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A new phenolic acid decarboxylase gene (blpad) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The full-length blpad encodes a 166-amino acid polypeptide with a predicted molecular mass and pI of 19,521 Da and 5.02, respectively. The recombinant BLPAD displayed maximum activity at 37 °C and pH 6.0. This enzyme possesses a broad substrate specificity and is able to decarboxylate p-coumaric, ferulic, caffeic, and sinapic acids at the relative ratios of specific activities 100:74.59:34.41:0.29. Kinetic constant Kₘvalues toward p-coumaric, ferulic, caffeic, and sinapic acids were 1.64, 1.55, 1.93, and 2.45 mM, and Vₘₐₓvalues were 268.43, 216.80, 119.07, and 0.78 U mg⁻¹, respectively. In comparison with other phenolic acid decarboxylases, BLPAD exhibited remarkable organic solvent tolerance and good thermal stability. BLPAD showed excellent catalytic performance in biphasic organic/aqueous systems and efficiently converted p-coumaric and ferulic acids into 4-vinylphenol and 4-vinylguaiacol. At 500 mM of p-coumaric and ferulic acids, the recombinant BLPAD produced a total 60.63 g l⁻¹4-vinylphenol and 58.30 g l⁻¹4-vinylguaiacol with the conversion yields 97.02 and 70.96 %, respectively. The low yield and product concentration are the crucial drawbacks to the practical bioproduction of vinyl phenol derivatives using phenolic acid decarboxylases. These unusual properties make BLPAD a desirable biocatalyst for commercial use in the bioconversion of hydroxycinnamic acids to vinyl phenol derivatives via enzymatic decarboxylation in a biphasic organic/aqueous reaction system.

Feruloyl esterase (FAE) is a critical enzyme in bio-extraction of ferulic acid (FA) from plant cell wall. A new FAE (EpFAE1) encoding gene was isolated from Eupenicillium parvum and heterologously expressed in Pichia pastoris cells. Based on phylogenetic tree analysis, the protein EpFAE1 belongs to type A of the seventh FAE subfamily. Using methyl ferulate as substrate, the optimum temperature and pH for the catalytic activity of EpFAE1 were 50 °C and 5.5, respectively. The enzyme exhibited high stability at 50 °C, in a wide pH range (3.0–11.0), or in the presence of 2 M of NaCl. Together with the endo-xylanase EpXYN1, EpFAE1 released 72.32% and 4.00% of the alkali-extractable FA from de-starched wheat bran (DSWB) or de-starched corn bran (DSCB), respectively. Meanwhile, the substrates were pretreated with 1.75% (for DSWB) or 1.0% (for DSCB) of phosphoric acid (PA) at 90 °C for 12 h, followed by enzymatic hydrolysis of the soluble and insoluble fractions. The release efficiencies of FA were up to 84.64% for DSWB and 66.73% for DSCB. Combined dilute PA pretreatment with enzymatic hydrolysis is a low-cost and highly efficient method for the extraction of FA from cereal brans.

Background Corn bran arabinoxylan (CBAX) is one of the most structurally complex xylans in nature. The bioconversion of CBAX into value-added products remains challenging because the substrate is resistant to pure xylanases and commercial enzyme cocktails. The carbohydrate-active enzymes (CAZymes) of Penicillium parvum 4–14 have been shown to efficiently hydrolyze CBAX. This study aimed to investigate the expression patterns and functional roles of CAZymes involved in the degradation of complex arabinoxylans in the fungus using transcriptomic and proteomic technologies. Results P. parvum 4–14 grew on CBAX and corn cob arabinoxylan (CCAX) with different substitution patterns and produced secretomes with varied compositions. The CBAX- or CCAX–induced fungal secretomes showed similar ratios (76.2% and 75.1%) on monosaccharide release from CBAX, but the former has a 12% higher ratio on monosaccharide release from CCAX than the latter. Integrated transcriptomic and proteomic analyses revealed distinct patterns of functional gene expression and CAZyme secretion in P. parvum cells induced by the two types of arabinoxylans, implying that the fungus has a complex regulatory system for CAZyme synthesis. A total of 26 CAZymes were inferred to be involved in the degradation of CBAX on the basis of multi-omics data and substrate structures. At the same fungal growth stage (48 h), 24 of these 26 CAZyme showed 0.42- to 5.74-fold higher gene transcription levels under CBAX culture than under CCAX culture. Conclusions Different sources of arabinoxylans significantly affect the production of extracellular CAZymes in P. parvum . These findings are valuable for understanding the key CBAX-degrading enzymes and engineering tailored enzyme systems to valorize complex hemicelluloses. Graphical abstract

