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WO2025117840A1 - Dégradation enzymatique de plla avec de l'afest - Google Patents

Dégradation enzymatique de plla avec de l'afest Download PDF

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Publication number
WO2025117840A1
WO2025117840A1 PCT/US2024/057883 US2024057883W WO2025117840A1 WO 2025117840 A1 WO2025117840 A1 WO 2025117840A1 US 2024057883 W US2024057883 W US 2024057883W WO 2025117840 A1 WO2025117840 A1 WO 2025117840A1
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pla
afest
activity
variant
variants
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Jared C. Lewis
Vikas Thakur
Qian Du
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Indiana University
Indiana University Bloomington
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Indiana University
Indiana University Bloomington
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J11/00—Recovery or working-up of waste materials
    • C08J11/04—Recovery or working-up of waste materials of polymers
    • C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
    • C08J11/105—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation by treatment with enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01001—Carboxylesterase (3.1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/50—Reuse, recycling or recovery technologies
    • Y02W30/62—Plastics recycling; Rubber recycling

Definitions

  • the present disclosure relates generally to improved A rchaeoglobus fulgidus esterases (Afest) engineered to degrade polyactic acid (PLA).
  • Afest A rchaeoglobus fulgidus esterases
  • Plastic waste is a major problem in society.
  • Polylactic acid (PLA) the second most produced biodegradable plastic in 2021 (18.9% of biodegradable plastic production), is a type of polyester.
  • PLA polylactic acid
  • biodegradable plastics like polylactic acid (PLA) can help avoid the plastic waste problem since biodegradable plastics could be composted.
  • the stability of PLA means that this process takes too long for commercial composting. This results in large amounts of PLA products being diverted to landfills.
  • Enzymatic degradation of PLA could be used to eliminate the plastic waste stream.
  • Complete PLA degradation generates its monomer building block lactic acid; a byproduct of anaerobic metabolism that is non-toxic to most flora and fauna.
  • an added benefit of degradation of PLA is generation of a low cost source of lactic acid, a valuable chiral chemical building block.
  • Enzymatic degradation of biodegradable plastics, including PLA have been reported. However, enzymes with improved activity are needed.
  • the present disclosure describes an engineered esterase with improved activity that overcome the PLA degradation challenges described above.
  • a first aspect of the invention includes variants of Archaeoglobus fulgidus esterase (Afest).
  • a second aspect of the invention includes host cells expressing the variants of Afest.
  • a third aspect of the invention includes methods of producing variants of Afest.
  • a fourth aspect of the invention includes compositions for decomposing polylactic acid (PLA) products.
  • a fifth aspect of the invention includes a method of decomposing polylactic acid (PLA) products using variants of Afest.
  • FIG. 1 is a graph of a lactic acid standard curve prepared using amplex red assay method
  • FIG. 2 is a graph of the screening for PLA depolymerizing enzymes and comparison of activities
  • FIG. 3 is a graph of the activity comparison of wild type AFest to its PROSS generated mutants
  • FIG. 4 is a graph of the activity profile of mutagenetic library generated for first round of evolution
  • FIG. 5 is a graph of the comparative representation of wild type Al to evolved versions during directed evolution
  • FIG. 6A is a graph of the biochemical characterization of purified 1KT at optimum pH conditions
  • FIG. 6B is a graph of the biochemical characterization of purified 1KT at optimum temperature conditions
  • FIG. 7 is a graph of the high-throughput degradation activity of 2820 variants from site saturation libraries using TMB assay for lactic acid detection
  • FIG. 8A is a heatmap showing activity of different variants in site saturation libraries
  • FIG. 8B is a heatmap showing activity of different variants in site saturation libraries
  • FIG. 8C is a heatmap showing activity of different variants in site saturation libraries.
  • FIG. 8D is a heatmap showing activity of different variants in site saturation libraries.
  • Archaeoglobus fitlgidus esterase degrades polylactic acid (PLA), a polyester, via a conventional serine hydrolase mechanism.
  • the present disclosure provides variants of Archaeoglobus fulgidus esterase (Afest) to provide for mutations in the 1KT enzyme that result in higher degradation of poly(lactic acid) (PLA).
  • Engineered variants for Afest disclosed herein include variants of SEQ ID NO: 4 having amino acid changes atP5E, F23P, C93G, I209G, S219W, E242R, V263A, F264Y, M267R, E274P, P292A (SEQ ID NO: 7), herein referenced as Pl .
  • Afest variants include amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or 100% homology/sequence identity to the full length sequence of SEQ ID NO: 7.
  • engineered Afest variants of the invention are enhanced variants of Pl, including but not limited to SEQ ID NO: 8, herein referenced at 1KT, as well as amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or 100% homology/sequence identity to the full length sequence to SEQ ID NO: 8.
  • Enhanced variants of SEQ ID NO: 8 include but are not limited to one of more of the following amino acid changes: 136, E51, G89, S97, and F218.
  • protein protein
  • peptide and “polypeptide” are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified.
  • nucleic acids or proteins of the invention refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection.
  • the gene encoding the nucleotide sequence corresponding to the amino acid sequence of the variant Afest may be incorporated into a host cell.
  • more than one copy of the gene may be expressed by a single recombinant vector introduced into a host cell.
  • more than one copy of the gene may be expressed by multiple recombinant vectors introduced into a host cell.
  • the recombinant vector comprises multiple expression sites, each site able to drive the expression of a different nucleotide sequence.
  • multiple copies of variant Afest gene can be expressed in a single cell.
  • Host cells utilized for the production of the disclosed variant Afest enzymes include eukaryotic cells, such as E. coli, though other host cells known in the art may also be utilized.
  • variant Afest enzymes of the present invention may be incorporated into compositions comprising PLA-containing materials for decomposing the PLA.
  • These compositions contain variant Afest enzymes disclosed herein, such as SEQ ID NO: 8 as well as amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or 100% homology/sequence identity to the full length sequence to SEQ ID NO: 8.
  • the compositions may also include buffers to maintain the pH of the composition between 6.0-9.0 during the decomposition process.
  • the decomposition process is maintained at a temperature between 50-90°C, between 60-80°C, or 60-70°C for optimal Afest enzyme activity.
  • the composition may include a variety of additives, such as heteropolymers.
  • the PLA-containing material for decomposition contains at least 70%, at least 80%, at least 90% or 100% PLA.
  • the lactic acid produced by the PLA decomposition process is collected.
  • a mastermix was formulated to estimate the lactate concentration in the degraded product of PLLA.
  • the mastermix comprises of 114.4pL water, 20pL buffer, 5 pL of 5mM Amplex red, 0.4pL of 200 U/mL lactate oxidase (LOD), and 0.2pL of 300 U/mL horseradish peroxidase (HRP).
  • LOD lactate oxidase
  • HRP horseradish peroxidase
  • a calibration curve was established by preparing lactic acid solutions with varying concentrations, ranging from 100 to 700 pM. 140ul of the master mix was added to lOpL of lactic acid and incubated at room temperature for 30 min. Post-incubation, absorbance measurements were taken at a wavelength of 571 nm. The measurements were assessed against the lactic acid standard curve to estimate the amount of the degradation products.
  • Proteinase K (P2308) from Tritirachium album (proK) enzyme was purchased from Sigma-Aldrich. Lactate Oxidase (LOD, LCO-301) was purchased from Fisher scientific. Peroxidase from horseradish (HRP, P6782), 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, 10102946001) were purchased from Sigma-Aldrich. Amplex Red (10-Acety 1 -3, 7-dihydroxyphen oxazine) was purchased from Cayman chemical. Poly (L-lactide) 1-5 kD and 45-55 kD are purchased from Akina, Inc.
  • the reaction was incubated at room temperature for 30 min. 10 pL of degraded product was transferred to new well, assay master mix was added to it, and incubated at room temperature for 30 min. The absorbance was taken at 571 nm post incubation. The specific activity calculated from the absorbance is defined as amount of lactic acid produced in micromoles per minute of reaction per milligram of protein.
