Degradation Of Polysaccharides Research Articles

Oxygen respiration and polysaccharide degradation by a sulfate-reducing acidobacterium

Sulfate-reducing microorganisms represent a globally important link between the sulfur and carbon cycles. Recent metagenomic surveys expanded the diversity of microorganisms putatively involved in sulfate reduction underscoring our incomplete understanding of this functional guild. Here, we use genome-centric metatranscriptomics to study the energy metabolism of Acidobacteriota that carry genes for dissimilation of sulfur compounds in a long-term continuous culture running under alternating anoxic and oxic conditions. Differential gene expression analysis reveals the unique metabolic flexibility of a pectin-degrading acidobacterium to switch from sulfate to oxygen reduction when shifting from anoxic to oxic conditions. The combination of facultative anaerobiosis and polysaccharide degradation expands the metabolic versatility among sulfate-reducing microorganisms. Our results highlight that sulfate reduction and aerobic respiration are not mutually exclusive in the same organism, sulfate reducers can mineralize organic polymers, and anaerobic mineralization of complex organic matter is not necessarily a multi-step process involving different microbial guilds but can be bypassed by a single microbial species.

Stable functional structure despite high taxonomic variability across fungal communities in soils of old-growth montane forests

BackgroundMajor advances over the past decade in molecular ecology are providing access to soil fungal diversity in forest ecosystems worldwide, but the diverse functions and metabolic capabilities of this microbial community remain largely elusive. We conducted a field survey in montane old-growth broadleaved and conifer forests, to investigate the relationship between soil fungal diversity and functional genetic traits. To assess the extent to which variation in community composition was associated with dominant tree species (oak, spruce, and fir) and environmental variations in the old-growth forests in the Jade Dragon Snow Mountain in Yunnan Province, we applied rDNA metabarcoding. We also assessed fungal gene expression in soil using mRNA sequencing and specifically assessed the expression of genes related to organic matter decomposition and nutrient acquisition in ectomycorrhizal and saprotrophic fungi.ResultsOur taxonomic profiling revealed striking shifts in the composition of the saprotrophic and ectomycorrhizal guilds among the oak-, fir-, and spruce-dominated forests. The core fungal microbiome comprised only ~ 20% of the total OTUs across all soil samples, although the overlap between conifer-associated communities was substantial. In contrast, seasonality and soil layer explained only a small proportion of the variation in community structure. However, despite their highly variable taxonomic composition, fungal guilds exhibited remarkably similar functional traits for growth-related and core metabolic pathways across forest associations, suggesting ecological redundancy. However, we found that the expression profiles of genes related to polysaccharide and protein degradation and nutrient transport notably varied between and within the fungal guilds, suggesting niche adaptation.ConclusionsOverall, our metatranscriptomic analyses revealed the functional potential of soil fungal communities in montane old-growth forests, including a suite of specialized genes and taxa involved in organic matter decomposition. By linking genes to ecological traits, this study provides insights into fungal adaptation strategies to biotic and environmental factors, and sheds light on the importance of understanding functional gene expression patterns in predicting ecosystem functioning.BiaDVnGNFcTVJp_FejwVZqVideo

Complete genome of Rossellomorea sp. DA94, an agarolytic and orange-pigmented bacterium isolated from mangrove sediment of the South China Sea.

