Polysaccharide Lyase Research Articles

Enhanced propagation of Granulicatella adiacens from human oral microbiota by hyaluronan

Host determinants for formation/composition of human oral microbiota remain to be clarified, although microorganisms entering the mouth cannot necessarily colonize the oral environment. Here we show that human oral-abundant bacteria degraded host glycosaminoglycans (GAGs) in saliva and gingiva, and certain bacteria significantly grew on hyaluronan (HA), a kind of GAGs. Microbial communities from teeth or gingiva of healthy donors assimilated HA. Metagenomic analysis of human oral microbiota under different carbon sources revealed HA-driven Granulicatella growth. HA-degrading bacterial strains independently isolated from teeth and gingiva were identified as Granulicatella adiacens producing extracellular 130 kDa polysaccharide lyase as a HA-degrading enzyme encoded in a peculiar GAG genetic cluster containing genes for isomerase KduI and dehydrogenase DhuD. These findings demonstrated that GAGs are one of the host determinants for formation/composition of oral microbiota not only for colonization but also for the adaptation to the host niche. Especially, HA enhanced the G. adiacens propagation.

Genome Analysis of a Novel Polysaccharide-Degrading Bacterium Paenibacillus algicola and Determination of Alginate Lyases.

Carbohydrate-active enzymes (CAZymes) are an important characteristic of bacteria in marine systems. We herein describe the CAZymes of Paenibacillus algicola HB172198T, a novel type species isolated from brown algae in Qishui Bay, Hainan, China. The genome of strain HB172198T is a 4,475,055 bp circular chromosome with an average GC content of 51.2%. Analysis of the nucleotide sequences of the predicted genes shows that strain HB172198T encodes 191 CAZymes. Abundant putative enzymes involved in the degradation of polysaccharides were identified, such as alginate lyase, agarase, carrageenase, xanthanase, xylanase, amylases, cellulase, chitinase, fucosidase and glucanase. Four of the putative polysaccharide lyases from families 7, 15 and 38 were involved in alginate degradation. The alginate lyases of strain HB172198T exhibited the maximum activity 152 U/mL at 50 °C and pH 8.0, and were relatively stable at pH 7.0 and temperatures lower than 40 °C. The average degree of polymerization (DP) of the sodium alginate oligosaccharide (AOS) degraded by the partially purified alginate lyases remained around 14.2, and the thin layer chromatography (TCL) analysis indicated that it contained DP2-DP8 oligosaccharides. The complete genome sequence of P. algicola HB172198T will enrich our knowledge of the mechanism of polysaccharide lyase production and provide insights into its potential applications in the degradation of polysaccharides such as alginate.

Rahnella sp., a Dominant Symbiont of the Core Gut Bacteriome of Dendroctonus Species, Has Metabolic Capacity to Degrade Xylan by Bifunctional Xylanase-Ferulic Acid Esterase.

Rahnella sp. ChDrAdgB13 is a dominant member of the gut bacterial core of species of the genus Dendroctonus, which is one of the most destructive pine forest bark beetles. The objectives of this study were identified in Rahnella sp. ChDrAdgB13 genome the glycosyl hydrolase families involved in carbohydrate metabolism and specifically, the genes that participate in xylan hydrolysis, to determine the functionality of a putative endo-1,4-β-D-xylanase, which results to be bifunctional xylanase–ferulic acid esterase called R13 Fae and characterize it biochemically. The carbohydrate-active enzyme prediction revealed 25 glycoside hydrolases, 20 glycosyl transferases, carbohydrate esterases, two auxiliary activities, one polysaccharide lyase, and one carbohydrate-binding module (CBM). The R13 Fae predicted showed high identity to the putative esterases and glycosyl hydrolases from Rahnella species and some members of the Yersiniaceae family. The r13 fae gene encodes 393 amino acids (43.5 kDa), containing a signal peptide, esterase catalytic domain, and CBM48. The R13 Fae modeling showed a higher binding affinity to ferulic acid, α-naphthyl acetate, and arabinoxylan, and a low affinity to starch. The R13 Fae recombinant protein showed activity on α-naphthyl acetate and xylan, but not on starch. This enzyme showed mesophilic characteristics, displaying its optimal activity at pH 6.0 and 25°C. The enzyme was stable at pH from 4.5 to 9.0, retaining nearly 66–71% of its original activity. The half-life of the enzyme was 23 days at 25°C. The enzyme was stable in the presence of metallic ions, except for Hg2+. The products of R13 Fae mediated hydrolysis of beechwood xylan were xylobiose and xylose, manifesting an exo-activity. The results suggest that Rahnella sp. ChDrAdgB13 hydrolyze xylan and its products could be assimilated by its host and other gut microbes as a nutritional source, demonstrating their functional role in the bacterial-insect interaction contributing to their fitness, development, and survival.

