CN116536391A - High-flux detection method for D-fructose and rare sugar - Google Patents

High-flux detection method for D-fructose and rare sugar Download PDF

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CN116536391A
CN116536391A CN202310605916.9A CN202310605916A CN116536391A CN 116536391 A CN116536391 A CN 116536391A CN 202310605916 A CN202310605916 A CN 202310605916A CN 116536391 A CN116536391 A CN 116536391A
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tagatose
fructose
psicose
epimerase
sugar
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刘展志
吴敬
郭雪红
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Jiangnan University
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Abstract

The invention discloses a high-throughput detection method of D-fructose and rare sugar, and belongs to the technical field of biology. The patent focuses on a mixed sugar system generated in the biosynthesis process of D-fructose to D-psicose or D-tagatose, and the D-fructose is consumed through the specific metabolism of active dry yeast, so that the D-psicose or D-tagatose in the reaction system is measured through a resorcinol method. The detection method is convenient and efficient and has low cost. The method is applied to high-throughput screening of rare sugar to prepare key enzyme, D-tagatose 4-epimerase mutants with improved heat stability and specific activity are screened, compared with wild type, the heat stability of the mutant I430P is improved by 1.83 times, and the specific activity of the G90S/T272A/I430P mutant is improved by 21.4%.

Description

High-flux detection method for D-fructose and rare sugar
Technical Field
The invention relates to a high-throughput detection method of D-fructose and rare sugar, belonging to the technical field of biology.
Background
In the carbohydrate research field, rare sugars are an important category of research. The definition of the international sugar society (ISRS) for rare sugars is: a class of monosaccharides and their derivatives that are found in nature but in very small amounts. Although the content is very small in nature, these substances play a very important role in the fields of diet, health care, medicine and the like. For example, D-psicose is a low energy, non-digestible sucrose substitute for humans; d-tagatose has the advantages of low calorie, low absorption, caries resistance, blood sugar reduction, intestinal flora improvement, good physicochemical properties and the like; d-allose has the functions of inhibiting active oxygen, inhibiting cancer cell proliferation and the like; the L-series sugar can be used as raw materials for producing various chemical products and medicines.
With the increasing global obesity patients, people's importance and demand for low calorie sweeteners are increasing, and natural healthy rare sugar has become a current research hotspot as a low calorie sweetener. The rare sugars which have been industrially produced at present mainly include D-psicose, D-tagatose, etc.
D-psicose is an epimer at the C-3 position of D-fructose, which is a six-carbon reducing ketose. D-psicose has a sweetness equivalent to 70% of sucrose, but a caloric value of only 0.4 kcal.g -1 Is 90% lower than sucrose. The U.S. food and drug administration (Food and Drug Administration, FDA) issues certification in month 6 of 2014, deems D-psicose to be a "generally recognized safe" (Generally Recognized as Safe, GRAS) grade food, and D-psicose was removed from the "total sugar" and "added sugar" portions of the food nutrition and additive label in 2019, and thus D-psicose is a potential sucrose substitute. D-psicose has no astringent taste and no intolerance symptoms such as diarrhea when compared with sugar alcohol, has stable property when compared with most other existing sweeteners, and can interact with amino acid and protein in food during food preparation and heating to generate Maillard reaction, thereby providing unique color and flavor for food.
D-tagatose is used as a naturally occurring hexulose, is an epimer of D-fructose at a C-4 position, has a relative molecular weight of 180.16, is white crystalline powder, is easy to cause caramelization reaction and Maillard browning reaction, has the sweetness of 92% of sucrose, has the energy of only 30% of sucrose, and belongs to a low-calorie sweetener. D-tagatose can meet the requirement of a large amount of sugar-containing products such as chocolate, chewing gum, cake, ice cream and the like on low-calorie sweeteners, and can also produce synergistic effect with some high-intensity sweeteners. In 2001, D-tagatose was approved by the FDA as a food additive and approved by the general accepted safety (GRAS) agency, and the world health organization associated with the food additive Committee recommended D-tagatose as a new low-calorie sweetener in the same year, and could be used as a food additive. In 2014, D-tagatose was approved by china as a new food material, and can be used in foods other than infant foods, and there is no limitation on the intake thereof.
The rare sugars are low in nature and are generally produced in large quantities by chemical or biological methods. The chemical method has certain disadvantages, such as severe reaction conditions, difficult control, more byproducts, unfavorable separation and purification, and the like. The biological method has the advantages of high conversion efficiency, strong specificity, few byproducts, simple separation and purification and the like, so the biological method becomes a main method for synthesizing rare sugar. The key enzyme for preparing the D-psicose is D-psicose 3-epimerase, and the D-psicose can be prepared from the D-fructose by epimerization reaction. Shin et al used Tagatose 3-epimerase (Tagaturonate 3-epomerase) derived from Thermotoga petroleum (Thermotoga petrophila) as a research material, and obtained an optimal mutant S125D/N129T/L140P/T181A/H362L by molecular engineering, which produced a 184-fold increase in D-Tagatose activity with D-fructose as a substrate, was designated Tagatose 4-epomerase, T4E (ACS Catalysis,2020,10 (20): 12212-12222). However, T4E still has the problem of poor thermal stability and poor specific activity, and limits industrial application.
In the mixed system of D-fructose and D-psicose and D-fructose and D-tagatose, as the D-fructose and the D-tagatose/D-psicose are ketoses, the conventional ketose detection method cannot distinguish the D-fructose and the D-tagatose from each other, but the conventional method for detecting the D-fructose by using the D-fructose-dehydrogenase has the defect of high cost, so that the detection means of the D-psicose and the D-tagatose are deficient.
Disclosure of Invention
The invention discloses a method for distinguishing and detecting specific sugar in a mixed system of D-fructose and D-psicose and D-fructose and D-tagatose, and a tagatose-4 epimerase mutant with improved heat stability and D-fructose conversion rate is screened by using the method. D-fructose in the reaction system is metabolized by adding a dry yeast solution to the scarce sugar mixture system, and then the residual sugar in the system is specifically detected by resorcinol reagent.
The invention provides a method for detecting rare sugar in a mixed system of D-fructose and rare sugar, which comprises the steps of digesting the D-fructose by utilizing yeast, and detecting residual sugar in the system by using resorcinol reagent, wherein the rare sugar comprises D-tagatose and D-psicose.
The invention also provides application of the method in preparing key enzyme by screening rare sugar in high throughput.
The invention also provides a method for preparing key enzyme by high-throughput screening of rare sugar, which comprises the following steps:
(1) Adding a buffer solution containing D-fructose into the enzyme solution;
(2) Adding yeast to the product obtained in the step (1);
(3) The resorcinol reagent was used to detect the remaining sugars in the system.
In one embodiment, the key enzyme comprises D-psicose 3-epimerase, D-tagatose 3-epimerase or D-tagatose 4-epimerase.
In one embodiment, the yeast of step (2) is Saccharomyces cerevisiae or Pichia pastoris at a concentration of 0.1 to 50 g.L -1 。
In one embodiment, the product obtained in step (2) is subjected to a temperature of from 25 to 40℃and a temperature of from 200 to 800 r.min -1 The reaction is carried out for 12-36h.
In one embodiment, the D-fructose-containing buffer of step (1) comprises 5 to 500 mmoL.L -1 D-fruitSugar.
The invention also provides the D-tagatose 4-epimerase mutant obtained by screening by the method, wherein the amino acid sequence shown in SEQ ID NO.1 is subjected to any mutation as follows:
(1) Mutating isoleucine at position 430 to proline;
(2) Glycine at position 90 was mutated to serine, threonine at position 272 was mutated to alanine, and isoleucine at position 430 was mutated to proline.
The invention also provides host cells expressing the mutants.
The invention also provides the use of said mutant, or said host cell, in the preparation of D-tagatose or a product comprising D-tagatose.
