[Biodegradation of polyethylene terephthalate: a review].

Plastics are widely used in human daily life, which bring great convenience. Nevertheless, the disposal of a large amount of plastic wastes also brings great pressure to the environment. Polyethylene terephthalate (PET) is a polymer thermoplastic material produced from petroleum. It has become one of the most commonly used plastics in the world due to its durability, high transparency, light weight and other characteristics. PET can exist in nature for a long time due to its complex structure and the difficulty in degradation, which causes serious pollution to the global ecological environment, and threatens human health. The degradation of PET wastes has since become one of the global challenges. Compared with physical and chemical methods, biodegradation is the greenest way for treating PET wastes. This review summarizes the recent advances on PET biodegradation including microbial and enzymatic degradation of PET, biodegradation pathway, biodegradation mechanisms, and molecular modification of PET-degrading enzymes. In addition, the prospect for achieveing efficient degradation of PET, searching and improving microorganisms or enzymes that can degrade PET of high crystallinity are presented, with the aimto facilitate the development, application and molecular modification of PET biodegradation microorganisms or enzymes.

Optimizing extraction conditions and isolation of bound phenolic compounds from corn silk (Stigma maydis) and their antioxidant effects.

During the processing of maize, Stigma maydis, also known as corn silk, is normally discarded as waste. Phytochemical research was carried out on the S. maydis to use it as a valuable source of bioactive components. This research aimed to maximize the recovery of free and bound phenolic compounds from corn silk under optimal experimental conditions. Response surface design was operated to optimize the alkaline hydrolysis extraction of bound phytochemicals from corn silk based on total phenolic content and DPPH radical scavenging activity. The optimum conditions (i.e., NaOH concentration 2 M, digestion time 135 min, digestion temperature of 37.5°C, the solid-to-solvent ratio of 1:17.5, and acetone) were obtained. The optimum parameters were used to extract the corn silk. The structures of two compounds isolated from ethyl acetate extracts were then identified as friedelin (1) and (E)-4-(4-hydroxy-3-methoxyphenyl) but-3-en-2-one (2). The DPPH, H2 O2 , and ABTS % inhibition of the compounds is as follows: compound (1) 74.81%, 76.8%, 70.33% and compound (2) 70.37%, 56.70% and 57.46%, respectively. The current study has opened previously unexplored perspectives of the composition of bound compounds in corn silk and established the foundations for more effective processing and utilization of corn waste. PRACTICAL APPLICATION: Bound phenolic compounds from corn silk under optimal experimental conditions were obtained. Corn silk can be utilized as a type of medicinal herb as well as a source of inexpensive natural antioxidants.

Identification and Characterization of a Novel Carboxylesterase Belonging to Family VIII with Promiscuous Acyltransferase Activity Toward Cyanidin-3-O-Glucoside from a Soil Metagenomic Library.

An alkaline esterase, designated as EstXT1, was identified through functional screening from a metagenomic library. Sequence analysis revealed that EstXT1 belonged to the family VIII carboxylesterases and contained a characteristic conserved S-x-x-K motif and a deduced catalytic triad Ser56-Lys59-Tyr165. EstXT1 exhibited the strongest activity toward methyl ferulate at pH 8.0 and temperature 55°C and retained over 80% of its original activity after incubation in the pH range of 7.0-10.6 buffers. Biochemical characterization of the recombinant enzyme showed that it was activated by Zn2+ and Co2+ metal ion, while inhibited by Cu2+ and CTAB. EstXT1 exhibited significant promiscuous acyltransferase activity preferred to the acylation of benzyl alcohol acceptor using short-chain pNP-esters (C2-C8) as acyl-donors. A structure-function analysis indicated that a WAG motif is essential to acyltransferase activity. This is the first report example that WAG motif plays a pivotal role in acyltransferase activity in family VIII carboxylesterases beside WGG motif. Further experiment indicated that EstXT1 successfully acylated cyanidin-3-O-glucoside in aqueous solution. The results from the current investigation provided new insights for the family VIII carboxylesterase and lay a foundation for the potential applications of EstXT1 in food and biotechnology fields.

Characterization of a novel carboxylesterase with catalytic activity toward di(2-ethylhexyl) phthalate from a soil metagenomic library.

A novel carboxylesterase gene estyz5 was isolated from a soil metagenomic library. The recombinant enzyme EstYZ5 is 298 amino acids in length with a predicted molecular weight of 32 kDa. Sequence alignment and phylogenetic analysis revealed that EstYZ5 belongs to the hormone-sensitive lipase (HSL) family with a deduced catalytic triad of Ser144-Glu238-His268. EstYZ5 contains two conserved motifs, a pentapeptide motif GDSAG and a HGGG motif, which are typically found in members of the HSL family. Esterolytic activity of the recombinant enzyme was optimal at 30 °C and pH 8.0, and the kcat/Km value of the enzyme for the optimum substrate p-nitrophenyl butyrate was as high as 1272 mM-1·s-1. Importantly, EstYZ5 showed activity toward di(2-ethylhexyl) phthalate with complex side chains, which is rare for HSLs. Molecular docking simulations revealed that the catalytic triad and an oxyanion hole likely play vital roles in enzymatic activity and specificity. The phthalate-degrading activity of EstYZ5, combined with its high levels of esterolytic activity, render this new enzyme a candidate for biotechnological applications.

