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Novel heat-resisting glucoside hydrolase MtCel1 and coding sequence and application thereof

A technology of glycoside hydrolase and coding sequence, which is applied to thermostable glycoside hydrolase MtCel1 and its coding sequence and application field, and can solve the problem of not finding DUF4360 superfamily hydrolysis activity and the like

Inactive Publication Date: 2016-03-23
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 MtCel1 produced by Myceliophthora thermophila belongs to the DUF4360 superfamily protein and has strong cellulase activity, but there has never been any report on the hydrolytic activity of the DUF4360 superfamily

Method used

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  • Novel heat-resisting glucoside hydrolase MtCel1 and coding sequence and application thereof
  • Novel heat-resisting glucoside hydrolase MtCel1 and coding sequence and application thereof
  • Novel heat-resisting glucoside hydrolase MtCel1 and coding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Isolation and Identification of Myceliophthorathermophila Myceliophthorathermophila

[0018] (1) Specimen collection: collected from compost.

[0019] (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.

[0020] (3) Identification: Refer to the documents of Cooney and Emerson (1964) and LaTouche (1950).

Embodiment 2

[0021] Embodiment 2: Cloning of thermostable glycoside hydrolase gene MtCel1

[0022] (1) Extraction of total RNA from Myceliophthorathermophila: refer to the instructions of the Trizol kit.

[0023] (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 minutes, immediately cool it in an ice bath for 5 minutes, then centrifuge slightly, and add the following components in sequence in the ice bath: 10mmol / LdNTPMixture2μL, 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, put it 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 to ...

Embodiment approach 3

[0044] Embodiment 3: Construction of expression vector

[0045] (1) Design expression primers according to the nucleotide sequence of the isolated MtCel1 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:

[0046] Upstream primer: 5'-CCG GAATTC GCCGTCATCACTCCG-3' (EcorI)

[0047] Downstream primer: 5'-TT GCGGCCGC TCAGTGGTGGTGGTGGTGGTGGGAAGAGGAATCAC-3'(NotI) (histidine sequence)

[0048] (2) Extraction of total RNA from Myceliophthora thermophila: extraction with Trizol reagent.

[0049](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.

[0050] (4...

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Abstract

The invention provides novel thermally stable Myceliophthora thermophila glucoside hydrolase MtCel1 and a coding sequence and application thereof. An RT-PCR method is adopted to obtain a DUF4360 superfamily glycoside hydrolase gene MtCel1 from Myceliophthora thermosphacta, the gene is cloned and inserted into a pichia pastoris integrated expression vector pPIC9K, then the obtained glycoside hydrolase gene MtCel1 expression vector pPIC9K / MtCel1 is introduced into pichia pastoris GS115, and then a yeast engineering bacterium GS-MT-Cel1 expressing the glycoside hydrolase gene MtCel1 is screened out and expresses that the produced glycoside hydrolase MtCel1 has very high incision hydrolase activity to cellulose. The MtCel1 has good thermal stability and strong cellulose hydrolysis capability, can be widely applied to fiber waste and other industrial fields and has great economic value and social value.

Description

(1) Technical field [0001] The invention relates to bioengineering, in particular to a heat-resistant glycoside hydrolase MtCel1 expressed by the DUF4360 superfamily gene MtCel1 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 only carbon source. [0004] Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56
Inventor 李多川韩超陈宸
Owner SHANDONG AGRICULTURAL UNIVERSITY
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