The beneficial effects are that:
the invention provides a method for detecting the rare sugar in a mixed system of D-fructose and the rare sugar, which utilizes yeast to digest the D-fructose and resorcinol to detect the residual rare sugar, and the detection method has high accuracy and low cost, and the cost of single detection of a single sample is not more than 0.1 yuan.
The method is applied to preparing key enzyme by high-throughput screening of rare sugar, the D-tagatose 4-epimerase mutant with improved heat stability and specific activity is screened, compared with a wild type, the heat stability of the mutant I430P is improved by 1.83 times, and the specific activity of the G90S/T272A/I430P mutant is improved by 21.4%.
Drawings
FIG. 1 is a graph showing the effect of yeast in digesting D-fructose, D-tagatose and D-psicose.
FIG. 2 is a graph of yeast digestion results; wherein, (a): d-fructose; (b): d-tagatose; (c): sugar is mixed.
FIG. 3 is a D-psicose standard curve.
FIG. 4 is a D-tagatose standard curve.
FIG. 5 is a high throughput screen of tagatose-4 epimerase (T4E).
FIG. 6 shows the thermostability of mutants I430P and G90S/T272A/I430 during shake flask rescreening.
FIG. 7 shows the results of D-tagatose synthesis from mutants I430P and G90S/T272A/I430.
Detailed Description
The invention is further illustrated below in conjunction with specific examples.
The reagents involved in the following examples were as follows:
the examples described below refer to 4- (2-hydroxyethyl) -1-piperazine propane sulfonic acid (HEPPS), D-psicose, D-tagatose, D-fructose all purchased from Milin Biochemical Co., ltd (Shanghai), resorcinol from Aldamasces reagent Co., ltd (Shanghai), active dry yeast from Angel Yeast Co., ltd (Shanghai), D-fructose, D-psicose, D-tagatose standard from Vita chemical Co., ltd (Shanghai). The construction host used was E.coli JM109, the expression host used was E.coli BL21 (DE 3), the plasmid vector used was pET-24a, and the resistance was kanamycin resistance.
The 96 shallow hole plates used in the high-throughput screening process are sterile 96 shallow hole plates, and the 96 deep hole plates are subjected to high-temperature and high-pressure steam sterilization (121 ℃ for 20 min) after being wrapped by newspapers. The split charging process of the sterilized culture medium is carried out in an ultra-clean workbench, and 100 mug/mL kanamycin is added before split charging of the culture medium. The seed culture medium is LB culture medium, and 200 mu L of seed culture medium is added into a 96 shallow hole plate; the fermentation medium is TB medium, and 1mL of the fermentation medium is added into a 96-deep well plate.
The following examples relate to the following media:
LB medium (g/L): peptone 10, yeast powder 5, naCl 10.
TB Medium (g/L): peptone 12, yeast powder 24, glycerol 10, K 2 HPO 4 ·3H 2 O 16.43，KH 2 PO 4 2.3。
The detection method involved in the following examples is as follows:
resorcinol detection method:
the digested shallow pore plate is processed at 4000 r.min -1 Centrifuging for 15min, collecting 100 μl of the supernatant, adding 100 μl of Seliwanoff reagent into each well, sealing, and accurately reacting in boiling water bathAfter the reaction was completed for 10min, the mixture was cooled in an ice bath, 150. Mu.L of the mixture was placed in a shallow well plate, and absorbance at 400nm was measured by using an ELISA reader.
Preparation of Seliwanoff reagent (as-prepared): weighing resorcinol with a certain mass, dissolving in 12% hydrochloric acid (diluted three times by concentrated hydrochloric acid), and the final concentration of resorcinol is 2g.L -1 。
The protein concentration detection method comprises the following steps:
protein gels were prepared according to the instructions of the Bio-Rad protein gel kit. First, a 12% separation gel was prepared, followed by a 5% concentration gel. Protein gel sample preparation: mu.L of SDS-PAGE protein loading buffer (5X) was added to 20. Mu.L of the sample. The treated sample was then placed in a metal bath at 100℃for 10min and centrifuged at 8000g for 30s. Protein gel electrophoresis was performed using Tris-HCl buffer (pH 8.3). And after the protein electrophoresis is finished, performing gel staining and gel decoloring, then using a gel imaging system to take pictures, and using imageJ software to perform image analysis to obtain the ratio of the target protein to the total protein. The total protein concentration was determined using Bradford protein concentration determination kit (beyotidme from bi-cloud) and the protein concentration of interest was calculated.
