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EP-4739773-A1 - THERMOSTABLE ENGINEERED ENZYME

EP4739773A1EP 4739773 A1EP4739773 A1EP 4739773A1EP-4739773-A1

Abstract

The invention relates to an engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 1. Preferably, the PETase enzyme comprises SEQ ID NO.1 with at least four mutations. The invention also refers to a composition and to a method for decomposing plastics.

Inventors

  • GAUTIERI, Alfonso
  • PARISINI, Emilio
  • ESTIRI, Hajar
  • CASTAGNA, Rossella
  • BHATTACHARYA, Shapla

Assignees

  • Politecnico di Milano
  • Latvian Institute Of Organic Synthesis

Dates

Publication Date
20260513
Application Date
20240708

Claims (11)

  1. 1. An engineered PETase enzyme comprising SEQ ID NO.1 or a composition comprising it, wherein SEQ ID NO. 1 comprises at least four mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K.
  2. 2. The enzyme according to claim 1 , wherein SEQ ID NO.1 comprises at least five mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K.
  3. 3. The enzyme according to claim 1 or 2, wherein SEQ ID NO.1 comprises at least six mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K.
  4. 4. The enzyme according to anyone of claims 1-3, wherein SEQ ID NO.1 comprises at least seven mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K.
  5. 5. The enzyme according to anyone of claims 1-4, wherein SEQ ID NO. 1 comprises the mutations: S48D, S57K, S145R, A209R, S241 D, N276D, N278K, Q293K.
  6. 6. The enzyme according to claim 5, consisting of SEQ ID NO.2.
  7. 7. The enzyme according to anyone of claims 1-4, wherein SEQ ID NO. 1 comprises the mutations: S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K, Q293K.
  8. 8. The enzyme according to claim 7 consisting of SEQ ID NO.3.
  9. 9. A method for decomposing plastics comprises at least one step of treating the plastics with the enzyme and/or with the composition according to anyone of claims 1-8.
  10. 10. The method according to claim 9, wherein the plastic is selected from: MHET, BHET, PET, preferably is PET.
  11. 11 . The method according to claim 9, or 10 wherein the enzyme is used in a concentration comprised between 20 and 60 nM, preferably comprised between 30 and 50 nM.

Description

Thermostable engineered enzyme FIELD OF THE INVENTION The present invention relates to a novel plastic degrading enzyme and a plastic decomposition method using the same. STATE OF THE ART PET (Polyethylene terephthalate) is a type of thermoplastic made by polymerization of TPA (terephthalic acid) and EG (ethylene glycol). PET has high transparency and excellent thermal insulation properties, so it is a polymer material commonly used in electric wire coverings, household goods, toys, and packaging materials, and is particularly widely used in the manufacture of bottles. However, since PET has a chemical structure that is not easily decomposed in nature, environmental pollution caused by the accumulation of PET in ecosystems including soil, rivers, and oceans is recognized as a serious social problem. In particular, the bioaccumulation problem caused by microplastics can adversely affect human health, so the need for recycling or environmentally friendly decomposition of PET is gradually increasing. However, when PET is recycled industrially, the recycling rate is not high due to poor quality and high cost, and when PET is decomposed through physical and chemical methods, endocrine-disrupting chemicals such as dioxins can be produced, which is a secondary problem. There is a problem that occurs. In 1977, Tokiwa and Suzuki proposed the idea of using enzymes to degrade polymeric materials. Indeed, enzymes work in mild conditions and can replace hazardous chemicals (a concept known as green chemistry). Since then, many PET-degrading enzymes from various microorganisms have been discovered and characterized, which has also led to the implementation of a series of strategies for enhancing their catalytic properties. Despite the availability of many suitable enzymes, their practical use for PET degradation is limited, mostly due to their low thermal stability. Indeed, the enzymatic degradation of PET by PETases does improve at high temperatures, where PET crystallinity is reduced. Hence, the design of a PETase with enhanced thermal stability is particularly relevant to the development of enzymatic PET-degrading strategies. SUMMARY OF THE INVENTION The first aspect of the invention relates to an engineered PETase enzyme. In one embodiment, the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 1 . Preferably, the PETase enzyme comprising SEQ ID NO.1 with at least four mutations, also referred as amino acid substitutions. In one embodiment, said SEQ ID NO.1 comprises at least four mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K. A second aspect of the present invention refers to a composition for decomposing plastics. The composition for decomposing plastic comprises the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 2 and/or the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 3. Another aspect of the present invention provides a method for decomposing plastics. The method for decomposing plastics includes treating the plastics with the composition for decomposing plastics of the present invention. The plastic may be MHET, BHET, PET, or the like, or derivatives thereof. BRIEF DESCRIPTION OF THE FIGURES The Figures show a comparison of PET-depolymerization over time for C09, C08 and ICCG (40 nM, 80 °C, pH 8). Means ± s.d (n=2) are shown. Figure 1 shows TPA production over time; Figure 2 shows TPA production at 4h and 144h. *P<0.05; **P<0.01 (one- side unpaired Welch’s t-test); and Figure 3 shows enzyme specific activity at the different time points. DETAILED DESCRIPTION OF THE INVENTION The first aspect of the invention relates to an engineered PETase enzyme. The term “PETase” or “polyethylene terephthalate hydrolases” refer to a class of hydrolases that have the property of catalysing the cleavage of PET (polyethylene terephthalate) into mono (2-hydroxyethyl) terephthalic acid. In one embodiment, there is provided an engineered thermostable PETase enzyme with a melting temperature (Tm) comprised between 80°C and 100°C, preferably, comprised between 82 and 98°C, that is higher than wild type enzyme. The engineered PETase enzyme may be active at higher temperatures (80-90 °C) enabling more efficient degradation of substrates. The thermostable PETase enzyme may also have longer shelf life at ambient temperatures. In one embodiment, the PETase enzyme has improved thermal stability. In one embodiment, the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 1 . Preferably, the PETase enzyme comprises SEQ ID NO.1 with at least four mutations, also referred as amino acid substitutions. In one embodiment, said SEQ ID NO.1