Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial α/β-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble α-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 Å resolution and revealed a classical α/β-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.

Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5°C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.

Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.

Peer reviewed versionCyswllt i'r cyhoeddiad / Link to publication Dyfyniad o'r fersiwn a gyhoeddwyd / Citation for published version (APA): Hajighasemi, M., Tchigvintsev, A. ABSTRACT 34The continuous growth of global plastics production for more than 50 years has resulted 35 in elevated levels of pollution and serious environmental problems. Enzymatic 36 depolymerization of synthetic polyesters represents an attractive approach for plastics 37 recycling and effective use of carbon resources. In this study, screening of over 200 38 purified uncharacterized hydrolases from environmental metagenomes and sequenced 39 microbial genomes identified 27 proteins with detectable activity and at least 10 proteins 40 with high hydrolytic activity against synthetic polyesters. The metagenomic esterases 41 GEN0105 and MGS0156 were active against a broad range of synthetic polyesters 42 including polylactic acid, polycaprolactone, and bis(benzoyloxyethyl)-terephthalate. With 43 solid polylactic acid as substrate, both enzymes produced a mixture of lactic acid 44 monomers, dimers, and higher oligomers. The crystal structure of MGS0156 was 45 determined at 1.95 Å resolution and revealed a modified α/β hydrolase fold, with a highly 46 hydrophobic active site and lid domain. Mutational studies of MGS0156 identified the 47 residues critical for hydrolytic activity against both monoester and polyester substrates, 48 and demonstrated a two-times higher polyesterase activity in the L169A mutant protein. 49 Thus, environmental metagenomes contain diverse polyesterases with high hydrolytic 50 activity against a broad range of synthetic polyesters with potential applications in 51 plastics recycling. 52 53 54 55 56 environmental conditions such as soils, sludges, composts, and marine water. 10 Many 102 6 aliphatic polyesters, including PLA and PCL, were found to be degraded by Aspergillus, 103 Penicillium, Pullularia, Trichoderma, and other fungal strains isolated from 104 environmental samples. 17, 18 Among bacteria, different strains of Bacillus, Pseudomonas, 105 Leptothrix, Roseateles, Corynebacterium, Streptomyces, and Enterobacter can efficiently 106 degrade both aliphatic and aliphatic-aromatic co-polyesters (e.g. PBAT). 10, 19 Most of the 107 biodegradable polyesters are degraded by serine-dependent hydrolases such as lipases, 108 esterases, proteases, and cutinases. 10 Several polyester degrading lipases and esterases 109 have been characterized biochemically, including Paenibacillus amylolyticus PlaA, 110 Thermobifida fusca TfH, ABO1197 and ABO1251 from Alcanivorax borkumensis, 111 several clostridial esterases (Chath_Est1, Cbotu_EstA, Cbotu_EstB), and the 112 metagenomics polyesterases PlaM4, EstB3, and EstC7. 20-25 Cutinases comprise a family 113 of serine hydrolases produced by bacteria, fungi, and plants, whose natural substrate is 114

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34 degrees C, pH 8.0-8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50 degrees C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0-10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.