Polypeptide label, highly soluble recombinant nitrilase and application thereof in synthesis of pharmaceutical chemicals

A technology of polypeptide labeling and nitrilase, which is applied in the fields of genetic engineering and protein engineering, can solve the problems of insufficient enzyme activity for large-scale industrial applications and poor thermal stability of enzymes, and achieve the effects of improving activity, excellent stability, and improving solubility

Active Publication Date: 2021-02-12
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solve the problems of poor thermal stability of enzymes and unsatisfactory enzyme activity for large-scale industrial applications

Method used

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  • Polypeptide label, highly soluble recombinant nitrilase and application thereof in synthesis of pharmaceutical chemicals
  • Polypeptide label, highly soluble recombinant nitrilase and application thereof in synthesis of pharmaceutical chemicals
  • Polypeptide label, highly soluble recombinant nitrilase and application thereof in synthesis of pharmaceutical chemicals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Construction of recombinant plasmids containing polypeptide tags

[0053] 1. The design principle is: the solubility of the protein is closely related to the hydrophobicity of the residues, and is also affected by the net charge of the protein or the ratio of helical residues. Polar amino acids have an important effect on the solubility of proteins. Palindromic element sequences usually consist of multiple repeating units containing one or two polar amino acids, have positive or negative charges, have been reported to facilitate protein folding, and are usually smaller than 15 residues.

[0054] Based on the above principles, we first designed a pentapeptide tag, in which both ends and the middle (i.e. 1, 3, 5 amino acids) are uncharged glycine (G), and the rest (i.e. 2, 4 amino acids) are glycine ( G), any one or two random combinations of histidine (H), glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), specifically Is one of: GDGDG, GDGEG, G...

Embodiment 2

[0065] Embodiment 2: Construction of recombinant escherichia coli

[0066] Axygen clean-up kit (purchased from Corning Life Science (Wujiang) Co., Ltd.) was used to purify (clean-up) the PCR product containing recombinant plasmid pET-28b(+) / tag-AcN-M in Example 1, The specific operation is: add three times the volume of PCR-A buffer to the 5 μL PCR product of Example 1 and mix well, transfer to the preparation tube, centrifuge at 12000 rpm for 1 min, discard the filtrate, add 700 μL W2 buffer to the preparation tube, and centrifuge at 12000 rpm 1min, discard the filtrate, wash twice with W2 buffer; add 5μL to pre-thawed competent cells E.coli BL21(DE3), ice-bath for 30min, then heat shock at 42°C for 90s, ice-bath again for 3-5min, add 700μL LB Liquid medium, incubate at 37°C for 1h. Inoculate 500 μL of the culture solution onto the LB solid medium with 0.5 μg / mL kana-resistance, spread evenly, culture at 37°C for 12-14 hours, and verify by sequencing to obtain recombinant Es...

Embodiment 3

[0067] Embodiment 3: Recombinant escherichia coli expresses nitrilase

[0068] 1. Resting cells: Take out the recombinant Escherichia coli E. coli BL21(DE3) / pET-28b(+) / tag-AcN-M strain constructed in Example 2 and inoculate it to a concentration of 0.5 Streak the μg / mL kana-resistant LB plate and culture at 37°C for 12-14 hours to obtain a single colony. Pick a single colony into 10mL LB test tube medium containing 0.5μg / mL kanamycin, culture at 37°C for 8h, then transfer 2mL to 100mL fermentation medium containing 0.5μg / mL kanamycin, 37°C After culturing for 2 hours, 100 μL of IPTG (final concentration 0.1 mM) was added to induce enzyme production at 28° C. for 12-14 hours. After centrifugation at 12,000 rpm for 10 min, the obtained bacteria were collected, washed twice with 0.9% normal saline, and then suspended to obtain a resting cell suspension, and the relative enzyme activity was determined.

[0069] 2. Pure enzyme: Take 1g of resting cells prepared by the method in s...

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Abstract

The invention discloses recombinant nitrilase and an application thereof in synthesis of pharmaceutical chemicals. The recombinant nitrilase is formed by connecting a polypeptide label to the N end ofan amino acid sequence of nitrilase, uncharged glycine G is arranged at the positions of the two ends of the polypeptide label, and the rest is any one or a random combination of more of glycine G, histidine H, glutamic acid E, aspartic acid D, lysine K and arginine R. When the recombinant nitrilase is used for preparing 1-cyan cyclohexyl acetic acid, the activity is as high as 3034.7 U / g dcw, the soluble expression of the nitrilase is remarkably improved due to the introduction of the polypeptide label, the hydrolysis of 1M substrates by using a whole-cell catalyst with the same concentration is completed 30 minutes faster than that of a female parent enzyme, and the stability is superior to that of the female parent enzyme. The recombinant nitrilase disclosed by the invention can be used for catalyzing and synthesizing other medical intermediates, improving the activity of the whole-cell catalyst in the reaction, improving the solubility of other different nitrilases and improving the activity of the corresponding whole-cell catalyst.

Description

[0001] (1) Technical field [0002] The invention relates to a polypeptide tag, in particular to a universal polypeptide tag capable of enhancing the soluble expression of nitrilase and other enzymes, and its application in the synthesis of pharmaceutical chemicals, belonging to the technical fields of genetic engineering and protein engineering. [0003] (2) Background technology [0004] Nitrilases (EC 3.5.5.1) are an important class of hydrolases with the Glu-Lys-Cys catalytic active center triplet, which can selectively and efficiently catalyze the hydrolysis of cyano groups to carboxyl groups in a one-step reaction. It is widely used in the synthesis of chemical products such as , amino acids, vitamins, and pharmaceutical chemicals such as gabapentin, clopidogrel, baclofen, and atorvastatin. [0005] So far, many effective methods have been reported to increase the soluble expression level of proteins, one is to construct recombinant periplasmic leaky strains or co-express...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K19/00C12N9/78C12N9/20C12N9/80C12N1/21C12P13/00C12P7/42C12R1/19
CPCC12N9/78C12P13/002C12P7/42C12Y305/05001C07K2319/35C07K7/06C12N9/20C07K2319/00C12N15/62C12R2001/19C12N9/1029C12P17/10
Inventor 薛亚平谢冬熊能郑裕国
Owner ZHEJIANG UNIV OF TECH
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