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Glucanases, nucleic acids encoding them and methods for making and using them

A technology of dextranase and nucleic acid, which is applied in the field of polynucleotides and polypeptides, and can solve problems affecting feed digestibility, loss of brittleness, shortened shelf life, etc.

Inactive Publication Date: 2014-01-01
BP CORP NORTH AMERICA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, this carbohydrate is strongly associated with rapid rehydration of baked products, leading to loss of crispness and reduced shelf life
For the application of monogastric animal feeds made from cereal foods, β-glucans are responsible for the stickiness of intestinal materials, thus adversely affecting the digestibility of the feed and the growth rate of the animals

Method used

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  • Glucanases, nucleic acids encoding them and methods for making and using them
  • Glucanases, nucleic acids encoding them and methods for making and using them
  • Glucanases, nucleic acids encoding them and methods for making and using them

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0718] Example 1: Plate-based Enzyme Discovery of Endoglycosidases: Expression Screening

[0719] The following examples demonstrate the enzymatic activity of exemplary enzymes of the invention and the isolation and identification of nucleic acids. These assays can also be used to determine whether a polypeptide has the requisite enzymatic (eg glucanase, mannanase or xylanase) activity within the scope of the invention.

[0720] Titration assay of the lambda library : To 600 μL E. coli MRF′ cells (OD 600 =1.0) by adding 1.0 μL of the Lambda Zap Express amplified library stock solution. with 10mM MgSO 4 Dilute the MRF' stock solution. Incubate the mixture at 37 °C for 15 min, then transfer the suspension to 5-6 mL of NZY top agar at 50 °C and mix gently. Quickly pour the agar solution onto a large (150 mm) NZY medium plate, allowing the top agar to solidify completely (approximately 30 minutes). Invert plate. Plates were incubated at 39°C for 8-12 hours. (Number of p...

Embodiment 2

[0727] Embodiment 2: activity assay

[0728] The following examples show the enzymatic activity of exemplary enzymes of the invention. These assays can also be used to determine whether a polypeptide possesses the requisite (eg, glucanase, mannanase, or xylanase) enzymatic activity within the scope of the invention.

[0729] Polypeptides of the invention having the sequences shown in the SEQ ID NO:s listed below were shown to have glucanase activity, as described below. Specific activity was determined on barley β-glucan (BBG) or carboxymethylcellulose (CMC) using the BCA reducing sugar assay. 1 unit (U) of dextranase activity = 1 μmol / min released at 37°C, pH 5.3 -1 Glucose reducing equivalent.

[0730]

[0731]

[0732] ND = not determined

Embodiment 3

[0735] Example 3: Cellulase activity assay: BCA reducing end assay

[0736] The following examples describe an assay, a cellulase activity assay (BCA reducing end assay), for determining whether a polypeptide has the necessary enzymes (e.g., glucanase, mannanase, or xylanase) within the scope of the invention. Glycanase) activity, such as alkaline endoglucanase / cellulase activity (see Example 2 above).

[0737] This assay is designed to measure the number of reducing ends generated during enzymatic degradation of carboxymethylcellulose (CMC) in a high-throughput multi-sample 96-well format.

[0738] Material:

[0739] Substrate solution:

[0740] 1%CMC

[0741] Dissolve 1 gm of CMC in 100 ml of 50 mM Britton-Robinson buffer at pH ~ 4, heat the CMC solution in a boiling water bath while mixing, and proceed for 20-40 minutes until it dissolves (the solution will still appear slightly milky, but translucent ). Adjust to desired pH with 1M NaOH or HCl.

[0742] Solut...

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PUM

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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 at increased pH and temperature.

Description

[0001] This application is a divisional application, the filing date of the original application is July 02, 2004, the application number is 200480025009.2 (PCT / US2004 / 021492), and the title of the invention is "glucanase, nucleic acid encoding them and preparation and use of them Methods". [0002] View the sequence listing submitted on CD-ROM [0003] This application includes a compact disc (filed in quadruplicate) containing the Sequence Listing. The entire content of the Sequence Listing is incorporated herein by reference. The sequence listing is identified on the disc as follows: [0004] field of invention [0005] In general, the present invention relates to enzymes, polynucleotides encoding the enzymes, uses of the polynucleotides and polypeptides; more specifically, the present invention relates to polypeptides with glucanase, such as endoglucanase activity ( eg enzyme, antibody), endoglucanase activity eg catalyzing the hydrolysis of internal endo-β-1,4- and / ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/11C12Q1/68C12Q1/34A23K1/00A23K1/165A23K1/18C12NC12N9/24
CPCA23K1/1653G01N33/53C12N11/16A23C9/1322A23K1/002C12Y302/01004Y02E50/17C12N11/02C12P7/10C12N11/14A23K1/1813D21C1/00G01N33/573G01N2333/924C12N9/2437G01N2500/00A23K1/1656A23L1/3053C12N9/2405A23L2/52A23C19/04A61K38/47A23K10/14A23K20/189A23K40/10A23K50/10A23L33/18A61P1/14A61P31/00A61P31/04A61P33/02Y02E50/10A23V2002/00A61K9/0056A23L29/10C09K8/62C11D3/38636C12P19/02C12P19/18D21C5/005
Inventor B·斯蒂尔W·卡伦S·希利D·普利安
Owner BP CORP NORTH AMERICA INC
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