CN114317477B

CN114317477B - Sucrose phosphorylase and glucose-1-phosphoric acid production process

Info

Publication number: CN114317477B
Application number: CN202111653419.3A
Authority: CN
Inventors: 丁雪峰; 李帅杰; 刘珊珊; 张文超; 王勇
Original assignee: Nanjing Nuoyun Biotechnology Co ltd
Current assignee: Nanjing Nuoyun Biotechnology Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-07-14
Anticipated expiration: 2041-12-30
Also published as: CN114317477A

Abstract

The invention relates to the technical field of enzyme catalysis, in particular to a sucrose phosphorylase and a glucose-1-phosphoric acid production process, wherein the amino acid sequence of the sucrose phosphorylase is shown as SEQ ID NO: 1. When in use, the sucrose mother solution is taken, the buffer solution and the pure water are added, the crude enzyme solution of the sucrose phosphorylase is added for reaction at 37 ℃. The invention has high conversion rate of the reaction system and high reaction speed, can still keep the conversion rate of the sucrose to be more than 85% in 3 hours reaction time when the high-concentration sucrose reacts, and can effectively inhibit the generation of byproduct glucose.

Description

Sucrose phosphorylase and glucose-1-phosphoric acid production process

Technical Field

The invention relates to the technical field of enzyme catalysis, in particular to a sucrose phosphorylase and a glucose-1-phosphoric acid production process.

Background

Glucose-1-phosphate (α -glucose-1-phosphate) is an important intermediate in carbohydrate metabolism, which is formed by phosphorylation of the terminal glucosyl linkages of a-1, 4-glucan. In order to produce energy and metabolites by catabolism of cells, G1P is converted to D-glucose-6-phosphate (G6P) by phosphoglucose isomerase, and then enters the glucose decomposition pathway.

G1P is also used as an important starting material for biosynthesis of sugar conjugates in cells, in particular for ribopyrophosphorylase-catalyzed synthesis of nucleotide diphosphate glucose (NDP-glucose). In addition, G1P may have medical value for the preparation of important raw materials for cytostatic agents, drugs for treating heart diseases, antibiotics and antitumor drugs.

Glucose-1-phosphate (α -glucose-1-phosphate) is widely present in animals, plants and microorganisms and plays an important role in organisms. G-1-P is the first product of degradation of polysaccharides (e.g., glycogen). It can produce glucose-6-phosphate under the action of glucose phosphate isomerase and can be fed into EMP pathway; it can also be used for nucleic acid diphosphate. NDP-glucose is synthesized under the catalysis of glucose pyrophosphorylase and is further used for synthesizing various saccharides. Is also a potential antibiotic or immunosuppressive drug for the production of linear amylose and other related glucose polymers for the synthesis of amylose-lipid complexes and glycoengineering.

In an organism, glucose-1-phosphate (G-1-P) is produced by the reaction of a polysaccharide such as glycogen, starch or maltodextrin with an inorganic phosphate, which is the first metabolite of the gluconeogenic pathway, and glucose-6-phosphate is produced by the action of glucose phosphomutase and enters glycolysis or other metabolic pathways.

As an activated glucose, glucose-1-phosphate (α -glucose-1-phosphate) is an important precursor in the synthesis of complex carbohydrate compounds such as glycolipids, oligosaccharides, sugar nucleotides. In addition, the compound can be used for synthesizing artificial starch, generating electricity through a biological sugar battery and generating hydrogen with high yield. The use of glucose 1-phosphate has not been fully explored to date, possibly due to its excessive production costs.

Glucose-1-phosphate (α -glucose-1-phosphate) can be produced by enzymatic hydrolysis of dextrins or other starch-based substances as valuable compounds. Glucose-1-phosphate is used as a precursor for synthesizing glucuronic acid, and can also be used as a substrate for synthesizing linear maltooligosaccharide and alpha, alpha-trehalose.

Currently, there are two main biological methods for the production of glucose-1-phosphate:

(1) Sucrose phosphorylase is used to catalyze the production of glucose-1-phosphate from phosphoric acid and sucrose.

(2) The starch or maltodextrin is used as a substrate, and is degraded by alpha-1, 4-glucan phosphorylase.

The cost of catalyzing and producing glucose-1-phosphate by using the glucan phosphorylase with the starch and the maltodextrin as substrates is lower, but the high-concentration glucose-1-phosphate production is difficult to achieve, the highest product in the existing report is only 0.2M product (equivalent to 52 g/L), and the method is far from industrial application.

