Val Gly Phe Pro Gln Glu Arg Leu Ile Leu Gly Gly Asp His Leu
85 90 95
Gly Pro Leu Pro Trp Ala His Glu Pro Ala Glu Glu Ala Met Gln Lys
100 105 110
Ala Ser Asp Leu Val Arg Ala Phe Val Gln Ala Gly Cys Thr Lys Ile
115 120 125
His Leu Asp Cys Ser Met Pro Leu Gly Gly Glu Thr Val Leu Pro Val
130 135 140
Glu Val Ile Ala Gln Arg Val Ala Arg Leu Ala Gln Val Ala Glu Glu
145 150 155 160
Ala Ala Gly Glu Arg Arg Gly Ala Leu Arg Tyr Val Ile Gly Ser Glu
165 170 175
Val Pro Pro Ala Gly Gly Ala Lys Ala Gly Glu Gly Pro Pro Ser Val
180 185 190
Thr Arg Pro Glu Asp Ala Ala Glu Ala Ile Glu Ala Thr His Arg Ala
195 200 205
Phe Arg Ala Leu Gly Leu Glu Glu Ala Trp Glu Arg Val Ile Ala Leu
210 215 220
Val Val Gln Pro Gly Val Glu Phe Gly Asp Glu Thr Ile His Glu Tyr
225 230 235 240
Asp Arg Ala Ala Ala Ala Pro Leu Val Arg Tyr Ile Glu Gly Val Pro
245 250 255
Gly Leu Val Tyr Glu Ala His Ser Thr Asp Tyr Gln Pro Leu Arg Ala
260 265 270
Leu Arg Ala Met Val Glu Asp His Phe Ala Ile Leu Lys Val Gly Pro
275 280 285
Ala Leu Thr Phe Ala Leu Arg Glu Ala Val Phe Ala Leu Ala Asp Ile
290 295 300
Glu Ala Ala Met Gly Leu Glu Pro Pro Ser Gly Ile Arg Glu Ala Phe
305 310 315 320
Glu Ala Ala Met Leu Ser Asn Pro Val His Trp Gln Arg Tyr Tyr Arg
325 330 335
Gly Asp Pro Met Ser Gln Lys Leu Ala Arg Gln Tyr Ser Leu Ser Asp
340 345 350
Arg Ile Arg Tyr Tyr Trp Thr Ala Pro Glu Val Gln Ala Ala Phe Ser
355 360 365
Arg Leu Met Arg Asn Leu Gly Asp Arg Pro Ile Pro Pro Gly Leu Leu
370 375 380
Ser Gln Tyr Met Pro Glu Glu Phe Arg Lys Val Arg Ala Gly Glu Leu
385 390 395 400
Lys Asn Arg Pro Asp Asp Leu Leu Leu Gly Arg Val Met Gly Val Leu
405 410 415
Glu Ala Tyr Arg Leu Ala Thr Gln Gly Val Pro Gly
420 425
<210> 3
<211> 405
<212> PRT
<213> Anaerolinea thermolimosa
<400> 3
Met Lys Pro Leu Lys Glu Val Val Arg Arg Leu Ile Glu Leu Arg Lys
1 5 10 15
Gln Gly Arg Lys Met Thr Leu Leu Ala Val Cys Pro Asn Ser Ser Ala
20 25 30
Val Leu Glu Ala Ala Val Gln Ser Ala Ala Leu His Arg Ser Val Met
35 40 45
Leu Phe Ala Ala Thr Leu Asn Gln Val Asp Arg Asp Gly Gly Tyr Thr
50 55 60
Gly Trp Thr Pro Glu Ser Phe Val Gln Glu Met Gln Arg Arg Ala Ala
65 70 75 80
Arg Ile Asn Trp Asn Gly Pro Leu Tyr Pro Cys Leu Asp His Gly Gly
85 90 95
Pro Trp Leu Lys Asp Asn Gln Ala Gln Phe Pro Tyr Ala Lys Thr Glu
100 105 110
