Endo-alginate lyase and coding gene and application thereof
A technology for endo-cutting alginate and cutting alginate is applied in the field of bioengineering, and can solve the problems of narrow action temperature range, industrial application limitation, interference with algal oligosaccharide correlation analysis and the like
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Embodiment 1
[0034] Embodiment 1 Preparation of endo-alginate lyase
[0035] Proceed as follows:
[0036] Experimental Materials
[0037] Vibrio sp.MCCC 1A13243 (preserved by China Marine Microorganism Culture Collection Management Center, the preservation number is 1A13243, which can be purchased), Escherichia coli E.coli TOP10, Escherichia coli E.coil BL21 (DE3) (purchased from ThermoFisher Company), expression vector pET-22b (purchased from ThermoFisher Company); Saibaisheng bacterial genomic DNA extraction kit (purchased from Xiamen Jingju Company); DNA polymerase (purchased from Quanshijin Company); restriction endonuclease Nde I and Xho I (purchased from Quanshijin Company); T4 ligase (purchased from Takara Company); LB medium (containing 10 g of peptone per liter, 5 g of yeast extract, and 10 g of NaCl); binding buffer (1× PBS buffer: 10mM phosphate, pH7.2~7.4); washing buffer (500mM NaCl, 10~20mM imidazole, 20mM phosphate, pH 7.4); elution buffer (500mM NaCl, 50~500mM imidazole, ...
Embodiment 2
[0049] Enzymatic properties of endo-alginate lyase
[0050] (1) Alginate lyase activity assay method
[0051] The content of reducing sugar was determined by DNS method. Mix 50 μL of diluted enzyme solution with 200 μL of 0.3% sodium alginate substrate, and react at 35° C. for 30 min. After the reaction is completed, add 500 μL of DNS reagent, boil for 5 minutes, immediately cool in ice water, take the supernatant after short centrifugation, and measure the OD 540 Value (with inactivated enzyme solution as a control), the amount of reducing sugar and enzyme activity were calculated according to the standard curve. Definition of enzyme activity unit: Under the above-mentioned measurement conditions, the amount of enzyme needed to crack sodium alginate to produce 1 μmol reducing sugar per minute is defined as an enzyme activity unit (U).
[0052] (2) The action temperature of the enzyme
[0053] Measure the enzyme activity of the diluted enzyme solution in the range of 0-90°...
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