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Novel heat-resistant dual-function glucoside hydrolase MtCel2 and encoding sequence and application thereof

A technology of glycoside hydrolase and coding sequence, applied in thermostable bifunctional glycoside hydrolase MtCel2 and its coding sequence and application field

Inactive Publication Date: 2016-03-30
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The thermostable glycoside hydrolase MtCel2 produced by Myceliophthora thermophila belongs to the DUF4360 superfamily protein, which has strong cellulase activity and xylanase activity, but there has never been any report on the hydrolytic activity of the DUF4360 superfamily

Method used

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  • Novel heat-resistant dual-function glucoside hydrolase MtCel2 and encoding sequence and application thereof
  • Novel heat-resistant dual-function glucoside hydrolase MtCel2 and encoding sequence and application thereof
  • Novel heat-resistant dual-function glucoside hydrolase MtCel2 and encoding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Isolation and Identification of Myceliophthorathermophila Myceliophthorathermophila

[0021] (1) Specimen collection: collected from compost.

[0022] (2) Separation and cultivation: 0.5 g of the collected specimens were placed on a PDA plate and cultured at 50° C. for 3 days before separation and purification. Operation steps refer to Cooney and Emerson (1964) literature.

[0023] (3) Identification: Refer to the documents of Cooney and Emerson (1964) and LaTouche (1950).

Embodiment 2

[0024] Embodiment 2: Cloning of thermostable glycoside hydrolase gene MtCel2

[0025] (1) Extraction of total RNA from Myceliophthorathermophila: refer to the instructions of the Trizol kit.

[0026] (2) Synthesis of the first strand of cDNA: according to the instructions of the TaKaRaRNAPCRkit (AMV) Ver3.0 kit from Takara Company: take 1-2 μg of total RNA, add RNaseFreeddH 2 From 0 to 9.5 μL, denature the RNA sample at 75°C for 5 min, immediately cool it in an ice bath for 5 min, then centrifuge slightly, and add the following components in sequence in the ice bath: 10 mmol / LdNTP Mixture 2 μL, 10×RTBuffer (Mg 2+ )2μL, 25mmol / LMgCl 2 4 μL, Oligod (T)-Adaptor Primer 1 μL, RNase Inhibiter 0.5 μL, AMVReverse Transcriptase 1 μL (Final Volume 20 μL), after mixing the reaction solution, let it stand at room temperature for 10 minutes, then incubate at 42°C for 60 minutes, and then boil for 5 minutes to inactivate the reverse transcriptase. Add 180 μL DEPC-treated ddH 2 O, dilute ...

Embodiment approach 3

[0046] Embodiment 3: Construction of expression vector

[0047] (1) Design expression primers according to the nucleotide sequence of the isolated MtCel2 gene, respectively introduce EcorI and NotI restriction sites at the 5' end of the primers, and introduce 6 histidine sequences into the downstream primers as purification tags:

[0048] Upstream primer: 5'-CCG GAATTC GCGCCCACGAACACT-3' (EcorI)

[0049] Downstream primer: 5'-TT GCGGCCGC TAGTGGTGGTGGTGGTGGTGCTCGCACTTGCGCCAAG-3'(NotI) (histidine sequence)

[0050] (2) Extraction of total RNA from Myceliophthora thermophila: extraction with Trizol reagent.

[0051] (3) Synthesize the first strand of cDNA by reverse transcription: take 2 μg total RNA, add 4 μL of 5× reaction buffer, 2 μL of 10 mMdNTP, 0.5 μL of ribonuclease inhibitor (40-200u / μL), primer oligodT (1 μg / μL) 1 μL, 2 μL of reverse transcriptase (10u / μL), react at 42°C for 60 minutes, then stop the reaction at 85°C for 10 minutes, and dilute to 200 μL.

[0052] (...

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Abstract

The invention relates to novel and heat-stable heat-resistant dual-function glucoside hydrolase MtCel2 of Myceliophthora thermophila and an encoding sequence and application thereof. A DUF4360 superfamily glucoside hydrolase gene MtCel2 is obtained from the Myceliophthora thermophila through a RT-PCR method and is cloned and inserted into a pichia pastoris integrative expression vector pPIC9K; then the obtained glucoside hydrolase gene MtCel2 expression vector pPIC9K / MtCel2 is transferred into pichia pastoris GS115, engineering saccharomycetes GS-MT-Cel2 expressing the glucoside hydrolase gene MtCel2 screened out, and the glucoside hydrolase gene MtCel2 generated by expression of the engineering saccharomycetes GS-MT-Cel2 has high endonuclease hydrolase activity to hydrolase and xylan. MtCel2 has good heat stability and high cellulose hydrolysis capacity, can be widely applied to the field of fiber waste materials and other industrial fields and has significant economic value and social value.

Description

(1) Technical field [0001] The invention relates to bioengineering, in particular to a thermostable bifunctional glycoside hydrolase MtCel2 expressed by the DUF4360 superfamily gene MtCel2 of Myceliophthorathermophila and its coding sequence and application. (2) Background technology [0002] Glycoside hydrolases (English: Glycosidehydrolases, also known as glycosidases) are enzymes that specifically hydrolyze glycosidic bonds and produce two smaller sugar molecules, and are also one of the common enzymes in nature. This type of protein is also used in human industry, for example, to degrade biomass such as cellulose and hemicellulose into usable small molecules. There are several families of glycoside hydrolases. [0003] Myceliophthorathermophila is a thermophilic fungus widely distributed in soil. The bacterium can produce thermostable cellulase and glycoside hydrolase under the condition of 50 ℃ in the medium with microcrystalline cellulose as the sole carbon source. ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81
CPCC12N9/2437C12N9/248C12Y302/01004
Inventor 李多川韩超陈宸
Owner SHANDONG AGRICULTURAL UNIVERSITY
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