Dual-enhanced acidic lactase Pichia pastoris expression strain, and construction method and fermentation technology thereof
A technology of acid lactase and Pichia pastoris, applied in the directions of fermentation, glycosylase, botanical equipment and methods, etc., can solve the problems of high cost, complicated production process, low unit yield of lactase, etc., and improve industrial production. The effect of improving the amount and expression
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Embodiment 1
[0037] Example 1 Construction and screening of inducible acid lactase Pichia pastoris expression strain
[0038]Synthesize the acid lactase integrated gene (ie Lac gene, refer to the gene with the sequence number XM_025613095 in the genebank of NCBI), and clone it into the PMD-18T vector. Inoculate a single colony of PMD-18T-Lac into 50ml LB medium for culture, and extract the plasmid. Inoculate a single colony of pPIC9K-DH5α into LB medium for culture, and extract the plasmid. According to the method steps of the plasmid extraction kit, the plasmids PMD-18T-Lac and pPIC9K were extracted. Plasmids pMD-18T-Lac and pPIC9K were cut with restriction endonucleases. The Lac fragment and the pPIC9K vector were recovered by tapping the rubber. Lac and pPIC9K were ligated with T4 DNA ligase. The ligation product was transformed into top10 k to be competent, spread on the LB Amp antibiotic screening plate, cultured overnight at 37°C, picked a single clone, and carried out colony PCR...
Embodiment 2
[0045] Example 2 Construction and screening of composition and induction double enhanced acid lactase Pichia pastoris expression strain
[0046] Synthetic ARS_OPT_BglII gene (the gene sequence refers to Liachko I, Dunham M J.Anautonomously replicating sequence for use in a wide range of budding yeasts [J]. FEMS Yeast Research, 2014, 14 (2), 364-367, page 365 2 paragraphs), cloned into the puc57 vector. Inoculate a single colony of puc57-ARS-top10 into 50mL LB medium for expansion, and extract the plasmid. ARS and pGAPZαA were ligated using a similar scheme as in Example 1, and transformed to obtain clones. Cultivate overnight at 37°C, pick a single colony, and perform colony PCR verification. Positive clones were inoculated with LLB (including ZEOCIN), and the extracted plasmids were sent to the sequencing company for sequencing verification. After the verification is correct, save Glycerol bacterium parsGAPzαA and store it at -70°C.
[0047] Then the plasmids parsGAPzαA a...
Embodiment 3
[0054] The fermentation process of the fermentor of the expression strain of embodiment 3 acid lactase recombinant Pichia pastoris
[0055] The glycerol bacterium of the recombinant yeast strain with the highest expression of enzyme activity screened in Example 1 and Example 2 was activated and cultured on YPD for 2-3 days until a single colony grew. Use an inoculation loop to pick a single colony into 50ml of YPD liquid medium for overnight culture on a shaking table (28°C, 245rpm), OD to 0.5-1; as a fermentation seed solution, use the flame inoculation method to inoculate. The fermentation parameters of the fermenter are set to PH4.0-6.5, the temperature is 25-32°C, and the speed, ventilation and feeding speed are constantly adjusted to maintain the dissolved oxygen at 0%-40%. Samples were taken every 12 hours after inoculation to determine lactase activity. Continue to culture until OD reaches 170-250. At this time, stop supplementing 50% glycerol, and start supplementing...
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