Rossellomorea sp. DA94, isolated from mangrove sediment in the South China Sea (Beihai, Guangxi province), is an agarolytic and orange-pigmented bacterium. Here, we present the complete genome sequence of strain DA94, which comprises 4.63Mb sequences with 43.5% GC content. In total, 4589 CDSs, 33 rRNA genes and 110 tRNA genes were obtained. Genomic analysis of strain DA94 revealed that 108 CAZymes were organized in 4578 PULs involved in polysaccharides degradation, transport, and regulation. Further, we performed the diversity of CAZymes and PULs comparison among Rossellomorea strains. Less CAZymes were organized more PULs, indicating highly efficiently polysaccharides utilization in Rossellomorea. Meanwhile, PUL0459, PUL0460 and PUL0316 related to agar degradation, and exolytic beta-agarase GH50, endo-type beta-agarase GH86 and arylsulfatase were identified in the genome of strain DA94. We verified that strain DA94 can degrade agar to form a bright clear zone around the bacterial colonies in the laboratory. Moreover, the carotenoid biosynthetic pathways were proposed, which may be responsible for orange-pigment of Rossellomorea sp. DA94. This study represents a thorough genomic characterization of CAZymes repertoire and carotenoid biosynthetic pathways of Rossellomorea, provides insight into diversity of related enzymes and their potential biotechnological applications.

Exploring the partial degradation of polysaccharides: Structure, mechanism, bioactivities, and perspectives.

Polysaccharides are promising biomolecules with lowtoxicity and diverse bioactivities in food processing and clinical drug development. However, an essential prerequisite for their applications is the fine structure characterization. Due to the complexity of polysaccharide structure, partial degradation is a powerful tool for fine structure analysis, which can effectively provide valid information on the structure of backbone and branching glycosidic fragments of complex polysaccharides. This review aims to conclude current methods of partial degradation employed for polysaccharide structural characterization, discuss the molecular mechanisms, and describe the molecular structure and solution properties of degraded polysaccharides. In addition, the effects of polysaccharide degradation on the conformational relationships between the molecular structure and bioactivities, such as antioxidant, antitumor, and immunomodulatory activities, are also discussed. Finally, we summarize the prospects and current challenges for the partial degradation of polysaccharides. This review will be of great value for the scientific elucidation of polysaccharide fine structures and potential applications.

Xylo-oligosaccharide-based prebiotics upregulate the proteins of the Sus-like system in caecal Bacteroidetes of the chicken: evidence of stimbiotic mechanism

The present study was conducted to investigate the stimbiotic mechanism of xylo-oligosaccharide (XOS) in degrading the complex polysaccharides by the caecal bacteria of the chicken, by applying a proteomic approach. A total of 800 as-hatched Ross 308 broiler chicks were equally divided into 4 experimental pens (200 chicks per pen) at a commercial poultry barn, allocating 2 pens per treatment. Birds were fed ad libitum with 2 dietary treatments; CON (without XOS) and XOS (with 0.1g XOS/kg diet) from d 0 to 35. Individual birds were weighed weekly whereas caecal content was obtained on d 35 from 10 of the individually weighed and cervically dislocated birds. The caecal bacteria were lysed and their proteins were quantified using label-free quantitative proteomic mass spectrometry. The results showed that XOS significantly increased (P < 0.05) bird weight on d 7, 14, 21, and 28, and body weight gain on d 7, 14, 21, and 35 compared to CON. However, no difference (P > 0.05) in body weight gain was observed from d 0 to 35 between CON and XOS. The proteomic analysis of caecal bacteria revealed that 29 proteins were expressed differently between the CON and the XOS group. Out of 29, 20 proteins were significantly increased in the XOS group compared to CON and 9 of those proteins belonged to the starch-utilizing system (Sus)-like system of the gram-negative Bacteroidetes. Bacteroides thetaiotaomicron (Bt) is a significant constituent of the human gut microbiota, known for its remarkable ability to hydrolyze most glycosidic bonds of polysaccharides. This microorganism possesses a 5-protein complex in its outer membrane, named the starch utilization system (Sus), responsible for adhering to, breaking down, and transporting starch into the cell. Sus serves as an exemplar system for numerous polysaccharide utilization loci that target glycans found in Bt and other members of the Bacteroidetes phylum. The proteins of the Sus-like system are involved in the degradation of complex polysaccharides and transportation of the oligosaccharides into the periplasm of the caecal bacteria where they are further broken down into smaller units. These smaller units are then transported into the cytoplasm of the cell where they are utilized in metabolic pathways leading to potential generation of short-chain fatty acids, thus improving the nutritive value of residual feed. In conclusion, XOS supplementation upregulates the expression of the proteins of the Sus-like system indicating its role as a stimbiotic.