The product detection method comprises the following steps:
sample after enzyme inactivation treatment is carried out at 12000 r.min -1 The mixture was centrifuged for 5min, and the supernatant was taken out and diluted to a certain concentration, after which the sample was filtered with a 0.22 μm filter membrane. Chromatographic conditions: agilent 1200HPLC chromatograph, agilent Hi-Plex Ca, 300X 7.7mm chromatographic column, agilent differential detector, agilent autosampler, mobile phase pure water, column temperature 80 ℃ and flow rate 0.5 mL-min -1 。
Example 1: digestion of rare sugars
Respectively preparing 25 g.L -1 D-fructose, D-tagatose and D-psicose solutions of (C) were added thereto at a final concentration of 5 g.L, respectively -1 Is dried in the presence of a yeast solution at 30℃and 200 r.min -1 Shake culturing for 24h. After digestion was complete, samples were taken and boiled for 10min for inactivation, followed by 12000 r.min -1 Centrifugation was performed for 10min, and the supernatant was assayed using resorcinol. As a result, the absorbance of D-fructose after digestion is shown in FIG. 1Significantly reduced, without substantial change in absorbance after digestion of D-tagatose and D-psicose, indicating that the yeast is able to digest D-fructose while having substantially no digestibility for D-tagatose and D-psicose.
Example 2: establishment of high throughput screening methods
Respectively preparing 25 g.L -1 D-fructose and 25 g.L -1 D-tagatose solution of (2) and mixing the two according to a ratio of 1:1 to prepare the solution containing 12.5 g.L -1 D-fructose of (C) and 12.5 g.L -1 D-tagatose of (a).
Yeast digestion experiments: respectively taking 10mL of the above solution in conical flask, adding 50mg of active dry yeast, sealing, and sealing at 30deg.C for 200 r.min -1 Shake culturing for 24 hr, sampling every 2 hr, boiling for 10min, inactivating, and sterilizing at 12000 r.min -1 Centrifuging for 10min, and detecting the supernatant in liquid phase. As shown in FIG. 2, the dry yeast can completely metabolize D-fructose in 12h without having a substantial metabolic capacity for D-tagatose, and in a mixed system of D-fructose and D-tagatose, D-fructose is completely metabolized without substantial change in D-tagatose concentration, and the result shows that the method can remove D-fructose in the system without affecting D-tagatose content.
Drawing a D-tagatose standard curve: as a result of measuring D-tagatose at various concentrations by the resorcinol method, the concentration of D-tagatose was 0 to 30 mmol.L as shown in FIG. 4 -1 In the range, the concentration of D-tagatose can be sensitively measured through resorcinol, and the linear relation between the concentration of D-tagatose and absorbance is good. The content of D-tagatose can be accurately measured through resorcinol, and the sensitivity of the method can be used for high-throughput screening.
Drawing a D-psicose standard curve: the results of measuring D-psicose at different concentrations by resorcinol method are shown in FIG. 3, in which D-psicose concentration is 0-20 mmol.L -1 In the range, the concentration can be measured sensitively by resorcinol, and the linear relation between the concentration and the absorbance is good. Proved by the fact that the content of D-psicose can be accurately measured by resorcinolIn amounts, and the sensitivity of the method can be used for high throughput screening.
Example 3: construction of tagatose-4 epimerase (T4E) mutation library
Since the crystal structure of T4E is not yet clear, random mutagenesis of T4E complete sequences was performed by error-prone PCR.
