CN117343976A - Method for coproducing glyceroglycosides and D-psicose by double enzyme cascading - Google Patents

Method for coproducing glyceroglycosides and D-psicose by double enzyme cascading Download PDF

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CN117343976A
CN117343976A CN202310956545.9A CN202310956545A CN117343976A CN 117343976 A CN117343976 A CN 117343976A CN 202310956545 A CN202310956545 A CN 202310956545A CN 117343976 A CN117343976 A CN 117343976A
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psicose
sucrose
glycerol
epimerase
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章文明
胡海波
姜岷
信丰学
王燕霞
蒋羽佳
姜万奎
陈帅强
钱刘骏
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Nanjing Tech University
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Abstract

The invention discloses a method for coproducing glyceroglycosides and D-psicose by double enzyme cascading, which takes sucrose and glycerol as co-substrates, and simultaneously adds sucrose phosphorylase and D-psicose-3-epimerase to perform a co-catalytic reaction to obtain the glyceroglycosides and the D-psicose. The invention realizes the multienzyme cascade reaction from sucrose to D-psicose for the first time, catalyzes more economic sucrose and glycerin as substrates to produce the D-psicose, has high raw material utilization rate, high D-psicose conversion rate, simple steps, low production cost, little pollution and little influence on environment, and can realize the large-scale production of the D-psicose. Sucrose phosphorylase and D-psicose-3-epimerase produced by recombinant escherichia coli take sucrose and glycerol as substrates, and after ultrasonic crushing, the glycerol glucoside yield of double-enzyme cascade catalytic reaction is 289.8g/L, the D-psicose yield is 93.4g/L, and the glycerol conversion rate is up to 75.6%.

Description

Method for coproducing glyceroglycosides and D-psicose by double enzyme cascading
Technical Field
The invention belongs to the technical fields of bioengineering and genetic engineering, and particularly relates to a research method for catalyzing sucrose and glycerol to co-produce glyceroglycosides and D-psicose by a double-enzyme cascade method.
Background
2-O-alpha-D-glyceroglycoside (2-O-alpha-D-glu-copyranosyl-sn-glycol, C) 9 H 18 O 8 ) Is a glycoside linked by a glucosyl group and a glyceryl group through a glycosidic bond, which is widely found in nature, particularly in salt tolerant cyanobacteria and south african density Luo Mu, and is the most predominant active substance of density Luo Mu capable of surviving and reactivating in extreme environments; in addition, 2-O-alpha-D-glyceroglycosides, which are active ingredients, are also found in Japanese sake. The 2-O-alpha-D-glyceroglycoside has protective effect on organisms, and can form protection on the cell surface when the cells are under severe conditions such as high osmotic pressure, strong ultraviolet rays or droughtThe membrane can effectively prevent the denaturation and inactivation of protein molecules, so that the 2-O-alpha-D-glyceroglycosides can be used as stabilizers of proteins and enzymes. In addition, 2-O- α -D-glyceroglycosides are often used as cosmetic raw materials for improving the moisturizing effect of the skin due to their high moisturizing property and low water absorption. The preparation method has the advantages of high moisture retention, prebiotic property, oxidation resistance, ultraviolet injury resistance and other functions, and has important application value in the fields of food, cosmetics, medicines and the like, so that the realization of the industrialized production of 2-alpha GG has great significance.
2-O-alpha-D-glyceroglycosides have various synthesis modes, in the reported synthesis methods of 2-alpha GG, the chemical method and the microbial method have low synthesis efficiency and complicated separation steps, and an enzyme catalysis method is attracting attention of more researchers as an economic and efficient preparation method, wherein sucrose phosphorylase (Sucrose phosphorylase, SPase) catalyzes sucrose and glycerol to prepare 2-alpha GG, which is the most promising method for realizing industrial production at present.
D-psicose (D-allose, C) 6 H 12 O 6 ) Is hexa-carbon hexulose with molecular weight of 180.16, melting point of 96 deg.C and density of 1.35g/cm 3 Is easily dissolved in water and is the C-3 epimer of D-fructose.
