CN115820761A - Method for catalytically synthesizing D-glucuronic acid by biological method - Google Patents
Method for catalytically synthesizing D-glucuronic acid by biological method Download PDFInfo
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- CN115820761A CN115820761A CN202211503997.3A CN202211503997A CN115820761A CN 115820761 A CN115820761 A CN 115820761A CN 202211503997 A CN202211503997 A CN 202211503997A CN 115820761 A CN115820761 A CN 115820761A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/584—Recycling of catalysts
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Abstract
The invention discloses a method for catalytically synthesizing D-glucuronic acid by a biological method. Expression of (+) -Mm-MIOX from mice by recombinant E.coli PET-28: (Marmota monax) The inositol oxygenase is repeatedly frozen and thawed, and then substrate inositol is added for whole-cell catalytic reaction to obtain D- (+) -glucuronic acid. Wherein the enzyme is derived from mouse (A)Marmota monax) The inositol oxygenase Mm-MIOX has 0.1M isopropyl-beta-D-thiogalactoside as inducer for inducing enzyme production, and can effectively improve the conversion rate of substrate inositol through repeated freezing and thawing, thereby improving the yield of D-glucuronic acid. The method has the advantages of high utilization rate of raw materials, high conversion rate of D-glucuronic acid and simple stepsThe method has the advantages of quickness, low production cost, little pollution and little influence on the environment, and can realize the large-scale production of the D-glucuronic acid. The inositol oxidase produced by the recombinant escherichia coli takes 8g/L inositol as a substrate, the yield of D-glucuronic acid of the whole-cell catalytic reaction can reach 5.62g/L, and the conversion rate is as high as 86.3%.
Description
Technical Field
The invention belongs to the field of enzyme catalysis production of D-glucuronic acid, and particularly relates to a method for synthesizing D-glucuronic acid under the catalysis of biological single enzyme.
Background
D-glucuronic acid (C) 6 H 10 O 7 D-glucuronic acid), which is a compound formed by oxidizing primary alcohol hydroxyl group of glucose to form carboxyl group, has poor stability in water and is easy to reach interconversion equilibrium with furan ring 3, 6-glucuronolactone in water.
Glucuronic acid is widely present in animals and plants in nature, and is present in the form of a tissue complex and α -uronic acid in animals and plants, respectively, such as chondroitin sulfate in connective tissues of the human body, carrageenan gum from india, and the like. D-glucuronic acid is also a very efficient liver antidote, glucuronic acid and derivatives thereof have glucuronic acid acidification characteristics, endogenous and exogenous toxic substances with phenolic hydroxyl, amino, alcoholic hydroxyl and other groups can be combined in the liver to generate glucuronic acid compounds, so that the water solubility of the glucuronic acid compounds is improved, and the glucuronic acid compounds are conveniently discharged through kidney urine or sweat. In addition, glucuronic acid has an anti-inflammatory and antibacterial effect, and can reduce high concentrations of cholesterol and triglycerides in blood, thus indirectly contributing to digestion, and is widely used in cosmetics and skin care products, as well as various beverages and foods. Glucuronic acid can also be used as a precursor for synthesizing ascorbic acid, glucosamine and D-glucaric acid, glucose can be converted into glucuronic acid under the action of related enzymes, and the glucuronic acid serving as an intermediate substance is subjected to configuration change under the catalysis of the enzymes and can be directly converted into the L-ascorbic acid. Therefore, D-glucuronic acid is considered as "a high value-added chemical of biomass".
The current production methods of D-glucuronic acid mainly comprise: chemical oxidation, polysaccharide hydrolysis and biological fermentation.
