WO2017167255A1 - 一种3-差向异构酶以及编码它的多核苷酸 - Google Patents

一种3-差向异构酶以及编码它的多核苷酸 Download PDF

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WO2017167255A1
WO2017167255A1 PCT/CN2017/078923 CN2017078923W WO2017167255A1 WO 2017167255 A1 WO2017167255 A1 WO 2017167255A1 CN 2017078923 W CN2017078923 W CN 2017078923W WO 2017167255 A1 WO2017167255 A1 WO 2017167255A1
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polypeptide
protein
epimerase
tagatose
psicose
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English (en)
French (fr)
Inventor
王三永
李春荣
韩诗蕾
严明
魏淼
陈圣
章志林
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L&p Food Ingredient Co Ltd
Langnai Biotech Co Ltd
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L&p Food Ingredient Co Ltd
Langnai Biotech Co Ltd
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Priority to KR1020187028940A priority Critical patent/KR102068113B1/ko
Priority to US16/088,821 priority patent/US10781467B2/en
Priority to EP17773285.6A priority patent/EP3438256B1/en
Priority to MX2018011980A priority patent/MX2018011980A/es
Publication of WO2017167255A1 publication Critical patent/WO2017167255A1/zh
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    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/33—Artificial sweetening agents containing sugars or derivatives
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
    • 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)
    • C12Y501/03001—Ribulose-phosphate 3-epimerase (5.1.3.1)
    • 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)
    • C12Y501/03022—L-Ribulose-5-phosphate 3-epimerase (5.1.3.22)

Definitions

  • the present invention relates to a polypeptide or protein having 3-epimerase activity, a polynucleotide sequence encoding a polypeptide or protein having 3-epimerase activity, a nucleic acid construct or expression comprising the polynucleotide sequence A carrier, a method of producing the enzyme, and the use of the enzyme in various industrial applications.
  • the wood invention relates to a method for producing D-psicose by using a polypeptide or protein having 3-epimerase activity to catalyze a monosaccharide or a mixed sugar such as fructose, glucose, or starch sugar, and using the polypeptide or protein A method for producing D-tagatose by catalyzing a monosaccharide or a mixed sugar such as sorbose or starch sugar.
  • D-psicose and D-tagatose have become natural hotspots for natural low-calorie functional sweeteners that are naturally occurring in nature but have little content.
  • D-allulose also known as D-ribose-2-hexulose
  • D-psicose is the epimer of the C-3 position of D-fructose.
  • D-psicose is a naturally occurring but very low-calorie functional sweetener with a sweetness of 70% of sucrose but only 0.3% of sucrose, which can be used as a low-calorie diet food.
  • the sweetener at the same time, D-psicose also has the function of inhibiting the activity of enzymes related to lipid synthesis in the liver, helping to reduce abdominal fat accumulation and controlling body weight to some extent, and can be used for health foods and the like.
  • a variety of functional foods can also improve the flavor, appearance, etc. of food, and extend the shelf life of food. Therefore, D-psicose, a healthy and safe low-calorie functional sweetener, has attracted more and more scholar' attention and become one of the most competitive new sweeteners in the market.
  • D-psicose Since D-psicose is a relatively rare natural monosaccharide, it has low yield and high cost from the natural environment, and it is difficult to meet the needs of people as low-calorie healthy sweeteners, and is not suitable for industrialized large-scale production. Therefore, in order to be applied to the food industry, there is a need for an efficient D-psicose production process.
  • the traditional D-psicose production process is mainly chemical, which requires a lot of cost, many by-products and pollutants.
  • the preparation of D-psicose by biological method has the characteristics of strong reaction specificity, simple and easy purification of product components and natural products, which has become a research hotspot.
  • the most effective way to prepare D-psicose by biological methods is to find an enzyme that converts fructose into D-psicose.
  • the current psicose-3-differential isomerase enzyme has problems such as reduced stability at high temperature or low activity and low reaction rate, which is not conducive to the cost control of industrial production of D-psicose. Therefore, there is a need to develop a psicose-3-epimerase having excellent high temperature stability and high activity to meet the demand for industrial production.
  • D-Tagatose is the epimer of the C-3 position of D-sorbose.
  • D-tagatose is a natural deposit
  • the low-calorie functional sweetener with a very low content has a sweetness of 92% of sucrose and a low absorption rate of only 20% to 25% in the human body, which does not cause significant changes in the blood sugar level of the body. It is very suitable for diabetics, and most of the tagatose directly enters the colon, which is selectively fermented by the microbial flora, promotes the proliferation of beneficial bacteria, inhibits the growth of harmful bacteria, and plays a significant role in improving the intestinal flora. It is a good probiotic and can be used in a variety of functional foods such as health foods.
  • D-tagatose can also improve food flavor, appearance and the like. Therefore, D-tagatose, a healthy and safe low-calorie functional sweetener, has attracted more and more scholars' attention and become one of the most promising new sweeteners.
  • Isomerization of sorbose to D-tagatose is one of the most effective methods for the preparation of D-tagatose by biological methods.
  • the key to this method is to find an enzyme that converts sorbose into D-tagatose.
  • the current tagatose-3-differential isomerase has problems such as reduced stability at high temperature or low activity and low reaction rate, which is not conducive to cost control and scale-up of industrial production of D-tagatose. Therefore, it is necessary to develop a tagatose-3-diffusion isomerase having excellent high temperature stability and high activity to meet the demand for industrial production.
  • the present inventors discovered a novel protein having 3-epimerase activity from the Thermomemmatispora carboxidivorans strain. Accordingly, the present invention provides novel proteins having 3-epimerase activity, and polynucleotides encoding the proteins or polypeptides.
  • the protein or polypeptide has psicose-3-epimerase and tagatose-3-epimerase activity.
  • the protein or polypeptide of the present invention has good activity and excellent thermal stability, both of which are excellent properties for producing D-psicose and D-tagatose at higher temperature conditions.
  • the present invention relates to an isolated protein or polypeptide having psicose-3-epimerase and tagatose-3-epimerase activity selected from the group consisting of
  • the invention also relates to nucleic acid constructs, expression vectors and recombinant host cells comprising the polynucleotides: A method of producing a polynucleotide.
  • the invention also relates to a process for the enzymatic production of D-psicose and D-tagatose.
  • Ipsicose-3-epimerase activity means catalyzing the conversion of the C-3 position of fructose to D-psicose active.
  • Tagatose-3-epimerase activity means the activity of catalyzing the conversion of the C-3 position of sorbose to D-tagatose.
  • Isolated protein or polypeptide means a polypeptide that has been human modified with respect to a protein or polypeptide as found in nature. In one aspect, the polypeptide is at least 1% pure, as determined by SDS-PAGE,
  • Non-limiting examples of isolated proteins or polypeptides include: (1) any non-naturally occurring protein or polypeptide, (2) any protein or polypeptide including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or accessory. Factors that are at least partially removed from one or more or all of the naturally occurring components that are associated with them in nature; (3) any protein or polypeptide that has been artificially modified relative to a protein or polypeptide found in nature: or (4) Any protein or polypeptide that is modified by increasing the amount of the protein or polypeptide relative to other components with which it is naturally associated.
  • An isolated protein or polypeptide can be present in the fermentation broth sample.
  • substantially pure protein or polypeptide means a protein or polypeptide preparation containing up to 10% by weight of its natural or recombinant association. Other protein or polypeptide materials. Preferably, the protein or polypeptide is at least 90% pure by weight of all protein or polypeptide material present in the preparation.
  • the protein or polypeptide of the invention is preferably in a substantially pure form, for example, this can be achieved by preparing the polypeptide by well known recombinant methods or by classical purification methods.
  • Sequence identity The parameter "sequence identity" describes the correlation between two amino acid sequences or between two nucleotide sequences.
  • the degree of sequence identity between two amino acid sequences is achieved by using, for example, the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends in Genetics 16: 276-277) (preferred)
  • EMBOSS software package EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends in Genetics 16: 276-277
  • the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) performed in the Needle program of version 3.0.0 or higher) was determined.
  • the optional parameters used were a gap open penalty of 10, a gap extension penalty of 0.5 and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • Use Needle to mark "highest identity” "The output (obtained with the -nobrief option) as a percentage of identity and calculated as follows:
  • the degree of sequence identity between two nucleotide sequences is achieved by using, for example, the EMBOSS software package (EMBOSS: The European Molecular Bi010 Open Software Suite, Rice et al, supra) (preferably version 3.0.0 or more).
  • EMBOSS European Molecular Bi010 Open Software Suite, Rice et al, supra
  • the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), performed in the higher version of the Needle program, was determined.
  • the optional parameters used were gap open penalty of 10, gap extension penalty of 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • fragment means a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has 3-epimerase activity.
  • allelic variant means any two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs naturally by mutation and can result in polymorphism within the population. A genetic mutation can be silent (no change in the encoded polypeptide) or can encode a polypeptide having an altered amino acid sequence.
  • An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of the gene.
  • Isolated polynucleotide means a human modified polynucleotide relative to a polynucleotide found in nature. In one aspect, the isolated polynucleotide is 1% to 95% pure as determined by agarose electrophoresis.
  • the polynucleotide can be of genomic, cDNA, RNA, semi-synthetic, synthetic origin, or any combination thereof.
  • a pure polynucleotide means a polynucleotide preparation free of other foreign or undesired nucleotides, and the polynucleotide preparation is suitable for A form used in genetically engineered protein production systems.
