Date nawah powder as a promising waste for ß-mannanase production from a new isolate Aspergillus niger MSSFW, statistically improving production and enzymatic characterization.

ß-Mannanase producing fungus was isolated from coffee powder waste and identified as Aspergillus niger MSSFW (Gen Bank accession number OR668928). Dates nawah powder as industrial and agricultural waste was the most inducer of ß-mannanase production. The Plackett-Burman and Central Composite designs were used to improve ß-mannanase titer. Optimization studies enhanced the enzyme yield with approximate 13.50-times. ß-Mannanase was purified by Sephadex G-150 gel filtration column and the molecular weight was estimated to be 60 kDa by SDS-PAGE. Crude and purified ß-mannanase displayed maximum activity at temperature 60 °C and 50 °C, respectively. Crude ß-mannanase showed an activation energy value 2.35-times higher than the purified enzyme. Activation energy for thermal denaturation of the purified ß-mannanase was 1.08-times higher than that of the crude enzyme. Purified ß-mannanase exhibited higher deactivation rate constant (Kd) and lower half-life (t0.5) and decimal reduction time (D-value) compared with the crude enzyme. Thermodynamic parameters of enthalpy, entropy, and free energy values for crude and purified ß-mannanase were calculated. Substrate kinetic parameters suggested that the purified ß-mannanase had a strong affinity toward locust bean gum by showing 3.44-times lower Km and 1.99-times higher Vmax compared to the crude enzyme.

Nutritional and functional roles of ß-mannanase on intestinal health and growth of newly weaned pigs fed two different types of feeds.

This study aimed to investigate the nutritional and functional roles of ß-mannanase on the intestinal health and growth of newly weaned pigs fed a typical or low-cost formulated feeds (LCF). Twenty-four newly weaned pigs at 6.2 kg ±â€…0.4 body weight (BW) were allotted to three dietary treatments based on a randomized complete block design with sex and initial BW as blocks. Three dietary treatments are as follows: Control, typical nursery feeds including animal protein supplements and enzyme-treated soybean meal; LCF with increased amounts of soybean meal, decreased amounts of animal protein supplements, and no enzyme-treated soybean meal; LCF+, low-cost formulated feed with ß-mannanase at 100 g/t, providing 800 thermostable ß-mannanase unit (TMU) per kg of feed. Pigs were fed based on a three-phase feeding program for a total of 37 d. On day 37 of feeding, all pigs were euthanized and the gastrointestinal tract was removed for sample collection to analyze intestinal health parameters, mucosa-associated microbiota, and gene expression of tight junction proteins. Pigs fed LCF increased (P < 0.05) the relative abundance of Proteobacteria and Helicobacter in the jejunal mucosa, tended to decrease (P = 0.097; P = 0.098) the concentration of malondialdehyde (MDA) and the expression of zona occluden 1 (ZO-1) gene in the jejunum, tended to decrease average daily gain (ADG; P = 0.084) and final BW (P = 0.090), and decreased (P < 0.05) average daily feed intake. Pigs fed LCF + tended to decrease (P = 0.088) digesta viscosity, decreased (P < 0.05) the relative abundance of Helicobacter, and increased (P < 0.05) Lactobacillus in the jejunal mucosa compared to LCF. Additionally, LCF + tended to increase final BW (P = 0.059) and ADG (P = 0.054), increased (P < 0.05) gain to feed ratio (G:F), and reduced (P < 0.05) fecal score compared to LCF. LCF with decreased amounts of animal protein supplements and increased amounts of soybean meal had negative effects on the composition of the mucosa-associated microbiota, intestinal integrity, and growth performance of nursery pigs. Beta-mannanase supplementation to LCF decreased digesta viscosity, increased the relative abundance of potentially health-benefitting microbiota such as Lactobacillus, and improved growth and fecal score, thus reflecting its efficacy in low-cost formulated feeds with increased amounts of soybean meal.

