Cellodextrin enzyme, encoding gene and application thereof
A technology encoding gene and dextrinase, applied in application, genetic engineering, plant gene improvement and other directions, can solve the problems of high cost, low efficiency and high price, and achieve the effect of high cellodextrinase activity
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Embodiment 1
[0039] Embodiment 1, the acquisition of cellodextrinase Umcel5C and its coding gene
[0040] 1. Construction of metagenomic library of buffalo rumen uncultured microorganisms
[0041] 1. Extraction of uncultured microbial metagenomics from buffalo rumen
[0042] Get 50g buffalo rumen contents (the source of the sample is the rumen of the buffalo just slaughtered by Nanning Meat Co., Ltd., and the collected sample is immediately preserved with liquid nitrogen), suspended in 200ml of 0.18M potassium phosphate buffer (pH6.5), Gently oscillate evenly, let stand for 10 minutes, let the cellulose particles in the rumen content automatically precipitate, discard the supernatant, and then add 200ml of 0.18M potassium phosphate buffer (pH6.5) to wash the sediment twice, and the last time Metagenomic DNA of microorganisms adsorbed to cellulose pellets was extracted by direct extraction from pellets. Add 100ml of extraction buffer (100mM sodium phosphate pH8.0; 100mM Tirs-HCl pH8.0; 10...
Embodiment 2
[0061] Embodiment 2, the expression of Umcel5C in escherichia coli
[0062] 1. Construction of a recombinant vector (pET-umcel5C) capable of expressing umcel5C
[0063] After the gene with coding signal peptide is expressed in vivo, the expression product is likely to be secreted outside the cell. In order to prevent the expression product from being secreted outside the cell, only the 306th to 1226th base of the 5' end of the artificially synthesized sequence 1 (that is, the first 25 amino acids of the amino terminal of the coding sequence 2 and the stop codon in the umcel5C gene are excluded. The DNA sequence is mainly to prevent the first 25 amino acids from forming a signal peptide), 921bp, the molecular weight of the encoded protein is estimated to be 35.39691KDa, and the isoelectric point pI is 4.65.
[0064] Artificially synthesized the entire sequence of the umcel5C gene except the first 25 amino acids at the amino terminal of the coding sequence 2 and the stop codon ...
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