Biochemical and Regulatory Analyses of Xylanolytic Regulons in Caldicellulosiruptor bescii Reveal Genus-Wide Features of Hemicellulose Utilization.

Caldicellulosiruptor species scavenge carbohydrates from runoff containing plant biomass that enters hot springs and from grasses that grow in more moderate parts of thermal features. While only a few Caldicellulosiruptor species can degrade cellulose, all known species are hemicellulolytic. The most well-characterized species, Caldicellulosiruptor bescii, decentralizes its hemicellulase inventory across five different genomic loci and two isolated genes. Transcriptomic analyses, comparative genomics, and enzymatic characterization were utilized to assign functional roles and determine the relative importance of its six putative endoxylanases (five glycoside hydrolase family 10 [GH10] enzymes and one GH11 enzyme) and two putative exoxylanases (one GH39 and one GH3) in C. bescii. Two genus-wide conserved xylanases, C. bescii XynA (GH10) and C. bescii Xyl3A (GH3), had the highest levels of sugar release on oat spelt xylan, were in the top 10% of all genes transcribed by C. bescii, and were highly induced on xylan compared to cellulose. This indicates that a minimal set of enzymes are used to drive xylan degradation in the genus Caldicellulosiruptor, complemented by hemicellulolytic inventories that are tuned to specific forms of hemicellulose in available plant biomasses. To this point, synergism studies revealed that the pairing of specific GH family proteins (GH3, -11, and -39) with C. bescii GH10 proteins released more sugar in vitro than mixtures containing five different GH10 proteins. Overall, this work demonstrates the essential requirements for Caldicellulosiruptor to degrade various forms of xylan and the differences in species genomic inventories that are tuned for survival in unique biotopes with variable lignocellulosic substrates. IMPORTANCE Microbial deconstruction of lignocellulose for the production of biofuels and chemicals requires the hydrolysis of heterogeneous hemicelluloses to access the microcrystalline cellulose portion. This work extends previous in vivo and in vitro efforts to characterize hemicellulose utilization by integrating genomic reconstruction, transcriptomic data, operon structures, and biochemical characteristics of key enzymes to understand the deployment and functionality of hemicellulases by the extreme thermophile Caldicellulosiruptor bescii. Furthermore, comparative genomics of the genus revealed both conserved and divergent mechanisms for hemicellulose utilization across the 15 sequenced species, thereby paving the way to connecting functional enzyme characterization with metabolic engineering efforts to enhance lignocellulose conversion.

Conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode.

Cellulosomes are multi-enzyme complexes produced by specialised micro-organisms. The spatial proximity of synergistically acting enzymes incorporated in these naturally occurring complexes supports the efficient hydrolysis of lignocellulosic biomass. Several functional designer cellulosomes, incorporating naturally non-cellulosomal cellulases, have been constructed and can be used for cellulose saccharification. However, in lignocellulosic biomass, cellulose is tightly intertwined with several hemicelluloses and lignin. One of the most abundant hemicelluloses interacting with cellulose microfibrils is xyloglucan, and degradation of these polymers is crucial for complete saccharification. Yet, designer cellulosome studies focusing on the incorporation of hemicellulases have been limited. Here, we report the conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode. Therefore, we constructed multiple docking enzyme variants of C. japonicus endoxyloglucanase, ß-1,2-galactosidase, α-1,6 xylosidase and ß-1,4-glucosidase, using the combinatorial VersaTile technique dedicated to the design and optimisation of modular proteins. We individually optimised the docking enzymes to degrade the xyloglucan backbone and side chains. Finally, we show that a purified designer xyloglucanosome comprising these docking enzymes was able to release xyloglucan oligosaccharides, galactose, xylose and glucose from tamarind xyloglucan. KEY POINTS: • Construction of xyloglucan-degrading designer cellulosome. • Conversion of free Cellvibrio japonicus enzymes to cellulosomal mode. • Type of linker inserted between dockerin and enzyme module affects docking enzyme activity.

Down-regulation of MANNANASE7 gene in Brassica napus L. enhances silique dehiscence-resistance.