  • a substantially pure polynucleotide contains from 0.5% to 10% by weight of other polynucleotide material with which it is naturally or recombinantly bound.
  • substantially pure polynucleotides can include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators.
  • the substantially pure polynucleotide is 90% to 99.5% pure by weight.
  • the polynucleotide of the present invention is preferably in a pure form on the basis of wood.
  • Coding sequence means a polynucleotide that directly specifies the amino acid sequence of a polypeptide.
  • the boundaries of the coding sequence are usually determined by an open reading frame, usually starting with the ATG start codon or alternative start codons such as GTG and TTG, and ending with stop codons such as TAA, TAG and TGA.
  • the coding sequence can be a DNA, cDNA, synthetic or recombinant polynucleotide.
  • cDNA means a DNA molecule capable of being prepared by reverse transcription from a mature, spliced mRNA molecule derived from a eukaryotic cell.
  • the cDNA lacks an intron sequence that is normally present in the corresponding genomic DNA.
  • the beginning (initial), a primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, and then appears as mature, spliced mRNA.
  • nucleic acid construct means a single-stranded or double-stranded nucleic acid molecule which is isolated from a naturally occurring gene or which is modified to contain a nucleic acid in a manner not inherent in nature. Section, or it is synthetic.
  • nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the regulatory sequences required for expression of a coding sequence of the invention.
  • Control sequence means all components necessary for expression of a polynucleotide encoding a polypeptide of the present invention.
  • Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or the individual regulatory sequences may be native or foreign to each other.
  • These regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator.
  • regulatory sequences include promoters as well as termination signals for transcription and translation.
  • the control sequences can be provided with a linker for the purpose of introducing a specific restriction site that facilitates ligation of the regulatory sequence to the coding region of the polynucleotide encoding the polypeptide.
  • operably linked means a configuration in which a regulatory sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the regulatory sequence directs expression of the coding sequence.
  • expression includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • Expression vector means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide, and the polynucleotide is operably linked to additional nucleotides provided for its expression.
  • host cell means any cell type that is susceptible to transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
  • host cell encompasses the progeny of any parental cell that differs from the parental cell due to mutations that occur during replication.
  • variant means a protein or polypeptide having 3-epimerase activity that comprises an alteration at one or more (several) positions, ie one or more (several) amino acid residues Substitutions, insertions and/or deletions. Substitution means replacing an amino acid occupying a position with a different amino acid: deletion means removal of an amino acid occupying a position; and insertion means addition of 1-3 amino acids to an amino acid adjacent to a position.
  • the present inventors studied the characteristics of an epimerase polypeptide derived from Thermomemmatispora carboxidivorans, and found that when converting fructose into D-psicose and converting sorbose into D-tagatose, The above polypeptide has a function of epimerization reaction of fructose and sorbose at the third carbon position to convert it into D-psicose or D-tagatose, which is carried out by the following steps: synthesis source Epimerase group from Thermomemmatispora carboxidivorans Therefore, the microorganism containing the gene expression vector is cultured and the polypeptide is overexpressed.
  • the polypeptide was found to have good psicose-3-epimerase and tagatose-3-epimerase activity and excellent thermal stability. Accordingly, the present invention provides a polypeptide having psicose-3-epimerase and tagatose-3-epimerase activity, and using the aldose--3-difference A method of producing D-psicose or D-tagatose to a polypeptide of isomerase and tagatose-3-epimerase activity.
  • the gene labeled as a polypeptide of the epimerase is obtained from the Thermomemmatispora carboxidivorans strain by gene synthesis, and the labeled epimerase gene is It is based only on the DNA base sequence, not on the results of its functional characterization.
  • the obtained epimerase gene is then inserted into a suitable expression vector to produce a recombinant vector containing the epimerase gene, and the recombinant vector is transformed into a suitable microorganism.
  • the transformed microorganism is cultured in a fermentation medium, and the polypeptide product of the epimerase gene is overexpressed in the microorganism, and then the polypeptide product of the epimerase gene is isolated and purified for use.
  • the polypeptide has psicose-3-epimerase and tagatose-3-epimerase activity, which can transform fructose to produce D-psicose, and can also transform sorbose. D-tagatose.
  • novel 3-epimerase-active polypeptide produced by the method of the present invention may have an amino acid sequence which is not limited to the amino acid sequence of SEQ ID No: 2 but includes the amino acid sequence of SEQ ID No: 2.
  • the expression vector which can be used to produce a recombinant expression vector can be any expression vector conventionally used in genetic recombination techniques, and can be, for example, pET-22b(+).
  • the microorganism capable of being transformed by the recombinant expression vector may be Escherichia coli BL21 (DE3).
  • the microorganism is not limited as long as it is any microorganism capable of overexpressing the gene after being transformed with a recombinant expression vector containing the desired gene, and capable of producing an active protein as a result of overexpression. Or a polypeptide.
  • the following process of culturing the transformed microorganism and inducing overexpression of the protein or polypeptide of the present invention can be carried out according to an exemplary experimental protocol of the present invention as described below.
  • the cryopreserved recombinant Escherichia coli was inoculated into a 250 mL flask containing 50 mL of LB medium, and the strain was cultured in a shaker maintained at 37 ° C until the absorbance at 600 nm reached 2.0.
  • the culture solution was added to a 7 L fermentor containing 5 L of fermentation broth, 25 g/L peptone, 25 g/L yeast extract, 10 g/L sodium chloride, 2 g/L glucose, 3 g.
  • the stirring rate is 500 rpm
  • the aeration rate is 1.0 vvm
  • the culture temperature is 37 ° C, and the above culture conditions are favorable for mass production. 3-epimerase.
  • the recombinant E. coli culture solution was centrifuged at 6,000 x g for 30 minutes at 4 ° C and then washed twice with 0.85% NaCl. Subsequently, the cells were resuspended in 50 mM sodium phosphate buffer solution (containing 300 mM NaCl) of pH 8.0, and the buffer solution containing the cells was placed in an ice bath for 30 minutes.
  • the cells in the buffer solution were disrupted by a high-pressure homogenizer, and the disrupted cells were centrifuged at 13,000 ⁇ g, 4 ° C for 20 minutes, and removed, and the supernatant was filtered through a filter having a pore size of 0.45 ⁇ m and passed through It is purified by rapid protein chromatography under low temperature conditions.
  • the filtrate containing the protein of the present invention was added to a HisTrap HP column equilibrated with 50 mM sodium phosphate buffer solution of pH 8.0 containing 300 mM NaCl and 10 mM imidazole.
  • the HisTrap HP column was washed with the same sodium phosphate buffer solution, and the protein attached to the column was eluted with the same sodium phosphate buffer solution containing imidazole having a concentration gradient of from 10 mM to 200 mM at a flow rate of 1 mL/min.
  • the eluate containing the protein of the present invention was added to a HiPrep 16/60 resin column which was equilibrated with a 50 mM sodium phosphate buffer solution of pH 7.5 and further at 6 mL/min. The rate of elution of the protein.
  • the protein solution thus collected was added to a Sephacryl S-100 HR column which was equilibrated with a pH 7.5 50 mM sodium phosphate buffer solution containing 0.15 M NaCl at a flow rate of At 6 mL/min, the eluted white matter was finally dialyzed against a 50 mM sodium phosphate buffer solution.
  • the protein of the present invention obtained as described above is a 3-epimerase, and the monomeric molecular weight of the 3-epimerase is 31,770 Da.
  • the 3-epimerase is a metalloenzyme, and the metal ion significantly promotes its activity.
  • the 3-epimerase is reacted in the presence of a metal ion to achieve the purpose of increasing the production of D-psicose from fructose, and also to improve the production of D- from sorbose.
  • the metal ion is selected from the group consisting of manganese, magnesium and cobalt in a concentration ranging from 0.5 to 5 mM, for example, 1 mM. When the concentration of the metal ions is less than 0.5 mM, the effect of increasing the conversion is not remarkable, and when the concentration of the metal ions is higher than 5 mM, there is no significant difference in the conversion rate.
  • the reaction between the 3-epimerase and fructose or sorbose can utilize a substrate having a concentration of 10-75% (w/w), pH 6-8 and a temperature of 50-90 ° C (ie fructose or sorbose solution). )get on.
  • concentration of the substrate, fructose or sorbose is in the range of 10-75% (w/w)
  • the yield of D-psicose or D-tagatose is good
  • the conversion rate is high
  • the pH and temperature conditions are the pH and temperature ranges that are optimal for 3-epimerase activity.
  • the 3-epimerase has excellent thermal stability. After the incubation at 60 ° C for 12 hours, the activity is still not detected. After incubation at 80 ° C for 12 hours, the activity remains above 80%, and the temperature is maintained at 90 ° C. There is still 50% residual activity after 8 hours.
  • the excellent thermal stability of the 3-phase isomerase is excellent for producing D-psicose and D-tagatose under higher temperature conditions.
  • the 3-epimerase conversion of fructose to produce D-psicose and the conversion of sorbose to produce D-tagatose can be achieved by 3-epimer isomerization during the reaction.
  • the enzyme immobilization is carried out on a carrier because the 3-epimerase immobilized on the carrier is capable of maintaining enzyme activity for a long period of time and is easy to reuse.
  • the carrier used in the present embodiment of the present invention may be any one known for its use in enzyme immobilization, and may be, for example, sodium alginate.
