CN113957065A - Sucrose isomerase with high conversion rate and application thereof - Google Patents
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Abstract
The invention relates to the technical field of enzyme catalysis, in particular to sucrose isomerase with high conversion rate and application thereof, wherein the amino acid sequence of the sucrose isomerase is shown as SEQ ID NO: 1 is shown. When in use, the cane sugar mother liquor is taken, PB and pure water are added, the crude enzyme liquid of the cane sugar isomerase is added, and the reaction is carried out at 30 ℃. Wherein the highest substrate concentration of the enzyme is less than or equal to 700g/L, and the substrate: the crude enzyme liquid feeding ratio is less than or equal to 100: 1. compared with wild sucrose isomerase, the high-conversion-rate sucrose isomerase has higher substrate concentration, higher reaction speed and low enzyme dosage, and can effectively reduce the cost; the conversion rate is high and is more than 95 percent.
Description
Technical Field
The invention relates to the technical field of enzyme catalysis, in particular to sucrose isomerase with high conversion rate.
Background
Sucrose is a commonly used sweetener. Once ingested, sucrose degrades into glucose and fructose, raising blood glucose levels. Excessive intake of sucrose is easily associated with obesity and health problems such as heart disease and type 2 diabetes. With the increasing level of living and health awareness, people are beginning to focus on healthier functional sweeteners that can replace sucrose.
With the improvement of living standard and the development of health consciousness, people pay more attention to diet. In the sweetener method, people prefer low-calorie and health-care functional products such as D-tagatose, psicose, isomaltulose, fructo-oligosaccharide, and the like. As an alternative to sucrose, isomaltulose has a number of health advantages, such as prolonged energy release, slower digestion, greater stability, reduced insulin levels, prevention of tooth decay and glycemic index. Therefore, isomaltulose is considered a parenteral nutrient for the treatment of diabetes. In addition, isomaltulose, as a reducing functional disaccharide, is currently the only sweetener that is not limited in amount.
Isomaltulose, commonly known as palatinose, is formed from d-glucose and d-fructose linked by an alpha-1, 6-glycosidic bond, unlike the alpha-1, 2-glycosidic bond in sucrose. Isomaltulose has similar physical and organoleptic properties as sucrose. However, it has several advantages over sucrose: isomaltulose is a low-calorie, non-cariogenic nutrient sugar, since it has less sweetness than sucrose (about 50% of sweetness); in addition, isomaltulose is slowly hydrolyzed by enzymes and absorbed into the body in the form of glucose and fructose, thereby reducing insulin levels and glycemic index. Therefore, isomaltulose has the potential to be a sucrose substitute for obesity and diabetes.
The synthesis of isomaltulose generally involves chemical or enzymatic methods. The chemical method is always accompanied with the formation of some byproducts, the color is brown, and the separation and purification difficulty of products is increased; in addition, these difficulties increase production costs and energy consumption. Therefore, the synthesis of isomaltulose using sucrose as a substrate by using a biotransformation technique has become a research hotspot. In addition, as an efficient and green biocatalyst, enzymes have the advantages of milder reaction conditions, easier processing, higher product yield and the like, so that the enzyme method is more and more important in various industries.
In industry, isomaltulose is produced mainly from sucrose by bacterial fermentation. These isomaltulose producing strains can synthesize sucrose isomerase, which is responsible for the isomerization of sucrose to isomaltulose and other by-products, such as trehalose, fructose and glucose. However, the concentration of sucrose substrate is limited, so that the yield of isomaltulose during fermentation is reduced.
At present, sucrose isomerase has been disclosed from various microbial sources, but according to the literature reports, the preparation of isomaltulose using sucrose isomerase has the following problems:
(1) as the reaction time is prolonged, the proportion of the heterosugar is increased, such as glucose, trehalulose and the like which account for 10-20 percent in total. In extreme cases, up to 50% of trehalulose has been observed.
(2) The reaction system of high concentration substrate can effectively increase the space-time production efficiency and reduce the energy consumption, and the microorganism can not grow under the high osmotic pressure of 800g/L sucrose, so the reaction system can be produced in open equipment.
Therefore, the development of a sucrose isomerase or mutant with high conversion rate shortens the reaction time, reduces the heterosugar ratio to a certain extent, and has great practical value.
Disclosure of Invention
The invention aims to provide a sucrose isomerase with high conversion rate and application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
a sucrose isomerase with high conversion rate has an amino acid sequence shown as SEQ ID NO: 1 is shown.
The substrate concentration and the reaction speed of the sucrose isomerase of the invention are higher than those of the wild sucrose isomerase. The conversion rate is more than 95%.