Metatranscriptomics Unravel Composition, Drivers, and Functions of the Active Microorganisms in Light-Flavor Liquor Fermentation.

ABSTRACTThe microbial community in the fermented pit determines the quantity and quality of light-flavor liquor. Genetic diversity and the potential functions of the microbial community are often analyzed by DNA-based omics sequencing. However, the features of the active microbial community have not been systematically studied. Here, metatranscriptomic analysis was performed to elucidate the active microbial composition, drivers, and their functions in light-flavor liquor fermentation. Bacterial genera, Lactobacillus, Streptococcus, Pediococcus, Thermotoga, and Faecalibacterium, and fungal genera, Saccharomyces, Talaromyces, Aspergillus, Clavispora, Rhizophagus, Cyberlindnera, and Wickerhamomyces, were the dominant active microorganisms during the fermentation process. Additionally, they dominated the three-stage fermentation successively. Redundancy analysis showed that pH, ethanol, moisture, and starch were the main driving forces of microbial succession. Among the genes for the respective carbohydrate-active enzyme families, those for the glycoside hydrolase family 23, the glycosyltransferase family 2, the carbohydrate-binding module family 50, the polysaccharide lyase family 4, the auxiliary activity family 1, and the carbohydrate esterase family 9 showed the highest expression level. Additionally, the highly expressed enzymes and their contributed microorganisms were found in the key KEGG pathways, including carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Based on these data, a functional model of carbohydrate hydrolysis, ethanol production, and flavor generation were proposed. Taken together, Saccharomyces, Lactobacillus, Wickerhamomyces, Pediococcus, Candida, and Faecalibacterium were suggested as the core active microorganisms. Overall, our findings provide new insights into the composition, drivers, and functions of the active microorganisms, which is crucial for improving the quality of light-flavor liquor.IMPORTANCE There is an urgent need for discovering the diversity and functions of the active microbial community in solid-state fermentation, especially in the pit of Chinese distilled liquor fermentation. Although the genetic composition of the microbial community has been clarified frequently by DNA-based sequencing, the composition and functions of the active microbial community have not been systematically revealed so far. Therefore, analysis of RNA-based data is crucial for discovering the functional microbial community. In this study, we employed metatranscriptomic analysis to elucidate the active microbial composition, successive drivers, and their functions in light-flavor liquor fermentation. The strategy can be broadly useful for discovering the active microbial community and exploring their functions in other types of flavor distilled liquor or other ecosystems. This study provides new insights into the understanding of the active microbial community composition and its functions.

An approach for evaluating the effects of dietary fiber polysaccharides on the human gut microbiome and plasma proteome

