A kind of genetically engineered bacteria with high yield of isoamylase and its fermentation process
A technology of genetically engineered bacteria and isoamylase, which is applied in genetic engineering, fermentation, plant gene improvement, etc., can solve problems such as no increase in expression, low yield of isoamylase, and influence of heterologous protein expression and secretion, and achieve the goal of solving enzyme low vigor effect
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Embodiment 1
[0037] Example 1: Construction of E.coli MDS42 (Tfu_1891 / pSX2)
[0038] 1. The Tfu_1891 / pT7-7 plasmid (application number 201110459137.X) constructed in the laboratory was used as a template to amplify the isoamylase Tfu_1891 gene (nucleotide sequence is SEQ ID NO.1) by PCR.
[0039] According to isoamylase Tfu_1891 and expression vector pSX2 gene design nucleotide sequence as primer Tfu_1891-F, Tfu_1891-R shown in SEQ ID NO.2, SEQID NO.3:
[0040] Tfu_1891-F: 5′- CATATG CCCATGGTGGAAGTCTG-3′contains NdeI restriction site
[0041] Tfu_1891-R: 5′- GGTACC TCAGGAGGGCAGGGCTTTC-3′contains KpnⅠ restriction site
[0042] Using the Tfu_1891 / pT7-7 plasmid as a template, the Tfu_1891 gene containing restriction sites was amplified by PCR. PCR system (50μL) is: ddH 2 034.5 μL, 4 μL dNTP mixture, 10 μL 5×PS buffer, 20 μM primers Tfu_1891-F and Tfu_1891-R 0.5 μL each, 1 μL PrimeStar polymerase, template 1 μL. PCR conditions: pre-denaturation at 94°C for 4min; denaturation at 98°C fo...
Embodiment 2
[0045] Embodiment 2 Shake flask fermentation produces enzyme
[0046] 1. Transfer the E.coli MDS42 (Tfu_1891 / pSX2) strain stored in a glycerol tube at -80°C to LB medium for 8-10 hours at 36-38°C, and then inoculate the obtained culture solution at an inoculum size of 4-6%. After culturing in TB fermentation medium at 36-38°C and 200r / min for 1.5-2.5h, induce with IPTG with a final concentration of 0.01-0.03mM, and continue culturing for 33-35h. The cell concentration was measured, and the 5OD fermented liquid was centrifuged by calculation to collect the bacteria. Suspend the cells with pH 5.520mM phosphate-citrate buffer, ultrasonically break, and centrifuge to measure the enzyme activity of the sample.
[0047] 2. Enzyme activity assay method: draw 71.4 μL of properly diluted broken-wall supernatant, add it into a 18 mm×18 cm test tube containing 429 μL of 0.833% amylopectin substrate (preheat at 50°C for 10 minutes), and incubate at 50°C for 30 minutes . After 30 minute...
Embodiment 3
[0049] The influence of different induction temperatures of embodiment 3 on the fermented enzyme of recombinant bacterium
[0050] 1. Seed culture: Inoculate the E.coli MDS42 (Tfu_1891 / pSX2) strain stored in a glycerol tube at -80°C into the seed medium, and cultivate it on a constant temperature shaker at a temperature of 37°C and a rotation speed of 200 rpm for 8 hours.
[0051] 2. Enzyme production by fermentation:
[0052] The seed solution was added to the fermentation medium with an inoculum of 8%. The dissolved oxygen was maintained at 30% by controlling the stirring speed and ventilation rate, the temperature was controlled at 37° C., and the pH was controlled at 7.0 by feeding 25% (v / v) ammonia water. After the initial glycerin is consumed and the dissolved oxygen rises to 80-100%, the batch fermentation culture ends. The feed medium was fed exponentially. When the cell concentration reached 23g / L, IPTG with a final concentration of 0.05mM was added once to induce ...
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