F85-20 protein, coding gene thereof and application of F85-20 protein as beta-glucosidase
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A protein and protein technology, applied in application, glycosylase, genetic engineering, etc., can solve problems such as restricting the efficient development of biofuel industry
Active Publication Date: 2015-03-04
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
In order to achieve h...
Method used
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Embodiment 1
[0040] Cloning of embodiment 1, F85-20 gene
[0041] In the previous study, the metagenomic library of the dry fermented sludge system was constructed using the Fosmid vector. The ORF was predicted by sequencing and softberry, and the NCBI and Pfam databases were annotated. Obtain the F85-20 gene, the nucleotide sequence of this gene is the 19-1404th nucleotide from the 5' end of sequence 1 in the sequence listing, and the protein encoded by it is named F85-20, and its amino acid sequence is the sequence in the sequence listing 2 Amino acids 1-462 from the N' terminal. After comparison, the F85-20 protein has the highest 60% similarity with the β-glucosidase from bacterium UASB270, and it is predicted to be a β-glucosidase.
Embodiment 2
[0042] Embodiment 2, the application of F85-20 as β-glucosidase
[0043] 1. Construction of recombinant vector
[0044] Using the artificially synthesized sequence 1 as a template, using F85-20F and F85-20R as primers, and using TAKARA's PrimeStar high-fidelity enzyme to perform PCR amplification, a 1413bp PCR product was obtained, and its nucleotide sequence was sequence 1.
[0045] F85-20F:5'-CATG CCATGG AGATACAAATGAATAAACAT-3' contains Nco I restriction site
[0046] F85-20R:5'-CCC AAGCTT GGCATAAAGGGCCTCC-3' contains a Hind III restriction site
[0047] The above PCR amplification system is as follows:
[0048]
[0049]
[0050] The PCR program is as follows:
[0051] Pre-denaturation at 94°C for 1 min
[0052] 98°C 10s
[0053] 68°C 90s
[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-...
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Abstract
The invention discloses an F85-20 protein, a coding gene thereof and an application of the F85-20 protein as beta-glucosidase. 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 beta-glucosidase F85-20 can still retain 80% of activity after being incubated at 50 DEG C for two hours under the conditions that p-nitrophenol glucoside (pNPG) is taken as the substrate and the pH value is 6.0, and the optimum temperature is 65 DEG C; new beta-glucosidase F85-20 can retain more than 70% of residual activity in a pH value range of 4.0-7.5 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 a F85-20 protein, its coding gene and its application as β-glucosidase. 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. However, in the process of cellulase hydrolysis, a large amount of cellobiose and oligosaccharides produced will strongly inhibit the activity of cellulase, resulting in a sharp drop in cellulose hydrolysis efficiency and a reduction in the overall ...
Claims
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