Increases in snack consumption associated with Westernized lifestyles provide an opportunity to introduce nutritious foods into poor diets. We describe two 10-wk-long open label, single group assignment human studies that measured the effects of two snack prototypes containing fiber preparations from two sustainable and scalable sources; the byproducts remaining after isolation of protein from the endosperm of peas and the vesicular pulp remaining after processing oranges for the manufacture of juices. The normal diets of study participants were supplemented with either a pea- or orange fiber-containing snack. We focused our analysis on quantifying the abundances of genes encoding carbohydrate-active enzymes (CAZymes) (glycoside hydrolases and polysaccharide lyases) in the fecal microbiome, mass spectrometric measurements of glycan structures (glycosidic linkages) in feces, plus aptamer-based assessment of levels of 1,300 plasma proteins reflecting a broad range of physiological functions. Computational methods for feature selection identified treatment-discriminatory changes in CAZyme genes that correlated with alterations in levels of fiber-associated glycosidic linkages; these changes in turn correlated with levels of plasma proteins representing diverse biological functions, including transforming growth factor type β/bone morphogenetic protein-mediated fibrosis, vascular endothelial growth factor-related angiogenesis, P38/MAPK-associated immune cell signaling, and obesity-associated hormonal regulators. The approach used represents a way to connect changes in consumer microbiomes produced by specific fiber types with host responses in the context of varying background diets.

YsHyl8A, an Alkalophilic Cold-Adapted Glycosaminoglycan Lyase Cloned from Pathogenic Yersinia sp. 298.

A high enzyme-yield strain Yersinia sp. 298 was screened from marine bacteria harvested from the coastal water. The screening conditions were extensive, utilizing hyaluronic acid (HA)/chondroitin sulfate (CS) as the carbon source. A coding gene yshyl8A of the family 8 polysaccharide lyase (PL8) was cloned from the genome of Yersinia sp. 298 and subjected to recombinant expression. The specific activity of the recombinase YsHyl8A was 11.19 U/mg, with an optimal reaction temperature of 40 °C and 50% of its specific activity remaining after thermal incubation at 30 °C for 1 h. In addition, its optimal reaction pH was 7.5, and while it was most stable at pH 6.0 in Na2HPO4-citric acid buffer, it remained highly stable at pH 6.0–11.0. Further, its enzymatic activity was increased five-fold with 0.1 M NaCl. YsHyl8A, as an endo-lyase, can degrade both HA and CS, producing disaccharide end-products. These properties suggested that YsHyl8A possessed both significant alkalophilic and cold-adapted features while being dependent on NaCl, likely resulting from its marine source. Yersinia is a typical fish pathogen, with glycosaminoglycan lyase (GAG lyase) as a potential pathogenic factor, exhibiting strong hyaluronidase and chondroitinase activity. Further research on the pathogenic mechanism of GAG lyase may benefit the prevention and treatment of related diseases.

An Improved Genome Sequence Resource of Bipolaris maydis, Causal Agent of Southern Corn Leaf Blight.

An Improved Genome Sequence Resource of Bipolaris maydis, Causal Agent of Southern Corn Leaf Blight.

Effects of Module Truncation of a New Alginate Lyase VxAly7C from Marine Vibrio xiamenensis QY104 on Biochemical Characteristics and Product Distribution.

Alginate lyase has received extensive attention as an important tool for oligosaccharide preparation, pharmaceutical production, and energy biotransformation. Noncatalytic module carbohydrate-binding modules (CBM) have a major impact on the function of alginate lyases. Although the effects of two different families of CBMs on enzyme characteristics have been reported, the effect of two combined CBM32s on enzyme function has not been elucidated. Herein, we cloned and expressed a new multimodular alginate lyase, VxAly7C, from Vibrio xiamenensis QY104, consisting of two CBM32s at N-terminus and a polysaccharide lyase family 7 (PL7) at C-terminus. To explore the function of CBM32s in VxAly7C, full-length (VxAly7C-FL) and two truncated mutants, VxAly7C-TM1 (with the first CBM32 deleted) and VxAly7C-TM2 (with both CBM32s deleted), were characterized. The catalytic efficiency of recombinant VxAly7C-TM2 was 1.82 and 4.25 times higher than that of VxAly7C-TM1 and VxAly7C-FL, respectively, indicating that CBM32s had an antagonistic effect. However, CBM32s improved the temperature stability, the adaptability in an alkaline environment, and the preference for polyG. Moreover, CBM32s contributed to the production of tri- and tetrasaccharides, significantly affecting the end-product distribution. This study advances the understanding of module function and provides a reference for broader enzymatic applications and further enzymatic improvement and assembly.

