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PET hydrolase IsPETase mutant enzyme, coding gene and engineering bacterium

A technology of mutant enzymes and hydrolytic enzymes, which is applied in the fields of enzyme engineering and bioengineering, can solve the problems of low stability, enzyme activity, and limited applications, and achieve the effect of improving hydrolysis efficiency

Active Publication Date: 2021-12-10
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because IsPETase only grows under mild conditions, its relatively low stability and enzymatic activity also limit its application in PET degradation.

Method used

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  • PET hydrolase IsPETase mutant enzyme, coding gene and engineering bacterium
  • PET hydrolase IsPETase mutant enzyme, coding gene and engineering bacterium
  • PET hydrolase IsPETase mutant enzyme, coding gene and engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Rational Design of Mutation Sites of PET Hydrolase IsPETase

[0035] (1) Signal peptide prediction of PET hydrolase IsPETase:

[0036] Use the SignalP-5.0Server online server to analyze whether there is a signal peptide and a suitable cleavage site in the original sequence (SEQ ID NO.3) of the PET hydrolase IsPETase.

[0037] (2) Simulation of the spatial structure of PET hydrolase IsPETase

[0038] Based on the IsPETase enzyme (PDB: 5XG0) analyzed by Xu Han et al. (PDB: 5XG0) as a model (SEQ ID NO.3), its structure was found to have a set of typical features:

[0039] ①IsPETase enzyme consists of three polypeptide chains, which are represented as A, B and C chains;

[0040] ②Belongs to the α / β hydrolase superfamily and presents a typical α / β hydrolase folding structure. The central β-sheet structure is formed by 9 mixed β strands (β1-β9), surrounded by 7 α-helices (α1-β9). α7) surround;

[0041] ③The catalytic triad (S131-H208-D177) is strictly conserved and locate...

Embodiment 2

[0051] Preparation of recombinant plasmids pET-22b-IsPETase-cSP and pET-22b-MHETase-cSP

[0052] The construction of the recombinant plasmid pET-22b-IsPETase was obtained by linking the wild-type PET hydrolase IsPETase gene (SEQ ID NO.4) to the pET-22b plasmid.

[0053] The construction of the recombinant plasmid pET-22b-MHETase was obtained by linking the wild-type MHETase gene (SEQ ID NO.8) to the pET-22b plasmid. The amino acid sequence of the wild-type MHETase enzyme is described in SEQ ID NO.7.

[0054] Wherein, the source of the wild-type PET hydrolase IsPETase gene: Ideonella sakaiensis (GI: 1028065175).

[0055] Among them, the source of the wild-type MHETase gene: Ideonella sakaiensis (GI: 1027923624).

[0056] The pET-22b plasmid is commercially available.

[0057] The preparation method of recombinant plasmid (recombinant plasmid pET-22b-IsPETase-cSP and pET-22b-MHETase-cSP), concrete steps are as follows:

[0058] According to the requirements of the seamless re...

Embodiment 3

[0068] Contains a mutant enzyme encoding PET hydrolase IsPETase (IsPETase R280A / S121K -cSP) gene recombinant plasmid (abbreviation: recombinant plasmid containing mutant gene) preparation

[0069] Based on the mutation site rationally designed in Example 1, the primers for site-directed mutagenesis were designed and synthesized as follows:

[0070] R280A-F: 5'-GAACAGCACCGCGGTGTCGGACTTCCGCACCGC (mutation introduced at position 280) (SEQID NO.13)

[0071] R280A-R: 5'-GTCCGACACCGCGGTGCTGTTCGGGTTCTCGC (mutation introduced at position 280) (SEQ ID NO.14)

[0072] S121K-F: 5'-GACCAGCCGAAAAGCCGCTCGTCGCAGCAG (mutation introduced at position 121) (SEQ ID NO.15)

[0073] S121K-R: 5'-GAGCGGCTTTTCGGCTGGTCGAGCGTGGAG (mutation introduced at position 121) (SEQ ID NO.16)

[0074] According to the requirements of the seamless recombination kit, the recombinant plasmid containing the mutant gene was constructed by seamless connection and reverse PCR technology, which is mainly divided into t...

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Abstract

The invention discloses a PET hydrolase IsPETase mutant enzyme, a coding gene and an engineering bacterium. The PET hydrolase IsPETase mutant enzyme is named as IsPETaseR280A / S121K-cSP, and the amino acid sequence of the IsPETaseR280A / S121K-cSP is as shown in SEQ ID NO.1. According to the invention, a rational design technology is adopted to perform molecular modification on PET hydrolase IsPETase, the PET hydrolase IsPETase mutant enzyme, namely the IsPETaseR280A / S121K-cSP, with improved hydrolysis efficiency is obtained by methods such as signal peptide prediction and homologous modeling, so that the hydrolysis efficiency of PET is improved, and besides, the PET hydrolase IsPETase mutant enzyme and MHETase-cSP are combined to improve the hydrolysis efficiency of PET.

Description

technical field [0001] The invention belongs to the technical fields of enzyme engineering and bioengineering, and in particular relates to a PET hydrolase IsPETase mutant enzyme, a coding gene, a plasmid containing the coding gene, and an engineering bacterium. Background technique [0002] With the continuous growth of global plastic production, the output in 2018 has reached 359 million tons, and the compound growth rate in the past ten years has reached 4.1%. The major types of plastic polymers currently produced include polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyethylene terephthalate (PET), polyurethane (PUR) and polystyrene (PS ). Among them, PET is the most abundant polyester plastic. [0003] PET is composed of terephthalic acid (TPA) obtained from crude oil and ethylene glycol (EG) linked by ester bonds. PET has the characteristics of simple synthesis, low price, and durability. PET products include fiber-grade PET and non-fiber-grade P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/18C12N15/70Y02W30/62
Inventor 齐崴尤生萍殷庆典苏荣欣
Owner TIANJIN UNIV
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