Enzymatic PLLA degradation with afest

Abstract

Disclosed herein are variants of Archaeoglobus fulgidus esterase (Afest), compositions for decomposing polylactic acid (PLA) products where the composition includes variants of Afest, and methods for decomposing PLA products using these compositions.

Description

ENZYMATIC PLLA DEGRADATION WITH AFESTSTATEMENT OF GOVERNMENTAL RIGHTS

[0001] This invention was made with government support under 2132025 awarded by the National Science Foundation. The Government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 604,410 entitled “ENZYMATIC PLLA DEGRADATION WITH AFEST”, filed on November 30, 2023, the entire disclosure of which is incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0003] A paper copy of the Sequence Listing and a computer readable form of the sequence containing the file named “IU202402902WOST26.xml”, which is 9,343 bytes in size (as measured in Microsoft WINDOWS® Explorer), are provided herein and are herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs:l-9.FIELD OF THE INVENTION

[0004] The present disclosure relates generally to improved A rchaeoglobus fulgidus esterases (Afest) engineered to degrade polyactic acid (PLA).BACKGROUND

[0005] 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. In theory, biodegradable plastics like polylactic acid (PLA) can help avoid the plastic wasteproblem since biodegradable plastics could be composted. Unfortunately, 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.

[0006] 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. Thus, 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.

[0007] The present disclosure describes an engineered esterase with improved activity that overcome the PLA degradation challenges described above.SUMMARY OF THE INVENTION

[0008] A first aspect of the invention includes variants of Archaeoglobus fulgidus esterase (Afest).

[0009] A second aspect of the invention includes host cells expressing the variants of Afest.

[0010] A third aspect of the invention includes methods of producing variants of Afest.

[0011] A fourth aspect of the invention includes compositions for decomposing polylactic acid (PLA) products.

[0012] A fifth aspect of the invention includes a method of decomposing polylactic acid (PLA) products using variants of Afest.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The above-mentioned and other features and advantages of this disclosure, and the manner of obtaining them, will become more apparent, and will be better understood by reference to the following description of the exemplary embodiments taken in conjunction with the accompanying drawings, wherein:

[0014] FIG. 1 is a graph of a lactic acid standard curve prepared using amplex red assay method;

[0015] FIG. 2 is a graph of the screening for PLA depolymerizing enzymes and comparison of activities;

[0016] FIG. 3 is a graph of the activity comparison of wild type AFest to its PROSS generated mutants;

[0017] FIG. 4 is a graph of the activity profile of mutagenetic library generated for first round of evolution;

[0018] FIG. 5 is a graph of the comparative representation of wild type Al to evolved versions during directed evolution;

[0019] FIG. 6A is a graph of the biochemical characterization of purified 1KT at optimum pH conditions;

[0020] FIG. 6B is a graph of the biochemical characterization of purified 1KT at optimum temperature conditions;

[0021] 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;

[0022] FIG. 8A is a heatmap showing activity of different variants in site saturation libraries;

[0023] FIG. 8B is a heatmap showing activity of different variants in site saturation libraries;

[0024] FIG. 8C is a heatmap showing activity of different variants in site saturation libraries; and

[0025] FIG. 8D is a heatmap showing activity of different variants in site saturation libraries.

[0026] The exemplification set out herein illustrates an embodiment of the invention, and such an exemplification is not to be construed as limiting the scope of the invention in any manner.DETAILED DESCRIPTION

[0027] Archaeoglobus fitlgidus esterase (Afest) 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).

[0028] Described herein are engineered variants of Archaeoglobus fulgidus esterase (Afest) with greater than lOx improved activity relative to wild type Afest. This increased activity is higher than any Afest identified to date. 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.

[0029] Preferably, 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 acidsequences 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.

[0030] 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.

[0031] The terms “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.

[0032] The term “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.

[0033] The terms “identical” or percent “identity,” in the context of two or more 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. See e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST / . Optimal alignment of such sequences can be carried out by any of the publicly available algorithms or programs for determining sequence identity and alignment, e.g., BLAST.

[0034] In some embodiments, the gene encoding the nucleotide sequence corresponding to the amino acid sequence of the variant Afest may be incorporated into a host cell. In some embodiments more than one copy of the gene may be expressed by a single recombinant vector introduced into a host cell. In some embodiments, more than one copy of the gene may be expressed by multiple recombinant vectors introduced into a host cell. In some embodiments, the recombinant vector comprises multiple expression sites, each site able to drive the expression of a different nucleotide sequence. By this method, 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.