Ala Glu Val Lys Glu Ser Leu Phe Ala Cys Leu His Ala Gly Tyr Ala
115 120 125
Leu Leu His Ile Asp Thr Thr Val Asp Arg Ser Leu Pro Pro Gly Met
130 135 140
Ala Pro Ala Ile Glu Val Val Val Asp Arg Ala Val Ser Leu Ile Gly
145 150 155 160
Ala Val Glu Lys Glu Arg Ile Glu His Gln Leu Pro Glu Ile Ala Tyr
165 170 175
Glu Val Gly Ser Asp Glu Val His Gly Gly Leu Val Glu Phe Asp Arg
180 185 190
Phe Arg Glu Phe Leu Ile Leu Leu Lys Glu Arg Leu Asp Arg Ala Gly
195 200 205
Leu Gly Glu Val Trp Pro Ala Phe Leu Val Thr Gln Val Gly Thr Asp
210 215 220
Leu His Thr Thr Arg Phe Asp Gly Glu Val Ala Lys Arg Leu Phe Asp
225 230 235 240
Leu Val Ser Pro Tyr Gly Ser Leu Ile Lys Gly His Tyr Thr Asp Trp
245 250 255
Val Glu Asn Pro Glu Met Tyr Pro Glu Thr Gly Met Gly Gly Ala Asn
260 265 270
Val Gly Pro Glu Phe Thr Thr Val Glu Tyr Leu Ala Leu Lys Glu Leu
275 280 285
Cys Ala Arg Glu Ser Glu Leu Leu Ala Gly Asn Pro Gly Lys Ala Ser
290 295 300
Glu Phe Leu Trp His Leu Glu His Ala Val Leu Asp Ser Gly Arg Trp
305 310 315 320
Lys Lys Trp Leu Phe Pro Glu Glu Arg Gly Leu Pro Phe Glu Glu Leu
325 330 335
Ser Lys Glu Arg Arg Glu Trp Leu Thr Gln Thr Gly Ala Arg Tyr Val
340 345 350
Trp Ser Gln Pro Val Val Arg Gln Ala Arg Ile Arg Leu Tyr Glu Asn
355 360 365
Leu Arg Gly Val Ile Ser Asp Pro His Ala Trp Val Val Arg Lys Ile
370 375 380
Gln Gln Ala Ile Asp Arg Tyr Ile Glu Ala Phe His Leu Thr Asp Ser
385 390 395 400
Ala Ser Leu Phe Glu
405
<210> 4
<211> 392
<212> PRT
<213> Ignisphaera aggregans
<400> 4
Met Ile Gly Val Arg Ser Thr Leu Asp Lys Ala Cys Lys Leu Pro Gly
1 5 10 15
Lys Ala Thr Leu Leu Cys Leu Ser Pro Ile Ser Arg His Val Val Gln
20 25 30
Ala Tyr Ile Lys Ile Ala Lys Glu Phe Asn Thr Pro Ile Cys Phe Ala
35 40 45
Thr Ser Leu Asn Gln Val Asp Arg Gly Gly Gly Tyr Thr Gly Trp Thr
50 55 60
Pro Ile Asp Phe Lys Asn Tyr Val Met Asp Met Ala Arg Glu Tyr Ser
65 70 75 80
Ile Ser Thr Pro Ile Ile Leu Gln Leu Asp His Gly Gly Pro Trp Leu
85 90 95
Lys Asp Glu His Ile Ala Lys Lys Tyr Ser Tyr Glu Glu Ala Leu Asn
100 105 110
Asp Phe Leu Lys Ser Leu Glu Leu Phe Ile Lys Ala Gly Phe Asp Val
115 120 125
Ile His Ile Asp Thr Thr Ile Asp Leu Asp Ser Arg Asp Gly Tyr Ala
130 135 140
Asp Val Glu Val Ala Ser Lys Arg Thr Ala Asp Leu Ile Met Tyr Ser
145 150 155 160
Glu Glu Ile Ala Ser Arg Tyr Gly Val Lys Leu Glu Tyr Glu Ile Gly
165 170 175
Ser Asp Arg Trp Gly Tyr Lys Pro Leu Glu Ile Val Glu Asn Phe Val
180 185 190
Ser Lys Ala Ile Ser Met Leu Arg Asp Arg Gly Phe Asp Ile Asn Arg
195 200 205
Leu Val Phe Gly Val Ala Asp Val Gly Thr Lys Val Cys Pro Gly Asn
210 215 220
Arg Val Asp Pro Val Ile Val Arg Glu Phe Ser Ser Leu Met Arg Arg
225 230 235 240
His Gly Leu Tyr Leu Lys Ile His Ser Gly Asp Tyr Leu Glu Asn Pro
245 250 255
Gly Glu Leu Pro Lys Asn Ser Val Gly Gly Val Asn Ile Gly Pro Met
260 265 270
Leu Ala His Ile Met Tyr Ser Thr Phe Lys Glu Ile Leu Tyr Glu Lys
275 280 285
Leu Asp Lys Asp Arg Ala Leu Glu Leu Leu Glu Glu Leu Asn Asn Phe
290 295 300
Ile Ala Ser Ser Asp Lys Leu Ala Lys Tyr Val Gly Lys Gly Leu Gly
305 310 315 320
Glu Ala Glu Glu Tyr Lys Leu Gly Leu Ala Ser Arg Tyr Ile Trp Ser
325 330 335
Thr Thr Lys Ala Lys Glu Phe Ile Asp Arg Ile Ser Lys Ile Ile Gly
340 345 350
Ile Asp Ile Glu Lys Leu Phe Ile Glu Lys Leu Ala Gln Thr Val Lys
355 360 365
Arg Tyr Val Ile Glu Leu Asn Ile Tyr Lys Leu Tyr Glu Thr Asn Lys
370 375 380
Lys His Ala Thr Leu Lys Gln Tyr
385 390
<210> 5
<211> 218
<212> PRT
<213> Thermomonospora curvata
<400> 5
Met Ala Gly Gly Gly Leu Gln Ala Val Leu Phe Asp Met Asp Gly Leu
1 5 10 15
Leu Ile Asp Ser Glu Pro Met Trp Leu Glu Val Glu Thr Glu Val Met
20 25 30
Ala Trp Leu Gly Gly Glu Trp Gly Pro Gln His Gln Gln Lys Leu Leu
35 40 45
Gly Gly Ser Val Thr Tyr Ala Ala His Tyr Met Leu Ser Leu Val Glu
50 55 60
Ala Thr Val Ala Pro Gln Glu Val Glu Arg Arg Leu Val Asp Gly Met
65 70 75 80
Ala Glu Arg Leu Ala Gly Ser Val Pro Leu Met Pro Gly Ala Lys Glu
85 90 95
Leu Leu Ala Glu Val Arg Ala Ala Gly Val Ala Thr Ala Leu Val Ser
100 105 110
Ser Ser Glu Arg Arg Leu Val Glu Ala Ala Leu Ala Gly Ile Gly Arg
115 120 125
Glu His Phe Asp Val Thr Val Ala Gly Asp Glu Val Ala Arg Arg Lys
130 135 140
Pro Asp Pro Glu Pro Tyr Leu Thr Ala Met Ala Arg Leu Gly Val Ser
145 150 155 160
Pro Gly Arg Cys Val Val Leu Glu Asp Ser Pro Thr Gly Leu Ala Ala
165 170 175
Ala Glu Ala Ala Gly Cys Val Thr Val Ala Val Pro Gly Val Val Pro
180 185 190
Val Pro Pro Ala Pro Gly Arg Thr Val Val Glu Ser Leu Arg Asn Val
195 200 205
Asp Leu Gln Met Leu Asn Gly Leu Leu Pro
210 215
<210> 6
<211> 237
<212> PRT
<213> Spirochaeta thermophila
