CN114395542B - Sucrose phosphorylase and application thereof - Google Patents
Sucrose phosphorylase and application thereof Download PDFInfo
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- CN114395542B CN114395542B CN202111658826.3A CN202111658826A CN114395542B CN 114395542 B CN114395542 B CN 114395542B CN 202111658826 A CN202111658826 A CN 202111658826A CN 114395542 B CN114395542 B CN 114395542B
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01007—Sucrose phosphorylase (2.4.1.7)
Abstract
The invention relates to the technical field of enzyme catalysis, in particular to sucrose phosphorylase and application thereof, 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, the glycerol and the pure water are added, and the crude enzyme solution of the sucrose phosphorylase is added for reaction at 37 ℃. The highest substrate concentration of the sucrose phosphorylase can reach 400g/L; and the conversion rate is greater than 90% in 3 hours. Compared with wild sucrose phosphorylase, the sucrose phosphorylase has higher reaction speed, can effectively shorten the reaction time, improve the production efficiency, reduce the energy consumption and comprehensively reduce the cost under high-concentration substrates; is especially suitable for industrial mass production of the glyceroglycosides.
Description
Technical Field
The invention relates to the technical field of enzyme catalysis, in particular to sucrose phosphorylase.
Background
2-O- α -D-glucopyranosyl glycerol is mainly found in cyanobacteria for combating the osmotic pressure of saline-alkaline environments; it was also found to be present in plants Myrothamnus flabellifolia and to have the effect of reviving the plants. Such desert plants can survive many years of complete desiccation without losing tissue integrity. Glucosyl glycerols also have an important function of protecting cells and tissues (e.g. skin), which offers the potential for use in cosmetics. Since glucosyl glycoside has a high water binding capacity, it gives a strong moisturizing effect when applied to skin. In addition to cosmetic applications, glucosyl glycerols have great potential as low calorie sweeteners, which may have prebiotic effects.
It has been reported to be useful as a substitute sweetener in foods because of its low cariogenicity and caloric value compared to sucrose. In addition, glucosyl glycerols and derivatives thereof have been studied as therapeutic agents for diseases caused by protein misfolding and cancer treatment. In cosmetics, glucosyl glycerols are used as anti-aging agents and as moisture regulating compounds.
In order to support the development of industrial applications, glucosyl glycerols must be efficiently provided on a large scale. Microbial production of glucosyl glycerols may be limited by the output parameters achievable by biosynthesis. Specifically, in the engineering corynebacterium glutamicum, the concentration of the product is less than or equal to 2g/L, the yield and the productivity on the used substrate are low, and in the engineering cyanobacteria polycystic strain, the yield is even lower.
The existing preparation methods mainly comprise chemical synthesis methods, enzymatic in-vitro catalysis and biological synthesis methods. The chemical process may involve various starting compounds such as maltitol, isomaltose, trehalose, etc. The use of sodium periodate and sodium borohydride has been reported to catalyze maltitol to glucosyl glycerol, but at 18% yield. The yield of the method for catalyzing isomaltose by using acetic acid, lead tetraacetate and sodium borohydride is 12%. The yield is only 5% by taking trehalose as a raw material. And the byproducts have larger influence on the subsequent purification. Biosynthesis can be achieved by treatment of microorganisms such as stenotrophomonas (Stenotrophomonas rhizophila DSM 14405) or cyanobacteria by salt stress, which has the disadvantage of large-scale production difficulties.
Enzymatic catalysis mainly involves alpha-glucosidase, cyclodextrin glucanotransferase, glycosyl-glycerol-phosphate synthase and sucrose phosphorylase.