The main component of common edible sugar in daily life is sucrose, typically representing white sugar, brown sugar and the like, the sugar has long been widely used in terms of pure sweetness, good taste and low cost, and is the most widely applied sweetener in the food industry. However, with the improvement of the living standard of people, the requirements on health and physical quality are continuously improved, the defect of sucrose is particularly obvious, and excessive intake of sucrose can cause obesity and blood sugar rise due to high calorie, so that the sucrose is especially unsuitable for diabetics to eat. In 2019, the international diabetes consortium (IDF) issued reports that 9.3% of adults 20-79 years old worldwide have diabetes, i.e., 4.63 million people have diabetes, and 7 hundred million people worldwide are expected to have diabetes in 2045 years, so that with the increasing importance of people on diet health, development of new sweetener substitutes is urgently needed. D-psicose is a rare sugar, the rare sugar is a sugar which naturally exists in the nature and is thinner, the sweetness of the sugar is about half of that of sucrose, unlike the traditional sweetener, when the D-psicose is taken into a human body, most of the sugar is directly discharged through urine or feces, the calorific value of the D-psicose is 0.029kJ/g, the energy generated by the D-psicose is only three percent of that of the equivalent sucrose, and the D-psicose can be used as a perfect substitute of the sucrose sweetener. It may also inhibit liver lipase activity; has the functions of reducing blood fat, reducing blood sugar and resisting oxidation, and can be widely applied to the fields of biological medicines and health care products; d-psicose also induces release of glucagon-like peptide-1 receptor, activates vagal afferent signals, reduces food intake, and is popular in the field of healthy diet; the U.S. food and drug administration has approved D-psicose as a generally recognized safety grade food (GRAS, generally recognized as safe) in 2011. D-psicose is extremely rare in natural world, the extraction amount in plants is extremely small, and trace D-psicose can be extracted in few bacteria, so that the D-psicose is not present in animals, and the large-scale preparation of the D-psicose becomes a main bottleneck at present. Currently, D-psicose is produced and sold by Ing Tay Co., korea Hijie first sugar manufacturing Co., indersen Global group, japanese Song Gum chemical, etc. enterprises exist worldwide. China starts later in the field, but has obvious research progress in recent years.
The current methods for producing D-psicose mainly comprise chemical synthesis methods and biological enzyme methods. The chemical synthesis of D-psicose is an earlier method, and comprises the steps of catalytic hydrogenation, selective aldol condensation synthesis, addition reaction, cyclic closure conversion synthesis, ferrier rearrangement, etc., such as Fang Zhijie, li Song, etc., by utilizing 1-deoxyiodo-ketose to generate reaction, then hydrolyzing under alkaline condition to obtain ketose intermediate, and selectively protecting and deprotecting hydroxyl group of the ketose intermediate to synthesize D-psicose. However, chemical methods are not neglected to the environment and human body, and are difficult to mass production due to low economic benefits. The bioenzyme method is researched and applied at present, and mainly takes fructose as a raw material to produce D-psicose through catalysis of D-psicose-3-epimerase. However, the substrate fructose of the method has higher cost and low economic benefit.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide a method for coproducing glyceroglycosides and D-psicose by double enzyme cascade.
The aim of the invention is achieved by the following technical scheme:
a method for coproducing glyceroglycosides and D-psicose by double enzyme cascade is characterized in that sucrose and glycerol are used as co-substrates, and sucrose phosphorylase and D-psicose-3-epimerase are added to perform a co-catalytic reaction to obtain the glyceroglycosides and the D-psicose.
As a preferred embodiment, recombinant expression strains of sucrose phosphorylase and D-psicose-3-epimerase are respectively constructed, and the two recombinant expression strains are subjected to induced expression and ultrasonic crushing, and then sucrose and glycerol are added for enzyme catalytic reaction to obtain glyceroglycoside and D-psicose.
As a preferred embodiment, recombinant expression strains of sucrose phosphorylase and D-psicose-3-epimerase are constructed using E.coli as a host strain, respectively.
As a preferred embodiment, the expression cassettes for sucrose phosphorylase and D-psicose-3-epimerase each use the T7 promoter of E.coli as a promoter and the T7 terminator of E.coli as a terminator.