The sucrose isomerase with high conversion rate can be used for catalyzing and producing isomaltulose, and the using method comprises the following steps: taking a sucrose mother liquor, adding PB and pure water, adding a crude enzyme solution of sucrose isomerase, and reacting at 30 ℃.
Through a large number of experiments, the highest substrate concentration of the sucrose isomerase is less than or equal to 700 g/L; substrate: the crude enzyme liquid feeding ratio is less than or equal to 100: 1.
wherein, the preparation method of the crude enzyme solution comprises the following steps:
(1) by a primer splicing method, the sequence shown in SEQ ID NO: 1, and cloning to a prokaryotic expression vector to realize high expression in escherichia coli;
(2) shake flask fermentation
Selecting a single escherichia coli colony containing an expression vector, inoculating the single escherichia coli colony into 10mL of culture medium A subjected to autoclaving, and carrying out overnight culture at 30 ℃ and 250 rpm;
taking a 1L triangular flask the next day, and carrying out the following steps: 100 of the inoculation ratio example is inoculated into 100mL of culture medium B after autoclaving, the culture is carried out at 30 ℃ until the thallus OD 5-6, and the triangular flask is immediately placed in a shaker at 25 ℃ and cultured for 1 hour at 250 rpm; IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 h at 250 rpm;
after the culture is finished, centrifuging the culture solution at 4 ℃ and 12000g for 20 minutes to collect wet thalli; then washing the thallus precipitate twice with distilled water, collecting thallus and preserving at-70 ℃; meanwhile, 2g of thalli is taken and added with 6mL of pure water for ultrasonic crushing, SDS-PAGE detection is carried out, and the crude enzyme liquid is preserved at the temperature of minus 20 ℃;
(3) fed-batch fermentation
Fed-batch fermentation is carried out in a bioreactor controlled by a computer, 200mL of culture is prepared by primary inoculation of strains, and the strains are inoculated when OD2.0 is reached; during the whole fermentation process, the temperature is kept at 37 ℃, the dissolved oxygen concentration during the fermentation process is automatically controlled at 30 percent by controlling the stirring speed and the aeration supply cascade, and the pH value of the culture medium is maintained at 7.0 by 50 percent of orthophosphoric acid and 30 percent of ammonia water;
during the fermentation, when a large dissolved oxygen rise occurs, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when OD600 was 35.0, induction with 0.2mM IPTG for 16 hours; taking 2g of thallus, adding 6mL of pure water, carrying out ultrasonic disruption, carrying out SDS-PAGE detection, and storing the crude enzyme liquid at-20 ℃.
Further, the culture medium A in the step (2) is: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.8g/L glucose, and kanamycin to 50 mg/L.
The culture medium B comprises: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.3g/L glucose, and kanamycin to 50 mg/L.
Further, the culture medium used in step (3) is: 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L catalase phosphate and 12.54g/L dipotassium phosphate, pH 7.0.
Compared with the prior art, the invention has the beneficial effects that:
compared with wild sucrose isomerase, the high-conversion-rate sucrose isomerase has higher substrate concentration, higher reaction speed and low enzyme dosage, and can effectively reduce the cost; the conversion rate is high and is more than 95 percent; is especially suitable for industrial large-scale production of isomaltulose, and can obtain better social benefit and economic value.
Drawings
FIG. 1 is a TLC detection profile of example 2; sequentially preparing a sucrose standard product, an isomaltose standard product and a reaction sample from left to right;
FIG. 2 is a 20g/L isomaltulose standard detection map; wherein the peak emergence time is 8.8 min;
FIG. 3 shows the detection result of example 3, wherein 8.7min represents the target product.
FIG. 4 shows the results of the detection in example 9, where 8.7min represents the objective product and 8.3min represents the residual substrate peak.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The detection conditions referred to in the following examples are as follows:
(1) conditions of liquid phase detection
Mobile phase: acetonitrile: 70 parts of water: 30
A detector: differential detector
Flow rate: 1mL/min
Column temperature: 40 deg.C
Differential detector cell temperature: 40 deg.C
A 250mm by 4.6um amino column was used.
(2) TLC detection conditions
Developing agent: and (2) adding n-butyl alcohol: acetone: mixing water at a ratio of 4:3:1, adding a drop of acetic acid, and running for 2 times (running once for blow drying and running once again).
Color developing agent: 4 percent of aniline, 4 percent of diphenylamine and 85 percent of phosphoric acid are mixed according to the volume ratio of 5:5:1 for use, the developing temperature is 85 ℃, and the developing time is 10 minutes.