KEY MESSAGE: MANNANASE7 gene in Brassica napus L. encodes a hemicellulose which located at cell wall or extracellular space and dehiscence-resistance can be manipulated by altering the expression of MANNANASE7. Silique dehiscence is an important physiological process in plant reproductive development, but causes heavy yield loss in crops. The lack of dehiscence-resistant germplasm limits the application of mechanized harvesting and greatly restricts the rapeseed (Brassica napus L.) production. Hemicellulases, together with cellulases and pectinases, play important roles in fruit development and maturation. The hemicellulase gene MANNANASE7 (MAN7) was previously shown to be involved in the development and dehiscence of Arabidopsis (Arabidopsis thaliana) siliques. Here, we cloned BnaA07g12590D (BnMAN7A07), an AtMAN7 homolog from rapeseed, and demonstrate its function in the dehiscence of rapeseed siliques. We found that BnMAN7A07 was expressed in both vegetative and reproductive organs and significantly highly expressed in leaves, flowers and siliques where the abscission or dehiscence process occurs. Subcellular localization experiment showed that BnMAN7A07 was localized in the cell wall. The biological activity of the BnMAN7A07 protein isolated and purified through prokaryotic expression system was verified to catalyse the decomposition of xylan into xylose. Phenotypic studies of RNA interference (RNAi) lines revealed that down-regulation of BnMAN7A07 in rapeseed could significantly enhance silique dehiscence-resistance. In addition, the expression of upstream silique development regulators is altered in BnMAN7A07-RNAi plants, suggesting that a possible feedback regulation mechanism exists in the regulation network of silique dehiscence. Our results demonstrate that dehiscence-resistance can be manipulated by altering the expression of hemicellulase gene BnMAN7A07, which could provide an available genetic resource for breeding practice in rapeseed which is beneficial to mechanized harvest.

ß-Xylosidases: Structural Diversity, Catalytic Mechanism, and Inhibition by Monosaccharides.

Xylan, a prominent component of cellulosic biomass, has a high potential for degradation into reducing sugars, and subsequent conversion into bioethanol. This process requires a range of xylanolytic enzymes. Among them, ß-xylosidases are crucial, because they hydrolyze more glycosidic bonds than any of the other xylanolytic enzymes. They also enhance the efficiency of the process by degrading xylooligosaccharides, which are potent inhibitors of other hemicellulose-/xylan-converting enzymes. On the other hand, the ß-xylosidase itself is also inhibited by monosaccharides that may be generated in high concentrations during the saccharification process. Structurally, ß-xylosidases are diverse enzymes with different substrate specificities and enzyme mechanisms. Here, we review the structural diversity and catalytic mechanisms of ß-xylosidases, and discuss their inhibition by monosaccharides.

Efficient Enzymatic Hydrolysis of Biomass Hemicellulose in the Absence of Bulk Water.

Current enzymatic methods for hemicellulosic biomass depolymerization are solution-based, typically require a harsh chemical pre-treatment of the material and large volumes of water, yet lack in efficiency. In our study, xylanase (E.C. 3.2.1.8) from Thermomyces lanuginosus is used to hydrolyze xylans from different sources. We report an innovative enzymatic process which avoids the use of bulk aqueous, organic or inorganic solvent, and enables hydrolysis of hemicellulose directly from chemically untreated biomass, to low-weight, soluble oligoxylosaccharides in >70% yields.

Biotransformation with cellulase, hemicellulase and Yarrowia lipolytica boosts health benefits of okara.