Extraction, isolation, characterization and antimicrobial activities of non-extractable polyphenols from pomegranate peel.

Non-extractable polyphenols (NEPPs) in pomegranate peel were released by acid hydrolysis followed by extraction using ethyl acetate (EtOAc). Ten NEPPs were identified in the hydrolysate using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Six compounds were then isolated from the EtOAc extracts whose structures were identified as ß-sitosterol-3-O-glycoside (1), ß-sitosterol (2), ursolic acid (3), corosolic acid (4), asiatic acid (5) and arjunolic acid (6) using a wide range of spectroscopic analyses. Compounds 4-6 were isolated for the first time from pomegranate peel. Antimicrobial experiments revealed that compound 3 and 5 showed significant antimicrobial activity against a range of pathogens, particularly compound 5 which exhibited selective inhibitive activity towards Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 16 µg/ml. The present study has provided new insights into the composition of bound chemicals in pomegranate peel and laid a foundation for improving its further processing and utilization.

Enhancing the activity and thermal stability of a phthalate-degrading hydrolase by random mutagenesis.

Our previous work has reported that EstJ6 was a phthalate-degrading hydrolase. In the study, a random mutant library was constructed by two rounds of error-prone PCR, three mutants (ET1.1, ET2.1, and ET2.2) with enhanced hydrolytic activity against dibutyl phthalate (DBP) were obtained. The best mutant ET2.2, accumulated three amino acid substitutions (Thr91Met, Ala67Val, and Val249Ile) and exhibited 2.8-fold increase enzyme activity and 2.3-fold higher expression level. Meanwhile, compared with EstJ6, ET2.2 showed over 50% improvement in thermostability (at 50 °C for 1 h) and 1.2-fold increase in 50% methanol tolerance. Kinetic parameters analysis revealed that the Km value for ET2.2 decreased by 60% and the kcat/Km value increased by 166%. The molecular docking indicated that the shortening of hydrogen bond between Ser146-OH and DBP-CO, which may led to an increase in enzyme activity and catalytic efficiency, the enhancement of hydrophobicity of hydrophobic pocket was related to the improvement of organic solvents tolerance, and three hydrophobic amino acid substitutions Thr91Met, Ala67Val, and Val249Ile facilitated to improve the thermal stability and organic solvents tolerance. These results confirmed that random mutagenesis was an effective tool for improving enzyme properties and lay a foundation for practical applications of phthalate-degrading hydrolase in biotechnology and industrial fields.

Characterization of XtjR8: A novel esterase with phthalate-hydrolyzing activity from a metagenomic library of lotus pond sludge.

A fosmid metagenomic library containing 9.7 × 104 clones was constructed. A novel esterase, XtjR8, was isolated through functional screening. XtjR8 shared the maximum amino acid identity (44%) with acetyl-hydrolase from Streptomyces hygroscopicus, and was classified into family IV esterase. XtjR8 exhibited the highest hydrolytic activity for p-nitrophenyl acetate at 40 °C and pH 8.0, and presented more than 40% activity from 20 °C to 80 °C. More importantly, XtjR8 displayed the ability to hydrolyze both phthalate monoesters and diesters, this feature is extremely rare among previously reported esterases. Site-directed mutagenesis experiments revealed that the catalytic triad residues were Ser152, Glu246, and His276. Among them, Ser152 formed a hydrogen bond with dibutyl phthalate (DBP) by molecular docking, Gly84, Gly85, and Leu248 of conserved motifs formed hydrophobic interactions with DBP, respectively, which were important for the catalytic activity. Considering its wide range of temperature and hydrolytic potential toward phthalate esters, XtjR8 will be served as an interesting candidate for biodegradation and industrial applications.

Identification and characterization of a novel phthalate-degrading hydrolase from a soil metagenomic library.

Phthalate esters have raised public concerns owing to their effects on the environment and human health. We identified a novel phthalate-degrading hydrolase, EstJ6, from a metagenomic library using function-driven screening. Phylogenetic analysis indicated that EstJ6 is a member of family IV esterases. EstJ6 hydrolyzed various dialkyl and monoalkyl phthalate esters, and exhibited high hydrolytic activity (128 U/mg) toward dibutyl phthalate at 40 °C and pH 7.5. EstJ6 hydrolyzed not only common phthalate esters with simple side chains but also diethylhexyl phthalate and monoethylhexyl phthalate, which have complex and long side chains. Site-directed mutagenesis indicated that the catalytic triad residues of EstJ6 consists of Ser146, Glu240, and His270. EstJ6 is therefore a promising biodegradation enzyme, and our study illustrates the advantages of a metagenomic approach in identifying enzyme-coding genes for agricultural, food, and biotechnological applications.

A Novel VIII Carboxylesterase with High Hydrolytic Activity Against Ampicillin from a Soil Metagenomic Library.

