CN114250207A - High-activity sucrose phosphorylase and application thereof - Google Patents
High-activity sucrose phosphorylase and application thereof Download PDFInfo
- Publication number
- CN114250207A CN114250207A CN202111661698.8A CN202111661698A CN114250207A CN 114250207 A CN114250207 A CN 114250207A CN 202111661698 A CN202111661698 A CN 202111661698A CN 114250207 A CN114250207 A CN 114250207A
- Authority
- CN
- China
- Prior art keywords
- sucrose phosphorylase
- sucrose
- glycerol
- asp
- leu
- Prior art date
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- Granted
Links
- 108020000005 Sucrose phosphorylase Proteins 0.000 title claims abstract description 42
- 230000000694 effects Effects 0.000 title claims abstract description 23
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 107
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 40
- 229930006000 Sucrose Natural products 0.000 claims abstract description 40
- 239000005720 sucrose Substances 0.000 claims abstract description 36
- 102000004190 Enzymes Human genes 0.000 claims abstract description 29
- 108090000790 Enzymes Proteins 0.000 claims abstract description 29
- 239000000758 substrate Substances 0.000 claims abstract description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000000243 solution Substances 0.000 claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- 230000036632 reaction speed Effects 0.000 claims abstract description 5
- 239000007853 buffer solution Substances 0.000 claims abstract description 3
- 239000012452 mother liquor Substances 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 238000006243 chemical reaction Methods 0.000 claims description 45
- 238000000855 fermentation Methods 0.000 claims description 18
- 230000004151 fermentation Effects 0.000 claims description 18
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 12
- 229940041514 candida albicans extract Drugs 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 12
- 239000012138 yeast extract Substances 0.000 claims description 12
- 229930182478 glucoside Natural products 0.000 claims description 11
- -1 glycerol glucoside Chemical class 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 7
- 230000014509 gene expression Effects 0.000 claims description 7
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 6
- 235000019270 ammonium chloride Nutrition 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 6
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 6
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims description 6
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 6
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 6
- 229930027917 kanamycin Natural products 0.000 claims description 6
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 6
- 229960000318 kanamycin Drugs 0.000 claims description 6
- 229930182823 kanamycin A Natural products 0.000 claims description 6
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 6
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 6
- 235000011152 sodium sulphate Nutrition 0.000 claims description 6
- 239000012137 tryptone Substances 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 238000010367 cloning Methods 0.000 claims description 4
- 230000009465 prokaryotic expression Effects 0.000 claims description 4
- 238000003786 synthesis reaction Methods 0.000 claims description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 102000016938 Catalase Human genes 0.000 claims description 3
- 108010053835 Catalase Proteins 0.000 claims description 3
- 238000005273 aeration Methods 0.000 claims description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 230000006698 induction Effects 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 235000011007 phosphoric acid Nutrition 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 230000003197 catalytic effect Effects 0.000 claims 6
- 238000006555 catalytic reaction Methods 0.000 abstract description 12
- 238000005265 energy consumption Methods 0.000 abstract description 3
- 230000035484 reaction time Effects 0.000 abstract description 3
- 229960004793 sucrose Drugs 0.000 description 36
- 235000011187 glycerol Nutrition 0.000 description 29
- NHJUPBDCSOGIKX-QMWFWAMKSA-N 1-O-(D-glucosyl)glycerol Chemical compound OCC(O)COC1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O NHJUPBDCSOGIKX-QMWFWAMKSA-N 0.000 description 7
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- BBPCSGKKPJUYRB-UVOCVTCTSA-N Thr-Thr-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O BBPCSGKKPJUYRB-UVOCVTCTSA-N 0.000 description 6
- HXXFSFRBOHSIMQ-VFUOTHLCSA-N alpha-D-glucose 1-phosphate Chemical compound OC[C@H]1O[C@H](OP(O)(O)=O)[C@H](O)[C@@H](O)[C@@H]1O HXXFSFRBOHSIMQ-VFUOTHLCSA-N 0.000 description 6
- 229950010772 glucose-1-phosphate Drugs 0.000 description 6
- 239000000370 acceptor Substances 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
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- IKKVASZHTMKJIR-ZKWXMUAHSA-N Ala-Asp-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O IKKVASZHTMKJIR-ZKWXMUAHSA-N 0.000 description 4
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- 229930091371 Fructose Natural products 0.000 description 4
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- SOEATRRYCIPEHA-BQBZGAKWSA-N Gly-Glu-Glu Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SOEATRRYCIPEHA-BQBZGAKWSA-N 0.000 description 4
- 239000002537 cosmetic Substances 0.000 description 4
- 108010050848 glycylleucine Proteins 0.000 description 4
- 108010054155 lysyllysine Proteins 0.000 description 4
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- 238000005259 measurement Methods 0.000 description 3
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- AQTKXCPRNZDOJU-ZEBDFXRSSA-N 2-O-(alpha-D-glucopyranosyl)glycerol Chemical compound OCC(CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O AQTKXCPRNZDOJU-ZEBDFXRSSA-N 0.000 description 2
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01007—Sucrose phosphorylase (2.4.1.7)
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Abstract
The invention relates to the technical field of enzyme catalysis, in particular to a sucrose phosphorylase with high activity and an application thereof, wherein the amino acid sequence of the sucrose phosphorylase is shown as SEQ ID NO: 1 is shown. When in use, the sucrose mother liquor is taken, added with buffer solution, glycerol and pure water, added with the crude enzyme solution of sucrose phosphorylase and reacted at 37 ℃. Compared with wild sucrose phosphorylase, the high-activity sucrose phosphorylase has higher reaction speed and higher activity, and can effectively shorten the reaction time, improve the production efficiency, reduce the energy consumption and comprehensively reduce the cost in a high-concentration substrate.
Description
Technical Field
The invention relates to the technical field of enzyme catalysis, in particular to sucrose phosphorylase with high activity.
Background
2-O-alpha-D-glucopyranosyl glycerol is mainly found in blue algae and is used for resisting osmotic pressure of saline-alkali environment; it was also found to be present in the plant Myrothamnus flabellifolia and has a rejuvenating effect on the plant. Such desert plants can survive years after being completely dry without losing tissue integrity. Glucosyl glycerol also has an important function of protecting cells and tissues (e.g., skin), which provides the possibility of potential applications in cosmetics. Since glycerol glucoside has a high water binding ability, it produces a strong moisturizing effect when applied to the skin. In addition to cosmetic applications, glucosylglycerols have great potential as low-calorie sweeteners, which may have prebiotic effects.
It has been reported to be useful as a replacement sweetener in food products because of its low cariogenicity and caloric value compared to sucrose. In addition, glucosylglycerols and derivatives thereof have been studied as therapeutic agents for diseases caused by protein misfolding and cancer treatment. In cosmetics, glucosyl glycerol is used as an anti-aging agent and a moisture regulating compound.
To support the development of industrial applications, glucosyl glycerol must be efficiently provided on a large scale. Microbial production of glucosylglycerols may be limited by the output parameters attainable for biosynthesis. In particular, the product concentration in the engineered Corynebacterium glutamicum is less than or equal to 2g/L, the yield and productivity on the substrate used are low, and even lower in the engineered cyanobacteria polycystic algae strains.
The existing preparation methods mainly comprise a chemical synthesis method, an enzymatic in vitro catalysis method and a biological synthesis method. Chemical methods may involve various starting compounds such as maltitol, isomalt, trehalose, and the like. It has been reported that sodium periodate and sodium borohydride can catalyze maltitol to glucosyl glycerol, but the yield is 18%. The yield of the method for catalyzing isomaltose by using acetic acid, lead tetraacetate and sodium borohydride is 12%. The yield was only 5% with trehalose as the starting material. And the byproducts have large influence on the subsequent purification. The biosynthesis method can be synthesized by treating microorganisms such as Stenotrophomonas rhizophila DSM14405 or blue algae by salt stress, and has the disadvantage of difficult scale-up.
Enzymatic catalysis mainly involves alpha-glucosidase, cyclodextrin glucanotransferase, glucosyl-glycerol-phosphate synthetase and sucrose phosphorylase.
Among them, sucrose phosphorylase (EC2.4.1.7) is a specific transglycosidase. Two reactions are mainly catalyzed: in the first category, the glucose group of glucose-1-phosphate can be transferred to an acceptor. For example, glucose-1-phosphate and D-fructose are capable of producing sucrose under the catalysis of sucrose phosphorylase. In the second category, the glucose group in sucrose is transferred to an acceptor. For example, sucrose and phosphate are capable of producing glucose-1-phosphate and D-fructose under the catalysis of sucrose phosphorylase. Sucrose phosphorylase catalyzes the reversible conversion of sucrose and phosphate to glucose 1-phosphate and D-fructose in the absence of external influences. In the absence of phosphate, glycerol can intercept the glucosylase intermediate that reacts with sucrose to produce glucosylglycerol, with minor hydrolysis side reactions occurring.
One effective route to glucosylglycerol production is sucrose phosphorylase. Sucrose is an excellent donor substrate for glycerol glycosylation, has high yield (more than or equal to 90 percent) and has better cost economy. Glucosyl glycerol was manufactured industrially by Bitop AG (dottmond, germany) using a biocatalytic process of LmSucP and formulated as a commercial product for cosmetic applications, sold as Glycoin (50% solution of GG).
Sucrose phosphorylase derived from Leuconostoc mesenteroides has a wide acceptor specificity when glucose-1-phosphate and sucrose are used as donors.
When glucose-1-phosphate is used as a glycosyl donor, only arabinose, arabitol and xylitol can be used as acceptors, and when sucrose is used as the glycosyl donor, the acceptors have wider range and higher activity.
WO2008034158 published by the university of Gredz technology is the first time sucrose phosphorylase (SPase) is used for catalyzing the conversion of sucrose and glycerol into glycerol glucoside, and under the reaction conditions of 0.3M sucrose and 2.0M glycerol, the concentration of the glucose-based glycerol product is about 0.29M, namely, the yield is about 70 g/L. However, the method has the disadvantages of large enzyme dosage, low product concentration and the like.
The patent CN109576239A utilizes Thermoanaerobacterium thermosaccharolyticum derived heat-resistant sucrose phosphorylase to carry out catalytic reaction under the high temperature condition, thereby avoiding the decomposition of substrates and products and the growth of miscellaneous bacteria by miscellaneous enzymes, and the highest concentration can reach 189.8g/L, and the highest conversion rate can reach 94%. However, high conversion (60g/L-200g/L sucrose) can be obtained only at low substrate concentration, and the enzyme dosage is higher, in the example, the highest concentration is 300g/L sucrose, the enzyme dosage is 150g/L, and the mass ratio of the substrate to the enzyme is 2: 1, the enzyme cost is comparatively 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 a sucrose phosphorylase with high activity and application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
a high-activity sucrose phosphorylase, the amino acid sequence of which is shown in SEQ ID NO: 1 is shown.
The reaction rate of the sucrose phosphorylase having high activity of the present invention is higher than that of a wild-type sucrose phosphorylase. The conversion rate is more than 90 percent under the concentration of 400g/L sucrose and is far higher than that of the wild type. The conversion rate is more than 75% at the sucrose concentration of 750g/L, which is much higher than that of the wild type.
The sucrose phosphorylase with high activity can be used for catalyzing and producing the glycerol glucoside, and the using method comprises the following steps: taking a sucrose mother liquor, adding a buffer solution, glycerol and pure water, adding the crude enzyme solution of sucrose phosphorylase, and reacting at 37 ℃.
Experiments prove that the substrate concentration of the sucrose phosphorylase can reach 400 g/L; and the conversion rate is more than 90% in 3 hours. The highest substrate concentration can reach 750g/L, and the conversion rate can reach 75%.
Wherein, the preparation method of the crude enzyme solution comprises the following steps:
(1) by a method of whole gene synthesis, the nucleotide sequence shown in SEQ ID NO: 1, and cloning to a prokaryotic expression vector for expression so as 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 phosphorylase, the high-activity sucrose phosphorylase has higher reaction speed and higher activity, and can effectively shorten the reaction time, improve the production efficiency, reduce the energy consumption and comprehensively reduce the cost in a high-concentration substrate.
The reaction system has high conversion rate, the concentration of the substrate reaches 400g/L, and the conversion rate is more than 90% in 3 hours; the highest substrate concentration can reach 750g/L, and the conversion rate can reach 75%. Is particularly suitable for the industrial large-scale production of the glycerol glucoside, and can obtain better social benefit and economic value.
Drawings
FIG. 1 is a substrate standard map; sucrose for 10 minutes and glycerol for 16.8 minutes.
FIG. 2 shows the results of the measurement in example 2, in which sucrose was used for 10 minutes, the objective product was used for 11.7 minutes, fructose was used for 12.8 minutes, and glycerol was used for 16.8 minutes.
Fig. 3 shows the results of the measurement in comparative example 1, sucrose at 10 minutes, the objective product at 11.7 minutes, fructose at 12.8 minutes, and glycerin at 16.8 minutes.
FIG. 4 shows the results of the measurement in example 3, in which sucrose was used for 10 minutes, the objective product was used for 11.7 minutes, fructose was used for 12.8 minutes, and glycerin was used for 16.8 minutes;
FIG. 5 shows the results of the test in example 4.
Fig. 6 is the result of the test of comparative example 2.
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.
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
A 250mm x 4.6um femomelatic acid column was used.
EXAMPLE 1 preparation of crude enzyme solution
(1) By a method of whole gene synthesis, the nucleotide sequence shown in SEQ ID NO: 1, or a corresponding coding polynucleotide sequence of the protein shown in SEQ ID NO: 3, assembling, cloning into a prokaryotic expression vector for expression so as to realize high expression in the Escherichia coli, and naming the ZY 2.
Likewise, a control wild-type sucrose phosphatase protein of SEQ ID NO: 2, the corresponding coding polynucleotide sequence of SEQ ID NO: 4, assembling, cloning to a prokaryotic expression vector for expression, and naming as ZY 0.
(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 2 application example of catalytic 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.
2ml of total reaction system with the final concentration of 50mM MES pH7.0, 0.9M sucrose and 1.8M glycerol is prepared in a 5ml centrifuge tube, water is supplemented to 1.8ml, the pH is adjusted to 7, then new enzyme preparation ZY20.2ml is added, and shaking table reaction at 37 ℃ is carried out. Sampling and detecting for 3 hours. Due to the high concentration of the substrate, the sample was diluted 20 times with pure water before injection. The HPLC results are shown in FIG. 2. At glycerol excess, sucrose conversion was greater than 90% at 3 hours.
Comparative example 1 control of catalytic reaction
2ml of total reaction system is prepared in a 5ml centrifuge tube, the final concentration is 50mM MES pH7.0, 0.9M sucrose and 1.8M glycerol, water is supplemented to 1.8ml, the pH is adjusted to 7, then new enzyme preparation ZY00.2ml is added, and shaking table reaction at 37 ℃ is carried out. Sampling and detecting for 3 hours. Due to the high concentration of the substrate, the sample was diluted 20 times with pure water before injection. The HPLC results are shown in FIG. 3. At glycerol excess, the 3 hour conversion of sucrose was about 68%.
Example 3 high concentration catalytic reaction example
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.
2ml of total reaction system is prepared in a 5ml centrifuge tube, the final concentration is 50mM MES pH7.0, 1.2M sucrose (equivalent to 408g/L substrate concentration) and 1.8M glycerol are supplemented with water to 1.8ml, after the pH is adjusted to 7, new enzyme preparation ZY20.2ml is added, and the shaking table reaction is carried out at 37 ℃. Sampling and detecting for 3 hours. Due to the high concentration of the substrate, the sample was diluted 20 times with pure water before injection. The HPLC results are shown in FIG. 4. At glycerol excess, the 3 hour conversion of sucrose was greater than 93%.
EXAMPLE 4 high concentration catalytic reaction example
2ml of total reaction system is prepared in a 5ml centrifuge tube, the final concentration is 50mM PB pH7.0, 2.2M sucrose (equivalent to 750g/L substrate concentration), 2.4M glycerol is supplemented with water to 1.8ml, after the pH is adjusted to 7, new enzyme preparation ZY20.2ml is added, and shaking table reaction is carried out at 37 ℃. Sampling and detecting for 25 hours. Due to the high concentration of the substrate, the sample was diluted 20 times with pure water before injection. The HPLC results are shown in FIG. 5. The sucrose conversion was about 75%.
In order to characterize the ultimate enzyme activity of the enzyme, the reaction system close to the saturated sucrose concentration is tried in the embodiment, the reaction solution is too viscous at the concentration, the requirement on the stirring hardware of a production reaction tank is higher, and although the conversion rate is reduced to some extent compared with the concentration of 1.2M, the reaction result is still greatly superior to that of the comparative example 2.
Comparative example 2 high concentration catalytic reaction control
2ml of total reaction system is prepared in a 5ml centrifuge tube, the final concentration is 50mM PB pH7.0, 2.2M sucrose (equivalent to 750g/L substrate concentration), 2.4M glycerol is supplemented with water to 1.8ml, after the pH is adjusted to 7, new enzyme preparation ZY00.2ml is added, and shaking table reaction is carried out at 37 ℃. Sampling and detecting for 25 hours. Due to the high concentration of the substrate, the sample was diluted 20 times with pure water before injection. The HPLC results are shown in FIG. 6. The sucrose conversion rate is about 20%, and a large amount of sucrose remains in the system.
The comparison of the experimental results shows that the sucrose phosphorylase has higher reaction speed than the wild sucrose phosphorylase, can effectively shorten the reaction time at high-concentration substrates, improves the production efficiency, reduces the energy consumption and comprehensively reduces the cost; the reaction system has high conversion rate and high reaction speed, when high-concentration sucrose is reacted, the concentration of a substrate reaches 400g/L, and the conversion rate is more than 90% within 3 hours; the highest substrate concentration can reach 750g/L, the conversion rate can reach 75%, and no other obvious by-products except fructose are generated. Is particularly suitable for the industrial large-scale production of the glycerol glucoside, and can obtain better social benefit and economic value.
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 phosphorylase with high activity and application thereof
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Asp Arg Gly Phe Ala Pro Ala Asp Tyr Thr Arg Val Asp Ala Ala Phe
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Gly Asp Trp Ala Asp Val Glu Ala Leu Gly Glu Glu Tyr Tyr Leu Met
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Asp Phe Lys Lys Asn His Asp Asp Ser Lys Tyr Lys Asp Phe Phe Leu
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Arg Trp Glu Lys Phe Trp Ala Lys Ala Gly Glu Asn Arg Pro Thr Gln
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Ala Asp Val Asp Leu Ile Tyr Lys Gln Lys Asp Lys Ala Pro Thr Gln
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Glu Ile Thr Phe Asp Asp Gly Thr Thr Glu Asn Leu Trp Asn Thr Phe
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Phe Ile Lys Thr Thr Leu Glu Asp Met Val Lys His Gly Ala Asn Leu
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Ile Arg Leu Asp Ala Phe Ala Tyr Ala Val Lys Lys Val Asp Thr Asn
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Asp Phe Phe Val Glu Pro Glu Ile Trp Asp Thr Leu Asn Glu Val Arg
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Glu Ile Leu Thr Pro Leu Lys Ala Glu Ile Leu Pro Glu Ile His Glu
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His Tyr Ser Ile Pro Lys Lys Ile Asn Asp His Gly Tyr Arg 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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Thr Asn Gln Leu Ala Lys Trp Leu Lys Met Ser Pro Met Lys Gln Phe
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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 Phe Lys Lys Asn His Asp Asp Ser Lys Tyr Lys Asp Phe Phe Ile
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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
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Ile Ala Leu Leu Glu Ser Thr Lys Glu Gly Arg Asn Ile Asn Arg His
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Ala Asn Leu Leu Lys Leu Leu Ser Trp Arg Asn Glu Ser Pro Ala Phe
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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 (9)
1. A highly active sucrose phosphorylase enzyme characterized by: the amino acid sequence is shown as SEQ ID NO: 1 is shown.
2. The high activity sucrose phosphorylase according to claim 1 wherein: the substrate concentration and the reaction speed are higher than those of the wild sucrose phosphorylase.
3. The high activity sucrose phosphorylase according to claim 1 wherein: the conversion rate is more than 90% under the concentration of 400g/L sucrose; the conversion rate is more than 75% under the sucrose concentration of 750 g/L.
4. Use of the high activity sucrose phosphorylase according to any one of claims 1 to 3 for the catalytic production of glycerol glucoside.
5. The use of a high activity sucrose phosphorylase according to claim 4 in the catalytic production of glycerol glucoside, wherein: taking a sucrose mother liquor, adding a buffer solution, glycerol and pure water, adding a crude enzyme solution of sucrose phosphorylase described in claims 1 to 3, and reacting at 37 ℃.
6. The use of a high activity sucrose phosphorylase according to claim 5 in the catalytic production of glycerol glucoside, wherein: the substrate concentration of the sucrose phosphorylase is less than or equal to 400g/L, and the conversion rate is more than 90% within 3 hours; the highest substrate concentration can reach 750g/L, and the conversion rate can reach 75%.
7. The use of the highly active sucrose phosphorylase enzyme according to claim 5 in the catalytic production of glycerol glucoside, wherein the preparation method of said crude enzyme solution comprises the steps of:
(1) by a method of whole gene synthesis, the nucleotide sequence shown in SEQ ID NO: 1, and cloning to a prokaryotic expression vector for expression so as 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 ℃.
8. The use of a high activity sucrose phosphorylase according to claim 7 in the catalytic production of glycerol glucoside, wherein: 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.
9. The use of a high activity sucrose phosphorylase according to claim 7 in the catalytic production of glycerol glucoside, wherein: 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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| CN109423485A (en) * | 2017-08-25 | 2019-03-05 | 中国科学院微生物研究所 | Saccharose phosphorylation enzyme mutant and its application |
| CN110358750A (en) * | 2019-08-06 | 2019-10-22 | 江苏诚信药业有限公司 | Novel type of sucrose phosphorylase mutant and its application in synthetic glycerine glucoside |
| CN111172127A (en) * | 2020-01-17 | 2020-05-19 | 浙江工业大学 | Application of sucrose phosphorylase in preparation of glycerol glucoside |
| US20210130797A1 (en) * | 2019-10-31 | 2021-05-06 | Jiangnan University | Sucrose Phosphorylase Mutant with Improved Enzyme Activity and Construction Method Thereof and Use Thereof |
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| CN109423485A (en) * | 2017-08-25 | 2019-03-05 | 中国科学院微生物研究所 | Saccharose phosphorylation enzyme mutant and its application |
| CN110358750A (en) * | 2019-08-06 | 2019-10-22 | 江苏诚信药业有限公司 | Novel type of sucrose phosphorylase mutant and its application in synthetic glycerine glucoside |
| US20210130797A1 (en) * | 2019-10-31 | 2021-05-06 | Jiangnan University | Sucrose Phosphorylase Mutant with Improved Enzyme Activity and Construction Method Thereof and Use Thereof |
| CN111172127A (en) * | 2020-01-17 | 2020-05-19 | 浙江工业大学 | Application of sucrose phosphorylase in preparation of glycerol glucoside |
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| ANDREAS KRUSCHITZ等: "Continuous process technology for glucoside production from sucrose using a whole cell-derived solid catalyst of sucrose phosphorylase", APPL MICROBIOL BIOTECHNOL, vol. 105, no. 13, pages 5383 - 5394, XP037510785, DOI: 10.1007/s00253-021-11411-x * |
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Denomination of invention: A highly active sucrose phosphorylase and its application Granted publication date: 20230811 Pledgee: Bank of China Limited Nanjing Chengdong Branch Pledgor: NANJING NUOYUN BIOTECHNOLOGY Co.,Ltd. Registration number: Y2024980013829 |