The chemical oxidation method is the most common method for producing glucuronic acid, which is most researched, and can be divided into a homogeneous catalytic oxidation method and a heterogeneous catalytic oxidation method according to different culture conditions, wherein the homogeneous catalytic oxidation method is that a catalyst and a reactant are in the same homogeneous system, so that the advantages of soft reaction, high reaction efficiency and high selectivity can be achieved, but the reaction process is lack of effective control, and the subsequent separation and purification operations are difficult. According to the difference of the catalyst, the method can be divided into an inorganic oxidation method and a catalytic oxidation method,
the inorganic oxidation method mainly utilizes an inorganic catalyst to catalyze a precursor or an intermediate of a reaction to produce glucuronic acid, and comprises a concentrated nitric acid oxidation method, a potassium permanganate oxidation method and the like, and the method for producing the glucuronic acid in China is mainly a starch concentrated nitric acid oxidation method.
The polysaccharide hydrolysis method is a process of hydrolyzing polysaccharide containing uronic acid by using enzyme, acid and alkali to obtain glucuronic acid. Yuanhua et al have reported the preparation of glucuronic acid by extracting holocellulose with water, hydrolyzing cotton and cellulose with alkali, oxidizing cellulose with an aqueous chlorine solution, and the like. However, the method needs high-concentration acid and alkali, has severe reaction conditions, certain pollution to the environment and certain damage to human health, is difficult to recover, and has low yield, so the method cannot be used for large-scale application.
The biological fermentation method is a hotspot for researching the production of the glucuronic acid product at present, the biological method is to catalyze precursors or original substrates of the glucuronic acid by utilizing biological cells or related enzymes so as to produce the glucuronic acid with high yield, and the method is relatively mild and environment-friendly and has little environmental pollution. Zhengshuxiang and the like utilize escherichia coli to express inositol oxidase MIOX in thermophilic bacteria and mouse kidney, the enzyme can catalyze inositol to produce glucuronic acid in one step, the operation is simple, the cost is low, but the stability in water of the MIOX enzyme and the activity of the enzyme per se need to be improved, the stability in water is not high, and the enzyme is easy to reach the interconversion balance with glucuronolactone in water. Kratzsch E et al first found UDP-glucose dehydrogenase in human amniotic membrane, which catalyzes UDP-glucuronic acid, which in turn efficiently synthesizes D-glucuronic acid. The recombinant escherichia coli expresses an inositol oxygenase MIOX gene from pichia pastoris for the first time, so that the biotransformation of inositol is realized, the glucuronic acid is generated, the yield of the glucuronic acid is 20mg/L, and a new thought is provided for producing the glucuronic acid by a single-enzyme biological method. Analysis by comparative experiments shows that inositol oxygenase MIOX is the key enzyme with lowest activity in the metabolic pathway from glucose to glucaric acid (the pathway mainly comprises three enzymes Ino1, MIOX and Udh), and the Eric Shiue group explores a protein fusion marker for increasing the MIOX solubility and directed evolution for increasing the MIOX activity. The fusion of N-terminal SUMO and MIOX leads to the increase of 75% of D-glucuronic acid produced by inositol, and 10.8g/L inositol can produce up to 4.85g/L D-glucuronic acid in recombinant Escherichia coli. An inositol oxidation pathway is constructed in Escherichia coli of Eric Shiue and the like for the first time, myo-inositol oxidase (MIOX) is over-expressed, and the yield of D-glucuronic acid obtained by catalyzing substrate inositol is 3.94g/L. The recombinant escherichia coli is used for expressing an inositol oxygenase MIOX gene derived from pichia pastoris for the first time in Kangzhen, chenjian and the like of Jiangnan university, so that the biotransformation of inositol is realized, glucuronic acid is generated, the yield of the glucuronic acid is 20mg/L, and the yield is lower, and therefore, a new method for producing the D-glucuronic acid with low cost, low pollution and higher yield is urgently needed to be developed.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art, selects the inositol oxidase aiming at the defect of low activity of the inositol oxidase, optimizes the fermentation and induction conditions of the inositol oxidase MIOX (as shown in figure 5), and provides a method for synthesizing D-glucuronic acid by biological catalysis.