Dissecting the Effect of Berberine on the Intestinal Microbiome in the Weaned Piglets by Metagenomic Sequencing.

This study aimed to investigate the microbial structure and function in the rectum of weaned piglets with berberine supplementation. Twelve healthy 21-day-old Duorc × (Landrace × Large White) weaned piglets (similar body weight) were evenly divided into control and berberine groups and were fed a basal diet supplemented with 0 and 0.1% berberine, respectively. After 21 days, metagenomic sequencing analysis was performed to detect microbial composition and function in the rectum of weaned piglets. Results showed that there were 10,597,721,931–14,059,392,900 base pairs (bp) and 10,186,558,171–15,859,563,342 bp of clean data in the control and berberine groups, respectively. The Q20s of the control and berberine groups were 97.15 to 97.7% and 96.26 to 97.68%, respectively. The microorganisms in the berberine group had lower (p < 0.05) Chao1, alternating conditional expectation, Shannon, and Simpson indices at the species levels than those in the control group. Analysis of similarity showed that there were significant differences (p < 0.01) between the control and berberine groups at the genus and species levels of the gut microorganisms. Dietary berberine significantly increased (p < 0.05) the abundance of Subdoligranulum variabile, but decreased (p < 0.05) the abundance of Prevotella copri compared with the control group. Carbohydrate-active enzymes analysis revealed that the levels of polysaccharide lyases and carbohydrate esterases were lower (p < 0.05) in the berberine group than that in the control group. Linear discriminant analysis effect size analysis showed that berberine supplementation could induce various significant Kyoto Encyclopedia of Genes and Genomes pathways, including carbohydrate metabolism, environmental information processing, and microbial metabolism in diverse environments. In conclusion, our findings suggest that berberine could improve the composition, abundance, structure, and function of gut microbiome in the weaned piglets, potentially providing a suitable approach for the application of berberine in human and animal health.

Degradation of Alginate by a Newly Isolated Marine Bacterium Agarivorans sp. B2Z047.

Alginate is the main component of brown algae, which is an important primary production in marine ecosystems and represents a huge marine biomass. The efficient utilization of alginate depends on alginate lyases to catalyze the degradation, and remains to be further explored. In this study, 354 strains were isolated from the gut of adult abalones, which mainly feed on brown algae. Among them, 100 alginate-degrading strains were gained and the majority belonged to the Gammaproteobacteria, followed by the Bacteroidetes and Alphaproteobacteria. A marine bacterium, Agarivorans sp. B2Z047, had the strongest degradation ability of alginate with the largest degradation circle and the highest enzyme activity. The optimal alginate lyase production medium of strain B2Z047 was determined as 1.1% sodium alginate, 0.3% yeast extract, 1% NaCl, and 0.1% MgSO4 in artificial seawater (pH 7.0). Cells of strain B2Z047 were Gram-stain-negative, aerobic, motile by flagella, short rod-shaped, and approximately 0.7–0.9 µm width and 1.2–1.9 µm length. The optimal growth conditions were determined to be at 30 °C, pH 7.0–8.0, and in 3% (w/v) NaCl. A total of 12 potential alginate lyase genes were identified through whole genome sequencing and prediction, which belonged to polysaccharide lyase family 6, 7, 17, and 38 (PL6, PL7, PL17, and PL38, respectively). Furthermore, the degradation products of nine alginate lyases were detected, among which Aly38A was the first alginate lyase belonging to the PL38 family that has been found to degrade alginate. The combination of alginate lyases functioning in the alginate-degrading process was further demonstrated by the growth curve and alginate lyase production of strain B2Z047 cultivated with or without sodium alginate, as well as the content changes of total sugar and reducing sugar and the transcript levels of alginate lyase genes. A simplified model was proposed to explain the alginate utilization process of Agarivorans sp. B2Z047.

