CN118562758A - Cellooligosaccharide phosphorylase StCDP and method for preparing glucose-1-phosphoric acid by enzymolysis - Google Patents
Cellooligosaccharide phosphorylase StCDP and method for preparing glucose-1-phosphoric acid by enzymolysis Download PDFInfo
- Publication number
- CN118562758A CN118562758A CN202410741154.XA CN202410741154A CN118562758A CN 118562758 A CN118562758 A CN 118562758A CN 202410741154 A CN202410741154 A CN 202410741154A CN 118562758 A CN118562758 A CN 118562758A
- Authority
- CN
- China
- Prior art keywords
- cellooligosaccharide
- stcdp
- phosphorylase
- glucose
- enzymolysis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- FYGDTMLNYKFZSV-ZWSAEMDYSA-N cellotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-ZWSAEMDYSA-N 0.000 title claims abstract description 125
- 108010073135 Phosphorylases Proteins 0.000 title claims abstract description 84
- 102000009097 Phosphorylases Human genes 0.000 title claims abstract description 83
- 229910000147 aluminium phosphate Inorganic materials 0.000 title claims abstract description 19
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 12
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
- 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 claims description 38
- 229950010772 glucose-1-phosphate Drugs 0.000 claims description 38
- 239000000047 product Substances 0.000 claims description 25
- 210000004027 cell Anatomy 0.000 claims description 20
- 108090000623 proteins and genes Proteins 0.000 claims description 18
- 238000003259 recombinant expression Methods 0.000 claims description 17
- 239000013604 expression vector Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- LUEWUZLMQUOBSB-ZLBHSGTGSA-N alpha-maltotetraose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](O[C@H](O[C@@H]3[C@H](O[C@H](O)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-ZLBHSGTGSA-N 0.000 claims description 6
- FTNIPWXXIGNQQF-XHCCAYEESA-N cellopentaose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3[C@H](O[C@@H](O[C@@H]4[C@H](OC(O)[C@H](O)[C@H]4O)CO)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FTNIPWXXIGNQQF-XHCCAYEESA-N 0.000 claims description 5
- 230000014509 gene expression Effects 0.000 claims description 5
- OCIBBXPLUVYKCH-FYTDUCIRSA-N beta-D-cellohexaose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3[C@H](O[C@@H](O[C@@H]4[C@H](O[C@@H](O[C@@H]5[C@H](O[C@@H](O)[C@H](O)[C@H]5O)CO)[C@H](O)[C@H]4O)CO)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O OCIBBXPLUVYKCH-FYTDUCIRSA-N 0.000 claims description 4
- OCIBBXPLUVYKCH-UHFFFAOYSA-N cellopentanose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(OC3C(OC(OC4C(OC(OC5C(OC(O)C(O)C5O)CO)C(O)C4O)CO)C(O)C3O)CO)C(O)C2O)CO)C(O)C1O OCIBBXPLUVYKCH-UHFFFAOYSA-N 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 241001052560 Thallis Species 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 230000006698 induction Effects 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 3
- 230000003115 biocidal effect Effects 0.000 claims 1
- 230000002255 enzymatic effect Effects 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 32
- 238000004519 manufacturing process Methods 0.000 abstract description 12
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 230000003197 catalytic effect Effects 0.000 abstract description 4
- 239000001913 cellulose Substances 0.000 abstract description 4
- 229920002678 cellulose Polymers 0.000 abstract description 4
- 239000007857 degradation product Substances 0.000 abstract description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 28
- 239000000872 buffer Substances 0.000 description 26
- 229910019142 PO4 Inorganic materials 0.000 description 24
- 241000588724 Escherichia coli Species 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 239000007788 liquid Substances 0.000 description 8
- 239000000758 substrate Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 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 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 3
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- PUWBXDQHRRUKNY-CKOVTTQDSA-N (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal phosphoric acid Chemical compound OP(O)(O)=O.OP(O)(O)=O.OP(O)(O)=O.OP(O)(O)=O.OP(O)(O)=O.OP(O)(O)=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O PUWBXDQHRRUKNY-CKOVTTQDSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 2
- 102000009569 Phosphoglucomutase Human genes 0.000 description 2
- 241000192029 Ruminococcus albus Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000589887 Spirochaeta thermophila Species 0.000 description 2
- 241000589970 Spirochaetales Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 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 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 108091000115 phosphomannomutase Proteins 0.000 description 2
- 239000008057 potassium phosphate buffer Substances 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- DBTMGCOVALSLOR-DEVYUCJPSA-N (2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](CO)O[C@H](O)[C@@H]2O)O)O[C@H](CO)[C@H]1O DBTMGCOVALSLOR-DEVYUCJPSA-N 0.000 description 1