1. Random mutation
The high throughput screening primers were:
forward primer F: ATATTTTCCGGATCTGAAACCGGTTAGA;
reverse primer R: TCATTTTGTTCCAGTGTTTTAAACAGTCTTTCTTT;
the mutant library is constructed by MEGAWHOP, and the reaction system and reaction conditions are as follows:
the T4E encoding gene is amplified by taking pET-24a (+) -T4E (after the company synthesizes the T4E encoding gene shown in SEQ ID NO.2, the vector is connected after BamHI and HindIII double enzyme digestion) as a template. PCR System (50. Mu.L): 50ng template, 5. Mu.L 10 XPCR buffer, 0.5. Mu.L rTaq enzyme, 1. Mu.L upstream and downstream primer (10. Mu.mol.L) -1 ) 4 mu L dNTP mix, final concentration of 0.15 mmoL.L -1 Mn of (2) 2+ ，ddH 2 And (3) supplementing O. Amplification procedure: 98 ℃ for 3min;30 cycles (98 ℃,30s;55 ℃,30s;72 ℃,1 min), 72 ℃,5min,4 ℃ heat preservation. Then, the PCR product is subjected to nucleic acid electrophoresis, and after the correct size of the strip is confirmed, the gel cutting and recovery are performed.
PCR was performed by MEGAWHOP method using the recovered target fragment as a primer to amplify the whole plasmid. The PCR system is the same as the system, the upstream and downstream primers are replaced by gel recovery fragments, and the procedure is as follows: 72 ℃ for 5min;98 ℃ for 3min; cycling 30 (98 ℃,30s;55 ℃,30s;72 ℃,7 min); 72 ℃ for 5min; preserving at 4 ℃.
2. Transformation of mutant library genes
mu.L of the PCR product was taken, 1. Mu.L of Dpn I enzyme and 1. Mu.L of buffer were added thereto, and the mixture was subjected to a water bath at 37℃for 2 hours, thereby performing a template digestion. After digestion of the template, the PCR products were transformed to e.coli BL21 (DE 3), spread on a resistance plate, and incubated at 37 ℃ at rest.
(1) After the colony size and number on the plate are proper, single colony is picked for library establishment.
(2) 200. Mu.L of LB liquid medium (Kan: 30. Mu.g.multidot.mL) was added to each well of a sterile 96-well plate -1 ) Single colonies on the plates were picked into each well of a shallow plate containing LB liquid medium using sterilized toothpicks, and a blank control and a wild type control were set, respectively.
(3) Sealing the 96 shallow hole plate at 37 deg.C and 700 r.min -1 Shake culturing for about 10 hr. Adding 50 mu L of sterilized 60% glycerol into the residual bacterial liquid in the shallow pore plate, sealing, and storing in a refrigerator at-80 ℃ for later use.
3. Fermentation of mutant library strains
(1) 1mL of TB medium (Kan: 30. Mu.g.mL) was added to each well of the sterilized 96-well plate -1 ) Transferring seed solution in shallow hole plate into deep hole plate at 5% inoculation amount, sealing deep hole plate with gauze, and transferring to 37deg.C, 750r.min -1 Shaking culture for 2 hr, transferring to 25deg.C, 750r.min -1 And (5) fermenting and inducing expression for 24h.
(2) After the deep-hole plate fermentation is finished, the deep-hole plate is subjected to the fermentation at 4 ℃ and 4000 r.min -1 Centrifuging for 15min, discarding the fermentation supernatant, and storing the rest thallus in a refrigerator at-20deg.C.
Example 4: screening mutants with improved D-fructose conversion by novel high throughput screening methods
(1) 300. Mu.L of HEPS buffer (containing 50 mol.L.) was added to each well of the deep well plate in which the cells were left -1 D-fructose and 1.5 mmoL.L -1 Ni of (2) 2+ ) Shaking, mixing, and heating to 60deg.C, 750r.min -1 The reaction was carried out for 2 hours.