[0035] In some embodiments, 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. Preferably, 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. Additionally, the composition may include a variety of additives, such as heteropolymers.

[0036] In some embodiments, the PLA-containing material for decomposition contains at least 70%, at least 80%, at least 90% or 100% PLA.

[0037] Preferably, the lactic acid produced by the PLA decomposition process is collected.Lactic acid collected from the decomposition process by be reused and / or repurposed in other chemical processes, including the production of new PLA materials.SEQUENCE LISTING

[0038] The following sequences correspond to the Afest variants described in the text and figures.

[0039] SEQUENCE ID NO: 1 - MGS0156

[0040] MAGRCCSRRRSGVTRLGREGCPRRQGASPFVSPGGEVFGATAGRLPRVRWTMRFPFATGGLPVWALGGLLLVAGLFPGCARRPVAPLAHAMSPSVLVPAGLAGIQDG RGRFREIMTAIMADHGAFLPGDRSSDGDGILWRLAGEPGPTGRPVPLGVSTAGIRLVLVP GLLAECVSESSLLFDDARPDVERYGYATTLVRTGGRWGSARNAAIIHEVVAKLPENDTI VFVTHSKGAVDVLEALVSYPDLAARTAAVVSVAGAIDGSPLAETFSDGLLRFAESMPLS SCPPGEGTEALDSLKRAYRLRFLAEHRLPARVRYYSLAAFASREETSAILRPFYDILAKT DALNDGLVIAADAIIPGGTLLGYPNADHLAVAMPFSKKPSLLTSVISKNSYPRPALLEAIA RYVEEDLGPEKGREN

[0041] SEQUENCE ID NO:2 - GEN0105

[0042] MSETS S AS ALP AY ARI VVDK APF IRAILYLILRYVII<RSMI<PDADILI<LRAMQLRADQKYAHPAADAVMTPVDCDGVKANWITLPGARPERVIFYLHGGAWMFNFPRT YAAMLGRWARLLNARVLMVDYRLAPEHRYPAGANDCETAYRWLLAQGIDSKQIVIGGDSAGGNLTLTTLLRLKSANQPLPACAVALSPFVDFTLSSPSMITNEKIDPMFTLEAMLGLRPHYLDPQDFLNVDASPIFGDFSGLPPIFFQSSNTEMLRDESVRAAARAHQHGVTVELEL WQHLPHVFQALQKLPQADAALQSIVRFINSHTGWQA

[0043] SEQUENCE ID NO:3 - EstlDM

[0044] MSVTTPRREASLLSRAVAVAAAAAATVALAAPAQAANPYERGPNPTESMLEARSGPFSVSEERASRLGADGFGGGTIYYPRENNTYGAIAISPGYTGTQSSIAWLGERIA SHGF VVIAIDTNTTLDQPD SRARQLNAALD YMLTD AS S S VRNRID ASRL AVMGHSMGG GGTLRLASQRPDLKAAIPLTPWHLNKSWRDITVPTLIIGADLDTIAPVSSHSEPFYNSIPSS TDKAYLELNNATHFAPNITNKTIGMYSVAWLKRFVDEDTRYTQFLCPGPRTGLLSDVDE YRSTCPF

[0045] SEQUENCE ID NO:4 - AFest

[0046] MLDMPIDPVYYQLAEYFDSLPKFDQFSSAREYREAINRIYEERNRQLSQHERVERVEDRTIKGRNGDIRVRVYQQKPDSPVLVYYHGGGFVICSIESHDALCRRIARLSNS TVVSVDYRLAPEHKFPAAVYDCYDATKWVAENAEELRIDPSKIFVGGDSAGGNLAAAV SIMARDSGEDFIKHQILIYPVVNFVAPTPSLLEFGEGLWILDQKIMSWFSEQYFSREEDKF NPLASVIFADLENLPPALIITAEYDPLRDEGEVFGQMLRRAGVEASIVRYRGVLHGFINY YPVLKAARDAINQIAALLVFD