<400> 6
Met Arg Met Arg Arg Glu Cys Ala Pro Pro Gly Ile Arg Ala Ala Ile
1 5 10 15
Phe Asp Met Asp Gly Thr Leu Val Asn Ser Glu Asp Val Tyr Trp Asp
20 25 30
Ala Asp Cys Ala Phe Leu Asp Arg Tyr Gly Ile Pro His Asp Asp Ala
35 40 45
Leu Arg Glu Tyr Met Ile Gly Arg Gly Thr Lys Gly Phe Ile Glu Trp
50 55 60
Met Arg Thr Gln Lys Glu Ile Pro Arg Ser Asp Glu Glu Leu Ala Arg
65 70 75 80
Glu Lys Ile Gly Leu Phe Leu Glu His Ala Arg Gly Arg Val Gln Val
85 90 95
Phe Pro Glu Met Arg Arg Leu Leu Gly Leu Phe Glu Glu Ala Gly Met
100 105 110
Ser Cys Ala Leu Ala Ser Gly Ser Pro Arg Arg Val Ile Glu Val Leu
115 120 125
Leu Glu Glu Thr Gly Leu Val Gly Phe Phe Arg Val Val Val Ser Ala
130 135 140
Asp Glu Val Ala Arg Pro Lys Pro Ala Pro Asp Val Phe Leu Glu Ala
145 150 155 160
Ala Gly Arg Leu Gly Val Glu Pro Gly Gly Cys Val Val Phe Glu Asp
165 170 175
Ser Glu Pro Gly Val Gln Ala Ala Leu Asp Ala Gly Met Val Cys Val
180 185 190
Ala Ile Pro Thr Leu Val Lys Asp Arg Tyr Pro Glu Val Phe Tyr Gln
195 200 205
Ala Asp Val Leu Phe Glu Gly Gly Met Gly Glu Phe Cys Ala Glu Arg
210 215 220
Val Trp Glu Trp Leu Gly Cys Gly Val Gly Val Arg Arg
225 230 235
<210> 7
<211> 223
<212> PRT
<213> Acetivibrio thermocellus
<400> 7
Met Lys Lys Val Lys Ala Val Ile Phe Asp Met Asp Gly Leu Met Ile
1 5 10 15
Asp Thr Glu Arg Leu Tyr Phe Glu Val Glu Arg Ile Met Ala Arg Lys
20 25 30
Phe Gly Lys Glu Val Lys Asp Glu Thr Leu Trp Lys Met Met Gly Arg
35 40 45
Lys Pro Leu Glu Ala Ile Thr Val Phe Ala Glu Asp Leu Glu Leu Asp
50 55 60
Ile Ser Pro Lys Lys Leu Leu Glu Ile Arg Asp Glu Leu Phe Val Lys
65 70 75 80
Lys Leu Val Asn Glu Val Glu Pro Met Pro Gly Leu Phe Asp Ile Leu
85 90 95
Asn Ile Leu Lys Gly Lys Val Lys Met Ala Ile Ala Thr Gly Ser Pro
100 105 110
Gln Lys Phe Leu Lys Ile Val Leu Asp Lys Leu Lys Ile Glu Ser Tyr
115 120 125
Phe Asp Val Phe Val Thr Ser Asp Glu Val Glu Lys Gly Lys Pro Asp
130 135 140
Pro Glu Val Tyr Asn Thr Ala Val Lys Arg Leu Lys Val Ala Pro Phe
145 150 155 160
Glu Cys Val Val Leu Glu Asp Ser Ser Asn Gly Ala Leu Ala Ala Val
165 170 175
Arg Ala Gly Cys Tyr Thr Ile Ala Val Pro Thr Val Tyr Thr Asn Lys
180 185 190
Gln Asp Phe Ser Phe Val Asn Tyr Val Ala Lys Asp Leu Lys Asp Ala
195 200 205
Ala Glu Lys Ile Asn Glu Phe Leu Cys Ser Gln Glu Ile Glu Phe
210 215 220

