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Beta-glucosaccharase and application thereof

The technology of glucosidase and crude enzyme liquid is applied to beta-glucosidase and its application field, which can solve the problems of unclear enzymatic properties and biological functions, and achieve the effect of improving the efficiency of saccharification and hydrolysis

Active Publication Date: 2014-04-09
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cel3E is the only extracellular β-glucosidase that has not been deeply studied. So far, it has never been isolated from fungal extracellular crude enzyme solution, and its enzymatic properties and biological functions are not clear.

Method used

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  • Beta-glucosaccharase and application thereof
  • Beta-glucosaccharase and application thereof
  • Beta-glucosaccharase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: The method steps of separating and purifying β-glucosidase are:

[0021] (1). Prepare the spore suspension of Penicillium juniper, and inoculate the spore suspension in a container with a fermentation broth, the fermentation broth contains a mass percentage of 2%-8% carbon source, 0.1%-5% Organic nitrogen source, and 0.0001%-10% inorganic salt, pH value is adjusted between 3.0-5.0. Place the container on a shaker for shaking culture;

[0022] (2). The fermented liquid is centrifuged at a high speed to remove impurities and keep the soluble protein part of the supernatant. Retain protein through 10kDa membrane, concentrate protein by ultrafiltration;

[0023] (3). Put the concentrated protein on a molecular sieve gel chromatography column Sephadex S-200 to collect the samples;

[0024] (4) The new type of β-glucosidase was identified by protein mass spectrometry MALDI-TOF;

[0025] (5). Using the glucoside pNPG as a substrate, the β-glucosidase enzyme a...

Embodiment 2

[0031] Embodiment 2: the research of the property of β-glucosidase

[0032] 1) Using conventional methods in the field to combine β-glucosidase with carboxymethylcellulose (CMC-Na), salicin (Salicin), PNPC, microcrystalline cellulose (Avicel), xylan (Xylan), PNPG, and cellobiose respectively carried out the enzyme activity test results as shown in Table 1: PpCel3E to carboxymethylcellulose sodium (CMC-Na), salicin (Salicin), PNPC, microcrystalline cellulose (Avicel), Xylan has no enzymatic activity. For PNPG, cellobiose has higher enzyme activities of 31.0IU / mg and 14.4IU / mg respectively. PpCel3E has the ability to hydrolyze cellooligosaccharides, and can decompose cellotriose into cellobiose and glucose, with an enzyme activity of up to 125IU / mg.

[0033]

[0034] Table 1

[0035] 2) Detection of the affinity between the novel β-glucosidase PpCel3E and the substrate:

[0036] With p-nitrophenyl-β-D-glucoside (PNPG) as substrate, PpCel3E K m The value is 0.0019mM, whic...

Embodiment 3

[0042] Example 3: Application of PpCel3E in inducing cellulase-producing bacteria to synthesize cellulase or hemicellulase.

[0043] With 40 g / L glucose as substrate, 100 μg PpCel3E was added, and reacted in a water-bath shaker at 50° C. for 72 h. After the reaction, the type and concentration of the product were analyzed using an Agilent ZORBAX Carbohydrate sugar analysis column. The detection conditions were acetonitrile as the mobile phase, the flow rate was 1.0 mL / min, and the detection temperature was 30°C. The detection standard selections are: sophorose and gentiobiose.

[0044] With xylotriose as the substrate, 100 μg PpCel3E was added, and reacted in a water-bath shaker at 50° C. for 72 hours. Aminex HPX-87H sugar analysis column was used to analyze the type and concentration of the product. The detection conditions were 5mM sulfuric acid as the mobile phase, the flow rate was 0.6mL / min, and the detection temperature was 60°C.

[0045]

[0046] table 3

[0047] ...

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Abstract

The invention discloses a beta-glucosaccharase and application thereof. The beta-glucosaccharase PpCel3E is separated from a cellulase producing bacterium extracellular crude enzyme solution for the first time. The amino acid sequence of the beta-glucosaccharase is disclosed as SEQ ID NO.1. The nucleotide sequence of the expression gene of the beta-glucosaccharase is disclosed as SEQ ID NO.2. The beta-glucosaccharase PpCel3E has unique actions on inducing cellulase or hemicellulase synthesis and enhancing saccharification and hydrolysis efficiency of cellulase producing fungus extracellular cellulase.

Description

technical field [0001] The invention relates to the field of microbial application, in particular to a β-glucosidase and its application. Background technique [0002] The conversion of biomass to glucose often requires the synergistic action of at least three different enzyme proteins, exoglucanase (cellobiohydrolase, CBH), acting on the end of the cellulose linear structure, hydrolyzing β-1,4 The glycosidic bond, in turn, cleaves cellobiose from the end of the cellulose. It can act on both crystalline and amorphous regions of cellulose. (2) Endoglucanase (EG). This kind of enzyme acts on the amorphous region inside the cellulose structure, randomly hydrolyzes the β-1,4 glucosidic bond, shortens the long-chain cellulose, and produces a large amount of small molecule cellulose with non-reducing and reducing ends. The role of endoglucanases is to provide a large number of reactive ends for exonucleases. (3) β-glucosidase (BG). These enzymes hydrolyze cellobiose, or short...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12R1/885
CPCC12N9/2437C12N9/2445C12N9/2477C12Y302/01021
Inventor 高乐张东远陈树林
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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