Patent ID: 11898182
Assignee: JIANGXI KEYUAN BIOPHARM CO., LTD.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method for preparing R-hydroxynitrile lyase, comprising:
mutating R-hydroxynitrile lyase wild-type gene, comprising: mutating the wild-type gene sequence of R-hydroxynitrile lyase set forth in SEQ ID NO. 1 to obtain a hydroxynitrile lyase mutant gene having the gene sequence set forth in SEQ ID NO. 2, wherein:
bases ATC at positions 325-327 of SEQ ID NO. 1 are substituted for bases ATG,
bases AAT at positions 328-330 of SEQ ID NO. 1 are substituted for bases AGT,
bases ATT at positions 961-963 of SEQ ID NO. 1 are substituted for bases ACT,
bases CCA at positions 1060-1062 of SEQ ID NO. 1 are substituted for bases ATA, and
bases TCT at positions 1063-1065 of SEQ ID NO. 1 are substituted for bases TTA;

adding an enzyme cleavage site, comprising: adding a double digestion site to the hydroxynitrile lyase mutant gene, wherein the double digestion site is Ndel/Hindllll;
preparing a recombinant plasmid, comprising: inserting the hydroxynitrile lyase mutant gene into an expression vector to obtain a recombinant plasmid, wherein the expression vector is pET26b(+), N-terminus of the expression vector is a signal peptide pelB leader having the gene sequence set forth in SEQ ID NO. 5;
introducing the recombinant plasmid into a strain, comprising: introducing the recombinant plasmid with the hydroxynitrile lyase mutant gene into the strain to obtain a recombinant expression strain, wherein the strain is E. coli BL21 (DE3); and
expressing and secreting the R-hydroxynitrile lyase in the strain, comprising: culturing the recombinant expression strain, and inducing the recombinant expression strain to express the enzyme of R-hydroxynitrile lyase in a culture medium and collecting a resulting enzyme liquid.