A novel carboxylesterase gene, named dlfae4, was discovered and sequenced from a soil metagenomic library. The dlfae4 gene was composed of 1017 base pairs encoding 338 amino acid residues with a predicted molecular mass of 37.2 kDa. DLFae4 exhibited strong hydrolytic activity towards methyl ferulate under optimum pH and temperature conditions (pH 8.6, 50 °C) and displayed remarkable thermostability, with residual activity as high as 50% after incubation for 3 h at 60 °C. A family VIII esterase DLFae4 was found to contain a typical serine residue within the S-X-X-K motif, which serves as a catalytic nucleophile in class C ß-lactamases and family VIII esterases. As a consequence of its high sequence similarity with ß-lactamases, DLFae4 exhibited significant hydrolytic activity towards ampicillin. In addition, DLFae4 was found to be the first known member of family VIII carboxylesterases with phthalate-degrading ability. Site-directed mutagenesis studies revealed that Ser11, Lys14, and Tyr121 residues play an essential catalytic role in DLFae4. These new findings, which are of great importance for further in-depth research and engineering development of carboxylesterases, should advance the implementation of biotechnological applications.

Molecular cloning, expression and characterization of a novel feruloyl esterase from a soil metagenomic library with phthalate-degrading activity.

OBJECTIVES: To discover novel feruloyl esterases (FAEs) by the function-driven screening procedure from soil metagenome. RESULTS: A novel FAE gene bds4 was isolated from a soil metagenomic library and over-expressed in Escherichia coli. The recombinant enzyme BDS4 was purified to homogeneity with a predicted molecular weight of 38.8 kDa. BDS4 exhibited strong activity (57.05 U/mg) toward methyl ferulate under the optimum pH and temperature of 8.0 and 37°C. Based on its amino acid sequence and model substrates specificity, BDS4 was classified as a type-C FAE. The quantity of the releasing ferulic acid can be enhanced significantly in the presence of xylanase compared with BDS4 alone from de-starched wheat bran. In addition, BDS4 can also hydrolyze several phthalates such as diethyl phthalate, dimethyl phthalate and dibutyl phthalate. CONCLUSION: The current investigation discovered a novel FAE with phthalate-degrading activity and highlighted the usefulness of metagenomic approaches as a powerful tool for discovery of novel FAEs.

An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification.

An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H2O2. The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5-104 CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry.

Application of femtosecond-laser induced nanostructures in optical memory.

The femtosecond laser induced micro- and nanostructures for the application to the three-dimensional optical data storage are investigated. We have observed the increase of refractive index due to local densification and atomic defect generation, and demonstrated the real time observation of photothermal effect after the femtosecond laser irradiation inside a glass by the transient lens (TrL) method. The TrL signal showed a damped oscillation with about an 800 ps period. The essential feature of the oscillation can be reproduced by the pressure wave creation and propagation to the outward direction from the irradiated region. The simulation based on elastodynamics has shown that a large thermoelastic stress is relaxed by the generation of the pressure wave. In the case of soda-lime glass, the velocity of the pressure wave is almost same as the longitudinal sound velocity at room temperature (5.8 microm/ns). We have also observed the localized photo-reduction of Sm3+ to Sm2+ inside a transparent and colorless Sm(3+)-doped borate glass. Photoluminescence spectra showed that some the Sm3+ ions in the focal spot within the glass sample were reduced to Sm2+ ions after femtosecond laser irradiation. A photo-reduction bit of 200 nm in three-dimensions can be recorded with a femtosecond laser and readout clearly by detecting the fluorescence excited by Ar+ laser (lambda = 488 nm). A photo-reduction bit can be also erased by photo-oxidation with a cw Ar+ laser (lambda = 514.5 nm). Since photo-reduction bits can be spaced 150 nm apart in a layer within glass, a memory capacity of as high as 1 Tbit can be achieved in a glass piece with dimensions of 10 mm x 10 mm x 1 mm. We have also demonstrated the first observation of the polarization-dependent periodic nanostructure formation by the interference between femtosecond laser light and electron acoustic waves. The observed nanostructures are the smallest embedded structures ever created by light. The period of self-organized nanostructures can be controlled from approximately 140 to 320 nm by the pulse energy and the number of irradiated pulses. Furthermore, we have also observed the self-assembled sub-wavelength periodic structures created in silica glass by femtosecond pulses on the plane of the propagation of light.

Self-organized nanogratings in glass irradiated by ultrashort light pulses.

Periodic nanostructures are observed inside silica glass after irradiation by a focused beam of a femtosecond Ti:sapphire laser. Backscattering electron images of the irradiated spot reveal a periodic structure of stripelike regions of approximately 20 nm width with a low oxygen concentration, which are aligned perpendicular to the laser polarization direction. These are the smallest embedded structures ever created by light. The period of self-organized grating structures can be controlled from approximately 140 to 320 nm by the pulse energy and the number of irradiated pulses. The phenomenon is interpreted in terms of interference between the incident light field and the electric field of the bulk electron plasma wave, resulting in the periodic modulation of electron plasma concentration and the structural changes in glass.