As a preferred embodiment, the sucrose phosphorylase is derived from leuconostoc mesenteroides Leuconostoc Mesenteroides (ATCC 12291); the D-psicose-3-epimerase is derived from agrobacterium tumefaciens A.Tumefaciens (ATCC 33970), and the recombinant escherichia coli BL21 (DE 3) is formed by inserting coding genes of sucrose phosphorylase and D-psicose-3-epimerase into plasmids pET-22b (+) and pET-Deut1 respectively to form recombinant plasmids pET-22b (+) -SPase and pET-Deut1-DPease.
As a preferred embodiment, the temperature of the enzyme-catalyzed reaction is 25-30℃and the reaction time is 20-25 hours. The reaction temperature is preferably 30 ℃.
As a preferred embodiment, the pH of the enzyme-catalyzed reaction is 7.0 to 8.0; the optimal pH was 7.0.
As a preferred embodiment, the reaction system of the enzyme-catalyzed reaction comprises a buffer, sucrose phosphorylase, D-psicose-3-epimerase, sucrose and glycerol.
As a preferred embodiment, the buffer is 50-60mM MES buffer.
As a preferred embodiment, the molar ratio of sucrose to glycerol is 1:2-3.
As a preferred embodiment, sucrose phosphorylase, D-psicose-3-epimerase is used in an amount of: 1.25-2.5 g sucrose phosphorylase is added to every 1mol sucrose; 1.25-2.5. 2.5g D-psicose-3-epimerase is added per 2mol of glycerol.
As a preferred embodiment, the two recombinant expression strains are induced to express in the following manner:
respectively inoculating sucrose phosphorylase recombinant expression strain and D-psicose-3-epimerase recombinant expression strain into LB culture medium for seed culture, inoculating the obtained seed solution into fermentation culture medium for fermentation culture, and growing to logarithmic phase (OD) 600 0.6 to 0.8), and an inducer 0.1mM isopropyl-beta-D-thiogalactoside (IPTG, C) 9 H 18 O 5 S) inducing enzyme production. Further, after induction of enzyme production, bacteria are harvested, repeatedly centrifuged and washed with 50mM MES buffer, and then OD is enriched 600 And (3) resuspension and concentration are carried out until about 60, the crude enzyme solution serving as double enzyme cascade is put into an ultrasonic crusher to be crushed for 30 minutes under low temperature, and then sucrose and glycerol are added to carry out enzyme catalytic reaction.
As a preferred embodiment, the temperature of the seed culture is 37 ℃; the incubation time was 12h.
As a preferred embodiment, the fermentation culture is aerobic fermentation at 37 ℃; the fermentation culture time is 3-4 h.
As a preferred embodiment, the induction enzyme production is aerobic induction, the induction temperature is 16-20 ℃, and the optimal induction temperature is 18 ℃; the induction culture time is 15-20h.
As a preferred embodiment, the seed solution is inoculated in an amount of 1% (v/v).
As a preferred embodiment, the inducer IPTG is used in an amount of 0.1-0.15 mM.
The invention has the following beneficial effects:
(1) Sucrose phosphorylase and D-psicose-3-epimerase produced by recombinant escherichia coli are adopted, 1mol/L sucrose and 2mol/L glycerol are taken as substrates, and the yield of the catalytic synthesis of the glycerol glucoside by the SPase is 295.4g/L, and the glycerol conversion rate is 78.77%; double enzyme cascade catalysis sucrose and glycerin reaction with coproduction of glycerin glucoside yield 289.8g/L, DThe yield of psicose is 93.4g/L, and the glycerol conversion rate is up to 75.6%.
(2) The method has the advantages of low cost and high conversion rate of the produced sucrose and glycerol which are cheaper, remarkably improves the economic benefits of the two products, effectively reduces the cost and has important application value.
(3) The method of the invention has the advantages of convenient and safe operation, no pollution and wide application range.
Drawings
FIG. 1 is a diagram of the construction of recombinant E.coli pET-Deut1-DPease and pET-22b (+) -SPase; the promoter is a T7 promoter.
FIG. 2 is a reaction equation for producing D-psicose by a one-pot double enzyme cascade method.