The purpose of the invention is realized by the following technical scheme:
a method for catalytically synthesizing D-glucuronic acid by biological method comprises expressing myoinositol oxygenase by recombinant Escherichia coli PET-28 (+) -Mm-MIOX, repeatedly freezing and thawing, and adding inositol (inositol, C) as substrate 6 H 12 O 6 ) Carrying out whole-cell catalytic reaction to obtain D- (+) -glucuronic acid.
The myo-inositol oxygenase is derived from mouse liver (Marmota monax), and the coding gene of the myo-inositol oxygenase is shown as SEQ ID NO:1 is shown.
The whole cell catalytic reaction temperature is 30-35 ℃, and the reaction time is 3-4 hours.
The whole-cell catalytic reaction system comprises: 50-80 mmol/L Tris-HCl buffer solution, 2-4 mmol/L L cysteine, 1-3 mmol/L ferrous ammonium sulfate, 5-10U/mL inositol oxidase, 8-10 g/L inositol, and the pH value is 7.5-8.0.
The repeated freezing and thawing step is to perform 3 to 4 times of freezing and thawing in a refrigerator at the temperature of minus 40 ℃.
The recombinant escherichia coli is constructed by inserting myo-inositol oxygenase into the genome of an initial strain BL21, and the promoter of the expression cassette is the T7 promoter of the escherichia coli; the terminator is the T7 terminator of Escherichia coli.
A method for synthesizing D-glucuronic acid by biological catalysis comprises culturing recombinant Escherichia coli strain with LB culture medium to obtain seed solution; transferring the seed liquid with MBL fermentation culture medium, and adding inducer 0.1m after the seed liquid grows to logarithmic phaseisopropyl-beta-D-thiogalactoside of M (IPTG, C) 9 H 18 O 5 S), inducing the recombinant strain to produce the inositol oxidase.
The recombinant Escherichia coli is constructed by inserting myo-inositol oxygenase into the genome of an initial strain BL21, wherein the myo-inositol oxygenase is derived from mouse liver (Marmota monax), and the coding gene of the myo-inositol oxygenase is shown as SEQ ID NO:1 is shown.
The recombinant Escherichia coli is applied to the production of D-glucuronic acid.
Inositol is used as a substrate, and inositol oxidase MIOX crude enzyme produced by recombinant escherichia coli is added to carry out whole-cell catalytic reaction to obtain D-glucuronic acid; wherein the enzyme is Myo-inositol oxidase (MIOX, EC: 1.13.99.1).
Inoculating LB seed liquid culture medium of recombinant Escherichia coli into MBL enrichment fermentation medium containing glucose to logarithmic growth phase (OD) 600 0.6 to 0.8), adding inducer IPTG (final concentration is 0.1 mM/L), and inducing for 12 to 15 hours to produce enzyme at the low temperature of 23 to 25 ℃. The late harvest strain is repeatedly centrifuged and washed by 50mM Tris-HCl, and OD is enriched after the repeated centrifugation 600 Concentrating to about 60 deg.C, freezing and thawing in a refrigerator at-40 deg.C for 3-4 times, dissolving, adding into a whole-cell reaction system with inositol as substrate, and reacting at 30 deg.C and 180rpm for 3-4 hr. Then, after the sample is processed by centrifugal filtration, the yield of the D-glucuronic acid is accurately measured by using high performance liquid chromatography.
The recombinant escherichia coli is PET-28a (+), and the cloning host is E.coil DH5 alpha; the expression host is E.coil BL21; the antibiotic is kanamycin, the inositol oxidase is Mm-MIOX from mice (Marmota monax), and the genes are shown as SEQ ID NO:1 is shown.