EXAMPLE 1 preparation of crude enzyme solution
(1) By a primer splicing method, the sequence shown in SEQ ID NO: 1, and cloning into a prokaryotic expression vector to realize high expression in Escherichia coli, and is named as PD 1. The specific operation is as follows:
primers PD1-1 to PD1-48 (shown as SEQ ID NO: 3-50) were dissolved in double distilled water and added to the reaction system so that the final concentration of each primer was 30nM and the final concentration of the head and tail primers was 0.6. mu.M.
| 2mM dNTP mix(2mM each dNTP) | 5μl |
| 10×Pfu buffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH2O | The total volume of the reaction system was adjusted to 50. mu.l |
The prepared PCR reaction system is placed in a Bori XP cycler gene amplification instrument and amplified according to the following procedures: 30s at 98 ℃, 45s at 55 ℃, 120s at 72 ℃ and 35 x. And (3) carrying out gel cutting, purifying, cloning and sequencing on the DNA fragment obtained by the PCR.
Likewise, the control wild-type protein SEQ ID NO: 2 and cloned into a prokaryotic expression vector, designated PD 2. The primers PD 2-1-PD 2-48 are shown as SEQ ID NO: 51-98.
(2) Shake flask fermentation
Coli single colonies containing the expression vector were picked and inoculated into 10mL of autoclaved medium: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.8g/L glucose, and kanamycin to 50 mg/L. The culture was carried out at 30 ℃ and 250rpm overnight. Taking a 1L triangular flask the next day, and carrying out the following steps: 100 inoculation ratio examples were inoculated into 100mL of autoclaved medium: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.3g/L glucose, and kanamycin to 50 mg/L. The cells were cultured at 30 ℃ until the OD 5-6 of the cells became zero, and the cells were immediately placed in a flask in a shaker at 25 ℃ and cultured at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 hours at 250 rpm. After completion of the culture, the culture was centrifuged at 12000g at 4 ℃ for 20 minutes to collect wet cells. Then the bacterial pellet is washed twice with distilled water, and the bacterial is collected and preserved at-70 ℃. Meanwhile, 2g of the thalli are added into 6mL of pure water for ultrasonic disruption, SDS-PAGE detection is carried out, and the crude enzyme solution is stored at the temperature of minus 20 ℃.
(3) Fed-batch fermentation:
the fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai Seisaku) with a reactor capacity of 15L and a working volume of 8L, using 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L catalase phosphate and 12.54g/L dipotassium hydrogen phosphate as the medium, pH 7.0. 200mL of the culture was prepared for the primary inoculum and inoculated at OD 2.0. Throughout the fermentation, the temperature was maintained at 37 ℃, the dissolved oxygen concentration during fermentation was automatically controlled at 30% by the agitation rate (rpm) and aeration supply cascade, while 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, when a large amount of dissolved oxygen rises, feeding is started. The feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol. When OD600 was about 35.0 (wet weight about 60g/L), induction was carried out with 0.2mM IPTG for 16 hours. Taking 2g of thallus, adding 6mL of pure water, carrying out ultrasonic disruption, carrying out SDS-PAGE detection, and storing the crude enzyme liquid at-20 ℃.
Example 2700 g/L sucrose catalyzed reaction
Firstly, preparing a cane sugar mother solution (800g/L), taking 800g of cane sugar, adding water to a constant volume of 1L, heating and dissolving assisting for later use.
1.75mL of sucrose stock solution (800g/L) was added with 0.1mL of 1M PB buffer solution (pH5.8), 56. mu.L of crude enzyme solution PD1 was added, and water was added to 2 mL. The reaction was allowed to proceed for 24 hours, and the dot plate showed that the substrate had reacted to completion. The TLC results are shown in FIG. 1.
Example 3700 g/L sucrose catalyzed reaction
1.75mL of sucrose stock solution (800g/L) was added with 0.1mL of 1M PB buffer solution (pH5.8), and 28. mu.L of crude enzyme solution PD1 was added, and water was added to 2 mL. The reaction was allowed to proceed for 24 hours, and the dot plate showed that the substrate had reacted to completion.
That is, at a substrate concentration of 700g/L, the substrate: when the dosage of the crude enzyme solution is 50:1, the substrate can be completely converted within 24 hours, and no sucrose residue exists. And the conversion rate of the target product is more than 95 percent. The liquid phase results are shown in FIG. 3.
Example 4700 g/L sucrose catalyzed reaction
1.75mL of sucrose stock solution (800g/L) was added with 0.1mL of 1M PB buffer solution (pH5.8), 14. mu.L of crude enzyme solution PD1 was added, and water was added to 2 mL. The reaction was carried out for 24 hours, and the dot plate showed that the substrate remained significantly.