FIG. 3 is the effect of sucrose phosphorylase induction conditions, including induction time, induction temperature, IPTG concentration, catalytic conditions on yield; catalytic conditions include catalytic temperature, catalytic pH and enzyme addition ratio.
FIG. 4 is the effect of catalytic time of sucrose phosphorylase on yield.
FIG. 5 is the effect of the induction conditions of D-psicose-3-epimerase on yield, including induction time, induction temperature, IPTG concentration.
FIG. 6 is the effect of catalytic conditions of the dual enzyme cascade on yield under optimal induction conditions, including pH, substrate addition, reaction temperature, reaction time.
Detailed Description
The invention will be further illustrated with reference to specific examples.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The HPLC of the example is the Siemens technology (Thermo Fisher) -DGP-3600SDN, S/N:8094557.
the parameters for detecting D-psicose by High Performance Liquid Chromatography (HPLC) are as follows: the detector is a refractive differential detector, the chromatographic column is an organic acid column (Aminex HPX-87H lon Exclusion Column), the column temperature is 55 ℃, the mobile phase is dilute sulfuric acid of 0.5g/L, the flow rate is 0.5ml/min, and the sample injection amount is 20 mu L.
The parameters for detecting the 2-O-alpha-D-glyceroglycosides by High Performance Liquid Chromatography (HPLC) are as follows: the detector is a refractive differential detector, the chromatographic column Shodex-Asahipak NH2P-50 4E, and the mobile phase is acetonitrile: water=80: 20 (v/v), flow rate was 0.8mL/min, detection temperature was 35℃and sample injection amount was 20. Mu.L.
Definition of SPase enzyme activity: the amount of enzyme required to hydrolyze 1. Mu. Mol of sucrose per minute to produce fructose is defined as one enzyme activity unit (U) of sucrose phosphorylase.
DPEase enzyme activity definition: the amount of enzyme required to hydrolyze 1. Mu. Mol of fructose per minute to produce D-psicose is defined as one enzyme activity unit (U) of D-psicose-3-epimerase.
Example 1
Preparation of target Gene and construction of recombinant plasmid
According to the nucleotide sequence of the sucrose phosphorylase encoding gene SPase provided on NCBI from Leuconostoc mesenteroides Leuconostoc Mesenteroides (ATCC 12291) (GenBank: D90314.1)) The plasmid pET-22b (+) -SPase is obtained by consignment of Nanjing Jinsri biotechnology Co., ltd, then the plasmid is firstly introduced into E.coli DH5 alpha for stable preservation, then the recombinant strain BL21 (DE 3)/pET-22 b (+) -SPase is constructed in E.coli BL21 (DE 3) for expression, the construction steps are referred toUltra One Step Cloning Kit kit instructions.
The plasmid pET-dur 1-DPease was obtained by the synthesis of Nanjing Jinsri Biotechnology Co., ltd, based on the nucleotide sequence (GenBank: KX 098480.1) of the gene encoding DPease of D-psicose-3-epimerase from Agrobacterium tumefaciens (ATCC 33970) provided on NCBI. The construction step is referred toUltra One Step Cloning Kit kit instructions.
Culturing engineering bacteria and induced expression of sucrose phosphorylase and D-psicose-3-epimerase
The recombinant strain BL21 (DE 3)/pET-22 b (+) -SPase and BL 21/pET-dure 1-DPease expression enzyme described in (I) are adopted to produce D-psicose, and the specific method is as follows: taking out the strain from the seed retaining tube, inoculating 1% of the strain into LB culture medium (test tube), and culturing at 37deg.C and 180rpm for 12 hr to obtain seed solution; the seed solution was inoculated into 200mL of the enriched fermentation medium at an inoculum size of 1%, and when the culture was carried out at 37℃and 180rpm, 0.1mM of the inducer IPTG was added and the culture was carried out at 18℃and 160rpm for 16 hours to induce enzyme production.
Wherein: the LB medium comprises the following components: 10g/L peptone, 5g/L, naCl g/L yeast powder and pure water as solvent.
