Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters.

The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 Å resolution and revealed a modified α/ß hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.

Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases.

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/ß-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/ß-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.

The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes.

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.

Production, optimization and purification of a novel extracellular protease from the moderately halophilic bacterium Halobacillus karajensis.

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.

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families.

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.

Salinicoccus iranensis sp. nov., a novel moderate halophile.

A novel moderately halophilic, Gram-positive bacterium, designated strain QW6(T), was isolated from textile industry wastewater in Qom, Iran. Strain QW6(T) was strictly aerobic, non-motile, non-sporulating and oxidase- and catalase-positive. It grew at salinities of 1-25% (w/v) NaCl, showing optimal growth at 7.5-10.0% (w/v). Growth occurred at 5.0-45.0 degrees C and over the pH range 6.5-10.0; growth was optimal at 35 degrees C and pH 7.5. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain QW6(T) is a member of the genus Salinicoccus. The organism possessed Lys- and Gly-containing peptidoglycan. Strain QW6(T) had iso-C(15:0) and anteiso-C(15:0) as the predominant fatty acids and MK-6 as the major respiratory lipoquinone. The chemotaxonomic profile of this organism was consistent with its classification in the genus Salinicoccus. The DNA G+C content of strain QW6(T) was 54.4 mol%. On the basis of phenotypic characteristics, 16S rRNA gene sequence analysis and DNA-DNA relatedness of less than 50% to species of the genus Salinicoccus, it is proposed that strain QW6(T) (=DSM 18903(T)=CCM 7448(T)) should be placed in the genus Salinicoccus as the type strain of a novel species, Salinicoccus iranensis sp. nov.

Salinivibrio proteolyticus sp. nov., a moderately halophilic and proteolytic species from a hypersaline lake in Iran.

A novel moderately halophilic, Gram-negative, curved-rod-shaped bacterium, designated strain AF-2004T, was isolated from Bakhtegan Lake, a hypersaline lake with 17 % (w/v) total salt located in the southern region of Iran. Strain AF-2004T was a facultative anaerobe, motile by one polar flagellum, non-sporulating and oxidase- and catalase-positive. It grew at salinities of 1-17 % (w/v) NaCl, showing optimal growth at 5 % (w/v). Growth occurred at 10.0-45.0 degrees C and pH 5.0-9.5, with optimum growth at 32.0-35.0 degrees C and pH 8.0-8.5. Phylogenetic analyses based on 16S rRNA gene sequence comparisons indicated that strain AF-2004T is a member of the genus Salinivibrio. Strain AF-2004T had C18 : 1omega7c (31.6 %), C16 : 1omega7c (22.1 %) and C16 : 0 (20.7 %) as the predominant fatty acids and Q-8 as the major respiratory lipoquinone. The DNA G+C content was 49.5 mol%, which is in the range of values for members of the genus Salinivibrio. On the basis of differentiation from Salinivibrio costicola by phenotypic and chemotaxonomic characteristics, 16S rRNA sequence analysis and DNA-DNA relatedness, it is proposed that strain AF-2004T (=DSM 19052T =CIP 109598T) should be placed in the genus Salinivibrio as the type strain of a novel species, Salinivibrio proteolyticus sp. nov.