Glucanases, Nucleic Acids Encoding Them and Methods for Making and Using Them

Abstract

The invention relates to polypeptides having glucanase, e.g., endoglucanase, mannanase, xylanase activity or a combination of these activities, and polynucleotides encoding them. In one aspect, the glucanase activity is an endoglucanase activity (e.g., endo-1,4-beta-D-glucan 4-glucano hydrolase activity) and comprises hydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (e.g., carboxy methyl cellulose and hydroxy ethyl cellulose) lichenin, beta-1,4 bonds in mixed beta-1,3 glucans, such as cereal beta-D-glucans or xyloglucans and other plant material containing cellulosic parts. In addition, methods of designing new enzymes and methods of use thereof are also provided. In alternative aspects, the new glucanases e.g., endoglucanases, mannanases, xylanases have increased activity and stability, including thermotolerance or thermostability, at increased or decreased pHs and temperatures.

Description

FIELD OF THE INVENTION[0001]This invention relates generally to enzymes used in food and feed compositions; and in alternative aspects provides novel enzymes, polynucleotides encoding these enzymes, and uses of these polynucleotides and polypeptides, and in alternative aspects provides polypeptides (e.g., enzymes, peptides, antibodies) having a glucanase activity, e.g., an endoglucanase, activity, e.g., catalyzing hydrolysis of internal endo-β-1,4- and / or β-1,3-glucanase linkages. In one aspect, the endoglucanase activity (e.g., endo-1,4-beta-D-glucan 4-glucano hydrolase activity) comprises hydrolysis of 1,4- and / or β-1,3-beta-D-glycosidic linkages in cellulose, cellulose derivatives (e.g., carboxy methyl cellulose and hydroxy ethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans, such as cereal beta-D-glucans or xyloglucans and other plant or organic material containing cellulosic parts. In one aspect, the polypeptides of the invention have a glucanase, xylanase and / ...

AI Technical Summary

Benefits of technology

[0195]In one aspect, the invention provides methods of dough conditioning comprising contacting a dough or a bread product with at least one polypeptide of the invention under conditions sufficient for conditioning the dough. In one aspect, the invention provides methods of beverage production comprising administration of at least one polypeptide of the invention to a beverage or a beverage precursor under conditions sufficient for decreasing the viscosity of the beverage.

[0360]The invention provides methods for improving texture and flavor of a dairy product comprising the following steps: (a) providing a polypeptide of the invention having a glucanase (or cellulase), e.g., endoglucanase, mannanase, xylanase, amylase, xanthanase and / or glycosidase, e.g., cellobiohydrolase, mannanase and / or beta-glucosidase activity, or a glucanase encoded by a nucleic acid of the invention; (b) providing a dairy product; and (c) contacting the polypeptide of step (a) and the dairy product of step (b) under conditions wherein the glucanase (or cellulase), e.g., endoglucanase, mannanase, xylanase, amylase, xanthanase and / or glycosidase, e.g., cellobiohydrolase, mannanase and / or beta-glucosidase can improve the texture or flavor of the dairy product. In one aspect, the dairy product comprises a cheese or a yogurt. The invention provides dairy products comprising a glucanase (or cellulase), e.g., endoglucanase, mannanase, xylanase, amylase, xanthanase and / or glycosidase, e.g., cellobiohydrolase, mannanase and / or beta-glucosidase of the invention, or is encoded by a nucleic acid of the invention.

[0375]In another aspect, the invention provides a method of dough conditioning comprising contacting dough with at least one polypeptide of the invention under conditions sufficient for conditioning the dough. Another aspect of the invention is a method of beverage production comprising administration of at least one polypeptide of the invention under conditions sufficient for decreasing the viscosity of wort or beer, or, increasing the clarity (e.g., clarification) of the beverage.

[0419]In alternative embodiments, advantages of practicing the compositions and methods of the invention can be: a) providing better distribution of the enzyme(s) within the mud cake (also known as “filter cake”) that will result in more uniform and effective mud cake removal, b) simplifying the operations by eliminating a separate enzyme delivery step (enzyme is included in the drilling fluid formulation, and / or in the fluids for oil and gas well washing and / or fracturing), and c) eliminating the need for buffering salts as the enzyme is not formulated with an acidic fluid.

[0420]In alternative embodiments, polymer-degrading enzymes, including amylases, glucanases, xanthanases, glycosidases, any starch degrading enzyme, any cellulase and / or protease, e.g., as described herein, are added to a drilling fluid and / or an oil and gas well washing and / or fracturing fluid that is used during an oil and gas well drilling operations or oil and gas well washing and / or fracturing processes. These fluids can contain starch as a viscosifier and can be formulated at relatively high alkalinity (pH=9-9.5). Because of the alkalinity of the fluid (in this embodiment), an acidic-to-neutral enzyme will remain dormant in the fluid and in the mud cake (“filter cake”) that is formed after the loss of water from the fluid onto the formation surface. In order to activate the enzyme, the mud cake can be washed with an acid solution. The acid will neutralize the alkalinity of the mud cake and will provide an acidic environment which will trigger the enzyme activity and hydrolytic function toward starch or other polymers. In this alternative embodiment, the “acid wash”’ is a necessary step, and can be applied during the well drilling operations and / or the well cleaning operations (including oil and gas well washing and / or fracturing processes) in order to remove calcium carbonate deposits from the formation. Once activated (by an acid environment), the enzyme will degrade the starch or other polymers, and will remove the mud cake from the well bore.

[0421]In one operation, this “washing” of the well bore is the final step in the drilling operation, and / or oil and gas well washing and / or fracturing operation, and a complete degradation of the mud cake (“filter cake”) by practicing the compositions and methods of the invention enables optimal productivity of the well.

Problems solved by technology

All of these characteristics present problems for several industries including brewing, baking, animal nutrition.

In brewing applications, the presence of glucan results in wort filterability and haze formation issues.

In baking applications (especially for cookies and crackers), glucans can create sticky doughs that are difficult to machine and reduce biscuit size.

In addition, this carbohydrate is implicated in rapid rehydration of the baked product resulting in loss of crispiness and reduced shelf-life.

For monogastric animal feed applications with cereal diets, beta-glucan is a contributing factor to viscosity of gut contents and thereby adversely affects the digestibility of the feed and animal growth rate.

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