The use of sucrose phosphorylase to catalyze the production of glucose-1-phosphate from phosphate and sucrose is a more efficient route and the production of 0.5M glucose-1-phosphate (130 g/L equivalent) has been reported. While sucrose phosphorylase processes have only 50% of theoretical yields and may have high product isolation costs, general process modifications (e.g., addition of fructose isomerase) may be used to re-enter the metabolic pathway (e.g., formation of glucose-6-phosphate into the metabolic pathway or further shift to regenerate glucose-1-phosphate).

Disclosure of Invention

The invention aims to provide a sucrose phosphorylase and a glucose-1-phosphate production process.

In order to achieve the above purpose, the present invention provides the following technical solutions:

a sucrose phosphorylase, the amino acid sequence of which is shown in SEQ ID NO: 1.

The reaction speed of the sucrose phosphorylase is higher than that of the wild type sucrose phosphorylase. The conversion rate is more than 85%, which is far higher than that of the wild type.

A production process of glucose-1-phosphoric acid specifically comprises the following steps: taking sucrose mother liquor, adding buffer solution and pure water, adding crude enzyme solution of sucrose phosphorylase, and reacting at 37 ℃.

Experiments prove that the highest substrate concentration of the sucrose phosphorylase can reach 342g/L; and the conversion rate is greater than 85% in 3 hours.

The preparation method of the crude enzyme liquid comprises the following steps:

(1) By total gene synthesis, SEQ ID NO:1, and cloning the corresponding coding polynucleotide sequence of the protein shown in the formula 1 into a prokaryotic expression vector for expression so as to realize high expression in escherichia coli;

(2) Shaking flask fermentation

E.coli single colony containing the expression vector is selected and inoculated in 10mL of culture medium A after autoclaving, and is cultured at 30 ℃ and 250rpm overnight;

taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1:100 inoculation ratio example 100 is inoculated into 100mL of medium B after autoclaving, cultured at 30 ℃ until the cell OD 5-6 is reached, immediately placing the triangular flask in a 25 ℃ shaker, and culturing at 250rpm for 1 hour; IPTG was added to a final concentration of 0.1mM and incubation was continued at 25℃and 250rpm for 16 hours;

after the culture, the culture solution was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells; then washing the bacterial precipitate twice with distilled water, collecting bacterial precipitate, and preserving at-70 ℃; simultaneously taking 2g of thalli, adding 6mL of pure water, performing SDS-PAGE detection after ultrasonic crushing, and preserving crude enzyme liquid at the temperature of minus 20 ℃;

(3) Fed-batch fermentation

Fed-batch fermentation was performed in a computer controlled bioreactor, with 200mL cultures prepared by primary inoculation of the strain, and inoculated at OD 2.0; the temperature was maintained at 37 ℃ throughout the fermentation, the dissolved oxygen concentration during the fermentation was automatically controlled at 30% by the stirring rate and aeration supply cascade, while the pH of the medium was maintained at 7.0 by 50% orthophosphoric acid and 30% aqueous ammonia;

during the fermentation, when the dissolved oxygen is greatly raised, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when the OD600 was 35.0, induction was performed with 0.2mM IPTG for 16 hours; 2g of the cells were sonicated in 6mL of pure water, and then subjected to SDS-PAGE, and the crude enzyme solution was stored at-20 ℃.

Further, the medium a in the step (2) is: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and kanamycin is added to 50mg/L.

The culture medium B is as follows: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and kanamycin is added to 50mg/L.

Further, the culture medium used in the step (3) is: 24g/L of yeast extract, 12g/L of peptone, 0.4% of glucose, 2.31g/L of phosphatase and 12.54g/L of dipotassium hydrogen phosphate, and the pH value is 7.0.

Compared with the prior art, the invention has the beneficial effects that:

the reaction system has high conversion rate and high reaction speed, can still keep the conversion rate of sucrose more than 85% in 3 hours reaction time when high-concentration sucrose reacts, and can effectively inhibit the generation of byproduct glucose; is especially suitable for the industrial mass production of glucose-1-phosphate, and can obtain better social benefit and economic value.

Drawings

FIG. 1 is a 0 hour HPLC plot of the substrate; 10 minutes is sucrose;

FIG. 2 shows the results of the test in example 2, sucrose for 10 minutes, target product for 8.2 minutes, glucose for 11.7 minutes, and fructose for 12.8 minutes;

FIG. 3 shows the results of comparative example 1, sucrose for 10 minutes, target product for 8.2 minutes, glucose for 11.7 minutes, and fructose for 12.8 minutes;

FIG. 4 shows the results of the test in example 3, sucrose for 10 minutes, target product for 8.2 minutes, glucose for 11.7 minutes, and fructose for 12.8 minutes;

FIG. 5 shows the results of comparative example 2, sucrose for 10 minutes, target product for 8.2 minutes, glucose for 11.7 minutes, and fructose for 12.8 minutes.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The detection conditions referred to in the following examples are as follows.