[0047] SEQUENCE ID NO : 5 - 2x mutant

[0048] MLDMPIDPVYYQLAEYFDSLPKFDQFSSAREYREAINRIAEERNRQLSQHERVERVEDRTIKGRNGDIRVRVYQQKPD SP VL VYYHGGGF VIGSIE SHD ALCRRIARL SNS TVVSVDYRLAPEHKFPAAVYDCYDATKWVAENAEELRIDPSKIFVGGDSAGGNLAAAV SIMARDSGEDFIKHQILIYPVVNFVAPTPSLLEFGEGLWILDQKIMSWFSEQYFSREEDKF NPLASVIFADLENLPPALIITAEYDPLRDEGEVFGQMLRRAGVEASIVRYRGVLHGFINYYPVLKAARDAINQIAALLVFD

[0049] SEQUENCE ID NO : 6 - P2

[0050] MLDME ID P V Y YQ L AE YFD SLPKPDQF S S ARE YREAINRIYEERNRQL SQHERVERVEDRTIKGRNGDIRVRVYQQKPD SPVL VYYHGGGF VIG SIETHD ALCRRIARL SNSTVVSVDYRLAPEHKFPAAVEDCYDATKWVAENAEELRIDPSRIFVGGDSAGGNLAAAVCIMARDSGEDFIKHQILIYPVVNFVFPTPSLLEFGEGLWGLDQKIMSWFWEQYFSREEDKFNPLASVIFADLRNLPPALIITAEYDPLRDEGEAYGQRLRRAGVPAEIVRYRGVLHGFINYYAVLKAARDAINQIAALLVFD

[0051] SEQUENCE ID NO:7 - Pl

[0052] MLDME ID P VYYQL AEYFD SLPKPDQF S S ARE YRE A I NRI YEERNRQL SQHERVERVEDRTIKGRNGDIRVRVYQQKPDSPVLVYYHGGGFVIGSIESHDALCRRIARLSNSTVVSVDYRLAPEHKFPAAVYDCYDATKWVAENAEELRIDPSKIFVGGDSAGGNLAAAVSIMARDSGEDFIKHQILIYPVVNFVAPTPSLLEFGEGLWGLDQKIMSWFWEQYFSREEDKFNPLASVIFADLRNLPPALIITAEYDPLRDEGEAYGQRLRRAGVPASIVRYRGVLHGFINYYAVLKAARDAINQIAALLVFD

[0053] SEQUENCE ID NO : 8 - 1 KT

[0054] MLDMEIDPVYYQLAEYFDSLPKPDQFSSAREYREAINRIYEERNRQLSQHERVERVEDRTIKGKNGDIRVRVYQQKPDSPVLVYYHGGGFVIGSIESHDALCRRIARLSNSTVVSVDYRLAPEHKFPAAVYDCYDATKWVAENAEELRIDPSKIFVGGDSAGGNLAAAVSIMARDSGEDFIKHQILIYPVVNFVAPTPSLLEFGEGLWGLDQKTMSWFWEQYFSREEDKFNPLASVIFADLRNLPPALIITAEYDPLRDEGEAYGQRLRRAGVPASIVRYRGVLHGFINYYAVLKAARDAINQIAALLVFD

[0055] SEQUENCE ID NO: 9 - 1KT DNA

[0056] ATGCTTGATATGGAAATCGACCCTGTTTACTACCAGCTTGCTGAGTATTTCGACAGTCTGCCGAAGCCGGACCAGTTTTCCTCGGCCAGAGAGTACAGGGAGGCGATAAATCGAATATACGAGGAGAGAAACCGGCAGCTGAGCCAGCATGAGAGGGTTGAAAGAGTTGAGGACAGGACGATTAAGGGGAAGAACGGAGACATCAGAGTCAGAGTTTACCAGCAGAAGCCCGATTCCCCGGTTCTGGTTTACTATCACGGTGGTGGATTTGTGATTGGTAGCATCGAGTCGCACGACGCCTTATGCAGGAGAATTGCGAGACTTTCAA ACTCTACCGTAGTCTCCGTGGATTACAGGCTCGCTCCTGAGCACAAGTTTCCCGCCG CAGTTTATGATTGCTACGATGCGACCAAGTGGGTTGCTGAGAACGCCGAGGAGCTG AGGATTGACCCGTCAAAAATCTTCGTTGGGGGGGACAGTGCGGGAGGGAATCTTGC CGCGGCGGTTTCAATAATGGCGAGAGACAGCGGAGAAGATTTCATAAAGCATCAAA TTCTAATTTACCCCGTTGTGAACTTTGTAGCCCCCACACCATCGCTTCTGGAGTTTGG AGAGGGGCTGTGGGGTCTCGACCAGAAGACAATGAGTTGGTTCTGGGAGCAGTACTTCTCCAGAGAGGAAGATAAGTTCAACCCCCTCGCCTCCGTAATCTTTGCGGACCTTC GTAACCTACCTCCTGCGCTGATCATAACCGCCGAATACGACCCGCTGAGAGATGAA GGAGAAGCATATGGGCAGCGTCTGAGAAGAGCCGGTGTTCCGGCGAGCATCGTCAG ATATAGAGGCGTGCTTCACGGATTCATCAATTACTATGCAGTGCTGAAGGCTGCGAG GGATGCGATAAACCAGATTGCCGCTCTTCTTGTGTTCGACExamplesExample 1

