Isolation and characterization of polyester polyurethane-degrading bacterium Bacillus sp. YXP1.

Microorganisms have the potential to be applied for the degradation or depolymerization of polyurethane (PU) and other plastic waste, which have attracted global attention. The appropriate strain or enzyme that can effectively degrade PU is the key to treat PU plastic wastes by biological methods. Here, a polyester PU-degrading bacterium Bacillus sp. YXP1 was isolated and identified from a plastic landfill. Three PU substrates with increasing structure complexities, including Impranil DLN, poly (1,4-butylene adipate)-based PU (PBA-PU), and polyester PU foam, were used to evaluate the degradation capacity of Bacillus sp. YXP1. Under optimal conditions, strain YXP1 could completely degrade 0.5% Impranil DLN within 7 days. After 30 days, the weight loss of polyester PU foam by strain YXP1 was as high as 42.1%. In addition, PBA-PU was applied for degradation pathway analysis due to its clear composition and chemical structure. Five degradation intermediates of PBA-PU were identified, including 4,4'-methylenedianiline (MDA), 1,4-butanediol, adipic acid, and two MDA derivates, indicating that strain YXP1 could depolymerize PBA-PU by the hydrolysis of ester and urethane bonds. Furthermore, the extracellular enzymes produced by strain YXP1 could hydrolyze PBA-PU to generate MDA. Together, this study provides a potential bacterium for the biological treatment of PU plastic wastes and for the mining of functional enzymes.

Biodegradation of Poly(ethylene terephthalate) by Bacillus safensis YX8.

Due to the extensive utilization of poly (ethylene terephthalate) (PET), a significant amount of PET waste has been discharged into the environment, endangering both human health and the ecology. As an eco-friendly approach to PET waste treatment, biodegradation is dependent on efficient strains and enzymes. In this study, a screening method was first established using polycaprolactone (PCL) and PET nanoparticles as substrates. A PET-degrading strain YX8 was isolated from the surface of PET waste. Based on the phylogenetic analysis of 16S rRNA and gyrA genes, this strain was identified as Bacillus safensis. Strain YX8 demonstrated the capability to degrade PET nanoparticles, resulting in the production of terephthalic acid (TPA), mono (2-hydroxyethyl) terephthalic acid (MHET), and bis (2-hydroxyethyl) terephthalic acid (BHET). Erosion spots on the PET film were observed after incubation with strain YX8. Furthermore, the extracellular enzymes produced by strain YX8 exhibited the ability to form a clear zone on the PCL plate and to hydrolyze PET nanoparticles to generate TPA, MHET, and BHET. This work developed a method for the isolation of PET-degrading microorganisms and provides new strain resources for PET degradation and for the mining of functional enzymes.

Improved Whole-Cell Biocatalyst for the Synthesis of Vitamin E Precursor 2,3,5-Trimethylhydroquinone.

2,3,5-Trimethylhydroquinone (2,3,5-TMHQ) is the key precursor in the synthesis of vitamin E. It is still a major challenge to produce 2,3,5-TMHQ under mild reaction conditions by chemical methods. The monooxygenase system MpdAB can specifically catalyze the conversion of 2,3,6-trimethylphenol (2,3,6-TMP) to 2,3,5-TMHQ. However, the weak catalytic capacity of wild-type MpdA and the cytotoxicity of the substrate limited the production efficiency of 2,3,5-TMHQ. Here, homologous modeling and saturation mutation were performed to increase the catalytic activity of MpdA. Two variants, L128A and L128K, with higher activity toward 2,3,6-TMP (1.86-1.87-fold) were obtained. On the other hand, an evolved strain B5-4M-evolved with enhanced resistance to 2,3,6-TMP (8.15-fold higher for 1000 µM 2,3,6-TMP) was obtained through adaptive laboratory evolution. Subsequently, a 5.29-fold (or 4.87-fold) improvement in 2,3,5-TMHQ production was achieved by a strain B5-4M-evolved harboring L128K (or L128A) and MpdB, in comparison with that of the wild type (strain B5-4M expressing MpdAB). This study provides better genetic resources for producing 2,3,5-TMHQ and proves that the synthesis efficiency of 2,3,5-TMHQ can be improved through enzyme modification and adaptive laboratory evolution.

Efficient secretion of mussel adhesion proteins using a chaperone protein Spy as fusion tag in Bacillus subtilis.

BACKGROUND: Mussel foot proteins (Mfps) are considered as remarkable materials due to their extraordinary adhesive capability. Recombinant expression is an ideal way to synthesis these proteins at large scale. However, secretory expression of Mfps into culture medium has not been achieved in a heterologous host. METHODS AND RESULTS: Here, to realize the secretion of Mfp3 and Mfp5 in Bacillus subtilis, signal peptide screening was first performed. Minimal Mfp3-6×His was targeted into the growth medium with AmyE signal peptide. We found that a small chaperone protein Spy was secreted efficiently in B. subtilis, and the fusion proteins Spy-Mfp3-6×His and Spy-Mfp5-6×His could also be delivered into growth medium well. The yield of Spy-Mfp3-6×His and Spy-Mfp5-6×His reached 255 and 119 mg L-1 at shake flask conditions, respectively. Mfp3-6×His and Mfp5-6×His were finally purified via TEV protease cleavage and NTA affinity chromatography. CONCLUSION: Mfp3-6×His and Mfp5-6×His could be efficiently secreted using a chaperone protein Spy as fusion tag in B. subtilis.

[Isolation and identification of a polyester-polyurethane degrading bacterium Bacillus altitudinis YX8-1].

Although polyurethane (PUR) plastics play important roles in daily life, its wastes bring serious environmental pollutions. Biological (enzymatic) degradation is considered as an environmentally friendly and low-cost method for PUR waste recycling, in which the efficient PUR-degrading strains or enzymes are crucial. In this work, a polyester PUR-degrading strain YX8-1 was isolated from the surface of PUR waste collected from a landfill. Based on colony morphology and micromorphology observation, phylogenetic analysis of 16S rDNA and gyrA gene, as well as genome sequence comparison, strain YX8-1 was identified as Bacillus altitudinis. The results of high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed that strain YX8-1 was able to depolymerize self-synthesized polyester PUR oligomer (PBA-PU) to produce a monomeric compound 4, 4'-methylene diphenylamine. Furthermore, strain YX8-1 was able to degrade 32% of the commercialized polyester PUR sponges within 30 days. This study thus provides a strain capable of biodegradation of PUR waste, which may facilitate the mining of related degrading enzymes.