Down-flow hanging sponge (DHS) reactors, employed in domestic wastewater treatment, have demonstrated efficacy in eliminating Escherichia coli and other potentially pathogenic bacteria. The aim of this study was to elucidate the mechanism of removal of E. coli by employing a cube-shaped polyurethane sponge carrier within a compact hanging reactor. An E. coli removal experiment was conducted on this prepared sponge. Escherichia. coli level was found to decrease by more than 2 logs after passing through five nutrient-restricted DHS sponges. Conversely, a newly introduced sponge did not exhibit a comparable reduction in E. coli level. Furthermore, under conditions of optimal nutritional status, the reduction in E. coli level was limited to 0.5 logs, underscoring the crucial role of nutrient restriction in achieving effective elimination. Analysis of the sponge-associated bacterial community revealed the presence of a type VI secretion system (T6SS), a competitive mechanism observed in bacteria. This finding suggests that T6SS might play a pivotal role in contributing to the observed decline in E. coli level.

The enzyme PETase fromIdeonella sakaiensis (IsPETase) strain 201-F6 can catalyze the hydrolysis of polyethylene terephthalate (PET), mainly converting it into mono(2-hydroxyethyl) terephthalic acid (MHET). In this study, we used quantum mechanics/molecular mechanics (QM/MM) simulations to explore the molecular details of the catalytic reaction mechanism of IsPETase in the formation of MHET. The QM region was described with AM1d/PhoT and M06-2X/6-31+G(d,p) potential. QM/MM simulations unveil the complete enzymatic PET hydrolysis mechanism and identify two possible reaction pathways for acylation and deacylation steps. The barrier obtained at M06-2X/6-31+G(d,p)/MM potential for the deacylation step corresponds to 20.4 kcal/mol, aligning with the experimental value of 18 kcal/mol. Our findings indicate that deacylation is the rate-limiting step of the process. Furthermore, per-residue interaction energy contributions revealed unfavorable contributions to the transition state of amino acids located at positions 200-230, suggesting potential sites for targeted mutations. These results can contribute to the development of more active and selective enzymes for PET depolymerization.

Enzyme-mediated polyethylene terephthalate (PET) depolymerization has recently emerged as a sustainable solution for PET recycling. Towards an industrial-scale implementation of this technology, various strategies are being explored to enhance PET depolymerization (PETase) activity and improve enzyme stability, expression, and purification processes. Recently, rational engineering of a known PET hydrolase (LCC-leaf compost cutinase) has resulted in the isolation of a variant harboring four-point mutations (LCC-ICCG), presenting increased PETase activity and thermal stability. Here, we revealed the enzyme\'s natural extracellular expression and used it to efficiently screen error-prone genetic libraries based on LCC-ICCG for enhanced activity toward consumer-grade PET. Following multiple rounds of mutagenesis and screening, we successfully isolated variants that exhibited up to a 60% increase in PETase activity. Among other mutations, the improved variants showed a histidine to tyrosine substitution at position 218, a residue known to be involved in substrate binding and stabilization. Introducing H218Y mutation on the background of LCC-ICCG (named here LCC-ICCG/H218Y) resulted in a similar level of activity improvement. Analysis of the solved structure of LCC-ICCG/H218Y compared to other known PETases featuring different amino acids at the equivalent position suggests that H218Y substitution promotes enhanced PETase activity. The expression and screening processes developed in this study can be further used to optimize additional enzymatic parameters crucial for efficient enzymatic degradation of consumer-grade PET.

Enzymatic depolymerization of PET waste emerges as a crucial and sustainable solution for combating environmental pollution. Over the past decade, PET hydrolytic enzymes, such as PETase from Ideonella sakaiensis (IsPETases), leaf compost cutinases (LCC), and lipases, have been subjected to rational mutation to enhance their enzymatic properties. ICCM, one of the best LCC mutants, was selected for overexpression in Escherichia coli BL21(DE3) for in vitro PET degradation. However, overexpressing ICCM presents challenges due to its low productivity. A new stress-inducible T7RNA polymerase-regulating E. coli strain, ASIAhsp, which significantly enhances ICCM production by 72.8 % and achieves higher enzyme solubility than other strains. The optimal cultural condition at 30 °C with high agitation, corresponding to high dissolved oxygen levels, has brought the maximum productivity of ICCM and high PET-hydrolytic activity. The most effective PET biodegradation using crude or pure ICCM occurred at pH 10 and 60 °C. Moreover, ICCM exhibited remarkable thermostability, retaining 60 % activity after a 5-day reaction at 60 °C. Notably, crude ICCM eliminates the need for purification and efficiently degrades PET films.

Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1-2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

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.

Polyethylene terephthalate (PET) is widely used for various industrial applications. However, owing to its extremely slow breakdown rate, PET accumulates as plastic trash, which negatively affects the environment and human health. Here, we report two novel PET hydrolases: PpPETase from Pseudomonas paralcaligenes MRCP1333, identified in human feces, and ScPETase from Streptomyces calvus DSM 41452. These two enzymes can decompose various PET materials, including semicrystalline PET powders (Cry-PET) and low-crystallinity PET films (gf-PET). By structure-guided engineering, two variants, PpPETaseY239R/F244G/Y250G and ScPETaseA212C/T249C/N195H/N243K were obtained that decompose Cry-PET 3.1- and 1.9-fold faster than their wild-type enzymes, respectively. The co-expression of ScPETase and mono-(2-hydroxyethyl) terephthalate hydrolase from Ideonella sakaiensis (IsMHETase) resulted in 1.4-fold more degradation than the single enzyme system. This engineered strain degraded Cry-PET and gf-PET by more than 40% and 6%, respectively, after 30 d. The concentrations of terephthalic acid (TPA) in the Cry-PET and gf-PET degradation products were 37.7% and 25.6%, respectively. The discovery of these two novel PET hydrolases provides opportunities to create more powerful biocatalysts for PET biodegradation.

Environmental pollution caused by plastic waste has become global problem that needs to be considered urgently. In the pursuit of a circular plastic economy, biodegradation provides an attractive strategy for managing plastic wastes, whereas effective plastic-degrading microbes and enzymes are required. In this study, we report that Blastobotrys sp. G-9 isolated from discarded plastic in landfills is capable of depolymerizing polyurethanes (PU) and poly (butylene adipate-co-terephthalate) (PBAT). Strain G-9 degrades up to 60% of PU foam after 21 days of incubation at 28 ℃ by breaking down carbonyl groups via secretory hydrolase as confirmed by structural characterization of plastics and degradation products identification. Within the supernatant of strain G-9, we identify a novel cutinase BaCut1, belonging to the esterase family, that can reproduce the same effect. BaCut1 demonstrates efficient degradation toward commercial polyester plastics PU foam (0.5 mg enzyme/25 mg plastic) and agricultural film PBAT (0.5 mg enzyme/10 mg plastic) with 50% and 18% weight loss at 37 ℃ for 48 h, respectively. BaCut1 hydrolyzes PU into adipic acid as a major end-product with 42.9% recovery via ester bond cleavage, and visible biodegradation is also identified from PBAT, which is a beneficial feature for future recycling economy. Molecular docking, along with products distribution, elucidates a special substrate-binding modes of BaCut1 with plastic substrate analogue. BaCut1-mediated polyester plastic degradation offers an alternative approach for managing PU plastic wastes through possible bio-recycling.

Two Gram-stain-negative bacterial strains, R39T and R73T, were isolated from the rhizosphere soil of the selenium hyperaccumulator Cardamine hupingshanesis in China. Strain R39T transformed selenite into elemental and volatile selenium, whereas strain R73T transformed both selenate and selenite into elemental selenium. Phylogenetic and phylogenomic analyses indicated that strain R39T belonged to the genus Achromobacter, while strain R73T belonged to the genus Buttiauxella. Strain R39T (genome size, 6.68 Mb; G+C content, 61.6 mol%) showed the closest relationship to Achromobacter marplatensis LMG 26219T and Achromobacter kerstersii LMG 3441T, with average nucleotide identity (ANI) values of 83.6 and 83.4 %, respectively. Strain R73T (genome size, 5.22 Mb; G+C content, 50.3 mol%) was most closely related to Buttiauxella ferragutiae ATCC 51602T with an ANI value of 86.4 %. Furthermore, strain A111 from the GenBank database was found to cluster with strain R73T within the genus Buttiauxella through phylogenomic analyses. The ANI and digital DNA-DNA hybridization values between strains R73T and A111 were 97.5 and 80.0% respectively, indicating that they belong to the same species. Phenotypic characteristics also differentiated strain R39T and strain R73T from their closely related species. Based on the polyphasic analyses, strain R39T and strain R73T represent novel species of the genera Achromobacter and Buttiauxella, respectively, for which the names Achromobacter seleniivolatilans sp. nov. (type strain R39T=GDMCC 1.3843T=JCM 36009T) and Buttiauxella selenatireducens sp. nov. (type strain R73T=GDMCC 1.3636T=JCM 35850T) are proposed.