Identification of Cutinolytic Esterase from Microplastic-Associated Microbiota Using Functional Metagenomics and Its Plastic Degrading Potential.

Plastic pollution has threatened biodiversity and human health by shrinking habitats, reducing food quality, and limiting the activities of organisms. Therefore, global interest in discovering novel enzymes capable of degrading plastics has increased considerably. Within this context, the functional metagenomic approach, which allows for unlocking the functional potential of uncultivable microbial biodiversity, was used to discover a plastic-degrading enzyme. First, metagenomic libraries derived from microplastic-associated microbiota were screened for esterases capable of degrading both tributyrin and polycaprolactone. Clone KAD01 produced esterase highly active against p-nitrophenyl esters (C2-C16). The gene corresponding to the enzyme activity showed moderate identity (≤ 55.94%) to any known esterases/cutinases. The gene was extracellularly expressed with a 6× histidine tag in E. coli BL21(DE3), extracellularly. Titer of the enzyme (CEstKAD01) was raised from 21.32 to 35.17 U/mL by the statistical optimization of expression conditions and media components. CEstKAD01 was most active at pH 7.0 and 30 °C. It was noteworthy stable over a wide pH (6.0-10.0) and temperature (20-50 °C). The enzyme was active and stable in elevated NaCl concentrations up to 12% (w/v). Pre-incubation of CEstKAD01 with Mg2+, Mn2+, and Ca2+ increased the enzyme activity. CEstKAD01 displayed an excellent tolerance against various chemicals and solvents. It was determined that 1 mg of the enzyme caused the release of 5.39 ± 0.18 mM fatty acids from 1 g apple cutin in 120 min. Km and Vmax values of CEstKAD01 against p-nitrophenyl butyrate were calculated to be 1.48 mM and 20.37 µmol/min, respectively. The enzyme caused 6.94 ± 0.55, 8.71 ± 0.56, 7.47 ± 0.47, and 9.22 ± 0.18% of weight loss in polystyrene, high-density polyethylene, low-density polyethylene, and polyvinyl chloride after 30-day incubation. The scanning electron microscopy (SEM) analysis indicated the formation of holes and pits on the plastic surfaces supporting the degradation. In addition, the change in chemical structure in plastics treated with the enzyme was determined by Fourier Transform Infrared Spectroscopy (FTIR) analysis. Finally, the degradation products were found to have no genotoxic potential. To our knowledge, no cutinolytic esterase with the potential to degrade polystyrene (PS), high-density polyethylene (HDPE), low-density polyethylene (LDPE), and polyvinyl chloride (PVC) has been identified from metagenomes derived from microplastic-associated microbiota.

Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes.

A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200 rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50-70°C) and over a wide pH range (5.5-9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent Km and Vmax values of the cutinase for p-nitrophenyl butyrate were 0.72 mM and 336.8 µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.

Microvirga makkahensis sp. nov., and Microvirga arabica sp. nov., isolated from sandy arid soil.

The taxonomic positions of two Gram-negative strains, SV1470(T) and SV2184P(T), isolated from arid soil samples, were determined using a polyphasic approach. Analysis of the 16S rRNA gene and the concatenated sequences of three housekeeping gene loci (dnaK, rpoB and gyrB) confirmed that the strains belong to the genus Microvirga. Strain SV1470(T) was found to be closely related to Microvirga vignae BR3299(T) (98.8 %), Microvirga flocculans TFB(T) (98.3 %) and Microvirga lupini Lut6(T) (98.2 %), whilst similarity to other type strains of the genus ranged from 97.8 to 96.3 %; strain SV2184P(T) was found to be closely related to Microvirga aerilata 5420S-16(T) (98.0 %), Microvirga zambiensis WSM3693(T) (97.8 %) and M. flocculans ATCC BAA-817(T) (97.4 %), whilst similarity to other type strains of the genus ranged from 97.2 to 95.9 %. The G + C content of the genomic DNA was determined to be 61.5 mol % for strain SV1470(T) and 62.1 mol % for strain SV2184P(T). Both strains were found to have the same quinone system, with Q-10 as the major ubiquinone. The polar lipid profile of strain SV1470(T) was found to consist of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and one unidentified aminolipid, while that of strain SV2184P(T) consisted of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, one unidentified aminolipid, one unidentified aminophospholipid and two unidentified phospholipids. DNA-DNA relatedness studies showed that the two strains belong to different genomic species. The strains were also distinguished using a combination of phenotypic properties. Based on the genotypic and phenotypic data, the novel species Microvirga makkahensis sp. nov. (type strain SV1470(T) = DSM 25394(T) = KCTC 23863(T) = NRRL-B 24875(T)) and Microvirga arabica sp. nov. (type strain SV2184P(T) = DSM 25393(T) = KCTC 23864(T) = NRRL-B 24874(T)) are proposed.