The enriched fermentation medium comprises the following components: 12g/L yeast powder, 15g/L peptone, 10g/L glycerin and 8.9g/L Na 2 HPO 4 ·12H 2 O、3.4g/L KH 2 PO 4 、2.67g/L NH 4 Cl、0.71g/L Na 2 SO 4 、0.3g/L MgSO 4 ·7H 2 O。
(three) one-pot double enzyme cascade catalysis sucrose and glycerol co-production of glyceroglycosides and D-psicose
(1) The bacterial solutions induced for 16h are respectively centrifuged, washed and resuspended (4 ℃,8000rpm,10 min), repeatedly washed for 2-3 times by using MES (morpholinoethanesulfonic acid) buffer solution with the concentration of 50-60mM, the supernatant is removed, and bacterial mud is remained in a centrifuge tube.
(2) The bacterial mud is resuspended in 40-50 mL 50-60mM MES buffer solution with pH7.0, put into an ultrasonic breaker for breaking wall for 30min, and broken after dissolution to be used as fermentation crude enzyme solution. Both enzymes are intracellular enzymes, so that the intracellular enzymes are released through wall breaking, and extracellular substrates and the intracellular enzymes can be combined and reacted more effectively during catalytic reaction, so that better yield and conversion rate are achieved, and the reaction efficiency is improved greatly.
(3) Adding the crude enzyme solution in the step (2) into a double-enzyme cascade catalytic system, wherein the catalytic reaction system is 100mL, and the composition of the catalytic reaction system is as follows: 50-60 mmol/L MES (morpholinoethanesulfonic acid) buffer pH7.0, 1.25mg/mL sucrose phosphorylase, 1.25mg/mL D-psicose-3-epimerase, 1mol/L sucrose and 2mol/L glycerol, pH 7.0-8.0, and optimal pH 7.0. The temperature of the double enzyme cascade catalytic reaction is 25-30 ℃, and the optimal temperature is 30 ℃; the catalytic reaction time is 20-25 hours.
(4) The reaction solution in (3) was heated in boiling water at 100℃for 15 to 20 minutes to stop the reaction, and then centrifuged (13000 rpm,2 min) to obtain a supernatant, and the contents of D-psicose and 2-O-alpha-D-glyceroglycosides were detected by high performance liquid chromatography.
The reaction of sucrose phosphorylase to produce glyceroglycosides with 1.25mg/mL (final concentration) of sucrose phosphorylase alone was used as a control group with 1mol/L sucrose and 2mol/L glycerol as substrates. In the detection result: the yield of the glycerol glucoside synthesized by the separate catalysis of the sucrose and the glycerol by the SPase is 295.4g/L, and the glycerol conversion rate is 78.77%; double enzyme cascade catalysis sucrose and glycerin reaction with coproduction of glycerin glucoside yield 289.8g/L, DThe yield of psicose is 93.4g/L, and the glycerol conversion rate is up to 75.6%.
Example 2 optimization of optimum Induction conditions and catalytic reaction conditions for sucrose phosphorylase
(1) Optimizing induction conditions;
experiments have found that the yield of the glyceroglycosides at 15 ℃, 18 ℃, 22 ℃, 25 ℃ and 30 ℃ is consistent under the four different induction temperatures, the other conditions are strictly controlled, the variable is strictly controlled, as shown in figure 3, the yield of the glyceroglycosides at the induction temperature of 18 ℃ is the highest, and then the optimal reaction temperature of 18 ℃ is selected.
Then, under the condition of the induction temperature of 18 ℃, five different induction times of 12h, 16h, 20h, 24h and 28h are explored for comparison, and as shown in figure 3, when the induction time is 16h, the yield of the glyceroglycosides is optimal.
Finally, the effect comparison of the inducer concentration of 0.05, 0.1, 0.15, 0.2mM was investigated at 18℃under 16h induction conditions, and as shown in FIG. 3, the production of glycerol glucoside was optimized at an IPTG concentration of 0.15mM.
When the induction condition is optimal, the protein expression amount is high, and according to detection, the enzyme expression amount of the sucrose phosphorylase under the optimal induction condition is about 5mg/mL, and when the sucrose phosphorylase is used, the substrate is added after concentration or dilution by a buffer solution according to the enzyme amount required by a reaction system. The measurement result of the enzyme activity of the SPase is 138.89U/mL.
