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Method for screening beta-glucosaccharase gene from mildewed sugarcane leaves based on metagenomic technology

A technology of glucosidase and screening method, which is applied in the field of beta-glucosidase gene and recombinant beta-glucosidase, and can solve the problems of sensitivity and low activity of hydrolyzed products.

Pending Publication Date: 2016-04-06
江西省农业科学院农业应用微生物研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The present invention also provides a screening method for recombinant β-glucosidase, which solves the problems in the prior art that the activity of the enzyme is not high under alkaline conditions and is very sensitive to hydrolyzed products

Method used

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  • Method for screening beta-glucosaccharase gene from mildewed sugarcane leaves based on metagenomic technology
  • Method for screening beta-glucosaccharase gene from mildewed sugarcane leaves based on metagenomic technology
  • Method for screening beta-glucosaccharase gene from mildewed sugarcane leaves based on metagenomic technology

Examples

Experimental program
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Embodiment 1

[0042] Example 1 Establishment of metagenomic library and acquisition of positive clones, gene cloning and expression

[0043] 1) Extraction of microbial metagenomic DNA from moldy sugarcane leaves: Collect 10 g of moldy sugarcane leaves, cut them to a length of 1-3 cm, and put them into a 250 ml Erlenmeyer flask. Add enrichment medium (g / L: sodium carboxymethylcellulose, 5; NaNO 3 ,2;K 2 PO 3 ,1; KCl,0.5; MgSO 4 ,0.5; FeSO 4 , 0.01.) 100ml, cultured with shaking at 30°C for 72h. The bacteria were collected by centrifugation, and the bacteria were resuspended in 1.35ml DNA extraction buffer (100mM Tris-HCl[pH8.0], 100mM EDTA-Na 2 [pH8.0], 100mM phosphate buffer [pH8.0], 1.5MNaCl, 1% CTAB), mix well, add 0.15ml 20% SDS, bathe in 65℃ water for 2 hours, during this period, turn it upside down 10 times every 20min and freeze it in liquid nitrogen 2min; centrifuge at 6000g for 10min, collect the supernatant, add equal v of chloroform, mix up and down; centrifuge at 6000g for ...

Embodiment 2

[0065] The study of the enzymatic properties of embodiment 2 recombinant β-glucosidase

[0066] Take 100μl crude enzyme solution, add 100μlpNPG, bathe in water at 35℃ for 15min, add 100μl0.5M Na2CO 3 , while using the inactivated crude enzyme solution as a control, measure the light absorbance at 405nm.

[0067] 1) Optimum reaction temperature and temperature stability of recombinant β-glucosidase Glu417

[0068] Under the condition of reaction temperature of 20-60° C., the enzyme activity was measured according to the above method, and the optimum reaction temperature was obtained (recorded as 100% when the enzyme activity was the highest). Incubate at 25, 30, 35, 40, 45, 50, 55, 60 and 65°C for 2 hours, measure the remaining enzyme activity at 35°C, and obtain the thermal stability of the enzyme. The result is as figure 2 with 3 As shown, the optimum reaction temperature of Glu417 is 35°C. When the temperature is lower than 35°C, Glu417 is stable. As the temperature rises...

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Abstract

The invention discloses a beta-glucosaccharase gene, wherein the nucleotide sequence of the beta-glucosaccharase gene is shown as the SEQ ID NO.1. The invention further discloses recombinant beta-glucosaccharase, wherein the amino acid sequence of the recombinant beta-glucosaccharase is shown as the SEQ ID NO.2. The invention also discloses a screening method of the recombinant beta-glucosaccharase. The screening method comprises the following steps: extraction of microbial metagenomic DNA from the mildewed sugarcane leaves; establishment of a metagenomic library; screening of the beta-glucosaccharase gene from the metagenomic library, wherein the nucleotide sequence of the beta-glucosaccharase gene is shown as the SEQ ID NO.1; and cloning and expression for the obtained beta-glucosaccharase gene, thus the recombinant beta-glucosaccharase is obtained, wherein the amino acid sequence of the recombinant beta-glucosaccharase is shown as the SEQ ID NO.2. The recombinant beta-glucosaccharase has extremely high activity and is not sensitive to hydrolysis products under the alkaline condition.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a β-glucosidase gene, and also relates to a recombinant β-glucosidase, and a method for the recombinant β-glucosidase. Background technique [0002] With the increasing consumption rate of fossil fuels and the aggravation of environmental and resource crises, the demand and development of alternative energy biomass energy is becoming increasingly urgent. Cellulose, as the main component of plant cell walls, is the most widely distributed and abundant carbohydrate on the earth. It is the most suitable raw material for the production of new energy such as bioethanol or other liquid fuels, which can effectively alleviate the energy crisis and environmental crisis. , to promote the sustainable development of the global economy. [0003] There are a variety of biological systems that efficiently degrade cellulose in nature, and using lignocellulolytic enzymes p...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/10C12Q1/68C12Q1/34
Inventor 姚健陈庆隆张诚钟国祥桂伦王洪秀马吉平陈柳萌
Owner 江西省农业科学院农业应用微生物研究所
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