Novel polyurethane-degrading cutinase BaCut1 from Blastobotrys sp. G-9 with potential role in plastic bio-recycling.

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 â„ƒ 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 â„ƒ 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.

Probing Enzymatic PET Degradation: Molecular Dynamics Analysis of Cutinase Adsorption and Stability.

Understanding the mechanisms influencing poly(ethylene terephthalate) (PET) biodegradation is crucial for developing innovative strategies to accelerate the breakdown of this persistent plastic. In this study, we employed all-atom molecular dynamics simulation to investigate the adsorption process of the LCC-ICCG cutinase enzyme onto the PET surface. Our results revealed that hydrophobic, π-π, and H bond interactions, specifically involving aliphatic, aromatic, and polar uncharged amino acids, were the primary driving forces for the adsorption of the cutinase enzyme onto PET. Additionally, we observed a negligible change in the enzyme's tertiary structure during the interaction with PET (RMSD = 1.35 Å), while its secondary structures remained remarkably stable. Quantitative analysis further demonstrated that there is about a 24% decrease in the number of enzyme-water hydrogen bonds upon adsorption onto the PET surface. The significance of this study lies in unraveling the molecular intricacies of the adsorption process, providing valuable insights into the initial steps of enzymatic PET degradation.

Multiple strategies to improve extracellular secretion and activity of feruloyl esterase.

Feruloyl esterase has a wide range of applications, but there are still problems with low enzyme yield and activity, and complex purification steps. Our previous research found Lactobacillus amylovorus feruloyl esterase could be secreted extracellular in Escherichia coli. In this study, multiple strategies were implemented to maximize the extracellular production of feruloyl esterase with improved activity in E. coli. Firstly, codon-optimized feruloyl esterase was obtained based on the preference of E. coli, resulting in 41.97 % increase in extracellular secretion. Furthermore, by cascading T7 promoters, replacing the 5' UTR, randomly mutating the N-terminal sequence, and co-expressing secretory cofactors, the extracellular secretion was increased by 36.46 %, 31.25 %, 20.66 % and 25.75 %, respectively. Moreover, the feruloyl esterase were mutated to improve the substrate affinity and activity. The catalytic efficiency of Fae-Q134T and Fae-Q198A increased by 4.62-fold and 5.42-fold. Combining above strategies, extracellular feruloyl esterase activity was increased from 2013.70 U/L to 10,349.04 U/L. These results indicated that the activity and yield of feruloyl esterase secreted by E. coli were significantly increased, which laid a foundation for its industrial application.

Characterization of a novel multifunctional ß-glucosidase/xylanase/feruloyl esterase and its effects on improving the quality of Longjing tea.

Herein, a novel multifunctional enzyme ß-glucosidase/xylanase/feruloyl esterase (GXF) was constructed by fusion of ß-glucosidase and bifunctional xylanase/feruloyl esterase. The activities of ß-glucosidase, xylanase, feruloyl esterase and acetyl xylan esterase displayed by GXF were 67.18 %, 49.54 %, 38.92 % and 23.54 %, respectively, higher than that of the corresponding single functional enzymes. Moreover, the GXF performed better in enhancing aroma and quality of Longjing tea than the single functional enzymes and their mixtures. After treatment with GXF, the grassy and floral odors of tea infusion were significantly improved. Moreover, GXF treatment could improve concentrations of flavonoid aglycones of myricetin, kaempferol and quercetin by 68.1-, 81.42- and 77.39-fold, respectively. In addition, GXF could accelerate the release of reducing sugars, ferulic acid and xylo-oligosaccharides by 9.48-, 8.25- and 4.11-fold, respectively. This multifunctional enzyme may have potential applications in other fields such as food production and biomass degradation.

ANCUT1, a novel thermoalkaline cutinase from Aspergillus nidulans and its application on hydroxycinnamic acids lipophilization.