Impact of feeding dried distillers' grains with solubles diet on microbiome and metabolome of ruminal and cecal contents in Guanling yellow cattle.

Dried distillers' grains with solubles (DDGS) are rich in nutrients, and partially alternative feeding of DDGS effectively reduces cost of feed and improves animals' growth. We used 16S rDNA gene sequencing and LC/MS-based metabolomics to explore the effect of feeding cattle with a basal diet (BD) and a Jiang-flavor DDGS diet (replaces 25% concentrate of the diet) on microbiome and metabolome of ruminal and cecal contents in Guanling yellow cattle. The results showed that the ruminal and cecal contents shared the same dominance of Bacteroidetes, Firmicutes and Proteobacteria in two groups. The ruminal dominant genera were Prevotella_1, Rikenellaceae_RC9_gut_group, and Ruminococcaceae_UCG-010; and the cecal dominant genera were Ruminococcaceae_UCG-005, Ruminococcaceae_UCG-010, and Rikenellaceae_RC9_gut_group. Linear discriminant analysis effect size analysis (LDA > 2, P < 0.05) revealed the significantly differential bacteria enriched in the DDGS group, including Ruminococcaceae_UCG_012, Prevotellaceae_UCG_004 and Anaerococcus in the ruminal contents, which was associated with degradation of plant polysaccharides. Besides, Anaerosporobacter, Anaerovibrio, and Caproiciproducens in the cecal contents were involved in fatty acid metabolism. Compared with the BD group, 20 significantly different metabolites obtained in the ruminal contents of DDGS group were down-regulated (P < 0.05), and based on them, 4 significantly different metabolic pathways (P < 0.05) were enriched including "Linoleic acid metabolism," "Biosynthesis of unsaturated fatty acids," "Taste transduction," and "Carbohydrate digestion and absorption." There were 65 significantly different metabolites (47 were upregulated, 18 were downregulated) in the cecal contents of DDGS group when compared with the BD group, and 4 significantly different metabolic pathways (P < 0.05) were enriched including "Longevity regulating pathway," "Bile secretion," "Choline metabolism in cancer," and "HIF-1 signaling pathway." Spearman analysis revealed close negative relationships between the top 20 significantly differential metabolites and Anaerococcus in the ruminal contents. Bacteria with high relevance to cecal differential metabolites were Erysipelotrichaceae_UCG-003, Dielma, and Solobacterium that affect specific metabolic pathways in cattle. Collectively, our results suggest that feeding cattle with a DDGS diet improves the microbial structure and the metabolic patterns of lipids and carbohydrates, thus contributing to the utilization efficiency of nutrients and physical health to some extent. Our findings will provide scientific reference for the utilization of DDGS as feed in cattle industry.

Metagenome-assembled genomes reveal carbohydrate degradation and element metabolism of microorganisms inhabiting Tengchong hot springs, China

A hot spring is a distinctive aquatic environment that provides an excellent system to investigate microorganisms and their function in elemental cycling processes. Previous studies of terrestrial hot springs have been mostly focused on the microbial community, one special phylum or category, or genes involved in a particular metabolic step, while little is known about the overall functional metabolic profiles of microorganisms inhabiting the terrestrial hot springs. Here, we analyzed the microbial community structure and their functional genes based on metagenomic sequencing of six selected hot springs with different temperature and pH conditions. We sequenced a total of 11 samples from six hot springs and constructed 162 metagenome-assembled genomes (MAGs) with completeness above 70% and contamination lower than 10%. Crenarchaeota, Euryarchaeota and Aquificae were found to be the dominant phyla. Functional annotation revealed that bacteria encode versatile carbohydrate-active enzymes (CAZYmes) for the degradation of complex polysaccharides, while archaea tend to assimilate C1 compounds through carbon fixation. Under nitrogen-deficient conditions, there were correspondingly fewer genes involved in nitrogen metabolism, while abundant and diverse set of genes participating in sulfur metabolism, particularly those associated with sulfide oxidation and thiosulfate disproportionation. In summary, archaea and bacteria residing in the hot springs display distinct carbon metabolism fate, while sharing the common energy preference through sulfur metabolism. Overall, this research contributes to a better comprehension of biogeochemistry of terrestrial hot springs.

The Ustilago maydis AA10 LPMO is active on fungal cell wall chitin.

Lytic polysaccharide monooxygenases (LPMOs) can perform oxidative cleavage of glycosidic bonds in carbohydrate polymers (e.g., cellulose, chitin), making them more accessible to hydrolytic enzymes. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. The AA10 LPMOs are active on chitin and/or cellulose and mostly found in bacteria and in some viruses and archaea. Interestingly, AA10-encoding genes are also encountered in some pathogenic fungi of the Ustilaginomycetes class, such as Ustilago maydis, responsible for corn smut disease. Transcriptomic studies have shown the overexpression of the AA10 gene during the infectious cycle of U. maydis. In fact, U. maydis has a unique AA10 gene that codes for a catalytic domain appended with a C-terminal disordered region. To date, there is no public report on fungal AA10 LPMOs. In this study, we successfully produced the catalytic domain of this LPMO (UmAA10_cd) in Pichia pastoris and carried out its biochemical characterization. Our results show that UmAA10_cd oxidatively cleaves α- and β-chitin with C1 regioselectivity and boosts chitin hydrolysis by a GH18 chitinase from U. maydis (UmGH18A). Using a biologically relevant substrate, we show that UmAA10_cd exhibits enzymatic activity on U. maydis fungal cell wall chitin and promotes its hydrolysis by UmGH18A. These results represent an important step toward the understanding of the role of LPMOs in the fungal cell wall remodeling process during the fungal life cycle. IMPORTANCE Lytic polysaccharide monooxygenases (LPMOs) have been mainly studied in a biotechnological context for the efficient degradation of recalcitrant polysaccharides. Only recently, alternative roles and paradigms begin to emerge. In this study, we provide evidence that the AA10 LPMO from the phytopathogen Ustilago maydis is active against fungal cell wall chitin. Given that chitin-active LPMOs are commonly found in microbes, it is important to consider fungal cell wall as a potential target for this enigmatic class of enzymes.

Enzyme-assisted nanofiltration to enrich tannins from softwood bark extract

Softwood bark is a rich source of renewable chemicals, including phenolic compounds known as tannins. Tannins can be extracted from bark with hot water, but the yield can be relatively low, leading to dilute extracts. Next to tannin, a considerable amount of extracted dry matter comprised of carbohydrates, and bark carbohydrates limit the applicability of the extract in applications that demand high purity. This study used membrane filtration to refine and concentrate tannins produced from hot water extracts of softwood bark. Enzymatic hydrolysis of bark carbohydrates prior to membrane filtration was assessed as means to improve the separation of bark sugars from bark phenols. Enzymatic hydrolysis using an optimised enzyme mixture liberated 55% of bark carbohydrates mainly by degradation of pectin and other polysaccharides in spruce unrefined tannins. Separation of bark phenols from the enzyme-treated extract by precipitation at acidic pH and subsequent microfiltration produced low tannin yield. Instead, keeping bark phenols soluble at alkaline pH allowed us to concentrate them 15-fold with good and stable flux using nanofiltration. Additionally, enzymatically liberated sugars permeated well through the nanofiltration membrane, decreasing the free sugar content of the concentrated tannin from 31% to 7.4%. However, diafiltration did not seem to decrease the proportion of free sugars in the concentrate further. This enzyme-assisted nanofiltration concept was successfully upscaled to a pilot scale which was a promising result for improved industrial applicability of softwood bark tannins in the future.

A meta-omics approach to explore the biofuel-producing enzyme potential from extreme environmental conditions

Geothermally warmed spring water contaminated with decomposed leaf biomass are unique hot spring ecosystems , are expected to support the recycling of various nutrients and to host lignocellulose degrading thermostable enzymes, genes and bacteria. An attempt is made in the present study to explore CAZymes in a carbohydrate-contaminated unique environment at Deulajhari spring through a multi-omics approach using an indigenous consortium developed from the spring sediment. Co-assembly of shotgun metagenome and metatranscriptome libraries from the Deulajhari hot spring consortia sample recovered seventeen refined, high-quality near-complete genomes. The predominant recovery of thermophilic aerobic chemo-heterotrophic Meiothermus and Rhodothermus has been observed through genome reconstruction. The reported production of an array of enzymes, including xylanase, β-xylosidase, endoglucanase and polysaccharide deacetylase, establishes their lignocellulose-degrading ability. The unexpected strong positive correlation between predominating Meiothermus and less prevailing members of Acetobacteraceae, unclassified Gaiellaceae, unclassified Burkholderiaceae and Tepidimonas of the consortium signifies their unexplored role in biomass degradation. Furthermore, the synergistic involvement of the diverse enzymes represented by a vast gene repertoire is responsible for the degradation of complex plant polysaccharides by the group of bacteria. The novelty of the present study stems from the identification of a diverse range of potential lignocellulose-digesting enzymes expressed by the bacterial consortium. This finding emphasizes the significant potential of these enzymes in facilitating industrial-scale production of biofuel, making it a notable contribution to the field.

Evaluation of the Deterioration State of Historical Palm Leaf Manuscripts from Burma

Palm leaf manuscripts were a prevalent literary medium from South Asia and Southeast Asia prior to the widespread use of paper. This study focuses on the analysis of historical palm leaf manuscripts from South and Southeast Asia. Sample palm leaf manuscripts from Burma were used as a case study; simulated palm leaf manuscripts were also created as a reference for comparison. The anatomy, chemical composition, and mechanical properties of the manuscripts were analyzed to find various forms of deterioration, including damage, fractures, pollution, acidification, and microbial deterioration. Specifically, the S1–S3 layers of the cell walls exhibited complete cracking, and the S2 layer showed numerous circular or nearly circular cavities caused by microbial erosion, while the middle lamella remained intact. The severe degradation of polysaccharides and pectin, accompanied by an increase in the relative content of lignin, caused the historical manuscripts to become more brittle. Additionally, the tensile strengths of historical palm leaf manuscripts were markedly reduced; their longitudinal tensile strength was significantly greater than their transverse tensile strength. This study can contribute to a better understanding of the deterioration process of historical palm leaf manuscripts and provide valuable insights for their restoration and preservation.

Advance in Heterologous Expression of Biomass-Degrading Auxiliary Activity 10 Family of Lytic Polysaccharide Monooxygenases

AA10 family lytic polysaccharide monooxygenases (AA10 LPMOs) are mainly distributed in bacteria. Because of their characteristics of oxidative degradation of crystalline polysaccharides, such as cellulose and chitin, they have great application potential in industrial biomass conversion and have attracted wide attention. Efficient heterologous expression of LPMOs by recombinant engineering bacteria has become the main strategy for the industrial production of enzymes. The research progress of AA10 LPMOs’ heterologous expression systems was reviewed in this paper. The construction strategies of its diversified heterologous expression system were introduced based on the design and processing of the expression host, vector, and LPMOs gene. The effects of different expression systems on the soluble expression of LPMOs and the development direction of the construction of LPMOs’ heterologous expression systems were discussed. The broad application prospect of LPMOs in the biomass conversion and biofuel industry has been prospected.

Strengthen high-loading operation of wastewater treatment plants by composite micron powder carrier: Microscale control of carbon, nitrogen, and sulfur metabolic pathways

The concentration of activated sludge is a crucial factor influencing the capacity and efficiency of sewage wastewater treatment plants (WWTPs). However, high sludge concentrations can lead to sludge loss in the secondary sedimentation tank, resulting in reduced processing capacity, particularly during low-temperature stages and sludge bulking. This study investigated the impact of adding composite micron powder carriers (CMPC) in high-concentration powder carrier biofluidized bed (HPB) technology to the biochemical units of WWTPs on sludge concentration and settling performance. For the traditional activated sludge method (ASM), its hydraulic retention time (HRT) was 8 h, with an average effluent total nitrogen (TN) of 15.14 mg/L. Sludge bulking was prone to occur in low-temperature environments, resulting in a high average sludge volume index (SVI) of 560 mL/g. Conversely, with a CMPC dosage of 4 g/L, the HRT of HPB technology was 4.8 h, and the average effluent TN was 11.40 mg/L, with a removal efficiency of 67.43 %. During operation of HPB technology under high sludge concentration conditions (8 g/L), the average SVI remained at 85 mL/g, indicating excellent settling characteristics. Moreover, in the sequencing batch reactor (SBR), the SVI value of bulking sludge decreased from the original 695 to 111 mL/g by the 9th day of operation with the CMPC dosage of 2 g/L. At the same time, the filamentous bacteria almost disappeared, suggesting that CMPC inhibit the growth of filamentous bacteria. Metagenomic analysis demonstrated that CPMC enhance the utilization of small molecular fatty acids in activated sludge and promote electron transfer between nitrate and nitrite, thereby improving wastewater treatment capacity. Additionally, CMPC enhanced the relative abundance of Saprospiraceae in sludge, which accelerate the degradation of polysaccharides in extracellular polymeric substances, weaken sludge's hydrophilic properties, and improve sludge's settling performance. Overall, these findings suggested that CMPC effectively strengthen the high-loading operation of WWTPs by improving sludge concentration and sedimentation performance.

A High-Quality Genome Sequence of the Penicillium oxalicum 5-18 Strain Isolated from a Poplar Plantation Provides Insights into Its Lignocellulose Degradation.

Studies on the degradation of plant cell wall polysaccharides by fungal extracellular enzymes have attracted recent attention from researchers. Xylan, abundant in hemicellulose, that play great role in connection between cellulose and lignin, has seen interest in its hydrolytic enzymatic complex. In this study, dozens of fungus species spanning genera were isolated from rotting leaves based on their ability to decompose xylan. Among these isolates, a strain with strong xylanase-producing ability was selected for further investigation by genome sequencing. Based on phylogenetic analysis of ITS (rDNA internal transcribed spacer) and LSU (Large subunit 28S rDNA) regions, the isolate was identified as Penicillium oxalicum. Morphological analysis also supported this finding. Xylanase activity of this isolated P. oxalicum 5-18 strain was recorded to be 30.83 U/mL using the 3,5-dinitro-salicylic acid (DNS) method. Further genome sequencing reveals that sequenced reads were assembled into a 30.78 Mb genome containing 10,074 predicted protein-encoding genes. In total, 439 carbohydrate-active enzymes (CAZymes) encoding genes were predicted, many of which were associated with cellulose, hemicellulose, pectin, chitin and starch degradation. Further analysis and comparison showed that the isolate P. oxalicum 5-18 contains a diverse set of CAZyme genes involved in degradation of plant cell wall components, particularly cellulose and hemicellulose. These findings provide us with valuable genetic information about the plant biomass-degrading enzyme system of P. oxalicum, facilitating a further exploration of the repertoire of industrially relevant lignocellulolytic enzymes of P. oxalicum 5-18.