Example 5500 g/L sucrose catalyzed reaction
500g/L sucrose reaction system: 1.25mL of sucrose stock solution (800g/L) was added to 0.1mL of 1M PB buffer (pH5.8), 0.55mL of purified water was added, and finally, 10.04mL of crude enzyme solution PD10 was added, in which case the total volume was about 2mL, and the shaking reaction was carried out at 30 ℃. Plate TLC was sampled at 24 hours for reaction to detect substrate residue. The results showed no sucrose residue.
Example 6500 g/L sucrose catalyzed reaction
500g/L sucrose reaction system: 1.25mL of sucrose mother liquor (800g/L) was added with 0.1mL of 1M PB buffer (pH5.8), 0.55mL of purified water, and finally, 10.02mL of crude enzyme solution PD10, which was about 2mL in total, and subjected to a shake reaction at 30 ℃. Plate TLC was sampled at 24 hours for reaction to detect substrate residue. The results showed no sucrose residue.
Example 7500 g/L sucrose catalyzed reaction
500g/L sucrose reaction system: 1.25mL of sucrose stock solution (800g/L) was added to 0.1mL of 1M PB buffer (pH5.8), 0.55mL of purified water was added, and 10.01mL of crude enzyme solution PDN was added, which was about 2mL in total, and the mixture was subjected to a shaking reaction at 30 ℃. Plate TLC was sampled at 24 hours for reaction to detect substrate residue. The results showed no sucrose residue.
That is, at a substrate concentration of 500g/L, the substrate: when the adding amount of the crude enzyme solution is 100:1, the substrate can be completely converted within 24 hours, no sucrose residue exists, and the conversion rate of the target product is more than 95%.
Example 8500 g/L sucrose catalyzed reaction
500g/L sucrose reaction system: 1.25mL of sucrose mother liquor (800g/L) was added with 0.1mL of 1M PB buffer (pH5.8), 0.55mL of purified water, and finally, crude enzyme solution PD 15. mu.L, which was about 2mL in total, and subjected to a shake reaction at 30 ℃. Plate TLC was sampled at 24 hours for reaction to detect substrate residue. The results showed that the substrate did not react completely.
Example 9700 g/L sucrose control experiment
1.75mL of sucrose stock (800g/L) was added to 0.1mL of 1M PB buffer (pH5.8), and the mixture was added to SEQ ID NO: 2, 28. mu.L of the control crude enzyme solution was supplemented with 2mL of water. The reaction was carried out for 24 hours, and the dot plate showed that the substrate did not react completely.
From this, it is understood that, at a substrate concentration of 700g/L, the substrate: complete conversion of the substrate in 24 hours was not achieved with the PD2 control crude enzyme solution at a dosage of 50: 1. The liquid phase results are shown in FIG. 4.
As can be seen from the comparison of the above experimental results, the sucrose isomerase of the present invention has a higher substrate concentration than the wild-type sucrose isomerase; compared with wild protein, the reaction speed is higher when the substrate is in high concentration, the enzyme dosage is low, and the cost can be effectively reduced; the conversion rate is high and is more than 95 percent.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments 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 Nuo cloud Biotechnology Ltd
<120> sucrose isomerase with high conversion rate and application thereof
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Leu Asp Arg Tyr Pro Asp Gln Arg Trp Arg Arg Lys Pro Trp Thr Leu
305 310 315 320
Ser Gln Phe Arg Gln Val Ile Ser Gln Thr Asp Arg Ala Ala Gly Glu
325 330 335
Phe Gly Trp Asn Ala Phe Phe Leu Asp Asn His Asp Asn Pro Arg Gln
340 345 350
Val Ser His Phe Gly Asp Asp Ser Pro Gln Trp Arg Glu Arg Ser Ala
355 360 365
Lys Ala Leu Ala Thr Leu Leu Leu Thr Gln Arg Ala Thr Pro Phe Ile
370 375 380
Phe Gln Gly Ala Glu Leu Gly Met Thr Asn Tyr Pro Phe Lys Asn Ile
385 390 395 400
Glu Glu Phe Asp Asp Ile Glu Val Lys Gly Phe Trp Asn Asp Tyr Val
405 410 415
Ala Ser Gly Lys Val Asn Ala Ala Glu Phe Leu Gln Glu Val Arg Met
420 425 430
Thr Ser Arg Asp Asn Ser Arg Thr Pro Met Gln Trp Asn Asp Ser Val
435 440 445
Asn Ala Gly Phe Thr Gln Gly Lys Pro Trp Phe His Leu Asn Pro Asn
450 455 460
Tyr Lys Gln Ile Asn Ala Ala Arg Glu Val Asn Lys Pro Asp Ser Val
465 470 475 480
Phe Ser Tyr Tyr Arg Gln Leu Ile Asn Leu Arg His Gln Ile Pro Ala
485 490 495
Leu Thr Ser Gly Glu Tyr Arg Asp Leu Asp Pro Gln Asn Asn Gln Val
500 505 510
Tyr Ala Tyr Thr Arg Ile Leu Asp Asn Glu Lys Tyr Leu Val Val Val