One of the four cutinases encoded in the Aspergillus nidulans genome, ANCUT1, is described here. Culture conditions were evaluated, and it was found that this enzyme is produced only when cutin is present in the culture medium, unlike the previously described ANCUT2, with which it shares 62% amino acid identity. The differences between them include the fact that ANCUT1 is a smaller enzyme, with experimental molecular weight and pI values of 22 kDa and 6, respectively. It shows maximum activity at pH 9 and 60 °C under assayed conditions and retains more than 60% of activity after incubation for 1 h at 60 °C in a wide range of pH values (6-10) after incubations of 1 or 3 h. It has a higher activity towards medium-chain esters and can modify long-chain length hydroxylated fatty acids constituting cutin. Its substrate specificity properties allow the lipophilization of alkyl coumarates, valuable antioxidants and its thermoalkaline behavior, which competes favorably with other fungal cutinases, suggests it may be useful in many more applications.

Production of tannase from a newly isolated yeast, Geotrichum cucujoidarum using agro-residues.

With an aim of producing commercially important tannase enzyme using cheap and readily available agro-residues, leaves of Indian Gooseberry (Phyllanthus emblica) and Jamun (Syzygium cumini), peels of Lemon (Citrus limon), and Pomegranate (Punica granatum) were screened. Newly isolated Geotrichum cucujoidarum was utilized for the study. Preliminary studies indicated that tannase titer obtained is not proportional to the tannin content of the agro-residues and solid state fermentation superior compared to submerged fermentation. Jamun mixed with lemon peel in equal proportion supplemented with minerals under solid-state fermentation gave a tannase titer of 15.46 U/g dry solids. Through successful implantation of Plackett-Burman design, yeast extract concentration, inoculum volume, and amount of substrate were found to be the most significant factors. Further optimization of these three factors through Response Surface Methodology resulted in the 1.7-fold increase in tannase titer. Validation experiments using 3.97 g of Jamun leaves + lemon peel powder mixed with a nutrient solution having (w/v) yeast extract - 1.1%, dextrose - 3%, Urea - 1.125%, potassium chloride - 0.1%, magnesium sulfate heptahydrate - 0.1% with the initial pH of 5, inoculated with 2.48 ml of inoculum gave a tannase titer of 26.43 U/g dry solids after 6 days of solid-state fermentation.

Arabidopsis PHYTOALEXIN DEFICIENT 4 promotes the maturation and nuclear accumulation of immune-related cysteine protease RD19.

Arabidopsis PHYTOALEXIN DEFICIENT 4 (PAD4) has an essential role in pathogen resistance as a heterodimer with ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1). Here we investigated an additional PAD4 role in which it associates with and promotes the maturation of the immune-related cysteine protease RESPONSIVE TO DEHYDRATION 19 (RD19). We found that RD19 and its paralog RD19c promoted EDS1- and PAD4-mediated effector-triggered immunity to an avirulent Pseudomonas syringae strain, DC3000, expressing the effector AvrRps4 and basal immunity against the fungal pathogen Golovinomyces cichoracearum. Overexpression of RD19, but not RD19 protease-inactive catalytic mutants, in Arabidopsis transgenic lines caused EDS1- and PAD4-dependent autoimmunity and enhanced pathogen resistance. In these lines, RD19 maturation to a pro-form required its catalytic residues, suggesting that RD19 undergoes auto-processing. In transient assays, PAD4 interacted preferentially with the RD19 pro-protease and promoted its nuclear accumulation in leaf cells. Our results lead us to propose a model for PAD4-stimulated defense potentiation. PAD4 promotes maturation and nuclear accumulation of processed RD19, and RD19 then stimulates EDS1-PAD4 dimer activity to confer pathogen resistance. This study highlights potentially important additional PAD4 functions that eventually converge on canonical EDS1-PAD4 dimer signaling in plant immunity.

Rational and random mutagenesis of GDEst-95 carboxylesterase: New functionality insights.

