f175-3 protein and its coding gene and application of hydrolyzed cellulose
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A technology of cellulase and protein, applied in the fields of application, hydrolase, genetic engineering, etc., can solve the problems that limit the efficient development of the biofuel industry
Active Publication Date: 2017-05-03
PEKING UNIV
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Problems solved by technology
This limits the efficient development of the biofuel industry and is also an important factor restricting the reduction of production costs of biofuels
Method used
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Embodiment 1
[0041] Cloning of embodiment 1, F175-3 gene
[0042] In the previous study, the metagenomic library of the dry fermented sludge system was constructed using the Fosmid vector. Using the Congo red-cellulose plate, an E. coli host clone containing cellulase activity was screened. The insert fragment contained in Fosmid is 37655bp, the ORF is predicted by softberry, and annotated by NCBI and Pfam database. Obtain the F175-3 gene, the nucleotide sequence of this gene is the 7-1293 nucleotides from the 5' end of sequence 1 in the sequence listing, the protein encoded by it is named F175-3, and its amino acid sequence is the sequence in the sequence listing 2 Amino acids 1-429 from the N' terminal. After comparison, the F175-3 protein had the highest similarity of 59% with the cellulase from uncultured bacteria, and it was predicted to be a cellulase.
Embodiment 2
[0043] Embodiment 2, the application of F175-3 as cellulase
[0044] 1. Construction of recombinant vector
[0045] Using the artificially synthesized sequence 1 as a template, using F175-3F and F175-3R as primers, and using TAKARA's PrimeStar high-fidelity enzyme to perform PCR amplification, a 1302bp PCR product was obtained, and its nucleotide sequence was sequence 1.
[0046] F175-3F:5'-CATG CCATGG CGAAAACCGGCGATCAG-3' contains Nco I restriction site
[0047] F175-3R:5'-CCC AAGCTT CTGGGCATATCTGATCATCG-3' contains Hind III restriction site
[0048] The above PCR amplification system is as follows:
[0049]
[0050] The PCR program is as follows:
[0051] Pre-denaturation at 94°C for 1 min
[0052] 98°C 10s
[0053] 68℃ 80s
[0054] Back to 2, 24 cycles
[0055] 72℃ 5min
[0056] end
[0057] The above PCR product was double digested with Nco I and Hind III, and the resulting digested product was ligated with the pET-28a vector (Novagen pET-28a DNA Cat. No. 6...
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Abstract
The invention discloses an F175-3 protein, a coding gene thereof and an application of the F175-3 protein to hydrolyzing cellulose. The invention provides the protein shown in the following 1) or 2): 1) a protein formed by amino acid residues shown in a sequence 2 in a sequence table; 2) a protein which is obtained by carrying out substitution and / or deletion and / or addition of one or more amino acid residues on an amino acid residue sequence of the sequence 2 in the sequence table, has the same functions as the protein shown in 1) and is derived from the protein shown in 1). Experiments prove that new cellulase F175-3 can still retain 88.5% of activity after being incubated at an optimum temperature of 60 DEG C for two hours under the conditions that cellulose is taken as the substrate and the pH value is 5.0; the new cellulase F175-3 can retain more than 80% of residual activity in a pH value range of 3.5-8.0 after being incubated in buffer solutions with different pH values for 24 hours at 4 DEG C.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to the application of F175-3 protein, its coding gene and hydrolyzed cellulose. Background technique [0002] According to statistics, plants around the world synthesize 5 billion tons of fibrous substances through photosynthesis every year. Cellulose is an important component of plant cell walls and the most abundant renewable resource in nature. Cellulase is a class of enzymes that can hydrolyze cellulose into oligoglucose and glucose. Many natural microorganisms contain cellulase. Because cellulase can be widely used in textile, paper, food and biofuel industries, it has attracted more and more research interests of scholars in recent years. In order to achieve high heat resistance and thermal stability of enzymes, many scholars screen new enzymes from thermophilic microorganisms, but they are rarely applicable to industrial conditions. In the biofuel industry, that is, the hydrol...
Claims
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