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Thermostable endo-beta-1,4-glucanase

A glucanase, endogenous technology, applied in the direction of glycosylase, enzyme, hydrolase, etc., can solve problems such as no further identification records

Inactive Publication Date: 2000-03-01
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, neither the enzyme nor the isolate has been further characterized (Bragger et al., 1989)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0194] A. Cloning of endo-β-1,4-glucanase from Pyrococcus furiosus DSM3638

[0195] A library derived from Pyrococcus furiosus DSM3638 was constructed and screened in E. coli as described in "Materials and Methods". A positive transformant isolated is DSM11203, which carries plasmid pSJ1678, which contains the DNA sequence encoding the cellulolytic enzyme of the present invention, namely SEQ ID NO:1.

[0196] B. Cloning of the Pyrococcus furiosus endo-β-1,4-glucanase gene from the deposited E. coli clone DSM 11203

[0197] According to the DNA sequence of SEQ ID NO: 1, a set of PCR primers was designed. These primers were designed to obtain a DNA sequence fragment encoding the most likely mature form of the endo-β-1,4-glucanase of the present invention. This fragment was cloned into the above vector pMB505 as a SacII-EagI fragment.

[0198] PCR was performed using the Expand High Fidelity PCR system (#1732650, BoehringerMannheim, GmbH). Use buffer 2 system, 200 μM each of ...

Embodiment 2

[0215] Purification and identification of Pyrococcus furious endo-beta-1,4-glucanase clone expressed in Example 2

[0216] Obtain the Bacillus 3600ml shake flask culture fluid containing the clone described in Example 1, adjust the 3200ml culture fluid to pH5.5 with HCl, then, at room temperature and under stirring, process with 150ml cationic coagulant, then use 300ml 0.1% Anionic coagulant solution for treatment. Agglomerated material was separated by centrifugation at 10,000 rpm for 30 minutes using a Sorval RC 3B. The supernatant was clarified using a Whatman F size glass filter. A total of 3400 ml was obtained with an activity of 8 CMCU / ml (activity measured at 90° C., pH 8.5).

[0217] The liquid was loaded into 3000 ml DEAE A-50 Sephadex equilibrated in 50 mM acetate buffer, pH 5.5. Unbound material contained a total of 18,000 CMCU.

[0218] Unbound material was concentrated to 1.4 L on a filter with a molecular weight cut-off of 10 kDa. Then, 2 parts of 400 ml liq...

Embodiment 3

[0231] Example 3 Biopolishing of cotton knitted fabrics with endo-β-1,4-glucanase of the present invention in a pad-steamer system

[0232] experimental method

[0233] Fabric and Solution Preparation: Bleached knitted cotton wool test fabric #460 (from TestFabrics Inc., Middlesex, NJ) was cut into approximately 20 x 30 cm pieces and placed in an artificial weather chamber (relative humidity 65 ± 2%, temperature 70 ± 3°F / 21+2°C) for at least 24 hours. All pieces of cloth were weighed to be 11.8-12.6g. Prepare a solution with buffer and enzyme so that it contains 0,2 CMCU / ml. Buffers were prepared by dissolving 1.5 g of tris(hydroxymethyl)aminomethane in 500 ml of deionized water and adjusting the pH to pH 8.9 and 7.3 with HCl. Endo-β-1,4-glucanase (MB473, #9730) prepared according to Examples 1 and 2 was used.

[0234] Padding: In a Mathis padding-steam unit (Werner Mathis USA Inc. Concord, NC), the cloth slabs were padded with an enzyme solution. Using the weight of the ...

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Abstract

An enzyme preparation having optimum endo- beta -1,4-glucanase activity at a temperature of at least 90 DEG C is obtainable from or endogeneous to a strain belonging to Archaea, e.g. an endo- beta -1,4-glucanase cloned from the species Pyrococcus furiosus, DSM 3638 and being encodable by the DNA sequence of SEQ ID NO:1.

Description

field of invention [0001] The present invention relates to an enzyme with cellulolytic activity at high temperature, in particular endo-beta-1,4-glucanase; a cloned DNA sequence encoding the enzyme with cellulolytic activity; A method for the gene of an enzyme; a method for producing the enzyme; an enzyme composition containing the enzyme having cellulolytic activity; and various industrial applications of the enzyme and the enzyme composition. Background of the invention [0002] Cellulases or cellulolytic enzymes are enzymes involved in the process of hydrolysis of cellulose. In the hydrolysis of natural cellulose, it is well known that there are three main types of cellulase involved in this process, namely cellobiohydrolase (1,4-β-D-glucan cellobiohydrolase, EC3.2.1. 91), endo-β-1,4-glucanase (endo-1,4-β-D-glucan 4-glucan hydrolase, EC3.2.1.4) and β-glucoside Enzymes (EC 3.2.1.21). [0003] In particular endo-beta-1,4-glucanases (EC 3.2.1.4) constitute an interesting ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C11D3/386C12H1/00C12N9/42C12R1/00C12R1/125D06M16/00D06P5/02
CPCD06M16/003C12H1/003C12Y302/01004C11D3/38645D06P5/02C12N9/2437
Inventor L·N·安德森M·E·布约恩瓦德M·舒莱恩
Owner NOVO NORDISK AS
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