Cloning, expression, and characterization of two pectate lyases isolated from the sheep rumen microbiome.

Pectate lyases (Pels) have a vital function in degradation of the primary plant cell wall and the middle lamella and have been widely used in the industry. In this study, two pectate lyase genes, IDSPel16 and IDSPel17, were cloned from a sheep rumen microbiome. The recombinant enzymes were expressed in Escherichia coli and functionally characterized. Both IDSPel16 and IDSPel17 proteins had an optimal temperature of 60 ℃, and an optimal pH of 10.0. IDSPel16 was relatively stable below 60 °C, maintaining 77.51% residual activity after preincubation at 60 °C for 1 h, whereas IDSPel17 denatured rapidly at 60 °C. IDSPel16 was relatively stable between pH 6.0 and 12.0, after pretreatment for 1 h, retaining over 60% residual activity. IDSPel16 had high activity towards polygalacturonic acid, with a Vmax of 942.90 ± 68.11, whereas IDSPel17 had a Vmax of only 28.19 ± 2.23 µmol/min/mg. Reaction product analyses revealed that IDSPel17 liberated unsaturated digalacturonate (uG2) and unsaturated trigalacturonate (uG3) from the substrate, indicating a typical endo-acting pectate lyase (EC 4.2.2.2). In contrast, IDSPel16 initially generated unsaturated oligogalacturonic acids, then converted these intermediates into uG2 and unsaturated galacturonic acid (uG1) as end products, a unique depolymerization profile among Pels. To the best of our knowledge, the IDSPel16 discovered with both endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities. These two pectate lyases, particularly the relatively thermo- and pH-stable IDSPel16, will be of interest for potential application in the textile, food, and feed industries. KEY POINTS: • Two novel pectate lyase genes, IDSPel16 and IDSPel17, were isolated and characterized from the sheep rumen microbiome. • Both IDSPel16 and IDSPel17 are alkaline pectate lyases, releasing unsaturated digalacturonate and unsaturated trigalacturonate from polygalacturonic acid. • IDSPel16, a bifunctional pectate lyase with endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities, could be a potential candidate for industrial application.

Microbial ß-glucanases: production, properties, and engineering.

Lignocellulosic biomass, which mainly consists of cellulose and hemicellulose, is the most abundant renewable biopolymer on earth. ß-Glucanases are glycoside hydrolases (GHs) that hydrolyze ß-glucan, one of the dominant components of the plant cell wall, into cello-oligosaccharides and glucose. Among them, endo-ß-1,4-glucanase (EC 3.2.1.4), exo-glucanase/cellobiohydrolase (EC 3.2.1.91), and ß-glucosidase (EC 3.2.1.21) play critical roles in the digestion of glucan-like substrates. ß-Glucanases have attracted considerable interest within the scientific community due to their applications in the feed, food, and textile industries. In the past decade, there has been considerable progress in the discovery, production, and characterization of novel ß-glucanases. Advances in the development of next-generation sequencing techniques, including metagenomics and metatranscriptomics, have unveiled novel ß-glucanases isolated from the gastrointestinal microbiota. The study of ß-glucanases is beneficial for research and development of commercial products. In this study, we review the classification, properties, and engineering of ß-glucanases.

Heterologous expression and characterization of two novel glucanases derived from sheep rumen microbiota.