Key roles of β-glucosidase BglA for the catabolism of both laminaribiose and cellobiose in the lignocellulolytic bacterium Clostridium thermocellum

The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides. The metabolism of β-1,4-linked cello-oligosaccharides is initiated by three enzymes, i.e., the cellodextrin phosphorylase (Cdp), the cellobiose phosphorylase (Cbp), and the β-glucosidase A (BglA), in C. thermocellum. In comparison, how the oligosaccharides containing other kinds of linkage are utilized is rarely understood. In this study, we found that BglA could hydrolyze the β-1,3-disaccharide laminaribiose with much higher activity than that against the β-1,4-disaccharide cellobiose. The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking. Genetic deletions of BglA and Cbp, respectively, and enzymatic analysis of cell extracts demonstrated that BglA is the key enzyme responsible for laminaribiose metabolism. Furthermore, the deletion of BglA can suppress the expression of Cbp and the deletion of Cbp can up-regulate the expression of BglA, indicating that BglA and Cbp have cross-regulation and BglA is also critical for cellobiose metabolism. These insights pave the way for both a fundamental understanding of metabolism and regulation in C. thermocellum and emphasize the importance of the degradation and utilization of polysaccharides containing β-1,3-linked glycosidic bonds in lignocellulose biorefinery.

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen's large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

Ultrasound- and Microwave-Assisted Extraction of Pectin from Apple Pomace and Its Effect on the Quality of Fruit Bars.

The article investigates the process of pectin extraction using ultrasonic and microwave techniques from apple pomace generated during juice production in the context of circular bioeconomy. The extraction yield, equivalent mass, content of methoxyl groups, content of anhydrogalacturonic acid, and degree of esterification of pectin were investigated. These indicators varied depending on the parameters and extraction method. The resulting pectin displayed a co-extracted total polyphenol content (TPC) ranging from 2.16 to 13.05 mg GAE/g DW and a DPPH radical inhibition capacity of 4.32-18.86 μmol TE/g. It was found that the antioxidant activity of raw pectin is correlated with TPC and with the content of terminal groups released during the polysaccharide degradation process. The extracted pectin was used as a binding and coating agent for dried fruit bars. Evaluation of water activity (aw), TPC and total flavonoid content (TFC), together with sensory and microbiological analyses of the fruit bars over a period of 360 days, revealed a protective effect of pectin: reducing moisture loss, minimizing the degradation of bioactive compounds during storage, and maintaining the potential antioxidant activity of the product.

Effect of vibration and broken cold chain on the evolution of cell wall polysaccharides during fruit cucumber (Cucumis sativus L.) shriveling under simulated transportation

The fruits and vegetables often result in shriveling, which can significantly affect the quality in terms of texture, appearance, and saleable weight during transportation and storage. In this study, fruit cucumbers (Cucumis sativus L.) were subjected to vibration followed by storing them under isothermal and non-isothermal conditions (simulated broken cold chain) for 15 days. Water movement, cell wall polysaccharides and degrading enzymes, cell wall structure of cucumbers were determined. The findings revealed that water initially migrated from the inner pericarp of cucumber. Vibration and broken cold chain damaged the fruit cell wall and cell wall polysaccharides, and increased degrading enzymes, which promoted the water movement from the tissues to the exterior, causing severe shriveling of cucumber. Firmness loss accompanied with cucumber shrivel appeared by as early as 6 days of storage after vibration (5.5 Hz), with decrease of 19.88–29.93 %. However, this phenomenon was not observed in the control cucumber until 12 days after storage with firmness of 8.22 N. Therefore, this study shows that simulated transport vibration and broken cold chain could cause intracellular damage in cucumber tissues, which expedites the process of water loss, degradation of cell wall polysaccharides and shriveling process. The study also reveals that precise temperature control and avoiding vibration of transportation in cold chain logistics of cucumbers are important in maintaining the product quality during storage and transport.