Liquid phase detection conditions:

mobile phase: 5mM sulfuric acid

A detector: differential detector

Flow rate: 0.6mL/min

Column temperature: 50 DEG C

Differential detector cell temperature: 50 DEG C

250mm 4.6um femomei organic acid column was used.

EXAMPLE 1 preparation of crude enzyme solution

(1) By total gene synthesis, SEQ ID NO:1, and the corresponding coding polynucleotide sequence of the protein shown in SEQ ID NO:3 and cloning to prokaryotic expression vector for high expression in colibacillus, named ZY4.

Likewise, the control wild-type sucrose phosphorylase protein SEQ ID NO:2, the corresponding coding polynucleotide sequence SEQ ID NO:4, and cloning to prokaryotic expression vector for expression, named ZY3.

(2) Shaking flask fermentation

E.coli single colonies containing the expression vector were picked and inoculated into 10mL of autoclaved medium: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and kanamycin is added to 50mg/L. Culturing at 30℃and 250rpm overnight. Taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1: inoculation ratio of 100 example 100mL of autoclaved medium was inoculated: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and kanamycin is added to 50mg/L. The cells were cultured at 30℃until the cell OD 5-6 was reached, and the flask was immediately placed in a 25℃shaker at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and the incubation was continued at 25℃and 250rpm for 16 hours. After the completion of the culture, the culture was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells. Then the bacterial cell precipitate is washed twice with distilled water, and the bacterial cells are collected and stored at-70 ℃. Meanwhile, 2g of thalli are added with 6mL of pure water for ultrasonic crushing, SDS-PAGE detection is carried out, and crude enzyme liquid is preserved at the temperature of minus 20 ℃.

(3) Fed-batch fermentation:

fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai state of China) with a capacity of 15L and a working volume of 8L, using 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L dihydrogenphosphate and 12.54g/L dipotassium hydrogen phosphate, pH 7.0. 200mL cultures were prepared from the primary seed strain and inoculated at OD 2.0. The temperature was maintained at 37℃throughout the fermentation, the dissolved oxygen concentration was automatically controlled at 30% by a stirring rate (rpm) and aeration supply cascade, and the pH of the medium was maintained at 7.0 by 50% (v/v) orthophosphoric acid and 30% (v/v) aqueous ammonia. During the fermentation process, when the dissolved oxygen is greatly raised, the feeding is started. The feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol. When the OD600 was about 35.0 (wet weight about 60 g/L), induction was performed with 0.2mM IPTG for 16 hours. 2g of the cells were sonicated in 6mL of pure water, and then subjected to SDS-PAGE, and the crude enzyme solution was stored at-20 ℃.

Example 2 catalytic reaction application example

Firstly, preparing sucrose mother liquor (800 g/L), taking 800g of sucrose, adding water to a constant volume to 1L, and heating to aid dissolution for later use.

In a 5ml centrifuge tube, a total reaction system 1ml,50mM MES pH6.5 was prepared, sucrose (about 274 g/L) was added to the final concentration 0.8M PB pH6.5,0.8M, water was added to the mixture to 0.9ml, pH was adjusted to 6.5, and then the mixture was added with an enzyme solution ZY 4.1 ml and reacted in a shaking table at 37 ℃. Sampling and detecting for 3 hours. The HPLC results are shown in FIG. 2. Sucrose had a conversion of 90% for 3 hours and the amount of glucose by-produced was small.

Comparative example 1 control of catalytic reactions

In a 5ml centrifuge tube, a total reaction system 1ml,50mM MES pH6.5, the final concentration of 0.8M PB pH6.5,0.8M sucrose, water addition to 0.9ml, pH adjustment to 6.5, and enzyme solution ZY 3.1 ml and shaking table reaction at 37 ℃ are prepared. Sampling and detecting for 3 hours. The HPLC results are shown in FIG. 3. Sucrose 3 hours conversion was 87%. And the concentration of the target product is only 83% of that of the example 2, and more by-product glucose is generated on the detection map, so that the effective conversion rate is reduced.

Example 3 high concentration catalytic reaction example

In a 5ml centrifuge tube, a total reaction system 1ml,50mM MES pH6.5 was prepared, the final concentration of 1M PB pH6.5,1M sucrose (about 342 g/L), water was added to 0.9ml, the pH was adjusted to 6.5, and then, an enzyme solution ZY 4.1 ml was added to carry out a shaking reaction at 37 ℃. Sampling and detecting for 3 hours. The HPLC results are shown in FIG. 4. Sucrose 3 hours conversion was 90%.

