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Multifunctional cellulase and application thereof

A cellulase, multi-functional technology, applied in the direction of application, enzymes, enzymes, etc., can solve the problems that cannot meet the needs of plant cellulose conversion into renewable energy

Inactive Publication Date: 2011-05-18
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

[0010] 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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  • Multifunctional cellulase and application thereof
  • Multifunctional cellulase and application thereof
  • Multifunctional cellulase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Purification of multifunctional cellulase EGX from gastric juice of apple snail

[0081] Apple snails are widely distributed in Fujian, Guangdong, Guangxi, Zhejiang, Jiangsu and other provinces in China. Apple snails were introduced from South America 20 years ago as food. They like to eat seedlings. Even if the water quality is poor, apple snails can multiply and grow, becoming rivers. and a scourge in rice fields. The apple snails purchased in the market were used as experimental materials.

[0082] (1) Extract gastric juice from apple snails and precipitate with 65% ammonium sulfate.

[0083] (2) Collect the precipitate, dissolve it with 10 mM phosphate buffer (containing 0.1 M sodium chloride), and dialyze. (3) Purify by DEAE-Sephadex A-50 column chromatography, collect the pNPC (p-Nitrophenylβ-D-cellobioside) hydrolysis activity peak, collect the obtained pNPC hydrolysis activity peak, purify it by molecular sieve column chromatography, and collect the pNPC hydro...

Embodiment 2

[0087] Determination of Enzyme Activity of Multifunctional Cellulase EGX from Apple Snail

[0088] 2.1 Determination of Hydrolytic Activity of Sodium Carboxymethyl Cellulose

[0089] 200μl 1% sodium carboxymethylcellulose (dissolved in 100mM pH4.8 sodium acetate buffer, containing 0.1M NaCl) preheated in a 50°C water bath for 10 minutes, added an appropriate amount of enzyme solution, reacted in a 50°C water bath for 10 minutes, added 0.5ml containing 40μg / ml Glucose DNS reagent, soak in boiling water for 6 minutes, immediately cool with cold water, add 0.5ml of double distilled water, and read the absorbance value at 540nm.

[0090] The hydrolytic activity of sodium carboxymethyl cellulose is defined as one unit (U) of the enzyme amount needed to hydrolyze 1 μmol of glucose reducing equivalent per minute under the above conditions.

[0091] 2.2 Determination of hydrolysis activity of xylan (Oat spelt)

[0092] 500μl 1% xylan (dissolved in 100mM pH4.8 sodium acetate buffer s...

Embodiment 3

[0103] Enzymatic properties of multifunctional cellulase EGX from apple snail

[0104] 3.1 Optimum pH value and pH stability of EGX

[0105] Take 1% soluble xylan, pNPC and sodium carboxymethyl cellulose respectively and mix them with 0.1M buffer solution (containing 0.1M NaCl) of different pH values, react with an appropriate amount of enzyme at 50°C for 10 minutes, and measure their activity.

[0106] The results showed that the optimum pH values ​​of EGX for the hydrolysis of substrates such as soluble xylan, pNPC and sodium carboxymethylcellulose were pH4.8-6.0, pH4.8-5.6 and pH4.4-4.8 ( figure 2 ).

[0107] The enzyme was incubated in 0.1M buffers with different pH values ​​(containing 0.1M NaCl) at 30°C for 24 hours, and then the residual activity of the enzyme activity was determined using pNPC as a substrate.

[0108] The results showed that the pH stability range of EGX was detected by pNPC hydrolysis activity, which showed higher stability in the range of pH5.0-11...

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Abstract

The invention provides novel multifunctional cellulase derived from Ampullaria crossean, polynucleotide for coding the enzyme, and a method for producing the enzyme by DNA recombination technology. The invention also discloses a vector and host cells containing the multifunctional cellulase, and the use in production of monosaccharides and glucose.

Description

technical field [0001] The present invention relates to the field of biology. More specifically, the present invention relates to a novel multifunctional cellulase, a polynucleotide encoding the enzyme and a method for producing the enzyme through DNA recombination technology. The invention also relates to a carrier and a host cell containing the multifunctional cellulase and its use in the production of simple sugars and glucose. Background technique [0002] Since the energy crisis in the 1970s, international oil prices have repeatedly hit new highs, and people have gradually begun to look for new renewable fuels to replace petroleum. Fuel ethanol is regarded as one of the most likely renewable energy sources to replace gasoline, and cellulosic ethanol The application prospect of fuel is widely optimistic. [0003] Plant dry matter is mainly composed of cellulose and hemicellulose. Cellulose is nature's most abundant renewable energy source. Bioconverting cellulose wit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12P19/14C12P19/12C12P19/02
Inventor 赵辅昆丁明许根俊
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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