  • sodium alginate is a natural colloidal polysaccharide which is abundant in algal cell walls and contains ⁇ -D-mannuronic acid and ⁇ -L-guluronic acid residues, said ⁇ -D-mannuronic acid And the ⁇ -L-guluronic acid residue is randomly linked by a ⁇ -1,4 bond. Therefore, sodium alginate allows for stable immobilization of the 3-epimerase and is advantageous for obtaining higher D-psicose or D-tagatose production.
  • sodium alginate can be used for the immobilization of 3-epimerase at a concentration of 1.5-4% (w/v), for example at a concentration of 2.5% (w/v).
  • the solution of the 3-epimerase is added to an aqueous solution of sodium alginate, and the volume of the aqueous solution of sodium alginate is 3 - one or two times the volume of the epimerase solution, and then the mixture is added dropwise to the 0.2 M calcium ion solution by means of a syringe pump and a vacuum pump to form a 3-epimerase-sodium alginate complex.
  • a syringe pump and a vacuum pump to form a 3-epimerase-sodium alginate complex.
  • These 3-epimerase-alginic acid complex spheres can be used in the conversion of fructose to produce D-psicose and the conversion of sorbose to produce D-tagatose.
  • the 3-epimerase of the present invention has good activity and excellent thermal stability for fructose and sorbose, both of which produce D-psicose and D-tagatose under higher temperature conditions. Words are excellent performance.
  • the method for producing D-psicose and D-tagatose according to an embodiment of the present invention is environmentally friendly because an enzyme derived from microorganisms is used, and the method requires a simple enzyme immobilization process, and Significantly increased production yield and production efficiency of D-psicose and D-tagatose, thereby reducing production costs and maximizing production.
  • the D-psicose and D-tagatose thus produced can be effectively used as an additive for foods or medicines.
  • the 3-epimerase of the present invention has good activity and excellent thermal stability for fructose and sorbose, both of which are for producing D-psicose and D-tagatose under high temperature conditions. It is excellent performance.
  • the method for producing D-psicose and D-tagatose according to an embodiment of the present invention is environmentally friendly because an enzyme derived from microorganisms is used, and the method requires a simple enzyme immobilization process, and Significantly increased production yield and production efficiency of D-psicose and D-tagatose, thereby reducing production costs and maximizing production.
  • the D-psicose and D-tagatose thus produced can be effectively used as an additive for foods or medicines.
  • Figure 1 is a flow chart showing the production of 3-epimerase in Example 1 of the present invention.
  • Figure 2-1 is a graph showing the effect of pH on the activity of 3-epimerase in Example 5 of the present invention.
  • Example 5 of the present invention is a graph showing the effect of temperature on the activity of 3-epimerase in Example 5 of the present invention.
  • Figure 3 is a graph showing the relationship of temperature activity in Example 6 of the present invention.
  • Figure 4 is a schematic diagram showing the conversion of D-psicose produced by the conversion of fructose by 3-epimerase in Example 7 of the present invention
  • Figure 5 is a graph showing the conversion of D-tagatose produced by the conversion of sorbose by 3-epimerase in Example 11 of the present invention.
  • the enzymatic activity was measured using fructose and sorbose as substrates.
  • 3-epimerase was mixed with 50 mM pH 7.5 sodium phosphate buffer solution containing 10% fructose or sorbose, reacted at 60 ° C for 20 minutes, and then heated at 100 ° C. 15 minutes to terminate the reaction.
  • the sodium phosphate buffer solution containing fructose or sorbose is prepared by dissolving fructose or sorbose in a sodium phosphate buffer solution of pH 7-8 to achieve a concentration of 60-70% (w/v), and The sodium phosphate buffer solution containing fructose or sorbose was continuously added to the bioreactor maintained at 60 °C.
  • one unit of psicose-3-epimerase is defined as required to produce 1 mole of D-psicose per minute at pH 7.5 and 60 °C.
  • Amount of psicose-3-epimerase; one unit of tagatose-3-epimerase is defined as producing 1 mole of D-tower per minute at pH 7.5 and 60 °C
  • the amount of tagatose-3-epimerase required for the sugar was measured by high performance liquid chromatography using a BP-100 calcium ion hydrocarbon column and an RI detector. The column temperature was 80 ° C, the mobile phase was ultrapure water, and the flow rate was 0.5 mL/min.
  • the 3-epimerase gene is obtained by synthesizing a gene of a polypeptide labeled as a epimerase in Thermogemmatispora carboxidivorans, which is based only on the sequence, not on the functional aspects thereof. Defined.
  • the obtained epimerase gene was inserted into the expression vector pET-22b(+) by using restriction enzymes NdeI and XhoI, thereby producing a recombinant expression vector pET-22b(+)/epimerase ( see picture 1).
  • the recombinant vector was transformed into E. coli BL21 (DE3) by a conventional transformation method.
  • the transformed recombinant Escherichia coli BL21 (DE3) was stored in an ultra-low temperature freezer at -80 °C.
  • the recombinant Escherichia coli was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of liquid LB medium, and culture-activated in a shaker at 37 ° C until the absorbance of the culture solution at a wavelength of 600 nm reached 2.0.
  • This culture solution was added to a 7 L fermentor containing 5 L of fermentation medium, and subjected to fermentation culture to mass-produce 3-epimerase.
  • the stirring rate was maintained at 500 rpm, the ventilation rate was 1.0 vvm, and the culture temperature was 37 °C.
  • the molecular weight of the purified 3-epimerase was measured, and it was found that the 3-epimerase monomer had a molecular weight of 31,770 Da.
  • the amino acid sequence of the 3-epimerase was confirmed to be identical to the amino acid sequence of NCBI Accession No. WP_052889376.
  • Example 3 in order to investigate the effect of metal ions on the 3-epimerase, the effect of fructose as a substrate on the activity of the substrate was determined in the presence of different metal ions, and the measurement was performed by EDTA. After the epimerization enzyme, 1 mM of various metal ions shown in Table 1 below was added to the 3-epimerase solution. The 3-epimerase reaction was carried out in a 50 mM Tris buffer solution of pH 7.5 at 60 ° C for 20 minutes, wherein the Tris buffer solution contained 0.04 U/mL of 3-epimerase and 10% ( The fructose of w/v) was further heated at 100 ° C for 15 minutes to terminate the reaction, and then the activity of the 3-epimerase was measured.
  • the 3-epimerase reaction was carried out in a 50 mM sodium phosphate buffer solution at pH 7.5 and 60 ° C for 20 minutes, wherein the sodium phosphate buffer solution contained 0.04 U/ml of the 3-epimer
  • Example 5 in order to investigate the effects of different pH and temperature on the activity of the 3-epimerase, the effect of fructose as a substrate on its activity was measured under different conditions of temperature and pH, and the differences were compared. Enzyme activity at temperature and pH.
  • the 3-epimerase reaction was carried out in a sodium phosphate buffer solution having a pH range of 6.0-8.5 50 mM, wherein the sodium phosphate buffer solution contained 0.04 U/mL of the 3-difference Enzyme and 10% (w/v) fructose.
  • the respective reactions were carried out at 60 ° C for 20 minutes without metal ion addition, followed by heating at 100 ° C for 15 minutes to terminate the reaction, and the enzyme activity was measured.
  • the result is illustrated in Figure 2-1.
  • the reaction was carried out in a 50 mM sodium phosphate buffer solution at a temperature ranging from 40 to 90 ° C for 20 minutes, wherein the sodium phosphate buffer solution contained 0.04 U/mL of the 3-epimerase. And 10% (w/v) fructose was reacted at pH 7.5. The reaction was terminated by heating at 100 ° C for 15 minutes, and its enzymatic activity was measured. The result is illustrated in Figure 2-2.
  • Figure 2-1 is a graph showing the effect of pH on 3-epimerase activity under the conditions of an embodiment of the invention.
  • Figure 2-2 is a graph showing the effect of temperature on 3-epimerase activity under the conditions of an embodiment of the invention.
  • Example 6 in order to study the thermal stability of the 3-epimerase, the 3-epimerase was separately incubated at different temperature conditions, and samples were taken at different times to measure fructose as a substrate. Residual activity. The measurement was carried out every 1 hour after the 3-epimerase was incubated at 50 ° C, 60 ° C, 70 ° C, 80 ° C and 90 ° C water bath conditions, respectively.
  • the 3-epimerase reaction was carried out in a sodium phosphate buffer solution of 50 mM pH 7.5 at 60 ° C for 20 minutes, wherein the sodium phosphate buffer solution contained 0.04 U/mL of 3-epimerase and 10
  • the reaction solution was heated at 100 ° C for 15 minutes to terminate the reaction, and then the activity of the 3-epimerase was measured. The result is illustrated in FIG.
  • Example 7 the 3-epimerase reaction was carried out in a pH 7.5 50 mM sodium phosphate buffer solution at a temperature range of 40-90 ° C. The reaction was allowed to proceed sufficiently for 12 hours, wherein the sodium phosphate buffer solution contained the 3-epimerase 0.04 U/mL, 1 mM cobalt ion, and 10% (w/v) fructose. . Then, the reaction was terminated by heating at 100 ° C for 15 minutes, and the contents of fructose and D-psicose in the sample were measured. The result is illustrated in FIG.
  • the reaction is carried out in a pH 7.5 50 mM sodium phosphate buffer solution at 60 ° C, wherein the sodium phosphate buffer solution contains 10 U/mL of the 3-differential Isomerase, 1 mM cobalt ion and 700 g/L fructose. Samples were then taken at different reaction time points, the reaction was stopped by heating at 100 ° C for 15 minutes, and the concentration of D-psicose in the sample was measured. The D-psicose yields for different reaction times are shown in Table 3 below.
  • the 3-epimerase was immobilized.
  • the yield of immobilized 3-epimerase production was measured and compared to the production yield of unfixed (free) 3-epimerase.
  • a 3-epimerase-alginate complex sphere was used, which was prepared by the following method: 3-epimerase solution Adding to 2.5% (w/v) sodium alginate solution, the volume of the sodium alginate solution is 1.5 times the volume of the 3-epimerase solution, and the mixture is added by a syringe pump and a vacuum pump. Into a 0.2M calcium ion solution.
  • This reaction was carried out in the same manner as in Example 7 except that a fixed 3-epimerase was used.
  • the amount of the 3-epimerase used in the reaction was 10 U/mL, and the D-psicose production rate was measured. The results are shown in Table 4 below.
  • Example 10 Production yield of D-psicose in a bioreactor
  • the immobilized 3-epimerase and fructose were prepared in the same manner as in Example 9, and fructose was added to the immobilized 3-epimerase, and the mixture was adjusted to a volume. 100 mL Subsequently, a bioreactor of 100 cm in height and 2.6 cm in diameter was filled with the mixture of the immobilized 3-epimerase and fructose, and the reaction was carried out at flow rates of 10 mL/h and 60 °C. The amount of 3-epimerase used was 500 U, and the concentration of fructose used was limited to 600 g/L due to precipitation of excess fructose during prolonged operation. The results are shown in Table 5 below.
  • the present invention can provide a D-psicose production system using a bioreactor capable of mass production on an industrial scale.
  • Example 11 the 3-epimerase reaction was carried out in a pH 7.5 50 mM sodium phosphate buffer solution at a temperature ranging from 40 to 90 ° C for 12 hours, thereby allowing the reaction to proceed sufficiently, wherein
  • the sodium phosphate buffer solution contained the 3-epimerase 0.04 U/mL, 1 mM cobalt ion, and 10% sorbose.
  • the reaction was terminated by heating at 100 ° C for 15 minutes, and the contents of sorbose and D-tagatose in the samples were measured. The result is illustrated in FIG. 5.
  • the reaction is carried out in a pH 7.5 50 mM sodium phosphate buffer solution at 60 ° C, wherein the sodium phosphate buffer solution contains 20 U/mL of the 3-differential Constructed enzyme, 1 mM cobalt ion and 500 g/L sorbose. Samples were then taken at different reaction time points, terminated by heating at 100 °C for 15 minutes, and the concentration of D-tagatose in the samples was measured. The D-tagatose yields for different reaction times are shown in Table 6 below.
  • the 3-epimerase was immobilized.
  • the production yield of the solid 3-densitized 3-epimerase was measured and compared with the production yield of the unfixed (free) 3-epimerase.
  • a 3-epimerase-alginate complex sphere was used, which was prepared by the following method: 3-epimerase solution Adding to 2.5% (w/v) sodium alginate solution, the volume of the sodium alginate solution is 1.5 times the volume of the 3-epimerase solution, and the mixture is added by a syringe pump and a vacuum pump. Into a 0.2M calcium ion solution.
  • This reaction was carried out in the same manner as in Example 12 except that a fixed 3-epimerase was used.
  • the amount of 3-epimerase used in the reaction was 20 U/mL, and D-tagatose productivity was measured. The results are shown in Table 7 below.
  • Example 14 Production yield of D-tagatose in a bioreactor
  • the immobilized 3-epimerase and sorbose were prepared in the same manner as in Example 13, sorbose was added to the immobilized 3-epimerase, and the mixture was adjusted.
  • a bioreactor 100 cm high and 2.6 cm in diameter was filled with the mixture of immobilized 3-epimerase and sorbose, and the reaction was carried out at flow rates of 10 mL/h and 60 °C. .
  • the amount of 3-epimerase used was 400 U, and the concentration of sorbose used was 400 g/L. The results are shown in Table 8 below.
  • the present invention can provide a D-tagatose production system using a bioreactor capable of mass production on an industrial scale.