Comparative example 2 high concentration catalytic reaction control example

In a 5ml centrifuge tube, a total reaction system 1ml,50mM MES pH6.5 was prepared, the final concentration of 1M PB pH was 6.5,1M sucrose was added to 0.9ml of water, the pH was adjusted to 6.5, and then, the mixture was added with an enzyme solution ZY 3.1 ml and subjected to shaking reaction at 37 ℃. Sampling and detecting for 3 hours. The HPLC results are shown in FIG. 5. Sucrose conversion was 88% for 3 hours. And the concentration of the target product is only 87% of that of the embodiment 3, so that more by-product glucose is generated on the detection map, and the effective conversion rate is reduced.

As can be seen from the comparison of the experimental results, the sucrose phosphorylase has the advantages of high effective conversion rate, less byproducts and higher reaction speed compared with the wild sucrose phosphorylase. The catalyst can catalyze the reaction in a short time under the condition of high concentration substrate, thereby obviously improving the production efficiency, reducing the energy consumption and comprehensively reducing the cost; the reaction system has high conversion rate and high reaction speed, can still keep the conversion rate of sucrose to be more than 85% in 3 hours reaction time when high-concentration sucrose reacts, and can effectively inhibit the generation of byproduct glucose. Is especially suitable for the industrial mass production of glucose-1-phosphate, and can obtain better social benefit and economic value.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

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accatccacg ctaacaccca cggtgaatct caggctgcta ccggtgctgc tgcttctaac 1020

ctggacctgt accaggttaa ctctacctac tactctgctc tgggttgcaa cgaccagcac 1080

tacatcgctg ctcgtgctgt tcagttcttc ctgccgggtg ttccgcaggt ttactacgtt 1140

ggtgctctgg ctggtaaaaa cgacatggaa ctgctgcgta aaaccaacaa cggtcgtgac 1200

atcaaccgtc actactactc taccgctgaa atcgacgaaa acctgaaacg tccggttgtt 1260

aaagctctga acgctctggc taaattccgt aacgaactgg acgctttcga cggtaccttc 1320

tcttacacca ccgacgacga cacctctatc tctttcacct ggcgtggtga aacctctcag 1380

gctaccctga ccttcgaacc gaaacgtggt ctgggtgttg acaacaccac cccggttgct 1440

atgctggaat gggaagactc tgctggtgac caccgttctg acgacctgat cgctaacccg 1500

ccggttgttg cttaa 1515

Claims

1. A production process of glucose-1-phosphoric acid is characterized in that: taking sucrose mother liquor, adding buffer solution and pure water, adding crude enzyme solution containing sucrose phosphorylase, and reacting at 37 ℃; the amino acid sequence of the sucrose phosphorylase is shown as SEQ ID NO: 1.

2. The process for producing glucose-1-phosphate according to claim 1, wherein the method for producing the crude enzyme solution comprises the steps of:

(2) Shaking flask fermentation

E.coli single colony containing the expression vector is selected and inoculated in 10mL of culture medium A after autoclaving, and is cultured at 30 ℃ and 250rpm overnight; the culture medium A is as follows: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and adding kanamycin to 50mg/L;

taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1: inoculating 100 to 100mL of autoclaved culture medium B, culturing at 30deg.C until the cell OD 5-6 is reached, immediately placing the triangular flask in 25 deg.C shaking table, and culturing at 250rpm for 1 hr; IPTG was added to a final concentration of 0.1mM and incubation was continued at 25℃and 250rpm for 16 hours; the culture medium B is as follows: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and adding kanamycin to 50mg/L;

alternatively, fed-batch fermentation:

fed-batch fermentation is carried out in a computer-controlled bioreactor, 200mL of culture is prepared by primary inoculation strain, and is connected to the bioreactor when the culture is OD2.0, wherein the strain is escherichia coli containing the expression vector and prepared in the step (1); the temperature was maintained at 37℃throughout the fermentation, the dissolved oxygen concentration during the fermentation was automatically controlled at 30% by the stirring rate and aeration supply cascade, and the pH of the medium was maintained at 7.0 by 50% v/v orthophosphoric acid and 30% v/v aqueous ammonia;

3. The process for producing glucose-1-phosphate according to claim 2, wherein: the culture medium used for fed-batch fermentation in the step (2) of the crude enzyme preparation method is as follows: 24g/L of yeast extract, 12g/L of peptone, 0.4% w/v glucose, 2.31g/L of phosphatase and 12.54g/L of dipotassium hydrogen phosphate, pH 7.0.