515 520 525
Asn Phe Lys Pro Glu Gln Leu His Tyr Ala Leu Pro Asp Asn Leu Thr
530 535 540
Ile Ala Ser Ser Leu Leu Glu Asn Val His Gln Pro Ser Leu Gln Glu
545 550 555 560
Asn Ala Ser Thr Leu Thr Leu Ala Pro Trp Gln Ala Gly Ile Tyr Lys
565 570 575
Leu Asn
<210> 3
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tgtttaactt taagaaggag atatacatat gaaatac 37
<210> 4
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
agcagaccag cagcagcggt cggcagcagg tatttcatat gtatatctcc ttcttaaagt 60
<210> 5
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gctgctgctg gtctgctgct cctcgctgcc cagccggcga tggccatggc ttctccgctg 60
<210> 6
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tttctggatg ttggtagcag cgatcggggt agacggtttg gtcagcggag aagccatggc 60
<210> 7
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gctgctacca acatccagaa atctgctgac ttcccgatct ggtggaaaca ggctgttttc 60
<210> 8
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
accgttagag tctttgaaag aacgcgggta gatctggtag aaaacagcct gtttccacca 60
<210> 9
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cgttctttca aagactctaa cggtgacggt atcggtgaca tcccgggtat catcgaaaaa 60
<210> 10
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cagatagcgt caacacccag cattttcagg tagtccagtt tttcgatgat acccgggatg 60
<210> 11
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ctgggtgttg acgctatctg gatcaacccg cactacgaat ctccgaacac cgacaacggt 60
<210> 12
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
accgtattct ttcatgattt tacggtagtc agagatgtcg taaccgttgt cggtgttcgg 60
<210> 13
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ccgtaaaatc atgaaagaat acggttctat ggctgacttc gaccgtctgg ttgctgaaat 60
<210> 14
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gataacgatg tcgatcatca gacgcatacc acgtttgttc atttcagcaa ccagacggtc 60
<210> 15
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gtctgatgat cgacatcgtt atcaaccaca cctctgaccg tcaccgttgg ttcgttcagt 60
<210> 16
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gaagtagtag tcacggtacg ggttgtcttt accagaacga gactgaacga accaacggtg 60
<210> 17
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cccgtaccgt gactactact tctggcgtga cggtaaacag ggtcaggctc cgaacaacta 60
<210> 18
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tttgtccagc tgccaagcag aaccaccgaa gaaagacggg tagttgttcg gagcctgacc 60
<210> 19
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gcttggcagc tggacaaaca gaccgaccag tactacctgc actacttcgc tccgcagcag 60
<210> 20
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
acagttcagc acgaactttc gggttgtccc agttcaggtc cggctgctgc ggagcgaagt 60
<210> 21
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cgaaagttcg tgctgaactg tacgacatcc tgcgtttctg gctggacaaa ggtgtttctg 60
<210> 22
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gggattttag agaaggtagc aacggtgtcg aaacgcagac cagaaacacc tttgtccagc 60
<210> 23
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gttgctacct tctctaaaat cccgggtttc ccggacctgt ctaaagctca gctgaaaaac 60
<210> 24
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gtggatgttc ggacctttgg tgtaagcttc agcgaagttt ttcagctgag ctttagacag 60
<210> 25
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ccaaaggtcc gaacatccac aaatacatcc acgaaatgaa ccgtcaggtt ctgtctaaat 60
<210> 26
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ggaacaccga agatttcacc agcggtagca acgttgtatt tagacagaac ctgacggttc 60
<210> 27
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tggtgaaatc ttcggtgttc cggtttctgc tatgccggac tacttcgacc gtcgtcgtga 60
<210> 28
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tccagacgga tcaggtcgaa ggtgaaagcg atgttcagtt cttcacgacg acggtcgaag 60
<210> 29
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tcgacctgat ccgtctggac cgttacccgg accagcgttg gcgtcgtaaa ccgtggaccc 60
<210> 30
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
agcacggtcg gtctgagaga taacctgacg gaactgagac agggtccacg gtttacgacg 60
<210> 31
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tctcagaccg accgtgctgc tggtgaattc ggttggaacg ctttcttcct ggacaaccac 60
<210> 32
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
agagtcgtca ccgaagtgag aaacagcacg cgggttgtcg tggttgtcca ggaagaaagc 60
<210> 33
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ctcacttcgg tgacgactct ccgcagtggc gtgaacgttc tgctaaagct ctggctaccc 60
<210> 34
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ccctggaaga tgaacggggt agcacgctgg gtcagcagca gggtagccag agctttagca 60
<210> 35
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