Among them, sucrose phosphorylase (EC2.4.1.7) is a specific transglycosidase. Mainly catalyzes two reactions: in the first category, the glucosyl group of glucose-1-phosphate may be transferred to a receptor. For example, glucose-1-phosphate and D-fructose are capable of producing sucrose under the catalysis of sucrose phosphorylase. Second, the glucosyl group in sucrose is transferred to the receptor. For example, sucrose and phosphoric acid are capable of producing glucose-1-phosphate and D-fructose under the catalysis of sucrose phosphorylase. In the absence of external influences, sucrose phosphorylase catalyzes the reversible conversion of sucrose and phosphate to glucose 1-phosphate and D-fructose. In the absence of phosphate, glycerol may intercept the glycosylase intermediate that reacts with sucrose to produce glycosylglycerol, with minimal hydrolysis side reactions occurring.
An efficient route for the preparation of glucosyl glycerols is sucrose phosphorylase. Sucrose is an excellent donor substrate for glycosylation of glycerol, has high yield (. Gtoreq.90%), and has good cost economy. Glucosyl glycerols are manufactured industrially by Bitop AG (dotmond, germany) using the biocatalytic process of lmSucp and formulated as commercial products for cosmetic applications, sold as Glycoin (50% solution of GG).
Leuconostoc mesenteroides-derived sucrose phosphorylase has a broad acceptor specificity when glucose-1-phosphate and sucrose are used as donors.
Bifidobacterium adolescentis sucrose phosphorylase uses only arabinose, arabitol and xylitol as acceptors when glucose-1-phosphate is used as glycosyl donor, and uses sucrose as glycosyl donor, the acceptors are wider and have higher activity.
Published patent WO2008034158 to the university of Gratz's technology is the first use of sucrose phosphorylase (SPase) to catalyze the conversion of sucrose and glycerol to glycerol glucoside at a concentration of about 0.29M for the glucosyl glycerol product at about 70g/L yield under reaction conditions of 0.3M sucrose and 2.0M glycerol. However, the method has the disadvantages of large enzyme dosage, low product concentration and the like.
The patent CN109576239A utilizes Thermoanaerobacterium thermosaccharolyticum source heat-resistant sucrose phosphorylase to carry out catalytic reaction under high temperature condition, avoids the decomposition of the substrate and the product by the enzyme and the growth of the bacteria, and has the highest concentration of 189.8g/L and the highest conversion rate of 94%. However, high conversions (60 g/L to 200g/L sucrose) are only obtained at low substrate concentrations, and the enzyme dosage is relatively high, in the case of a maximum concentration of 300g/L sucrose, the enzyme dosage is 150g/L, the mass ratio of substrate to enzyme is 2:1, the enzyme cost is relatively high, and the conversion rate of 28 hours of reaction under the condition is only 85.2 percent.
Disclosure of Invention
The invention aims to provide sucrose phosphorylase and application thereof. The present invention relates to a simple single enzyme reaction for directly producing glucosyl glycerine from sucrose and glycerine, and is characterized by that it can shorten reaction time, raise substrate concentration, raise production efficiency and reduce production cost.
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 90 percent and is far higher than that of the wild type.
The sucrose phosphorylase can be used for catalyzing and producing the glyceroglycosides, and the using method is as follows: taking sucrose mother liquor, adding buffer solution, glycerol and pure water, adding the crude enzyme solution of sucrose phosphorylase, and reacting at 37 ℃.
Experiments prove that the highest substrate concentration of the sucrose phosphorylase can reach 400g/L; and the conversion rate is greater than 90% 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:
compared with wild sucrose phosphorylase, the sucrose phosphorylase has higher reaction speed, can effectively shorten the reaction time, improve the production efficiency, reduce the energy consumption and comprehensively reduce the cost under high-concentration substrates; the conversion rate of the reaction system is high and is more than 90 percent. Is especially suitable for the industrial mass production of the glyceroglycosides, and can obtain better social benefit and economic value.
Drawings
FIG. 1 is a map of a substrate standard; sucrose for 10 minutes and glycerin for 16.8 minutes.