Lipolytic enzymes are important contributors in industrial processes from lipid hydrolysis to biofuel production or even polyester biodegradation. While these enzymes can be used in numerous applications, the genotype-phenotype space of certain promising enzymes is still poorly explored. This limits the effective application of such biocatalysts. In this work the genotype space of a 55 kDa carboxylesterase GDEst-95 from Geobacillus sp. 95 was explored using site-directed mutagenesis and directed evolution methods. In this study four site-directed mutants (Gly108Arg, Ala410Arg, Leu226Arg, Leu411Ala) were created based on previous analysis of GDEst-95 carboxylesterase. Error-prone PCR resulted three mutants: two of them with distal mutations: GDEst-RM1 (Arg75Gln), GDEst-RM2 (Gly20Ser Arg75Gln) and the third, GDEst-RM3, with a distal (Ser210Gly) and Tyr317Ala (amino acid position near to the active site) mutation. Mutants with Ala substitution displayed approximately twofold higher specific activity. Arg mutations lead a reduced specific activity, retaining 2.86 % (Gly108Arg), 10.95 % (Ala410Arg), and 44.23 % (Leu226Arg) of lipolytic activity. All three random mutants displayed increased specific activity as well as improved catalytic properties. This research provides the first deeper insights into the functionality of understudied Geobacillus spp. carboxylesterases with 55 kDa in size.

Functional characterization and structural insights of three cutinases from the ascomycete Fusarium verticillioides.

Cutinases are serine esterases that belong to the α/ß hydrolases superfamily. The natural substrates for these enzymes are cutin and suberin, components of the plant cuticle, the first barrier in the defense system against pathogen invasion. It is well-reported that plant pathogens produce cutinases to facilitate infection. Fusarium verticillioides, one important corn pathogens, is an ascomycete upon which its cutinases are poorly explored. Consequently, the objective of this study was to perform the biochemical characterization of three precursor cutinases (FvCut1, FvCut2, and FvCut3) from F. verticillioides and to obtain structural insights about them. The cutinases were produced in Escherichia coli and purified. FvCut1, FvCut2, and FvCut3 presented optimal temperatures of 20, 40, and 35 °C, and optimal pH of 9, 7, and 8, respectively. Some chemicals stimulated the enzymatic activity. The kinetic parameters revealed that FvCut1 has higher catalytic efficiency (Kcat/Km) in the p-nitrophenyl-butyrate (p-NPB) substrate. Nevertheless, the enzymes were not able to hydrolyze polyethylene terephthalate (PET). Furthermore, the three-dimensional models of these enzymes showed structural differences among them, mainly FvCut1, which presented a narrower opening cleft to access the catalytic site. Therefore, our study contributes to exploring the diversity of fungal cutinases and their potential biotechnological applications.

Structural insights into the molecular mechanisms of substrate recognition and hydrolysis by feruloyl esterase from Aspergillus sydowii.

The depolymerization of lignocellulosic biomass is facilitated by feruloyl esterases (FAEs), which hydrolyze ester bonds between lignin and polysaccharides. Fungal FAEs belonging to subfamily (SF) 6 release precursors such as ferulic acid derivatives, attractive for biochemical production. Among these, Aspergillus sydowii FAE (AsFaeE), an SF6 FAE, exhibits remarkable activity across various substrates. In this study, we conducted X-ray crystallography and kinetic analysis to unravel the molecular mechanisms governing substrate recognition and catalysis by AsFaeE. AsFaeE exhibits a typical α/ß-hydrolase fold, characterized by a catalytic triad of serine, aspartate, and histidine. Comparative analysis of substrate-free, ferulic acid-bound, and sinapic acid-bound forms of AsFaeE suggests a conformational change in the loop covering the substrate-binding pocket upon binding. Notably, Pro158 and Phe159 within this loop cover the phenolic part of the substrate, forming three layers of planar rings. Our structure-based functional mutagenesis clarifies the roles of the residues involved in substrate binding and catalytic activity. Furthermore, distinct substrate-binding mechanisms between AsFaeE and other studied FAEs are identified. This investigation offers the initial structural insights into substrate recognition by SF6 FAEs, equipping us with structural knowledge that might facilitate the design of FAE variants capable of efficiently processing a wider range of substrate sizes.