Genomic and phylotypic properties of three novel marine Bacteroidota from bare tidal flats reveal insights into their potential of polysaccharide metabolism

Special geographical location and abundant organic matter profiles in tidal flats have resulted in great microbial diversity, in which Bacteroidota strains are considered as one of the primary degraders of polysaccharides, playing a crucial role in the carbon cycle. In this study, we collected sediment or sand samples from 34 bare tidal flats in China and investigated the profile of culturable bacteria, selected three Bacteroidota for polyphasic taxonomic analysis and revealed their polysaccharide metabolic potential. Totally, we isolated 352 pure cultured bacteria and they mainly distributed in Bacteroidota, Pseudomonadota, Bacillota, and Actinomycetota. It is shown that the bare tidal flats contained a large number of potential novel species, mainly distributed in Flavobacteriales and Cytophagales within Bacteroidota. Three Bacteroidota strains, M17T, M82T, and M415T, isolated from mudflat were selected for polyphasic taxonomic analysis. The 16S rRNA gene sequence similarity between strain M17T and Mangrovivirga cuniculi KCTC 72349T was 99.28%, and less than 90.09% with other species; strain M82T shared the highest 16S rRNA gene sequence similarity of 97.85% with Pontibacter litorisediminis KCTC 52252T, and less than 97.43% with other species; strain M415T had higher 16S rRNA gene sequence similarities with type species of genera Eudoraea (92.62-93.68%), Zeaxanthinibacter (92.02-92.91%), and Muriicola (92.21-92.83%). Phylogenetic analysis based on 16S rRNA gene sequences and single-copy orthologous clusters showed that strains M17T and M82T represent novel species within the genus Mangrovivirga and Pontibacter, respectively, and strain M415T represents a novel species of a novel genus within the family Flavobacteriaceae. The potential in polysaccharide metabolism of all these three strains was analyzed by genomes. The analysis revealed that glycoside hydrolases and glycosyltransferases account for more than 70% of the total CAZymes. Additionally, the numbers of polysaccharide utilization loci (PULs) and annotated CAZymes in Cytophagales spp. M17T and M82T were found to be higher than those in Flavobacteriales sp. M415T. Highly specialized saccharolytic systems and the presence of numerous diversified CAZymes for obtaining energy through polysaccharide metabolism were speculated to help the three novel strains adapt to the utilization of both terrestrial and marine polysaccharides.

The glycoside hydrolase gene family profile and microbial function of Debaryomyces hansenii Y4 during South-road dark tea fermentation.

Microbes are crucial to the quality formation of Sichuan South-road Dark Tea (SSDT) during pile-fermentation, but their mechanism of action has not yet been elucidated. Here, the glycoside hydrolase (GH) gene family and microbial function of Debaryomyces hansenii Y4 during solid-state fermentation were analyzed, and the results showed that many GH genes being distributed in comparatively abundant GH17, GH18, GH76, GH31, GH47, and GH2 were discovered in D. hansenii. They encoded beta-galactosidase, alpha-D-galactoside galactohydrolase, alpha-xylosidase, mannosidase, etc., and most of the GHs were located in the exocellular space and participated in the degradation of polysaccharides and oligosaccharides. D. hansenii Y4 could develop the mellow mouthfeel and "reddish brown" factors of SSDT via increasing the levels of water extracts, soluble sugars and amino acids but decreasing the tea polyphenols and caffeine levels, combined with altering the levels of thearubiins and brown index. It may facilitate the isomerization between epicatechin gallate and catechin gallate. Moreover, the expression levels of DEHA2G24860g (Beta-galactosidase gene) and DEHA2G08602g (Mannan endo-1,6-alpha-mannosidase DFG5 gene) were sharply up-regulated in fermentative anaphase, and they were significantly and negatively correlated with epicatechin content, especially, the expression of DEHA2G08602g was significantly and negatively correlated with catechin gallate level. It was hypothesized that D. hansenii Y4 is likely to be an important functional microbe targeting carbohydrate destruction and catechin transformation during SSDT pile-fermentation, with DEHA2G08602g as a key thermotolerant functional gene.