FIG. 2 shows the reaction results of example 2, sucrose for 10 minutes, the target product for 11.7 minutes, fructose for 12.8 minutes, and glycerol for 16.8 minutes.
FIG. 3 shows the results of the reaction of the comparative example, sucrose for 10 minutes, target product for 11.7 minutes, fructose for 12.8 minutes, and glycerol for 16.8 minutes.
FIG. 4 shows the results of the test in example 3, sucrose for 10 minutes, the target product for 11.7 minutes, fructose for 12.8 minutes, and glycerin for 16.8 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 expression to realize high expression in Escherichia coli, and named ZY1.
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 ZY0.
(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.
2ml of the total reaction system, 50mM MES pH7.0,0.6M sucrose with final concentration and 1.8M glycerol are prepared in a 5ml centrifuge tube, water is added to 1.8ml, pH is adjusted to 7, and then 0.2ml of new enzyme preparation ZY is added to perform shaking table reaction at 37 ℃. Sampling and detecting for 3 hours. The HPLC results are shown in FIG. 2. Sucrose 3 hours conversion was greater than 95% at glycerol excess.
Comparative example control of catalytic reactions
2ml of the total reaction system, 50mM MES pH7.0,0.6M sucrose with final concentration and 1.8M glycerol are prepared in a 5ml centrifuge tube, water is added to 1.8ml, pH is adjusted to 7, and then 0.2ml of new enzyme preparation ZY is added to perform shaking table reaction at 37 ℃. Sampling and detecting for 3 hours. The HPLC results are shown in FIG. 3. At glycerol overdose, sucrose conversion was less than 70% for 3 hours.
Example 3 high concentration catalytic reaction 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.
2ml of the total reaction system, 50mM MES pH7.0,1.2M sucrose (reduced to 408g/L substrate concentration), 1.8M glycerol, water addition to 1.8ml, pH adjustment to 7, and addition of 1.2 ml of fresh enzyme solution ZY, and shaking reaction at 37 ℃. Sampling and detecting for 3 hours. Because of the high substrate concentration, the sample is diluted 20 times with pure water before injection. The HPLC results are shown in FIG. 4. Sucrose 3 hours conversion was greater than 95% at glycerol excess.
As can be seen from the comparison of the experimental results, the sucrose phosphorylase has a faster reaction speed than the wild sucrose phosphorylase, and can still effectively shorten the reaction time at high concentration of substrate, improve the production efficiency, reduce the energy consumption and comprehensively reduce 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 90% in 3 hours reaction time when high-concentration sucrose reacts, and has no obvious byproducts except fructose. Is especially suitable for the industrial mass production of the glyceroglycosides, 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
<110> Nanjing Nocloud biotechnology Co., ltd
<120> sucrose phosphorylase and use thereof
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His Tyr Ser Ile Pro Lys Lys Ile Asn Asp His Gly Tyr Phe Thr Tyr
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Asp Phe Ala Leu Pro Met Thr Thr Leu Tyr Thr Leu Tyr Ser Gly Lys
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Ile Leu Thr Asp Asp Glu Ile Asp Tyr Ala Ser Glu Gln Leu Tyr Lys
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Val Gly Ala Asn Val Lys Lys Thr Tyr Thr Ser Ala Ser Tyr Asn Asn