The High Performance Liquid Chromatography (HPLC) is a Saimer Feishell technology (Thermo Fisher) -DGP-3600SDN, S/N:8094557. the detector is a refractive index difference detector, the chromatographic Column is an organic acid Column (Aminex HPX-87H lon Exclusion Column), the Column temperature is 40 ℃, the mobile phase is 0.5g/L dilute sulfuric acid, the flow rate is 0.5ml/min, and the sample injection amount is 20 mu L.
The temperature of the seed liquid in the test tube culture is 37 ℃; the culture time is 12h.
The fermentation culture is aerobic fermentation, and the fermentation temperature is 37 ℃; the fermentation culture time is 3-4 h.
The induced enzyme production is aerobic fermentation, the induction temperature is 23-25 ℃, and the optimal induction temperature is 25 ℃; the induction culture time is 12-15 h. The inoculum size of the fermentation culture was 1% (v/v).
The inoculation amount of the inducer IPTG is 1 per mill (v/v).
The seed culture medium is an LB culture medium and comprises the following components: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl and a solvent of pure water.
The fermentation medium is an MBL medium and comprises the following components: 10g/L of peptone, 10g/L of yeast powder, 10g/L of NaCl, (NH) 4 ) 2 HPO 4 3.5g/L、KH 2 PO 4 3.5g/L、K 2 HPO 4 5g/L、MgSO 4 ·7H 2 O7 g/L, glucose 10g/L and pure water as solvent.
The whole-cell catalytic reaction system is a 50mL catalytic reaction system and comprises the following components: 50-80 mmol/L Tris-HCl buffer solution, 2-4 mmol/L L cysteine, 1-3 mmol/L ferrous ammonium sulfate, 5-10U/mL inositol oxidase, 8-10 g/L inositol, 8g/L optimal substrate addition, 7.5-8.0 pH and 7.5 optimal pH. The temperature of the whole-cell catalytic reaction is 30-35 ℃, and the optimal temperature is 30 ℃; the time of the whole cell catalytic reaction is 3 to 4 hours.
Has the advantages that:
the invention adopts the inositol oxidase produced by recombinant escherichia coli, takes 8-10 g/L inositol as a substrate, the yield of D-glucuronic acid of the whole-cell catalytic reaction can reach 5.62g/L, and the conversion rate reaches 86.3 percent.
The method has the advantages of convenient and safe operation and wide application range, remarkably improves the yield of D-glucuronic acid, and has important application value.
Drawings
FIG. 1: and (3) performing protein electrophoresis pattern of the purified inositol oxidase, wherein the purification tag is a His protein tag, 1: 5-8 tubes of pure enzyme; 2: 10-14 tubes of pure enzyme (10 mu L of addition); 3: 10-14 tubes of pure enzyme (addition amount is 15 mu L); 4: 16-18 tubes of pure enzyme (the addition amount is 10 mu L); 5: pure enzyme 16-18 tubes (addition amount 15. Mu.L).
FIG. 2: the enzymatic properties of the purified inositol oxidase were investigated, with an optimum pH of 7.5 and an optimum temperature of 30 ℃.
FIG. 3: the structure of recombinant Escherichia coli PET-28a (+) -MIOX structure, and the promoter is T7 promoter.
FIG. 4: three crushing methods: comparing the yield and effect of the whole-cell catalysis method, the freeze-thaw disruption method and the ultrasonic disruption method (because the stability of D-glucuronic acid in water is not good and the D-glucuronic acid lactone is easy to reach interconversion balance in water, the yield of D-glucuronic acid is reduced when the optimal catalysis time is exceeded), the three methods are all under the optimal catalysis condition: the fermentation temperature of the seed liquid is 37 ℃, the IPTG concentration is 0.1M, the induction temperature is 25 ℃, the induction time is 12h, the whole-cell catalysis temperature is 30 ℃, the whole-cell catalysis time is 3-4 h, the initial PH is 7.5, and the addition amount of substrate inositol is 8g/L.
FIG. 5: optimal fermentation conditions and induction conditions of the mouse-derived inositol oxidase Mm-MIOX are optimized and screened.
