Plastic pollution presents a global challenge, impacting ecosystems, wildlife, and economies. Polyethylene terephthalate (PET), widely used in products like bottles, significantly contributes to this issue due to poor waste collection. In recent years, there has been increasing interest in plant biomass-degrading enzymes for plastic breakdown, due to the structural and physicochemical similarities between natural and synthetic polymers. Filamentous fungi involved in hemicellulose degradation have developed a complex mode of action that includes not only enzymes but also biosurfactants; surface-active molecules that facilitate enzyme-substrate interactions. For this reason, this study aimed to mimic the mechanism of biomass degradation by repurposing plant cell wall degrading enzymes including a cutinase and three esterases to cooperatively contribute to PET degradation. Surfactants of different charge were also introduced in the reactions, as their role is similar to biosurfactants, altering the surface tension of the polymers and thus improving enzymes\' accessibility. Notably, Fusarium oxysporum cutinase combined with anionic surfactant exhibited a 2.3- and 1.6-fold higher efficacy in hydrolyzing amorphous and semi-crystalline PET, respectively. When cutinase was combined with either of two ferulic acid esterases, it resulted in complete conversion of PET intermediate products to TPA, increasing the overall product release up to 1.9- fold in presence of surfactant. The combination of cutinase with a glucuronoyl esterase demonstrated significant potential in plastic depolymerization, increasing degradation yields in semi-crystalline PET by up to 1.4-fold. The approach of incorporating enzyme cocktails and surfactants emerge as an efficient solution for PET degradation in mild reaction conditions, with potential applications in eco-friendly plastic waste management.

The lignocellulolytic accessory enzyme, Feruloyl esterase C (FE_5DR), encoded in the genome of thermotolerant Myceliophthora verrucosa was successfully cloned and heterologously expressed in Pichia pastoris. The expressed FE_5DR was purified using UNOsphere™ Q anion exchange chromatography column, exhibiting a homogeneous band of ~ 39 kDa. Its optimum temperature was determined to be 60 °C, with an optimal pH of 6.0. Additionally, the enzyme activity of FE_5DR was significantly enhanced by preincubation in a buffer containing Mg2+, Cu2+ and Ca2 metal ions. Enzyme kinetic parameters, computed from double reciprocal Lineweaver-Burk plots, yielded observed Vmax and Km values of 0.758 U/mg and 0.439 mM, respectively. Furthermore, the potential of custom-made cocktails comprising FE_5DR and benchmark cellulase derived from the developed mutant strain of Aspergillus allahabadii MAN 40, as well as the biorefinery-relevant lignocellulolytic enzyme Cellic CTec 3, resulted in improved saccharification of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw when compared to benchmark MAN 40 and Cellic CTec 3.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s13205-024-04013-7.

BACKGROUND: Xylans are polysaccharides that are naturally abundant in agricultural by-products, such as cereal brans and straws. Microbial degradation of arabinoxylan is facilitated by extracellular esterases that remove acetyl, feruloyl, and p-coumaroyl decorations. The bacterium Ruminiclostridium cellulolyticum possesses the Xua (xylan utilization associated) system, which is responsible for importing and intracellularly degrading arabinoxylodextrins. This system includes an arabinoxylodextrins importer, four intracellular glycosyl hydrolases, and two intracellular esterases, XuaH and XuaJ which are encoded at the end of the gene cluster.
RESULTS: Genetic studies demonstrate that the genes xuaH and xuaJ are part of the xua operon, which covers xuaABCDD\'EFGHIJ. This operon forms a functional unit regulated by the two-component system XuaSR. The esterases encoded at the end of the cluster have been further characterized: XuaJ is an acetyl esterase active on model substrates, while XuaH is a xylan feruloyl- and p-coumaryl-esterase. This latter is active on oligosaccharides derived from wheat bran and wheat straw. Modelling studies indicate that XuaH has the potential to interact with arabinoxylobiose acylated with mono- or diferulate. The intracellular esterases XuaH and XuaJ are believed to allow the cell to fully utilize the complex acylated arabinoxylo-dextrins imported into the cytoplasm during growth on wheat bran or straw.
CONCLUSIONS: This study reports for the first time that a cytosolic feruloyl esterase is part of an intracellular arabinoxylo-dextrin import and degradation system, completing its cytosolic enzymatic arsenal. This system represents a new pathway for processing highly-decorated arabinoxylo-dextrins, which could provide a competitive advantage to the cell and may have interesting biotechnological applications.

