Thermophilic thermostable glyceric acid kinase, preparation method and application thereof

A technology of glyceric acid and kinase, applied in the field of bioengineering, can solve the problems of time-consuming and many processes

Inactive Publication Date: 2009-08-19
SHANDONG UNIV
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the organic synthesis process of this compound has the disadvantages of time-consuming and many procedures, while the enzymatic conversion faces the separation and purification of 2-PGA and 3-PGA and other difficult-to-obtain enzymes (such as phosphoglycerate mutase, ene Alcoholase) and substrates (such as 3-phosphoglycerate, phosphoenolpyruvate) and other issues

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Thermophilic thermostable glyceric acid kinase, preparation method and application thereof
  • Thermophilic thermostable glyceric acid kinase, preparation method and application thereof
  • Thermophilic thermostable glyceric acid kinase, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Embodiment 1. A thermophilic and high temperature resistant glycerate kinase, the DNA sequence encoding the glycerate kinase is as follows:

[0092]gtgattgcca tggatattag ggagataggc ctgagattgg ttggagaagc aataaaagct 60

[0093] gcagatccat acagggcagt cctaaatgca gttaaagtga gtgatgacaa gataattgtt 120

[0094] caaggtaagg aatttgaaat caagggaaag gtgtacgtaa tagccctggg gaaagcagca 180

[0095] tgtgaaatgg caagggccat agaggatata ctcgatgttg aggatggcgt tgcggtaact 240

[0096] aagtacggtt atggaaaaga gttaaagagg ataaaggtga ttgaagcggg acacccaata 300

[0097] cctgatgaga aatccatatt gggagctaaa gaggccctat cgatattgaa tagagctagg 360

[0098] gaaaatgaca tagttttcat cctaatttca ggtggaggtt cggcactatt tgagcttcca 420

[0099] gaagaaggta tttccctaga agatcttaag ctaaccacgg atctccttct aaagagcggg 480

[0100] cttgcgaaga tgatcaaggg gaccggaata gtactgataa tttcggatgt agttggcgat 600

[0101] aacctagaag ccatagcctc agggcccacc gttaaggatc caactacctt cgaagatgcg 660

[0102] aagaggatcc tggaactcta cgacatctgg gagaagg...

Embodiment 2

[0114] Embodiment 2, a glycerate kinase, is characterized in that, has the amino acid sequence as follows:

[0115] MIAMD IREIG LRLVG EAIKA ADPYR AVLNA VKVSD DKIIV QGKEF EIKGK VYVIA LGKAA

[0116] 5 10 15 20 25 30 35 40 45 50 55 60

[0117] CEMAR AIEDI LDVED GVAVT KYGYG KELKR IKVIE AGHPI PDEKS ILGAK EALSI LNRAR

[0118] 65 70 75 80 85 90 95 100 105 110 115 120

[0119] ENDIV FILIS GGGSA LFELP EEGIS LEDLK LTTDL LLKSG AKIHE INTVR KHISK VKGGK

[0120] 125 130 135 140 145 150 155 160 165 170 175 180

[0121] LAKMI KGTGI VLIIS DVVGD NLEAI ASGPT VKDPT TFEDA KRILE LYDIW EKVPE SVRLH

[0122] 185 190 195 200 205 210 215 220 225 230 235 240

[0123] IERGL RGEVE ETLKE DLPNV HNFLI ASNSI SCEAI AREAQ RLGFK AYIMT TTLEG EAKDA

[0124] 245 250 255 260 265 270 275 280 285 290 295 300

[0125] GLFIG SIVQE IAERG RPFEP PVVLV FGGET TVTIE GKGGK GGPNQ EIALS ATRKI SDLEA

[0126] 305 310 315 320 325 330 335 340 345 350 355 360

[0127] LIVAF DTDGT DGPTD AAGGI VDGTT YKKLR EKGID V...

Embodiment 3

[0131] Embodiment 3, the preparation of thermophilic high temperature resistant glycerate kinase

[0132] The preparation method of thermophilic and high temperature resistant glyceric acid kinase includes bacterial strain, plasmid, enzyme and culture medium, PCR amplification and construction of recombinant plasmid, induced expression of gene and purification of protein. Specific steps are as follows:

[0133] (1) Strain, plasmid, enzyme and culture medium:

[0134] Escherichia coli (Escherichia coli) DH5α, restriction endonuclease, Pyrobest DNA polymerase, DNA ligation kit ver.2.1 were purchased from TakaRa company; clone and expression plasmid vector pET15b, Escherichia coli (Escherichia coli) BL21-codonPlus(DE3)- RIL was purchased from Novagen; LB medium: containing 10 g of Tryptone, 5 g of Yeastextract, 10 g of NaCl, 100 mg / L of ampicillin and 34 mg / L of chloramphenicol per liter.

[0135] (2) PCR amplification and construction of recombinant plasmids:

[0136] Design ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention is one kind of thermophilic heat resisting glyceratekinase and its preparation process and application, and belongs to the field of bioengingeering technology. The glyceratekinase has optimal activity temperature of 45 deg.c, relatively wide adapting temperature range, very high activity in 20-50 deg.c and acidic condition, and optimal activity pH of 2.5. The glyceratekinase may have ATP, GTP, CTP, UTP or ADP as phospho donor. The preparation process of the glyceratekinase includes the steps of selecting plasmid, strain and culture medium, PCR amplification and recombinant plasmid constitution, gene inducing expression and protein purification. The glyceratekinase has very high heat stability, and may be used for preparing 2-phosphoglyceric acid in medicine and bioengineering.

Description

(1) Technical field [0001] The invention relates to a novel thermophilic and high temperature-resistant glycerate kinase and a preparation method thereof, belonging to the technical field of bioengineering. (2) Background technology [0002] According to the adaptability of organisms to the temperature of the surrounding living environment, organisms can be divided into psychrophilic organisms (<15°C), mesophilic organisms (15-60°C), thermophilic organisms (60-80°C), extreme thermophilic organisms ( 80-110°C). The optimum activity temperature of proteins in most organisms is generally at or close to the optimum growth temperature (optimum growth temperature, OGT) of the organism. Therefore, proteins can be divided into psychrophilic proteins, mesophilic proteins, thermophilic proteins, and extreme thermophilic proteins according to their dependence on temperature. Since most thermophilic and extreme thermophilic proteins are some enzymes (such as glycoside hydrolase, DN...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/12C12N1/21C12P7/42
Inventor 申玉龙刘波
Owner SHANDONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap