Isolation of four xylanases capable of hydrolyzing corn fiber xylan from Paenibacillus sp. H2C.

Corn fibre xylan (CX) shows high resistance to enzymatic hydrolysis due to its densely decorated side chains. To find enzymes capable of hydrolyzing CX, we isolated a bacterial strain (named H2C) from soil, by enrichment culture using non-starch polysaccharides of corn as the sole carbon source. Analysis based on the 16S rRNA sequence placed strain H2C within genus Paenibacillus. Enzymes were purified from supernatant of culture broth of strain H2C based on solubilizing activities toward CX. Four enzymes, Xyn5A, Xyn10B, Xyn11A, and Xyn30A, were successfully identified, which belong to glycoside hydrolase (GH) families, 5, 10, 11, and 30, respectively. Phylogenetic analysis classified Xyn5A in subfamily 35 of GH family 5, a subfamily of unknown function. Their activities toward beechwood xylan and/or wheat arabinoxylan indicated that these enzymes are ß-1,4-xylanases. They showed high solubilizing activities toward a feed material, corn dried distiller's grains with solubles, compared to five previously characterized xylanases.Abbreviations : CX: corn fibre xylan; DDGS: corn dried distiller's grains with solubles.

Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase.

BACKGROUND: A mesophilic xylanase PjxA from Penicillium janthinellum MA21601 has high specific activity under acidic condition and holds great potential for applications in the animal feed industry. To enhance the thermostability of xylanase PjxA, two mutation strategies in the N-terminal region were examined and then integrated into the xylanase to further improvement. The recombinant xylanase PTxA-DB (The meaning of DB is disulfide-bridge.) was constructed by replacement of five residues in the mutated region in TfxA (T10Y, N11H, N12D, Y15F, N30 L), combined with an additional disulfide bridge in the N-terminal region. RESULTS: The Tm value of mutant PTxA-DB was improved from 21.3 °C to 76.6 °C, and its half-life was found to be 53.6 min at 60 °C, 107-fold higher than the wild type strain. The location of the disulfide bridge (T2C-T29C) was between the irregular loop and the ß-strand A2, accounting for most of the improvement in thermostability of PjxA. Further analysis indicated T2C, T29C, N30 L and Y15F lead to increase N-terminal hydrophobicity. Moreover, the specific activity and substrate affinity of PTxA-DB were also enhanced under the acidic pH values. CONCLUSIONS: These results indicated PTxA-DB could be a prospective additive to industrial animal feeds.

Variation in levels of non-starch polysaccharides and endogenous endo-1,4-ß-xylanases affects the nutritive value of wheat for poultry.

1. Endo-1,4-ß-xylanase is known to improve the nutritive value of wheat-based diets for poultry by degrading dietary arabinoxylans. However, broilers' response to supplementation of wheat-based diets with exogenous endo-1,4-ß-xylanase is not always observed. 2. In this study, 108 different wheat lots were analysed for levels of extract viscosity as well as for endogenous endo-1,4-ß-xylanase activity, and the impact of these two variables in animal performance was tested. 3. Results revealed that endogenous endo-1,4-ß-xylanase activity and extract viscosity content varied widely among different wheat lots. Thus, a trial was conducted to evaluate the efficacy of exogenous enzyme supplementation in broiler diets using wheats with different levels of extract viscosity and endogenous endo-1,4-ß-xylanase activity. 4. The data revealed that exogenous enzyme supplementation was only effective when the wheat present in the diet had high levels of extract viscosity (14.8 cP) with low endogenous endo-1,4-ß-xylanase activity (347.0 U/kg). Nevertheless, it is apparent that exogenous microbial xylanases reduce digesta extract viscosity and feed conversion ratio independently of the endogenous properties presented by different wheat lots. 5. The data suggest that extract viscosity and/or endogenous endo-1,4-ß-xylanase activity affect the response to enzyme supplementation by poultry fed on wheat-based diets.

Co-production of Xylooligosaccharides and Xylose From Poplar Sawdust by Recombinant Endo-1,4-ß-Xylanase and ß-Xylosidase Mixture Hydrolysis.

As is well-known, endo-1,4-ß-xylanase and ß-xylosidase are the rate-limiting enzymes in the degradation of xylan (the major hemicellulosic component), main functions of which are cleavaging xylan to release xylooligosaccharides (XOS) and xylose that these two compounds have important application value in fuel, food, and other industries. This study focuses on enzymatic hydrolysis of poplar sawdust xylan for production of XOS and xylose by a GH11 endo-1,4-ß-xylanase MxynB-8 and a GH39 ß-xylosidase Xln-DT. MxynB-8 showed excellent ability to hydrolyze hemicellulose of broadleaf plants, such as poplar. Under optimized conditions (50°C, pH 6.0, dosage of 500 U/g, substrate concentration of 2 mg/mL), the final XOS yield was 85.5%, and the content of XOS2-3 reached 93.9% after 18 h. The enzymatic efficiency by MxynB-8 based on the poplar sawdust xylan in the raw material was 30.5%. Xln-DT showed excellent xylose/glucose/arabinose tolerance, which is applied as a candidate to apply in degradation of hemicellulose. In addition, the process and enzymatic mode of poplar sawdust xylan with MxynB-8 and Xln-DT were investigated. The results showed that the enzymatic hydrolysis yield of poplar sawdust xylan was improved by adding Xln-DT, and a xylose-rich hydrolysate could be obtained at high purity, with the xylose yield of 89.9%. The enzymatic hydrolysis yield was higher (32.2%) by using MxynB-8 and Xln-DT together. This study provides a deep understanding of double-enzyme synergetic enzymolysis of wood polysaccharides to valuable products.

Enzymatic Properties of endo-1,4-ß-xylanase from Wheat Malt.

BACKGROUND: Arabinoxylan (AX) is the main non-starch polysaccharide in wheat. Wheat malts are traditional raw materials for beer brewing. AX is divided into water-soluble arabinoxylan (WEAX) and waterinsoluble arabinoxylan (WUAX). In the mashing stage of beer production, WUAX in malt is degraded by arabinoxylanase to WEAX, which is further degraded to smaller molecules and retained in the final beer. The viscosity of WEAX is related to its molecular weight. WEAX with higher molecular weight and viscosity can increase viscosity and turbidity and reduce filtration speed of wort and beer; WEAX with moderate molecular weight and viscosity contributes to the foaming characteristics and foam stability, and promotes the taste and texture of a beer; WEAX with small molecular weight has the functions of anti-tumor and lowering blood pressure and is regarded as a prebiotic. Because WEAXs with different molecular weight and properties have different impacts on the beer brewing process and qualities of the final beer, it becomes more important to control the degradation of AX during the brewing process of a beer. Endo-1,4-ß-xylanase (EC 3.2.1.8) is the most important AX degrading enzyme, which cleaves the ß -xylosidic bond between two d-xylopyranosyl residues linked in ß-(1,4). The study of enzymatic properties of endo-1,4-ß-xylanase from wheat malt is very important for the rational formulation of the content and molecular weight of WEAX in wort and beer during the mashing procedure when using wheat malt as the main raw materials. OBJECTIVE: In this article, our motivation is to study the enzymatic properties (including optimum pH and temperature, pH and temperature stability, the effect of inhibitors) of wheat malt endo-1,4-ß-xylanase. METHODS: In this article, we prepared crude enzyme according to the method of Guo with minor modifications. The endo-1,4-ß-xylanase activity was determined according to the method of Biely in the previous report with minor modifications. The 0.5 mL crude enzyme sample was mixed with 0.5 mL 1 mg/mL 4-O-methyl-dglucurono- d-xylan dyed with Remazol Brilliant Blue R (RBBR-Xylan) solution, intensively mixed, and incubated at 40 °C for exactly 90 min. The reaction was stopped by precipitation using 2 mL absolute ethanol, and the reaction mixture was stirred acutely and placed at room temperature for 30 min. Then, the mixture was mixed again and centrifuged at 6000 g for 10 min. The supernatant was collected and the absorbance was measured at 590 nm. Absolute ethanol and RBBR-Xylan were added to the control tubes first, and after the reaction was completed, the crude enzyme sample was added. One unit of endo-1,4-ß-xylanase was defined as at pH 5.5 and 40 °C liberate 1 µmol xylose equivalents in 1 min per g dry wheat malt. RESULTS: The results showed that the optimal activity of endo-1,4-ß-xylanase was achieved at pH 5.5-6.0, and the enzyme was extremely stable at pH 4.5, 5.5 and 6.5 after incubation for 30, 50 and 60 min, respectively. The optimal temperature was 40-45 °C and the deactivation temperature was 75 °C. Endo-1,4-ß-xylanase was stable at 20 °C and 40 °C; the stability was slightly decreased at 50 °C and rapidly decreased at 55 °C. The enzyme activity was mildly inhibited by K+, Na+, and Pb2+, moderately inhibited by Ca2+, Mg2+ and Mn2+ and severely inhibited by Cu2+, Ag+ and EDTA. CONCLUSION: We have got the enzymatic properties of endo-1,4-ß-xylanase from wheat malt, so during wort mashing, we could apply this research result to carry out the rational formulation of the content and molecular weight of WEAX in wort and beer during the mashing procedure when using wheat malt as the main raw materials. Expected to solve the technical problems such as high viscosity, slow filtration speed and so on, but also highlight the typical flavors of WEAX such as rich and persistent foam and mellow texture during the brewing process of a beer.

Partial Optimization of Endo-1, 4-Β-Xylanase Production by Aureobasidium pullulans Using Agro-Industrial Residues.

UNLABELLED: Objective(s) : Although bacteria and molds are the pioneering microorganisms for production of many enzymes, yet yeasts provide safe and reliable sources of enzymes with applications in food and feed. MATERIALS AND METHODS: Single xylanase producer yeast was isolated from plant residues based on formation of transparent halo zones on xylan agar plates. The isolate showed much greater endo-1, 4-ß-xylanase activity of 2.73 IU/ml after optimization of the initial extrinsic conditions. It was shown that the strain was also able to produce ß-xylosidase (0.179 IU/ml) and α-arabinofuranosidase (0.063 IU/ml). Identification of the isolate was carried out and the endo-1, 4-ß-xylanaseproduction by feeding the yeast cells on agro-industrial residues was optimized using one factor at a time approach. RESULTS: The enzyme producer strain was identified as Aureobasidiumpullulans. Based on the optimization approach, an incubation time of 48 hr at 27°C, inoculum size of 2% (v/v), initial pH value of 4 and agitation rate of 90 rpm were found to be the optimal conditions for achieving maximum yield of the enzyme. Xylan, containing agricultural residues, was evaluated as low-cost alternative carbon source for production of xylanolytic enzymes. The production of xylanase enzyme in media containing wheat bran as the sole carbon source was very similar to that of the medium containing pure beechwoodxylan. CONCLUSION: This finding indicates the feasibility of growing of A. pullulans strain SN090 on wheat bran as an alternate economical substrate in order for reducing the costs of enzyme production and using this fortified agro-industrial byproduct in formulation of animal feed.