	US 11,898,182 B2
	Method for preparing r-hydroxynitrile lyase and use thereof
Jinhui Cao, Jiujiang (CN); Kuang Zong, Jiujiang (CN); Hailiang Yu, Jiujiang (CN); Peng Zeng, Jiujiang (CN); Jianming Liu, Jiujiang (CN); and Wenhuan Chen, Jiujiang (CN)
Assigned to JIANGXI KEYUAN BIOPHARM CO., LTD., Jiujiang (CN)
Filed by JIANGXI KEYUAN BIOPHARM CO., LTD., Jiujiang (CN)
Filed on Jul. 27, 2022, as Appl. No. 17/815,447.
Claims priority of application No. 202110985869.6 (CN), filed on Aug. 26, 2021.
Prior Publication US 2023/0072514 A1, Mar. 9, 2023
Int. Cl. C12N 9/88 (2006.01); C12N 1/20 (2006.01); C12N 15/52 (2006.01); C12P 13/00 (2006.01)

CPC C12N 9/88 (2013.01) [C12N 1/20 (2013.01); C12N 15/52 (2013.01); C12P 13/004 (2013.01); C12Y 401/02046 (2013.01)]

4 Claims

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.