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 1
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- 102000005731 Glucose-6-phosphate isomerase Human genes 0.000 description 1
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 102000051366 Glycosyltransferases Human genes 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 229920001543 Laminarin Polymers 0.000 description 1
- 239000005717 Laminarin Substances 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 241000113606 Ruminiclostridium Species 0.000 description 1
- HIWPGCMGAMJNRG-ACCAVRKYSA-N Sophorose Natural products O([C@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HIWPGCMGAMJNRG-ACCAVRKYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- HIWPGCMGAMJNRG-UHFFFAOYSA-N beta-sophorose Natural products OC1C(O)C(CO)OC(O)C1OC1C(O)C(O)C(O)C(CO)O1 HIWPGCMGAMJNRG-UHFFFAOYSA-N 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 108010048610 cellobiose phosphorylase Proteins 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 235000021310 complex sugar Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002303 glucose derivatives Chemical class 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 238000006303 photolysis reaction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- PZDOWFGHCNHPQD-VNNZMYODSA-N sophorose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](C=O)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O PZDOWFGHCNHPQD-VNNZMYODSA-N 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a cellooligosaccharide phosphorylase StCDP and a method for preparing glucose-1-phosphoric acid by enzymolysis, belonging to the technical field of engineering enzymes. The invention provides a cellooligosaccharide phosphorylase StCDP, and the amino acid sequence of the cellooligosaccharide phosphorylase StCDP is shown as SEQ ID No. 1. The cellooligosaccharide phosphorylase StCDP has excellent heat resistance, has high catalytic activity in the temperature range of 25-75 ℃, belongs to thermophilic cellooligosaccharide phosphorylase, and has wider optimal reaction temperature. The invention uses cellulose degradation products and cellooligosaccharide as raw materials, can realize the high-efficiency production of glucose-1-phosphoric acid by simple cellooligosaccharide phosphorylase StCDP treatment, and has the advantages of high raw material utilization rate, high glucose-1-phosphoric acid yield (17.6-19.7 g/L), low production cost and the like.
Description
Technical Field
The invention belongs to the technical field of engineering enzymes, and particularly relates to a cellooligosaccharide phosphorylase StCDP and a method for preparing glucose-1-phosphate by enzymolysis.
Background
In vivo, glucose-1-phosphate is an important intermediate, produced by the phosphorylation of glycogen or starch by glucan phosphorylase, which is then converted to glucose-6-phosphate by phosphoglucose isomerase, and enters the glucose cleavage pathway. Glucose-1-phosphate is used as an activated glucose, and is an important precursor for synthesizing complex sugar compounds such as glycolipid, oligosaccharide, sugar nucleotide and the like in organisms. Along with the development of biosynthesis technology, glucose-1-phosphate can be applied to biological manufacturing processes such as synthesis of artificial starch, laminarin, sophorose and the like. Therefore, development of an efficient glucose-1-phosphate production method has wide application value.
Cellulose is the most abundant natural renewable resource in nature, and a large amount of cellobiose and cellooligosaccharide can be produced by cellulose hydrolysis, and the products can produce glucose-1-phosphate under the action of polysaccharide phosphorylase. Compared with the glucose-1-phosphate production pathway which takes starch and sucrose as substrates, the production pathway which takes cellobiose and cellooligosaccharide as substrates does not compete with food. Cellobiose is used as a substrate, and cellobiose phosphorylase is used, so that the yield is only 50%. And the yield of glucose-1-phosphoric acid can be effectively improved by phosphorylating cellooligosaccharide with cellooligosaccharide as a substrate. For example, glucose-1-phosphoric acid is produced by taking cellotriose, cellotetraose and cellopentaose as substrates, and the yields can reach 67%, 75% and 80% respectively. Therefore, the glucose-1-phosphate can be efficiently produced by coupling the cellulase-cellooligosaccharide phosphorylase with high cellooligosaccharide production, and the production cost is effectively reduced. If Sawano is equal to 2013, using 5.2g/L cellotriose as substrate, using RaCDP (FEBS Journal 2013,280 (18), 4463-4473) from Ruminococcus albus (Ruminococcus albus) with a conversion efficiency of 46%; liu was equal to 2019, and glucose-1-phosphate was produced using cellooligosaccharide phosphorylase CdpC derived from (Ruminiclostridium cellulolyticum) with a conversion efficiency of 55.7% (Biotechnology for Biofuels 2019,12 (1), 208) using 0.26g/L cellotriose as a substrate, but the conversion efficiency was low.
