F85-20 protein, coding gene thereof and application of F85-20 protein as beta-glucosidase
A protein and protein technology, applied in application, glycosylase, genetic engineering, etc., can solve problems such as restricting the efficient development of biofuel industry
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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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