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Positioning mutant gene of Trichoderma viride endoglucanase and application thereof

A kind of technology of endoglucanase, Trichoderma viride

Inactive Publication Date: 2012-10-17
林忠平
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese invention patent 201010173947.4 discloses a Trichoderma viride endoglucan gene egVII and its encoded amino acid sequence, without modification of the gene and without showing the advantage of enzyme activity

Method used

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  • Positioning mutant gene of Trichoderma viride endoglucanase and application thereof
  • Positioning mutant gene of Trichoderma viride endoglucanase and application thereof
  • Positioning mutant gene of Trichoderma viride endoglucanase and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The isolation and identification of embodiment 1 Trichoderma viride

[0032] 1. Materials and methods

[0033] 1 material

[0034] 1.1 Strains and plasmids

[0035] Escherichia coli (Escherichia coli) DH5α strain; plasmid pMD18T vector was purchased from Takara Company; Pichia pastoris (Pichia pastoris) GS115 and pPIC9K expression vectors were purchased from Invitrogen Company, and its plasmid map is as follows: Figure 9 shown.

[0036] 1.2 Reagents

[0037] Restriction endonuclease and T4-DNA ligase were purchased from TaKaRa Company; reverse transcriptase was purchased from Promega Company; TRIZol was purchased from Invitrogen Company; agarose gel purification and recovery kit, plasmid extraction kit, yeast DNA extraction kit Purchased from Biomed Company; PCR primer synthesis was completed by Sangon Company; DNA sequencing was completed by Biosune Company and Biomed Company.

[0038] 1.3 Medium

[0039] Trichoderma viride medium is CYM medium maltose 10g / L, gl...

Embodiment 2

[0044] Cloning of embodiment 2 Trichoderma viride endoglucanase gene

[0045] The endoglucanase gene (EG) of Trichoderma viride was cloned by RT-PCR.

[0046] Trichoderma viride total RNA was extracted by TRIzol reagent.

[0047] Synthesis of cDNA: Mix 7 μL of total RNA, 1 μL of Olig(dT)18, 2 μL of Nuclease-Free Water, bathe in 70°C water for 5 minutes, and after 10 minutes on ice, add 4 μL of M-MLV 5×Reation Buffer, 1 μL of M-MLV RT, dNTP 1 μL, Ribonuclease Inhibition 0.5 μL, Nuclease-Free Water 3.5 μL, mix well, 37 ℃ water bath for 1 hour, 70 ℃ water bath for 15 minutes to inactivate enzyme activity.

[0048] According to the endoglucanase sequence published by GenBank, design specific primers:

[0049] Upstream primer R1: 5′-ATGAACAGGACCATGGCTCCATTG-3′

[0050] Downstream primer F1: 5′-TTACTTCCTGGCGAGACACGAGCT-3′

[0051] PCR amplification of the target gene: 95°C 5min pre-denaturation; 95°C 30S, 55°C 30S, 72°C 1min30S, 30 cycles; 72°C extension 10min.

[0052] PCR ampli...

Embodiment 3

[0055] Example 3 Site-directed mutation of EG gene

[0056] Site-directed mutagenesis of EG gene was performed by overlapping primer extension method.

[0057] Design 2 mutant primers RM and FM, and use R1 and F1 as the full-length primers.

[0058] RM: 5′-GATCTTCGATCTCCACAAGTACTTAG-3′

[0059] FM: 5′-CTAAGTACTTGTGGAGATCGAAGATC-3′

[0060] The mutation site (base G at position 922 is changed to C, and amino acid at position 308 is changed from V to L) is included in the primer RM, and FM is the reverse complementary sequence of RM. Two rounds of PCR conditions for the whole mutation process: 95°C 5min pre-denaturation; 95°C 30S, 55°C 30S, 72°C 1min 30 cycles; 72°C extension 10min. The first-round PCR amplification system is the same as 1.2.2; the second-round PCR amplification system: Pyrobest DNA Polymerase 0.5 μL, 10×PyrobestBuffer II 5 μL, dNTP 4 μL, upstream primer 2 μL, downstream primer 2 μL, substrate 4 μL, ddH2O 32.5 μL .

[0061] The obtained EG gene was subjecte...

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Abstract

The invention relates to the field of gene engineering, and specially relates to a positioning mutant gene of Trichoderma viride endoglucanase, wherein the nucleotide sequence of the mutant gene is shown in SEQ ID NO : 3. The Trichoderma viride endoglucanase gene is subjected to a site-directed mutagenesis to get the positioning mutant gene of the Trichoderma viride endoglucanase, named as EG-mut and having a nucleotide sequence shown in SEQ ID NO : 3. Pichia yeast secretory-type expression vectors pPIC9K-EG and pPIC9K-EG-mut are constructed respectively by using the Trichoderma viride endoglucanase gene (EG) and the positioning mutant gene of Trichoderma viride endoglucanase (EG-mut) and are respectively transformed into the pichia yeasts to get recombinant pichia yeasts which are induced to express, and the enzyme activities are determined. The result shows that the enzyme activity of exocytosis endoglucanase of the recombinant pichia yeast strain with the mutant sequence EG-mut is increased by 15.27% compared with that of recombinant pichia yeast strain with the original sequence EG.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a Trichoderma viride endoglucanase gene and its application in producing cellulase endoglucanase. Background technique [0002] Cellulose resources are the most abundant renewable resources on the earth. Effective use of cellulose resources can alleviate the increasingly prominent food and energy shortages worldwide. Cellulase passes through three components in cellulase: endoglucanase (EG), exoglucanase (cellubiohydralases, CBH), and β-glucosidases (BG ) synergistically hydrolyzes cellulose to glucose. Among them, endoglucanase is the most important component of the cellulase system. It first acts on cellulose, randomly hydrolyzes the glycosidic bonds inside the cellulose molecule, and breaks the cellulose chain. It degrades the entire cellulose play a key role. [0003] Trichoderma viride is one of the strains with the highest cellulase activity. It is widely distributed i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/63C12N15/81
Inventor 林忠平杜娟胡鸢雷徐志超陆晓菡
Owner 林忠平
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