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.

Heterologous expression, purification, and characterization of thermo- and alkali-tolerant laccase-like multicopper oxidase from Bacillus mojavensis TH309 and determination of its antibiotic removal potential.

Laccases or laccase-like multicopper oxidases have great potential in bioremediation to oxidase phenolic or non-phenolic substrates. However, their inability to maintain stability in harsh environmental conditions and against non-substrate compounds is one of the main reasons for their limited use. The gene (mco) encoding multicopper oxidase from Bacillus mojavensis TH309 were cloned into pET14b( +), expressed in Escherichia coli, and purified as histidine tagged enzyme (BmLMCO). The molecular weight of the enzyme was about 60 kDa. The enzyme exhibited laccase-like activity toward 2,6-dimethoxyphenol (2,6-DMP), syringaldazine (SGZ), and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The highest enzyme activity was recorded at 80 °C and pH 8. BmLMCO showed a half-life of ~ 305, 99, 50, 46, 36, and 20 min at 40, 50, 60, 70, 80, and 90 °C, respectively. It retained more than 60% of its activity after pre-incubation in the range of pH 5-12 for 60 min. The enzyme activity significantly increased in the presence of 1 mM of Cu2+. Moreover, BmLMCO tolerated various chemicals and showed excellent compatibility with organic solvents. The Michaelis constant (Km) and the maximum velocity (Vmax) values of BmLMCO were 0.98 mM and 93.45 µmol/min, respectively, with 2,6-DMP as the substrate. BmLMCO reduced the antibacterial activity of cefprozil, gentamycin, and erythromycin by 72.3 ± 1.5%, 79.6 ± 6.4%, and 19.7 ± 4.1%, respectively. This is the first revealing shows the recombinant production of laccase-like multicopper oxidase from any B. mojavensis strains, its biochemical properties, and potential for use in bioremediation.

Antimicrobial, antibiofilm, and anticancer potential of silver nanoparticles synthesized using pigment-producing Micromonospora sp. SH121.

Silver nanoparticles (AgNPs) have gained interest as an alternative pharmaceutical agent because of antimicrobial resistance and drug toxicity. Considering the increasing request, eco-friendly, sustainable, and cost-effective synthesis of versatile AgNPs has become necessary. In this study, green-made AgNPs were successfully synthesized using Micromonospora sp. SH121 (Mm-AgNPs). Synthesis was verified by surface plasmon resonance (SPR) peak at 402 nm wavelength in the UV-Visible (UV-Vis) absorption spectrum. Scanning electron microscopy (SEM) analysis depicted that Mm-AgNPs were in the size range of 10-30 nm and spherical. Fourier transform infrared spectroscopy (FTIR) confirmed the existence of bioactive molecules on the surface of nanoparticles. The X-ray diffraction (XRD) analysis revealed the face-centered cubic (fcc) structure of the Mm-AgNPs. Their polydispersity index (PDI) and zeta potential were 0. 284 and -35.3 mV, respectively. Mm-AgNPs (4-32 µg/mL) exhibited strong antimicrobial activity against Bacillus cereus, Enterococcus faecalis, Enterococcus hirae, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas putida, Staphylococcus epidermidis, Streptococcus pneumoniae, and Aspergillus flavus. Mm-AgNPs partially inhibited the biofilm formation in Acinetobacter baumannii, E. coli, K. pneumoniae, and Pseudomonas aeruginosa. Furthermore, results showed that low concentrations of Mm-AgNPs (1 and 10 µg/mL) caused higher cytotoxicity and apoptosis in DU 145 cells than human fibroblast cells. Based on the results, Mm-AgNPs have an excellent potential for treating infectious diseases and prostate cancer.

Synthesis of graphite/rGO-modified fungal hyphae for chromium (VI) bioremediation process.

Bioremediation is a promising technology that can eliminate the drawbacks of conventional treatment methods in removing harmful toxic metals including chromium(VI). Therefore, in this study, fungal hyphae modified with graphite and reduced graphene oxide were synthesized and assessed for their potential to bioremediate heavy metals for the first time in the literature. The effects of the carbon-based materials on microbial structure were characterized using scanning electron microscopy analysis. Thermogravimetric, RAMAN, X-ray diffraction, and enzymatic analyzes were performed to determine the role of functional groups. In addition, batch adsorption experiments utilizing response surface methodology were conducted to optimize operating parameters such as time (1-11 h), chromium (10-50 mg/L), and graphite/reduced graphene oxide (0.1-1 g/L). The maximum adsorption capacity with the graphene fungal hyphae was determined to be 568 mg.g-1, which is 9.7 times that of the crude fungal hyphae. The Cr(VI) removal for fungal hyphae-graphite and fungal hyphae-reduced graphene oxide biocomposites was 98.25% and 98.49%, respectively. The isothermal and kinetic results perfectly matched the 2nd order pseudo-model and Langmuir model in terms of the nature of the adsorption process. The laboratory scale test results indicate that fungal hyphae modified with graphite and reduced graphene oxide have a high adsorption capacity, suitable for the removal of chromium (VI) from wastewater.

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.