Glycolate oxidase (GO) isozyme with high specific activity (75.0～279.0 U/mg) is purified quickly on DEAE- Cellulose column from Brassica parachinensis Bailey. Its pI is greater than 10.0 assayed by acetate cellulose ...Glycolate oxidase (GO) isozyme with high specific activity (75.0～279.0 U/mg) is purified quickly on DEAE- Cellulose column from Brassica parachinensis Bailey. Its pI is greater than 10.0 assayed by acetate cellulose membrane electrophoresis for 1 hour. In view of about ten kinds of pI varied from 4.5 to 10.0 are observed when the same GO isozyme is assayed in IEF for 14 hours, it is obvious that its pI decreases in IEF. Its pI also decreases when this GO isozyme is assayed in PAGE for 14 hours. Based on the results in SDS-PAGE, CGE-SDS, and IEF, it is most likely that this GO isozyme comprises two noncovalently associated 66 kD basic subunit and 40 kD acidic subunit, the phenomenon of pI change is related to subunit dissociation. The basic/acidic amino acid residues ratios in GO isozyme and its 40 kD acidic subunit are detected to be 0.66 and 0.54, respectively, a value much lower than that (0.96) in 40 kD peptide encoded by GO cDNA reported previously, indicating neither M r nor charge characteristic of this 40 kD peptide is similar to that of GO isozyme subunits, two subunits of GO isozyme may be the modified products of the same GO gene after post-translation.展开更多
对来自新疆天山冻土的Rahnella sp．R3所产的胞内低温β-半乳糖苷酶进行纯化，并对其酶学性质进行研究。采用硫酸铵分级沉淀、Phenyl Sepharose CL-4B疏水层析、QSepharose High Performance阴离子交换层析、Sephacryl S-200 High Resol...对来自新疆天山冻土的Rahnella sp．R3所产的胞内低温β-半乳糖苷酶进行纯化，并对其酶学性质进行研究。采用硫酸铵分级沉淀、Phenyl Sepharose CL-4B疏水层析、QSepharose High Performance阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析，得到电泳纯酶。酶的活性回收率为21．3％，纯化倍数为35．6，比酶活由1．28U／mg提高到45．54U／mg。SDS—PAGE电泳显示其表观分子量为57．3kDa。酶学性质研究表明，该酶最适反应温度为45℃，在15℃时的酶活为最高酶活的40％，4℃时的酶活为最高酶活的23％。该酶对热敏感，45℃保温45min酶活全部丧失。纯酶的最适PH为7．0，在pH6．5～7．5时保持稳定。5mmol／LNa^＋、Ca^2＋、Cu^2＋、Al^3＋、Zn^2＋对酶活力有不同程度的抑制作用，其中Al^3＋抑制K^＋作用最强，Na^＋、Ca^2＋抑制作用不明显。5mmol／LMg^2＋、K^＋对酶活力具有促进作用，其中Mg^2＋促进作用较强，使酶活提高到1．19倍。25℃以ONPG为底物的Vmax，Km值分别为7．19mol／（min·mL）、4．64mmol／L。展开更多
文摘Glycolate oxidase (GO) isozyme with high specific activity (75.0～279.0 U/mg) is purified quickly on DEAE- Cellulose column from Brassica parachinensis Bailey. Its pI is greater than 10.0 assayed by acetate cellulose membrane electrophoresis for 1 hour. In view of about ten kinds of pI varied from 4.5 to 10.0 are observed when the same GO isozyme is assayed in IEF for 14 hours, it is obvious that its pI decreases in IEF. Its pI also decreases when this GO isozyme is assayed in PAGE for 14 hours. Based on the results in SDS-PAGE, CGE-SDS, and IEF, it is most likely that this GO isozyme comprises two noncovalently associated 66 kD basic subunit and 40 kD acidic subunit, the phenomenon of pI change is related to subunit dissociation. The basic/acidic amino acid residues ratios in GO isozyme and its 40 kD acidic subunit are detected to be 0.66 and 0.54, respectively, a value much lower than that (0.96) in 40 kD peptide encoded by GO cDNA reported previously, indicating neither M r nor charge characteristic of this 40 kD peptide is similar to that of GO isozyme subunits, two subunits of GO isozyme may be the modified products of the same GO gene after post-translation.
文摘对来自新疆天山冻土的Rahnella sp．R3所产的胞内低温β-半乳糖苷酶进行纯化，并对其酶学性质进行研究。采用硫酸铵分级沉淀、Phenyl Sepharose CL-4B疏水层析、QSepharose High Performance阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析，得到电泳纯酶。酶的活性回收率为21．3％，纯化倍数为35．6，比酶活由1．28U／mg提高到45．54U／mg。SDS—PAGE电泳显示其表观分子量为57．3kDa。酶学性质研究表明，该酶最适反应温度为45℃，在15℃时的酶活为最高酶活的40％，4℃时的酶活为最高酶活的23％。该酶对热敏感，45℃保温45min酶活全部丧失。纯酶的最适PH为7．0，在pH6．5～7．5时保持稳定。5mmol／LNa^＋、Ca^2＋、Cu^2＋、Al^3＋、Zn^2＋对酶活力有不同程度的抑制作用，其中Al^3＋抑制K^＋作用最强，Na^＋、Ca^2＋抑制作用不明显。5mmol／LMg^2＋、K^＋对酶活力具有促进作用，其中Mg^2＋促进作用较强，使酶活提高到1．19倍。25℃以ONPG为底物的Vmax，Km值分别为7．19mol／（min·mL）、4．64mmol／L。
