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Plant nitrilase mutant, coding gene and application of plant nitrilase mutant

A technology of nitrilase and coding gene, which is applied in the field of bioengineering, can solve the problems of inability to meet the needs of industrial production, low catalytic activity and stereoselectivity of nitrilase, and achieve the effect of improving catalytic activity and solubility

Active Publication Date: 2020-01-21
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The compound described in this patented patents allows plants to make certain chemical products faster without losing their ability to produce them due to its unique properties that increase it's effectiveness compared to other methods like ammoniacal cyanide method or molded case hardening process. This makes these processes easier but still produces better results when used on specific types of molecules.

Problems solved by technology

This patents discuss how chemistry plays major roles during modifying biochemically active compounds like nitrogen bases into different types of substances called quaternary salts. These modifications help make them more effective at various reactions involving nucleation processes. Additionally, current methods require expensive equipment and involve complicated steps making their use difficult without causing harmful side effects on the surrounding environments.

Method used

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  • Plant nitrilase mutant, coding gene and application of plant nitrilase mutant
  • Plant nitrilase mutant, coding gene and application of plant nitrilase mutant
  • Plant nitrilase mutant, coding gene and application of plant nitrilase mutant

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Experimental program
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Effect test

Embodiment 1

[0064] Construction of parental nitrilase gene

[0065] By comparing the nucleotide and amino acid sequences of cruciferous plant nitrilase, the key peptide 225-285 region was identified. See GenBank accession number: ABM55734.1 for the wild-type turnip nitrilase (BrNIT) sequence; see GenBank accession number: KFK44999 for the wild-type Alpine Arabidopsis thaliana nitrilase (AaNIT) sequence.

[0066] A one-step cloning method was used to insert the nucleotide sequence corresponding to the 225-285 peptides of Alpine Arabidopsis thaliana nitrilase (AaNIT) into the turnip nitrile hydrolase (BrNIT) plasmid fragment that lacked the corresponding peptide. Design primers are shown in Table 1.

[0067] Table 1: BaNIT chimeric enzyme primer design table

[0068]

[0069] Using the AaNIT nucleotide sequence as a template, clone the 225-285 peptide DNA fragment. PCR reaction system (50μL): Template DNA Master Mix, 0.2μM upstream and downstream primers, the rest ddH 2 O make up to the total vo...

Embodiment 2

[0076] Site-directed saturation mutations at positions 223, 263 and 279 of nitrilase

[0077] In order to mutate Leu(L) at position 223, His(H) at position 263 and Gln(Q) at position 279 in the parent amino acid sequence, corresponding primers were designed, as shown in Table 2. Show.

[0078] Table 2: Primer design table

[0079]

[0080] Note: N=A / G / C / T, K=G / T, M=A / C.

[0081] The recombinant plasmid pET28b-BaNIT containing the target gene fragment was used as the template, and the template was amplified by the whole plasmid according to the method of overlap extension PCR.

[0082] PCR amplification system (50μL): template DNA 0.1ng-1ng, 2×Phanta Max Buffer 25μL, dNTPs (10mM each) 1μL, mutation primer upstream and downstream each 1μL, Phanta Max Super-Fidelity DNA Polymerase 1U, the rest ddH 2 O make up to the total volume.

[0083] PCR reaction parameters: (1) 95°C pre-denaturation for 30s; (2) 95°C denaturation for 15s; (3) 63°C annealing for 15s; (4) 72°C extension for 6min, steps...

Embodiment 3

[0089] Construction of combined mutants of nitrilase

[0090] Using the expression plasmid pET28b-BaNIT-L223Q or pET28b-BaNIT-H263D as a template, site-directed mutagenesis was performed through whole plasmid amplification.

[0091] PCR amplification system (50μL): template DNA 0.1ng-1ng, 2×Phanta Max Buffer 25μL, dNTPs (10mM each) 1μL, mutation primer upstream and downstream each 1μL, Phanta Max Super-Fidelity DNA Polymerase 1μL, the rest ddH 2 O make up to the total volume.

[0092] PCR reaction parameters: (1) 95°C pre-denaturation for 30s; (2) 95°C denaturation for 15s; (3) 60°C annealing for 15s; (4) 72°C extension for 6min, steps (2)-(4) cycle 30 times; 5) Extend thoroughly at 72°C for 5 minutes and store at 4°C.

[0093] After the PCR product was positively analyzed by 0.9% agarose gel electrophoresis, take 20μL of PCR reaction solution, add 1μL of endonuclease Dpn I, digest the template plasmid DNA at 37℃ for 3h, and inactivate it at 65℃ for 10min. Transform into E.coli BL21(...

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Abstract

The invention discloses a cruciferous plant nitrilase mutant, and belongs to the technical field of biological engineering. According to the cruciferous plant nitrilase mutant, peptide fragments of sites 225-285 of the amino acid sequences of an arabis alpina nitrilase are embedded into a turnip nitrilase lacking the corresponding peptide fragments to obtain a turnip/arabis alpina nitrilase chimera, and then a coding gene of the chimeric BaNIT is subjected to site-specific saturation mutation to obtain the plant nitrilase mutant, and the plant nitrilase mutant is one or more of the following:(1) L at the site 223 mutates into Q; (2) H at the site 263 mutates into D; and (3) Q at the site 279 mutates into E. The catalytic activity of the plant nitrilase mutant is increased by 2.23 times, the solubility of a recombinant protein is greatly improved, the enantioselectivity E value is maintained at 400 or above, and the plant nitrilase mutant has good application prospects in efficient catalysis of racemic isobutylsuccinonitrile to synthesize (S)-3-cyano-5-methylhexanoic acid.

Description

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Claims

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Application Information

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Owner ZHEJIANG UNIV OF TECH
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