Two recombinant cutinases, AfCutA and AfCutB, derived from Aspergillus fumigatus, were heterologously expressed in Pichia pastoris and systematically characterized for their biochemical properties and polyester-degrading capabilities. AfCutA demonstrated superior catalytic performance compared with AfCutB, displaying higher optimal pH (8.0–9.0 vs. 7.0–8.0), higher optimal temperature (60 °C vs. 50 °C), and greater thermostability. AfCutA exhibited increased hydrolytic activity toward p-nitrophenyl esters (C4–C16) and synthetic polyesters. Additionally, AfCutA released approximately 3.2-fold more acetic acid from polyvinyl acetate (PVAc) hydrolysis than AfCutB. Quartz crystal microbalance with dissipation monitoring (QCM-D) revealed rapid adsorption of both enzymes onto polyester films. However, their adsorption capacity on poly (ε-caprolactone) (PCL) films was significantly higher than on polybutylene succinate (PBS) films, and was influenced by pH. Comparative modeling of catalytic domains identified distinct structural differences between the two cutinases. AfCutA possesses a shallower substrate-binding cleft, fewer acidic residues, and more extensive hydrophobic regions around the active site, potentially explaining its enhanced interfacial activation and catalytic efficiency toward synthetic polyester substrates. The notably superior performance of AfCutA suggests its potential as a biocatalyst in industrial applications, particularly in polyester waste bioremediation and sustainable polymer processing.

Background The non-productive adsorption of cellulases onto lignin in biomass is a key issue for the biofuel process economy. It would be helpful to reduce the inhibitory effect of lignin on enzymatic hydrolysis by engineering weak lignin-binding cellulases. Cellulase linkers are highly divergent in their lengths, compositions, and glycosylations. Numerous studies have revealed that linkers can facilitate optimal interactions between structured domains. Recently, efforts have focused on the contributions and mechanisms of carbohydrate-binding modules and catalytic domains that affect lignin affinity and processivity of cellulases, but our understanding of the effects of the linker regions on lignin adsorption and processivity of GH5 processive endoglucanases is still limited. Results Eight GH5 endoglucanase 1 variants of varying length, flexibility, and sequence in the linker region were constructed. Their characteristics were then compared to the wild-type enzyme (EG1). Remarkably, significant differences in the lignin adsorption profiles and processivities were observed for EG1 and other variants. Our studies suggest that either the length or the specific amino acid composition of the linker has a prominent influence on the lignin-binding affinity of the enzymes. Comparatively, the processivity may depend primarily on the length of the linker and less so on the specific amino acid composition. EG1-ApCel5A, a variant with better performance in enzymatic hydrolysis in the presence of lignin, was obtained by replacing a longer, flexible linker. In total, up to between 28.2 and 30.1% more reducing sugars were generated from filter paper by EG1-ApCel5A in the presence of lignin compared to EG1. Conclusions Our results highlight the relevance of the linker region in the lignin adsorption and processivity of a processive endoglucanase. Our findings suggest that the linker region may be used as a target for the design of more active and weaker lignin-binding cellulases.

The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named lac3 and lac4, encoding proteins of 547 and 532-amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete Coprinus comatus. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in Pichia pastoris by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser) tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest Km and highest kcat/Km value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of kcat/Km) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes.

L-Ribulose and L-ribose are two high-value unnatural sugars that can be biosynthesized by sugar isomerases. In this paper, an L-arabinose isomerase (BvAI) from Bacillus velezensis CICC 24777 was cloned and overexpressed in Escherichia coli BL21 (DE3) strain. The maximum activity of recombinant BvAI was observed at 45 °C and pH 8.0, in the presence of 1.0 mM Mn2+. Approximately 207.2 g/L L-ribulose was obtained from 300 g/L L-arabinose in 1.5 h by E. coli harboring BvAI. In addition, approximately 74.25 g/L L-ribose was produced from 300 g/L L-arabinose in 7 h by E. coli co-expressing BvAI and L-RI from Actinotalea fermentans ATCC 43279 (AfRI). This study provides a feasible approach for producing L-ribose from L-arabinose using a co-expression system harboring L-Al and L-RI.