FIG. 6: under the optimal conditions, the test chart shows that the highest yield of D-glucuronic acid produced by inositol and inositol oxidase is obtained.
Detailed Description
The present invention will be further described with reference to the following specific examples.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Preparation of target gene and construction of recombinant plasmid
According to the nucleotide sequence of the inositol oxidase encoding gene MIOX from Marmota monax provided on NCBI, the plasmid PET-28a (+) -MIOX is obtained by entrusting the synthesis of Nanjing Kingsry Biotech Co., ltd, the structure diagram of the plasmid is shown in figure 3, the gene sequence of Mm-MIOX is shown in SEQ ID No:1, then firstThe plasmid is introduced into Escherichia coli DH5 alpha for stability preservation, and then introduced into Escherichia coli BL21 to construct a recombinant strain BL21/PET-28a (+) -MIOX for expression, wherein the construction steps refer to
Instructions for the UltraOne Step Cloning Kit.
(II) culturing engineering bacteria and inducing expression of inositol oxidase
The recombinant strain BL21/PET-28a (+) -MIOX expresses enzyme to produce D-glucuronic acid by the following specific method: taking out the strain from the seed preservation tube, inoculating the strain into an LB test tube with the inoculation amount of 1 percent, and carrying out shaking culture at 37 ℃ and 180rpm for 12 hours to obtain seed liquid; inoculating the seed solution into 300ml MBL fermentation medium at an inoculum size of 1%, adding 0.1mM inducer IPTG during shaking culture at 37 deg.C and 180rpm until logarithmic phase, and shaking culture at 25 deg.C and 160rpm for 12 hr to induce enzyme production.
Wherein: the seed culture medium is LB culture medium, comprising the following components: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl and pure water as a solvent.
The MBL fermentation medium comprises the following components: 10g/L of peptone, 10g/L of yeast powder, 10g/L of NaCl, (NH) 4 ) 2 HPO 4 3.5g/L、KH 2 PO 4 3.5g/L、K 2 HPO 4 5g/L、MgSO 4 ·7H 2 O7 g/L, glucose 10g/L and pure water as solvent.
(III) production of D-glucuronic acid by whole-cell catalysis method
(1) And (3) centrifugally washing and re-rotating the bacteria liquid for 12h induction (4 ℃,8000rpm, 10min), wherein the buffer solution is 50mM Tris-HCl buffer solution, repeatedly washing for 2-3 times, removing supernate, and leaving bacterial sludge in a centrifugal tube.
(2) The bacterial sludge is re-spun in 40-50mL PH7.5 50mM Tris-HCl buffer solution, and is put into a refrigerator with the temperature of-40 ℃ for repeated freeze thawing and wall breaking for 3 times, the freezing temperature is-40 ℃, the thawing temperature is 35 ℃ and constant temperature water bath is carried out for 1-2 h, and the bacterial sludge is dissolved for the last time to be used as a fermentation crude enzyme solution. The freeze-thaw method is a mild cell wall breaking method, and cell walls of cells can be weakened through repeated freeze-thaw, so that extracellular substrates and intracellular enzymes can be effectively combined and reacted during catalytic reaction, and further, better yield and conversion rate are achieved.
(3) Adding the crude enzyme solution obtained in the step (2) into a whole-cell catalytic system, wherein the whole-cell catalytic reaction is a 50mL catalytic reaction system and comprises the following components: 50mmol/L Tris-HCl buffer solution with pH7.5, 2mmol/L L-cysteine, 1mmol/L ferrous ammonium sulfate, 5-10U/mL inositol oxidase, 8g/L inositol, and pH 7.5. The temperature of the whole-cell catalytic reaction is 30 ℃; the time of the whole cell catalytic reaction is 180rpm for 3-4 hours.