(2) After the reaction is finished, the deep pore plate is positioned at 4000 r.min -1 Centrifuging for 15min, collecting 200 μl of the supernatant, adding 20 μl of dry yeast solution (dry yeast concentration 50g.L) into each well of the shallow plate -1 ) Purging, mixing, sealing at 30deg.C, 750r.min -1 And (3) reacting for 24 hours, and performing yeast digestion treatment.
(3) The digested shallow pore plate is processed at 4000 r.min -1 Centrifuging for 15min, collecting 100 μl of the supernatant, placing in each well of the deep-well plate, and adding 100 μl of Seliwan into each welloff reagent, accurately reacting in boiling water bath for 10min after sealing, cooling in ice bath after the reaction is finished, taking 150 mu L of the solution in a shallow pore plate, and measuring absorbance at 400nm by using an enzyme-labeling instrument.
(4) And selecting mutants with higher absorbance values than wild type to perform 96-well plate rescreening, wherein the rescreening method is basically consistent with that of the primary screening, three groups of mutants are arranged in parallel, and the screening process is repeated.
(5) Shaking and re-screening: sequencing the mutant with good expression in 96-well plate, adding the mutant strain with mutation into LB culture medium (kanamycin 30 μg.mL) at 2% -1 ) And at 37deg.C, 200r.min -1 Shake culturing for about 10 hr. The cultured bacterial liquid was added to TB medium (kanamycin 30. Mu.g.mL) at a concentration of 5% -1 ) And at 37 ℃ and 200 r.min -1 Shake culturing for 2 hr to OD 600 About 0.6, and then changing the culture temperature at 25deg.C, 200r.min -1 Culturing for 24h, and performing induced expression. After fermentation, the obtained bacteria are re-suspended to 50OD, high-pressure homogenizing and wall breaking are carried out, and nickel column purification is carried out, so that pure enzyme is prepared.
(6) Protein purification
a, using HEPPS buffer solution with the pH of 8.5 to suspend the bacterial body, opening a high-pressure homogenizer in advance to pre-cool and clean, adding the bacterial liquid after the suspension into a sample tank, and breaking the cell wall of the bacterial liquid under 800-900bar, wherein the wall breaking time of each sample is 3min.
b, breaking cell wall of the bacterial liquid at 4 ℃ and 8000 r.min -1 The supernatant was collected by centrifugation at low temperature for 20min, and the supernatant after centrifugation was filtered using a 0.22 μm filter head to remove impurities.
c, washing the nickel column with 100mL of deionized water to remove residual ethanol, washing the nickel column with 100mL of buffer A to perform column balancing, and then hanging the crude enzyme solution on the column at a low flow rate, and repeating the column hanging for three times.
Washing the nickel column with 100mL of buffer A to remove the impurity proteins with weak binding capacity, and then respectively using 60mL of 30 mmol.L -1 Is 60mL 60 mmol.L -1 Eluting the hybrid protein from the imidazole solution; then respectively using 60mL 90 mmol.L -1 、60mL 150mmol·L -1 Eluting the target protein from the imidazole solution.
e adding deionized water into ultrafiltration tube with 10kDa size at 4deg.C and 3500 r.min -1 And (3) centrifuging for 20min to carry out ultrafiltration cleaning, adding the eluent containing the target protein into an ultrafiltration tube to carry out ultrafiltration, adding a certain volume of HEPPS buffer solution into the ultrafiltration tube to carry out ultrafiltration so as to replace the buffer solution after the ultrafiltration is finished, and collecting and storing the enzyme solution for later use.
Measurement of enzyme Activity: 200. Mu.L of an enzyme solution having an equal protein concentration was added to 800. Mu.L of a solution containing 50 mmol.L -1 HEPPS buffer of D-fructose (50 mmol.L) -1 ，pH 8.5，1.5mmol·L -1 Ni 2+ ) The buffer solution is preheated in advance, mixed uniformly and then placed at 70 ℃ for reaction for 30min, and then boiled water is used for 10min for enzyme deactivation.
Definition of enzyme activity: the amount of enzyme used to produce 1. Mu. Mol of D-tagatose per minute was one enzyme activity unit (U).
