Carbonic anhydrase with heat stability and high activity and application thereof

A carbonic anhydrase, high activity technology, applied in the field of biochemistry, can solve the problem that human carbonic anhydrase II cannot meet the use requirements

Inactive Publication Date: 2018-12-21
ANHUI UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

while for CO 2 Adsorption, usually the operating temperature is 50 ℃, or even above, so human carbonic anhydrase II can not meet the use requirements

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] (1) The 65-position alanine and 203-position leucine of human carbonic anhydrase are mutated and replaced by glutamic acid and lysine.

[0024] (2) After designing primers according to the target gene, extract RNA and reverse transcribe to obtain cDNA; then use the designed primers to perform PCR and agarose gel electrophoresis to amplify and separate to obtain the target gene.

[0025] (3) The gene was then connected to a cloning vector, and transformed into Escherichia coli DH5α. After the transformant was identified, the plasmid was extracted; it was then transformed into Escherichia coli BL21, and the transformant was identified.

[0026] (4) Finally, the BL21 bacteria were expanded and cultured, and an appropriate concentration of IPTG was added to induce expression; the bacteria were collected and lysed, and the lysate and precipitate were obtained, and the fusion protein was identified by SDS-PAGE and Western blot.

[0027] (5) The expressed protein was separate...

Embodiment 2

[0030] (1) The leucine at position 202 and the asparagine at position 243 of human carbonic anhydrase are mutated and replaced by arginine and threonine.

[0031] (2) Design primers according to the target gene, extract RNA, and obtain cDNA by reverse transcription; then use the designed primers to perform PCR and agarose gel electrophoresis to amplify and separate to obtain the target gene.

[0032] (3) The gene was then connected to a cloning vector, and transformed into Escherichia coli DH5α. After the transformant was identified, the plasmid was extracted; it was then transformed into Escherichia coli BL21, and the transformant was identified.

[0033] (4) Finally, the BL21 bacteria were expanded and cultured, and an appropriate concentration of IPTG was added to induce expression; the bacteria were collected and lysed, and the lysate and precipitate were obtained, and the fusion protein was identified by SDS-PAGE and Western blot.

[0034] (5) The expressed protein was s...

Embodiment 3

[0037] (1) The 198-position leucine and the 245-position glycine of human carbonic anhydrase are mutated and replaced by arginine and glycine.

[0038] (2) Design primers according to the target gene, extract RNA, reverse transcribe to obtain cDNA; then use the designed primers to perform PCR and agarose gel electrophoresis, amplify and separate to obtain the target gene.

[0039] (3) The gene was then connected to a cloning vector, and transformed into Escherichia coli DH5α. After the transformant was identified, the plasmid was extracted; it was then transformed into Escherichia coli BL21, and the transformant was identified.

[0040] (4) Finally, the BL21 bacteria were expanded and cultured, and an appropriate concentration of IPTG was added to induce expression; the bacteria were collected and lysed, and the lysate and precipitate were obtained, and the fusion protein was identified by SDS-PAGE and Western blot.

[0041] (5) The expressed protein was separated using a nic...

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PUM

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Abstract

The invention provides a carbonic anhydrase with heat stability and high activity and application thereof, and relates to the field of biological chemistry. The carbonic anhydrase is used for enhancing the CO2 catalytic conversion activity of a heat stability type carbonic anhydrase. In the invention, one or more of alanine 65, leucine 198, leucine 202, leucine 203, asparaginate 243, histidine 244and arginine 245 of a carbonic anhydrase II of human is/are mutated, and one or more of mutation sites exists/exist in the mutation; the operation steps are described as follows: firstly, analyzing the structural property of the carbonic anhydrase, mutating a residue on the basis and constructing a vector containing target genes; then, expressing proteins, and performing separation and purification and renaturation; finally, determining the activity of the carbonic anhydrase. According to the carbonic anhydrase, the capture and conversion efficiency of CO2 at high temperature can be effectively improved.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a carbonic anhydrase with thermal stability and high activity and its application. Background technique [0002] Carbonic anhydrase (CA) is a Zn-containing 2+ metalloenzyme that converts CO 2 Catalytic conversion to HCO 3- hydratase catalyst. CO 2 The reversible hydration reaction of CO is very slow in the absence of catalyst, on the contrary, in the presence of carbonic anhydrase, CO 2 The rate of the hydration reversibility reaction can be increased from 15 seconds to 10 per second 6 responses. It is precisely because CA has the characteristics of high efficiency, specificity, and environmental friendliness. In recent years, the use of carbonic anhydrase for CO 2 Capture technology has received more and more attention. [0003] in CO 2 The use of human carbonic anhydrase II in the adsorption process can greatly reduce the energy consumption of the chemisorption desorption p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12P3/00
CPCC12N9/88C12P3/00C12Y402/01001
Inventor 徐霞陈金瑞徐建
Owner ANHUI UNIVERSITY OF TECHNOLOGY
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