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Abstract

提供了一种3-差向异构酶和编码该酶的多核苷酸，包含该多核苷酸的核酸构建体、载体和宿主细胞，以及生产该3-差向异构酶的方法，还提供了该3-差向异构酶的用途。

Description

[根据细则37.2由ISA制定的发明名称]　一种3-差向异构酶以及编码它的多核苷酸 技术领域
本发明涉及具有3-差向异构酶活性的多肽或蛋白质，编码具有3-差向异构酶活性的多肽或蛋白质的多核苷酸序列，包含所述多核苷酸序列的核酸构建体或表达载体，产生所述酶的方法，和所述酶在多种工业应用中的用途。
此外，木发明涉及通过使用具有3-差向异构酶活性的多肽或蛋白质催化果糖、葡萄糖、淀粉糖等单糖或混合糖生产D-阿洛酮糖的方法，以及使用所述多肽或蛋白质催化山梨糖、淀粉糖等单糖或混合糖生产D-塔格糖的方法。
背景技术
随着人们对健康饮食的日益重视，研发健康安全的低热量功能性甜味剂成为食品行业研究的重点。D-阿洛酮糖和D-塔格糖作为自然界中天然存在但含量极少的天然低热量功能性甜味剂，已经成为人们研究的热点。
D-阿洛酮糖(D-allulose)又名D-核糖-2-己酮糖，是D-果糖的C-3位的差向异构体。D-阿洛酮糖是一种天然存在但有含量极少的低热量功能性甜味剂，其甜度为蔗糖的70％，但能量仅为蔗糖的0.3％，可以用作低卡路里减肥食品的甜味剂，同时，D-阿洛酮糖还具有抑制肝中脂质合成有关酶活性的功能，有助于减少腹部脂肪堆积，并能一定程度上控制体重，可以用于健康食品等各种各样的功能性食品。此外，D-阿洛酮糖还能够改善食品风味、外观等，延长食品的保存期限。因此，D-阿洛酮糖这种健康安全的低热量功能性甜味剂已获得越来越多学者的关注，成为最具市场竞争力的新型甜味剂之一。
由于D-阿洛酮糖是一种较为稀有的天然单糖，从自然界中分离提取产量低，成本高，难以满足人们作为低热量健康甜味剂的需要，也不适宜工业化大生产的要求，因此为了应用于食品工业，需要有高效的D-阿洛酮糖生产方法。传统的D-阿洛酮糖生产方法主要为化学法，生产过程需要消耗大量费用、产生许多副产物和污染物。而生物法制备D-阿洛酮糖具有反应专一性强，产物组分简单易纯化且属于天然产品等特点，已经成为研究的热点。
生物法制备D-阿洛酮糖最为有效的方法是寻找能使果糖转变为D-阿洛酮糖的酶。然而，目前的阿洛酮糖-3-差相异构酶酶存在高温下稳定性降低或者活性低反应速度慢等问题，不利于工业化生产D-阿洛酮糖的成本控制。因此，需要开发高温稳定性优良、活性高的阿洛酮糖-3-差向异构酶以适应工业化生产的需求。
D-塔格糖(D-Tagatose)，是D-山梨糖的C-3位的差向异构体。D-塔格糖是一种天然存 在但有含量极少的低热量功能性甜味剂，其甜度为蔗糖的92％，在人体内吸收率较低，仅为20％～25％，不会引起机体血糖水平的明显变化,很适合糖尿病人食用，而且绝大部分塔格糖直接进入结肠,被其中微生物菌群所选择性发酵,促进有益菌增殖,抑制有害菌的生长,起到明显的改善肠道菌群的作用,是一种很好的益生素，可以用于健康食品等各种各样的功能性食品。此外，D-塔格糖还能够改善食品风味、外观等。因此，D-塔格糖这种健康安全的低热量功能性甜味剂已获得越来越多学者的关注，成为极具市场前景的新型甜味剂之一。
使山梨糖异构为D-塔格糖是生物法制备D-塔格糖最为有效的方法之一，这种方法的关键是寻找能使山梨糖转变为D-塔格糖的酶。目前的塔格糖-3-差相异构酶存在高温下稳定性降低或者活性低反应速度慢等问题，不利于工业化生产D-塔格糖的成本控制和规模放大。因此，需要开发高温稳定性优良、活性高的塔格糖-3-差相异构酶以适应工业化生产的需求。
发明内容
本发明人从Thermogemmatispora carboxidivorans菌种发现了一种新型具有3-差向异构酶活性的蛋白质。相应地，本发明提供了具有3-差向异构酶活性的新型蛋白质，和编码所述蛋白质或多肽的多核苷酸。所述蛋白质或多肽具有阿洛酮糖-3-差向异构酶及塔格糖-3-差向异构酶活性。本发明的蛋白质或多肽具有良好的活性和优秀的热稳定性，这两点对较高温度条件下生产D-阿洛酮糖和D-塔格糖而言是优良的性能。
本发明涉及具有阿洛酮糖-3-差向异构酶及塔格糖-3-差向异构酶活性的分离的蛋白质或多肽，其选自下组：
(a)蛋白质或多肽，其与SEQ ID No：2的氨基酸序列具有至少70％序列同一性；
(b)蛋白质或多肽，其由多核苷酸编码，所述多核苷酸在中等-高严格条件下与以下杂交：(i)SEQ ID No：1的多肽编码序列，(ii)包含SEQ ID No：1的多肽编码序列的基因组DNA序列，或(iii)(i)或(ii)的全长互补链；
(c)蛋白质或多肽，其由多核苷酸编码，所述多核苷酸与SEQ ID NO：1的蛋白质或多肽编码序列具有至少70％序列同一性；
(d)SEQ ID NO：2的蛋白质或多肽包含一个或多个(几个)氨基酸的取代、缺失和/或插入的变体；和
(e)任何(a)、(b)或(c)的蛋白质或多肽，其包含SEQ ID No：2或由SEQ ID No：2组成，和
(f)(a)，(b)，(c)，(d)，或(e)的蛋白质或多肽的片段，其具有阿洛酮糖-3-差向异构酶及塔格糖-3-差向异构酶活性。
本发明亦涉及包含所述多核苷酸的核酸构建体、表达载体和重组宿主细胞：并涉及产 生多核苷酸的方法。
本发明亦涉及酶法生产D-阿洛酮糖和D-塔格糖的方法。
定义
阿洛酮糖-3-差向异构酶活性：术语“阿洛酮糖-3-差向异构酶活性”意指催化果糖的C-3位异构转化为D-阿洛酮糖的活性。
塔格糖-3-差向异构酶活性：术语“塔格糖-3-差向异构酶活性”意指催化山梨糖的C-3位异构转化为D-塔格糖的活性。
分离的蛋白质或多肽：术语“分离的蛋白质或多肽”意指相对于如见于自然界的蛋白质或多肽经人力修饰的多肽。在一个方面，多肽如通过SDS-PAGE测定的，为至少1％纯，
例如至少5％纯，至少10％纯，至少20％纯，至少40％纯，至少60％纯，至少80％纯，并且最优选至少90％纯。
分离的蛋白质或多肽的非限定性实例包括：(1)任何非天然存在的蛋白质或多肽，(2)任何蛋白质或多肽，其包括但不限于任何酶、变体、核酸、蛋白质、肽或辅因子，它们至少部分地从与其在自然界结合的一种或多种或所有天然存在的组分移出；(3)任何蛋白质或多肽，其相对于见于自然界的蛋白质或多肽经人工修饰：或(4)任何蛋白质或多肽，其通过相对于与其天然结合的其它组分增加所述蛋白质或多肽的量而受修饰。分离的蛋白质或多肽可存在于发酵液样品。
基本上纯的蛋白质或多肽：术语“基本上纯的蛋白质或多肽”意指蛋白质或多肽制备物，所述蛋白质或多肽制备物含有按重量计至多10％的与其天然或重组结合的(associated)的其它蛋白质或多肽材料。优选地，所述蛋白质或多肽是按存在于制备物中的全部蛋白质或多肽材料的重量计至少90％纯。本发明的蛋白质或多肽优选是基本上纯的形式，例如，这能够通过以下实现：通过公知的重组方法或由经典纯化方法制备多肽。
序列同一性：参数“序列同一性"描述两个氨基酸序列之间或两个核苷酸序列之间的相关性。
就本发明而言，两个氨基酸序列之间的序列同一性程度通过使用如EMBOSS软件包(EMBOSS：The European Molecular Biology Open Software Suite,Rice等，2000，Trends in Genetics 16：276-277)(优选3.0.0版或更高版木)的Needle程序中执行的Needleman-Wunsch算法(Needleman和Wunsch,1970，J.Mol.Biol.48：443-453)来测定。使用的可选参数为缺口开放罚分(gap open penalty)为10，缺口延伸罚分(gap extensionpenalty)为0.5和EBLOSUM62(BLOSUM62的EMBOSS版)取代矩阵。使用Needle标记为“最高同一性(longest identity) "的输出结果(使用-nobrief选项获得)作为同一性百分比，并计算如下：
(同样的残基×100)/(比对长度一比对中缺口的总数)
就本发明而言，两个核苷酸序列之间的序列同一性程度通过使用如EMBOSS软件包(EMBOSS：The European Molecular Bi010Open Software Suite,Rice等，见上文)(优选3.0.0版或更高版本)的Needle程序中执行的Needleman-Wunsch算法(Needleman和Wunsch,1970，见上文)来测定。使用的可选参数为缺口开放罚分为10，缺口延伸罚分为0.5和EDNAFULL(NCBI NUC4.4的EMBOSS版)取代矩阵。使用Needle标记为“最高同一性”的输出结果(使用-nobrief选项获得)作为同一性百分比，并计算如下：
(同样的脱氧核糖核苷酸×100)/(比对长度一比对中缺口的总数)
片段：术语“片段”意指从成熟多肽的氨基和(或)羧基末端缺失一个或多个(几个)氨基酸的多肽；其中所述片段具有3-差向异构酶活性。
等位变体(allelic variant)：术语“等位变体”意指占据相同染色体基因座的基因的任何两种或两种以上可选形式。等位变异通过突变天然地发生，并且可导致种群内的多态性。基因突变可以是沉默的(在编码的多肽中无变化)或可以编码具有改变的氨基酸序列的多肽。多肽的等位变体是由基因的等位变体编码的多肽。
分离的多核苷酸：术语“分离的多核苷酸”意指相对于见于自然界的多核苷酸经人力修饰的多核苷酸。在一个方面，分离的多核苷酸如通过琼脂糖电泳测定的，为1％至95％纯。所述多核苷酸可为基因组、cDNA、RNA、半合成、合成来源，或其任意组合。