Claims (16)

1. Use of a polypeptide selected from any one of the following groups (i) - (iv) as tagatose 6-phosphate epimerase, wherein the polypeptide:
(i) Has the sequence shown in SEQ ID NO: 1-4;
(ii) Has at least 70% sequence identity to the sequence set forth in (i) and excludes the sequence set forth in SEQ ID NO: 1-4;
(iii) A polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with a polynucleotide as set forth in (a) or (b):
(a) A polynucleotide encoding a polypeptide of the amino acid sequence shown in (i);
(b) The full-length complementary polynucleotide of (a);
(iv) Fragments of the polypeptides shown by (i), (ii), (iii) and which still have tagatose 6-phosphate epimerase activity.
2. Use according to claim 1, wherein the tagatose 6-phosphate epimerase is derived from a thermostable microorganism; preferably, the thermostable microorganism is selected from Rhodothermus, anaerorinea, ignosphaera or thermostremia; 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 following groups (v) - (viii) as tagatose 6-phosphate phosphatase, wherein the polypeptide:
(v) Has the sequence shown in SEQ ID NO: 5-7;
(vi) Has at least 70% sequence identity to the sequence set forth in (v) and excludes the sequence set forth in SEQ ID NO: 5-7;
(vii) A polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with a polynucleotide as set forth in (a) or (b):
(a) A polynucleotide encoding a polypeptide of the amino acid sequence shown in (v);
(b) The full-length complementary polynucleotide of (a);
(viii) Fragments of the polypeptides shown by (v), (vi), (vii), and which still have tagatose 6-phosphate phosphatase activity.
4. The use according to claim 3, wherein the tagatose 6-phosphate phosphatase is derived from a thermostable microorganism; preferably, the heat-resistant microorganism is selected from the group consisting of thermospora, spiraeta or huntateiclotriostrichum; 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 comprises tagatose 6-phosphate epimerase and/or tagatose 6-phosphate phosphatase.
6. The enzyme composition according to claim 5, wherein the tagatose 6-phosphate epimerase is the polypeptide in the use according to any one of claims 1 to 2, and the tagatose 6-phosphate phosphatase is the polypeptide in the use according to any one of claims 3 to 4.
7. The enzyme composition according to any one of claims 5-6, wherein the enzyme composition further comprises one or more of the group consisting of: starch branching enzymes (including isoamylase and pullulanase), alpha-glucan phosphorylase, glucose phosphomutase, glucose phosphoisomerase, maltose phosphorylase, beta-glucose phosphomutase, polyphosphate glucokinase, cellodextrin phosphorylase, cellobiose phosphorylase, sucrose phosphorylase, glucose isomerase, 4-glucan transferase, alpha-amylase, beta-amylase.
8. A strain or strain composition expressing the enzyme composition of any one of claims 5-7.
9. The strain or strain composition according to claim 8, wherein the host cell of the strain or strain composition is derived from corynebacterium, brevibacterium, arthrobacter, microbacterium or escherichia; preferably, the host cell is Bacillus subtilis, corynebacterium glutamicum or Escherichia coli.
10. The strain or strain composition of any one of claims 8-9, wherein the strain or strain composition converts an expression vector of:
an expression vector comprising a nucleic acid encoding a tagatose 6-phosphate epimerase for use according to any one of claims 1 to 2; and/or
An expression vector comprising a nucleic acid encoding tagatose 6-phosphate phosphatase for use according to any one of claims 3 to 4.
11. The strain or strain composition of claim 10, wherein the strain or strain composition further comprises transformed therein an expression vector comprising a nucleic acid encoding a starch branching enzyme (including isoamylase and pullulanase), an alpha-glucan phosphorylase, a glucose phosphomutase, a glucose phosphoisomerase, a maltose phosphorylase, a beta-glucose phosphomutase, a polyphosphate glucokinase, a cellodextrin phosphorylase, a cellobiose phosphorylase, a sucrose phosphorylase, a glucose isomerase, a 4-glucan transferase, an alpha-amylase, or a beta-amylase.
12. Use of the enzyme composition of any one of claims 5-7 or the strain or strain composition of any one of claims 8-11 in the production of tagatose.
13. A process for producing tagatose, wherein the process comprises: a step of converting a substrate to tagatose by adding the enzyme composition of any one of claims 5 to 7 or inoculating the strain or strain composition of any one of claims 8 to 11;
optionally, the method further comprises the step of pre-treating the substrate; or (b)
And purifying or separating the tagatose.
14. The method of claim 13, wherein the method comprises the step of further adding a metal ion or metal salt to the reaction; preferably, the metal is selected from metals capable of forming divalent cations; more preferably, the metal is selected from one or more of the group consisting of magnesium, nickel, manganese, zinc, cobalt, iron, copper, calcium, molybdenum, selenium.
15. The method according to any one of claims 13-14, wherein the substrate is selected from saccharides or derivatives thereof; preferably, the fermentation substrate is selected from one or more of the group consisting of: starch or its derivative, cellulose or its derivative, fructose, glucose, sucrose, maltose.
16. The method according to any one of claims 13-15, wherein the method is selected from one or more of the group consisting of: multienzyme catalysis, whole cell catalysis, ferment catalysis containing enzymes/whole cells, immobilized multienzyme catalysis, immobilized whole cell catalysis.
CN202210730019.6A 2022-06-24 2022-06-24 Enzyme for preparing tagatose, composition and application thereof Pending CN117327684A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202210730019.6A CN117327684A (en) 2022-06-24 2022-06-24 Enzyme for preparing tagatose, composition and application thereof
PCT/CN2023/073198 WO2023246099A1 (en) 2022-06-24 2023-01-19 Enzyme for preparing tagatose, composition and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210730019.6A CN117327684A (en) 2022-06-24 2022-06-24 Enzyme for preparing tagatose, composition and application thereof