Improvement of α-amino Ester Hydrolase Stability via Computational Protein Design.

Amino ester hydrolases (AEHs) are capable of rapid synthesis of cephalexin but suffer from rapid deactivation even at low temperatures. Previous efforts to engineer AEH have generated several improved variants but have been limited in scope in part due to limitations in activity assay throughput for ß-lactam synthesis reactions. Rational design of 'whole variants' was explored to rapidly improve AEH thermostability by mutating between 3-15% of residues. Most variants were found to be inactive due to a mutated calcium binding site, the function of which has not previously been described. Four active variants, all with improved melting temperatures, were characterized in terms of synthesis and hydrolysis activity, melting temperature, and deactivation at 25°C. Two variants were found to have improved total turnover numbers relative to the initial AEH variant; however, a clear tradeoff exists between improved stability and overall activity of each variant.

Computational analysis of carboxylesterase genes and proteins in non-pathogenic food bacterium Alicyclobacillus acidocaldarius: insights from proteogenomics.

Over recent years, Alicyclobacillus acidocaldarius, a Gram-positive nonpathogenic rod-shaped thermo-acid-tolerant bacterium, has posed numerous challenges for the fruit juice industry. However, the bacterium's unique characteristics, particularly its nonpathogenic and thermophilic capabilities, offer significant opportunities for genetic exploration by biotechnologists. This study presents the computational proteogenomics report on the carboxylesterase (CE) enzyme in A. acidocaldarius, shedding light on structural and evolutional of CEs from this bacterium. Our analysis revealed that the average molecular weight of CEs in A. acidocaldarius was 41 kDa, with an isoelectric point around 5. The amino acid composition favored negative amino acids over positive ones. The aliphatic index and hydropathicity were approximately 88 and - 0.15, respectively. While the protein sequence showed no disulfide bonds in the CEs' structure, the presence of Cys amino acids was observed in the structure of CEs. Phylogenetic analysis presented more than 99% similarity between CEs, indicating their close evolutionary relationship. By applying homology modeling, the 3-dimensional structural models of the carboxylesterase were constructed, which with the help of structural conservation and solvent accessibility analysis highlighted key residues and regions responsible for enzyme stability and conformation. The specific patterns presented the total solvent accessibility of less than 25 (Å2) was in considerable position as well as Gly residues were noticeably have high accessibility to solvent in all structures. Ala was the more frequent amino acids in the conserved-SASA of carboxylesterases. Furthermore, unsupervised agglomerative hierarchical clustering based on solvent accessibility feature successfully clustered and even distinguished this enzyme from proteases from the same genome. These findings contribute to a deeper understanding of the nonpathogenic A. acidocaldarius carboxylesterase and its potential applications in biotechnology. Additionally, structural analysis of CEs would help to address potential solutions in fruit juice industry with utilization of computational structural biology.

The structure of a Lactobacillus helveticus chlorogenic acid esterase and the dynamics of its insertion domain provide insights into substrate binding.

Chlorogenic acid esterases (ChlEs) are a useful class of enzymes that hydrolyze chlorogenic acid (CGA) into caffeic and quinic acids. ChlEs can break down CGA in foods to improve their sensory properties and release caffeic acid in the digestive system to improve the absorption of bioactive compounds. This work presents the structure, molecular dynamics, and biochemical characterization of a ChlE from Lactobacillus helveticus (Lh). Molecular dynamics simulations suggest that substrate access to the active site of LhChlE is modulated by two hairpin loops above the active site. Docking simulations and mutational analysis suggest that two residues within the loops, Gln145 and Lys164 , are important for CGA binding. Lys164 provides a slight substrate preference for CGA, whereas Gln145 is required for efficient turnover. This work is the first to examine the dynamics of a bacterial ChlE and provides insights on substrate binding preference and turnover in this type of enzyme.

Structural insights into the oligomeric effects on catalytic activity of a decameric feruloyl esterase and its application in ferulic acid production.