  • the PROSS server was employed to identify potential thermostability-conferring residues in the AFest (Al) protein sequence (Goldenzweig et al. 2016). The predicted mutations were then incorporated into the sequence, and the resultant variants were synthesized through TWIST Bioscience. These sequences were subsequently inserted into the pET 28a (+) vector and expressed in E. coli BL21 DE3 cells. Following expression, the target proteins were purified using Ni-NTA affinity chromatography. The PLA degradation activities of the synthesized variants were assessed and compared to the wild-type AFest.
  • the method was automated to pick colonies using colony picker and inoculate in 96 well deepwell plates. These plates were grown overnight and inoculated into fresh 1ml LB media in the 2ml deep well plates. The plates were then subjected to expression using 500 pM IPTG as the OD reaches 0.6.
  • the expressed cells were spun down at 3500 rpm for 10 min and supernatant was discarded. 125 pl lysozyme was added to each well and vortexed vigorously. 10 pl DNase was added and incubated at 37 °C for 15 min. The plate was spun at 3500 rpm for 10 min and the supernatant was used for enzyme reactions. Again, the robot was used to setup the degradation reactions followed by Amplex red assay.
  • the triple mutant 1VKT underwent a deconvolution process, where one mutation was systematically removed at a time utilizing splicing overhang expression (SOE) PCR. The resulting double mutants were subsequently expressed, purified, and evaluated for activity. Additionally, mutations from hit 2 (1HDG) were incrementally introduced into 1KT, generating triple mutants. To ensure the accuracy of these genetic modifications, all newly created variants underwent sanger sequencing to validate the successful reversion and insertion of mutations.
  • SOE splicing overhang expression
  • the One Factor at a Time (OF AT) approach was employed to investigate the biochemical characteristics of purified 1KT and determine its maximum activity.
  • the pH range for the reaction was explored using diverse buffer systems: sodium citrate (pH 3.0-5.0), potassium phosphate (pH 6.0-7.0), Tris-HCl (pH 8.0-10.0), and sodium carbonate-bicarbonate (pH 11.0— 12.0).
  • the reaction parameters were set as follows: temperature at 25°C, time for 30 minutes, and 20ul of PLA substrate at 0.1% (w/v).
  • the buffer system exhibiting the highest specific activity was identified as the optimal pH for the reaction.
  • the optimal reaction temperature was determined under the previously optimized pH conditions, with a 30-minute incubation time and 0.1% (w/v) PLA.
  • the reactions were conducted over a temperature range of 4-100 °C.
  • PLA polylactic acid
  • the developed enzyme-coupled assay successfully enabled the quantification of lactic acid concentration in the degraded product of PLLA.
  • the absorbance values obtained from the samples were plotted against the concentrations of the lactic acid standards, yielding a calibration curve with a high degree of linearity (Fig. 1).
  • Fig. 1 concentration of lactic acid in the degraded product of PLLA was determined.
  • the assay provided precise and reliable quantification, showcasing its effectiveness in analyzing lactic acid content in degraded PLLA samples.
  • PLA depolymerizing enzymes were primarily identified in a limited number of bacterial sources, such as Amycolatopsis sp. strain K104-1, which exhibited a specific activity of 25.7 lU/mg (Nakamura et al., 2001), Amycolatopsis orientalis with a reported activity of 0.35 lU/mg (Li et al., 2008), and Pseudomonas tamsuii TKU015 showing an activity of 0.008 lU/mg (Liang et al., 2016).
  • Al esterase has demonstrated significantly higher activity compared to these previously reported PLA depolymerases, it is crucial to note that direct comparisons with these studies may be challenging due to variations in reaction conditions.
  • the Pl enzyme demonstrated a 2-fold increase in activity when compared to Al, underscoring the significant enhancement achieved through strategic mutations.
  • the Pl gene was amplified under the presence of varied concentrations of MnCh. The 0.4 mM concentration was selected based on the 3-4 mutations observed in the gene. A total of 672 colonies were picked after transformation including 30 parent colonies. The expression of all these colonies and further activity analysis showed enhancement of activity in few variants compared to parent (Fig. 4). The top five hits from first round of evolution were then expressed and purified followed by PLA degradation activity.
  • the purified 1KT was subjected to biochemical characterization such as buffer pH and temperature optimization.
  • the PLA-degrading enzyme 1KT demonstrates promising characteristics for industrial applications in the bioprocessing of polylactic acid (PLA).
  • the enzyme displays a broad pH functionality, maintaining activity from pH 4.0 to 11.0, with optimal activity at pH 7.0.
  • the enzyme retains 40% and 25% activity, respectively (Fig. 6A).