ß-Glucanases are a suite of glycoside hydrolases that depolymerize ß-glucan into cellooligosaccharides and/or monosaccharides and have been widely used as feed additives in livestock. In this study, two novel glucanase genes, IDSGluc5-26 and IDSGluc5-37, derived from sheep rumen microbiota, were expressed and functionally characterized. The optimal temperatures/pH of recombinant IDSGLUC5-26 and IDSGLUC5-37 were 50 °C/5.0 and 40 °C/6.0, respectively. Notably, IDSGLUC5-26 showed considerable stability under acidic conditions. Both IDSGLUC5-26 and IDSGLUC5-37 showed the highest activities toward barley ß-glucan, with Vmax values of 89.96 ± 9.19 µmol/min/mg and 459.50 ± 25.02 µmol/min/mg, respectively. Additionally, these two glucanases demonstrated hydrolysis of Icelandic moss lichenan and konjac gum, IDSGLUC5-26 releasing cellobiose (G2; occupying 17.37% of total reducing sugars), cellotriose (G3; 23.97%), and cellotetraose (G4; 30.93%) from barley ß-glucan and Icelandic moss lichenan after 10 min and suggestive of a typical endo-ß-1,4-glucanase (EC.3.2.1.4). In contrast, IDSGLUC5-37 was capable of liberating dominant G3 (64.11% or 67.55%) from barley ß-glucan or Icelandic moss lichenan, suggesting that the enzyme was likely an endo-ß-1,3 - 1,4-glucanases/lichenase (EC3.2.1.73). These findings describe the expression and characterization of two novel glucanase genes from sheep rumen microbiota. The two recombinant enzymes, particularly the acid-stable IDSGLUC5-26, will be of interest for potential application in food-/feed-additive development.

Enhanced stability of a rumen-derived xylanase using SpyTag/SpyCatcher cyclization.

Microbiota from herbivore rumen is of great interest for mining glycoside hydrolases for lignocellulosic biomass biorefinement. We previously isolated a highly active but poorly thermostable xylanase (LXY) from a rumen fluid fosmid library of Hu sheep, a local high-reproductive species in China. In this study, we used a universal enzyme-engineering strategy called SpyTag/SpyCatcher molecular cyclization to improve LXY stability via isopeptide-bond-mediated ligation. Both linear and cyclized LXY (L- and C-LXY, respectively) shared similar patterns of optimal pH and temperature, pH stability, and kinetic constants (km and Vmax). However, the C-LXY showed enhanced thermostability, ion stability, and resilience to aggregation and freeze-thaw treatment than L-LXY, without compromise of its catalytic efficiency. Circular dichroism and intrinsic and 8-anilino-1-naphthalenesulfonic acid-binding fluorescence analysis indicated that the cyclized enzyme was more capable of maintaining its secondary and tertiary structures than the linear enzyme. Taken together, these results promote the cyclized enzyme for potential applications in the feed, food, paper pulp, and bioenergy industries.

Dynamics Analysis of a New Fractional-Order Hopfield Neural Network with Delay and Its Generalized Projective Synchronization.

In this paper, a new three-dimensional fractional-order Hopfield-type neural network with delay is proposed. The system has a unique equilibrium point at the origin, which is a saddle point with index two, hence unstable. Intermittent chaos is found in this system. The complex dynamics are analyzed both theoretically and numerically, including intermittent chaos, periodicity, and stability. Those phenomena are confirmed by phase portraits, bifurcation diagrams, and the Largest Lyapunov exponent. Furthermore, a synchronization method based on the state observer is proposed to synchronize a class of time-delayed fractional-order Hopfield-type neural networks.

Metabolomics of four biofluids from dairy cows: potential biomarkers for milk production and quality.

The fundamental understanding of the mechanisms regulating milk protein synthesis is limited. This study aimed to elucidate the metabolic mechanisms of milk production affected by forage quality through studying metabolites from four biofluids (rumen fluid, milk, serum, and urine) collected from 16 lactating cows fed alfalfa hay (AH, high-quality, n = 8) and corn stover (CS, low-quality, n = 8) using gas chromatography-time-of-flight/mass spectrometry. The cows fed AH exhibited higher milk yield (P < 0.01), milk protein yield (P = 0.04), and milk efficiency (P < 0.01) than those fed CS. A total of 165, 195, 218, and 156 metabolites were identified in the rumen fluid, milk, serum, and urine, respectively, while 29 metabolites were found in all four biofluids. In addition 55, 8, 28, and 31 metabolites in each biofluid were significantly different (VIP > 1 and P < 0.05) between the AH- and CS-fed animals. These metabolites were involved in glycine, serine, and threonine metabolism; tyrosine metabolism; and phenylalanine metabolism. Further integrated key metabolic pathway analysis showed that the AH-fed cows may have more comprehensive amino acid metabolisms, suggesting that these metabolite-associated pathways may serve as biomarkers for higher milk yield and better milk protein quality.