Oligomeric feruloyl esterase (FAE) has great application prospect in industry due to its potentially high stability and fine-tuned activity. However, the relationship between catalytic capability and oligomeric structure remains undetermined. Here we identified and characterized a novel, cold-adapted FAE (BtFae) derived from Bacteroides thetaiotaomicron. Structural studies unraveled that BtFae adopts a barrel-like decameric architecture unique in esterase families. By disrupting the interface, the monomeric variant exhibited significantly reduced catalytic activity and stability toward methyl ferulate, potentially due to its impact on the flexibility of the catalytic triad. Additionally, our results also showed that the monomerization of BtFae severely decreased the ferulic acid release from de-starched wheat bran and insoluble wheat arabinoxylan by 75 % and 80 %, respectively. Collectively, this study revealed novel connections between oligomerization and FAE catalytic function, which will benefit for further protein engineering of FAEs at the quaternary structure level for improved industrial applications.

Harnessing extremophilic carboxylesterases for applications in polyester depolymerisation and plastic waste recycling.

The steady growth in industrial production of synthetic plastics and their limited recycling have resulted in severe environmental pollution and contribute to global warming and oil depletion. Currently, there is an urgent need to develop efficient plastic recycling technologies to prevent further environmental pollution and recover chemical feedstocks for polymer re-synthesis and upcycling in a circular economy. Enzymatic depolymerization of synthetic polyesters by microbial carboxylesterases provides an attractive addition to existing mechanical and chemical recycling technologies due to enzyme specificity, low energy consumption, and mild reaction conditions. Carboxylesterases constitute a diverse group of serine-dependent hydrolases catalysing the cleavage and formation of ester bonds. However, the stability and hydrolytic activity of identified natural esterases towards synthetic polyesters are usually insufficient for applications in industrial polyester recycling. This necessitates further efforts on the discovery of robust enzymes, as well as protein engineering of natural enzymes for enhanced activity and stability. In this essay, we discuss the current knowledge of microbial carboxylesterases that degrade polyesters (polyesterases) with focus on polyethylene terephthalate (PET), which is one of the five major synthetic polymers. Then, we briefly review the recent progress in the discovery and protein engineering of microbial polyesterases, as well as developing enzyme cocktails and secreted protein expression for applications in the depolymerisation of polyester blends and mixed plastics. Future research aimed at the discovery of novel polyesterases from extreme environments and protein engineering for improved performance will aid developing efficient polyester recycling technologies for the circular plastics economy.

Sequence, structure and functionality of pectin methylesterases and their use in sustainable carbohydrate bioproducts: A review.

Pectin methylesterases (PMEs) are enzymes that play a critical role in modifying pectins, a class of complex polysaccharides in plant cell walls. These enzymes catalyze the removal of methyl ester groups from pectins, resulting in a change in the degree of esterification and consequently, the physicochemical properties of the polymers. PMEs are found in various plant tissues and organs, and their activity is tightly regulated in response to developmental and environmental factors. In addition to the biochemical modification of pectins, PMEs have been implicated in various biological processes, including fruit ripening, defense against pathogens, and cell wall remodelling. This review presents updated information on PMEs, including their sources, sequences and structural diversity, biochemical properties and function in plant development. The article also explores the mechanism of PME action and the factors influencing enzyme activity. In addition, the review highlights the potential applications of PMEs in various industrial sectors related to biomass exploitation, food, and textile industries, with a focus on development of bioproducts based on eco-friendly and efficient industrial processes.

A near-infrared fluorescent probe based on a hemi-cyanine skeleton for detecting CES1 activity and evaluating pesticide toxicity.

A novel near-infrared (NIR) fluorescent probe CHC-CES1 based on a hemi-cyanine skeleton for detecting carboxylesterase 1 (CES1) activity was developed. Herein, CHC-CES1 could be specifically hydrolysed to CHC-COOH along with a significant NIR fluorescence signal enhancement at 670 nm. Systematic evaluation indicated that CHC-CES1 possessed an outstanding selectivity and sensitivity towards CES1, and possessed good chemical stability in complex biosamples. Finally, CHC-CES1 was successfully used for the real-time imaging of endogenous CES1 activity in living cells. Moreover, CHC-CES1 was applied to evaluate the inhibitory effects of various pesticides towards CES1, and visually revealed the inhibitory effect of combined residue pesticides.