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Phe Asp Phe Met Ile Asn His Ile Ser Arg Glu Ser Val Met Tyr Gln
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Asp Phe Lys Lys Asn His Asp Asp Ser Lys Tyr Lys Asp Phe Phe Ile
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His Tyr Ser Ile Pro Lys Lys Ile Asn Asp His Gly Tyr Phe Thr Tyr
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Thr Thr Leu Asp Thr His Asp Gly Ile Gly Val Val Asp Ala Arg Asp
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Ile Leu Thr Asp Asp Glu Ile Asp Tyr Ala Ser Glu Gln Leu Tyr Lys
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Val Gly Ala Asn Val Lys Lys Thr Tyr Ser Ser Ala Ser Tyr Asn Asn
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Leu Asp Ile Tyr Gln Ile Asn Ser Thr Tyr Tyr Ser Ala Leu Gly Asn
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Asp Asp Ala Ala Tyr Leu Leu Ser Arg Val Phe Gln Val Phe Ala Pro
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Gly Ile Pro Gln Ile Tyr Tyr Val Gly Leu Leu Ala Gly Glu Asn Asp
370 375 380
Ile Ala Leu Leu Glu Ser Thr Lys Glu Gly Arg Asn Ile Asn Arg His
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Tyr Tyr Thr Arg Glu Glu Val Lys Ser Glu Val Lys Arg Pro Val Val
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Ala Asn Leu Leu Lys Leu Leu Ser Trp Arg Asn Glu Ser Pro Ala Phe
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Val Val Thr Arg Gln Asp Glu Asn Gly Gln Asn Lys Ala Val Leu Thr
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Ala Asp Ala Ala Asn Lys Thr Phe Glu Ile Val Glu Asn Gly Gln Thr
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Val Met Ser Ser Asp Asn Leu Thr Gln Asn
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aacgaagttc gtgaaatcct gaccccgctg aaagctgaaa tcctgccgga aatccacgaa 720
cactactcta tcccgaaaaa aatcaacgac cacggttact tcacctacga cttcgctctg 780
ccgatgacca ccctgtacac cctgtactct ggtaaaacca accagctggc taaatggctg 840
aaaatgtctc cgatgaaaca gttcaccgtt ctggacaccc acgacggtat cggtgttgtt 900
gacgctcgtg acatcctgac cgacgacgaa atcgactacg cttctgaaca gctgtacaaa 960
gttggtgcta acgttaaaaa aacctacacc tctgcttctt acaacaacct ggacatctac 1020
cagatcaact ctacctacta ctctgctctg ggtaacgacg acgctgctta cctgctgtct 1080
cgtgttttcc aggttttcgc tccgggtatc ccgcagatct actacgttgg tctgctggct 1140
ggtgaaaacg acatcgctct gctggaatct accaaagaag gtcgtaacat caaccgtcac 1200
tactacaccc gtgaagaagt taaatctgaa gttaaacgtc cggttgttgc taacctgctg 1260
aaactgctgt cttggcgtaa cgaatctccg gctttcgacc tggctggttc tatcaccgtt 1320
gacaccccga ccgacaccac catcgttgtt acccgtcagg acgaaaacgg tcagaacaaa 1380
gctgttctga ccgctgacgc tgctaacaaa accttcgaaa tcgttgaaaa cggtcagacc 1440
gttatgtctt ctgacaacct gacccagaac taa 1473
<210> 4
<211> 1473
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atggaaatcc agaacaaagc tatgctgatc acctacgctg actctctggg taaaaacctg 60
aaagacgttc accaggttct gaaagaagac atcggtgacg ctatcggtgg tgttcacctg 120
ctgccgttct tcccgtctac cggtgaccgt ggtttcgctc cggctgacta cacccgtgtt 180
gacgctgctt tcggtgactg ggctgacgtt gaagctctgg gtgaagaata ctacctgatg 240
ttcgacttca tgatcaacca catctctcgt gaatctgtta tgtaccagga cttcaaaaaa 300
aaccacgacg actctaaata caaagacttc ttcatccgtt gggaaaaatt ctgggctaaa 360
gctggtgaaa accgtccgac ccaggctgac gttgacctga tctacaaacg taaagacaaa 420
gctccgaccc aggaaatcac cttcgacgac ggtaccaccg aaaacctgtg gaacaccttc 480
ggtgaagaac agatcgacat cgacgttaac tctgctatcg ctaaagaatt catcaaaacc 540