Disclosure of Invention
The invention aims to provide a cellooligosaccharide phosphorylase StCDP and a method for preparing glucose-1-phosphoric acid by enzymolysis, which can realize the efficient production of glucose-1-phosphoric acid by using cellooligosaccharide as a raw material and treating with simple cellooligosaccharide phosphorylase StCDP, and have the advantages of high raw material utilization rate, high glucose-1-phosphoric acid yield, low production cost and the like.
The invention provides a cellooligosaccharide phosphorylase StCDP, and the amino acid sequence of the cellooligosaccharide phosphorylase StCDP is shown as SEQ ID No. 1.
The invention also provides a coding gene StCDP of the cellooligosaccharide phosphorylase, and a nucleotide sequence of the coding gene StCDP is shown as SEQ ID No. 2.
The invention also provides a recombinant expression vector containing the coding gene StCDP and expressing the cellooligosaccharide phosphorylase StCDP.
The invention also provides a recombinant expression cell containing the recombinant expression vector.
Preferably, the recombinant expression cell uses a prokaryotic cell as a host cell.
The invention also provides a preparation method of the cellooligosaccharide phosphorylase StCDP, which comprises the steps of culturing the recombinant expression cells into seed liquid, inoculating the seed liquid into a liquid culture medium containing corresponding antibiotics for induction expression, and collecting thalli;
After the cells are broken, collecting a supernatant, wherein the supernatant contains the cellooligosaccharide phosphorylase StCDP.
The invention also provides application of the cellooligosaccharide phosphorylase StCDP in preparing glucose-1-phosphate by enzymolysis.
The invention also provides a method for preparing glucose-1-phosphoric acid by enzymolysis, which comprises the following steps: the cellooligosaccharide is taken as a raw material, the cellooligosaccharide phosphorylase StCDP is utilized for enzymolysis, and the enzymolysis product comprises the glucose-1-phosphoric acid.
Preferably, the cellooligosaccharide comprises at least one of the following: cellotriose, cellotetraose, cellopentaose and cellohexaose.
Preferably, the enzymolysis temperature is 25-75 ℃, and the enzymolysis time is 10 min-48 h.
The beneficial effects are that: the invention provides a cellooligosaccharide phosphorylase StCDP, and the amino acid sequence of the cellooligosaccharide phosphorylase StCDP is shown as SEQ ID No. 1. The cellooligosaccharide phosphorylase StCDP of the invention is obtained by codon optimization of a cellooligosaccharide phosphorylase gene (WP_ 014625716.1) from a thermophilic spirochete (Spirochaeta thermophila), synthesis of the protein gene and prokaryotic expression. The cellooligosaccharide phosphorylase StCDP has excellent heat resistance, has high catalytic activity in the temperature range of 25-75 ℃, belongs to thermophilic cellooligosaccharide phosphorylase, has wider optimal reaction temperature, has no loss of catalytic activity at 75 ℃, and has better thermal stability.
The invention uses cellulose degradation products and cellooligosaccharide as raw materials, can realize the high-efficiency production of glucose-1-phosphoric acid by simple cellooligosaccharide phosphorylase StCDP treatment, and has the advantages of high raw material utilization rate, high glucose-1-phosphoric acid yield, low production cost and the like. In the embodiment of the invention, 1000U of cellooligosaccharide phosphorylase StCDP can be used for converting cellooligosaccharide to prepare 17.6-19.7 g/L glucose-1-phosphoric acid after 10 hours at the temperature of 25-75 ℃.
Detailed Description
The invention provides a cellooligosaccharide phosphorylase StCDP, the amino acid sequence of which is shown as SEQ ID No.1 of the cellooligosaccharide phosphorylase StCDP ：MSYGHFEGREYVITNPRTPVKWINYVGTIDFGGFVDHTGGLLVCKGDPALNRITKYITQLPSSEFKGSTIYLRGRRGDRSFLYSPYYVPTLHEYDRYECRIGLGYSRFLMEYVGIRTEITVFVPEGESVVLQDVRIRNTGSDTVEVLDLIPVVEYSHFDALKQLTNADWVPQTMTSKAIGRPGGLLVLRQCAFMQTGRAENFLTSNSPVSSFEADRRRFLGENEYGTWKMPFSLVDGREFSNYEPLRGDNVGALMHHVGPLAPGEERRVIVQLGQVESVEKAMPLIERFREPEEVDAAFARMQAFWEEYLNVCQVETPDPEFDLLVNTHNPRQCYVTLNWSRYLSYYQLGYGARGIGVRDSSQDVMAVVAGAPGRAKKLLRKLLSVQKRNGSSMHQFNPKTMEATMGDAREMEDRPQYYGDDHLWLVFAVCHYIAETGDYAFLEEEIPFYEKDKEGRPLESARVLEHLERALAFTWNDTGVHGIPHLGFADWNDTVNLKIGAESFFIAHQFSKAARDLADLMDHLGKKEKAETYRSYAEEMKRRVNEVGWDGEWYLRYFDWDGSPIGSRGNVHGKIYTNAQSWAVISGNATPERARKALDAVYTHLNTRYGIKLSTPGYDHYDPNLGGVTTYPPGAKENGGIFLHANPWVIIAECMMGNGERAYQYHRQVNPIKKNEIIDIYEVEPYVFCQNILGDEHPQFGLGRNSWLTGTASWMYQAATQYILGVRVTHSGIVIDPCLPSEWEGFRMRRKVRGTWYEITVRNPEHVSRGVQSCTLDGEAVEVSGGAARIPFDTEGKDHVVEVILGA.