Okara (soybean residue) is a highly perishable food processing by-product from soymilk and tofu manufacture. It contains a large proportion of insoluble dietary fibre (40-60% on a dry basis), as well as digestion-resistant proteins, trypsin inhibitors and phytic acid. These factors contribute lead to the under-utilisation of okara. To improve the overall nutritional quality of okara, sequential saccharification of okara by Celluclast® 1.5L (cellulase) or Viscozyme® L (cellulase and hemicellulase) and fermentation by the yeast Yarrowia lipolytica were performed. The changes in the antioxidant capacity, amino acids, phenolic acids, isoflavones, phytic acid and dietary fibre during biotransformation were studied. Carbohydrase pre-treatment increased the amounts of monosaccharides, trans-cinnamic acid and aglycone isoflavones in okara. After fermentation, the okara had higher antioxidant activity and greater amounts of total amino acids and ferulic acid. Some positive interactions between the carbohydrase and Y. lipolytica were hypothesised: the carbohydrase and Y. lipolytica proteases could have synergised with each other to break down the okara secondary cell wall more efficiently. After 52 h, Celluclast® 1.5 L and Viscozyme® L significantly reduced the insoluble dietary fibre content from 61.9 ± 0.6 to 45.6 ± 3.0% and 24.7 ± 0.3%, respectively (all w/w, dry basis), while increasing the soluble dietary fibre content by about onefold. Both carbohydrases also increased the amounts of monosaccharides, trans-cinnamic acid, and aglycone isoflavones in okara. The addition of Y. lipolytica led to a higher antioxidant capacity and greater amounts of total amino acids and ferulic acid in okara. The overall improvements in the digestibility and potential health benefits of okara highlight the promising applicability of biotransformation in okara valorisation.

Enzymatic Hydrolysis of Water Extractable Polysaccharides from Leaves of Plantago major L.

BACKGROUND: Plantago major L. leaves have been used for centuries by the traditional medicine in the treatment of infectious disorders of the respiratory, urinary and digestive tracts. Researchers have reported that hot water extracts of Plantago major possess a broad-spectrum of anticancer, antioxidant and antiviral activities, as well as activities which modulate cell-mediated immunity. Their beneficial properties may be due to the significant content of polysaccharides. The polysaccharides that have been isolated from the leaves of Plantago major L. have different structures - pectic substances, galactans, arabinogalactans, glucomannans. AIM: The aim of this paper was to study the correlation between the structure of the water extractable polysaccharides isolated from Plantago major L. leaves and their enzymatic hydrolysis with different carbohydrate hydrolases. MATERIALS AND METHODS: The hydrolysis reactions were performed with the enzymes hemicellulase and mannanase. Spectrophotometric total reducing sugars assay was used to examine the hydrolysis yield. The monosaccharide and oligosaccharide compositions were determined using HPLC analysis. RESULTS: The highest hydrolysis yield of the water extractable polysaccharides from Plantago major leaves was obtained by treatment with hemicellulase. The hydrolysis yield increased with the augmentation of the ratio of enzyme to polysaccharide. Galactose was the prevalent monosaccharide identified in the composition of the isolated polysaccharides. Oligosaccharides with different degree of polymerization were also detected. CONCLUSION: The enzymatic hydrolysis of water extractable polysaccharides from Plantago major leaves allows us to obtain different types of oligosaccharides with beneficial effects on both human health and industry.

Quantitative Site-Specific Phosphoproteomics of Trichoderma reesei Signaling Pathways upon Induction of Hydrolytic Enzyme Production.

The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed.

Penicillium oxalicum PoFlbC regulates fungal asexual development and is important for cellulase gene expression.

Filamentous fungi can initiate vegetative growth on complex plant polysaccharides in nature through secreting a large amount of lignocellulose-degrading enzymes. These fungi develop a large amount of asexual spores to disperse and survive under harsh conditions, such as carbon and nitrogen depletion. Numerous studies report the presence of a cross-talk between asexual development and extracellular enzyme production, especially at the regulation level. This study identified and characterized a C2H2-type transcription factor called PoFlbC, which is an Aspergillus FlbC ortholog, in cellulolytic fungus Penicillium oxalicum. Results showed that the native level of PoFlbC was crucial for the normal growth and asexual development of P. oxalicum. Importantly, deletion of the PoflbC gene substantially reduced cellulase and hemicellulase productions. Comparative transcriptome analysis by RNA sequencing revealed a global downregulation of genes encoding cellulases, hemicellulases, and other proteins with functions in lignocellulose degradation. A similar defect was also observed in the OEPoflbC strain, suggesting that the production of cellulolytic enzymes was maintained by native expression of the PoflbC. In this study, an essential activator for both fungal asexual development and cellulase production was established in P. oxalicum.

Involvement of an SRF-MADS protein McmA in regulation of extracellular enzyme production and asexual/sexual development in Aspergillus nidulans.