  • Fig. 6A the enzyme retains 40% and 25% activity, respectively
  • Fig. 6B the enzyme retains over 60% activity at 40°C and over 90% activity until 90°C, indicating versatility and thermostability.
  • the activity drastically drops to 34% at 100°C (Fig. 6B).
  • the 1KT enzyme presented an even more expansive pH and temperature range than the previously discussed reports. Moreover, it displayed notably higher relative activity, positioning it as an exceptionally versatile candidate for a spectrum of industrial applications involving PLA degradation.
  • the adaptability of this enzyme to diverse pH and temperature conditions underscores its potential utility in various industrial processes, further emphasizing its significance in the field of PLA degradation.
  • AFest (Al) is identified as a PLA-degrading enzyme, and the results above show that variant 1KT is highly active with PLA-degradation. This variant exhibited remarkable adaptability, undergoing advantageous mutations that resulted in a considerable increase in enzymatic activity.
  • the biochemical characterization of 1KT revealed its versatility, displaying a broad pH range (4.0 to 11.0) with optimal activity at pH 7.0, and a wide temperature range (4°C to 100°C) with optimal activity at 70°C. Furthermore, the confirmed efficacy of the enzyme in degrading commercial PLA cups underscores its potential for large-scale industrial applications.
  • SSVL site saturation variant libraries
  • Figure 8 shows the degradation activity of 2820 variants from site saturation libraries using TMB assay for lactic acid detection.
  • the middle horizontal line represents average parent activity (1KT), with the top and bottom lines indicating standard deviations.
  • a calibration curve was established by preparing lactic acid solutions with varying concentrations, ranging from 100 to 700 pM. 140ul of the master mix was added to lOpL of lactic acid and incubated at room temperature for 30 min. Post-incubation, absorbance measurements were taken at a wavelength of 571 nm. The measurements were assessed against the lactic acid standard curve to estimate the amount of the degradation products. [00100] Reaction conditions
  • Low molecular weight PLLA ranging from 1-5 kDa
  • acetone a stock solution with a concentration of 10 mg/ml.
  • 10 pL of the prepared PLLA stock solution was added to each well of a 96-well plate. The plate was then left at room temperature to facilitate the evaporation of acetone, ensuring the substrate's proper deposition.
  • 145 pL of lOmM Na-phosphate buffer (pH 8.0) was added to wells containing the PLLA substrate.
  • 5 pL of purified protein enzymes were added to the reaction mixture. The reaction was incubated at room temperature for 1 hour.
  • Site saturation variant libraries on 1KT [00102] Screening of site saturation variant libraries (SSVL) was conducted to identify enzyme variants with enhanced activity for the degradation of poly(lactic acid) (1-5 kDa).
  • the variant libraries were cloned into BL21 cells and expressed across three batches.
  • the expressed variants were lysed using lysozyme and DNase, after which PLA degradation reactions were carried out with the resulting lysates. These reactions were set up using an automated liquid handling system and screened in a high-throughput format. They were incubated overnight at room temperature, and PLA degradation, indicated by lactic acid production, was measured using the TMB assay.
  • AA numbers are amino acid positions based on 1KT protein sequence.
  • the left side column represents the location of variants on an assay plate.

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Abstract

La présente invention concerne des variants d'estérase de Archaeoglobus fulgidus (Afest), des compositions pour la décomposition de produits à base d'acide polylactique (PLA) dans lesquelles la composition comprend des variants d'Afest, et des procédés pour la décomposition de produits PLA par l'utilisation de ces compositions.
PCT/US2024/057883 2023-11-30 2024-11-27 Dégradation enzymatique de plla avec de l'afest Pending WO2025117840A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020146799A1 (en) * 1996-02-16 2002-10-10 Robertson Dan E. Enzymes having esterase activity and methods of use thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020146799A1 (en) * 1996-02-16 2002-10-10 Robertson Dan E. Enzymes having esterase activity and methods of use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MA ET AL.: "Efficient molecular evolution to generate enantioselective enzymes using a dual- channel microfluidic droplet screening platform", NATURE COMMUNICATIONS, vol. 9, 12 March 2018 (2018-03-12), pages 1030, XP055915256, DOI: 10.1038/s41467-018-03492-6 *

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