The cellooligosaccharide phosphorylase StCDP of the invention is obtained by codon optimization of a cellooligosaccharide phosphorylase (glycosyl transferase, glycosyl hydrolase family) gene (WP_ 014625716.1) from a thermophilic spirochete (Spirochaeta thermophila) and synthesis of the protein gene (StCDP).
The invention also provides a coding gene StCDP of the cellooligosaccharide phosphorylase, and the nucleotide sequence of the coding gene StCDP is shown as SEQ ID No.2 ：ATGAGCTATGGCCATTTTGAAGGCCGCGAATATGTGATTACCAACCCGCGCACCCCGGTGAAATGGATTAACTATGTGGGCACCATTGATTTTGGCGGCTTTGTGGATCATACCGGCGGCCTGCTGGTGTGCAAAGGCGATCCGGCGCTGAACCGCATTACCAAATATATTACGCAGCTGCCGAGCAGCGAATTTAAAGGCAGCACCATTTATCTGCGCGGCCGCCGCGGCGATCGCAGCTTTCTGTATAGCCCGTATTATGTGCCGACCCTGCATGAATATGATCGCTATGAATGCCGCATTGGCCTGGGCTATAGCCGCTTTCTGATGGAATATGTGGGCATTCGCACCGAAATTACCGTGTTTGTGCCGGAAGGCGAAAGCGTGGTGCTGCAAGATGTGCGCATTCGCAACACCGGCAGCGATACCGTGGAAGTGCTGGATCTGATTCCGGTGGTGGAATATAGCCATTTTGATGCGCTGAAACAGCTGACCAACGCGGATTGGGTGCCGCAGACCATGACGAGCAAAGCGATTGGCCGCCCGGGCGGTTTACTGGTGCTGCGTCAGTGCGCGTTTATGCAGACCGGCCGCGCGGAAAACTTTCTGACGAGCAACAGCCCGGTGAGCAGCTTTGAAGCGGATCGCCGTCGCTTTCTGGGCGAAAACGAATATGGCACCTGGAAAATGCCGTTTAGCCTGGTGGATGGCCGCGAATTTAGCAACTATGAACCGCTGCGCGGCGATAACGTGGGCGCGCTGATGCATCATGTGGGCCCGCTGGCGCCGGGCGAAGAACGCCGCGTGATTGTTCAGCTGGGCCAAGTGGAAAGCGTGGAAAAAGCGATGCCGCTGATTGAACGCTTTCGCGAACCGGAAGAAGTGGATGCGGCGTTTGCGCGCATGCAAGCGTTTTGGGAAGAATATCTGAACGTGTGCCAAGTGGAAACCCCGGACCCGGAATTTGATCTGCTGGTGAACACCCATAACCCGCGTCAGTGCTATGTGACCCTGAACTGGAGCCGCTATCTGAGCTATTATCAGCTGGGCTATGGCGCGCGCGGCATTGGCGTGCGCGATAGCAGCCAAGATGTGATGGCGGTGGTGGCGGGCGCGCCGGGCCGCGCGAAAAAACTGCTGCGCAAACTGCTGAGCGTGCAGAAACGCAACGGCAGCAGCATGCATCAGTTTAACCCGAAAACCATGGAAGCGACCATGGGCGATGCGCGCGAAATGGAAGATCGCCCGCAGTATTATGGCGATGATCATCTGTGGCTGGTGTTTGCGGTGTGCCATTATATTGCGGAAACCGGCGATTATGCGTTTCTGGAAGAGGAAATTCCGTTTTATGAAAAAGATAAAGAAGGCCGCCCGCTGGAAAGCGCGCGCGTGCTGGAACATCTGGAACGCGCGCTGGCGTTTACCTGGAACGATACCGGCGTGCATGGCATTCCGCATCTGGGCTTTGCGGATTGGAACGACACCGTGAACCTGAAAATTGGCGCGGAGAGCTTTTTTATTGCGCATCAGTTTAGCAAAGCGGCGCGCGATCTGGCGGATCTGATGGATCATCTGGGCAAAAAAGAAAAAGCGGAAACCTATCGCAGCTATGCGGAAGAAATGAAACGCCGTGTGAACGAAGTGGGCTGGGATGGCGAATGGTATCTGCGCTATTTTGATTGGGATGGCAGCCCGATTGGCAGCCGCGGCAACGTGCATGGCAAAATTTATACCAACGCGCAGAGCTGGGCGGTGATTAGCGGCAACGCGACCCCGGAACGCGCGCGCAAAGCGCTGGATGCGGTGTATACCCATCTGAACACCCGCTATGGCATTAAACTGAGCACCCCGGGCTATGATCATTATGATCCGAACCTGGGCGGCGTGACCACCTATCCGCCGGGCGCGAAAGAAAACGGCGGCATTTTTCTGCATGCGAACCCGTGGGTGATTATTGCGGAATGCATGATGGGCAACGGCGAACGCGCGTATCAGTATCATCGCCAAGTGAACCCGATTAAAAAAAACGAAATTATTGATATTTATGAAGTGGAACCGTATGTGTTTTGTCAGAACATTCTGGGCGATGAACATCCGCAGTTTGGCCTGGGCCGCAACAGCTGGCTGACCGGCACCGCGAGCTGGATGTATCAAGCGGCGACGCAGTATATTCTGGGCGTGCGCGTGACCCATAGCGGCATTGTGATTGATCCGTGCCTGCCGAGCGAATGGGAAGGCTTTCGCATGCGCCGCAAAGTGCGCGGCACCTGGTATGAAATCACCGTGCGTAATCCGGAACATGTGAGCCGCGGCGTGCAGAGCTGCACCCTGGATGGCGAAGCGGTGGAAGTGAGCGGCGGCGCGGCGCGCATTCCGTTTGATACCGAAGGCAAAGATCATGTGGTGGAAGTGATTCTGGGCGCGCATCACCATCACCATCATTAA.