[0057] Development of Enzyme-Coupled Assay for Lactic Acid Quantification

[0058] An enzyme-coupled assay for lactic acid quantifications was developed. 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). 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 takenat a wavelength of 571 nm. The measurements were assessed against the lactic acid standard curve to estimate the amount of the degradation products.

[0059] 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.

[0060] Screening for PLA Degrading Enzymes

[0061] A comprehensive literature and sequence-based survey was conducted for selection of ideal PLA degrading enzymes. Five different enzyme sources were chosen from Table 1. The proteinase K (PK) was purchased from Sigma Aldrich, MGS0156 and GEN0105 plasmids were provided by Dr. Alexander F. Yakunin (Hajighasemi et al. 2018), while the remaining plasmids were procured by synthesizing them as E. coli codon-optimized gene constructs on the pET-28a (+) vector through Twist Bioscience. These proteins were expressed in E. coli BL21 DE3 cells with 500 pM IPTG and purified using HisPure Ni-NTA resin in a gravity flow column. The enzyme activities were carried out with the purified proteins at 25 °C.Table 1 : Screening Various Esterases for PLA Degradation Screening

[0062] Low molecular weight PLLA, ranging from 1-5 kDa, was dissolved in acetone to create a stock solution with a concentration of 0.1 mg / ml. 20 pL of the prepared PLLA stock solution was carefully 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. 140 pL of lOmM Na-phosphate buffer (pH 8.0) was added to wells containing the PLLA substrate. Subsequently, 10 pL of purified protein enzymes were added to the reaction mixture. 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.

[0063] Protein Repair One Stop Shop (PROSS Server)

[0064] 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.

[0065] Automating Directed Evolution and High Throughput Enzyme Screening

[0066] In the pursuit of the automating the first round of evolution for Pl enzyme, the liquid handling robot system was used for error-prone mutagenesis library preparation and enzyme assay. This process involved the meticulous design of mutagenic primers targeting the Pl gene,followed by the preparation of error-prone PCR reactions utilizing a DNA polymerase. Error-prone PCR was executed using MnCh concentrations ranging from O. lmM to 0.5mM to introduce mutations into the Pl gene. The inserts with mutations were assembled into the pET 28 a(+) vector using Gibson assembly mixture. The library of Pl mutant plasmids was generated by transforming these into an E. colt host strain and plating them on LB agar. After incubation, a diverse mutant library was established. 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.

[0067] 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.

[0068] Expression, and Purification of Top Hits from the First Round of Evolution

[0069] The top six candidates from the initial round of evolution were expressed and purified. Subsequently, the purified proteins underwent assessment for their PLLA degrading activity, with a comparison made against the parent strain P 1. The candidate exhibiting the highest activity among these hits was singled out for further in-depth studies.

[0070] Deconvolutions of Pl Variant and Combining New Mutations

[0071] 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.

[0072] Biochemical Characterization

[0073] 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. Subsequently, 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.

[0074] Commercial PLA degradation

[0075] The analysis of the polylactic acid (PLA) degradation ability of 1KT involved treating a commercial PLA cup with the purified enzyme. The PLA cup was cut into small squares of 5mg each, and the degradation reaction was initiated with 200 pL of reaction mixture at 25°C for 30 minutes. Subsequently, an Amplex red assay was conducted to assess the extent of PLA degradation.

[0076] Results and discussion

[0077] A. Assay development for lactate quantification

[0078] The developed enzyme-coupled assay successfully enabled the quantification of lactic acid concentration in the degraded product of PLLA. The absorbance values obtained fromthe samples were plotted against the concentrations of the lactic acid standards, yielding a calibration curve with a high degree of linearity (Fig. 1). Using this calibration curve, the 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.

