Oxidative degradation of UV-irradiated polyethylene by laccase-mediator system.

Polyethylene (PE) is one of the most widely used plastics. However, the chemical inertness, inefficient recycling, and random landfilling of PE waste have caused serious pollution to the natural environment. In this study, a series of laccase-mediator systems (LMS) were constructed by combination of two laccases from Botrytis aclada (BaLac) and Bacillus subtilis (BsLac) with three synthetic mediators (ABTS, HBT, and TEMPO) to oxidize LDPE films (UVPE) pretreated with high-temperature UV irradiation. Scanning electron microscopy showed aging phenomena such as etching, fragmentation, and cracking on the surface of the UVPE films after LMS incubation. The FTIR results showed that LMS-UVPE added new oxygen-containing functional groups such as -OH, -CO, and CC. High-temperature gel chromatography confirmed that the average reduction in weight-average molecular weight (Mw) was approximately 40% for the BaLac experimental group. GC-MS analysis showed the presence of oxygen-containing products, such as aldehydes, ketones, and alcohols, in the reaction mixture. To verify the oxidation process UVPE degradation by LMS, we inferred three possible pathways by combined analysis of the oxidation products of LMS on UVPE and model substrates oleic acid and squalene.

A novel salt-tolerant GH42 ß-galactosidase with transglycosylation activity from deep-sea metagenome.

ß-Galactosidase is a widely adopted enzyme in the food and pharmaceutical industries. Metagenome techniques have the advantage of discovering novel functional genes, particularly potential genes from uncultivated microbes. In this study, a novel GH42 ß-galactosidase isolated from a deep-sea metagenome was overexpressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. The optimal temperatures and pH of the enzyme for o-nitrophenyl-ß-D-galactopyranoside (oNPG) and lactose were 40 â„ƒ, 6.5 and 50 â„ƒ, 7, respectively. The enzyme was stable at temperatures between 4 and 30 â„ƒ and within the pH range of 6-9. Moreover, it was highly tolerant to salt and inhibited by Zn2+ and Cu2+. The kinetic values of Km and kcat of the enzyme against oNPG were 1.1 mM and 57.8 s-1, respectively. Furthermore, it showed hydrolysis and transglycosylation activity to lactose and the extra monosaccharides could improve the productivity of oligosaccharides. Overall, this recombinant ß-galactosidase is a potential biocatalyst for the hydrolysis of milk lactose and synthesis of functional oligosaccharides.

Oxidative degradation of pre-oxidated polystyrene plastics by dye decolorizing peroxidases from Thermomonospora curvata and Nostocaceae.

Biodegradation of PS has attracted lots of public attentions due to its environmental friendliness. However, no specific PS degrading enzyme has been identified yet. Dye decolorizing peroxidases (DyPs) are heme-containing peroxidases named for the ability to degrade a variety of organic dyes. Herein, the abilities of two DyPs from Thermomonospora curvata (TcDyP) and Nostocaceae (AnaPX) to degrade PS were evaluated. Preoxidation methods by ultraviolet (UV) irradiation and chemical oxidants were developed to initially activate C-C bonds in the PS skeleton. DyPs degradation caused obvious etching and enhanced hydrophilicity of UV-PS films, and also generated new CO and C-OH groups. The cleavage of activated C-C bonds by DyPs was experimentally proven by analyzing the degradation products of UV-PS and model substrates. Furthermore, better pre-oxidation was obtained by using chemical oxidants KMnO4/H2SO4 and mCPBA to oxidize PS materials in dissolved state. And AnaPX exhibited stronger degradation effects on KMnO4/H2SO4-PS and mCPBA-PS by causing greater changes in functional groups CO, C-O, -OH groups and substituted benzenes and higher molecular weight reductions of 19.7% and 31.0%, respectively. To our knowledge, this is the first report on the identification of PS-degrading enzymes that provides experimental evidence.

Degradation of UV-pretreated polyolefins by latex clearing protein from Streptomyces sp. Strain K30.

Plastic products made of polyethylene (PE), polypropylene (PP), and polystyrene (PS) are widely used in daily life and industrial production. Polyolefins-which have a very stable structure and do not contain any active molecular groups-are difficult to degrade and pose a serious global environment threat. This study selected latex clearing protein (LcpK30) derived from Streptomyces sp. Strain K30. The natural substrate of the enzyme is rubber (cis-1, 4-polyisoprene), and the site of action is the carbon­carbon double bond. LcpK30 was incubated with UV-irradiated polyolefin PE, PP and PS (UV-PE, UV-PP, and UV-PS containing carbon­carbon double bonds) for 5 d at 37 °C. The results showed that UV-PE-LcpK30 was more fragmented than UV-PE-blank; the Fourier transform infrared spectroscopy results showed that UV-PE-LcpK30 and UV-PP-LcpK30 produced new active groups (e.g., -OH and -C=O); however, the effect on UV-PS was not significant. Scanning electron microscopy results showed that the treated group had more obvious roughness, cracks, and pits than the control group. The results of high-temperature gel permeation chromatography showed that the average molecular weight (Mw) of UV-PE-LcpK30 and UV-PP-LcpK30 decreased; the Mw of UV-PE5-LcpK30 was reduced by 42.02%. The results of gas chromatography-mass spectrometry showed the production of ketones. Therefore, the LcpK30 latex clearing protein degrade UV-oxidized polyolefin plastics and has great potential for PE and PP degradation but may not be suitable for PS. Furthermore, other Lcps (such as LcpNRRL, LcpNVL3) can also degrade UV-PE.

Cloning, Expression and Characterization of a Novel Cold-adapted ß-galactosidase from the Deep-sea Bacterium Alteromonas sp. ML52.

The bacterium Alteromonas sp. ML52, isolated from deep-sea water, was found to synthesize an intracellular cold-adapted ß-galactosidase. A novel ß-galactosidase gene from strain ML52, encoding 1058 amino acids residues, was cloned and expressed in Escherichia coli. The enzyme belongs to glycoside hydrolase family 2 and is active as a homotetrameric protein. The recombinant enzyme had maximum activity at 35 °C and pH 8 with a low thermal stability over 30 °C. The enzyme also exhibited a Km of 0.14 mM, a Vmax of 464.7 U/mg and a kcat of 3688.1 S-1 at 35 °C with 2-nitrophenyl-ß-d-galactopyranoside as a substrate. Hydrolysis of lactose assay, performed using milk, indicated that over 90% lactose in milk was hydrolyzed after incubation for 5 h at 25 °C or 24 h at 4 °C and 10 °C, respectively. These properties suggest that recombinant Alteromonas sp. ML52 ß-galactosidase is a potential biocatalyst for the lactose-reduced dairy industry.

Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 ß-glucosidase from the marine bacterium Alteromonas sp. L82.

A novel gene (bgl) encoding a cold-adapted ß-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni2+ affinity chromatography. The purified recombinant ß-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant ß-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant ß-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a Km of 0.55 mM, a Vmax of 83.6 U/mg, a kcat of 74.3 s-1 and kcat/Km of 135.1 at 40°C, pH 7 with 4-nitrophenyl-ß-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.