Alginate lyase and application thereof
The disclosure discloses an alginate lyase and application thereof, and belongs to the technical field of biology. The alginate lyase provided by the disclosure has high degradation activity, and the enzyme activity reaches 65 U/mg; the alginate lyase is stable in nature, and the enzyme activity remains 98% or higher of the initial enzyme activity after storage at 4° C. for 18 months; and the alginate lyase has high product specificity. The disclosure uses E. coli as a host to express the alginate lyase derived from V. natriegens , the obtained recombinant E. coli can produce the alginate lyase secreted extracellularly in a conventional LB medium without adding an induction substrate sodium alginate, so the downstream processing technology of protein is simplified, and the disclosure has great industrial application potential.
1. A recombinant cell, wherein the recombinant cell expresses alginate lyase from Vibrio natriegens SK42.001, wherein the amino acid sequence of the alginate lyase is set forth in SEQ ID NO: 1.
2. The recombinant cell of claim 1 , wherein the recombinant cell is an Escherichia coli cell.
3. The recombinant cell of claim 2 , wherein the recombinant cell is Escherichia coli -BL21.
4. The recombinant cell of claim 3 , comprising a recombinant expression vector wherein the expression vector is pET-28a(+).
5. The recombinant cell of claim 1 , wherein the recombinant cell is Escherichia coli BL21 comprising pET-28a(+) as an expression vector and wherein the cell expresses a gene with the nucleotide sequence set forth in SEQ ID NO: 2 encoding the alginate lyase.
6. A method for producing the alginate lyase of claim 2 , comprising inoculating a seed solution of the recombinant cell of claim 2 into a medium, and after a period of incubation, adding IPTG to induce expression of the alginate lyase.
7. The method of claim 6 , wherein the seed solution is inoculated into an LB medium at an inoculum amount of 2-5%, and incubated at 35-370 C and 180-200 rpm until OD 600 is 0.6-0.8, and 0.5-1 mM IPTG is added to induce incubation at 15-180 C and 180-200 rpm for 45-48 h to obtain a fermentation broth.
8. The method of claim 7 , wherein the fermentation broth is centrifuged to remove thallus to obtain a crude enzyme preparation of the alginate lyase, wherein the crude enzyme preparation is subjected to nickel column affinity chromatography and dialysis to obtain a pure enzyme liquid, and wherein the pure enzyme liquid is freeze-dried to obtain an enzyme powder.
9. The method of claim 6 , wherein a single colony of the recombinant cell is picked into an LB medium containing kanamycin, and incubated to obtain a seed solution.
10. A method for producing the alginate lyase of claim 5 , lyase, comprising inoculating a seed solution of the recombinant cell of claim 5 into a medium, and after a period of incubation, adding IPTG to induce expression of the alginate lyase.
11. The method of claim 10 , wherein the seed solution is inoculated into an LB medium at an inoculum amount of 2-5%, and incubated at 35-370 C and 180-200 rpm until OD 600 is 0.6-0.8, and 0.5-1 mM IPTG is added to induce incubation at 15-180 C and 180-200 rpm for 45-48 h to obtain a fermentation broth.
12. The method of claim 11 , wherein the fermentation broth is centrifuged to remove thallus to obtain a crude enzyme preparation of the alginate lyase, wherein the crude enzyme preparation is subjected to nickel column affinity chromatography and dialyzed to obtain a pure enzyme liquid, and wherein the pure enzyme liquid is freeze-dried to obtain enzyme powder.
13. The method of claim 10 , wherein a single colony of the recombinant cell is picked into an LB medium containing kanamycin, and incubated to obtain a seed solution.