Investigating functional mechanisms in the Co-biodegradation of lignite and guar gum under the influence of salinity.

The biodegradation of guar gum by microorganisms sourced from coalbeds can result in low-temperature gel breaking, thereby reducing reservoir damage. However, limited attention has been given to the influence of salinity on the synergistic biodegradation of coal and guar gum. In this study, biodegradation experiments of guar gum and lignite were conducted under varying salinity conditions. The primary objective was to investigate the controlling effects and mechanisms of salinity on the synergistic biodegradation of lignite and guar gum. The findings revealed that salinity had an inhibitory effect on the biomethane production from the co-degradation of lignite and guar gum. The biomethane production declined with increasing salinity levels, decreasing from 120.9 mL to 47.3 mL. Even under 20 g/L salt stress conditions, bacteria in coalbeds could effectively break the gel and the viscosity decreased to levels below 5 mPa s. As salinity increased, the removal rate of soluble chemical oxygen demand (SCOD) decreased from 55.63% to 31.17%, and volatile fatty acids (VFAs) accumulated in the digestion system. High salt environment reduces the intensity of each fluorescence peak. Alterations in salinity led to changes in microbial community structure and diversity. Under salt stress, there was an increased relative abundance of Proteiniphilum and Methanobacterium, ensuring the continuity of anaerobic digestion. Hydrogentrophic methanogens exhibited higher salt tolerance compared to acetoclastic methanogens. These findings provide experimental evidence supporting the use of guar gum fracturing fluid in coalbeds with varying salinity levels.

Metabolism mechanisms of biogenic methane production by synergistic biodegradation of lignite and guar gum.

Coalbed methane (CBM) presents a promising energy source for addressing global energy shortages. Nonetheless, challenges such as low gas production from individual wells and difficulties in breaking gels at low temperatures during extraction hinder its efficient utilization. Addressing this, we explored native microorganisms within coal seams to degrade guar gum, thereby enhancing CBM production. However, the underlying mechanisms of biogenic methane production by synergistic biodegradation of lignite and guar gum remain unclear. Research results showed that the combined effect of lignite and guar gum enhanced the production, yield rate and concentration of biomethane. When the added guar gum content was 0.8 % (w/w), methane production of lignite and guar gum reached its maximum at 561.9 mL, which was 11.8 times that of single lignite (47.3 mL). Additionally, guar gum addition provided aromatic and tryptophan proteins and promoted the effective utilization of CC/CH and OCO groups on the coal surface. Moreover, the cooperation of lignite and guar gum accelerated the transformation of volatile fatty acids into methane and mitigated volatile fatty acid inhibition. Dominant bacteria such as Sphaerochaeta, Macellibacteroides and Petrimonas improved the efficiency of hydrolysis and acidification. Electroactive microorganisms such as Sphaerochaeta and Methanobacterium have been selectively enriched, enabling the establishment of direct interspecies electron transfer pathways. This study offers valuable insights for increasing the production of biogenic CBM and advancing the engineering application of microbial degradation of guar gum fracturing fluid. Future research will focus on exploring the methanogenic capabilities of lignite and guar gum in in-situ environments, as well as elucidating the specific metabolic pathways involved in their co-degradation.

Fermenter scale production of recombinant beta-mannanase by E. coli BL21 cells under microaerobic environment.

Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides.

Effects of partially hydrolyzed guar gums of different molecular weights on a human intestinal in vitro fermentation model.

Partially hydrolyzed guar gums (PHGGs) are prebiotic soluble dietary fibers. High molecular-weight PHGGs have rapid fermentation and high short-chain fatty acid (SCFA) productivity rates, compared to low molecular-weight PHGGs. Therefore, low molecular-weight PHGGs may have less pronounced prebiotic effects than high molecular-weight PHGGs. However, the effects of PHGGs of different molecular weights on the human intestinal microbiota, as well as their fermentation ability and prebiotic effects, have not been investigated. The aim of this study was to evaluate the effects of two PHGGs of different molecular weights, Sunfiber-R (SF-R; 20 kDa) and Sunfiber-V (SF-V; 5 kDa), on human colonic microbiota and SCFA production. A human intestinal in vitro fermentation model was operated by fecal samples with and without the PHGGs. The addition of 0.2% SF-R or SF-V increased the relative abundance of Bacteroides spp., especially that of Bacteroides uniformis. This increase corresponded to a significant (p = 0.030) and non-significant (p = 0.073) increase in propionate production in response to SF-R and SF-V addition, respectively. Both fibers increased the relative abundance of Faecalibacterium and stimulated an increase in the abundance of unclassified Lachnospiraceae and Bifidobacterium. In conclusion, the low molecular-weight PHGG exerted prebiotic effects on human colonic microbiota to increase SCFA production and bacteria that are beneficial to human health in a manner similar to that of the high molecular-weight forms of PHGG.