Immediately following weaning, pigs are exposed to various stressors associated with environmental, social, and dietary shifts that can result in depressed growth, high incidence of diarrhea, and increases in mortality. Major plant-based feedstuffs such as soybean meal contain anti-nutritional compounds including nonstarch polysaccharides causing negative impacts on the intestinal health and growth of nursery pigs due to the immaturity of the intestine and intestinal immune system. ß-mannanase hydrolyzes ß-mannans, nonstarch polysaccharides, resulting in elimination of anti-nutritional properties and the release of mannooligosaccharides. The aim of this study was to investigate the nutritional and functional roles of ß-mannanase on intestinal health and growth of newly weaned pigs fed a low-cost formulated feed (LCF). This study showed that increased amounts of soybean meal in LCF increased pathogenic bacteria associated to the jejunal mucosa of nursery pigs, impaired intestinal integrity, and decreased growth. Supplementation of ß-mannanase to LCF decreased digesta viscosity, increased Lactobacillus in the jejunal mucosa, reduced diarrhea, and improved growth performance of nursery pigs.

The effects of ß-mannanase supplementation on growth performance, digestive enzyme activity, cecal microbial communities, and short-chain fatty acid production in broiler chickens fed diets with different metabolizable energy levels.

This study assessed the effects of ß-mannanase (BM) supplementation on growth performance, digestive enzyme activity, cecal microbial communities, and short-chain fatty acid (SCFA) production in broiler chickens fed diets with different metabolizable energy (ME) levels. A total of 1,296 male 1-d-old Cobb 500 broilers were randomly distributed in a 3 × 2 factorial arrangement (3 ME levels × 0 or 200 g/ton BM), with 6 replicates per treatment combination. The 3 ME levels were 3,000 (ME1), 2,930 (ME2), and 2,860 (ME3) kcal/kg, respectively, during the 0 to 3 wk-old stages and 3,150 (ME1), 3,080 (ME2), and 3,010 (ME3) kcal/kg, respectively, during the 3 to 6 wk-old stages. Reducing ME levels increased broiler feed intake (P = 0.036) and decreased average daily gain (ADG, P = 0.002) during the entire period. While BM supplementation increased ADG (P = 0.002) and improved the feed conversion ratio (P = 0.001) during the 0 to 3 wk-old stages, with no effect during the 3 to 6 wk-old stages. Overall, reducing ME levels increased pancreatic lipase (P = 0.045) and amylase (P = 0.013) activity and duodenal amylase activity (P = 0.047). Notably, BM supplementation significantly increased pancreatic lipase activity (P = 0.015) and increased lipase (P = 0.029) and amylase (P = 0.025) activities in the jejunal chyme. Although diet or enzyme supplementation did not affect microbial diversity, significant differences in microbial communities were observed. At the genus level, decreasing ME levels significantly affected the average abundances of Tyzzerella (P = 0.028), Candidatus_Bacilloplasma (P = 0.001), Vibrio (P = 0.005), and Anaerotruncus (P = 0.026) among groups, whereas BM supplementation reduced the average abundances of Escherichia-Shigella (P = 0.048) and increased the average abundances of Barnesiella (P = 0.047), Ruminococcus (P = 0.020), Alistipes (P = 0.050), and Lachnospiraceae_unclassified (P = 0.009). SCFA concentrations strongly depended on bacterial community composition, and BM supplementation increased acetic acid (P = 0.004), propionic acid (P = 0.016), and total SCFA concentrations. In conclusion, BM supplementation improved the performance of younger broilers, and both enzyme supplementation and reduced ME levels positively affected digestive enzyme activity and intestinal microflora.

The broiler industry currently faces numerous challenges, especially the significant rise in feedstuff prices, and the economic considerations with regard to feedstuff formulation have become important factors for reducing costs and enhancing benefits. Low-energy diets have been increasingly suggested to control feed costs; however, lower nutrient concentrations may result in reduced performance, thereby increasing attention toward the potential use of feed enzymes. In this study, we have assessed the impacts of ß-Mannanase (BM), the major ß-mannan degrading enzyme, supplementation on poultry health and digestion. Our study involved a total of 1,296 Cobb broilers, randomly distributed into a 3 × 2 factorial arrangement to evaluate the effects of BM supplementation on growth, digestive enzyme activity, cecal microbial communities, and SCFA production in broiler chickens fed diets with varying ME levels. Our findings confirmed previous research by showing that BM-supplemented diets are effective in improving performance in younger broilers. In addition, we found that both BM supplementation and reduced ME levels also have positive impacts on digestive enzyme activity and intestinal flora in chickens, which might account for improving production performance. These results will be a crucial refence for the scientific implementation of BM supplementation, which may help achieve better performance and intestinal microecology, resulting in higher economic productivity of broiler farms.

Simulated Swine Digestion and Gut Microbiota Fermentation of Hydrolyzed Copra Meal.

This study aimed to compare the effects of hydrolyzed copra meal (HCM) inclusion at 1% on its in vitro digestibility and the microbiota and cecum fermentation using the gut microbiota of weaned swine, targeting microbial community and short-chain fatty acids (SCF). For this reason, three treatments were considered: control (no copra meal), 1% non-hydrolyzed copra meal (CM), and 1% HCM. Non-defatted copra meal was hydrolyzed and analyzed (reducing sugars and total carbohydrates) in our laboratory. For digestion, microbiota identification, and fermentation assays, fresh fecal samples from two weaned pigs (1 month old) were used. Three replicates of each treatment were employed. HCM was more digestible, with approximately 0.68 g of hydrolysate recovered after simulated digestion compared to 0.82 g of hydrolysate recovered from CM. This was shown by Scanning Electron Microscope (SEM) images. Also, the three swine shared the majority of microbial species identified at the phylum and family levels. There were no differences (p > 0.05) between treatments in the microbial community and SCFA during fermentation. However, higher Chao-1 and Shannon indexes were observed in CM and HCM treatments. HCM was also found to be capable of preserving Actinobacterota and Proteobacteria at the phylum level, while at the family level, both treatments may help Lactobacillaceae, Peptostreptococcaceae, Lachnospiraceae, and Ruminococcaceae survive in the long term. Also, there was a potential trend of increasing acetic acid and butyric acid in the CM and HCM treatments. While HCM shows promise in potentially modulating the gut microbiota of weaned swine, additional research is required to investigate the effects of higher doses of HCM on swine performance parameters.

Unravelling biochemical and structural features of Bacillus licheniformis GH5 mannanase using site-directed mutagenesis and high-resolution protein crystallography studies.

Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-ß-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.

Mannooligosaccharide production from açaí seeds by enzymatic hydrolysis: optimization through response surface methodology.

Recognized for its bioactive compounds, açaí has become a functional food, but it has a low pulp yield, and the seeds are the main waste. This study investigates the potential of açaí seeds (Euterpe oleracea Mart.) to produce mannooligosaccharides (MOS) through enzymatic hydrolysis. Using response surface methodology (RSM), the research optimizes MOS extraction while minimizing mannose production and reducing processing time, achieving MOS production of about 10 g/L, a value within the range of similar investigations. The RSM quadratic models establish correlations between MOS production (M2-M5) and enzymatic hydrolysis conditions, with R2 values ranging from 0.6136 to 0.9031. These models are used to emphasize MOS performance (M2-M5) while reducing mannose production, which also promotes profitability by reducing time. Experimental validation agrees with model predictions, highlighting optimal conditions near 40 °C, intermediate enzyme loading, and basic pH that effectively promotes MOS generation on mannose within an accelerated processing time frame. With predictions of experimental results within a margin of error of < 9%, the validity of the models was acceptable. This research contributes to the advancement of the understanding of the enzymatic hydrolysis of açaí seeds, which is a step toward the sustainable use of resources with a focus on process engineering aspects.

Optimized production and characterization of endo-ß-mannanase by Aspergillus niger for generation of prebiotic mannooligosaccharides from guar gum.

Optimized production of Aspergillus niger ATCC 26011 endo-ß-mannanase (ManAn) on copra meal resulted in 2.46-fold increase (10,028 U/gds). Purified ManAn (47 kDa) showed high affinity towards guar gum (GG) as compared to konjac gum and locust bean gum with Km 2.67, 3.25 and 4.07 mg/mL, respectively. ManAn efficiently hydrolyzed GG and liberated mannooligosaccharides (MOS). Changes occurring in the rheological and compositional aspects of GG studied using Differential scanning calorimetry (DSC), Thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) revealed increased thermal stability and crystallinity of the partially hydrolyzed guar gum (PHGG). Parametric optimization of the time and temperature dependent hydrolysis of GG (1% w/v) with 100 U/mL of ManAn at 60 °C and pH: 5.0 resulted in 12.126 mg/mL of mannotetraose (M4) in 5 min. Enhanced growth of probiotics Lactobacilli and production of short chain fatty acids (SCFA) that inhibited enteropathogens, confirmed the prebiotic potential of PHGG and M4.

Surface-Displayed Mannanolytic and Chitinolytic Enzymes Using Peptidoglycan Binding LysM Domains.

Using Lactiplantibacillus plantarum as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30 kDa) from Bacillus subtilis and a mannanase (ManB, 40 kDa) from B. licheniformis on the surface of L. plantarum WCFS1 using either a single LysM domain derived from the extracellular transglycosylase Lp_3014 or a double LysM domain derived from the muropeptidase Lp_2162. ManB and CsnA were fused with the LysM domains of Lp_3014 or Lp_2162, produced in Escherichia coli and anchored to the cell surface of L. plantarum. The localization of the recombinant proteins on the bacterial cell surface was successfully confirmed by Western blot and flow cytometry analysis. The highest immobilization yields (44-48%) and activities of mannanase and chitosanase on the displaying cell surface (812 and 508 U/g of dry cell weight, respectively) were obtained when using the double LysM domain of Lp_2162 as an anchor. The presence of manno-oligosaccharides or chito-oligosaccharides in the reaction mixtures containing appropriate substrates and ManB or CsnA-displaying cells was determined by high-performance anion exchange chromatography. This study indicated that non-GMO Lactiplantibacillus chitosanase- and mannanase-displaying cells could be used to produce potentially prebiotic oligosaccharides.

Carbohydrate polymer degradation derivatives as possible natural mannanase inhibitors.

The role of mannanases is diverse and they are used in many industrial applications, in animal feed, in the food industry and in healthcare. They are also applied in biomass processing, because they play an important role in the breakdown of hemicellulose. Among the mannanase inhibitors, heavy metal ions and general enzyme inhibitors are mainly mentioned. Unfortunately, almost no data are available on carbohydrate-based natural inhibitors of mannanases. According to the literature, carbohydrates do not play an important role in the inhibition of mannanases, so neither do oligosaccharides. This is in contrast to the action and inhibition of other O-glycosyl hydrolases. My hypothesis is that mannanases, like other polysaccharide-degrading enzymes, work in the same way and can be inhibited by oligosaccharides. Evidence from docking and modeling results supports and makes probable the hypothesis that oligosaccharides can inhibit the activity of mannanases, similar to the inhibition of other O-glycosyl hydrolases. Among natural carbohydrate oligomers, several potential mannanase inhibitors have been identified and characterized. In addition to expensive research, it is very important to use research based on cheaper modeling to explore the processes. The results obtained are novel and forward-looking, enabling in-depth and targeted research to be carried out.

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.

The Discovery of a Multidomain Mannanase Containing Dual-Catalytic Domain of the Same Activity: Biochemical Properties and Synergistic Effect.

In recent years, the wide application of mannan has driven the demand for the exploration of mannanase. As one of the main components of hemicellulose, mannan is an important polysaccharide that ruminants need to degrade and utilize, making rumen a rich source of mannanases. In this study, gene mining of mannanases was performed using bioinformatics, and potential dual-catalytic domain mannanases were heterologously expressed to analyze their properties. The hydrolysis pattern and enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). A dual-catalytic domain mannanase Man26/5 with the same function as the substrate was successfully mined from the genome of cattle rumen microbiota. Compared to the single-catalytic domain, its higher thermal stability (≤50 °C) and catalytic efficiency confirm the synergistic effect between the two catalytic domains. It exhibited a unique "crab-like" structure where the CBM located in the middle is responsible for binding, and the catalytic domains at both ends are responsible for cutting. The exploration of its multidomain structure and synergistic patterns could provide a reference for the artificial construction and molecular modification of enzymes.

Efficient production of High-Purity manno-oligosaccharides from guar gum by citric acid and enzymatic hydrolysis.

Currently, the production of manno-oligosaccharides (MOS) from guar gum faces challenges of low oligosaccharide enzymatic hydrolysis yield and complicated steps in separation and purification. In this work, a potential strategy to address these issues was explored. By combining citric acid pretreatment (300 mM, 130 °C, 1 h) with ß-mannanase hydrolysis, an impressive MOS yield of 61.8 % from guar gum (10 %, w/v) was achieved. The key success lay in the optimizing conditions that completely degraded other galactomannans into monosaccharides, which could be easily removable through Saccharomyces cerevisiae fermentation (without additional nutrients). Following ion exchange chromatography for desalination, and concluding with spray drying, 4.57 g of solid MOS with a purity of 90 % was obtained from 10 g of guar gum. This method offers a streamlined and effective pathway for obtaining high-yield and high-purity MOS from guar gum by combining citric acid pretreatment and enzymatic hydrolysis.

Mannans: Structural carbohydrates produced during seed maturation in Euterpe edulis Martius, an Atlantic Forest species vulnerable to extinction.

Palm seedlings are visually selected from mature fruits in a slow process that leads to nonuniform germination and high embryo mortality. In this study, we determined the levels of monosaccharides, their crystallinity, and their role in the formation of Euterpe edulis endosperm during seed maturation. Seeds harvested from 108 to 262 days after anthesis (DAA) were analyzed morphologically, physiologically, and chemically to measure soluble and insoluble lignins, ashes, structural carbohydrates, degree of crystallinity, and endo-ß-mannanase. The seeds achieved maximum germination and vigor at 164 DAA. During the early stages, only compounds with a low structural order were formed. The contents of soluble and insoluble lignins, ashes, glucans, and galactans decreased during maturation. Those of mannans, the main structural carbohydrate in the endosperm, increased along with the degree of crystallinity, as suggested by a mannan-I-type X-ray diffraction pattern. Similarly, endo-ß-mannanase activity peaked at 262 DAA. The superior physiological outcome of seeds and seedlings at 164 DAA implies a 98-day shorter harvesting time. The state of mannans during seed maturation could be used as a marker to improve seedling production by E. edulis.

Research and application progress of microbial ß-mannanases: a mini-review.

Mannan is a predominant constituent of cork hemicellulose and is widely distributed in various plant tissues. ß-Mannanase is the principal mannan-degrading enzyme, which breaks down the ß-1,4-linked mannosidic bonds in mannans in an endo-acting manner. Microorganisms are a valuable source of ß-mannanase, which exhibits catalytic activity in a wide range of pH and temperature, making it highly versatile and applicable in pharmaceuticals, feed, paper pulping, biorefinery, and other industries. Here, the origin, classification, enzymatic properties, molecular modification, immobilization, and practical applications of microbial ß-mannanases are reviewed, the future research directions for microbial ß-mannanases are also outlined.

Analysis of the effects of ß-mannanase on immune function and intestinal flora in broilers fed the low energy diet based on 16S rRNA sequencing and metagenomic sequencing.