Complete genome sequence of Kordiimonas pumila N18T sheds light on biogeochemical roles of the genus Kordiimonas

Kordiimonas pumila N18T (=KCTC 62164T=MCCC 1K03436T) was isolated from coastal sediments collected in Zhoushan, P. R. China. Ensuing genome sequencing revealed that it consists of a single chromosome with a size of 4,041,658bp and a DNA G+C content of 47.4%. The genomic annotations further identified 3724 open reading frames (ORFs), 4 rRNA genes, and 43 tRNA genes in the Kordiimonas pumila N18T genome. The percentages of ORFs assigned to the CAZy, COG, GO, and KEGG databases were 1.0, 84.8, 19.9, and 59.3%, respectively. Comparative genomic analysis with four other Kordiimonas genomes indicated that the pan- and core-genomes of the genus Kordiimonas consists of 8985 and 1470 orthologous clusters, respectively. Functional annotations of the genus Kordiimonas core genome further revealed that they utilize monosaccharides, peptides, and esters as energy substrates because of the absence of several glycoside hydrolases, glycosyl transferases, aminopeptidases, carbohydrate esterase coding genes, and polysaccharide lyases coding genes in their genomes. Moreover, the genus Kordiimonas possesses survival advantages in ammonium-, phosphorus-, and iron- deficient environments, which are vital for inhabiting marine environments. Overall, this study reports the complete genome of the order Kordiimonadales and broadens the understanding of its ecological roles.

Whole-Genome Sequence Resource of Fusarium oxysporum Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from Tetrastigma hemsleyanum

Whole-Genome Sequence Resource of <i>Fusarium oxysporum</i> Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from <i>Tetrastigma hemsleyanum</i>

Cloning, expression and improvement of catalytic activity of alginate lyase by site-directed mutation

Alginate lyase mainly produces active alginate oligosaccharides (AOS) by degrading alginate via β-elimination process. In this study, the Pseudoalteromonas sp. Alg6B alginate lyase-encoding gene alg6B-7 from polysaccharide lyase (PL)-7 family was successfully cloned, sequenced, expressed in Escherichia coli. Based on rational design and amino acid sequence alignment of the alginate lyase from various sources, four positive mutants were obtained. The specific enzyme activities of four mutants I62A, A99K, V132S, and L157T were 38.84%, 42.85%, 75.8% and 51.83% higher than that of the wild enzyme, respectively. The Kcat/Km values of the four mutants were both increased, and the catalytic efficiency of V132S was 1.92-fold higher than that of the wild enzyme, especially. The rational design that was employed in this study achieved the dramatic improvement of catalytic activity, which may provide the application potential in industrial production.

Biochemical Properties of a New Polysaccharide Lyase Family 25 Ulvan Lyase TsUly25B from Marine Bacterium Thalassomonas sp. LD5.

Marine macroalgae, contributing much to the bioeconomy, have inspired tremendous attention as sustainable raw materials. Ulvan, as one of the main structural components of green algae cell walls, can be degraded by ulvan lyase through the β-elimination mechanism to obtain oligosaccharides exhibiting several good physiological activities. Only a few ulvan lyases have been characterized until now. This thesis explores the properties of a new polysaccharide lyase family 25 ulvan lyase TsUly25B from the marine bacterium Thalassomonas sp. LD5. Its protein molecular weight was 54.54 KDa, and it was most active under the conditions of 60 °C and pH 9.0. The Km and kcat values were 1.01 ± 0.05 mg/mL and 10.52 ± 0.28 s−1, respectively. TsUly25B was salt-tolerant and NaCl can significantly improve its thermal stability. Over 80% of activity can be preserved after being incubated at 30 °C for two days when the concentration of NaCl in the solution is above 1 M, while 60% can be preserved after incubation at 40 °C for 10 h with 2 M NaCl. TsUly25B adopted an endolytic manner to degrade ulvan polysaccharides, and the main end-products were unsaturated ulvan disaccharides and tetrasaccharides. In conclusion, our research enriches the ulvan lyase library and advances the utilization of ulvan lyases in further fundamental research as well as ulvan oligosaccharides production.

Cloning, expression, and characterization of a novel endo-type alginate lyase from Microbulbifer sp. BY17.

