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Highly catalytically active pet hydrolase mutants

A mutant, hydrolase technology, applied in hydrolase, biochemical equipment and methods, fermentation and other directions, can solve the problems of low efficiency of PET plastic degradation, insufficient catalytic function of key enzyme Ec_PETase, difficult industrial application, etc. Degradation cost, high enzymatic activity, effect of simplifying degradation steps

Active Publication Date: 2020-09-18
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the discovery of Ideonella sakaiensis 201-F6 strain and the functional analysis of the key enzyme PET hydrolase (PETase) have brought hope for the biodegradation of PET plastics, the efficiency of Ideonella sakaiensis 201-F6 wild-type strains for the degradation of PET plastics is low, The catalytic function of the key enzyme Ec_PETase involved in PET degradation metabolism is not high enough
It is difficult to directly realize the industrial application of PET plastic biodegradation

Method used

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  • Highly catalytically active pet hydrolase mutants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1, Escherichia coli secreted often expressed PET hydrolase (Ec_PETase) mutation hotspot screening

[0035] 1. Construction of 3D model of PETase

[0036]Enter the amino acid sequence of PETase: (GenBank accession number, GAP38373.1) into the three websites I-TASSER, MULTICOM, and ROBETTA to obtain the 3D model of PETase, use Rampage to evaluate the obtained model, and select 3 models for follow-up Docking experiment.

[0037] 2. Use Auto Dock software for substrate docking

[0038] Since the pNPB method is a commonly used lipase activity detection method, pNPB, the product of lipase hydrolysis, has an absorption peak at 405nm under the conditions of 34°C and pH=7.4. Therefore, we used pNPB as the substrate for molecular docking. GaussView was used to draw its 3D structure, and dynamic optimization was performed to finally obtain the most stable conformation of pNPB in space. The best results were obtained after docking simulation analysis.

[0039] 3 mutati...

Embodiment 2

[0041] Example 2. Construction of site-directed mutants of mutation hotspots Trp159, Gln182, Ala183, Ile208 and Ser238.

[0042] The primers used in the experiment are listed in Table 1 below:

[0043] Table 1 Primers used for construction of site-directed mutants

[0044] Primer serial number Primer sequence (5'-3') W159H-Forward SEQ ID No.9 TATGGGTCATAGCATGGGTGGCGGTGGCAGC W159H-Reverse SEQ ID No.10 CACCCATGCTATGACCCATAACGCCCATACG Q182L-F SEQ ID No.11 GGCGCCGCTGGCGCCGTGGGACAGCAGCTTC Q182L-R SEQ ID No.12 CACGGCGCCAGCGGCGCCGCAGCTTTCAGGCT A183T-F SEQ ID No.13 CGCCGCAAACCCCCGTGGGACAGCAGCTTCAGC A183T-R SEQ ID No.14 TCCCACGGGGTTTGCGGCGCCGCAGCTTTCAG I208V-F SEQ ID No.15 ACGATAGCGTTGCGCCGGTGAACAGCAGCGCG I208V-R SEQ ID No.16 ACCGGCGCAACGCTATCGTTCTCGCACGCAAA S238F-F SEQ ID No.17 GCAGCCACTTCTGCGCGAACAGCGGTAACAGC S238F-R SEQ ID No. 18 GTTCGCGCAGAAGTGGCTGCCACCGTTAATTTC

[0045] The n...

Embodiment 3

[0047] Example 3, EcPETase recombinant expression and activity evaluation

[0048] 1. Take pUC57-EcPETase genetically engineered Escherichia coli mutants respectively, culture them with shaking in LB liquid medium at 37°C and 180rpm for 4 hours (OD600=1.5), centrifuge to separate the protein secreted into the extracellular space and concentrate it to 0.025mg / ml is used for the determination of enzyme catalytic activity.

[0049] 2. Obtain a concentrated supernatant sample of the overnight culture product of the pUC57-EcPETase genetically engineered E. coli strain, using pNPB as the substrate. References: "oshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, Toyohara K , Miyamoto K, Kimura Y, Oda K.2016.A bacterium that degrades and assimilates poly(ethylene terephthalate).Science,351(6278):1196-1199."The experimental method disclosed in the test pUC57-EcPETase genetically engineered Escherichia coli strain Catalytic activity of EcPETase in culture medium. Add 100 ...

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Abstract

The invention belongs to the field of protein engineering, in particular to a PET hydrolase mutant. The technical problem to be solved by the present invention is that the activity of the PET hydrolase (PETase) currently derived from the Ideonella sakaiensis 201-F6 strain is not ideal. The technical solution of the present invention to solve the technical problem is to provide a PET hydrolase mutant. In the present invention, 5 mutation hotspots are obtained through a large number of researches on PET hydrolase (PETase); 14 mutants are constructed by applying site-directed mutation technology, and the activity of 2 mutant ETase enzymes is finally screened out compared with the wild type Ec_PETase. , has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of Escherichia coli genetic engineering, in particular to a PET hydrolase (Ec_PETase) mutant derived from Escherichia coli engineering bacteria with improved catalytic activity. Background technique [0002] Polyethylene terephthalate (PET) is one of the most common plastic materials. In 2013, the annual output of PET reached 56 million tons, accounting for about 1 / 5 of the global plastic output. According to statistics, PET is currently the most recycled plastic material, and its recycling rates in the United States and the European Union are 31% and 50% respectively. Although the PET polymer is only composed of two simple monomers connected by an ester bond, the ester bond is relatively strong and difficult to degrade naturally. Tens of millions of tons of PET plastic waste are difficult to be effectively disposed of every year, which is extremely harmful to the environment. [0003] Currently existing PE...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N15/55
CPCC12N9/18
Inventor 汤丽霞李思扬张勇郑雪莲刘炳麟邹佳佳
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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