The invention also provides a recombinant expression vector containing the coding gene StCDP and expressing the cellooligosaccharide phosphorylase StCDP.
The recombinant expression vector of the present invention is preferably based on a prokaryotic expression vector, and in the examples, pET-SUMO vector is exemplified, but it is not intended to be construed as a complete scope of the present invention. In the embodiment of the invention, the gene fragment shown in SEQ ID No.2 is preferably inserted into the multiple cloning sites (BamHI and BpuEI) of the pET-SUMO vector, so as to construct the expression vector pET-StCDP of the cellooligosaccharide phosphorylase StCDP.
The invention also provides a recombinant expression cell containing the recombinant expression vector.
The recombinant expression cell of the invention preferably uses a prokaryotic cell as a host cell, and in the embodiment, pET-StCDP plasmid is preferably transferred into E.coli BL21 to construct E.coli BL21 (pET-StCDP), and the cellooligosaccharide phosphorylase StCDP is expressed and purified by the cultured cells.
The invention also provides a preparation method of the cellooligosaccharide phosphorylase StCDP, which comprises the steps of culturing the recombinant expression cells into seed liquid, inoculating the seed liquid into a liquid culture medium containing corresponding antibiotics for induction expression, and collecting thalli;
After the cells are broken, collecting a supernatant, wherein the supernatant contains the cellooligosaccharide phosphorylase StCDP.
The strain E.coli BL21 (pET-StCDP) is preferably stored in an LB solid medium containing kanamycin, wherein the composition of the LB solid medium preferably comprises 0.5% of yeast powder, 1% of tryptone, 1% of sodium chloride and 2% of agar powder, and the balance of the LB liquid medium is the same except that the LB liquid medium does not contain the agar powder.
In the present invention, E.coli BL21 (pET-StCDP) single colonies are preferably selected and cultured in LB liquid medium containing kanamycin (50. Mu.g/mL) at 37℃and 220rpm for 10 to 14 hours as seed solution. 1% of the inoculum size was inoculated into 1LTB medium containing kanamycin (50. Mu.g/mL), cultured with shaking at 37℃and 220rpm for 3 hours, then IPTG (0.15 mM) was added, cellooligosaccharide phosphorylase StCDP was induced at 18℃for 12 hours, and after centrifugation (5500 rpm) at 4℃for 5 minutes, E.coli BL21 (pET-StCDP) cells were harvested. The components of the TB medium of the invention are preferably: 24g/L yeast powder, 12g/L tryptone, 2.3g/L potassium dihydrogen phosphate and 16.4g/L dipotassium hydrogen phosphate.