(4) And (3) heating the reaction solution in the step (3) in boiling water at 100 ℃ for 15-20 min to stop the reaction, centrifuging (13000rpm, 2min) to obtain a supernatant, and carrying out quantitative detection by using a high performance liquid chromatography with a differential detector. The High Performance Liquid Chromatography (HPLC) is a Saimer Feishell technology (Thermo Fisher) -DGP-3600SDN, S/N:8094557. the detector is a refractive index difference detector, the chromatographic Column is an organic acid Column (Aminex HPX-87H lon Exclusion Column), the Column temperature is 40 ℃, the mobile phase is 0.5g/L dilute sulfuric acid, the flow rate is 0.5ml/min, and the sample injection amount is 20 mu L.
The inositol oxidase produced by the recombinant escherichia coli takes 8g/L inositol as a substrate, the yield of D-glucuronic acid of the whole-cell catalytic reaction can reach 5.62g/L, and the conversion rate is as high as 86.3%.
Example 2 investigation of the enzymatic Properties of purified inositol oxidase
And (3) purification conditions: strains and plasmids: using a strain E coil BL21 (DE 3)/pET 28a (+) -MIOX; the growth culture conditions of the escherichia coli are 37 ℃ and 12 hours; the culture conditions for induced expression are 25 ℃ and 12h.
The main reagents are as follows: buffer solution: 0.05mM PBS buffer pH8.0; eluent: (gradient elution) 30mM/50mM/100mM/200mM PBS (containing imidazole) pH8.0; concentration buffer solution: 50mM Tris-HCl pH 7.0; purifying the column: ni-His Trap FF; purifying a tube: soaking in 20% ethanol, and storing at 4 deg.C.
The main apparatus is as follows: an ultrasonic crusher; a protein purification instrument; a high-speed centrifuge: (separation washing) 8000rpm/10min 2-3 times; (concentration) 4000rpm/15min 4-5 times to concentrate to 1.5-2.0mL
After purification and concentration, SDS-PAGE protein electrophoresis is carried out for verification (shown in figure 1), after protein expression, SDS-PAGE electrophoresis is carried out, and obvious foreign protein expression is carried out at 37.5kDa, so that the verification is correct. Then, the enzymology property of the pure enzyme is explored, and the exploration conditions are as follows: the fermentation temperature of the seed liquid is 37 ℃, the IPTG concentration is 0.1M, the induction temperature is 25 ℃, the induction time is 12h, the whole-cell catalysis temperature is 30 ℃, the whole-cell catalysis time is 3-4 h, the initial PH is 7.5, and the addition amount of substrate inositol is 8g/L. (in protein purification, the imidazole removal step is a considerable impairment of the enzymatic activity, and therefore the yield of pure enzyme is low, but does not affect the study of the enzymatic properties of the enzyme for the inositol oxidase MIOX itself). It was found that the pH optimum of the pure enzyme is 7.5 to 8.0, and the temperature optimum is 30 deg.C (see FIG. 2).
Example 3 optimization of optimal fermentation and Induction conditions
The experiment explored the production of D-glucuronic acid at four different induction temperatures, the remaining conditions were kept consistent, the variables were strictly controlled, as shown in FIG. 4, the yields at 16 ℃ and 25 ℃ were close, but considering that too low a temperature would affect the enzyme activity, thus making the reaction time too long, the optimal induction temperature was chosen to be 25 ℃. Then, optimization of different inducer IPTG concentrations is researched under the condition of induction temperature of 25 ℃, and the experiment researches the yield of D-glucuronic acid with final concentration of 0.05, 0.1, 0.2 and 0.4mM/L, as shown in figure 4, when the final concentration is 0.1mM/L, the yield of D-glucuronic acid is optimal. Then, under the conditions of induction temperature of 25 ℃ and IPTG concentration of 0.1mM, the comparison of four different induction times of 8h, 10h, 12h and 14h is researched, and as shown in FIG. 4, when the induction time is 12h, the yield of D-glucuronic acid is optimal. Under the condition of determining the optimal induction conditions, the optimal catalytic time of the enzyme reaction (2 h, 3h, 4h and 5 h), the optimal pH (7, 7.5, 8 and 8.5) and the optimal addition amount of substrate inositol (6 g/L, 8g/L, 10g/L and 12 g/L) are explored, and as shown in a figure 4, all the optimal conditions of the whole set of reaction are finally determined:
the fermentation temperature of the seed liquid is 37 ℃, the IPTG concentration is 0.1M, the induction temperature is 25 ℃, the induction time is 12h, the whole-cell catalysis temperature is 30 ℃, the whole-cell catalysis time is 3-4 h, the initial PH is 7.5, and the addition amount of substrate inositol is 8g/L. The catalytic reaction is a 50mL catalytic reaction system, which comprises the following components: 50mmol/L Tris-HCl buffer solution with pH of 7.5, 2mmol/L L-cysteine, 1mmol/L ferrous ammonium sulfate, 5-10U/mL inositol oxidase, 8g/L inositol and pH of 7.5. The temperature of the catalytic reaction is 30 ℃; the time of the catalytic reaction is 180rpm for 3-4 hours.
Example 4 comparison of the effectiveness of different crushing methods
The crushing method is divided into a whole cell catalysis method, an ultrasonic crushing method, a freeze-thaw cell wall-breaking method and the like. The three methods were compared (see FIG. 4), and the optimal conditions were used for the three methods according to the principle of controlling variables, except for the treatment of the enzyme-producing cells, as follows:
the fermentation temperature of the seed liquid is 37 ℃, the IPTG concentration is 0.1M, the induction temperature is 25 ℃, the induction time is 12h, the whole-cell catalysis temperature is 30 ℃, the whole-cell catalysis time is 3-4 h, the initial PH is 7.5, and the addition amount of substrate inositol is 8g/L. The catalytic reaction is a 50mL catalytic reaction system, which comprises the following components: 50mmol/L Tris-HCl buffer solution with pH of 7.5, 2mmol/L L-cysteine, 1mmol/L ferrous ammonium sulfate, 5-10U/mL inositol oxidase, 8g/L inositol and pH of 7.5. The temperature of the catalytic reaction is 30 ℃; the time of the catalytic reaction is 180rpm for 3-4 hours.
Under the control test of control variables, the method for freezing and thawing the wall with the highest yield and the best conversion rate is obtained. The enzyme-producing bacteria are repeatedly frozen and thawed at the temperature of minus 40 ℃, so that the cell wall of the cell becomes fragile, and the contact reaction of an extracellular substrate and intracellular enzyme is more facilitated, and the method has the advantages of high yield of D-glucuronic acid, high conversion rate of raw materials, low production cost, no pollution and the like.
