A high-temperature acidic β-mannanase man5dw1 and its gene and application
A mannanase and acidic technology, applied in the field of genetic engineering, can solve the problems of unsatisfactory application, poor thermal stability, low expression level, etc., and achieve the effect of good resistance to metal ions and good heat resistance
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Embodiment 1
[0056] Cloning of embodiment 1β-mannanase encoding gene man5DW1
[0057] Genomic DNA extraction
[0058] The bacteria cultured in liquid for 3 days were centrifuged at 12,000rpm for 10min, and the collected mycelium was added to a high-temperature sterilized mortar, and quickly ground to powder with liquid nitrogen, and then the ground bacteria were transferred to a new, packed Put 15ml of CTAB lysate in a 50mL centrifuge tube, mix it up and down gently, place it in a 70°C water bath for 3 hours, and mix it upside down and gently once every 20 minutes, so as to fully lyse the bacteria. Centrifuge at 12,000 rpm at 4°C for 10 min, pipette the supernatant into a new centrifuge tube, add an equal volume of chloroform for extraction, and place at room temperature for 5 min. Centrifuge at 12,000 rpm for 10 min at 4°C. Take the supernatant and add an equal volume of phenol / chloroform for extraction, and place it at room temperature for 5 minutes. Centrifuge at 12,000 rpm for 10 mi...
Embodiment 2
[0064] Example 2 Obtaining of β-mannanase cDNA
[0065] Total RNA was extracted using Oligo(dT) 20 and reverse transcriptase to obtain a strand of cDNA, then design primers F and R (see Table 1) for amplifying the open reading frame, amplify the single-stranded cDNA, obtain the cDNA sequence of mannanase, and amplify to obtain the product After recovery, they were sent to Ruibo Biotechnology Co., Ltd. for sequencing.
[0066] After comparing the genome sequence and cDNA sequence of mannanase, it is found that the gene contains an intron, the cDNA is 1122bp long, encodes 373 amino acids and a stop codon, and the N-terminal 15 amino acids are its signal peptide sequence , the comparison proves that the gene encoding mannanase isolated and cloned from Alternariasp. is a new gene.
Embodiment 3
[0067] The construction of embodiment 3 β-mannanase engineering strains
[0068] (1) Construction of expression vector and expression in yeast
[0069] Using the correctly sequenced mannanase Man5DW1 cDNA as a template, primers F and R with EcoRI and NotI restriction sites were designed and synthesized (see Table 1) to amplify the coding region of the mature protein of Man5DW1 . And use EcoRI and NotI to digest the PCR product, connect it into the expression vector pPIC9 (Invitrogen, SanDiego), and the sequence of the mature protein of β-mannanase Man5DW1 is inserted into the downstream of the signal peptide sequence of the above expression vector to form a correct sequence with the signal peptide. The reading frame was constructed into a yeast expression vector pPIC9-man5DW1, and transformed into Escherichia coli competent cell JM109. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-scale prepar...
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