[0079] B. Screening for PLA degrading enzymes

[0080] The PLA degradation by purified 05 proteins (Table 1) revealed that AFest (Al) has the highest activity of 2200 ± 124 lU / mg (Fig. 2). Al is a thermostable, 35.4kDa protein obtained from Archaeglobus fulgisidus and concluded as a potential candidate for PLA degradation. While proteinase K (PK) has been previously explored for PLA degradation, the findings of the present disclosure indicate a lack of observable activity. One plausible explanation for this result could be attributed to the proteolytic impact of proteinase K on the assay enzymes, specifically LOD and HRP. The proteolytic nature of proteinase K may interfere with the functionality of these enzymes, potentially leading to a lack of measurable activity in the context of PLA degradation. Previously, 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). Although 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. Nevertheless, the study introduces a novel approach that not only surpasses the efficacy of previously employed methods but also boasts enhanced speed, reliability, and sensitivity. Thisadvancement in methodology opens new avenues for exploring PLA depolymerization and sets a higher standard for future investigations in this domain.

[0081] Additionally, we employed the PROSS server to synthesize three variants of AFest, namely the 2x mutant with two key mutations (Y40A, C93G), Pl with an impressive 11 mutations (P5E, F23P, C93G, I209G, S219W, E242R, V263A, F264Y, M267R, E274P, P292A), and P2, encompassing all the Pl mutations plus an additional six (S97T, Y131E, K153R, S170C, A195F, S276E). The overexpressed and purified proteins derived from these variants exhibited distinct activities, with Pl showcasing the highest enzymatic activity compared to the wild type (Fig. 3).

[0082] Remarkably, the Pl enzyme demonstrated a 2-fold increase in activity when compared to Al, underscoring the significant enhancement achieved through strategic mutations. These findings not only provide valuable insights into the structure-function relationship of AFest but also present promising opportunities for the optimization of enzymatic activities in various applications.

[0083] C. First round of directed evolution and high throughput enzyme screening

[0084] 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.

[0085] The highest specific activity of 12528± 162 International Unit (IU) / mg was observed in a triple mutant, 1VKT (E51V, R64K, and I2 I4T). This evolved variant was furthersubjected to deconvolutions to assess the individual impact of these 3 mutations. The deconvolution using SOE PCR resulted in generation of a double mutants 1KT, 1VT, and 1VK. These mutants were again expressed, purified, and assessed for activities. The variant 1KT showed specific activity of 35613 ± 245 lU / mg, a 2.5-fold enhancement in activity compared to the parent Pl . 1KT was attempted to be deconvoluted further to IK and IT, but the activity declined. Further mutations were introduced from 2ndtop hit 1HDG (H, D, G) to 1KT one by one creating triple mutants 1HKT, 1DKT, and 1KGT. These variants showed less specific activity compared to 1KT (Fig. 5). The double mutant 1KT emerged as a particularly promising variant, showcasing significantly improved specific activity in the degradation of polylactic acid. Further attempts at deconvolution and introducing additional mutations demonstrated the complexity of optimizing enzyme performance, with 1KT standing out as a highly active variant in the context of PLA degradation. In conclusion, the evidence from the study strongly suggests that the protein encoded by the gene is highly evolvable. Its ability to undergo beneficial mutations, resulting in a substantial increase in enzymatic activity, underscores its adaptability and potential for optimization through evolutionary processes.

[0086] D. Biochemical characterization of 1KT

[0087] The purified 1KT was subjected to biochemical characterization such as buffer pH and temperature optimization.

[0088] E. pH and temperature optimization

[0089] 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. At pH 4.0 and 11.0, the enzyme retains 40% and 25% activity, respectively (Fig. 6A). Additionally, itexhibits a wide temperature range from 4°C to 100°C, with optimal activity at 70°C. Notably, the enzyme retains over 60% activity at 40°C and over 90% activity until 90°C, indicating versatility and thermostability. However, the activity drastically drops to 34% at 100°C (Fig. 6B).

[0090] In contrast to prior studies on PLA depolymerases, the enzyme derived from Amycolatopsis orientalis exhibited optimal activity under alkaline conditions and within a temperature range of 30-70 °C (Li et al., 2008). Meanwhile, the PLA depolymerase sourced from Pseudomonas tarn suit TKU015 demonstrated a broad pH range spanning from 4 to 11, with an optimum activity observed at pH 10. However, its efficacy markedly diminished under acidic conditions. Notably, this enzyme showcased versatility with a wide temperature range from 25 to 90 °C, reaching its peak activity at 60 °C (Liang et al., 2016).

