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Mutant of PET (polyethylene terephthalate) degrading enzyme and application of PET degrading enzyme

A mutant and degrading enzyme technology, applied in the field of genetic engineering, can solve the problem of low activity of IsPETase, achieve the effect of improved hydrolysis activity and efficient degradation

Active Publication Date: 2018-09-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of this, the purpose of the present invention is to provide a mutant of new PETase in order to overcome the problem of low activity of the original IsPETase

Method used

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  • Mutant of PET (polyethylene terephthalate) degrading enzyme and application of PET degrading enzyme
  • Mutant of PET (polyethylene terephthalate) degrading enzyme and application of PET degrading enzyme

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Experimental program
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Effect test

Embodiment 1

[0042] The wild-type IsPETase was cloned into a commercially available pET28a+ vector by restriction enzyme ligation. Then, IsPETase was subjected to site-directed mutation using a commercially available site-directed mutagenesis reagent, and transformed into E. coli BL21 (DE3) to obtain an E. coli strain of PET degrading enzyme.

Embodiment 2

[0044]In order to obtain wild-type IsPETase and its mutant proteins, the wild-type IsPETase and mutant Escherichia coli strains obtained in Example 1 were cultured at 37° C. in the medium of LB plus ampicillin, and grown to an OD600 of about 0.6-0.8. Afterwards, the culture was cooled to 16 °C and induced with IPTG at a final concentration of 0.1 mM at 16 °C and 160 rpm for 20 h. Cells were harvested by centrifugation (25 min, 10°C, 4,000 rpm). Resuspend the cell pellet from 200 mL of cell culture in 10 mL of Ni-NTA lysis buffer (50 mM NaH 2 PO 4 , 300mM NaCl, 10mM imidazole, pH8). Cells were disrupted by sonication on ice and lysates were centrifuged (30 min, 10°C, 4,000 rpm). The supernatant was applied to a His-Accept nickel column (Beyotime). After washing unbound protein with 10 mL of lysis buffer, the bound protein was eluted with elution buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 200 mM imidazole), buffer exchanged to 50 mM Na 2 HPO 4 - HCl, pH 7.0 and 100 mM NaC...

Embodiment 3

[0045] Embodiment 3: relative activity comparison

[0046] All PET membranes were washed in three consecutive steps: first in 1% SDS solution, then with ethanol, and finally with deionized water, before hydrolysis of the PET membranes with wild-type IsPETase and the respective mutant protein PETases. Each step lasts 30 minutes.

[0047] Hydrolysis of PET film: Take four PET films (1.5cm×1.0cm, about 36mg) and place them in a reaction containing 50μg wild-type IsPETase and three mutant protein PETases and 1.5ml 50mM Bicine-NaOH (pH8.5) respectively solution, reacted at 30°C for 48 hours. The aqueous solution was diluted with 1.0M NaOH, followed by heat treatment (50°C, 10 minutes) to terminate the reaction. The final products of each group were analyzed by reverse phase HPLC, the results are shown in figure 1 . The changes in the quality of PET films in each group are as follows: figure 2 (PET film mass change / mg).

[0048] figure 1 The results showed that compared with...

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Abstract

The invention belongs to the field of gene engineering, and discloses a mutant of a PET (polyethylene terephthalate) degrading enzyme and the application of the PET degrading enzyme. The mutant of thePET degrading enzyme is obtained by point mutation at a substrate binding site position of IsPETase, and comprises an IsPETase mutant M1, wherein arginine at the site 89 of IsPETase is mutated into alanine, an IsPETase mutant M2 where arginine at the site 89 of IsPETase is mutated into alanine and leucine at the site 117 is mutated into phenylalanine, and an IsPETase mutant M3 where arginine at the site 89 of IsPETase is mutated into alanine and leucine at the site 208 is mutated into phenylalanine. An experiment shows that compared with wild IsPETase, the three PETase mutants have the advantages that the hydrolysis activity is obviously improved, and PET can be efficiently degraded at a medium temperature (30 DEG C).

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to PET degrading enzyme mutants, coding genes and applications thereof. Background technique [0002] Polyethylene terephthalate (PET) is a high molecular weight polymer composed of terephthalate (TPA) and ethylene glycol (EG) linked by ester bonds. It has excellent physical and mechanical properties in a wide temperature range. The long-term use temperature can reach 120 ° C. It has excellent electrical insulation. Even at high temperature and high frequency, its electrical properties are still good, but its corona resistance is poor. Creep resistance, fatigue resistance, friction resistance and dimensional stability are all very good. Durability and other favorable physical properties make PET one of the most widely used plastics. However, a large amount of PET entering and accumulating in the ecosystem poses a huge challenge to the environment, and the problem of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/55C12N1/21A62D3/02A62D101/28
CPCA62D3/02A62D2101/28C12N9/14
Inventor 元英进马渊丁明珠姚明东何博
Owner TIANJIN UNIV
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