Definition of specific activity: unit of enzyme activity per unit weight (mg) of enzyme.
Thermal stability determination: taking a certain amount of T4E enzyme liquid, placing at 70 ℃ for incubation, sampling at intervals, measuring residual enzyme activity according to the method, defining the enzyme activity at 0h of reaction as 100%, and calculating the relative enzyme activities of different samples.
Screening results: T4E mutant strain 3000 strains were co-screened, wherein D-tagatose synthesis capacity was improved for mutant strain 2 strains, and the results are shown in FIGS. 6 to 7. Wherein the half life of the I430P mutant is 1.83 times of that of the wild type at 70 ℃, and the synthesis capability of D-tagatose is improved to a certain extent. The specific activity of the G90S/T272A/I430P mutant strain is improved by 21.4% compared with that of the wild type strain, and the synthesis capability of D-tagatose is also improved to a certain extent.
Comparative example 1:
the reference example 4 is different in that the yeast digestion step is omitted, and the result shows that D-fructose and D-tagatose and OD cannot be distinguished after the enzymatic reaction 400nm All are about 1.8, and D-fructose and D-tagatose cannot be distinguished, so that subsequent experiments cannot be performed.
Comparative example 2:
specific embodiment referring to example 4, the difference is that D-fructose is specifically detected by using a commercial fructose detection kit, and the result shows that D-fructose can be detected to some extent by comparing the concentration change values of D-fructose before and after the enzymatic reaction to reflect the enzymatic reaction rate, but the screening application is limited due to the high price (usually about 5000 price and 100 times of detection) of the commercial kit, and the cost of single detection of a single sample by the method of the present invention is not more than 0.1 yuan.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A method for detecting the rare sugar in a mixed system of D-fructose and the rare sugar is characterized in that the D-fructose is digested by yeast, and then the residual sugar in the system is detected; the rare sugars include D-tagatose and D-psicose.
2. A method for preparing a key enzyme by high throughput screening of rare sugars using the method of claim 1, wherein said key enzyme is capable of using D-fructose, said method comprising the steps of:
(1) Adding a buffer solution containing D-fructose into an enzyme solution of a key enzyme;
(2) Adding yeast to the product obtained in the step (1);
(3) The resorcinol reagent was used to detect the remaining sugars in the system.
3. The method of claim 2, wherein the key enzyme comprises D-psicose 3-epimerase, D-tagatose 3-epimerase, or D-tagatose 4-epimerase; when the key enzyme is D-psicose 3-epimerase or D-tagatose 3-epimerase, the remaining sugar is D-psicose; when the key enzyme is D-tagatose 4-epimerase, the remaining sugar is D-tagatose.
4. The method of claim 2, wherein the yeast in step (2) is Saccharomyces cerevisiae or Pichia pastoris at a final concentration of 0.1-50 g.L -1 。
5. The process according to claim 2, wherein the product obtained in step (2) is subjected to a temperature of 25 to 40℃and a temperature of 200 to 800 r.min -1 The reaction is carried out for 12-36h.
6. The method of claim 2, wherein the D-fructose-containing buffer of step (1) comprises 5-500 mmol.l -1 D-fructose.
7. Use of the method of claim 1 for high throughput screening of rare sugars for the production of key enzymes.
8. The D-tagatose 4-epimerase mutant obtained by screening by the method according to any one of claims 2 to 6, characterized in that the amino acid sequence represented by SEQ ID No.1 is subjected to any one of the following mutations:
(1) Mutating isoleucine at position 430 to proline;
(2) Glycine at position 90 was mutated to serine, threonine at position 272 was mutated to alanine, and isoleucine at position 430 was mutated to proline.
9. A host cell expressing the mutant of claim 8.
10. Use of the mutant of claim 8, or the host cell of claim 9, for the preparation of D-tagatose or a D-tagatose-containing product.
CN202310605916.9A 2023-05-26 2023-05-26 High-flux detection method for D-fructose and rare sugar Pending CN116536391A (en)

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