[0091] In comparison, 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.

[0092] Conclusions

[0093] 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°Cto 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.Example 2

[0094] TWIST Screening 1KT

[0095] An extensive 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). Using 1KT as the parent enzyme, 47 specific sites were selected based on their potential to significantly impact enzyme performance, considering factors such as structural importance and proximity to the active site. For each site, site saturation variant libraries were created, systematically mutating each amino acid position to all possible alternatives.

[0096] High-throughput screening with a liquid handler system (robot) were utilized to efficiently manage the large number of samples. From these libraries, 60 colonies per site were picked and screened, resulting in a total of 2820 variants. Each variant was evaluated for its ability to degrade PLA. By targeting these key sites and screening a large number of colonies, mutations that could enhance the enzyme's efficiency in breaking down PLA may be identified.

[0097] 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.Example 3

[0098] Development of Enzyme-Coupled Assay for Lactic Acid Quantification

[0099] Carboxylesterases, including AfEst, can cleave Amplex Red, leading to false positives in assays using this reagent. To address this issue, we developed a second assay using TMB (3,3',5,5'-Tetramethylbenzidine). A mastermix was formulated to estimate the lactic acid concentration in the degraded product of PLLA. The mastermix comprises of 24pL water, 50pL buffer, 20ul pL of 20mM TMB, 0.4pL of 300 U / mL lactate oxidase (LOD), and 0.2pL of 300 U / mL horseradish peroxidase (HRP). 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.

[0100] Reaction conditions

[0101] Low molecular weight PLLA, ranging from 1-5 kDa, was dissolved in acetone to create 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. Subsequently, 5 pL of purified protein enzymes were added to the reaction mixture. The reaction was incubated at room temperature for 1 hour. 50 pL of degraded product was transferred to new well, lOOul assay master mix was added to it, and incubated at room temperature for 2 min. The absorbance was taken at 370 nm post incubation. The specific activity was calculated from the absorbance is defined as amount of lactic acid produced in micromoles per minute of reaction per milligram of protein.

[0102] Site saturation variant libraries on 1KT

[0103] 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.

[0104] Using 1KT as the parent enzyme, 47 specific sites were selected based on their potential to significantly impact enzyme performance, considering factors such as structural importance and proximity to the active site. For each site, site saturation variant libraries were created, systematically mutating each amino acid position to all possible alternatives. The liquid handler system (robot) was used for high-throughput screening to efficiently manage the large number of samples. From these libraries, 60 colonies per site (57 variants and 3 parents) were picked and screened, resulting in a total of 2820 variants. These variants were expressed in 3 batches and evaluated for their ability to degrade PLA with lysates (Figs. 9A, 9B, and 9C). The heatmaps show activity of different variants (Y-axis) in site saturation libraries (X-axis). Activity was normalized to parent. Dark blue indicates lower activity than parent; red indicates higher activity than parent. The results show that mutations at many of the active site residues targeted can improve PLA degradation activity. The AA numbers are amino acid positions based on 1KT protein sequence. The left side column represents the location of variants on an assay plate.

Claims

CLAIMSI claim1. A variant of Archaeoglobus fulgidus esterase (Afest) comprising 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.

2. The variant Afest of claim 1 being identical to the entire length of SEQ ID NO: 8.

3. The variant Afest of claim 1 further comprising one or more amino acid change from the group of : 136, E51, G89, S97, and F218.

4. An expression cassette comprising a regulatory sequence operably linked to a nucleotide sequence which encodes the variant Afest of claims 1-3.

5. The expression cassette of claim 4 wherein the nucleotide sequence is SEQ ID NO: 9.

6. The expression cassette of claim 4 or 5 wherein the expression cassette is integrated into the genomic DNA of a host cell.

7. A host cell comprising the expression cassette of claim 4 or 5.

8. The host cell of claim 7 wherein the host cell produces the variant Afest of claim 1.

9. A composition for decomposing polylactic acid (PLA)-containing material comprising the variant Afest of claims 1-3.

10. The composition of claim 9 wherein the composition further contains a buffer.11 . The composition of claim 9 wherein the PLA-containing material contains at least 70% PLA.

12. A method of decomposing PLA-containing materials using the composition of claim 9.

13. The method of claim 12 further comprising recovering the lactic acid monomers.

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