本上纯的多核苷酸：术语“基本上纯的多核苷酸"意指不含其它外来的或不期望的核苷酸的多核苷酸制备物，并且所述多核苷酸制备物处于适合于在遗传工程的蛋白质生产体系中使用的形式。因此，基本上纯的多核苷酸含有按重量计0.5％至10％的与其天然或重组结合的其它多核苷酸材料。然而，基本上纯的多核苷酸可以包括天然存在的5’和3’非译区，如启动子和终止子。优选地，基本上纯的多核苷酸是按重量计90％至99.5％纯的。本发明所述多核苷酸优选为基木上纯的形式。
编码序列：术语“编码序列”意指直接指定多肽氨基酸序列的多核苷酸。编码序列的边界通常由开放阅读框决定，所述开放阅读框通常以ATG起始密码子或可供选择的起始密码子例如GTG和TTG开始，并且以终止密码子如TAA、TAG和TGA结束。编码序列可以是DNA、cDNA、合成的或重组的多核苷酸。
cDNA：术语“cDNA”意指能够通过反转录从得自真核细胞的成熟的、己剪接的mRNA分子制备的DNA分子。cDNA缺少通常存在于相应基因组DNA中的内含子序列。起始的 (initial)、初级的RNA转录物是mRNA的前体，其通过一系列的步骤(包括剪接)加工然后作为成熟的己剪接的mRNA出现。
核酸构建体：术语“核酸构建体”意指单链或双链的核酸分子，其分离自天然存在的基因，或将其以本来不存在于(not otherwise exist)自然界中的方式修饰以含有核酸的区段，或其为合成的。当所述核酸构建体含有表达本发明的编码序列所需的调控序列时，术语核酸构建体与术语“表达盒”同义。
调控序列(control sequence)：术语“调控序列”意指对编码本发明多肽的多核苷酸表达是必需的所有成分。各个调控序列对于编码所述多肽的核苷酸序列可以是天然的或外源的，或各个调控序列对于彼此可以是天然的或外源的。这些调控序列包括但不限于前导序列、聚腺苷酸化序列、前肽序列、启动子、信号肽序列和转录终止子。最低限度，调控序列包括启动子以及转录和翻译的终止信号。调控序列可以和用于引入特异性限制位点目的的接头一起提供，所述特异性限制位点促进调控序列与编码多肽的多核苷酸的编码区的连接。
可操作地连接：术语“可操作地连接”意指这样的构型，其中将调控序列置于相对于多核苷酸的编码序列的适当位置，使得调控序列指导编码序列的表达。
表达：术语“表达"包括涉及多肽产生的任何步骤，其包括但不限于转录、转录后修饰、翩译、翻译后修饰和分泌。
表达载体：术语“表达载体"意指线性的或环状的DNA分子，其包含编码多肽的多核苷酸，并且所述多核苷酸与提供用于其表达的额外核苷酸可操作地连接。
宿主细胞：术语“宿主细胞"意指任何细胞类型，所述细胞类型对于使用包含本发明多核苷酸的核酸构建体或表达载体的转化、转染、转导等是易感的(susceptible)'术语“宿主细胞"涵盖任何亲本细胞的后代，其由于在复制过程中发生的突变而不同于亲本细胞。
变体：术语“变体"意指具有3-差向异构酶活性的蛋白质或多肽，其在一个或多个(几个)位置包含改变，即一个或多个(几个)氨基酸残基的取代、插入和(或)缺失。取代意指用不同的氨基酸替代占据某位置的氨基酸：缺失意指移出占据某位置的氨基酸；和插入意指紧邻占据某位置的氨基酸添加1-3个氨基酸。
发明详述
本发明人研究了源自Thermogemmatispora carboxidivorans菌的差向异构酶多肽的特性，在对其进行将果糖转化为D-阿洛酮糖及将山梨糖转化为D-塔格糖的实验时发现，上述多肽具有使果糖及山梨糖第3个碳位发生差向异构化反应、使其转化为D-阿洛酮糖或D-塔格糖的功能，这是通过以下步骤进行的：合成源自Thermogemmatispora carboxidivorans菌的差向异构酶基 因，培养包含所述基因表达载体的微生物，并过表达该多肽。经过实验，发现该多肽具有良好的阿洛酮糖-3-差向异构酶及塔格糖-3-差向异构酶活性和优秀的热稳定性。因此，本发明提供了一种具有阿洛酮糖-3-差向异构酶及塔格糖-3-差向异构酶活性的多肽，并利用所述具有阿洛酮糖-3-差向异构酶及塔格糖-3-差向异构酶活性的多肽生产D-阿洛酮糖或D-塔格糖的方法。
为了确定该差向异构酶的多肽的特性，在本发明中，通过基因合成从Thermogemmatispora carboxidivorans菌株中获得该标注为差向异构酶的多肽的基因，所标注的差向异构酶基因是仅根据DNA碱基序列，而不是根据其功能方面的表征结果所定义的。然后，将所获得的差向异构酶基因插入到适合的表达载体中，从而产生含有差向异构酶基因的重组载体，并且将所述重组载体转化到适合的微生物中。将该转化的微生物培养在发酵培养基中，并在该微生物中过表达所述差向异构酶基因的多肽产物，然后分离和纯化所述差向异构酶基因的多肽产物，以备用。经过实验，发现该多肽具有阿洛酮糖-3-差向异构酶及塔格糖-3-差向异构酶活性，能够转化果糖生成D-阿洛酮糖，也能转化山梨糖生成D-塔格糖。
由本发明的方法所产生的新型3-差向异构酶活性的多肽可具有这样的氨基酸序列，该序列不仅限于SEQ ID No：2的氨基酸序列，而且包括由SEQ ID No：2的氨基酸序列中一些氨基酸残基的替换、插入或缺失所形成的氨基酸序列，只要这些氨基酸修饰过的蛋白质或多肽具有阿洛酮糖-3-差向异构酶及塔格糖-3-差向异构酶活性，能够转化果糖或山梨糖生成D-阿洛酮糖或D-塔格糖。
在本发明的方法中，能用于产生重组表达载体的表达载体可以是任何常规用于遗传重组技术的表达载体，并且可以是，例如，pET-22b(+)。能够通过重组表达载体转化的微生物可以是大肠杆菌(E.coli)BL21(DE3)。然而，所述微生物是不受限制的，只要它是任何这样的微生物，即该微生物能够在经过包含所需要基因的重组表达载体转化后过表达该基因，并且作为过表达的结果能够产生活性蛋白质或多肽。
更详细地，以下培养转化的微生物和诱导本发明的蛋白质或多肽过表达的过程可以根据本发明的示范的实验方案如以下描述进行。将低温保存的重组大肠杆菌接种到装有50mL LB培养基的250mL的烧瓶中，并将该菌株在维持在37℃的摇床中培养，直到600nm处的吸光度达到2.0。将该培养溶液加入到装有5L发酵培养基的7L的发酵罐中，所述发酵培养基由15g/L蛋白胨，25g/L酵母提取物，10g/L氯化钠，2g/L葡萄糖，3g/L乳糖组成，并将该混合物在所述发酵罐中培养，以诱导本发明的蛋白质的过表达。在发酵过程中，搅拌速率为500rpm，通气量为1.0vvm，及培养温度为37℃，而且上述培养条件有利于大规模生产 3-差向异构酶。
为了纯化由过表达所产生的蛋白质，将所述重组大肠杆菌培养溶液在6,000×g，4℃离心30分钟，然后用0.85％的NaCl洗涤两次。随后，将该细胞重新悬浮于50mM pH8.0的磷酸钠缓冲溶液(含有300mM NaCl)中，并将含有所述细胞的缓冲溶液置于冰浴中30分钟。利用高压均质机破碎该缓冲溶液中的细胞，将破碎的细胞在13,000×g，4℃离心20分钟，并将其除去，而使上清液通过孔径0.45um的滤膜进行过滤，并通过低温条件下的快速蛋白质色谱法对其进行纯化。将含有本发明的蛋白质的滤出液加入HisTrap HP柱，所述HisTrap HP柱是经过50mM pH8.0的磷酸钠缓冲溶液平衡的，其中所述磷酸钠缓冲溶液含有300mM NaCl和10mM咪唑。随后，用相同的磷酸钠缓冲溶液洗涤该HisTrap HP柱，并且再用含有浓度梯度为从10mM到200mM的咪唑的相同磷酸钠缓冲溶液洗脱附着于该柱的蛋白质，其流速为1mL/min。将含有本发明蛋白质的洗脱液加入到HiPrep 16/60树脂柱中去除咪唑，所述HiPrep 16/60树脂柱是经过pH7.5的50mM的磷酸钠缓冲溶液平衡的，并且再以6mL/min的速率洗脱该蛋白质。将由此所收集的蛋白质溶液加入到Sephacryl S-100HR柱中以洗脱该蛋白质，所述Sephacryl S-100HR柱是经过pH7.5的含有0.15MNaCl的50mM的磷酸钠缓冲溶液平衡的，其流速为6mL/min，最后将洗脱的白质在50mM的磷酸钠缓冲溶液中进行透析。
如上述所获得的本发明的蛋白质是3-差向异构酶，所述3-差向异构酶的单体分子量为31770Da。该3-差向异构酶是金属酶，金属离子对其活性有明显促进。
根据本发明的另一个实施方案，3-差向异构酶在金属离子的存在下反应，可实现提高由果糖生产D-阿洛酮糖产量的目的，也可实现提高由山梨糖生产D-塔格糖产量的目的。所述金属离子选自下列各项组成的组：锰、镁和钴，其浓度范围为0.5–5mM，例如，1mM。当所述金属离子的浓度低于0.5mM时.提高转化率的作用并不明显，而当所述金属离子的浓度高于5mM时，转化率并没有显著的差异。
该3-差向异构酶和果糖或山梨糖间的反应可利用浓度为10-75％(w/w)，pH6-8和温度为50-90℃的底物(即果糖或山梨糖溶液)进行。当该底物，即果糖或山梨糖的浓度在10-75％(w/w)的范围内时，D-阿洛酮糖或D-塔格糖的产量好，转化率高，且上述范围的pH和温度条件是对3-差向异构酶活性最佳的pH和温度范围。
该3-差向异构酶具有优良的热稳定性，在60℃保温12小时后活性仍没有检测到活性下降，在80℃保温12小时后，活性仍然保持在80％以上，在90℃保温8小时后仍然有50％的残余活性。该3-差相异构酶优良的热稳定性对较高温度条件下生产D-阿洛酮糖和D-塔格糖而言是优良的性能。