Progresses in chitin, chitosan, starch, cellulose, pectin, alginate, gelatin and gum based (nano)catalysts for the Heck coupling reactions: A review.

Heck cross-coupling reaction (HCR) is one of the few transition metal catalyzed CC bond-forming reactions, which has been considered as the most effective, direct, and atom economical synthetic method using various catalytic systems. Heck reaction is widely employed in numerous syntheses including preparation of pharmaceutical and biologically active compounds, agrochemicals, natural products, fine chemicals, etc. Commonly, Pd-based catalysts have been used in HCR. In recent decades, the application of biopolymers as natural and effective supports has received attention due to their being cost effective, abundance, and non-toxicity. In fact, recent studies demonstrated that biopolymer-based catalysts had high sorption capacities, chelating activities, versatility, and stability, which make them potentially applicable as green materials (supports) in HCR. These catalytic systems present high stability and recyclability after several cycles of reaction. This review aims at providing an overview of the current progresses made towards the application of various polysaccharide and gelatin-supported metal catalysts in HCR in recent years. Natural polymers such as starch, gum, pectin, chitin, chitosan, cellulose, alginate and gelatin have been used as natural supports for metal-based catalysts in HCR. Diverse aspects of the reactions, different methods of preparation and application of polysaccharide and gelatin-based catalysts and their reusability have been reviewed.

Synthesis, characterization and use of enzyme cashew gum nanoparticles for biosensing applications.

This research reports, for the first time, the immobilization of an enzyme - Rhus vernificera laccase - on cashew gum (CG) nanoparticles (NPs) and its application as a biological layer in the design and development of an electrochemical biosensor. Laccase-CG nanoparticles (LacCG-NPs) were prepared by the nanoprecipitation method and characterized by UV-Vis spectrophotometry, atomic force microscopy, scanning electron microscopy, attenuated total reflectance-Fourier-transform infrared spectroscopy, circular dichroism, cyclic voltammetry, and electrochemical impedance spectroscopy. The average size and stability of the NPs were predicted by DLS and zeta potential. The ATR-FTIR results clearly demonstrated an interaction between -NH and -OH groups to form LacCG-NPs. The average size found for LacCG-NPs was 280 ± 53 nm and a polydispersity index of 0.309 ± 0.08 indicated a good particle size distribution. The zeta potential shows a good colloidal stability. The use of a natural product to prepare the enzymatic nanoparticles, its easy synthesis and the immobilization efficiency should be highlighted. LacCG-NPs were successfully applied as a biolayer in the development of an amperometric biosensor for catechol detection. The resulting device showed a low response time (6 s), good sensitivity (7.86 µA µM-1 cm-2), wide linear range of 2.5 × 10-7-2.0 × 10-4 M, and low detection limit (50 nM).

Physical, sensory, and simulated mastication properties of texture-modified Spanish sauce using different texturing agents.

This study aims to evaluate the colour texture, flow, viscoelastic, sensory, and simulated mastication properties, in the presence and absence of artificial saliva, of texture-modified Spanish sauce at different temperatures (25 °C, 37 °C and/or 55 °C). Sauce texture was modified using five hydrocolloids (modified starch (MS), guar gum (GG), tara gum (TG), sodium carboxymethylcellulose (CMC), and chia seed mucilage (CSM) as an alternative texturing agent), achieving two well-differentiated consistencies: honey-like and pudding-like. The MS, GG, TG and CSM sauces showed greater consistency, firmness, stiffness, and resistance to flow than the CMC samples. Furthermore, the internal structure of CMC sauces was the most affected by temperature changes. The addition of saliva decreased the apparent viscosity, consistency, and adhesiveness of the sauces. Among the samples studied, the GG and CSM texture-modified sauces would be suitable for dysphagic patients because of their good elasticity, relatively high resistance to deformation and structural stability, as well as better resistance to salivary α-amylase action. However, CSM sauces obtained the lowest sensory attribute scores. This work opens the door to the use of CSM as a texturing agent and demonstrates the importance of considering not only the hydrocolloid type and consistency level, but also the administration temperature of dysphagia-oriented products. Selecting a suitable texturing agent is of great importance for safe and easy swallowing by dysphagic patients.

Identification of Key Metabolic Pathways and Biomarkers Underlying Flowering Time of Guar (Cyamopsis tetragonoloba (L.) Taub.) via Integrated Transcriptome-Metabolome Analysis.

Guar (Cyamopsis tetragonoloba (L.) Taub.) is an annual legume crop native to India and Pakistan. Seeds of the plant serve as a source of galactomannan polysaccharide (guar gum) used in the food industry as a stabilizer (E412) and as a gelling agent in oil and gas fracturing fluids. There were several attempts to introduce this crop to countries of more northern latitudes. However, guar is a plant of a short photoperiod, therefore, its introduction, for example, to Russia is complicated by a long day length during the growing season. Breeding of new guar varieties insensitive to photoperiod slowed down due to the lack of information on functional molecular markers, which, in turn, requires information on guar genome. Modern breeding strategies, e.g., genomic predictions, benefit from integration of multi-omics approaches such as transcriptome, proteome and metabolome assays. Here we present an attempt to use transcriptome-metabolome integration to understand the genetic determination of flowering time variation among guar plants that differ in their photoperiod sensitivity. This study was performed on nine early- and six delayed-flowering guar varieties with the goal to find a connection between 63 metabolites and 1,067 differentially expressed transcripts using Shiny GAM approach. For the key biomarker of flowering in guar myo-inositol we also evaluated the KEGG biochemical pathway maps available for Arabidopsis thaliana. We found that the phosphatidylinositol signaling pathway is initiated in guar plants that are ready for flowering through the activation of the phospholipase C (PLC) gene, resulting in an exponential increase in the amount of myo-inositol in its free form observed on GC-MS chromatograms. The signaling pathway is performed by suppression of myo-inositol phosphate kinases (phosphorylation) and alternative overexpression of phosphatases (dephosphorylation). Our study suggests that metabolome and transcriptome information taken together, provide valuable information about biomarkers that can be used as a tool for marker-assisted breeding, metabolomics and functional genomics of this important legume crop.

Effect of starch-hydrocolloid complexes with heat-moisture treatment on in vivo digestibility.

The purpose of this study was to investigate the effect of starch-hydrocolloid (gum arabic, xanthan gum, and guar gum) complexes with heat-moisture treatment (HMT) on in vivo digestibility. In vivo digestibility experiments revealed that the body weight, liver weight, and fat index of mice in the intervention group were significantly reduced compared with those in the high-fat group. Glucose tolerance improved, and blood lipid levels, liver and adipose tissue morphology returned to normal. The results of mRNA expression levels showed that the intervention of corn starch-hydrocolloid complexes after HMT down-regulated the expression level of genes related to fat synthesis compared with the high-fat group, which could decrease lipid deposition and stabilize blood lipid levels. Results revealed that starch-xanthan gum complex (1 : 40 ratio) with HMT could markedly reduce the digestibility of starch. Overall, this study provides new ideas for the application of low-glycemic-index and functional foods.

Comparative transcriptome and metabolome profiling in the maturing seeds of contrasting cluster bean (Cyamopsis tetragonoloba L. Taub) cultivars identified key molecular variations leading to increased gum accumulation.

Cluster bean (Guar) is the major source of industrial gum. Knowledge on the molecular events regulating galactomannan gum accumulation in guar will pave way for accelerated development of gummy guar genotypes. RNA Seq analysis in the immature seeds of contrasting cluster bean genotypes HGS 563 (gum type) and Pusa Navbahar (vegetable type) resulted in the generation of 19,855,490 and 21,488,472 quality reads. Data analysis identified 4938 differentially expressed genes between the gummy vs vegetable genotypes. A set of 2241 genes were up-regulated and 2587 genes were down-regulated in gummy guar. Significant up-regulation of genes involved in the biosynthesis of galactomannan and cell wall storage polysaccharides was observed in the gummy HGS 563. Genes involved in carotenoids, flavonoids, non mevalonic acid, terpenoids, and wax metabolism were also up-regulated in HGS 563. Mannose and galactose were the major nucleotide sugars in Pusa Navbahar and HGS 563 immature seeds. Immature seeds of HGS 563 showed high concentration of mannose and galactose accumulation compared to Pusa Navbahar. qRT-PCR analysis of selected genes confirmed the findings of transcriptome data.

Mannose- and Mannobiose-Specific Responses of the Insect-Associated Cellulolytic Bacterium Streptomyces sp. Strain SirexAA-E.

The cellulolytic insect symbiont bacterium Streptomyces sp. strain SirexAA-E secretes a suite of carbohydrate-active enzymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. Using genomic analysis, we found a unique 12-bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and thus they are likely under the regulation of both SsManR and SsCebR. IMPORTANCEStreptomyces sp. SirexAA-E, a microbial symbiont of biomass-harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.

Frequent consumption of sap suggests that omnivory is widespread among Australian geckos.

The diets of many animals are influenced by resource availability, competition, and evolutionary selected traits enabling the utilization of palatable foods. Omnivores are species that maintain their macronutrient balance by supplementing highly abundant but poor nutritional quality food items, with sporadically available but high nutritional quality food items. Although there are anecdotal observations of Australian geckos (Lacertilia: Gekkonidae) consuming plant exudates, the consumption of plant material has long been considered to be anomalous behavior among Australian geckos. Here, we test the idea that sap feeding may not be anomalous behavior but instead a dietary niche of geckos that has gone unappreciated due to constraints on the methods used to quantify geckos' diets. We tested this idea by investigating the consumption of Acacia victoriae gum by the gecko Gehyra versicolor using timed searches and time-lapse photography. We found that geckos frequently consumed gum, and G. versicolor numbers were five times greater on A. victoriae trees that exhibited significant gum bleeds compared to gecko numbers on non-bleeding trees. Taken together, our observations that G. versicolor spp. frequently feed on gum along with anecdotal reports of geckos consuming gum provide compelling evidence that gum/sap feeding is not anomalous behavior and suggest that many Australian gecko species are omnivores whose diets include plant exudates and animal prey.

Carob kibbles as an alternative raw material for production of kvass with probiotic potential.

BACKGROUND: Non-diary beverages with probiotic properties are of great interest nowadays. In this research, we evaluated the suitability of carob kibbles in the manufacture of kvass. Kvass is a low-alcohol drink popular in Central and Eastern Europe and indicated as a potential non-diary beverage with probiotic properties. Therefore, the viability of probiotic strains of Lactobacillus plantarum and Saccharomyces boulardii during 4 weeks' storage in manufactured beverages was tested. RESULTS: Carob kibbles introduced significant amounts of phenolic compounds into kvasses, especially gallic acid (up to 117.45 ± 10.56 mg L-1 ), and improved antiradical activity up to 78% after fermentation. Moreover, fermentation efficiently reduced furfural and hydroxymethylfurfural content in samples up to 12.9% and 29.9%, respectively. Kvasses with rye malt extract possessed coffee-like, chocolate-like, roasted and caramel-like odours and a more bitter taste. Whereas kvass with carob kibbles was characterized by fruit-like odour and sweeter taste. Fermentation contributed to a creation as well as degradation of volatiles. L. plantarum exhibited higher general mortality during storage, whereas, in the case of S. boulardii, the viability was significantly higher regardless of the sample composition. CONCLUSION: This is the first study reporting the use of carob kibbles for kvass production. The obtained results showed that carob kibbles can replace rye malt extract, at least partially, in the production of kvass, giving to the product added health benefits. Moreover, S. boulardii is a better choice for production of kvass with probiotic properties. © 2021 Society of Chemical Industry.

CYP3A4 mediated pharmacokinetics drug interaction potential of Maha-Yogaraj Gugglu and E, Z guggulsterone.

Maha yogaraja guggulu (MYG) is a classical herbomineral polyherbal formulation being widely used since centuries. The aim of this study was to investigate the effect of MYG formulation and its major constituents E & Z guggulsterone on CYP3A4 mediated metabolism. In vitro inhibition of MYG and Guggulsterone isomers on CYP3A4 was evaluated by high throughput fluorometric assay. Eighteen Adult male Sprague-Dawley rats (200 ± 25 g body weight) were randomly divided into three groups. Group A, Group B and Group C were treated with placebo, MYG and Standard E & Z guggulsterone for 14 days respectively by oral route. On 15th day, midazolam (5 mg/kg) was administered orally to all rats in each group. Blood samples (0.3 mL) were collected from the retro orbital vein at 0.25, 0.5, 0.75, 1, 2, 4, 6, 12 and 24 h of each rat were collected. The findings from the in vitro & in vivo study proposed that the MYG tablets and its guggulsterone isomers have drug interaction potential when consumed along with conventional drugs which are CYP3A4 substrates. In vivo pharmacokinetic drug interaction study of midazolam pointed out that the MYG tablets and guggulsterone isomers showed an inhibitory activity towards CYP3A4 which may have leads to clinically significant interactions.

Lignolytic and hemicellulolytic enzyme cocktail production from Bacillus tequilensis LXM 55 and its application in pulp biobleaching.

Bioprocessing of pulp requires lignolytic as well as hemicellulolytic enzymes. The present study is the first report of a cocktail of laccase (L), xylanase (X), and mannanase (M), from a single bacterium for pulp biobleaching. A novel strain Bacillus tequilensis LXM 55 produced thermo-alkali stable L + X + M. On optimization higher enzyme yield (IUml-1/fold increase) of laccase (396.35/24.16), xylanase (212.95/81.90) and mannanase (153.33/102.90) were achieved in the cocktail. Treatment of pulp with cocktail of enzymes led to 49.35% reduction in kappa number and considerable enhancement in the brightness (11.59%), whiteness (4.11%), and other pulp properties. Most importantly, no mediator system was required for the application of laccase. 40% less chlorine consumption was required to obtain the paper of the same quality as that of pulp treated without enzyme but with 100% chlorine. Therefore, this cocktail of enzymes is highly suitable for pulp biobleaching in the paper mill.

Characterization of a thermotolerant and acidophilic mannanase producing Microbacterium sp. CIAB417 for mannooligosachharide production from agro-residues and dye decolorization.

Mannanases are ubiquitous enzymes and are being explored for diverse industrial applications. In this study, a novel bacterial strain Microbacterium sp. CIAB417 was identified and characterized for extracellular production of mannanase. Microbacterium sp. CIAB417 was found to produce maximum mannanase after 36 h of incubation at 37 °C. Mannanase produced by the isolate was observed for maximum activity at optimum pH of 6 and optimum temperature of 50 °C. Crude mannanase was found to be capable of producing mannooligosachharides (MOS) by hydrolyzing hemicellulose from locust bean gum and Aloe vera. The produced MOS was characterized and found to be mixture of mannobiose to mannohexose units. Mannanase was also explored for decolorization of dyes. Bromophenol blue and coomassie blue R-250 were observed to be decolorized to the extent of 45.40 and 42.75%, respectively. Hence, the identified bacterial strain producing mannanase could be of great significance for applications in food and textile industry.

Dietary galactosyl and mannosyl carbohydrates: In-vitro assessment of prebiotic effects.

The prebiotic activities of hydrolyzed guar gum (GMOS, <1 kDa; GMPS, 1-10 kDa), manno-oligosaccharide (MOS, <1 kDa), and galacto-oligosaccharide (GOS, <1 kDa) were evaluated by in vitro fermentation. The tested carbohydrates showed selective prebiotic effects on bacterial growth, short-chain fatty acid (SCFA)-production, and substrate consumption. GOS and GMOS markedly promoted the growth of bifidobacteria and Clostridium butyricum, respectively, whereas MOS showed the strongest butyrogenic effect. Moreover, SCFA production in the hydrolyzed guar gum groups was closely related to the varied molecular weight (Mw) of the hydrolysate. During in vitro fermentation with human fecal inocula, GMOS gave the highest yields of lactate, propionate, and butyrate after 48 h fermentation. Combined application of MOS and C. butyricum increased the abundance of Clostridiaceae_1. Overall, our results indicate that galactosyl and mannosyl carbohydrates have individualized prebiotic effects which are associated with their chemical structures including their glycoside composition and Mw.

Properties of hydrolyzed guar gum fermented in vitro with pig fecal inocula and its favorable impacts on microbiota.

In order to identify an appropriate substitute for antibiotic use in livestock production, this study investigates the fermentation of guar gum and its low molecular weight hydrolyzed derivatives (GMLP-1, 1-10 kDa; GMLP-2, < 1 kDa) in pig fecal cultures and the associated effects on the intestinal microbiota. Both the non-hydrolyzed guar gum and GMLP were quickly utilized by fecal microbiota. GMLP-2 showed the most rapid SCFA-producing activity and produced higher concentrations of lactate, acetate, and propionate. However, GMLP-1 showed the highest yield of total SCFAs and butyrate. Both the guar gum and GMLP groups improved the abundance of Clostridium sensu stricto 1 and Bifidobacterium, but the most significant enhancement was observed with GMLP-1. This study showed that by associating with its chemical structure, GMLP-1 can be utilized to direct a targeted promotion of the intestinal microbiota and may offer the most favorable effects in livestock production.