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.

The microbial consortium FA12 that can release ferulic acid (FA) by fermenting distiller\'s grains was screened from Daqu. Taibaiella, Comamonadaceae, and Ochrobacum were highly abundant in FA12 by 16S rRNA gene sequencing. In the process of long-term acclimation with distiller\'s grains as a medium, the biomass of FA12 remained stable, and the pH value of fermentation liquid was also relatively stable. Meanwhile, the activities of cellulase, xylanase, and feruloyl esterase secreted by FA12 were stable in the ranges of 0.2350-0.4470, 0.1917-0.3078, and 0.1103-0.1595 U/mL, respectively, and the release of FA could reach 133.77 μg/g. It is proven that the microbial consortium has good genetic stability. In addition, the structural changes of lignocellulose in distiller\'s grains before and after fermentation were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR), and the changes of distiller\'s grains weight and lignocellulose content before and after fermentation were also detected. These results all confirmed that FA12 had the function of degrading distiller\'s grains. In this study, we explored a method to use microbial communities to release FA from distiller\'s grains and degrade lignocellulose in the waste, which opened up a new way for the application of the high value of lost waste.

The use of host to secrete several hemicellulase is a cost-effective way for hemicellulose degradation. In this study, the xylose utilization gene xylAB of Escherichia coli BL21 was knocked out, and the xylanase (N20Xyl), β-xylosidase (Xys), and feruloyl esterase (FaeLam) were co-expressed in this strain. By measuring the content of reducing sugars generated by enzymatic hydrolysis of wheat bran in the fermentation supernatant, the order of the three enzymes was screened to obtain the optimal recombinant strain of E. coli BL21/∆xylAB/pDIII-2. Subsequently, fermentation conditions including culture medium, inducer concentration, induction timing, metal ions, and glycine concentration were optimized. Then, different concentrations of wheat bran and xylan were added to the fermentation medium for degradation. The results showed that the extracellular reducing sugars content reached the highest value of 33.70 ± 0.46 g/L when 50 g/L xylan was added. Besides, the scavenging rates of hydroxyl radical by the fermentation supernatant was 81.0 ± 1.41 %, and the total antioxidant capacity reached 2.289 ± 0.55. Furthermore, it showed the growth promotion effect on different lactic acid bacteria. These results provided a basis for constructing E. coli strain to efficiently degrade hemicellulose, and the strain obtained has great potential application to transform hemicellulose into fermentable carbon source.

In this study, strains producing feruloyl esterase were screened by Oxford Cup clear zones method and by evaluating the ability to decompose hydroxycinnamoyl esters. The strain was identified by 16S rDNA molecular biology. The contents of dietary fiber, reducing sugar, water-extractable arabinoxylans, phytic acid, total phenolics, total flavonoid, phenolic compounds composition, microstructure and antioxidant activity in bran before and after fermentation were studied. Eight strains producing feruloyl esterase were screened, among which strain P1 had the strongest ability to decompose hydroxycinnamoyl esters. The strain was identified and named L. fermentum NB02. Compared with unfermented bran, fermented bran exhibited higher contents of soluble dietary fiber, reducing sugar, water-extractable arabinoxylans, total phenolics, total flavonoid, and lower insoluble dietary fiber and phytic acid content. The dense surface structure of bran was destroyed, forming a porous structure. The release of phenolic compounds increased significantly. L. fermentum NB02 fermentation improved the antioxidant capacity of bran.

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/β-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.

The feruloyl esterase B gene (faeB) is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in faeB induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 Aspergillus niger strains for mutants unable to utilize ferulic acid as a carbon source. The ΔfarA transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express faeB. For this screen a PfaeB-amdS and PfaeB-lux613 dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces amdS (acetamidase) expression via the faeB promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce faeB are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the farA mutant and sequencing the farA gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the farA mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)2Cys6 domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)2Cys6 domain and possesses only the fungal specific transcription factor domain.

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.