Molecular engineering of PETase for efficient PET biodegradation.

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 µM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 µM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

[Synthesis and characterization of dihydroeugenol acrylate].

Dihydroeugenol acrylate was synthesized by the reaction of acryloyl chloride (AC) with lignin mode compound dihydroeugenol (DH) in the presence of TEA and characterized by using FTIR, GC/MS, 1H-NMR and GPC. FTIR spectra showed that, after the esterification with acryloyl chloride, the intensity of stretching vibration peak of O-H (centered at 3 495 cm(-1)) of DH was disappeared. At the same time, a new peak appeared at 1 762 cm(-1) which was assigned to ester group. Additionally, the appearance of 1 631 and 981 cm(-1) were attributed to the carbon - carbon double bond confirmed the success in the synthesis of DH-AC. 1H-NMR spectra showed that, after the esterification with acryloyl chloride, the proton signal of O-H at 5.5 ppm was disappeared. Meanwhile, the appearance of three new proton signals at 6.0 ppm, 6.4 and 6.7 ppm, attributed to the vinylic protons, indicated that acryloyl chloride was successfully grafted onto DH. The results further confirmed the structures of the DH-AC. GC-MS results showed the DH-AC had a high purity of 98.63%. GPC results showed that dihydroeugenol acrylate could polymerize in the 1,4-dioxane using a thermal initiator of AIBN (2.0 Wt% of total monomers). The weight average molecular mass (Mw) of the homopolymer is 37 400 g x mol(-1), and the number average molecular mass is 23 400 g x mol(-1)' with a polydispersity index Mw/Mn of 1.60, indicating that the dihydroeugenol acrylate has high polymerization activity. This strategy provides a novel approach for extending the comprehensive utilization of lignin.

[Chemical structure of bioethanol lignin by low-temperature alkaline catalytic hydrothermal treatment].

In order to improve the reaction activity of bioethanol lignin, we investigated the activation of bioethanol lignin by a hydrothermal treatment method. Catalytic hydrothermal treatment of bioethanol lignin was performed at 180 degrees C for 3 h in the presence of alkaline solutions (NaOH, Na2 CO3, KOH and K2 CO3), the change in bioethanol lignin structures was studied comparatively by FTIR, 1H NMR,GPC and elemental analysis. FTIR spectra showed that after alkali hydrothermal treatment, the band at 1 375 cm(-1) attributed to the phenolic hydroxyl groups increased, and the band intensity at 1 116 cm(-1) attributed to the ether bond decreased. On the other hand, the band at 1 597 and 1 511 cm(-1) attributed to aromatic skeletal vibration remained almost unchanged. 1H NMR spectra showed that after alkali hydrothermal treatment, the number of aromatic methoxyl is increased, and based on the increment of the content of phenolic hydroxyl, the catalytic activity can be ranked as follows: KOH > NaOH > K2 CO3 > Na2 CO3. Especially for KOH, the increment of the content of phenolic hydroxyl was 170%, because the ion radius of potassium cation is bigger than sodium cation, so the potassium cations more easily formed cation adducts with lignin. GPC results showed that the molecular weight of alkali hydrothermal treatment lignin decreased and the molecular distribution got wider. Elemental analysis showed that hydrothermal treatment could break the interlinkage between lignin and protein, which can reduce the protein content and increase the purity of lignin, meanwhile, the content of O and H both decreased,while C fell, indicating that the bioethanol lignin had suffered a decarbonylation reaction. This is the most benefit of the lignin as a substitute for phenol.