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Immobilized nitrilase induced by self-aggregating short peptide and its preparation method and application

A technology of nitrilase and self-aggregation, applied in the fields of hydrolase, immobilized on/in organic carriers, chemical industry, etc., to achieve good substrate tolerance, save cumbersome operations, and facilitate long-term storage

Inactive Publication Date: 2016-03-02
SOUTH CHINA UNIV OF TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The object of the present invention is to overcome the shortcoming of existing nitrilase immobilization method, provide a kind of nitrilase immobilization method based on self-aggregation short peptide induced aggregation

Method used

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  • Immobilized nitrilase induced by self-aggregating short peptide and its preparation method and application
  • Immobilized nitrilase induced by self-aggregating short peptide and its preparation method and application
  • Immobilized nitrilase induced by self-aggregating short peptide and its preparation method and application

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Embodiment 1

[0040] 1. Construction of expression vectors containing coding short peptides

[0041] According to the amino acid sequence of PT-linker and 18A, the corresponding DNA sequence PT-linker+18A was synthesized in Gene Synthesis Company, and the HindIII restriction site was introduced upstream of the PT-linker+18A DNA sequence, and the XhoI enzyme site was introduced downstream. Plasmid pET-30a(+) was used as the basic plasmid, double-digested with HindIII and XhoI, connected into the corresponding PT-linker+18A sequence, transformed into E.coliDH5α competent cells, and passed colony PCR, enzyme digestion and sequencing Analyze and identify positive transformants to obtain plasmid pET-aIB, such as figure 1 shown.

[0042] 2. Construction of engineering bacteria expressing nitrilase active aggregates in Escherichia coli cells

[0043] Using the genome of Alcaligenes faecalis JM3 as a template, under the action of primer 1 and primer 2, a 1 kb nitrilase gene fragment was amplified...

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Abstract

The invention discloses a self-aggregation short-peptide induction based immobilized nitrilase and a preparation method and application thereof. The preparation method comprises the following steps: (1) splicing connecting peptides and self-aggregation short peptides, so as to construct an expression vector; (2) connecting a nitrilase gene and the expression vector, and transforming into a recipient strain, so as to obtain an engineering strain; (3) carrying out induced expression on the engineering strain, carrying out wall breaking treatment on cells, and centrifuging and / or filtrating to obtain a precipitate, namely a nitrilase endoenzyme aggregate; (4) immobilizing the obtained nitrilase endoenzyme aggregate in alginate, thereby obtaining the immobilized nitrilase. According to the immobilized nitrilase aggregate obtained by the method, the particle diameter of the immobilized nitrilase is about 1mm. According to the immobilized nitrilase, particles can be subjected to freeze-drying preservation, and the enzyme activity is reserved to 90% when the immobilized nitrilase is preserved for 20 days at the temperature of 37 DEG C; the activity of the immobilized nitrilase is high, and the operating flow is simple.

Description

technical field [0001] The invention belongs to the fields of bioengineering and biocatalysis. The invention relates to a method for aggregation, expression and immobilization of nitrilase induced by self-aggregation short peptides. More specifically, the present invention constructs a nitrilase genetically engineered bacterium expressed in the form of enzyme aggregates in vivo, the aggregates with nitrilase activity are fixed in sodium alginate solution, and the racemization is catalyzed in aqueous solution The nitriles are converted to the corresponding carboxylic acids. Background technique [0002] Nitrilase can convert nitriles into corresponding carboxylic acids and ammonia through a one-step catalytic reaction in aqueous solution. In recent years, many experts and scholars have used nitrilase to catalyze the biotransformation process of fatty nitriles, aromatic nitriles and alicyclic nitriles into corresponding organic carboxylic acids, and there have been very succ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N9/78C12N11/10C12P7/42
CPCY02P20/50
Inventor 李爽杨晓锋林章凛王菊芳王小宁
Owner SOUTH CHINA UNIV OF TECH
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