SEQ ID NO：1：
ATGAAGGTCGATGTGGGCCCAGACCCTTCCCTGGTCTATCGACCCGATGTGGACCCAG
AGATGGCCAAAAGCAAGGACAGCTTCCGAAACTATACTTCAGGCCCGCTGCTGGATCG
TGTCTTTACCACATACAAGCTCATGCACACTCACCAGACTGTGGACTTCGTCAGCAGG
AAGCGCATCCAGTATGGAAGCTTCTCTTACAAGAAGATGACCATCATGGAGGCTGTGG
GCATGCTGGATTATCTGGTGGACGAATCTGACCCAGACGTAGATTTCCCCAACTCCTTC
CACGCGTTCCAGACCGCGGAGGGCATCCGGAAAGCCCACCCGGACAAGGACTGGTTC
CACCTGGTCGGACTTTTGCACGATCTGGGGAAAATTATGGCTCTGTGGGGGGAACCTC
AGTGGGCTGTTGTTGGAGACACGTTCCCCGTGGGCTGCCGTCCCCAGGCCTCTGTGGT
GTTCTGTGACTCTACTTTCCAGGACAATCCTGACCTCCAGGATCCTCGATACAACACAG
AACTCGGCATGTACCAGCCTCACTGTGGACTAGAGAACGTCCTTATGTCCTGGGGCCAT
GATGAGTACCTATACCAGATGATGAAGTTCAACAAGTTCTCCCTGCCTTCAGAGGCCTT
CTACATGATCCGATTCCACTCCTTCTATCCGTGGCACACCGGCGGTGACTACCGGCAGC
TGTGCAGCCAGCAGGACCTGGATATGCTGCCCTGGGTGCAAGAGTTCAACAAGTTTGA
TCTCTACACGAAGTGCCCTGACCTACCGGATGTGGAGAGCCTGCGGCCCTACTATCAA
GGGCTGATTGACAAGTACTGCCCGGGCACCCTGAGCTGGTGA。
Claims (10)
1. A method for catalytically synthesizing D-glucuronic acid by a biological method is characterized in that recombinant escherichia coli PET-28 (+) -Mm-MIOX is used for expressing myo-inositol oxygenase, and substrate myo-inositol is added after repeated freeze thawing for whole-cell catalytic reaction to obtain the D-glucuronic acid.
2. The method for the biological catalytic synthesis of D-glucuronic acid according to claim 1, wherein the myo-inositol oxygenase is derived from mouse liver (Marmota monax), and the coding gene of the myo-inositol oxygenase is shown as SEQ ID NO:1 is shown.
3. The method for the catalytic synthesis of D-glucuronic acid by the biological method according to claim 1, wherein the whole-cell catalytic reaction temperature is 30-35 ℃ and the reaction time is 3-4 hours.
4. The method for the biological catalytic synthesis of D-glucuronic acid according to claim 1, wherein the whole-cell catalytic reaction system is as follows: 50-100 mmol/L Tris-HCl buffer solution, 2-4 mmol/L L cysteine, 1-3 mmol/L ferrous ammonium sulfate, 5-10U/mL inositol oxidase, 8-10 g/L inositol, and the pH value is 7.5-8.0.
5. The method for synthesizing D-glucuronic acid by biological catalytic method according to claim 1, wherein the repeated freezing and thawing step is freezing and thawing for wall breaking, and 3-4 times of freezing and thawing are carried out in a refrigerator at-40 ℃ to-80 ℃.
6. The method for the catalytic synthesis of D-glucuronic acid by the biological method according to claim 1, wherein the recombinant Escherichia coli is constructed by inserting myo-inositol oxygenase into the genome of an initial strain BL21, and the promoter of the expression cassette is the T7 promoter of Escherichia coli; the terminator is the T7 terminator of Escherichia coli.
7. The method for synthesizing D-glucuronic acid through biological catalytic synthesis according to claim 1, wherein an LB culture medium is adopted to culture a recombinant Escherichia coli strain to obtain a seed solution; transferring the seed solution with MBL fermentation medium, growing to logarithmic phase, adding 0.1mM inducer isopropyl-beta-D-thiogalactoside (IPTG, C) 9 H 18 O 5 S), inducing the recombinant strain to produce the inositol oxidase.
8. The method for the biological catalytic synthesis of D-glucuronic acid according to claim 4, wherein the whole-cell catalytic reaction system is as follows: 50mmol/L Tris-HCl buffer solution, 2mmol/L L-cysteine, 1mmol/L ferrous ammonium sulfate, 5-10U/mL inositol oxidase, 8g/L inositol and pH 7.5-8.0.
9. A recombinant Escherichia coli is characterized in that the recombinant strain is constructed by inserting myo-inositol oxygenase into the genome of an initial strain BL21, the myo-inositol oxygenase is derived from mouse liver (Marmota monax), and the encoding gene of the myo-inositol oxygenase is shown as SEQ ID NO:1 is shown.
10. Use of the recombinant E.coli of claim 9 for the production of D-glucuronic acid.
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