HIGH FIDELITY RESTRICTION ENDONUCLEASE

• Main Authors: XU SHUANG-YONG, GUAN SHENGXI, SUN DAPENG, HUANG YISHU, ZHU ZHENYU, ZHANG CHUNHUA, LAI XUHUI, AINE QUIMBY, LI XIANGHUI, CHAN SIU-HONG
• Format: Patent
• Language: English; Japanese
• Published: 18.01.2018
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; COMPOSITIONS THEREOF; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; CULTURE MEDIA; ENZYMOLOGY; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROCESSES OF PREPARING SUCH COMPOSITIONS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

PROBLEM TO BE SOLVED: To provide a method and composition provided for engineering a mutant enzyme with reduced star activity where the mutant enzyme has a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer.SOLUTION: According to the present invention, there are provided a composition comprising a restriction endonuclease variant in which one or more amino acids are mutated, and a method for identifying a fidelity index (FI) of a restriction endonuclease and a variant thereof by including a step of making the restriction endonuclease diluted sequentially and the variant thereof cut a DNA substrate. Preferably, the method further comprises comparing the FI for the restriction endonuclease and the variant thereof to obtain an improvement factor exceeding two-folds for the variant. In the method, a reaction buffer is potassium acetate, Tris acetate and magnesium acetate, or magnesium chloride.SELECTED DRAWING: Figure 1B 【課題】スター活性が低く、特定緩衝液中の忠実度指数（ＦＩ）が同一緩衝液中の非変異酵素のＦＩよりも高い変異体酵素を作製するための方法及び組成物の提供。【解決手段】１カ所以上のアミノ酸を変異させた制限エンドヌクレアーゼ変異体を含む組成物、及び順次希釈した制限エンドヌクレアーゼ又はその変異体にＤＮＡ基質を切断させる段階を含むことによって、制限エンドヌクレアーゼ及びその変異体の忠実度指数（ＦＩ）の同定方法。制限エンドヌクレアーゼとその変異体のＦＩを比較して、変異体の改善倍率を得て、改善倍率が２倍を上廻ることが好ましい、方法。反応バッファーが酢酸カリウム、トリス酢酸及び酢酸マグネシウム又は塩化マグネシウムである、方法。【選択図】図１Ｂ