The present invention preferably uses potassium phosphate buffer (0.1M, pH 7.0) containing 10% glycerol, 0.1mM DTT and 0.5M KCl to resuspend the E.coli BL21 (pET-StCDP) cells, and then after disruption, centrifugation, collect the supernatant, which contains cellooligosaccharide phosphorylase StCDP. The invention preferably further comprises purifying by using a nickel affinity chromatography column after collecting the supernatant.
The invention also provides application of the cellooligosaccharide phosphorylase StCDP in preparing glucose-1-phosphate by enzymolysis.
The invention preferably takes cellooligosaccharide as a raw material, utilizes cellooligosaccharide phosphorylase StCDP to catalyze the photolysis reaction, and separates and purifies reaction products to obtain glucose-1-phosphoric acid. The cellooligosaccharide according to the invention preferably comprises at least one of the following: cellotriose, cellotetraose, cellopentaose and cellohexaose. The cellooligosaccharide phosphorylase StCDP has excellent heat resistance and high catalytic activity in the temperature range of 25-75 ℃, and belongs to thermophilic cellooligosaccharide phosphorylase.
The invention also provides a method for preparing glucose-1-phosphoric acid by enzymolysis, which comprises the following steps: the cellooligosaccharide is taken as a raw material, the cellooligosaccharide phosphorylase StCDP is utilized for enzymolysis, and the enzymolysis product comprises the glucose-1-phosphoric acid.
The cellooligosaccharide according to the invention preferably comprises at least one of the following: cellotriose, cellotetraose, cellopentaose and cellohexaose. The temperature of the enzymolysis is preferably 25-75 ℃, and the time of the enzymolysis is preferably 10 min-48 h. In carrying out the enzymatic hydrolysis, the reaction system of the enzymatic hydrolysis preferably comprises, in 20 mL: the final concentrations were 0.2M Tris-HCl (pH 7.5), 0.1MNaH 2PO4 and 1000U cellooligosaccharide phosphorylase StCDP.
In order to further illustrate the present invention, the cellooligosaccharide phosphorylase StCDP and the method for preparing glucose-1-phosphate by enzymolysis provided in the present invention are described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the embodiment of the invention, the detection method of glucose 1-phosphate is preferably as follows: the 240. Mu.L assay contained 0.8. Mu.L of glucose phosphomutase (Tris-HCl buffer, pH 7.5,4U/mL), 0.4. Mu.L of glucose hexaphosphate dehydrogenase (Tris-HCl buffer, pH 7.5,2U/mL), 198.8. Mu. LNADP + (3 mM) and 40. Mu.L of glucose 1-phosphate test sample. The reaction was carried out at 30℃for 15min, and the increase in NADPH was detected at 340 nm.
Example 1
According to the codon preference of the escherichia coli, the cellooligosaccharide phosphorylase StCDP gene (WP_ 014625716.1) is subjected to codon optimization, the gene (StCDP) is synthesized, and then the synthesized gene fragment is inserted into a pET-SUMO vector multiple cloning site (between BamHI and BpuEI), so as to construct StCDP expression vector pET-StCDP. pET-StCDP was transferred into E.coli BL21 strain to obtain expression strain E.coli BL21 (pET-StCDP), which was stored on LB solid medium containing kanamycin (50. Mu.g/mL).
Single colonies of E.coli BL21 (pET-StCDP) were picked and cultured in LB liquid medium containing kana (50. Mu.g/mL) at 37℃and 220rpm for 13h as seed solution. Inoculating 1% of the strain into TB medium, culturing at 37deg.C with shaking at 220rpm for 3 hr, adding IPTG (0.15 mM), inducing StCDP expression at 18deg.C, freezing at 4deg.C for 12 hr, centrifuging at 5500rpm, and collecting thallus.
Collected 20g of freshly cultured E.coli BL21 (pET-StCDP) cells were resuspended in 80mL of 10% glycerol, 0.1mM DTT,0.5M KCl potassium phosphate buffer (0.1M, pH 7.0), the cells were disrupted twice with low temperature and ultra high pressure (4 ℃,1000 bar), centrifuged at 4℃and 12000rpm for 20min, and the supernatant was collected.