Pectin induces an in vitro rumen microbial population shift attributed to the pectinolytic Treponema group.

Pectin is a non-fiber carbohydrate (NFC) that exists in forages, but it is not clear how pectin exerts its effect on populations of either known microbial species or uncultured ruminal bacteria. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR analysis were used in the present study to investigate the effects of pectin on microbial communities in an in vitro rumen fermentation system. The fermentations were conducted using forage (corn stover or alfalfa), an NFC source (pectin or corn starch), or their combination as the substrates. Addition of pectin increased acetate (P < 0.05), whereas inclusion of starch increased butyrate production (P < 0.05). The pectate lyase activity was higher with alfalfa than with corn straw, or with pectin than with corn starch (P < 0.05), while the amylase activity was higher in corn starch-included treatments than the others (P < 0.05). The cluster analysis of the bacterial 16S rRNA gene showed that the DGGE banding patterns differed significantly between the treatments and led to the identification of three groups that were highly associated with the NFC sources. The specific bands associated with pectin-rich treatments were identified to be dominated by members of the Treponema genus. The growth of the Treponema genus was remarkably supported by the inclusion of pectin, highlighting their specific ability to degrade pectin. The results from the present study expand our knowledge of the microbial populations associated with pectin digestion, which may not only facilitate future research on utilization of pectin in feeds, but also improve our understanding of pectin digestion with respect to the rumen micro-ecosystem.

Expression and characterization of a novel microbial GH9 glucanase, IDSGLUC9-4, isolated from sheep rumen.

Objective: This study aimed to identify and characterize a novel endo-ß-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep. Methods: A novel endo-ß-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley ß-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates. Results: The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40 °C and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley ß-glucan (109.59 ± 3.61 µmol·mg-1·min-1) and Icelandic moss lichenan (35.35 ± 1.55 µmol·mg-1·min-1). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity. Conclusion: The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.

BsLPMO10A from Bacillus subtilis boosts the depolymerization of diverse polysaccharides linked via ß-1,4-glycosidic bonds.

Lytic polysaccharide monooxygenase (LPMO) is known as an oxidatively cleaving enzyme in recalcitrant polysaccharide deconstruction. Herein, we report a novel AA10 LPMO derived from Bacillus subtilis (BsLPMO10A). A substrate specificity study revealed that the enzyme exhibited an extensive active-substrate spectrum, particularly for polysaccharides linked via ß-1,4 glycosidic bonds, such as ß-(Man1 â†’ 4Man), ß-(Glc1 â†’ 4Glc) and ß-(Xyl1 â†’ 4Xyl). HPAEC-PAD and MALDI-TOF-MS analyses indicated that BsLPMO10A dominantly liberated native oligosaccharides with a degree of polymerization (DP) of 3-6 and C1-oxidized oligosaccharides ranging from DP3ox to DP6ox from mixed linkage glucans and beechwood xylan. Due to its synergistic action with a variety of glycoside hydrolases, including glucanase IDSGLUC5-38, xylanase TfXYN11-1, cellulase IDSGLUC5-11 and chitinase BtCHI18-1, BsLPMO10A dramatically accelerated glucan, xylan, cellulose and chitin saccharification. After co-reaction for 72 h, the reducing sugars in Icelandic moss lichenan, beechwood xylan, phosphoric acid swollen cellulose and chitin yielded 3176 ± 97, 7436 ± 165, 649 ± 44, and 2604 ± 130 µmol/L, which were 1.47-, 1.56-, 1.44- and 1.25-fold higher than those in the GHs alone groups, respectively (P < 0.001). In addition, the synergy of BsLPMO10A and GHs was further validated by the degradation of natural feedstuffs, the co-operation of BsLPMO10A and GHs released 3266 ± 182 and 1725 ± 107 µmol/L of reducing sugars from Oryza sativa L. and Arachis hypogaea L. straws, respectively, which were significantly higher than those produced by GHs alone (P < 0.001). Furthermore, BsLPMO10A also accelerated the liberation of reducing sugars from Celluclast® 1.5 L, a commercial cellulase cocktail, on filter paper, A. hypogaea L. and O. sativa L. straws by 49.58 % (P < 0.05), 72.19 % (P < 0.001) and 54.36 % (P < 0.05), respectively. This work has characterized BsLPMO10A with a broad active-substrate scope, providing a promising candidate for lignocellulosic biomass biorefinery.

Epidemiology and outcome of individuals with intraductal papillary neoplasms of the bile duct.

BACKGROUND: Intraductal papillary neoplasm of the bile duct (IPNB) is a rare distinct subtype of precursor lesions of biliary carcinoma. IPNB is considered to originate from luminal biliary epithelial cells, typically displays mucin-hypersecretion or a papillary growth pattern, and results in cystic dilatation[1]. IPNB develops anywhere in the intrahepatic and extrahepatic biliary tracts, and can occur in various pathological stages from low-grade dysplasia to invasive carcinoma. IPNBs have similar phenotypic changes in the occurrence and development of all subtypes, and the prognosis is significantly better than that of traditional (non-papillary) cholangiocarcinoma. AIM: To evaluate the clinicopathological features of IPNB to provide evidence-based guidance for treatment. METHODS: Invasive IPNB, invasive intraductal papillary mucinous neoplasm of the pancreas (IPMN), and traditional cholangiocarcinoma data for affected individuals from 1975 to 2016 were obtained from the Surveillance, Epidemiology, and End Results (SEER) database. Annual percentage changes (APCs) in the incidence and incidence-based (IB) mortality were calculated. We identified the independent predictors of overall survival (OS) and cancer-specific survival (CSS) in individuals with invasive IPNB. RESULTS: The incidence and IB mortality of invasive IPNB showed sustained decreases, with an APC of -4.5% (95%CI: -5.1% to -3.8%) and -3.3% (95%CI: -4.1% to -2.6%) (P < 0.001), respectively. Similar decreases in incidence and IB mortality were seen for invasive IPMN but not for traditional cholangiocarcinoma. Both OS and CSS for invasive IPNB were better than for invasive IPMN and traditional cholangiocarcinoma. A total of 1635 individuals with invasive IPNB were included in our prognosis analysis. The most common tumor sites were the pancreaticobiliary ampulla (47.9%) and perihilar tract (36.7%), but the mucin-related subtype of invasive IPNB was the main type, intrahepatically (approximately 90%). In the univariate and multivariate Cox regression analysis, age, tumor site, grade and stage, subtype, surgery, and chemotherapy were associated with OS and CSS (P < 0.05). CONCLUSION: Incidence and IB mortality of invasive IPNB trended steadily downward. The heterogeneity of IPNB comprises site and the tumor's mucin-producing status.

Effects of tea saponins on rumen microbiota, rumen fermentation, methane production and growth performance--a review.

Reducing methane emission from ruminant animals has implications not only for global environmental protection but also for efficient animal production. Tea saponins (TS) extracted from seeds, leaves or roots of tea plant are pentacyclic triterpenes. They have a lasting antiprotozoal effect, but little effect on the methanogen population in sheep. There was no significant correlation between the protozoa counts and methanogens. The TS decreased methanogen activity. It seems that TS influenced the activity of the methanogens indirectly via the depressed ciliate protozoal population. The TS addition decreased fungal population in the medium containing rumen liquor in in vitro fermentation, but no such effect was observed in the rumen liquor of sheep fed TS. Tea saponins had a minor effect on the pattern of rumen fermentation and hence on nutrient digestion. When added at 3 g/day in diets, TS could improve daily weight gain and feed efficiency in goats. No positive associative effect existed between TS and disodium fumarate or soybean oil on methane suppression. Inclusion of TS in diets may be an effective way for improving feed efficiency in ruminants.

Safety, Tolerability, and Pharmacokinetic Study of 101BHG-D01 Nasal Spray, a Novel Long-Acting and Selective Cholinergic M Receptor Antagonist, in Healthy Chinese Volunteers: A Randomized, Double-Blind, Placebo-Controlled, Single-Dose Escalation, First-In-Human Study.

