黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAE Sepharose Fast Flow阴离子交换层析和Sephadex G-75凝胶过滤层析得到电泳纯的脂肪酶，纯化倍数为73．71倍，活性回收率为34％。对纯化脂肪酶性质研究表明：该脂肪酶分子量约为35～...黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAE Sepharose Fast Flow阴离子交换层析和Sephadex G-75凝胶过滤层析得到电泳纯的脂肪酶，纯化倍数为73．71倍，活性回收率为34％。对纯化脂肪酶性质研究表明：该脂肪酶分子量约为35～40kD，水解橄榄油的最适温度和最适pH分别为45℃和7．0，在60℃以下和pH2．0～9．0之间有很好的稳定性。该脂肪酶的水解活性对Ca^2＋表现明显的依赖性，而Mn^2＋、Fe^2＋和Zn^2＋对脂肪酶则有显著的抑制作用。在最适条件下水解pNPP的Km和Vmax分别为7．37mmol／L和25．91μmol／（min·mg）。其N-端的15个氨基酸序列为Ser（Glu／His）-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln。展开更多
采用中空纤维膜超滤和葡聚糖凝胶层析相结合的方法对黑曲霉N5-5单宁酶进行纯化,然后对纯酶性质进行测定。结果显示,黑曲霉N5-5单宁酶用该方法纯化后,可纯化近20倍,酶活力可回收23.30%。对纯酶作十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,...采用中空纤维膜超滤和葡聚糖凝胶层析相结合的方法对黑曲霉N5-5单宁酶进行纯化,然后对纯酶性质进行测定。结果显示,黑曲霉N5-5单宁酶用该方法纯化后,可纯化近20倍,酶活力可回收23.30%。对纯酶作十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,可知黑曲霉N5-5单宁酶为分子质量64.2 k D的单肽链蛋白。纯酶的酶促反应最适温度为45℃,且在25~45℃范围内热稳定性良好;酶促反应最适p H值为5.0,且在p H 5.0~5.5范围内酸碱稳定性良好。另外,反应动力学测定结果表明,该酶对底物没食子酸丙酯的米氏常数K_m为0.916 mmol/L,最大反应速率vmax为0.877 mmol/(L·min)。展开更多
对蜡状芽孢杆菌Bc-05菌株的发酵上清液经硫酸铵分级沉淀、DEAE Sepharose Fast Flow阴离子交换层析所获得的纤溶酶进行性质研究.结果表明:经纤维蛋白平板法检测该酶有直接水解纤维蛋白和激活纤溶酶原的双重作用,最适作用温度37℃,最适pH...对蜡状芽孢杆菌Bc-05菌株的发酵上清液经硫酸铵分级沉淀、DEAE Sepharose Fast Flow阴离子交换层析所获得的纤溶酶进行性质研究.结果表明:经纤维蛋白平板法检测该酶有直接水解纤维蛋白和激活纤溶酶原的双重作用,最适作用温度37℃,最适pH=8.0,在pH=8.0条件下25℃和37℃放置24 h酶活力仍保持77.52%和78.96%,该酶体外对兔血凝块有明显的溶解作用;Ca2+,Mn2+离子对该酶具有激活作用,而Cu2+,Fe3+完全抑制其纤溶活性,PMSF,EDTA和DTT对该酶有抑制作用,说明活性中心含有二硫键、金属离子和丝氨酸;测其N端10个氨基酸序列为NH2-Val-Thr-Pro-Thr-Asn-Ala-Val-Asn-Thr-Gly,与其他生物来源的纤溶酶相比较没有同源性.展开更多
Extracellular xylanase XYNB from Streptomy-ces olivaceoviridis A1 has been purified and characterized. The optimal pH value and temperature of XYNB for its ac-tivity are 5.2 and 60℃, respectively. The specific activi...Extracellular xylanase XYNB from Streptomy-ces olivaceoviridis A1 has been purified and characterized. The optimal pH value and temperature of XYNB for its ac-tivity are 5.2 and 60℃, respectively. The specific activity of XYNB is as high as 2869.78 U/mg. Metal cations, EDTA and SDS have no effects on enzyme activity of XYNB. The gene xynB coding mature protein of XYNB has been cloned by PCR. The forward oligonucleotide primer used in the PCR reaction was synthesized based on the N-terminal amino acid sequence of XYNB mature protein, and the reverse oligonu-cleotide primers are random oligonucleotide. The cloned gene xynB is 576 bp long and its G+C content is 64.3%. The xynB encodes 191 amino acid residues, and the putative mo-lecular weight of XYNB is 20.839 kD. The xynB has been expressed in E. coli, and the expressed xylanase has normal bioactivity.展开更多
文摘黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAE Sepharose Fast Flow阴离子交换层析和Sephadex G-75凝胶过滤层析得到电泳纯的脂肪酶，纯化倍数为73．71倍，活性回收率为34％。对纯化脂肪酶性质研究表明：该脂肪酶分子量约为35～40kD，水解橄榄油的最适温度和最适pH分别为45℃和7．0，在60℃以下和pH2．0～9．0之间有很好的稳定性。该脂肪酶的水解活性对Ca^2＋表现明显的依赖性，而Mn^2＋、Fe^2＋和Zn^2＋对脂肪酶则有显著的抑制作用。在最适条件下水解pNPP的Km和Vmax分别为7．37mmol／L和25．91μmol／（min·mg）。其N-端的15个氨基酸序列为Ser（Glu／His）-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln。
文摘采用中空纤维膜超滤和葡聚糖凝胶层析相结合的方法对黑曲霉N5-5单宁酶进行纯化,然后对纯酶性质进行测定。结果显示,黑曲霉N5-5单宁酶用该方法纯化后,可纯化近20倍,酶活力可回收23.30%。对纯酶作十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,可知黑曲霉N5-5单宁酶为分子质量64.2 k D的单肽链蛋白。纯酶的酶促反应最适温度为45℃,且在25~45℃范围内热稳定性良好;酶促反应最适p H值为5.0,且在p H 5.0~5.5范围内酸碱稳定性良好。另外,反应动力学测定结果表明,该酶对底物没食子酸丙酯的米氏常数K_m为0.916 mmol/L,最大反应速率vmax为0.877 mmol/(L·min)。
文摘对蜡状芽孢杆菌Bc-05菌株的发酵上清液经硫酸铵分级沉淀、DEAE Sepharose Fast Flow阴离子交换层析所获得的纤溶酶进行性质研究.结果表明:经纤维蛋白平板法检测该酶有直接水解纤维蛋白和激活纤溶酶原的双重作用,最适作用温度37℃,最适pH=8.0,在pH=8.0条件下25℃和37℃放置24 h酶活力仍保持77.52%和78.96%,该酶体外对兔血凝块有明显的溶解作用;Ca2+,Mn2+离子对该酶具有激活作用,而Cu2+,Fe3+完全抑制其纤溶活性,PMSF,EDTA和DTT对该酶有抑制作用,说明活性中心含有二硫键、金属离子和丝氨酸;测其N端10个氨基酸序列为NH2-Val-Thr-Pro-Thr-Asn-Ala-Val-Asn-Thr-Gly,与其他生物来源的纤溶酶相比较没有同源性.
基金This work was supported by the State"863"High-Tech Programs(Grant No.2001AA214041).
文摘Extracellular xylanase XYNB from Streptomy-ces olivaceoviridis A1 has been purified and characterized. The optimal pH value and temperature of XYNB for its ac-tivity are 5.2 and 60℃, respectively. The specific activity of XYNB is as high as 2869.78 U/mg. Metal cations, EDTA and SDS have no effects on enzyme activity of XYNB. The gene xynB coding mature protein of XYNB has been cloned by PCR. The forward oligonucleotide primer used in the PCR reaction was synthesized based on the N-terminal amino acid sequence of XYNB mature protein, and the reverse oligonu-cleotide primers are random oligonucleotide. The cloned gene xynB is 576 bp long and its G+C content is 64.3%. The xynB encodes 191 amino acid residues, and the putative mo-lecular weight of XYNB is 20.839 kD. The xynB has been expressed in E. coli, and the expressed xylanase has normal bioactivity.