As an enzyme, ß-mannanase (BM) can be widely used as feed additive to improve the growth performance of animals. This experiment aimed to determine the effect of the addition of BM to low-energy diet on the immune function and intestinal microflora of broiler chickens. In this study, 384 one-day-old Arbor Acres broilers were randomly divided into 3 groups (8 replicates per group): positive control (PC, received a corn-soybean meal basal diet), negative control (NC, received a low-energy diet with Metabolizable Energy (ME) reduced by 50 kcal/kg) and NC + BM group (NC birds + 100 mg/kg BM). All birds were raised for 42 d. The results showed that BM mitigated the damage of immune function in peripheral blood of broilers caused by the decrease of dietary energy level by increasing the Concanavalin A (Con A) index of stimulation (SI) and macrophages phagocytic activity in the peripheral blood of broilers at 42 d (P < 0.05). The analysis of cecum flora showed that the low-energy diet significantly reduced the observed_species index (P < 0.01), Chao1 index and ACE index (P < 0.05), which reduced the abundance and evenness of species in the cecum of broilers at 21 d. It also significantly reduced the relative abundance of Candidatus_Arthromitus and significantly increased the relative abundance of Pseudomonas in the cecum of broilers at 21 d, while also significantly increasing the relative abundance of Monoglobus at 42 d. BM significantly increased the relative abundance of Lachnospiraceae_UCG-001 and Lachnospiraceae_bacterium_615 in the cecum of broilers at 21 d. In addition, BM inhibited microbial Fatty acid degradation by decreasing the activity of glutaryl-CoA dehydrogenase. Collectively, BM could improve intestinal health by enhancing the immune function of broilers, promoting the proliferation of beneficial bacteria and reducing the number of harmful bacteria, regulating intestinal flora, thereby alleviating the adverse effects of lower dietary energy levels.

Thermophilic ß-mannanases from bacteria: production, resources, structural features and bioengineering strategies.

ß-mannanases are pivotal enzymes that cleave the mannan backbone to release short chain mannooligosaccharides, which have tremendous biotechnological applications including food/feed, prebiotics and biofuel production. Due to the high temperature conditions in many industrial applications, thermophilic mannanases seem to have great potential to overcome the thermal impediments. Thus, structural analysis of thermostable ß-mannanases is extremely important, as it could open up new avenues for genetic engineering, and protein engineering of these enzymes with enhanced properties and catalytic efficiencies. Under this scope, the present review provides a state-of-the-art discussion on the thermophilic ß-mannanases from bacterial origin, their production, engineering and structural characterization. It covers broad insights into various molecular biology techniques such as gene mutagenesis, heterologous gene expression, and protein engineering, that are employed to improve the catalytic efficiency and thermostability of bacterial mannanases for potential industrial applications. Further, the bottlenecks associated with mannanase production and process optimization are also discussed. Finally, future research related to bioengineering of mannanases with novel protein expression systems for commercial applications are also elaborated.

Exploiting Systematic Engineering of the Expression Cassette as a Powerful Tool to Enhance Heterologous Gene Expression in Trichoderma reesei.

Many endeavors in expressing a heterologous gene in microbial hosts rely on simply placing the gene of interest between a selected pair of promoters and terminator. However, although the expression efficiency could be improved by engineering the host cell, how modifying the expression cassette itself systematically would affect heterologous gene expression remains largely unknown. As the promoter and terminator bear plentiful cis-elements, herein using the Aspergillus niger mannanase with high application value in animal feeds and the eukaryotic filamentous fungus workhorse Trichoderma reesei as a model gene/host, systematic engineering of an expression cassette was investigated to decipher the effect of its mutagenesis on heterologous gene expression. Modifying the promoter, signal peptide, the eukaryotic-specific Kozak sequence, and the 3'-UTR could stepwise improve extracellular mannanase production from 17 U/mL to an ultimate 471 U/mL, representing a 27.7-fold increase in expression. The strategies can be generally applied in improving the production of heterologous proteins in eukaryotic microbial hosts.

Mannanase improves the growth performance of broilers by alleviating inflammation of the intestinal epithelium and improving intestinal microbiota.

This experiment aimed to discuss and reveal the effect and mechanism of mannanase on intestinal inflammation in broilers triggered by a soybean meal diet. In this experiment, 384 Arbor Acres broilers at 1 d old were randomly divided into 3 treatment groups. The broilers were fed a corn-soybean meal basal diet, a low-energy diet (metabolizable energy reduced by 50 kcal/kg), and a low-energy diet supplemented with 100 mg/kg mannanase for 42 d. The low-energy diet increased feed conversion ratio from 0 to 42 d, reduced ileal villus height and villus height-to-crypt depth ratio and upregulated the expression of nuclear factor kappa B (NF-κB) in the ileum (P < 0.05). It also reduced cecal short-chain fatty acids (SCFA), such as acetic acid (P < 0.05). Compared with low-energy diets, the addition of mannanase increased body weight at 42 d, promoted the digestibility of nutrients, and maintained the morphology and integrity of the intestinal epithelium of broilers (P < 0.05). In addition, mannanase upregulated the expression of claudin-1 (CLDN1) and zonula occludens-1 (ZO-1) in the jejunum at 21 d, downregulated the expression of ileal NF-κB, and increased the content of isobutyric acid in the cecum of broilers (P < 0.05). The results for the ileal microbiota showed that a low-energy diet led to a decrease in the relative abundance of Lactobacillus reuteri in the ileum of broilers. The addition of mannanase increased the relative abundance of Lactobacillus-KC45b and Lactobacillus johnsonii in broilers. Furthermore, a low-energy diet reduced the relative abundance of Butyricicoccus in the intestine of broilers and inhibited oxidative phosphorylation and phosphoinositol metabolism. Mannanase increased the relative abundance of Odoribacter, promoted energy metabolism and N-glycan biosynthesis, and increased the activities of GH3 and GH18. It is concluded that mannanase could improve the growth performance of broilers by reducing the expression of NF-κB in the ileum, increasing the production of SCFA in the cecum, suppressing intestinal inflammation, balancing the intestinal microbiota, reducing damage to the intestinal barrier, and improving the efficiency of nutrient utilization to alleviate the adverse effects caused by the decrease in dietary energy level.

Effects of ß-mannanase supplementation on intestinal health and growth of nursery pigs.

Two experiments were conducted using 120 pigs to test the hypothesis that supplementation of ß-mannanase could reduce digesta viscosity, enhance nutrient digestion, and improve intestinal health and growth of nursery pigs. In experiment 1, 48 crossbred barrows were randomly allotted to four treatments with increasing levels of ß-mannanase at 0, 200, 400, and 600 U/kg in feeds. All pigs were euthanized on day 12 to collect jejunal digesta to measure digesta viscosity and ileal digesta to measure apparent ileal digestibility (AID) of dry matter (DM), gross energy (GE), neutral detergent fiber (NDF), and acid detergent fiber (ADF). In experiment 2, 72 nursery pigs were randomly allotted to three treatments with increasing levels of ß-mannanase at 0, 400, and 600 U/kg in feeds. Plasma collected on day 9 was used to measure tumor necrosis factor-α (TNF-α), immunoglobulin G (IgG), malondialdehyde (MDA), and protein carbonyl (PC). All pigs were euthanized on day 10 to collect duodenal and jejunal tissues to evaluate the production of TNF-α, IL-6, and MDA, morphology, crypt cell proliferation, and expression of tight junction proteins in the jejunum. Data were analyzed using the MIXED procedure for polynomial contrasts and the NLMIXED procedure for broken-line analysis of SAS. In experiment 1, ß-mannanase supplementation tended to have quadratic effects on digesta viscosity (P = 0.085) and AID of GE (P = 0.093) in the pigs. In experiment 2, jejunal digesta viscosity of the pigs was reduced (P < 0.05) when ß-mannanase was supplemented at 360 U/kg of feed. ß-Mannanase supplementation linearly reduced (P < 0.05) TNF-α, IgG, MDA, and PC in the duodenum, and TNF-α, IgG, and MDA in the jejunum of the pigs. ß-Mannanase supplementation linearly increased (P < 0.05) villus height to crypt depth ratio and crypt cell proliferation in the jejunum. ß-Mannanase supplementation tended to linearly improve (P = 0.083) expression of zonula occludens-1 in the jejunum. In conclusion, supplementation of ß-mannanase at 360 U/kg reduced the digesta viscosity and up to 600 U/kg positively affected intestinal health and growth of pigs by reducing inflammation and oxidative stress whilst enhancing structure and barrier function in the jejunum.

Nursery pigs face challenges in digesting complex carbohydrates in their feeds, which can negatively affect their growth and intestinal health. In particular, ß-mannans can increase digesta viscosity and hinder nutrient digestion of nursery pigs. ß-Mannanase, an enzyme that breaks down ß-mannans, has been used in nursery feeds to alleviate negative impacts on nutrient utilization and intestinal health of nursery pigs. This study investigated the effects of increasing supplementation levels of ß-mannanase on intestinal health, nutrient utilization, and growth of nursery pigs. The results showed that supplementation of ß-mannanase at 360 U/kg in the feed reduced the digesta viscosity in the jejunum and up to 600 U/kg positively had beneficial effects on the intestinal health and growth of nursery pigs by reducing inflammation and oxidative stress through improving structure and barrier function in the jejunum.

Effects of ß-mannanase supplementation on productive performance, inflammation, energy metabolism, and cecum microbiota composition of laying hens fed with reduced-energy diets.

The objective of this study is to investigate the beneficial effects and underlying mechanism of dietary ß-mannanase supplementation on the productive performance of laying hens fed with metabolic energy (ME)-reduced diets. A total of 448 Hy-Line gray laying hens were randomly assigned to seven groups. Each group had 8 replicates with 8 hens. The groups included a control diet (CON) with a ME of 2750 kcal/Kg, diets reduced by 100 kcal/Kg or 200 kcal/Kg ME (ME_100 or ME_200), and diets with 0.15 g/Kg or 0.2 g/Kg ß-mannanase (ME_100+ß-M_0.15, ME_100+ß-M_0.2, ME_200+ß-M_0.15, and ME_200+ß-M_0.2). The productive performance, egg quality, intestinal morphology, inflammatory response, mRNA expression related to the Nuclear factor kappa B (NF-κB) and AMPK pathway, and cecum microbiome were evaluated in this study. ME-reduced diets negatively impacted the productive performance of laying hens. However, supplementation with ß-mannanase improved FCR, decreased ADFI, and restored average egg weight to the level of the CON group. ME-reduced diets increased the levels of interleukin-1ß (IL-1ß) and IL-6 while decreasing the levels of IL-4 and IL-10 in the jejunum of laying hens. However, dietary ß-mannanase supplementation improved jejunum morphology, reduced pro-inflammatory cytokine concentrations, and increased levels of anti-inflammatory factors in laying hens fed with ME-reduced diets. The mRNA levels of IL-6, IFN-γ, TLR4, MyD88, and NF-κB in the jejunum of ME-reduced diets were significantly higher than that in CON, dietary ß-mannanase supplementation decreased these genes expression in laying hens fed with ME-reduced diets. Moreover, dietary ß-mannanase supplementation also decreased the mRNA levels of AMPKα and AMPKγ, and increased the abundance of mTOR in the jejunum of laying hens fed with ME-reduced diets. Cecum microbiota analysis revealed that dietary ß-mannanase increased the abundance of various beneficial bacteria (e.g., g_Pseudoflavonifractor, g_Butyricicoccus, and f_Lactobacillaceae) in laying hens fed with ME-reduced diets. In conclusion, dietary ß-mannanase supplementation could improve the productive performance of laying hens fed with a ME-reduced diet by improving intestinal morphology, alleviating intestinal inflammation, changing energy metabolism-related signaling pathways, and increasing cecum-beneficial microbiota.