Alginate oligosaccharides (AOS), with various physiological effects, have been widely used in the food, agricultural, and pharmaceutical industries. The biological enzymatic method of preparing AOS, using alginate lyase, has more advantages compared with physical and chemical methods. Cloning and heterologously expressing alginate lyase are therefore very important. A novel alginate lyase, BY17PV7, from Microbulbifer sp. BY17, isolated from Gracilaria, was cloned and expressed in Escherichia coli BL21(DE3). BY17PV7 was about 27 KDa. BY17PV7 showed the greatest activity (150.42 3.32 U/mg) at 43 °C and pH8.9. It could be activated by Ca2+ , Mn2+ , Co2+ , Fe3+ , Na+ , and inhibited by Mg2+ , Zn2+ , Ba2+ , Cu2+ , sodium dodecyl sulfate (SDS), ethylene diamine tetraacetic acid (EDTA). BY17PV7 had a wide range of substrate specificity and good degradation effects for poly β-D-mannuronate (polyM) and poly α-L-guluronate (polyG), demonstrating that it is a bifunctional alginate lyase. The kinetic parameters showed that BY17PV7 had a greater affinity for polyG. BY17PV7 released AOS with a degree of polymerization (DP) of 3-4 in an endolytic manner from sodium alginate. Alginate oligosaccharides showed strong antioxidant ability of reducing Fe3+ and scavenging radicals such as hydroxyl, 2,2-azion-bia (3-ethylbenzo-thiazoline-6-sulfonic acid diammonium salt) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). A novel bifunctional alginate lyase, BY17PV7, was expressed and characterized in Escherichia coli BL21(DE3). The results were helpful for the analysis of the molecular mechanisms of degrading patterns in the polysaccharide lyase (PL) family. © 2022 Society of Chemical Industry.

In-silico analysis of the regulatory region of effector protein genes in Verticillium dahliae

Verticillium dahliae is a soil-borne fungal plant pathogen that infects more than 400 plant species throughout the world, causing huge economic losses annually. Due to its wide host range and adaptation strategy of producing resting structures, Verticillium infection and spread are difficult to control. The resting dormant melanized structures called microsclerotia, produced during the dormant stage of the pathogen can persist in soil for years and are the primary source of infection as they germinate after being stimulated by root exudates. The fungus secrets effector proteins that facilitate the primary infection and subsequent spread. In the current study, we used in silico tools to analyze effector proteins belonging to hydrophobin, necrosis, and ethylene inducing like-protein and polysaccharide lyase 3 protein family which are required for virulence and pathogenicity of the pathogen. Our study showed that the genes for these protein families consist of many paralogs. Some of these paralogous proteins display similar functions but differential expression during pathogenicity establishment. This study will provide information about cis-acting factors regulating differential regulation of paralogs during infection.

Structural and biological insights into Klebsiella pneumoniae surface polysaccharide degradation by a bacteriophage K1 lyase: implications for clinical use

BackgroundK1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge.MethodsWe analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes.ResultsWe found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1–3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric β-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose.ConclusionsOur results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.

Recalcitrant cell wall of Ulva lactuca seaweed is degraded by a single ulvan lyase from family 25 of polysaccharide lyases.