Selection and Molecular Response of AHL-lactonase (aiiA) Producing Bacillus sp. Under Penicillin G-induced Conditions.

Quorum sensing (QS) is the process by which microorganisms employ chemicals called autoinducers (AIs) to communicate with their population. The QS mechanism generally controls the expression of the virulence related genes in bacteria. N-acyl homoserine lactones (AHLs) are the most widespread QS molecules. Due to their diverse AHL-lactonase activities, Bacillus species make particularly suitable candidates for procedures such as demolition of pathogenic bacterial QS signals and bioremediation of ß-lactam antibiotics from contaminated environments. In this study, seven Bacillus strains with Quorum quenching (QQ) activity were isolated using an enrichment medium supplemented with Penicillin G (PenG). The AHL-lactonase encoding gene (aiiA) was amplified by PCR and sequenced. Amino acid sequences underwent multiple sequence alignment. Docking studies were carried out with both C6HSL and PenG ligand using AutoDock tools. The aiiA amino acid sequences of the isolates were found to be well conserved. Furthermore, amino acid sequence alignment revealed that 74.9% of amino acid sequences were conserved in the genus Bacillus. Docking of the C6HSL to wild type (3DHA) and H97D variant reduced the docking score by only 0.1 kcal/mol for the mutated protein. When PenG docked with a higher (1.5 kcal/mol) score as a ligand to wild-type and mutant receptors, the docking score for the mutated protein likewise decreased by 0.1 kcal/mol. This research contributed to the diversification of organisms with QQ activity and beta-lactam antibiotic resistance. It also clarified the binding score of the PenG ligand to the Bacillus AHL lactonase molecule for the first time.

Biochemical Characterization and NMR Study of a PET-Hydrolyzing Cutinase from Fusarium solani pisi.

In recent years, the drawbacks of plastics have become evident, with plastic pollution becoming a major environmental issue. There is an urgent need to find solutions to efficiently manage plastic waste by using novel recycling methods. Biocatalytic recycling of plastics by using enzyme-catalyzed hydrolysis is one such solution that has gained interest, in particular for recycling poly(ethylene terephthalate) (PET). To provide insights into PET hydrolysis by cutinases, we have here characterized the kinetics of a PET-hydrolyzing cutinase from Fusarium solani pisi (FsC) at different pH values, mapped the interaction between FsC and the PET analogue BHET by using NMR spectroscopy, and monitored product release directly and in real time by using time-resolved NMR experiments. We found that primarily aliphatic side chains around the active site participate in the interaction with BHET and that pH conditions and a mutation around the active site (L182A) can be used to tune the relative amounts of degradation products. Moreover, we propose that the low catalytic performance of FsC on PET is caused by poor substrate binding combined with slow MHET hydrolysis. Overall, our results provide insights into obstacles that preclude efficient PET hydrolysis by FsC and suggest future approaches for overcoming these obstacles and generating efficient PET-hydrolyzing enzymes.

Crystal structure of the Fusarium oxysporum tannase-like feruloyl esterase FaeC in complex with p-coumaric acid provides insight into ligand binding.

Feruloyl esterases (FAEs) hydrolyze the ester bonds between hydroxycinnamic acids and arabinose residues of plant cell walls and exhibit considerable diversity in terms of substrate specificity. Here, we report the crystal structure of an FAE from Fusarium oxysporum (FoFaeC) at 1.7 Å resolution in complex with p-coumaric acid, which is the first ligand-bound structure of a tannase-like FAE. Our data reveal local conformational changes around the active site upon ligand binding, suggesting alternation between an active and a resting state of the enzyme. A swinging tyrosine residue appears to be gating the substrate binding pocket, while the lid domain of the protein exerts substrate specificity by means of a well-defined hydrophobic core that encases the phenyl moiety of the substrate.