ccccgttcat cttccagggt gctgaactgg gtatgaccaa ctacccgttc aaaaacatcg 60
<210> 36
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ttccagaaac ctttaacttc gatgtcgtcg aattcttcga tgtttttgaa cgggtagttg 60
<210> 37
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
catcgaagtt aaaggtttct ggaacgacta cgttgcttct ggtaaagtta acgctgctga 60
<210> 38
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gagttgtcac gagaggtcat acgaacttcc tgcaggaatt cagcagcgtt aactttacca 60
<210> 39
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gtatgacctc tcgtgacaac tctcgtaccc cgatgcagtg gaacgactct gttaacgctg 60
<210> 40
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tcgggttcag gtggaaccac ggtttaccct gggtgaaacc agcgttaaca gagtcgttcc 60
<210> 41
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ggttccacct gaacccgaac tacaaacaga tcaacgctgc tcgtgaagtt aacaaaccgg 60
<210> 42
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
caggttgatc agctgacggt agtaagagaa aacagagtcc ggtttgttaa cttcacgagc 60
<210> 43
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
ccgtcagctg atcaacctgc gtcaccagat cccggctctg acctctggtg aataccgtga 60
<210> 44
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
cgggtgtaag cgtaaacctg gttgttctgc gggtccaggt cacggtattc accagaggtc 60
<210> 45
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
caggtttacg cttacacccg tatcctggac aacgaaaaat acctggttgt tgttaacttc 60
<210> 46
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gtccggcaga gcgtagtgca gctgttccgg tttgaagtta acaacaacca ggtatttttc 60
<210> 47
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
actacgctct gccggacaac ctgaccatcg cttcttctct gctggaaaac gttcaccagc 60
<210> 48
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ggagccaggg tcagggtaga agcgttttcc tgcagagacg gctggtgaac gttttccagc 60
<210> 49
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
accctgaccc tggctccgtg gcaggctggt atctacaaac tgaactaact cgagcaccac 60
<210> 50
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cggatctcag tggtggtggt ggtggtgctc gagttagttc ag 42
<210> 51
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tgtttaactt taagaaggag atatacatat gaaatac 37
<210> 52
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
agcagaccag cagcagcggt cggcagcagg tatttcatat gtatatctcc ttcttaaagt 60
<210> 53
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
gctgctgctg gtctgctgct cctcgctgcc cagccggcga tggccatggc ttctccgctg 60
<210> 54
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tttctggatg ttggtagcag cgatcggggt agacggtttg gtcagcggag aagccatggc 60
<210> 55
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gctgctacca acatccagaa atctgctgac ttcccgatct ggtggaaaca ggctgttttc 60
<210> 56
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
accgttagag tctttgaaag aacgcgggta gatctggtag aaaacagcct gtttccacca 60
<210> 57
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
cgttctttca aagactctaa cggtgacggt atcggtgaca tcccgggtat catcgaaaaa 60
<210> 58
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
cagatagcgt caacacccag cattttcagg tagtccagtt tttcgatgat acccgggatg 60
<210> 59
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
ctgggtgttg acgctatctg gatcaacccg cactacgaat ctccgaacac cgacaacggt 60
<210> 60
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
accgtattct ttcatgattt tacggtagtc agagatgtcg taaccgttgt cggtgttcgg 60
<210> 61
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ccgtaaaatc atgaaagaat acggttctat ggctgacttc gaccgtctgg ttgctgaaat 60
<210> 62
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gataacgatg tcgatcatca gacgcatacc acgtttgttc atttcagcaa ccagacggtc 60
<210> 63
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
gtctgatgat cgacatcgtt atcaaccaca cctctgaccg tcaccgttgg ttcgttcagt 60
<210> 64
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
gaagtagtag tcacggtacg ggttgtcttt accagaacga gactgaacga accaacggtg 60
<210> 65
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
cccgtaccgt gactactact tctggcgtga cggtaaacag ggtcaggctc cgaacaacta 60
<210> 66
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
tttgtccagc tgccaagcag aaccaccgaa gaaagacggg tagttgttcg gagcctgacc 60
<210> 67
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
gcttggcagc tggacaaaca gaccgaccag tactacctgc actacttcgc tccgcagcag 60
<210> 68
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
acagttcagc acgaactttc gggttgtccc agttcaggtc cggctgctgc ggagcgaagt 60
<210> 69
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