Green macroalgae, e.g., Ulva lactuca, are valuable bioactive sources of nutrients; but algae recalcitrant cell walls, composed of a complex cross-linked matrix of polysaccharides, can compromise their utilization as feedstuffs for monogastric animals. This study aimed to evaluate the ability of pre-selected Carbohydrate-Active enZymes (CAZymes) and sulfatases to degrade U. lactuca cell walls and release nutritive compounds. A databank of 199 recombinant CAZymes and sulfatases was tested in vitro for their action towards U. lactuca cell wall polysaccharides. The enzymes were incubated with the macroalga, either alone or in combination, to release reducing sugars and decrease fluorescence intensity of Calcofluor White stained cell walls. The individual action of a polysaccharide lyase family 25 (PL25), an ulvan lyase, was shown to be the most efficient in cell wall disruption. The ulvan lyase treatment, in triplicate measures, promoted the release of 4.54 g/L (P < 0.001) reducing sugars, a mono- and oligosaccharides release of 11.4 and 11.2 mmol/100 g of dried alga (P < 0.01), respectively, and a decrease of 41.7% (P < 0.001) in cell wall fluorescence, in comparison to control. The ability of ulvan lyase treatment to promote the release of nutritional compounds from alga biomass was also evaluated. A release of some monounsaturated fatty acids was observed, particularly the health beneficial 18:1c9 (P < 0.001). However, no significant release of total fatty acids (P > 0.05), proteins (P = 0.861) or pigments (P > 0.05) was found. These results highlight the capacity of a single recombinant ulvan lyase (PL25 family) to incompletely disrupt U. lactuca cell walls. This enzyme could enhance the bioaccessibility of U. lactuca bioactive products with promising utilization in the feed industry.

The Function of CBM32 in Alginate Lyase VxAly7B on the Activity on Both Soluble Sodium Alginate and Alginate Gel.

Carbohydrate-binding modules (CBMs), as an important auxiliary module, play a key role in degrading soluble alginate by alginate lyase, but the function on alginate gel has not been elucidated. Recently, we reported alginate lyase VxAly7B containing a CBM32 and a polysaccharide lyase family 7 (PL7). To investigate the specific function of CBM32, we characterized the full-length alginate lyase VxAly7B (VxAly7B-FL) and truncated mutants VxAly7B-CM (PL7) and VxAly7B-CBM (CBM32). Both VxAly7B-FL and native VxAly7B can spontaneously cleavage between CBM32 and PL7. The substrate-binding capacity and activity of VxAly7B-CM to soluble alginate were 0.86- and 1.97-fold those of VxAly7B-FL, respectively. Moreover, CBM32 could accelerate the expansion and cleavage of alginate gel beads, and the degradation rate of VxAly7B-FL to alginate gel beads was threefold that of VxAly7B-CM. Results showed that CBM32 is not conducive to the degradation of soluble alginate by VxAly7B but is helpful for binding and degradation of insoluble alginate gel. This study provides new insights into the function of CBM32 on alginate gel, which may inspire the application strategy of CBMs in insoluble substrates.

Identification and Expression of Some Plant Cell Wall-Degrading Enzymes Present in Three Ontogenetics Stages of Thecaphora frezii, a Peanut (&amp;lt;i&amp;gt;Arachis hypogaea&amp;lt;/i&amp;gt; L.) Pathogenic Fungus

Peanuts can be affected by the presence of pathogenic microorganisms. The fungus Thecaphora frezii (T. frezii), which belongs to the taxonomic class Ustilaginomycetes, is the causal agent of the disease known as “peanut smut”. The life cycle of this fungus includes three stages, namely teliospores, basidiospores and hyphae. In the hyphae stage, infection occurs in the peanut plant, which requires the involvement of some enzymes secreted by the fungus. These include the Plant Cell Wall-Degrading Enzymes (PCWDEs), which degrade various polysaccharides. This study aimed to identify the presence of transcript for enzymes belonging to the PCWDEs from three stages of T. frezii. For this, total RNA was extracted from the three ontogenetic stages of T. frezii. These samples were analyzed using an RNA-Seq approach and some transcripts were quantified using Real Time PCR. The analysis of the data provided by the RNA-Seq of the three T. frezii stages, it was possible to identify some transcripts that could encode enzymes compatible with polysaccharides degradation that are part of the plant cell wall. In T. frezii transcriptome, 40 deduced proteins would be enzymes with functions of PCWDEs were identified. They were divided into 27 glycoside hydrolases; two polysaccharide lyases; three carbohydrate esterases and eight enzymes with auxiliary activities. In addition, the fungal SNF1 gene was identified whose activity could be affected by high glucose level, and indirectly influence the levels of some PCWDEs. The analysis of the PCWDEs could help to understand part of the fungal infection process and possibly find substances that can control its development.