BACKGROUND AND OBJECTIVE: 101BHG-D01 nasal spray is the first novel long-acting cholinergic M receptor antagonist under development to treat rhinorrhea in rhinitis. This first-in-human study aimed to evaluate the safety, tolerability, and pharmacokinetics of 101BHG-D01 nasal spray following single intranasal doses in healthy Chinese subjects. METHODS: A randomized, double-blind, placebo-controlled, single-dose escalation study was conducted in healthy Chinese volunteers after intranasal doses of 101BHG-D01 nasal spray or placebo ranging from 40 µg to 960 µg (total of six doses). Blood samples were collected at scheduled time points, and plasma concentrations were determined using a validated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. A non-compartmental method was used to calculate the main pharmacokinetic parameters, including the area under the plasma concentration-time curve from time zero to the time of the last measurable concentration (AUC0-t), the area under the plasma concentration-time curve from time zero to infinity (AUC0-∞), the maximum plasma concentration (Cmax), the time to maximum plasma concentration (Tmax), and the elimination half-life (t1/2). Safety was evaluated by monitoring adverse events, laboratory assays, vital signs, physical examinations, 12-lead electrocardiograms (ECGs), anterior rhinoscopy, ophthalmic examination, and ambulatory ECG monitoring. RESULTS: Following single intranasal dosing, 101BHG-D01 was rapidly absorbed with a median Tmax of 0.34-0.50 h and eliminated slowly with a mean t1/2 ranging from 4.29 to 46.76 h for different dose groups. The Cmax and AUC of 101BHG-D01 increased linearly across the examined dose range of 40-960 µg. 101BHG-D01 nasal spray was well tolerated, all AEs were mild, and no serious adverse events occurred during the study. CONCLUSIONS: 101BHG-D01 nasal spray was safe and well tolerated in healthy Chinese subjects when administered intranasally in single escalating doses. The mean Cmax and AUC increased proportionally to the studied dose. The pharmacokinetic, safety, and tolerability profiles of 101BHG-D01 nasal spray indicate that it is a good candidate for further development as a treatment for rhinorrhea in rhinitis.

A novel bifunctional glucanase exhibiting high production of glucose and cellobiose from rumen bacterium.

Herbivores gastrointestinal microbiota is of tremendous interest for mining novel lignocellulosic enzymes for bioprocessing. We previously reported a set of potential carbohydrate-active enzymes from the metatranscriptome of the Hu sheep rumen microbiome. In this study, we isolated and heterologously expressed two novel glucanase genes, Cel5A-h38 and Cel5A-h49, finding that both recombinant enzymes showed the optimum temperatures of 50 °C. Substrate-specificity determination revealed that Cel5A-h38 was exclusively active in the presence of mixed-linked glucans, such as barley ß-glucan and Icelandic moss lichenan, whereas Cel5A-h49 (EC 3.2.1.4) exhibited a wider substrate spectrum. Surprisingly, Cel5A-h38 initially released only cellotriose from lichenan and further converted it into an equivalent amount of glucose and cellobiose, suggesting a dual-function as both endo-ß-1,3-1,4-glucanase (EC 3.2.1.73) and exo-cellobiohydrolase (EC 3.2.1.91). Additionally, we performed enzymatic hydrolysis of sheepgrass (Leymus chinensis) and rice (Orysa sativa) straw using Cel5A-h38, revealing liberation of 1.91 ± 0.30 mmol/mL and 2.03 ± 0.09 mmol/mL reducing sugars, respectively, including high concentrations of glucose and cellobiose. These results provided new insights into glucanase activity and lay a foundation for bioconversion of lignocellulosic biomass.

SpyTag/SpyCatcher molecular cyclization confers protein stability and resilience to aggregation.

The capacities for thermal and inhibitor tolerance are critical for industrial enzymes and loss of activity is a major challenge in deploying natural enzymes for commercial applications. Protein engineering approaches, such as site-directed mutagenesis and directed evolution, have been devoted to modifying natural enzymes. Recently, a post-translation protein engineering strategy, the SpyTag/SpyCatcher system, was introduced. Here, we have generated a thermo- and ion-tolerant cyclized xylanase (C-TFX) by fusing the SpyTag and SpyCatcher peptides to its N- and C- terminus respectively. Compared with the linear enzyme, C-TFX retained greater residual activity after heating or metal ion exposure. Intrinsic fluorescence and circular dichroism analysis revealed that the isopeptide bond mediated by SpyTag/SpyCatcher cyclization contributed to enhanced thermo- and ion-stability, probably by stabilizing its secondary and conformational structure. In addition, the heat-challenged C-TFX was observed to degrade natural lignocellulosic substrates efficiently. The cyclized xylanase was more stable and resistent to denaturation and aggregation than the linear enzyme. The "superglue" SpyTag/SpyCatcher cyclization system enables the enzyme to maintain its structural conformation, which will be of particular interest in engineering of enzymes for industrial application such as feed additives and functional oligosaccharides production.

Characterization of Thermostable and Chimeric Enzymes via Isopeptide Bond-Mediated Molecular Cyclization.

Mannooligosaccharides are released by mannan-degrading endo-ß-1,4-mannanase and are known as functional additives in human and animal diets. To satisfy demands for biocatalysis and bioprocessing in crowed environments, in this study, we employed a recently developed enzyme-engineering system, isopeptide bond-mediated molecular cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results revealed that the cyclized enzymes showed enhanced thermostability and ion stability and resilience to aggregation and freeze-thaw treatment by maintaining their conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic activity in substrate deconstruction without compromising substrate affinity. Interestingly, the dual-enzyme ring conformation was observed to be more robust than the linear enzyme but inferior to the single-enzyme ring conformation. Taken together, these findings provided new insights into the mechanisms of molecular cyclization on stability improvement and will be useful in the production of new functional oligosaccharides and feed additives.

Effect of changing forage on the dynamic variation in rumen fermentation in sheep.

To better understand rumen adaptation during dietary transitions between high- and low-quality forages, 10 rumen-cannulated Hu sheep were randomly allocated to two dietary treatments (five sheep each) with the same concentrate-to-forage ratio and concentration mixture, but different forage sequences: (i) alfalfa hay (AH) to corn stover (CS) and back to AH; and (ii) CS to AH and back to CS. A significant decrease in the rumen microbial protein concentration was observed on day 6 after dietary transition whether the transition was from AH to CS or from CS to AH, and this was accompanied by an increase in the ammonia nitrogen concentration as well as a decrease in the total volatile fatty acids concentration and pH. However, after transitioning back to the original forage, the rumen fermentation parameters returned to their initial levels within 2 weeks. Our findings suggest that abrupt substitutions of forages with large nutrient differences could influence rumen function to some extent, but recovery can occur within 2 weeks without detrimental effects. Furthermore, we speculate that the variation of fermentation in the first 6 days may indicate an important rumen transition stage that requires further study.

Characterization of a novel xylanase gene from rumen content of Hu sheep.

A novel xylanase gene, xyn-lxy, was cloned from a metagenomic fosmid library, which was previously constructed from the rumen contents of Hu sheep and was functionally characterized in Escherichia coli. The open reading frame was composed of 1923 bp and encoded for 640 amino acids, including a catalytic domain of glycosyl hydrolase family 10 and carbohydrate-binding module 9. The gene showed 97 % identity with uncultured bacterium Contig1552 but low similarity with xylanases from known cellulolytic-degrading microorganisms in the rumen. The recombinant XYN-LXY showed a specific activity of 664.7 U mg(-1). The optimal temperature and pH of the enzyme were 50 °C and 6.0, respectively. Specifically, XYN-LXY was exclusively activated by Mn(2+) among all of the cations and reducing agents tested in this study. An enzymatic hydrolysis assay revealed that XYN-LXY degraded birchwood xylan into xylooligosaccharide with a low degree of polymerization. After incubation for 4 h, the concentration of the dominant product, xylobiose, was 2.297 ± 0.175 mg ml(-1) (74.07 % of total product) followed by xylose with a concentration of 0.656 ± 0.010 mg ml(-1) (21.14 % of total product). The XYN-LXY exhibited deep degradation effects on the xylan substrate, which were rarely observed with endo-xylanase, making it a promising candidate for industrial application, especially in biofuel production.

Recombination of thermo-alkalistable, high xylooligosaccharides producing endo-xylanase from Thermobifida fusca and expression in Pichia pastoris.

For xylooligosaccharide (XO) production, endo-xylanase from Thermobifida fusca was modified by error-prone PCR and DNA shuffling. The G4SM1 mutant (S62T, S144C, N198D, and A217V) showed the most improved hydrolytic activity and was two copies expressed in Pichia pastoris under the control of GAP promoter. The maximum xylanase activity in culture supernatants was 165 ± 5.5 U/ml, and the secreted protein concentration reached 493 mg/l in a 2-l baffled shake flask. After 6× His-tagged protein purification, the specific activity of G4SM1 was 2036 ± 45.8 U/mg, 2.12 times greater than that of wild-type enzyme. Additionally, G4SM1 was stable over a wide pH range from 5.0 to 9.0. Meanwhile, half-life of G4SM1 thermal inactivation at 70 °C increased 8.5-fold. Three-dimensional structures suggest that two amino acid substitutions, S62T and S144C, located at catalytic domain may be responsible for the enhanced activity and thermostability of xylanase. Xylobiose was the dominant end product of xylan hydrolysis by G4SM1. Due to its attractive biochemical properties, G4SM1 has potential value in commercial XO production.

Rumen fermentation and acetogen population changes in response to an exogenous acetogen TWA4 strain and Saccharomyces cerevisiae fermentation product.

The presence of yeast cells could stimulate hydrogen utilization of acetogens and enhance acetogenesis. To understand the roles of acetogens in rumen fermentation, an in vitro rumen fermentation experiment was conducted with addition of acetogen strain (TWA4) and/or Saccharomyces cerevisiae fermentation product (XP). A 2×2 factorial design with two levels of TWA4 (0 or 2×10(7) cells/ml) and XP (0 or 2 g/L) was performed. Volatile fatty acids (VFAs) were increased (P<0.05) in XP and TWA4XP, while methane was increased only in TWA4XP (P<0.05). The increase rate of microorganisms with formyltetrahydrofolate synthetase, especially acetogens, was higher than that of methanogens under all treatments. Lachnospiraceae was predominant in all acetogen communities, but without close acetyl-CoA synthase (ACS) amino acid sequences from cultured isolates. Low-Acetitomaculum ruminis-like ACS was predominant in all acetogen communities, while four unique phylotypes in XP treatment were all amino acid identified low-Eubacterium limosum-like acetogens. It differs to XP treatment that more low-A. ruminis-like and less low-E. limosum-like sequences were identified in TWA4 and TWA4XP treatments. Enhancing acetogenesis by supplementation with an acetogen strain and/or yeast cells may be an approach to mitigate methane, by targeting proper acetogens such as uncultured low-E. limosum-like acetogens.

Monitoring the rumen pectinolytic bacteria Treponema saccharophilum using real-time PCR.

Treponema saccharophilum is a pectinolytic bacterium isolated from the bovine rumen. The abundance of this bacterium has not been well determined, reflecting the lack of a reliable and accurate detection method. To develop a rapid method for monitoring T. saccharophilum, we performed pyrosequencing of genomic DNA isolated from rumen microbiota to explore the 16S rRNA gene sequences of T. saccharophilum candidates. Species-specific primers were designed based on fifteen sequences of partial 16S rRNA genes generated through pyrosequencing with 97% or higher similarity with T. saccharophilum DSM2985 along with sequence from type strain. The relative abundance of T. saccharophilum was quantified in both in vitro and in vivo rumen systems with varied pectin-containing forages using real-time PCR. There was a clear association of T. saccharophilum with alfalfa hay, which contains more pectin than Chinese wild rye hay or corn stover. The relative abundance of T. saccharophilum was as high as 0.58% in vivo, comparable with the population density of other common rumen bacteria. It is recognized that T. saccharophilum plays an important role in pectin digestion in the rumen.