(2) Under the optimal induction condition, exploring the influence of the catalytic condition and optimizing the catalytic condition;
first, the effect of five different catalytic temperatures of 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ on the yield was investigated, and as shown in fig. 3, the yield of the glyceroglycosides reached the highest when the catalytic temperature was 25 ℃.
Then, the influence of three different buffers on the enzymatic reaction was investigated, namely morpholinoethanesulfonic acid (MES), PBS and physiological saline (9% NaCl), and as shown in FIG. 3, the product yield of MES was relatively high, so MES was selected as the buffer for the enzymatic reaction.
Then, under the condition of the reaction temperature of 25 ℃, seven different catalytic reaction times of 18h, 19h, 20h, 21h, 22h, 23h and 24h are explored, and as shown in fig. 3, when the catalytic time is 24h, the product yield is optimal.
Five different pH effects on the yield were investigated at 25℃for 24h, 5, 6, 7, 8, 9, and the product yield was optimized when the pH was 6.0, as shown in FIG. 3.
Under the optimal catalytic conditions, the optimal substrate sucrose and glycerol addition amounts (sucrose: glycerol 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, 1:3 mol/L) were explored, as shown in FIG. 3, and finally all the optimal conditions for the complete set of catalytic reactions were determined:
the concentration of IPTG is 0.15mM, the induction temperature is 18 ℃, the induction time is 16 hours, and the enzyme activity of sucrose phosphorylase is 1.25-2.5 mg/mL under the optimal induction condition. Catalytic temperature 25 ℃, catalytic time 24h, substrate 1mol/L sucrose and 2mol/L glycerol. The catalytic reaction is 100mL of catalytic reaction system, and the composition of the catalytic reaction system is as follows: 50-60 mmol/L MES (morpholinoethanesulfonic acid) buffer, 1.25mg/mL sucrose phosphorylase, 1mol/L sucrose and 2mol/L glycerol. The reaction was carried out at pH 6.0 and a catalytic temperature of 25℃for 40 hours, and the results are shown in FIG. 4, and at 24 hours, the maximum production of glycerol glucoside was substantially achieved.
Example 3 optimization of optimal Induction conditions for D-psicose-3-epimerase
Experiments prove that the yield of D-psicose produced by substrate fructose at the temperature of 12 ℃, 18 ℃, 22 ℃, 25 ℃ and 30 ℃ is consistent under five different induction temperatures, the other conditions are all kept the same, the variables are strictly controlled, as shown in figure 5, the yield of D-psicose at the induction temperature of 18 ℃ is the highest, and then the optimal reaction temperature is 18 ℃.
Then, under the condition of the induction temperature of 18 ℃, five different induction times of 12h, 16h, 20h, 24h and 28h are explored, and as shown in figure 5, when the induction time is 20h, the yield of D-psicose is optimal.
The effect of inducer concentrations of 0.05, 0.1, 0.15 and 0.2mM was examined and compared at 18℃for 20 hours, and as shown in FIG. 5, the yield of D-psicose was optimized at an IPTG concentration of 0.1 mM.
According to the detection, the enzyme expression amount of the D-psicose-3-epimerase under the optimal induction condition is about 5mg/mL, and when the enzyme is used, a substrate is added after concentration or dilution with a buffer solution according to the required enzyme amount of a reaction system. The DPease enzyme activity was measured at 41.67U/mL.
Example 4 optimization of optimal catalytic conditions for one-pot two-enzyme cascades of sucrose phosphorylase and D-psicose-3-epimerase
The catalytic reaction is 100mL of catalytic reaction system, and the composition of the catalytic reaction system is as follows: 50-60 mmol/L MES (morpholinoethanesulfonic acid) buffer, 1.25mg/mL sucrose phosphorylase, 1.25mg/mL D-psicose-3-epimerase, 1mol/L sucrose and 2mol/L glycerol.
Experiments prove that the yields of the substrate sucrose and the glycerol coproduced with the glyceroglycosides and the D-psicose are consistent under four different catalysis temperatures of 20 ℃, 25 ℃, 30 ℃ and 35 ℃, and the other conditions are kept consistent, the variables are strictly controlled, as shown in figure 6, the yield of the D-psicose is highest at the catalysis temperature of 30 ℃, so that the optimal catalysis reaction temperature is 30 ℃.