Publications (1)

Publication Number Publication Date
CN117327684A true CN117327684A (en) 2024-01-02

Family

ID=89276061

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210730019.6A Pending CN117327684A (en) 2022-06-24 2022-06-24 Enzyme for preparing tagatose, composition and application thereof

Country Status (2)

Country Link
CN (1) CN117327684A (en)
WO (1) WO2023246099A1 (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399427B (en) * 2016-11-01 2018-11-13 中国科学院天津工业生物技术研究所 The preparation method of Tagatose
CN109750024B (en) * 2017-11-02 2021-12-14 中国科学院天津工业生物技术研究所 6-tagatose phosphate 4-position epimerase and application thereof
KR102328650B1 (en) * 2018-09-27 2021-11-19 씨제이제일제당 주식회사 A Novel Fructose C4 epimerases and Preparation Method for producing Tagatose using the same

Also Published As

Publication number Publication date
WO2023246099A1 (en) 2023-12-28

Similar Documents

Publication Publication Date Title
CN111394330B (en) Cyclodextrin glucosyltransferase mutant T168A and preparation method thereof
CN111534503B (en) Application of Haloferrila sp
KR20190054187A (en) Enzymatic synthesis of d-tagatose
KR101521711B1 (en) Novel β-agarooligosaccharide hydrolase and enzymatic production method of 3,6-anhydro-L-galactose and galactose from agarose by using the same
KR20010081985A (en) Starch debranching enzymes
EP1661985A1 (en) Method of making sucrose phosphorylase(sp) heat-stable
JP7566993B2 (en) Motif for ribulose phosphate 3-epimerase with low side reactivity and enzyme containing same
WO2020126613A1 (en) Enzymatic hexosaminidation of lactose
WO1995034642A1 (en) Novel transferase and amylase, process for producing the enzymes,use thereof, and gene coding for the same
CN110157688B (en) Linear maltooligosaccharide-producing enzyme mutant with improved maltopentaose production capacity
CN110885809A (en) α -L-fucosidase and related biological material and application thereof
CN105647888B (en) Endo-chitinase, coding gene thereof and application of endo-chitinase in production of chitobiose
CN110229800B (en) Linear maltooligosaccharide-producing enzyme mutant with improved maltohexaose production capacity
CN116622747A (en) Gene for coding dextran sucrase and application thereof
CN114790469B (en) Method for enzymatic synthesis of psicose
CN107779443B (en) Cellobiohydrolase mutants and uses thereof
WO2023246099A1 (en) Enzyme for preparing tagatose, composition and use thereof
CN114231511B (en) Difructosan hydrolase mutant E160F with improved thermostability
TW201943854A (en) Enzymatic production of D-allulose
CN111534498B (en) Cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield
EP3359656A1 (en) Compositions and methods comprising the use of exiguobacterium acetylicum and bacillus coagluans alpha-glucanotransferase enzymes
JPWO2004113525A1 (en) Method for heat-resistant α-glucan phosphorylase (GP)
KR20210096084A (en) Enzymatic production of tagatose
CN118406672B (en) Chitosan CsnQB-E203A and application thereof
KR102300386B1 (en) Use of alpha-L-fucosidase having dual enzymatic activity for cleaving alpha- and beta-1,4-glycosidic linkages

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination