Beta-glucosidase Mut1b as well as expressed gene and application thereof

A glucosidase and gene technology, applied to β-glucosidase Mut1b and its expression genes and application fields, can solve the problems of low enzyme content and low activity, and achieve the effects of improving efficiency, important economic value and social benefits

Inactive Publication Date: 2012-06-27
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

β-glucosidase is an important member of the cellulase system of Trichoderma reesei, and the enzyme content is the least in the cellulase component, and the activity is generally low, so it becomes the bottleneck of cellulose enzymatic hydrolysis

Method used

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  • Beta-glucosidase Mut1b as well as expressed gene and application thereof
  • Beta-glucosidase Mut1b as well as expressed gene and application thereof
  • Beta-glucosidase Mut1b as well as expressed gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Gene isolation, site-directed mutagenesis, heterologous expression and purification of β-glucosidase Mut1b

[0019] (1) Inoculate 10 spores of Trichoderma reesei (Trichoderma reesei) in Trichoderma reesei MM medium 6 one, cultivated at 30°C for 2-3 days, collected mycelia, extracted RNA, and obtained cDNA by reverse transcription;

[0020] Trichoderma reesei MM medium components:

[0021] Peptone, 2g; Glucose, 20g; (NH 4 ) 2 SO 4 , 5g; K 2 HPO 4 , 15g with 1M NaOH to adjust the pH to 5.5, after the constant volume value is 1L, divide into 200ml per bottle, add 200×MgSO to each bottle after sterilization 4 (120g / l) 1ml; 200×CaCl 2 (120g / l) 1ml; 100× uridine (1M), 2ml; 1000× trace elements (FeSO 4 ·7H 2 O, 5g / l; MnSO 4 ·H 2 O, 1.6g / l; ZnSO 4 , 1.4g / l; CoCl 2 , 2g / l), 200 μl.

[0022] The RNA was extracted using the Trizol method, and the Trizol reagent was purchased from Invitrogen, and the steps were referred to in the product manual.

[0023] Fo...

Embodiment 2

[0043] Example 2 Properties of β-glucosidase Cel1b and Mutated β-glucosidase Mut1b

[0044] (1) Determination of enzyme activity of β-glucosidase Cel1b and mutated β-glucosidase Mut1b

[0045] 1) 5mM p-nitrophenyl-6-D-glucopyranoside (pNPG) was dissolved in 50mM citric acid buffer (pH6.0);

[0046] 2) The 200 μl reaction solution contains 50 μl of appropriately diluted enzyme solution, 100 μl of 50 mM HAc-NaAc buffer solution, 50 μl of substrate, and reacts at 45°C for 30 minutes;

[0047] 3) Add 10% Na 2 CO 3 50 μl to stop the reaction, measure the amount of p-nitrophenol produced at 420nm;

[0048] 4) With 10μM p-nitrophenol plus 10% Na in the same proportion 2 CO 3 Make a standard curve to calculate the amount of cellobiose and the unit of enzyme activity, and the conversion of 1 nmol of substrate per mg of protein per minute is defined as an enzyme activity unit (1U).

[0049] The results are shown in Table 1, and it was found that the specific enzyme activity of Mut...

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Abstract

The invention relates to beta-glucosidase Mut1b as well as an expressed gene and application thereof, which belong to the technical field of biological engineering. The invention provides gene mut1b for expressing the beta-glucosidase Mut1b, wherein the nucleotide sequence of the gene mut1b is shown as SEQ ID NO.1; and the invention also provides the beta-glucosidase Mut1b, wherein the amino acidsequence of the beta-glucosidase Mut1b is shown as SEQ ID NO.2. Mutant Mut1b with high hydrolyzing activity for the beta-glucosidase can be used for constructing a high-yield cellulase strain and a high-activity cellulase system in an industry, and the efficiency of enzymolysis of cellulose is effectively enhanced; and furthermore, the beta-glucosidase Mut1b is beneficial to the high-efficiency conversion of agricultural waste resources and has important economic value and social benefit.

Description

technical field [0001] The invention relates to beta-glucosidase Mut1b and its expression gene and application, belonging to the technical field of bioengineering. Background technique [0002] As the main component of the cell wall of higher plants, cellulose accounts for 35% to 50% of the dry weight of the plant. It is a linear polymer composed of β-1,4-glucosidic bonds connecting glucose residues. It is insoluble in water but can be absorbed by fiber. enzyme degradation. It is the most widely distributed and abundant carbohydrate on earth. For human beings, it is also the most abundant renewable resource on earth. According to statistics, the total annual output of lignocellulose accounts for about 50% of all biological resources, or about 10-50 billion tons. The use of cellulase to degrade lignocellulose to produce glucose, and then to ferment and produce bio-based products including ethanol has important practical significance for social and economic development. Du...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/63C12N1/21C12P19/14C12R1/885C12R1/19
Inventor 周庆新刘巍峰陈冠军徐金涛吕新星
Owner SHANDONG UNIV
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