A thermophilic fungal GH36 α-galactosidase from Lichtheimia ramosa and its synergistic hydrolysis of locust bean gum.

A novel GH36 α-galactosidase gene (LrAgal36A) from Lichtheimia ramosa was synthesized and highly expressed in Pichia pastoris. The enzyme titer and protein yield for high-density fermentation in a 5 L fermentor were up to 953.6 U mL-1 and 4.36 g L-1. Purified recombinant LrAgal36A showed the maximum activity at pH 6.0 and 65 °C and was thermostable with a half-life of 70 min at 60 °C. LrAgal36A displayed the highest specific activity (353.17 ± 4.19 U mg-1) toward p-nitrophenyl-α-d-galactopyranoside (pNPGal) followed by galacto-oligosaccharides and could act slightly on galactomannans. The Km and catalytic efficiency (kcat/Km) of LrAgal36A for pNPGal were 0.33 mM and 1569.50 mM-1 s-1, respectively. LrAgal36A and GH5 ß-mannanase from L. ramosa showed a significant synergistic effect on the degradation of locust bean gum (LBG), resulting in release more reducing sugars (1.56 folds) and galactose (7.6 folds) by simultaneous or sequential reactions. Due to its hydrolysis properties, LrAgal36A might have potential applications in the area of pulp biobleaching, feed and food processing.

A Multifunctional Polysaccharide Utilization Gene Cluster in Colwellia echini Encodes Enzymes for the Complete Degradation of κ-Carrageenan, ι-Carrageenan, and Hybrid ß/κ-Carrageenan.

Algal cell wall polysaccharides constitute a large fraction in the biomass of marine primary producers and are thus important in nutrient transfer between trophic levels in the marine ecosystem. In order for this transfer to take place, polysaccharides must be degraded into smaller mono- and disaccharide units, which are subsequently metabolized, and key components in this degradation are bacterial enzymes. The marine bacterium Colwellia echini A3T is a potent enzyme producer since it completely hydrolyzes agar and κ-carrageenan. Here, we report that the genome of C. echini A3T harbors two large gene clusters for the degradation of carrageenan and agar, respectively. Phylogenetical and functional studies combined with transcriptomics and in silico structural modeling revealed that the carrageenolytic cluster encodes furcellaranases, a new class of glycoside hydrolase family 16 (GH16) enzymes that are key enzymes for hydrolysis of furcellaran, a hybrid carrageenan containing both ß- and κ-carrageenan motifs. We show that furcellaranases degrade furcellaran into neocarratetraose-43-O-monosulfate [DA-(α1,3)-G4S-(ß1,4)-DA-(α1,3)-G], and we propose a molecular model of furcellaranases and compare the active site architectures of furcellaranases, κ-carrageenases, ß-agarases, and ß-porphyranases. Furthermore, C. echini A3T was shown to encode κ-carrageenases, ι-carrageenases, and members of a new class of enzymes, active only on hybrid ß/κ-carrageenan tetrasaccharides. On the basis of our genomic, transcriptomic, and functional analyses of the carrageenolytic enzyme repertoire, we propose a new model for how C. echini A3T degrades complex sulfated marine polysaccharides such as furcellaran, κ-carrageenan, and ι-carrageenan.IMPORTANCE Here, we report that a recently described bacterium, Colwellia echini, harbors a large number of enzymes enabling the bacterium to grow on κ-carrageenan and agar. The genes are organized in two clusters that encode enzymes for the total degradation of κ-carrageenan and agar, respectively. As the first, we report on the structure/function relationship of a new class of enzymes that hydrolyze furcellaran, a partially sulfated ß/κ-carrageenan. Using an in silico model, we hypothesize a molecular structure of furcellaranases and compare structural features and active site architectures of furcellaranases with those of other GH16 polysaccharide hydrolases, such as κ-carrageenases, ß-agarases, and ß-porphyranases. Furthermore, we describe a new class of enzymes distantly related to GH42 and GH160 ß-galactosidases and show that this new class of enzymes is active only on hybrid ß/κ-carrageenan oligosaccharides. Finally, we propose a new model for how the carrageenolytic enzyme repertoire enables C. echini to metabolize ß/κ-, κ-, and ι-carrageenan.