根据本发明另一个实施方案，该3-差向异构酶转化果糖生产D-阿洛酮糖和转化山梨糖生产D-塔格糖的反应可通过在反应过程中将3-差向异构酶固定在载体上进行，这是因为固定在载体上的3-差向异构酶能够长期地保持酶活性且便于重复使用。用于本发明目前的实施方案中的载体可以是任意己知其在酶固定化中用途的载体，并且可以是例如，海藻酸钠。海藻酸钠是天然胶体多糖，它在藻类细胞壁中很丰富，且含了β-D-甘露糖醛酸和α-L-古洛糖醛酸残基，所述β-D-甘露糖醛酸和α-L-古洛糖醛酸残基通过β-1,4键随机连接。因此，海藻酸钠容许3-差向异构酶稳定的固定，且有利于获得较高的D-阿洛酮糖或D-塔格糖产量。为了获得D-阿洛酮糖或D-塔格糖的最大产量，海藻酸钠可用于进行3-差向异构酶的固定，其浓度为1.5-4％(w/v)，例如浓度为2.5％(w/v)。当海藻酸钠用作固定3-差向异构酶的载体时，将所述3-差向异构酶的溶液加入到海藻酸钠水性溶液中，所述海藻酸钠水性溶液的体积是3-差向异构酶溶液体积的一倍或两倍，然后利用注射器泵和真空泵将该混合物逐滴地加入到0.2M钙离子溶液中，从而形成3-差向异构酶-海藻酸钠复合体球。这些3-差向异构酶-海藻酸复合体球可接用于转化果糖生产D-阿洛酮糖以及转化山梨糖生产D-塔格糖的反应中。
本发明的3-差向异构酶对果糖和山梨糖均具有良好的活性和优良的热稳定性，这两点对较高温度条件下生产D-阿洛酮糖和D-塔格糖而言是优良的性能。根据本发明实施方案的生产D-阿洛酮糖和D-塔格糖的方法是对环境友好的，这是因为使用了源自微生物的酶，并且该方法要求简单的酶固定化过程，以及显著地增加了D-阿洛酮糖和D-塔格糖的生产产量和生产效率，因此降低了生产成本，而最大化了生产的效果。
由此生产的D-阿洛酮糖和D-塔格糖能够有效地用作食品或药物的添加剂。
有利作用
本发明的3-差向异构酶对果糖和山梨糖均具有良好的活性和优良的热稳定性，这两点对高温度条件下生产D-阿洛酮糖和D-塔格糖而言是优良的性能。根据本发明实施方案的生产D-阿洛酮糖和D-塔格糖的方法是对环境友好的，这是因为使用了源自微生物的酶，并且该方法要求简单的酶固定化过程，以及显著地增加了D-阿洛酮糖和D-塔格糖的生产产量和生产效率，因此降低了生产成本，而最大化了生产的效果。
由此生产的D-阿洛酮糖和D-塔格糖能够有效地用作食品或药物的添加剂。
附图说明
图1是本发明实施例1中3-差向异构酶的生产流程图；
图2-1是本发明实施例5中pH对于3-差向异构酶活性影响的曲线图；
图2-2是本发明实施例5中温度对于3-差向异构酶活性影响的曲线图；
图3是本发明实施例6中温度活性关系图；
图4是本发明实施例7中利用3-差向异构酶所转化果糖生产D-阿洛酮糖的转化率示意图；
图5是本发明实施例11中利用3-差向异构酶所转化山梨糖生产D-塔格糖的转化率示意图。
具体实施方式
下文中，将参考具体实施例对本发明进行更详细地描述。这些实施例仅仅是为了例证的目的，而非意欲限制本发明的范围。
在目前的试验实施例中，利用果糖和山梨糖作为底物测量了其酶活性。为了测量其酶活性，将3-差向异构酶与含有10％果糖或山梨糖的50mM pH7.5磷酸钠缓冲溶液混合，在60℃条件下反应20分钟，再在100℃加热该反应溶液15分钟，以终止该反应。所述含有果糖或山梨糖的磷酸钠缓冲溶液是通过将果糖或山梨糖溶解于pH7-8的磷酸钠缓冲溶液中，以达到浓度60-70％(w/v)配制而成的，并且将该含有果糖或山梨糖的磷酸钠缓冲溶液持续地加入到维持在60℃的生物反应器中。为了实现方便比较其酶活性的目的，将一单位的阿洛酮糖-3-差向异构酶定义为在pH7.5和60℃条件下每分钟生产1摩尔D-阿洛酮糖所需要的阿洛酮糖-3-差向异构酶的量；将一单位的塔格糖-3-差向异构酶定义为在pH7.5和60℃条件下每分钟生产1摩尔D-塔格糖所需要的塔格糖-3-差向异构酶的量。果糖、D-阿洛酮糖、山梨糖、D-塔格糖的浓度是利用高效液相色谱法测量的，该高效液相色谱法使用BP-100钙离子碳氢化合物柱和RI检测器，柱温80℃，流动相为超纯水，流速0.5mL/min。
实施例1：3-差向异构酶的生产
3-差向异构酶基因是通过合成Thermogemmatispora carboxidivorans中标注为差向异构酶的多肽的基因获得的，所述差向异构酶基因是仅根据序列，而不是根据其功能方面的表征结果所定义的。通过利用限制性内切酶NdeI和XhoI，将获得的差向异构酶基因插入到表达载体pET-22b(+)中，从而产生重组表达载体pET-22b(+)/差向异构酶(见图1)。通过常规转化方法，将该重组达载体转化到大肠杆菌BL21(DE3)中。将转化得到的重组大肠杆菌BL21(DE3)保藏在-80℃的超低温冰箱中。
此后，将该重组大肠杆菌接种在装有50mL液体LB培养基的250mL三角烧瓶中，并在37℃的摇床中培养活化，直到培养液在波长600nm处的吸光度达到2.0。将该培养液加入到装有5L发酵培养基的7L的发酵罐中，进行发酵培养以大规模生产3-差向异构酶。在发酵过程中，维持搅拌速率为500rpm，通风率为1.0vvm，培养温度为37℃。
实施例2：3-差向异构酶的纯化
为了对3-差向异构酶的性质进行表征，利用亲合色谱法(HisTrap HP柱)，HiPrep 16/60柱和 Sephacryl S-100HR柱纯化3-差向异构酶。
测量了该纯化的3-差向异构酶的分子量，发现该3-差向异构酶单体分子量为31770Da的。证实了该3-差向异构酶的氨基酸序列与NCBI登记号WP_052889376的氨基酸序列相同。
实施例3：3-差向异构酶的金属依赖性
在实施例3中，为了研究金属离子的对该3-差向异构酶的影响，在不同金属离子存在条件下将果糖作为底物测定对其活性的影响，测量是在用EDTA处理该3-差向异构酶后，向3-差向异构酶溶液中加入1mM下表1中所示的各种金属离子后进行的。该3-差向异构酶反应在50mMpH7.5的Tris缓冲溶液中，60℃条件下进行20分钟，其中所述Tris缓冲溶液含有0.04U/mL的3-差向异构酶和10％(w/v)的果糖，再将该反应溶液在100℃加热15分钟，以终止该反应，然后测量该3-差向异构酶的活性。
结果显示该3-差向异构酶具有金属依赖性，如下表1所示，镁、锰和钴离子增强其酶活性，而铜和锌离子抑制其酶活性。
表1
金属离子 相对活性(％)
无 100
Co2+ 170
Ca2+ 65
Mn2+ 168
Mg2+ 130
Zn2+ 0
Cu2+ 0
Na+ 120
K+ 113
实施例4：3-差向异构酶的底物特异性
该3-差向异构酶反应在50mM的磷酸钠缓冲溶液中，pH7.5和60℃条件下进行20分钟，其中所述磷酸钠缓冲溶液含有0.04U/ml的该3-差向异构酶和10mM下表2中所示的各种单独的单糖，再将每个反应溶液在100℃加热15分钟，以终止该反应，然后，测量了每种反应溶液中该3-差向异构酶的酶活性。
结果显示该3-差向异构酶对D-果糖、D-阿洛酮糖、D-山梨糖、D-塔格糖均具有活性，该3-差向异构酶除可用于生产D-阿洛酮糖，也可用于生产D-塔格糖。
表2
  相对活性(％)
果糖 63.9
D-阿洛酮糖 100
山梨糖 21
D-塔格糖 40.7
实施例5：pH和温度和对3-差向异构酶活性的影响
在实施例5中，为了研究不同pH和温度对该3-差向异构酶活性的影响，在不同的温度和pH条件下将果糖作为底物测定对其活性的影响，且比较了在不同温度和pH下的酶活性。为了研究pH的作用，该3-差向异构酶的反应在pH范围6.0-8.5 50mM的磷酸钠缓冲溶液中进行，其中所述磷酸钠缓冲溶液含有0.04U/mL的该3-差向异构酶和10％(w/v)的果糖。在此，各自的反应在60℃、无金属离子加入的条件下进行20分钟，然后在100℃加热15分钟来终止该反应，并测量了其酶活性。其结果在图2-1中所说明。
为了研究温度的作用，该反应在50mM的磷酸钠缓冲溶液中，温度范围40-90℃中进行了20分钟，其中所述磷酸钠缓冲溶液含有0.04U/mL的该3-差向异构酶和10％(w/v)的果糖，在pH7.5条件下进行反应。通过在100℃加热15分钟来终止该反应，并测量了其酶活性。其结果在图2-2中所说明。
结果显示该3-差向异构酶的最佳pH和温度分别为7.5和90℃。
图2-1是显示在本发明实施方案条件下pH对于3-差向异构酶活性影响的曲线图。
图2-2是显示在本发明实施方案条件下温度对于3-差向异构酶活性影响的曲线图。
实施例6:3-差向异构酶的热稳定性
在实施例6中，为了研究该3-差向异构酶的热稳定性，将该3-差向异构酶分别置于不同温度条件下保温，在不同时间取样将果糖作为底物测定其残余活性。测量是在将该3-差向异构酶分别置于50℃、60℃、70℃、80℃和90℃水浴条件下保温后，每隔1小时取样进行的。该3-差向异构酶反应在50mMpH7.5的磷酸钠缓冲溶液中，60℃条件下进行20分钟，其中所述磷酸钠缓冲溶液含有0.04U/mL的3-差向异构酶和10％(w/v)的果糖，再将该反应溶液在100℃加热15分钟，以终止该反应，然后测量该3-差向异构酶的活性。其结果在图3中所说明。
结果显示该3-差向异构酶具有优良的热稳定性，在60℃保温12小时后活性仍没有检测到活性下降，在80℃保温12小时后，活性仍然保持在80％以上，在90℃保温8小时后仍然有50％的残余活性。
实施例7：利用3-差向异构酶所转化果糖生产D-阿洛酮糖的转化率
在实施例7中，该3-差向异构酶的反应在pH7.5 50mM的磷酸钠缓冲溶液中，温度范围40-90℃ 条件下进行12小时，从而容许该反应充分地进行，其中所述磷酸钠缓冲溶液含有该3-差向异构酶0.04U/mL，1mM的钴离子，和10％(w/v)的果糖。然后，通过在100℃加热15分钟来终止该反应，并测量样品中果糖和D-阿洛酮糖的含量。其结果在图4中所说明。
结果显示在12小时后该3-差向异构酶转化果糖生产D-阿洛酮糖的转化率在90℃时转化率最高，为39％，50℃时转化率最低，为22％，而在60℃时转化率为37％。
实施例8：利用3-差向异构酶生产D-阿洛酮糖
