The capacity of combinations of feed enzymes, natural betaine and a probiotic, combined with alternative plant-based ingredients, to totally replace soybean meal (SBM) in a broiler diet was evaluated. Day-old Ross 308 males (2,574) were assigned to 9 treatments (13 pens/treatment, 22 birds/pen) in a completely randomized design. All diets were pelleted and fed ad libitum in 4 phases: starter, grower, finisher 1, finisher 2 (0-10, 10-21, 21-35, and 35-42 d of age, respectively). Treatments included: 1) control diet containing SBM (SBM control), supplemented with phytase (PhyG), at 2,000, 1,500, 1000 and 1,000 FTU/kg in each phase and xylanase (X) at 750 U/kg, [crude protein (CP): 23.5%, 22.0%, 20.2% and 19.3% in each phase]; 2) to 5), alternative (ALT), SBM-free diets, containing the same CP level as the control (\"CP high\"), supplemented with PhyG as in the control, protease (P, 800 U/kg) and in 2) xylanase (750 U/kg) (ALT+PhyG+P+X), 3) xylanase-β-glucanase (XB, 1,200 U/kg and 152 U/kg) (Alt+PhyG+P+XB), 4) XB plus betaine (800 g/ton) (ALT+PhyG+P+XB+Bet), and 5) XB plus a probiotic [150,000 colony forming units (CFU)/g] (ALT+PhyG+P+XB+Prob); 6) to 9) as treatments 2) to 5) but with CP reduced by -2.0 to -1.5% points vs. control (\'CP low\'). Final (d 42) BW and overall (d 0-42) feed conversion ratio (FCR) of birds fed the SBM control exceeded breeder objectives (+3.8% and -1.9%, respectively). Overall FCR was reduced and d 42 BW increased in birds fed \"low\" vs. \"high\" CP (P < 0.01). Overall FCR and feed intake were not different in ALT+PhyG+XB+P+Bet and ALT+PhyG+XB+P+Prob vs. the control, whereas final BW was reduced (P < 0.05) in all ALT treatments but close to breeder objectives (98.3%) in ALT+PhyG+XB+P+Prob. Feed costs of this treatment were similar to the control. Total replacement of SBM with alternative plant-based ingredients in a CP-low diet supplemented with hydrolytic enzymes and probiotics can achieve growth performance outcomes close to commercial breeder objectives.

The effect of applying an energy and nutrient matrix to a wheat-corn-soybean meal-based diet supplemented with a novel consensus bacterial 6-phytase variant (PhyG) and xylanase-β-glucanase on growth performance, bone mineralization, carcass weights, feed costs, and carbon footprint was evaluated. A randomized complete block design (3,300 Ross 308 mixed-sex birds; 60 pens, 12 pens per treatment) tested 5 treatments: 1) a positive control diet (PC), containing 0.92, 0.84, 0.71% Ca and 0.43, 0.38, 0.30% digestible P during 1 to 10, 11 to 21, and 22 to 32 d of age, respectively; 2) a negative control reduced in Ca, digestible P, digestible AA, ME, and Na by phase based on the PhyG dosing regimen (NC1); 3) NC1 supplemented with PhyG at 2,000, 1,500, and 1,000 FTU/kg by phase (NC1+PhyG); 4) as NC1 but additionally reduced in ME (NC2); and 5) NC2 supplemented with PhyG as in 3) plus 1,220 U/kg of xylanase and 152 U/kg of β-glucanase (NC2+PhyG+XB). Final (d 32) BW, overall (0-32 d of age) ADFI, FCR, d 10 and 32 tibia ash and carcass part weights were reduced or impaired (P < 0.05) in NC1 and NC2 vs. PC (d 32 BW -477 g/bird (23.4%) and -422 g/bird (20.7%), respectively). Growth performance (all measures, all phases) was improved and tibia ash (at 10 and 32 d of age), total carcass thigh, breast and leg weights were increased (P < 0.05) in NC1+PhyG vs. NC1, and NC2+PhyG+XB vs. NC2. Overall growth performance outcomes in NC1+PhyG and NC2+PhyG+XB were not different (P > 0.05) from the PC. Total feed cost and carbon footprint per kilogram BW gain (BWG) were reduced (P < 0.05) vs. PC in NC2+PhyG+XB [-0.052 € and -376 g CO2 eq./kg BWG, respectively] and NC1+PhyG [-0.038 € and -260 g CO2 eq./kg BWG, respectively]. The results validated the nutrient matrices in the test diets and highlighted a potential feed cost and environmental sustainability benefit which was greatest when the enzymes were applied in combination.

High throughput screening approaches can significantly speed up the identification of novel enzymes from natural microbial consortiums. A two-step high throughput screening process was proposed and explored to screen lignin-degrading microorganisms. By employing this modified culture enrichment method and screening based on enzyme activity, a total of 82 bacterial and 46 fungal strains were isolated from fifty decayed wood samples (100 liquid cultures) collected from the banks of the Ottawa River in Canada. Among them, ten bacterial and five fungal strains were selected and identified based on their high laccase activities by 16S rDNA and ITS gene sequencing, respectively. The study identified bacterial strains from various genera including Serratia, Enterobacter, Raoultella, and Bacillus, along with fungal counterparts including Mucor, Trametes, Conifera and Aspergillus. Moreover, Aspergillus sydowii (AORF21), Mucor sp. (AORF43), Trametes versicolor (AORF3) and Enterobacter sp. (AORB55) exhibited xylanase and β- glucanase activities in addition to laccase production. The proposed approach allowed for the quick identification of promising consortia and enhanced the chance of isolating desired strains based on desired enzyme activities. This method is not limited to lignocellulose and lignin-degrading microorganisms but can be applied to identify novel microbial strains and enzymes from different natural samples.

Species of the genus Plenodomus (Leptosphaeria) are phytopathogens of the Brassicaceae family, which includes oilseed rape. The spores of these fungi spread by airborne transmission, infect plants, and cause crop losses. The secondary metabolism of P. lingam and P. biglobosus was studied and compared, with the main focus being on the ability to produce Extracellular Polymeric Substances (EPS). In spite of the 1.5-2-fold faster growth rate of P. biglobosus on Czapek-Dox and other screening media, the average yield of EPS in this fungus was only 0.29 g/L, compared to that of P. lingam (0.43 g/L). In turn, P. biglobosus showed a higher capacity to synthesise IAA, i.e., 14 µg/mL, in contrast to <1.5 µg/mL produced by P. lingam. On the other hand, the P. lingam strains showed higher β-glucanase activity (350-400 mU/mL), compared to 50-100 mU/mL in P. biglobosus. Invertase levels were similar in both species (250 mU/mL). The positive correlation between invertase activity and EPS yield contrasted with the absence of a correlation of EPS with β-glucanase. Plenodomus neither solubilised phosphate nor used proteins from milk. All strains showed the ability to synthesise siderophores on CAS agar. P. biglobosus exhibited the highest efficiency of amylolytic and cellulolytic activity.

Rice sheath blight (ShB) caused by Rhizoctonia solani is one of the most serious diseases that threatens rice (Oryza sativa) production. However, the mechanisms of defense against ShB in rice remain largely unknown. In this study, we identified that the expression levels of β-glucanase (OsBGL) family genes sensitively respond to infection by R. solani, and OsBGLs positively regulate rice resistance to ShB. In addition, OsBGL2 colocalized with AtPDCB1 at the plasmodesmata (PD) and limited the PD permeability. The level of callose accumulation in osbgls mutants and overexpressors was examined, and OsBGLs were found contribute to callose accumulation. Taken together, these data suggest that OsBGLs can regulate the deposition of callose at the PD to reduce its permeability to defend itself against ShB. Through the identification of these genes and the elucidation of their functions, this research fills the gap in the mechanism of PD permeability in rice ShB resistance.

Three experiments evaluated effects of adaptation diet and exogenous β-glucanase and xylanase on TMEn of barley and rye. Single Comb White Leghorn roosters were fed adaptation diets based on corn/soybean meal (SBM), barley/SBM with and without β-glucanase, or rye/corn/SBM with and without xylanase for 4 wk. In Experiments 1 and 2, after the adaptation period, TMEn was determined using a 48 h precision-fed rooster assay for 100% barley or 100% rye diets with or without β-glucanase or xylanase, respectively. Experiment 3 consisted only of feeding adaptation diets for 4 wk. Cecal samples were collected at the end of experiments for microbial ecology, short-chain fatty acid (SCFA) profiles, and enzyme activity analyses. In Experiments 1 and 2, β-glucanase increased (P < 0.05) TMEn of barley, and there was no significant effect of adaptation diet on TMEn values. Total cecal Eubacteria and Ruminococcaceae were decreased (P < 0.05) and Escherichia coli were increased (P < 0.05) at the end of the TMEn assay compared with the end of the adaptation period (with no TMEn assay). There was a large decrease (P < 0.05) for most cecal SCFA at the end of the TMEn assay compared with the end of the adaptation period. Both cecal β-glucanase and xylanase activity were increased for birds fed adaptation diets containing the respective enzyme. In Experiment 3, there were no consistent effects of adaptation diet on cecal microbial profiles or SCFA but cecal β-glucanase activity was increased (P < 0.05) by exogenous β-glucanase for barley and cecal xylanase activity was increased (P < 0.05) by exogenous xylanase for rye. Overall, the results indicated that TMEn of barley was increased by exogenous β-glucanase, adaptation diet did not significantly influence the TMEn response to the dietary enzymes, and cecal fermentation (based on cecal SCFA) was greatly reduced by the TMEn assay. Cecal β-glucanase and xylanase activity, however, were often increased by feeding high barley and high rye diets containing exogenous enzymes.

BACKGROUND: The combination of cellulase and lytic polysaccharide monooxygenase (LPMO) is known to boost enzymatic saccharification of cellulose. Although the synergy between cellulases (GH5, 6 or 7) and LPMOs (AA9) has been extensively studied, the interplay between other glycoside hydrolase and LPMO families remains poorly understood.
RESULTS: In this study, two cellulolytic enzyme-encoding genes SmBglu12A and SmLpmo10A from Streptomyces megaspores were identified and heterologously expressed in Escherichia coli. The recombinant SmBglu12A is a non-typical endo-β-1,4-glucanase that preferentially hydrolyzed β-1,3-1,4-glucans and slightly hydrolyzed β-1,4-glucans and belongs to GH12 family. The recombinant SmLpmo10A belongs to a C1-oxidizing cellulose-active LPMO that catalyzed the oxidation of phosphoric acid swollen cellulose to produce celloaldonic acids. Moreover, individual SmBglu12A and SmLpmo10A were both active on barley β-1,3-1,4-glucan, lichenan, sodium carboxymethyl cellulose, phosphoric acid swollen cellulose, as well as Avicel. Furthermore, the combination of SmBglu12A and SmLpmo10A enhanced enzymatic saccharification of phosphoric acid swollen cellulose by improving the native and oxidized cello-oligosaccharides yields.
CONCLUSIONS: These results proved for the first time that the AA10 LPMO was able to boost the catalytic efficiency of GH12 glycoside hydrolases on cellulosic substrates, providing another novel combination of glycoside hydrolase and LPMO for cellulose enzymatic saccharification.

In the genome of Cellulomonas flavigena, two genes that potentially encode endoglucanases - Cfla_2912 and Cfla_2913 were identified. We cloned the genes and created Pichia pastoris-based recombinant producers of two proteins that were expressed from the AOX1 promoter. Each of the endoglucanase molecules contains a GH6 catalytic domain, CBM2 carbohydrate-binding module, and TAT signal peptide. The fermentation of the producers was carried out in a 10 L fermenter; Cfla_2912 and Cfla_2913 were purified using affinity chromatography. The yield comprised 10.3 mg/ml (430 U/ml) for Cfla_2913 and 9 mg/ml (370 U/ml) for Cfla_2912. Cfla_2912 and Cfla_2913 were found to have a high activity against barley β-glucan and lichenan, a weak activity against carboxymethyl cellulose (CMC), phosphoric-acid treated cellulose, and no activity against laminarin, xylan, soluble starch, microcrystalline cellulose, cellobiose, and cellotriose. Thus, the proteins exhibited β-glucanase activity. Both proteins had a neutral pH optimum of about 7.0 and were more stable at neutral and slightly alkaline pH ranging from 7.0 to 9.0. Cfla_2912 and Cfla_2913 showed a moderate thermal stability. The products of barley β-glucan hydrolysis by Cfla_2912 and Cfla_2913 were trisaccharide, tetrasaccharide, and cellobiose. Cfla_2912 and Cfla_2913 efficiently hydrolyzed cereal polysaccharides, which indicate that they may have biotechnological potential.

Lignocellulosic biomass, which mainly consists of cellulose and hemicellulose, is the most abundant renewable biopolymer on earth. β-Glucanases are glycoside hydrolases (GHs) that hydrolyze β-glucan, one of the dominant components of the plant cell wall, into cello-oligosaccharides and glucose. Among them, endo-β-1,4-glucanase (EC 3.2.1.4), exo-glucanase/cellobiohydrolase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21) play critical roles in the digestion of glucan-like substrates. β-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 β-glucanases. Advances in the development of next-generation sequencing techniques, including metagenomics and metatranscriptomics, have unveiled novel β-glucanases isolated from the gastrointestinal microbiota. The study of β-glucanases is beneficial for research and development of commercial products. In this study, we review the classification, properties, and engineering of β-glucanases.

UNASSIGNED: Alternative sweeteners, such as steviol glucosides from the plant Stevia rebaudiana Bertoni, are becoming increasingly popular for the design of next-generation foodstuffs. However, the bitter aftertaste of native steviol glucosides is one of the main reasons behind consumer reluctance towards stevia-containing products. Biocatalysis could be a sustainable solution to this problem, through addition of glucosyl moieties to the molecule. Glycoside hydrolases are enzymes performing transglycosylation reactions, and they can be exploited for such modifications. In the present work, the commercial β-glucanase Finizym 250L® was employed for the transglycosylation of stevioside. After optimization of several reaction parameters, the maximal reaction yield obtained was 19%, with barley β-glucan as the glycosyl donor. With the aim to develop a sustainable process, β-glucan extracts from different fungal sources were prepared. Pulsed Electric Field pretreatment of mycelial biomass resulted in extracts with higher β-glucan content. The extracts were tested as alternative glucosyl donors, reaching up to 15.5% conversion yield, from Pleurotus-extracted β-glucan. Overall, in the present work a novel enzymatic process for the modification of stevioside is proposed, with concomitant valorization of β-glucans extracted from fungal biomass, potentially generated as a byproduct from other applications, in concert with the principles of circular economy.
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