cgaaagttcg tgctgaactg tacgacatcc tgcgtttctg gctggacaaa ggtgtttctg 60
<210> 70
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
gggattttag agaaggtagc aacggtgtcg aaacgcagac cagaaacacc tttgtccagc 60
<210> 71
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
gttgctacct tctctaaaat cccgggtttc ccggacctgt ctaaagctca gctgaaaaac 60
<210> 72
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
gtggatgttc ggaccttcgg tgtaagcttc agcgaagttt ttcagctgag ctttagacag 60
<210> 73
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
cgaaggtccg aacatccaca aatacatcca cgaaatgaac cgtcaggttc tgtctaaata 60
<210> 74
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
ggaacaccga agatttcacc agcggtagca acgttgtatt tagacagaac ctgacggttc 60
<210> 75
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
tggtgaaatc ttcggtgttc cggtttctgc tatgccggac tacttcgacc gtcgtcgtga 60
<210> 76
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
tccagacgga tcaggtcgaa ggtgaaagcg atgttcagtt cttcacgacg acggtcgaag 60
<210> 77
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
tcgacctgat ccgtctggac cgttacccgg accagcgttg gcgtcgtaaa ccgtggaccc 60
<210> 78
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
agcacggtcg gtctgagaga taacctgacg gaactgagac agggtccacg gtttacgacg 60
<210> 79
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
tctcagaccg accgtgctgc tggtgaattc ggttggaacg ctttcttcct ggacaaccac 60
<210> 80
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
agagtcgtca ccgaagtgag aaacctgacg cgggttgtcg tggttgtcca ggaagaaagc 60
<210> 81
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
ctcacttcgg tgacgactct ccgcagtggc gtgaacgttc tgctaaagct ctggctaccc 60
<210> 82
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
ccctggaaga tgaacggggt agcacgctgg gtcagcagca gggtagccag agctttagca 60
<210> 83
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
ccccgttcat cttccagggt gctgaactgg gtatgaccaa ctacccgttc aaaaacatcg 60
<210> 84
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
ttccagaaac ctttaacttc gatgtcgtcg aattcttcga tgtttttgaa cgggtagttg 60
<210> 85
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
catcgaagtt aaaggtttct ggaacgacta cgttgcttct ggtaaagtta acgctgctga 60
<210> 86
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gagttgtcac gagaggtcat acgaacttcc tgcaggaatt cagcagcgtt aactttacca 60
<210> 87
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gtatgacctc tcgtgacaac tctcgtaccc cgatgcagtg gaacgactct gttaacgctg 60
<210> 88
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
tcgggttcag gtggaaccac ggtttaccct gggtgaaacc agcgttaaca gagtcgttcc 60
<210> 89
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
ggttccacct gaacccgaac tacaaacaga tcaacgctgc tcgtgaagtt aacaaaccgg 60
<210> 90
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
caggttgatc agctgacggt agtaagagaa aacagagtcc ggtttgttaa cttcacgagc 60
<210> 91
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
ccgtcagctg atcaacctgc gtcaccagat cccggctctg acctctggtg aataccgtga 60
<210> 92
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
cgggtgtaag cgtaaacctg gttgttctgc gggtccaggt cacggtattc accagaggtc 60
<210> 93
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
caggtttacg cttacacccg tatcctggac aacgaaaaat acctggttgt tgttaacttc 60
<210> 94
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
gtccggcaga gcgtagtgca gctgttccgg tttgaagtta acaacaacca ggtatttttc 60
<210> 95
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
actacgctct gccggacaac ctgaccatcg cttcttctct gctggaaaac gttcaccagc 60
<210> 96
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
ggagccaggg tcagggtaga agcgttttcc tgcagagacg gctggtgaac gttttccagc 60
<210> 97
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
accctgaccc tggctccgtg gcaggctggt atctacaaac tgaactaact cgagcaccac 60
<210> 98
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
cggatctcag tggtggtggt ggtggtgctc gagttagttc ag 42
Claims (10)
1. A sucrose isomerase enzyme with high conversion rate, which is characterized in that: the amino acid sequence is shown as SEQ ID NO: 1 is shown.
2. The high conversion sucrose isomerase enzyme according to claim 1, wherein: the substrate concentration and the reaction speed are higher than those of the wild sucrose isomerase.
3. The high conversion sucrose isomerase enzyme according to claim 1, wherein: the conversion rate is more than 95%.
4. Use of a high conversion sucrose isomerase as claimed in any one of claims 1 to 3 for the catalytic production of isomaltulose.
