Preliminary Study on Selection and Identification of a Mannanase-producing Strain and Its Enzymatic Properties

[Objective] This study aimed to identify a maunanase-producing strain isolated from soil. [Method] With kanjac powder as the substrate, a man- nanase-producing dominant strain was iselated from the soil samples collected from Kunyu Mountain by using plate selection method. Sequence analysis of the 16SrDNA fragment of the strain was conducted, and the strain was identified as Bacillus subtilis. Fermentation conditions and enzymatic characteristics were studied preliminarily. [ Result] Experimental result showed that enzyme yield of this strain was different in different medium and in the same medium at different tempera- ture. Enzyme yield of this strain in LB medium was higher when incubated at 27 ℃ than at 30 ℃ ; however, incubation at 30 ℃ was more conducive to the enzyme production than incubation at 27 ℃ in SOC medium. The optimal reaction pH was 7.0 and the optimal reaction temperature was 55 ℃ for enzyme production of this strain. When the temperature was above 55 ℃, enzyme activity declined sharply with the raise of temperature. Under the optimum conditions, enzyme activity could achieve 95.3 U. [ Conclusion] This study provided reference for the industrial application of degradation products of mannan.

Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA, an Endoglucanase with Cellulase and Mannanase Activity

The discovery of new, highly active, biomass-degrading enzymes is important to the development of a sustainable biofuels industry. Dictyoglomus turgidum, a thermophilic, anaerobic eubacterium that ferments cellulose and produces ethanol and hydrogen, was chosen as a candidate to screen for novel enzymes. A novel thermostable endoglucanase, CelA, was identified and purified during screening of a shotgun library of Dic（yoglomus turgidum and subsequently subcloned and expressed in E. coli. The celA gene coding for a 312 amino acid protein showed low homology to proteins outside the genus Dictoglomi and lacked an apparent signal peptide. CelA had a broad substrate range, possessing both endo and exo activity on soluble and insoluble β-（1,4）-Iinked glucose-containing substrates as well as endo activity on soluble and insoluble β-（1,4）-linked mannose containing substrates. The specific activity of CelA was 226 U/rag using β-glucan, 66 U/mg using glucomannan, and 63 U/mg using CMC as substrates. The high temperature optimum of 70 ℃ to 80 ℃ and wide substrate range of the enzyme might make it an excellent tool for biomass degradation at high temperature.

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 at1 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 from0 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.

In vitro Evaluation of Feed Enzyme Compound Produced by Aspergillus sulphureus in Solid-state Fermentation

ABSTRACT： Three trials were conducted to analyze a multi-enzyme compound produced by Aspergillus sulphureus in solid-state fermentation （SSF） as a po- tential feed additive. The results of the first trial showed that there were at least 5 non-starch polysac- charide enzymes： xylanase, 13-ghicanase, pectinase, mannase and carboxy methyl cellulase （CMCase） contained in the compound. Xylanase and fl-glucanase showed good activities at pH 2.5-7.0, which were in the range of 649-1046 U/g and 444-648 U/g, respec- tively. Pectinase showed good activity in acidic solu- tion （pH 2.5-3.0）,which ranged from 195 to 917 U/g. Mannase showed high activity of 235-298 U/g at pH 3.5-4.5 and the activity of CMCase was relatively constant at pH 2.5-7.0, which was in the range of 38.2-78.6 U/g. The second trial was aimed to test the stability of the enzymes in gastric liquor （pH 2.6） of finishing pigs and Na2 HPO4-gastric liquor （ pH 5.5 ）.After 6 h incubation at 40℃ in gastric liquor,the re- tained activity of xylanase, 13-glucanase, pectinase, mannase and CMCase was 26.3% ,65.0% ,71.0%, 74.8% and 85.6%, respectively. While after 6 h in- cubation at 40℃ in Na2I-IPO4-gastric liquor, the re- tained activity of xylanase, [3-glucanase, pectinase, mannase and CMCase was 87.9% ,91.1% ,92.3%, 95.0%, and 97.5%, respectively. The third trial was carried out in a jejunum liquor （ pH 5.8,200 mL）, which contained 0.2 g of the multi-enzyme compound and 10 g of soybean hull or wheat bran, respectively. After 8 h incubation at 40℃, 18.7% of soybean hull and 20.1% of wheat bran could be degraded to solu- ble saccharide, respectively. Compared with the tradi- tional methods for feed enzyme testing which involve feeding animals for 1-3 months, enzyme assay in this way was relatively convenient.

Non-starch polysaccharide-degrading enzymes may improve performance when included in wheat- but not maize-based diets fed to broiler chickens under subclinical necrotic enteritis challenge

The present study investigated whether supplementing fibre-degrading enzymes can ameliorate the severity of subclinical necrotic enteritis(NE)in broiler chickens offered wheat-or maize-based diets.A total of 1,544 mixed-sex broiler chickens were assigned to 16 experimental treatments as a 2×2×4 factorial arrangement of treatments.The factors were the following:NE challenge,yes or no;diet type,wheat-or maize-based;and enzyme supplementation,control(no enzyme),family 10 xylanase(XYN10),family 11 xylanase(XYN11)or b-mannanase(MAN).Each treatment was replicated 6 times,with 16 birds per replicate pen.A three-way challenge×diet type×enzyme interaction occurred for body weight at 21 d of age(P=0.025)and overall feed conversion ratio(P=0.001).In the non-challenged birds fed the wheat-based diet,supplementing MAN increased d 21 body weight compared to the control.In challenged birds fed the maize-based diet,supplemental XYN11 impeded body weight and overall FCR compared to the control.Birds offered the maize-based diet presented heavier relative gizzard weights at both 16 and 21 d of age(P<0.001)and reduced liveability(P=0.046)compared to those fed the wheatbased diet.Enzyme supplementation reduced ileal and jejunal digesta viscosity at 16 d of age only in birds fed the wheat-based diet(P<0.001).XYN11 increased ileal digesta viscosity in birds fed the maizebased diet,and MAN reduced it in birds fed the wheat-based diet at 21 d of age(P=0.030).Supplementing XYN11 improved ileal soluble non-starch polysaccharides(NSP)digestibility in birds fed the wheat-based diet compared to non-supplemented birds(P<0.001).Birds fed the wheat-based diet displayed a higher abundance of Bifidobacterium,Lactobacillus and Enterobacteriaceae and butyric acid in the caeca at 16 d of age compared to birds fed the maize-based diet(P<0.05).In conclusion,supplemental XYN11 exacerbated the negative impact of NE on growth performance in birds fed the maizebased diet.Supplementing wheat-based diets with fibre-degrading enzymes ameliorates production losses induced by NE.