accctggaag acatggttaa acacggtgct aacctgatcc gtctggacgc tttcgcttac 600
gctgttaaaa aagttgacac caacgacttc ttcgttgaac cggaaatctg ggacaccctg 660
aacgaagttc gtgaaatcct gaccccgctg aaagctgaaa tcctgccgga aatccacgaa 720
cactactcta tcccgaaaaa aatcaacgac cacggttact tcacctacga cttcgctctg 780
ccgatgacca ccctgtacac cctgtactct ggtaaaacca accagctggc taaatggctg 840
aaaatgtctc cgatgaaaca gttcaccacc ctggacaccc acgacggtat cggtgttgtt 900
gacgctcgtg acatcctgac cgacgacgaa atcgactacg cttctgaaca gctgtacaaa 960
gttggtgcta acgttaaaaa aacctactct tctgcttctt acaacaacct ggacatctac 1020
cagatcaact ctacctacta ctctgctctg ggtaacgacg acgctgctta cctgctgtct 1080
cgtgttttcc aggttttcgc tccgggtatc ccgcagatct actacgttgg tctgctggct 1140
ggtgaaaacg acatcgctct gctggaatct accaaagaag gtcgtaacat caaccgtcac 1200
tactacaccc gtgaagaagt taaatctgaa gttaaacgtc cggttgttgc taacctgctg 1260
aaactgctgt cttggcgtaa cgaatctccg gctttcgacc tggctggttc tatcaccgtt 1320
gacaccccga ccgacaccac catcgttgtt acccgtcagg acgaaaacgg tcagaacaaa 1380
gctgttctga ccgctgacgc tgctaacaaa accttcgaaa tcgttgaaaa cggtcagacc 1440
gttatgtctt ctgacaacct gacccagaac taa 1473
Claims (5)
1. A sucrose phosphorylase, characterized in that: the amino acid sequence is shown in SEQ ID NO: 1.
2. Use of the sucrose phosphorylase according to claim 1 in the catalytic production of glycerol glucoside.
3. Use of sucrose phosphorylase according to claim 2 in the catalytic production of glycerol glucoside, characterized in that: taking sucrose mother liquor, adding buffer solution, glycerol and pure water, adding the crude enzyme solution of the sucrose phosphorylase in claim 1, and reacting at 37 ℃.
4. Use of sucrose phosphorylase in the catalytic production of glycerol glucoside according to claim 3, wherein the method for preparing the crude enzyme solution comprises the steps of:
(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; 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;
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 ℃;
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;
during fermentation, when a large rise of dissolved oxygen occurs, feeding is started, 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 ℃.
5. The use of sucrose phosphorylase according to claim 4 in the catalytic production of glycerol glucoside, 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.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110438100A (en) * | 2019-08-02 | 2019-11-12 | 中国科学院天津工业生物技术研究所 | A kind of method of biocatalysis synthetic glycerine glucoside |
| CN111621483A (en) * | 2020-06-05 | 2020-09-04 | 江南大学 | Sucrose phosphorylase mutant and application thereof |
| CN113383072A (en) * | 2018-10-29 | 2021-09-10 | 博努莫斯股份有限公司 | Enzyme method for producing hexose |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113383072A (en) * | 2018-10-29 | 2021-09-10 | 博努莫斯股份有限公司 | Enzyme method for producing hexose |
| CN110438100A (en) * | 2019-08-02 | 2019-11-12 | 中国科学院天津工业生物技术研究所 | A kind of method of biocatalysis synthetic glycerine glucoside |
| CN111621483A (en) * | 2020-06-05 | 2020-09-04 | 江南大学 | Sucrose phosphorylase mutant and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| sucrose phosphorylase [Leuconostoc pseudomesenteroides];NCBI Reference Sequence: WP_010279952.1;《GenBank》;参见序列及相关信息 * |
| 蔗糖磷酸化酶及其在糖基化反应中的应用;江瑞妮等;《生物工程学报》;第37卷(第1期);第112-129页，参见全文 * |
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