5. Use of a high conversion sucrose isomerase according to claim 4 for the catalytic production of isomaltulose, characterized in that: taking a sucrose mother liquor, adding PB and pure water, adding a crude enzyme solution of the sucrose isomerase in the claims 1-3, and reacting at 30 ℃.
6. Use of a high conversion sucrose isomerase according to claim 5 for the catalytic production of isomaltulose, characterized in that: the highest substrate concentration of the enzyme is less than or equal to 700 g/L.
7. Use of a high conversion sucrose isomerase according to claim 5 for the catalytic production of isomaltulose, characterized in that: substrate: the crude enzyme liquid feeding ratio is less than or equal to 100: 1.
8. the use of the sucrose isomerase enzyme with high conversion rate in the catalytic production of isomaltulose according to claim 5, wherein the crude enzyme solution is prepared by the following steps:
(1) by a primer splicing method, the sequence shown in SEQ ID NO: 1, and cloning to a prokaryotic expression vector to realize high expression in escherichia coli;
(2) shake flask fermentation
Selecting a single escherichia coli colony containing an expression vector, inoculating the single escherichia coli colony into 10mL of culture medium A subjected to autoclaving, and carrying out overnight culture at 30 ℃ and 250 rpm;
taking a 1L triangular flask the next day, and carrying out the following steps: 100 of the inoculation ratio example is inoculated into 100mL of culture medium B after autoclaving, the culture is carried out at 30 ℃ until the thallus OD 5-6, and the triangular flask is immediately placed in a shaker at 25 ℃ and cultured for 1 hour at 250 rpm; IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 h at 250 rpm;
after the culture is finished, centrifuging the culture solution at 4 ℃ and 12000g for 20 minutes to collect wet thalli; then washing the thallus precipitate twice with distilled water, collecting thallus and preserving at-70 ℃; meanwhile, 2g of thalli is taken and added with 6mL of pure water for ultrasonic crushing, SDS-PAGE detection is carried out, and the crude enzyme liquid is preserved at the temperature of minus 20 ℃;
(3) fed-batch fermentation
Fed-batch fermentation is carried out in a bioreactor controlled by a computer, 200mL of culture is prepared by primary inoculation of strains, and the strains are inoculated when OD2.0 is reached; during the whole fermentation process, the temperature is kept at 37 ℃, the dissolved oxygen concentration during the fermentation process is automatically controlled at 30 percent by controlling the stirring speed and the aeration supply cascade, and the pH value of the culture medium is maintained at 7.0 by 50 percent of orthophosphoric acid and 30 percent of ammonia water;
during the fermentation, when a large dissolved oxygen rise occurs, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when OD600 was 35.0, induction with 0.2mM IPTG for 16 hours; taking 2g of thallus, adding 6mL of pure water, carrying out ultrasonic disruption, carrying out SDS-PAGE detection, and storing the crude enzyme liquid at-20 ℃.
9. Use of a high conversion sucrose isomerase according to claim 8 for the catalytic production of isomaltulose, characterized in that: in the step (2) of the crude enzyme solution preparation method, the culture medium A is: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.8g/L glucose, and kanamycin to 50 mg/L;
the culture medium B comprises: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.3g/L glucose, and kanamycin to 50 mg/L.
10. Use of a high conversion sucrose isomerase according to claim 8 for the catalytic production of isomaltulose, characterized in that: the culture medium used in step (3) of the crude enzyme solution preparation method is as follows: 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L catalase phosphate and 12.54g/L dipotassium phosphate, pH 7.0.
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| WO2019175633A1 (en) * | 2018-03-15 | 2019-09-19 | Petiva Private Limited | Methods for refolding sucrose isomerase |
| CN112063666A (en) * | 2020-08-05 | 2020-12-11 | 浙江工业大学 | Application of recombinant sucrose isomerase in preparation of isomaltulose by converting sucrose |
| CN113174385A (en) * | 2021-04-13 | 2021-07-27 | 苏州朗邦营养科技有限公司 | Sucrose isomerase mutant with high activity and high conversion rate and application thereof |
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| WO2019175633A1 (en) * | 2018-03-15 | 2019-09-19 | Petiva Private Limited | Methods for refolding sucrose isomerase |
| CN112063666A (en) * | 2020-08-05 | 2020-12-11 | 浙江工业大学 | Application of recombinant sucrose isomerase in preparation of isomaltulose by converting sucrose |
| CN113174385A (en) * | 2021-04-13 | 2021-07-27 | 苏州朗邦营养科技有限公司 | Sucrose isomerase mutant with high activity and high conversion rate and application thereof |
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| Title |
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| LIU LI 等: "A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigenaMT翻译", APPLIED SCIENCES, vol. 11, no. 12, pages 5521 * |
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