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.

Effects of neutral phytase on growth performance and phosphorus utilization in crucian carp (Carassius auratus).

A feeding trial was conducted for nine weeks to investigate the effects of partially replacing Ca(H2PO4)2 with neutral phytase on the growth performance, phosphorus utilization, nutrient digestibility, serum biochemical parameters, bone and carcass mineral composition, and digestive-enzyme-specific activity in crucian carp (Carassius auratus). The diets prepared with 0.8%, 0%, and 1.8% Ca(H2PO4)2 (1%=1 g/100 g) supplements were regarded as the P1E0, negative control (NC), and positive control (PC) groups, respectively; the other three experimental diets were prepared with the addition of 200, 300, and 500 U/kg of neutral phytase, respectively, based on the P1E0 group. Three hundred and eighty-four fish ((1.50±0.01) g) were randomly distributed in the six treatments with four replicates each. The fish were initially fed with 2%-3% diets of their body weight per day, with feeding twice daily (08:00 and 16:00), under a 12-h light/12-h dark cycle at the temperature of (27.56±0.89) °C. The results showed that supplemental phytase at different levels in the diet improved the final body weight, average daily gain, feed conversion ratio, phosphorus utilization, and protein efficiency ratio of crucian carp (P<0.05). Phytase supplementation increased the mineral content in serum (P), bone (P, Ca), and carcass (P, Ca, Zn, Na, and Mg) (P<0.05); the trypsin and chymotrypsin activity soared when fed with the phytase-supplemented diets (P<0.05). We may conclude that supplemental dietary neutral phytase improved the growth performance, phosphorus utilization as well as nutrient utilization in crucian carp, and it can be considered an important nutritional replacement for Ca(H2PO4)2.

Characterization of ß-glucosidase from Aspergillus terreus and its application in the hydrolysis of soybean isoflavones.

An extracellular ß-glucosidase produced by Aspergillus terreus was identified, purified, characterized and was tested for the hydrolysis of soybean isoflavone. Matrix-assisted laser desorption/ionization with tandem time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) revealed the protein to be a member of the glycosyl hydrolase family 3 with an apparent molecular mass of about 120 kDa. The purified ß-glucosidase showed optimal activity at pH 5.0 and 65 °C and was very stable at 50 °C. Moreover, the enzyme exhibited good stability over pH 3.0-8.0 and possessed high tolerance towards pepsin and trypsin. The kinetic parameters Km (apparent Michaelis-Menten constant) and Vmax (maximal reaction velocity) for p-nitrophenyl-ß-D-glucopyranoside (pNPG) were 1.73 mmol/L and 42.37 U/mg, respectively. The Km and Vmax for cellobiose were 4.11 mmol/L and 5.7 U/mg, respectively. The enzyme efficiently converted isoflavone glycosides to aglycones, with a hydrolysis rate of 95.8% for daidzin, 86.7% for genistin, and 72.1% for glycitin. Meanwhile, the productivities were 1.14 mmol/(L·h) for daidzein, 0.72 mmol/(L·h) for genistein, and 0.19 mmol/(L·h) for glycitein. This is the first report on the application of A. terreus ß-glucosidase for converting isoflavone glycosides to their aglycones in soybean products.

Chitosan-zinc chelate improves intestinal structure and mucosal function and decreases apoptosis in ileal mucosal epithelial cells in weaned pigs.

The present study was conducted to investigate the effects of chitosan (CS)-Zn on intestinal morphology, mucosal epithelial cell apoptosis and mucosal immune function in weanling pigs. A total of 150 weanling barrows with a body weight of 7.2 kg were randomly allocated into five groups. A basal diet without Zn supplementation was used as the control and other four groups were fed the control diet supplemented with 50 or 100 mg/kg of Zn as CS-Zn, 100 mg/kg of Zn as ZnSO4 and 3000 mg/kg of Zn as ZnO, respectively. The feeding trial lasted for 28 d. The results showed that serum diamine oxidase activities, d-lactate levels and endotoxin contents were lower in pigs fed dietary 100 mg/kg of Zn as CS-Zn or 3000 mg/kg of Zn as ZnO than in pigs fed the control or 100 mg Zn/kg as ZnSO4 diet. The ratios of the villus height:crypt depth of the duodenum, jejunum and ileum were higher in pigs that received 100 mg/kg of Zn as CS-Zn or a high level of Zn as ZnO than in pigs fed the control diet. Moreover, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL)-stained ileal epithelial cells were found in the control group, and apoptotic cells did not appear prominently in pigs that received the 100 mg/kg of CS-Zn or ZnO diet. Secretory IgA concentration in ileal mucus was increased in the dietary group that received 100 mg/kg of CS-Zn or ZnO. These results indicated that dietary 100 mg CS-Zn/kg had similar biological effects to dietary 3000 mg ZnO/kg on intestinal morphology, mucosal epithelial cell apoptosis and mucosal immune function.

Effects of zinc on hepatic antioxidant systems and the mRNA expression levels assayed by cDNA microarrays in rats.

BACKGROUND: This study evaluated effects of zinc on the hepatic lipid peroxidation, antioxidant components and mRNA expression levels in rats. METHODS: Three diets with different Zn levels including Zn adequacy (ZA; 34.50 mg/kg, control), Zn deficiency (ZD; 3.30 mg/kg), and Zn overdose (ZO; 345.45 mg/kg) were fed to rats for 6 weeks. The mRNA expression levels were analyzed by cDNA microarrays. RESULTS: The body weight of rats fed the ZD diet was less (p < 0.01) than that of rats fed the ZA diet. Zn overdose elevated body weight, but the increase was not detected (p > 0.05) at week 6. Although copper and iron status in serum were declined (p < 0.01), those in liver were not affected (p > 0.05) by the high intake of zinc. The glutathione peroxidase (GPx) and glutathione (GSH) remained unchanged (p > 0.05) by zinc treatment. Rats fed the ZD diet showed reductions(p < 0.01) in the Cu-Zn superoxide dismutase (Cu-Zn SOD) and catalase (CAT) activity, and increases (p < 0.01) in the malondialdehyde and hydrogen peroxide (H(2)O(2)) contents. Rats fed the ZO diet particularly had higher Cu-Zn SOD (p < 0.01) activity. The mRNA expression levels of SOD were upregulated in the ZO group, and CAT was downregulated in the ZD group, while no changes in GPx mRNA levels were found after zinc treatment. CONCLUSION: The study suggested that zinc deficiency largely decreased body weight; zinc overdose, however, moderately stimulated growth in the early growing phase of rats. High dietary zinc did not compete with liver copper and iron status. Although Zn deficiency impaired antioxidant functions, zinc overdose hardly enhanced the antioxidant systems of animals.

Distribution and characteristics of endogenous digestive enzymes in the red-eared slider turtle, Trachemys scripta elegans.

Distribution and properties of the main digestive enzymes including protease and amylase, from stomach, pancreas and the anterior, middle and posterior intestine of the adult red-eared slider turtle Trachemys scripta elegans were studied at various pHs and temperatures. The optimum temperature and pH for protease in stomach, pancreas and the anterior, middle and posterior intestine were 40 degrees C, 2.5; 50 degrees C, 8.0; 50 degrees C, 7.0; 50 degrees C, 8.0; and 50 degrees C, 8.5; respectively. The optimum temperature and pH for amylase in stomach, pancreas and anterior, middle and posterior intestine were 40 degrees C, 8.0; 30 degrees C, 7.5; 40 degrees C, 7.0; 50 degrees C, 8.0; and 50 degrees C, 8.0; respectively. Under the optimum conditions, the order of protease activity from high to low was of pancreas, stomach and the anterior, posterior and middle intestine; the activity of amylase in descending order was of anterior intestine, pancreas, posterior intestine, middle intestine and stomach.