EG1 from Volvariella volvacea is a processive endoglucanase belonging to glycoside hydrolase family 5. The impacts of cotton linter pulp characteristics, such as degree of polymerization (DP), crystallinity, and the initial cellulose-reducing ends, on the processivity of EG1 were investigated. Three commercial cotton linter pulp with different DP were used in present study. Ball milling was used to alter the crystallinity and DP of cellulose. The results indicate that the crystallinity has the most significant impact on enzyme processivity followed by initial cellulose-reducing ends. Whereas the DP indirectly affects the enzymatic hydrolysis and influenced by the pulp preparation method and conditions. The initial cellulose-reducing ends also affect enzyme adsorption but their impact is not obvious when the crystallinity is very low. These results also demonstrate the endo- and exo-action are both exist for EG1. The processive exo-action can start from the newly created cellulose-reducing ends by endo-action as well as the initial cellulose-reducing ends. The contribution of initial cellulose-reducing ends is affected by its quantity and cellulose crystallinity. A plausible action mode for EG1 is also proposed.

Background The recently discovered Pc AA14A and B from white-rot basidiomycete Pycnoporus coccineus enriched our understanding of the oxidative degradation of xylan in fungi, however, the unusual mode of action of AA14 LPMOs has sparked controversy. The substrate specificity and functionality of AA14 LPMOs still remain enigmatic and need further investigation. Results In this study, a novel AA14 LPMO was characterized from the ascomycete Talaromyces rugulosus . Tr AA14A has a broad substrate specificity with strong oxidative activity on pure amorphous cellulose and xyloglucan. It could simultaneously oxidize cellulose, xylan and xyloglucan in natural hemi/cellulosic substrate such as fibrillated eucalyptus pulp, and released native and oxidized cello-oligosaccharides, xylo-oligosaccharides and xyloglucan oligosaccharides from this substrate, but its cellulolytic/hemicellulolytic activity became weaker as the contents of xylan increase in the alkaline-extracted hemi/cellulosic substrates. The dual cellulolytic/hemicellulolytic activity enables Tr AA14A to possess a profound boosting effect on cellulose hydrolysis by cellulolytic enzymes. Structure modelling of Tr AA14A revealed that it exhibits a relatively flat active-site surface similar to the active-site surfaces in AA9 LPMOs but quite distinct from Pc AA14B, despite Tr AA14A is strongly clustered together with AA14 LPMOs. Remarkable difference in electrostatic potentials of L2 and L3 surfaces was also observed among TrAA14A, Pc AA14B and Nc LPMO9F. We speculated that the unique feature in substrate-binding surface might contribute to the cellulolytic/hemicellulolytic activity of Tr AA14A. Conclusions The extensive cellulolytic/hemicellulolytic activity on natural hemi/cellulosic substrate indicated that Tr AA14A from ascomycete is distinctively different from previously characterized xylan-active AA9 or AA14 LPMOs. It may play as a bifunctional enzyme to decompose some specific network structures formed between cellulose and hemicellulose in the plant cell walls. Our findings shed new insights into the novel substrate specificities and biological functionalities of AA14 LPMOs, and will contribute to developing novel bifunctional LPMOs as the booster in commercial cellulase cocktails to efficiently break down the hemicellulose-cellulose matrix in lignocellulose.

d-Mannose and l-ribose are two important monosaccharides, which have attracted public attention recently because of their great application potentials in food, cosmetic and pharmaceutical industries. Sugar isomerases catalyze the sugar isomerization and therefore can be used as the biocatalysts for production of the high-value sugars from inexpensive sugars. l-arabinose isomerase catalyzes the conversion of l-arabinose to l-ribulose, while d-lyxose isomerase catalyzes l-ribulose and d-fructose to l-ribose and d-mannose, respectively. In this paper, a putative d-LI from Bacillus velezensis (BvLI) was identified, characterized and used to produce d-mannose and l-ribose from d-fructose and l-arabinose, respectively. The recombinant BvLI exhibited a maximum activity at 55 °C and pH 6.5, in the presence of 0.1 mM Co2+. Approximately 110.75 g/L d-mannose was obtained from 500 g/L d-fructose in 6 h by the recombinant BvLI, and approximately 105 g/L l-ribose was obtained from 500 g/L l-arabinose in 8 h by the successive biocatalysis of l-arabinose isomerase from Bacillus licheniformis (BlAI) and BvLI.