A method for increasing the yield of maltogenic amylase
A technology of maltose amylase and Bacillus subtilis is applied in the field of increasing the yield of maltose amylase, and can solve the problems of lowering the final yield of maltose, lowering product purity and the like
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Embodiment 1
[0023] Embodiment 1: the construction of the recombinant bacterium producing maltogenic amylase
[0024] (1) Codon optimization is performed on the maltose amylase shown in SEQ ID NO.1, and the gene sequence amyMT of the maltose amylase is synthesized by a chemical total synthesis method. The pMA5 plasmid and the codon-optimized gene sequence were subjected to NcoI and HindIII double enzyme digestion respectively. After the digestion products were recovered by gel, they were ligated with T4 ligase overnight, and the ligated products were transformed into E. coli JM109 competent cells, and the transformed products were coated On the LB plate containing 100mg / L kanamycin, cultivate overnight at 37°C, pick a single colony, insert it into LB liquid medium, extract the plasmid after 8 hours for verification, the result is correct, and the enriched pMA5-amyMT plasmid is obtained . According to the same method as above, the maltogenic amylase with NCBI accession number: AAA22233.1 w...
Embodiment 2
[0026] Embodiment 2: Shake flask fermentation produces enzyme and the mensuration of maltogenic amylase enzyme activity
[0027] The recombinant Bacillus subtilis strain obtained in Example 1 was inoculated in LB medium, and after culturing at 37°C for 8-10 hours, it was transferred to TB fermentation medium with a 5% inoculation amount, and cultivated at 37°C and 200rpm for 2h, Then move to 33 ℃ constant temperature culture for 48h to produce enzyme. After the fermentation, the supernatant collected by centrifugation is the crude enzyme liquid.
[0028] Taking Bacillus subtilis pMA5-amyMT without signal peptide as a control, the enzyme activity of recombinant bacteria after introducing different signal peptides is shown in Table 1.
[0029] Table 1 The maltogenic amylase output of different recombinant bacteria
[0030]
Embodiment 3
[0031] Embodiment 3: Shake flask fermentation produces enzyme and the mensuration of maltose amylase enzyme activity
[0032] With the recombinant bacteria containing the pMA5-wapA-amyMT recombinant plasmid as the fermentation strain, in the fermentation medium (yeast extract 25g / L, soybean peptone 5g / L, soluble starch 5g / L, KH 2 PO 4 2.3g / L, K 2 HPO 4 16.4g / L, initial pH7.0), cultured at 200rpm40°C for 48 hours, the obtained fermentation broth was centrifuged at 12000rpm for 5min to remove the bacteria, the obtained fermentation supernatant was the crude enzyme liquid, and the enzyme activity of the obtained fermentation supernatant was 462.8U / mL.
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