为了生产高浓度的D-阿洛酮糖，该反应在pH7.5 50mM的磷酸钠缓冲溶液中，60℃条件下进行反应，其中所述磷酸钠缓冲溶液含有10U/mL的该3-差向异构酶，1mM的钴离子和700g/L的果糖。然后在不同反应时间点取样，通过在100℃加热15分钟终止该反应，并测量样品中D-阿洛酮糖的浓度。不同反应时间的D-阿洛酮糖产量显示在下表3中。
表3
  D-阿洛酮糖(g/L)
1 98
2 154
3 182
4 210
6 259
8 257
结果显示，反应6小时，产生了259g/L的D-阿洛酮糖，转化率约为37％。
实施例9：通过固定化酶生产D-阿洛酮糖
为了研究该生产D-阿洛酮糖方法的效率，对该3-差向异构酶进行了固定化。测量了固定化的3-差向异构酶生产产量，将其与未固定的(游离的)3-差向异构酶的生产产量进行比较。
对于固定在载体上的3-差向异构酶，使用了3-差向异构酶-海藻酸钠复合体球，该小球是通过如下方法制备的：将3-差向异构酶溶液加入到2.5％(w/v)的海藻酸钠溶液中，所述海藻酸钠溶液的体积是所述3-差向异构酶溶液体积的1.5倍，再利用注射器泵和真空泵将该混合物加入到0.2M钙离子溶液中。
除了使用了固定的3-差向异构酶之外，该反应以与实施例7中相同的方式进行。该反应中所用的3-差向异构酶的量为10U/mL，并测量了D-阿洛酮糖生产率。其结果显示在下表4中。
表4
反应时间(小时) D-阿洛酮糖(g/L)
1 89
2 141
3 165
4 190
6 231
8 258
10 260
结果显示，固定化的3-差向异构酶在反应8小时后达到最大产量258g/L，转化率约为37％，比游离的3-差向异构酶反应速度稍慢，但是固定化的3-差向异构酶更利于连续化生产，实现D-阿洛酮糖的高效生产。
实施例10：D-阿洛酮糖在生物反应器中的生产产量
以下反应在生物反应器中进行，从而检验实施例9的固定化的3-差向异构酶生产产量。
首先，该固定化的3-差向异构酶和果糖以与实施例9中相同的方式制备，将果糖加入到该固定化的3-差向异构酶中，并将该混合物调节为体积100mL随后，将高100cm和直径2.6cm的生物反应器中充满所述固定化的3-差向异构酶和果糖的混合物，且该反应在流速10mL/h和60℃条件下进行。所用的3-差向异构酶的量为500U，且所用果糖的浓度限制为600g/L，这归因于长时间操作过程中过量果糖的沉淀问题。其结果显示在下表5中。
表5
Figure PCTCN2017078923-appb-000001
结果显示，3-差向异构酶和果糖间的反应在整个30天的试验周期中是稳定的，从果糖向D-阿洛酮糖的转化率为37％，产物D-阿洛酮糖浓度为220g/L。其产量能够满足糖类大规模生产的需要。
因此，本发明能够提供利用生物反应器的D-阿洛酮糖生产系统，所述生物反应器能够进行工业规模的大规模生产。
实施例11：利用3-差向异构酶所转化山梨糖生产D-塔格糖的转化率
在实施例11中，该3-差向异构酶的反应在pH7.5 50mM的磷酸钠缓冲溶液中，温度范围40-90℃条件下进行12小时，从而容许该反应充分地进行，其中所述磷酸钠缓冲溶液含有该3-差向异构酶0.04U/mL，1mM的钴离子，和10％的山梨糖。然后，通过在100℃加热15分钟来终止该反应，并测量样品中山梨糖和D-塔格糖的含量。其结果在图5中所说明。
结果显示在12小时后该3-差向异构酶转化山梨糖生产D-塔格糖的转化率在90℃时转 化率最高，为36％，50℃时转化率最低，为29％，而在60℃时转化率为34％。
实施例12：利用3-差向异构酶生产D-塔格糖
为了生产高浓度的D-塔格糖，该反应在pH7.5 50mM的磷酸钠缓冲溶液中，60℃条件下进行反应，其中所述磷酸钠缓冲溶液含有20U/mL的该3-差向异构酶，1mM的钴离子和500g/L的山梨糖。然后在不同反应时间点取样，通过在100℃加热15分钟终止该反应，并测量样品中D-塔格糖的浓度。不同反应时间的D-塔格糖产量显示在下表6中。
表6
反应时间(小时) D-塔格糖(g/L)
1 65
2 101
3 120
4 139
6 171
8 170
结果显示，反应8小时，产生了171g/L的D-塔格糖，转化率约为34％。
实施例13：通过固定化酶生产D-塔格糖
为了研究该生产D-塔格糖方法的效率，对该3-差向异构酶进行了固定化。测量了固3定化的3-差向异构酶生产产量，将其与未固定的(游离的)3-差向异构酶的生产产量进行比较。
对于固定在载体上的3-差向异构酶，使用了3-差向异构酶-海藻酸钠复合体球，该小球是通过如下方法制备的：将3-差向异构酶溶液加入到2.5％(w/v)的海藻酸钠溶液中，所述海藻酸钠溶液的体积是所述3-差向异构酶溶液体积的1.5倍，再利用注射器泵和真空泵将该混合物加入到0.2M钙离子溶液中。
除了使用了固定的3-差向异构酶之外，该反应以与实施例12中相同的方式进行。该反应中所用的3-差向异构酶的量为20U/mL，并测量了D-塔格糖生产率。其结果显示在下表7中。
表7
反应时间(小时) D-塔格糖(g/L)
1 59
2 95
3 108
4 125
6 153
8 170
10 171
结果显示，固定化的3-差向异构酶在反应10小时后达到最大产量170g/L，转化率约为34％，与游离的3-差向异构酶相比反应速度稍慢，但是固定化的3-差向异构酶更利于连续化生产，实现D-塔格糖的高效生产。
实施例14：D-塔格糖在生物反应器中的生产产量
以下反应在生物反应器中进行，从而检验实施例13的固定化的3-差向异构酶生产产量。
首先，该固定化的3-差向异构酶和山梨糖以与实施例13中相同的方式制备，将山梨糖加入到该固定化的3-差向异构酶中，并将该混合物调节为体积100mL随后，将高100cm和直径2.6cm的生物反应器中充满所述固定化的3-差向异构酶和山梨糖的混合物，且该反应在流速10mL/h和60℃条件下进行。所用的3-差向异构酶的量为400U，且所用山梨糖的浓度为400g/L。其结果显示在下表8中。
表8
Figure PCTCN2017078923-appb-000002
结果显示，3-差向异构酶和山梨糖间的反应在整个30天的试验周期中是稳定的，从山梨糖向D-塔格糖的转化率为34％，产物D-塔格糖浓度为170g/L。其产量能够满足糖类大规模生产的需要。
因此，本发明能够提供利用生物反应器的D-塔格糖生产系统，所述生物反应器能够进行工业规模的大规模生产。

Claims (13)

  1. 一种新型3-差向异构酶，其选自下组：
    (a)多肽或蛋白质，其氨基酸序列与SEQ ID No：2具有至少70％，例如至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或甚至100％序列同一性；
    (b)多肽或蛋白质，其由多核苷酸编码，所述多核苷酸在中等-高严格条件下与以下杂交：(i)SEQ ID No：1的多肽编码序列，(ii)包含SEQ ID No：1的多肽编码序列的基因组DNA序列，或(iii)(i)或(ii)的全长互补链；
    (c)多肽或蛋白质，其由多核苷酸编码，所述多核苷酸与SEQ ID NO：1的多肽编码序列具有至少70％，例如至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或甚至100％序列同一性；
    (d)SEQ ID NO：2的多肽或蛋白质包含一个或多个(几个)氨基酸的取代、缺失和/或插入的变体；
    (e)任何(a)、(b)或(c)的多肽或蛋白质，其氨基酸序列包含SEQ ID No：2或由SEQ ID No：2组成，和
    (f)(a)，(b)，(c)，(d)，或(e)的多肽或蛋白质的片段，其具有3-差向异构酶活性。
  2. 权利要求1的分离的多肽或蛋白质，其与SEQ ID No：2的多肽或蛋白质的氨基酸序列具有至少70％，例如至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或甚至100％序列同一性。
  3. 权利要求1或2任一项的分离的多肽或蛋白质，其包含SEQ ID No：2或由SEQID No：2组成。
  4. 一种分离的多核苷酸，其编码权利要求1至3任一项的多肽或蛋白质。
  5. 一种核酸构建体或表达载体，其包含权利要求4的多核苷酸，所述多核苷酸可操作地连接于一个或多个(几个)调控序列，所述调控序列指导所述多肽或蛋白质在表达载体中的产生。
  6. 一种重组宿主细胞，其包含权利要求4的多核苷酸，所述多核苷酸可操作地连接于一个或多个调控序列，所述调控序列指导所述多肽或蛋白质的产生。
  7. 一种产生权利要求1至3任一项的多肽或蛋白质的方法，其包括：
    (a)在有助于所述多肽或蛋白质的产生的条件下培养细胞，其以其野生型形式产生所述多肽或蛋白质；
    和
    (b)回收所述多肽或蛋白质。
  8. 一种产生权利要求1至3任一项的多肽或蛋白质的方法，其包括：
    (a)在有助于所述多肽或蛋白质的产生的条件下培养权利要求6的宿主细胞；和
    (b)回收所述多肽或蛋白质。
  9. 一种组合物，其包含权利要求1至3任一项的多肽或蛋白质和其它酶。
  10. 权利要求1至3任一项的多肽或蛋白质或权利要求9的组合物在用于生产D-阿洛酮糖的工艺中的用途。
  11. 权利要求1至3任一项的多肽或蛋白质或权利要求9的组合物在用于生产D-塔格糖的工艺中的用途。
  12. 一种用于生产D-阿洛酮糖的方法，其包括将果糖、葡萄糖及淀粉糖等混合糖与权利要求1至3任一项的多肽或蛋白质的水性溶液相接触生产D-阿洛酮糖。
  13. 一种用于生产D-塔格糖的方法，其包括将山梨糖及淀粉糖等混合糖与权利要求1至3任一项的多肽或蛋白质的水性溶液相接触生产D-塔格糖。
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