Then, five different catalytic times of 6h, 12h, 18h, 24h and 30h were examined under the condition of the catalytic temperature of 30 ℃, and as shown in fig. 6, when the catalytic time is 30h, the yield of D-psicose reaches the best, but when the catalytic time is 24h, the yield is basically close to the maximum yield (30 h yield), so the best catalytic time is selected to be 24h.
The effect comparison of five catalytic pH values of 5, 6, 7, 8 and 9 was investigated at 30℃for 24 hours, and as shown in FIG. 6, the yield of D-psicose was optimized when the pH was 7. Therefore, the optimum catalytic pH was chosen to be 7.0.
Therefore, the temperature of the double enzyme cascade catalytic reaction is 25-30 ℃, and the optimal temperature is 30 ℃; the catalytic reaction time is 20-25 hours.

Claims (10)

1. A method for coproducing glyceroglycosides and D-psicose by double enzyme cascading is characterized in that sucrose and glycerol are taken as co-substrates, and sucrose phosphorylase and D-psicose-3-epimerase are added to perform a co-catalytic reaction to obtain the glyceroglycosides and the D-psicose.
2. The method of claim 1, wherein the recombinant expression strains of sucrose phosphorylase and D-psicose-3-epimerase are respectively constructed, and the two recombinant expression strains are subjected to induced expression and ultrasonic disruption, and then sucrose and glycerol are added for enzyme catalytic reaction to obtain the glyceroglycoside and the D-psicose.
3. The method according to claim 1, wherein the recombinant expression strains of sucrose phosphorylase and D-psicose-3-epimerase are constructed using escherichia coli as a host strain, respectively; the expression cassettes of sucrose phosphorylase and D-psicose-3-epimerase both use the T7 promoter of escherichia coli as a promoter and the T7 terminator of escherichia coli as a terminator.
4. The method according to claim 1, wherein the temperature of the enzyme-catalyzed reaction is 25-30 ℃ and the reaction time is 20-25 hours; the pH of the enzyme catalytic reaction is 7.0-8.0.
5. The method according to claim 1, wherein the reaction system of the enzyme-catalyzed reaction comprises a buffer, sucrose phosphorylase, D-psicose-3-epimerase, sucrose and glycerol.
6. The method of claim 5, wherein the buffer is 50-60mM MES buffer.
7. The method according to claim 1 or 5, wherein the molar ratio of sucrose to glycerol is 1:2-3.
8. The method according to claim 1 or 5, wherein the sucrose phosphorylase, D-psicose-3-epimerase is used in an amount of: 1.25-2.5 g of sucrose phosphorylase is added to every 1mol of sucrose; 1.25-2.5 g of D-psicose-3-epimerase is added per 2mol of glycerol.
9. The method of claim 1, wherein the two recombinant expression strains are induced to express in the following manner:
and respectively inoculating the sucrose phosphorylase recombinant expression strain and the D-psicose-3-epimerase recombinant expression strain to an LB culture medium for seed culture, inoculating the cultured seed solution to a fermentation culture medium for culture to a logarithmic growth phase, and adding IPTG to induce enzyme production.
10. A method according to claim 9, wherein IPTG is added to induce enzyme production at 18 ℃, after which bacteria are harvested and washed centrifugally with MES buffer, then enriched, resuspended and concentrated to obtain a crude enzyme solution, which is subjected to an enzymatic reaction by adding sucrose and glycerol after ultrasonication.
CN202310956545.9A 2023-08-01 2023-08-01 Method for coproducing glyceroglycosides and D-psicose by double enzyme cascading Pending CN117343976A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114736942A (en) * 2022-03-25 2022-07-12 上海龙殷生物科技有限公司 Preparation method of alpha-glycerol glucoside

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114736942A (en) * 2022-03-25 2022-07-12 上海龙殷生物科技有限公司 Preparation method of alpha-glycerol glucoside
CN114736942B (en) * 2022-03-25 2024-04-02 上海龙殷生物科技有限公司 Preparation method of alpha-glyceroglycosides

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