A recombinant yeast strain producing chondroitin sulfate abc lyase and its construction method and application
A technology of chondroitin sulfate and lyase, which is applied in the biological field, can solve the problems of increasing production costs and low enzyme activity of ChSaseABC, and achieve the effects of saving resources, reducing production costs, and simple operation
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Embodiment 1
[0065] Embodiment 1: Construction of recombinant Pichia pastoris GS115
[0066] Amplification of the chondroitin sulfate ABC lyase gene: the genome of Proteus vulgaris ATCC33420 was used as a template to amplify the chondroitin sulfate ABC lyase gene fragment. The PCR amplification system was 1 μL of genomic DNA, 4 μL each of primer 1 and primer 2, 50 μL of KOD polymerase, ddH 2 O 41 μL, PCR reaction program: 94°C pre-denaturation for 4 min, 94°C denaturation for 2 min; then annealing at 60°C for 30 s, extension at 72°C for 3 min, cycle 35 times; extension at 72°C for 1 min. The PCR product was subjected to gel electrophoresis and then gel-cut, and recovered with a column-type gel-tapping recovery kit to obtain a purified chondroitin sulfate ABC lyase gene fragment.
[0067] Table 1 Primers required for the construction of recombinant Pichia pastoris GS115
[0068]
[0069] Double digestion of chondroitin sulfate ABC lyase gene: the double digestion system is 10×QuickCut ...
Embodiment 2
[0073] Embodiment 2: the induced expression of chondroitin sulfate ABC lyase (see attached figure 2 )
[0074] The constructed recombinant Pichia pastoris GS115-pPIC9K-csl ABC was inoculated in YPD liquid medium and cultured overnight at 28°C; then transferred to 1L fermentation medium with 4% inoculum size, and fermented to OD 660 At 5 o'clock, centrifuge the fermentation broth under sterile conditions, discard the fermentation supernatant, resuspend the recombinant Pichia pastoris GS115-pPIC9K-csl ABC cell enzyme production medium, add 10g / L methanol (supplemented every day) to induce sulfate cartilage Secreted expression of ABC lyase, after 5 days of culture, the enzyme activity in the fermentation broth can reach 8U / mL.
Embodiment 3
[0075] Embodiment 3: the chondroitin sulfate ABC lyase enzyme activity assay of fermented liquid
[0076] Take 1mL of fermentation broth and centrifuge, take 0.1mL supernatant and 7.9mL 1g / L chondroitin sulfate A (prepared with 0.02mol / LTris-HCL, pH7.5) respectively, add them to a 15mL colorimetric tube, place at 28 React in a water bath at ℃ for 20 minutes, immediately place it in a boiling water bath and boil for 5 minutes, add inactivated fermentation broth supernatant to the control tube under the same conditions, and measure the light absorption value at 232 nm. The enzyme activity unit U is defined as the amount of enzyme required to catalyze the formation of 1 μmol of unsaturated disaccharide per minute at 28°C.
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