Microbially expresses xylanases and their use as feed additives and other uses

The current invention relates to codon-optimized xylanase programming sequences as well as the expression of xylanases in microbes and yeast. The invention further relates to utilizing numerous copies of the xylanase expression construct for elevated levels of protein expression. The invention also relates to the use of xylanases as feed or food additives. The invention also relates to means of expression of enzymes to increase thermotolerance by expressing them in organisms which glycosylate proteins in comparison to saying that the exact same enzyme with no glycosylation. Moreover, the invention relates to methods for feed, enzyme feed additives, and methods of reducing the feed conversion ration or raising weight gain of creatures.

 

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BACKGROUND

 

 
Non-starch polysaccharides (NSP) have been implicated in the variability of the nutritional quality of cereals such as chickens, associated with changes in viscosity of digesta (Bedford, M. R. & H. L. Classen (1993)”An in vitro Assay for Predictionof Broiler Intestinal Viscosity and Development When Fed Rye-Based Diets in the existence of Exogeneous Enzymes” Poult. Sci. 72, 137-143). Arabinoxylans are the significant NSP of wheat and several commercially available xylanase enzyme products, produced fromTrichoderma, Humicola and Aspergillus spp are shown to reduce digesta viscosity and to improve the nutritive value of diets.
 

 
Xylans are linear polysaccharides shaped from beta-1,4-connected D-xylopyranoses. In cereals, xylans often contain side chains of alpha-1,2, alpha-1,3, or alpha-1,2 and alpha-1,3 connected L-arabinofuranoside. These substituted xylans arecommonly known as arabinoxylans. Xylanases (e.g., endo-1,4-beta-xylanase, EC 3.2.2.8) hydrolyze internal beta-1,4-xylosidic linkages in xylan to Generate smaller molecular weight xylo-oligomers
 

 
Xylanases may be used, e.g., in animal feed compositions which are full of arabinoxylans and glucoxylans, in baking, in brewing, and in pulp and paper applications, e.g. to improve the bleachability of pulps. When added to feeds (e.g. formonogastric animals, including poultry or swine) which include cereals (e.g. wheat, barley, maize, rye, triticale or oats) or cereal by-products, a hemicellulolytic enzyme improves the break-down of plant cell walls which leads to better utilization ofthe plant nutrients from the animal. This leads to enhanced growth rate and feed conversion. Additionally, the viscosity of the feeds containing xylan can be reduced.
 

 
In a number of the practical applications, bodily conditions (e.g., temperature and pH) interfere with the use of xylanases; the xylanases must be active in the temperatures and pH conditions of the procedure in which they’re used. Formulation ofcommercial feed with pelleting, extrusion or expanding, often contains steps between large temperatures (70-180. degree. C.). Enzymes added to the formula procedure should defy these conditions. On the other hand, the corresponding temperaturein the intestine of animals is about 40. degree. C. Therefore, perfect xylanases for feed compositions should withstand the above-mentioned intense temperatures. In whitening software, xylanase program is not quite as straightforward as incorporating a xylanase treatmentstep. Since the whitening procedure, and even the arrangement of the steps utilized in the whitening procedure varies in different pulp mills, there’s thus a continuous need to locate new xylanases active in different temperatures and pH conditions.
 

 
Most commercial xylanases designed for feed applications aren’t very thermotolerant, particularly when neutral or alkaline pH requirements are utilized. In training, xylanases are usually ineffective or inactive at temperatures higher than60.degree. C. and often these enzymes operate under contaminated conditions. Normally, you will find differences in the physiological characteristics of xylanases of bacteria and parasites (for review, see Wong et al., Microbiol. Rev. 52:305-317 (1988)). Typically,fungal xylanases have a temperature optimum at about 50. degree. C. and reduced pH optimum than those of bacterial source. Xylanases of bacterial source generally have a temperature optimum in the range of 50 to 70. degree. C. Numerous xylanases fromfungal and bacterial germs are identified and distinguished. (See, e.g., U.S. Pat. No. 5,437,992; Coughlin, M. P.; Biely, P. et al.,”Proceedings of the second TRICEL symposium on Trichoderma reesei Cellulases and Other Hydrolases,”Espoo 1993, P. Souminen and T. Reinikainen eds., Foundation for Biotechnical and Industrial Fermentation Research 8:125-135 (1993) and also WO03/16654). Specifically, three particular xylanases (XYL-I, XYL-II, and XYL-III) have been identified in T. reesei(Tenkanen, et al., Enzyme Microb. Technol. 14:566 (1992); Torronen, et al., Bio/Technology 10:1461 (1992); and Xu, et al., Appl. Microbiol. Biotechnol. 49:718 (1998)). Although numerous xylanases are explained in the literature, the needstill is present to determine novel xylanases which are effective in applications like those relating to animal feed and grain processing, biofuels, cleaning, fabric maintenance, substances, plant processing, and delignifying and brightening of paper and pulp.
 

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