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Novel cellulase and uses thereof

A cellulase, cellulose technology, applied in the application, enzyme, enzyme and other directions, can solve problems such as inability to adapt

Inactive Publication Date: 2008-08-20
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current cellulase enzymes are far from being able to meet the needs of converting plant cellulose into simple sugars as a renewable energy source

Method used

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  • Novel cellulase and uses thereof
  • Novel cellulase and uses thereof
  • Novel cellulase and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Separation and purification of apple snail EG65

[0090] 1. Materials and Reagents

[0091] Apple snails are widely distributed in Fujian, Guangdong, Guangxi, Zhejiang, Jiangsu and other provinces in China. Apple snails purchased from Xiamen, Fujian were used as experimental materials. In the experiment, the chromatography media DEAE-Sepharose CL-6B, DEAE-Sepharose fastflow DEAE-Sephadex A-50, Phenyl-Sepharose CL-4B were purchased from Pharmacia AB (Sweden), and Bio-gel P-100 was purchased from Bio-Rad (USA) Company. The electrophoresis reagents acrylamide, methylene bisacrylamide and sodium dodecylsulfonate (SDS) were purchased from Pharmacia AB (Sweden), and protein molecular weight standards were purchased from Shanghai Sebastian Company. Bioassay reagents pNPC, Sigmacell 101, xylan (xylan from birchwood or oat spellt), CMC-Na (medium viscosity), starch (from potato) were purchased from Sigma Company; β-salicin, Avicel pH101 were purchased from Fluka Co...

Embodiment 2

[0107] Example 2: Activity assay of EG65

[0108] 1. Activity assay method

[0109] (1) Glycoprotein identification

[0110] Fluorescent hydrazide (DNS-Gly-NHNH2) was used to specifically label sugar chains on glycoproteins, according to W.Y.Liu, R.Q.Jiang in "The fluorescent labeling of glycoproteins on sodium dodecyl sulfate-polyacrylamide gel" (Bioorg.Chem.22(1994) 29-35) to identify glycoproteins.

[0111] (2) Determination of optimum pH, optimum temperature and pH stability and temperature stability

[0112] 1) Optimum pH: Take an appropriate amount of EG65 and add it to 200μl of different pH values, dissolve it in 100mMC at pH3.6-7.2 6 h 8 o 7 (citric acid) / Na 2 HPO 4 2H 2 The concentration of O was 1% (w / v) in CMC-Na, and the activity was measured according to the standard method.

[0113] 2) Optimum temperature: measure the activity according to the standard method within the range of 25°C to 70°C.

[0114] 3) pH stability: the EG65 enzyme was incubated at 50...

Embodiment 3

[0133] Embodiment 3: Cloning and expression of the gene of EG65 and its related genes

[0134] 1. Materials, Reagents and Methods

[0135] (1) Materials and reagents

[0136] 3-cyclohexylamino-1-propanesulfonic acid (CAPS) transfer buffer was purchased from Amresco; V8 proteolytic enzyme was purchased from Pierce; dithiothreitol (DTT) was purchased from Sigma; PVDF membrane was purchased from Millipore company; Trizol was purchased from Shanghai Sangong (Canada BioBasic Inc. imported packaging); reverse transcription reagents were purchased from Promega; , Pyrobest E, La-Taq E, etc.) and the carrier pMD18-T Vector were purchased from Takara; the gel recovery kit and Ponceau were purchased from Shanghai Huashun Bioengineering Co., Ltd.; the plasmid extraction kit was purchased from Broadtech; IPTG Purchased from BBI Company; X-gal was purchased from Takara Company; the peptone (Tryptone) and yeast extract (Yeast Extract) used in the cultivation of E.coli were purchased from O...

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Abstract

The invention relates to a new cellulose enzyme, a polynucleotide for encoding the enzyme and a method for producing the enzyme by DNA recombinant technology. The invention further relates to a vector containing the cellulose enzyme and a host cell, as well as the usage in the aspects of producing simple sugar and glucose.

Description

technical field [0001] The present invention relates to the field of biology. More specifically, the present invention relates to a novel cellulase, a polynucleotide encoding the enzyme and a method for producing the enzyme by DNA recombination technology. The invention also relates to vectors and host cells containing the cellulase and its use in the production of simple sugars and glucose. Background technique [0002] Cellulose is nature's most abundant renewable energy source. Bioconverting cellulose without any chemical treatment into simple sugar or glucose as a fermentative carbon source to produce ethanol is one of the most ideal and effective methods for utilizing natural cellulose resources. [0003] It is generally believed that the biotransformation of cellulose to produce glucose requires the synergy of at least three different enzymes: (1) endoglucanase (Endo-1, 4-β-D-glucanase, E.C.3.2.1.4, also known as for endocellulase). It acts on the non-crystalline r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12P19/02C12P19/12
Inventor 赵辅昆李燕红丁明袁泉许根俊
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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