Transferring the supernatant to a Ni affinity chromatographic column, and performing gradient elution by using a phosphate buffer solution containing 20-200mM imidazole to obtain the pure cellooligosaccharide phosphorylase StCDP. The StCDP protein samples were concentrated and imidazole removed. The concentration of StCDP protein can reach 8g/L.
Pure enzyme StCDP enzyme activity assay: first, 40. Mu.L of the reaction system contained: 200mM Tris-HCl (pH 7.5), 100mMNaH 2PO4 and 10. Mu.L cellooligosaccharide phosphorylase StCDP, at 45℃for 10min; next, the 240 μl assay system comprises: 0.8. Mu.L of glucose phosphomutase (Tris-HCl buffer, pH7.5,4U/mL), 0.4. Mu.L of glucose hexaphosphate dehydrogenase (Tris-HCl buffer, pH7.5,2U/mL), 198.8. Mu. LNADP + (3 mM) and 40. Mu. LStCDP catalyze the phospholysis reaction, the reaction is carried out at 30℃for 15min, and the amount of NADPH activity is measured at 340 nm. The phospholysis activity of the StCDP enzyme solution prepared is as follows: 18400U/mL.
Subsequent experiments were performed using cellooligosaccharide phosphorylase StCDP prepared in example 1:
example 2
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.0) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 30℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 16.2g/L, and the conversion efficiency is 78%.
Example 3
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 30℃for 10 hours. The glucose-1-phosphate concentration of the obtained product was 19.6g/L, and the conversion efficiency was 94%.
Example 4
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 8.0) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 30℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 18.1g/L, and the conversion efficiency is 87%.
Example 5
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 8.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 30℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 17.7g/L, and the conversion efficiency is 85%.
Example 6
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 9.0) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 30℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 16.8g/L, and the conversion efficiency is 81%.
Example 7
Into a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 9.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added to obtain a glucose-1-phosphate concentration of 15.4g/L, and a conversion efficiency of 74%.
Example 8
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 25℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 17.6g/L, and the conversion efficiency is 85%.
Example 9
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 35℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 18.5g/L, and the conversion efficiency is 89%.
Example 10
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 37℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 18.5g/L, and the conversion efficiency is 89%.
Example 11
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 40℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 19.1g/L, and the conversion efficiency is 92%.
Example 12
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 45℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 19.7g/L, and the conversion efficiency is 95%.
Example 13
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 50℃for 10 hours. The glucose-1-phosphate concentration of the obtained product was 19.6g/L, and the conversion efficiency was 94%.
Example 14
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 60℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 19.7g/L, and the conversion efficiency is 95%.
Example 15
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 70℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 19.1g/L, and the conversion efficiency is 92%.
Example 16
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 75℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 12.9g/L, and the conversion efficiency is 62%.
Example 17
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.5g of cellotetraose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 45℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 23.4g/L, and the conversion efficiency is 79%.
Example 18
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.5g of cellopentasaccharide and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 45℃for 10 hours. The concentration of the obtained glucose-1-phosphate product is 21.8g/L, and the conversion efficiency is 70%.
Example 19
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 45℃for 10min. The concentration of the obtained glucose-1-phosphate product is 0.33g/L, and the conversion efficiency is 2%.
Example 20
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 45℃for 2 hours. The concentration of the obtained glucose-1-phosphate product is 3.8g/L, and the conversion efficiency is 23%.
Example 21
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 45℃for 5 hours. The concentration of the obtained glucose-1-phosphate product is 9.8g/L, and the conversion efficiency is 46%.
Example 22
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 45℃for 24 hours. The concentration of the obtained glucose-1-phosphate product is 19.9g/L, and the conversion efficiency is 98%.
Example 23
In a 125mL Erlenmeyer flask, 20mL of Tris-HCl buffer (0.2M, pH 7.5) containing 0.1MNaH 2PO4, 0.4g of cellotriose and 1000U of cellooligosaccharide phosphorylase StCDP were added and reacted at 45℃for 48 hours. The concentration of the obtained glucose-1-phosphate product is 19.9g/L, and the conversion efficiency is 98%.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The cellooligosaccharide phosphorylase StCDP is characterized in that the amino acid sequence of the cellooligosaccharide phosphorylase StCDP is shown in SEQ ID No. 1.
2. The cellooligosaccharide phosphorylase encoding gene StCDP of claim 1, wherein the nucleotide sequence of encoding gene StCDP is shown in SEQ ID No. 2.
3. A recombinant expression vector comprising the coding gene StCDP of claim 2 and expressing the cellooligosaccharide phosphorylase StCDP of claim 1.
4. A recombinant expression cell comprising the recombinant expression vector of claim 3.
5. The recombinant expression cell of claim 4, wherein the recombinant expression cell comprises a prokaryotic cell as a host cell.
6. The method for preparing cellooligosaccharide phosphorylase StCDP according to claim 1, which comprises culturing the recombinant expression cell according to claim 4 or 5 into a seed solution, inoculating the seed solution into a liquid culture medium containing a corresponding antibiotic for induction expression, and collecting thalli;
After the cells are broken, collecting a supernatant, wherein the supernatant contains the cellooligosaccharide phosphorylase StCDP.
7. The use of cellooligosaccharide phosphorylase StCDP according to claim 1 in the enzymatic preparation of glucose-1-phosphate.
8. The method for preparing glucose-1-phosphate by enzymolysis is characterized by comprising the following steps: the cellooligosaccharide is taken as a raw material, the cellooligosaccharide phosphorylase StCDP in claim 1 is utilized for enzymolysis, and the enzymolysis product comprises the glucose-1-phosphoric acid.
9. The method of claim 8, wherein the cellooligosaccharide comprises at least one of: cellotriose, cellotetraose, cellopentaose and cellohexaose.
10. The method according to claim 8, wherein the temperature of the enzymolysis is 25-75 ℃, and the time of the enzymolysis is 10 min-48 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410741154.XA CN118562758A (en) | 2024-06-07 | 2024-06-07 | Cellooligosaccharide phosphorylase StCDP and method for preparing glucose-1-phosphoric acid by enzymolysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410741154.XA CN118562758A (en) | 2024-06-07 | 2024-06-07 | Cellooligosaccharide phosphorylase StCDP and method for preparing glucose-1-phosphoric acid by enzymolysis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118562758A true CN118562758A (en) | 2024-08-30 |
Family
ID=92476113
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410741154.XA Pending CN118562758A (en) | 2024-06-07 | 2024-06-07 | Cellooligosaccharide phosphorylase StCDP and method for preparing glucose-1-phosphoric acid by enzymolysis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118562758A (en) |
-
2024
- 2024-06-07 CN CN202410741154.XA patent/CN118562758A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102581107B1 (en) | Enzymatic production of D-allulose | |
| CN111534503B (en) | Application of Haloferrila sp | |
| EP2516663B1 (en) | Methods for improving the efficiency of simultaneous saccharification and fermentation reactions | |
| KR20190054187A (en) | Enzymatic synthesis of d-tagatose | |
| CN107988286B (en) | Method for preparing tagatose by whole-cell catalysis | |
| EP4276171A1 (en) | Bacillus subtilis genetically engineered bacterium for producing tagatose and method for preparing tagatose | |
| JP7569565B2 (en) | Enzymatic production of mannose. | |
| Krishnareddy et al. | Cellodextrin phosphorylase from Clostridium thermocellum YM4 strain expressed in Escherichia coli | |
| JP2024075570A (en) | Enzymatic production of tagatose | |
| JP2023526624A (en) | Enzymatic production of allulose | |
| CN117587085A (en) | Method for synthesizing lactoyl-N-trisaccharide II by coupling fermentation of three bacteria | |
| CN118562758A (en) | Cellooligosaccharide phosphorylase StCDP and method for preparing glucose-1-phosphoric acid by enzymolysis | |
| CN114277043B (en) | Heat-resistant mannosidase gene, and expression protein and application thereof | |
| TW201943854A (en) | Enzymatic production of D-allulose | |
| CN104232605B (en) | A kind of xylosidase Xyl_S and encoding gene thereof and application | |
| CN118126988B (en) | Wide Wen Naqie beta-1, 4-glucanase and application thereof | |
| AU2015261652B2 (en) | Methods for improving the efficiency of simultaneous saccharification and fermentation reactions | |
| CN113897405B (en) | Method for synthesizing 3' -sialyllactose by three-strain coupling fermentation at low cost | |
| CN116286770B (en) | D-psicose-3-epimerase from clostridium and application thereof | |
| CN113366112B (en) | Enzymatic production of tagatose | |
| CN118165950A (en) | Application of laminaria oligosaccharide phosphorylase in synthesis of laminaria oligosaccharide and multienzyme catalytic system | |
| Turner et al. | Expression, purification, crystallization and preliminary X-ray diffraction analysis of Thermotoga neapolitana β-glucosidase B | |
| CN117568422A (en) | Method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn straw hydrolysate | |
| CN118581075A (en) | D-psicose-3-epimerase mutant, related biological material, preparation method and application thereof | |
| CN117925756A (en) | Preparation method of high-purity citrus flavonoid glycoside |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |