Construction method for co-expression hemoglobin VHb and cellulase protein in pichia pastoris

A Pichia pastoris and hemoglobin technology, applied in the field of recombinant Pichia pastoris system construction, can solve the problems of low enzymatic hydrolysis efficiency, lack of exonuclease, core enzyme components, etc. The effect of industrial production potential, improving enzyme production efficiency, and improving expression efficiency

Inactive Publication Date: 2016-03-23
JIANGNAN UNIV
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Problems solved by technology

However, these self-produced enzymes are born with certain proportion defects. As far as Trichoderma reesei, the most widely used cellulase-producing fungus in industry, its cellulase system lacks exonuclease CBHII and β- Glucosidase BG, resulting in low enzymatic hydrolysis efficiency of its cellulase system
In addition, whether it is a self-produced enzyme system derived from organisms or an artificially produced commercial cellulase system, there are as many as 80 or more proteins contained in it, resulting in a decrease in the specific enzyme activity of the core enzyme components, which is also It is also the reason for the low efficiency of enzymatic hydrolysis of cellulase
More importantly, the proportions of cellulose, hemicellulose and lignin in different lignocellulosic materials are significantly different, resulting in the same type of commercial cellulase preparations or self-produced enzymes not being able to exert the best enzymatic hydrolysis effect

Method used

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  • Construction method for co-expression hemoglobin VHb and cellulase protein in pichia pastoris
  • Construction method for co-expression hemoglobin VHb and cellulase protein in pichia pastoris
  • Construction method for co-expression hemoglobin VHb and cellulase protein in pichia pastoris

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Example 1: Trichoderma reesei endoglucanase II (EGII) gene codon optimization and expression vector construction.

[0063] 1, the present invention utilizes GeneDesigner (DNA2.0, MenloPark, CA, USA) to realize as shown in SEQIDNO.1 EGII (GenBankAccessionNo.DQ178347.1) nucleotide sequence codon bias optimization, optimized nucleotide sequence such as SEQIDNO .3 shown. The codon-optimized amino acid sequence is consistent with the original amino acid sequence, as shown in SEQ ID NO.2. Using Pichiapastoris GS115 as the host, the nucleotide sequence after codon bias optimization and the original nucleotide sequence are compared, see figure 2 , compare the amino acid sequence after codon bias optimization with the original amino acid sequence, see image 3 .

[0064] 2. With EcoRI as the 5'-end restriction site and NotI as the double-restriction site as the 3'-end restriction site, insert the pPIC9K plasmid vector downstream of the AOX1 promoter to construct the expressi...

Embodiment 2

[0065] Example 2: Linearizing expression vectors and performing electrotransformation and screening high-yielding strains.

[0066] 1. Use SacI as the linearization site of the pPIC9K-eg2 expression vector to realize the linearization of the expression vector, and verify the linearized plasmid by nucleic acid electrophoresis, see Figure 5 . Transform into Pichiapastoris GS115 by electroporation to obtain recombinant strains, use geneticin G-418 concentration gradient to screen multi-copy transformants, and obtain high-yield strains by shake-flask fermentation of colonies that can grow normally on high-concentration resistance plates. Determination of CMC enzyme activity in fermentation supernatant by DNS method, see Figure 6 , wherein, 1 unit of enzyme activity is defined as the amount of enzyme that can convert 1 μmol of substrate in 1 minute under the condition of 50°C water bath.

Embodiment 3

[0067] Example 3: Acquisition and expression vector construction of Vitella hyaline hemoglobin (VHb) gene.

[0068] 1. Obtain the Vitiligo hyaline hemoglobin gene (GenBankAccessionNo.M30794.1) from NCBI, add an EcoRI restriction site at the 5' end, add a NotI restriction site at the 3' end, and obtain the target gene by artificial synthesis.

[0069] 2. With EcoRI as the 5'-end restriction site and NotI as the 3'-end restriction site as the double restriction site, insert the pPICZαA plasmid vector downstream of the AOX1 promoter to construct the expression vector pPICZαA-vgb. See the expression vector map Figure 7 . The expression vector was verified by double enzyme digestion to confirm that the expression vector pPICZαA-vgb was successfully constructed. See the nucleic acid electrophoresis diagram after enzyme digestion Figure 8 .

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Abstract

The invention relates to a construction method for co-expression hemoglobin VHb and cellulase protein in pichia pastoris, in particular to cellulase protein gene codon optimization and construction of a cellulase protein and vitreoscilla hemoglobin protein (VHb)-co-expressed pichia pastoris system. According to the construction method, codon bias optimization is performed on the nucleotide sequence of EG II (GenBank Accession No.DQ178347.1) through Gene Designer (DNA2.0, Menlo Park, CA, USA) software, a pPIC9K-eg2 expression vector is constructed, and a recombined pichia pastoris strain is obtained by taking Pichiapastoris GS115 as a host through electrotransformation. In addition, the nucleotide sequence of the VHb (GenBank Accession No.M30794.1) is obtained from NCBI and artificially synthesized into a gene, then a pPICZalphaA-vgb expression vector is constructed, and the co-expressed pichia pastoris strain is obtained by taking the recombined pichia pastoris strain containing the EG II gene as a host through electrotransformation. Detection shows that the co-expressed strain is improved on the aspects of bacterial concentration growth and enzyme activity, wherein the OD600 value is increased by 7.2%, and the enzyme activity is improved by 2.2%.

Description

technical field [0001] The invention relates to the construction of a recombinant Pichia pastoris system co-expressing Vitella hyaline hemoglobin gene (vgb) and cellulase protein, and belongs to the technical field of bioengineering. Background technique [0002] Cellulase is a general term for a class of multicomponent enzymes, which can hydrolyze cellulose, which is difficult to be bioavailable, into glucose, which is convenient for bioavailability, so it is considered to be one of the enzyme preparations with great potential. Cellulase belongs to glycoside hydrolase, and the cellulase system capable of degrading cellulose includes at least three types of components, namely exoglucanase (CBH), endoglucanase (EG) and β-glucoside enzyme (BG). Among them, EG acts on the non-crystalline region of cellulose, hydrolyzes the β-1,4 glycosidic bond, and can truncate the linear cellulose polymer to generate a large number of small cellulose molecules; CBH hydrolyzes the 1,4-β-D-gly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12R1/84
Inventor 孙付保白仁惠张震宇许银彪王春迪
Owner JIANGNAN UNIV
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