SRF-MADS proteins are transcription factors conserved among eukaryotes that regulate a variety of cellular functions; however, their physiological roles are still not well understood in filamentous fungi. Effects of a mutation in mcmA gene that encodes the sole SRF-MADS protein in the fungus Aspergillus nidulans were examined by RNA sequencing. Sequencing data revealed that expression levels of cellulase genes were significantly decreased by the mutation as reported previously. However, expression levels of various hemicellulolytic enzyme genes, several extracellular protease genes, the nosA and rosA genes involved in sexual development, and AN4394 encoding an ortholog of EcdR involved in Aspergillus oryzae conidiation, were also significantly decreased by the mutation. As expected from the RNA sequencing data, the mcmA mutant had reduced protease production, cleistothecial development, and conidiation. This is the first report describing the involvement of SRF-MADS proteins in protease production in fungi, and asexual and sexual development in Aspergillus.

Production of ß-xylosidase from Trichoderma asperellum KIF125 and its application in efficient hydrolysis of pretreated rice straw with fungal cellulase.

On-site cellulase and hemicellulase production is a promising way to reduce enzyme cost in the commercialization of the lignocellulose-to-ethanol process. A hemicellulase-producing fungal strain suitable for on-site enzyme production was selected from cultures prepared using wet disc-milling rice straw (WDM-RS) and identified as Trichoderma asperellum KIF125. KIF125 hemicellulase showed uniquely high abundance of ß-xylosidase in the xylanolytic enzyme system compared to other fungal hemicellulase preparations. Supplementation of Talaromyces cellulolyticus cellulase with KIF125 hemicellulase was more effective than that with the hemicellulases from other fungal sources in reducing the total enzyme loading for the improvement of xylose yield in the hydrolysis of ball-milling RS, due to its high ß-xylosidase dominance. ß-Xylosidase in KIF125 hemicellulase was purified and classified as a glycosyl hydrolase family 3 enzyme with relatively high specificity for xylobiose. The production of KIF125 ß-xylosidase in the fermentor was estimated as 118 U/g-WDM-RS (2350 U/L culture) at 48 h. These results demonstrate that KIF125 is promising as a practical hemicellulase source to combine with on-site cellulase production using T. cellulolyticus.

First structural insights into α-L-arabinofuranosidases from the two GH62 glycoside hydrolase subfamilies.

α-L-arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-L-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-L-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-L-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed ß-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-L-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family.

The genus Geobacillus and their biotechnological potential.

The genus Geobacillus comprises a group of Gram-positive thermophilic bacteria, including obligate aerobes, denitrifiers, and facultative anaerobes that can grow over a range of 45-75°C. Originally classified as group five Bacillus spp., strains of Bacillus stearothermophilus came to prominence as contaminants of canned food and soon became the organism of choice for comparative studies of metabolism and enzymology between mesophiles and thermophiles. More recently, their catabolic versatility, particularly in the degradation of hemicellulose and starch, and rapid growth rates have raised their profile as organisms with potential for second-generation (lignocellulosic) biorefineries for biofuel or chemical production. The continued development of genetic tools to facilitate both fundamental investigation and metabolic engineering is now helping to realize this potential, for both metabolite production and optimized catabolism. In addition, this catabolic versatility provides a range of useful thermostable enzymes for industrial application. A number of genome-sequencing projects have been completed or are underway allowing comparative studies. These reveal a significant amount of genome rearrangement within the genus, the presence of large genomic islands encompassing all the hemicellulose utilization genes and a genomic island incorporating a set of long chain alkane monooxygenase genes. With G+C contents of 45-55%, thermostability appears to derive in part from the ability to synthesize protamine and spermine, which can condense DNA and raise its Tm.

High Genetic Diversity of Microbial Cellulase and Hemicellulase Genes in the Hindgut of Holotrichia parallela Larvae.

In this study, we used a culture-independent method based on library construction and sequencing to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of Holotrichia parallela larvae. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela. The total of 101 distinct gene fragments (similarity <95%) of glycosyl hydrolase families including GH2 (24 genes), GH8 (27 genes), GH10 (19 genes), GH11 (14 genes) and GH36 (17 genes) families was retrieved, and certain sequences of GH2 (10.61%), GH8 (3.33%), and GH11 (18.42%) families had <60% identities with known sequences in GenBank, indicating their novelty. Based on phylogenetic analysis, sequences from hemicellulase families were related to enzymes from Bacteroidetes and Firmicutes. Fragments from cellulase family were most associated with the phylum of Proteobacteria. Furthermore, a full-length endo-xylanase gene was obtained, and the enzyme exhibited activity over a broad range of pH levels. Our results indicate that there are large number of cellulolytic and xylanolytic bacteria in the hindgut of H. parallela larvae, and these symbiotic bacteria play an important role in the degradation of roots and other organic matter for the host insect.

Tailoring a cellulolytic enzyme cocktail for efficient hydrolysis of mildly pretreated lignocellulosic biomass.

Commercially available cellulase cocktails frequently demonstrate high efficiency in hydrolyzing easily digestible pretreated biomass, which often lacks hemicellulose and/or lignin fractions. However, the challenge arises with enzymatic hydrolysis of mildly pretreated lignocellulosic biomasses, which contain cellulose, hemicellulose and lignin in high proportions. This study aimed to address this question by evaluating the supplementation of a commercial cellulolytic cocktail with accessory hemicellulases and two additives (H2O2 and Tween® 80). Statistical optimization methods were employed to enhance the release of glucose and xylose from mildly pretreated sugarcane bagasse. The optimized supplement composition resulted in the production of 304 and 124 mg g-1 DM of glucose and xylose, respectively, significantly increasing glucose release by 84% and xylose release by 94% compared to using only the cellulolytic cocktail. This enhancement might be attributed to a coordinated hemicellulases action degrading hemicellulose, creating more space for cellulase activity, potentially boosted by the presence of H2O2 and Tween® 80. However, the addition of different concentrations of H2O2 in combination with hemicellulase and Tween® 80 did not result a significant difference on sugar release, which could be attributed to the limited range of concentrations studied (5 to 65 µM). The results obtained in this study using the mix of three supplements were also compared to the addition of only hemicellulase and only Tween® 80 to the cellulolytic cocktail. A significant increase in glucose release of 39% and 41%, respectively, was observed when using the optimized combination. For xylose, the increase was 38% and 41%, respectively. This study underscores the substantial potential in optimizing enzyme cocktails for the hydrolysis of mildly pretreated lignocellulosic biomass by using enzymes and additive combinations tailored to the specific biomass composition.

Deciphering the complex interplay between gut microbiota and crop residue breakdown in forager and hive bees (Apis mellifera L.).

This study investigates A. mellifera gut microbiota diversity and enzymatic activities, aiming to utilize identified isolates for practical applications in sustainable crop residue management and soil health enhancement. This study sampled honey bees, analyzed gut bacterial diversity via 16S rRNA gene, and screened isolates for cellulolytic, hemicellulolytic, and pectinolytic activities, with subsequent assessment of enzymatic potential. The study reveals that cellulolytic and hemicellulolytic bacterial isolates, mainly from γ-Proteobacteria, Actinobacteria, and Firmicutes, have significant potential for crop residue management. Some genera, like Aneurinibacillus, Bacillus, Clostridium, Enterobacter, Serratia, Stenotrophomonas, Apilactobacillus, Lysinibacillus, and Pseudomonas, are very good at breaking down cellulose and hemicellulase. Notable cellulose-degrading genera include Cedecea (1.390 ± 0.57), Clostridium (1.360 ± 0.86 U/mg), Enterobacter (1.493 ± 1.10 U/mg), Klebsiella (1.380 ± 2.03 U/mg), and Serratia (1.402 ± 0.31 U/mg), while Aneurinibacillus (1.213 ± 1.12 U/mg), Bacillus (3.119 ± 0.55 U/mg), Enterobacter (1.042 ± 0.14 U/mg), Serratia (1.589 ± 0.05 U/mg), and Xanthomonas (1.156 ± 0.08 U/mg) excel in hemicellulase activity. Specific isolates with high cellulolytic and hemicellulolytic activities are identified, highlighting their potential for crop residue management. The research explores gut bacterial compartmentalization in A. mellifera, emphasising gut physiology's role in cellulose and hemicellulose digestion. Pectinolytic activity is observed, particularly in the Bacillaceae clade (3.229 ± 0.02), contributing to understanding the honey bee gut microbiome. The findings offer insights into microbiome diversity and enzymatic capabilities, with implications for biotechnological applications in sustainable crop residue management. The study concludes by emphasizing the need for ongoing research to uncover underlying mechanisms and ecological factors influencing gut microbiota, impacting honey bee health, colony dynamics, and advancements in crop residue management.

Combinational manipulation of transcription factors, CreA and ClbR, is a viable strategy to improve cellulolytic enzyme production in Aspergillus aculeatus.

The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in Aspergillus aculeatus. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor creA and the overexpression of the transcriptional activator clbR in enzyme production in A. aculeatus. Here, we verified that a combination of creA deletion and clbR overexpression (Δc&OE) improved cellulase, ß-1,4-xylanase, and ß-glucosidase production. Cellulase and ß-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&OE compared with the host strain (MR12) at 96-h incubation, respectively. ß-Glucosidase production in ΔcreA and Δc&OE increased approximately 5.0-fold compared with that in MR12 at 240-h incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-ß-1,4-glucanase, ß-1,4-xylanase, and ß-glucosidase 1 (bgl1). Interestingly, bgl1 expression in ΔcreA increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and ß-glucosidase production in A. aculeatus.

Biochemical unravelling of the endoxylanase activity in a bifunctional GH39 enzyme cloned and expressed from thermophilic Geobacillus sp. WSUCF1.

The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable ß-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg-1) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion.

Cumulative expression of heterologous XlnR regulatory modules and AraRA731V in Penicillium oxalicum enhances saccharification efficiency of corn stover and corn fiber.

Penicillium oxalicum engineered strain DB2 and its mutant strains with multiple regulatory modules were constructed. Mutant strain RE-4-2 with two regulatory modules showed a significant increase in the reducing sugar released from corn stover and corn fiber as well as in the conversion of cellulose than DB2. RE-5-2 with three regulatory modules showed a further increase in reducing sugar released from corn stover and the conversion of cellulose on the basis of RE-4-2. RE-4-2-AraRA731V constructed by overexpressing AraRA731V in RE-4-2 showed an increase of 7.2 times and 1.2 times in arabinofuranosidase and xylosidase activities, respectively. Reducing sugar yield and cellulose conversion of corn stover and corn fiber by RE-4-2-AraRA731V were further increased.

Combined Effect of Protease, Hemicellulase and Pectinase on the Quality of Hemp Seed Oil (Cannabis sativa L.) Obtained by Aqueous Enzymatic Extraction as an Eco-friendly Method.

The objective of this research was to evaluate the efficiency of aqueous enzymatic extraction (AEE) to obtain oil from hemp seeds (Cannabis sativa L.) grown in northern Morocco. Optimisation of AEE extraction parameters, including pH, enzyme concentration (hemicellulase, protease and pectinase), temperature and incubation time, to maximize oil yield was achieved using response surface methodology with a central composite design. For comparison, the solvent extraction (Soxhlet) (SE) method was also used. Optimized hydrolysis conditions involved incubation for 4 hours at 60°C with a pH of 6.5, using a multi-enzyme preparation comprising protease, hemicellulase and pectinase at concentrations of 55, 202.5 and 234 U/mg, respectively. Referring to the conventional Soxhlet extraction (SE), Aqueous Enzymatic Extraction (AEE) achieved a 30.65% oil recovery rate under the optimized parameters mentioned above. The use of enzymes produced an oil that was more stable against oxidation than the solvent-extracted oil, with a peroxide value (PV) of 19.54 and 47.87 meq O 2 /kg, respectively. Furthermore, HPLC-DAD analysis of tocopherol content indicated a higher total tocopherol content (547.2 mg/kg) in Aqueous Enzymatic Extraction (AEE) compared to Soxhlet Extraction (SE) (513.51 mg/kg), with γ-tocopherol being the predominant form. No significant differences in fatty acid composition were observed between the two extraction methods with linoleic acid and alpha-linolenic acid being the predominant constituents.