A strain HB-03 to produce alkaline extracellular lipase was isolated from oil-rich soil samples and identified as Aspergillus awamori.The growth conditions and nutritional factors for lipase production by strain HB-03...A strain HB-03 to produce alkaline extracellular lipase was isolated from oil-rich soil samples and identified as Aspergillus awamori.The growth conditions and nutritional factors for lipase production by strain HB-03 were optimized,and the maximum lipase production of (45.9±2.3) U/mL was obtained at 30 °C and pH 7.0 after 36 h using olive oil (1%) and sucrose (0.5%) as carbon sources and combination of peptone (2%),yeast extract (0.5%) and ammonium sulfate (0.1%) as nitrogen sources.The lipase was purified to homogeneity with 10.6-fold,18.84% yield and a specific activity of 1 862.2 U/mg using ammonium sulfate precipitation followed by SephadexG-75 gel filtration chromatography.The purified lipase with molecular mass of 68 ku was estimated by SDS-PAGE.The optimum pH and temperature for the purified lipase were found to be 8.5 and 40 °C,respectively.The lipase kept more than 80% of activity in pH 7.0-10.0 and temperatures up to 45 °C.The metal ions of Mn2+,Ba2+ significantly enhanced the lipase activity,whereas Cu2+,Fe3+ and Mg2+ strongly reduced the lipase activity.The Km and Vmax values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mmol/L and 60.6 mmol/(L-min),respectively.The results show that this novel lipase has potential industrial applications.展开更多
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
文摘A strain HB-03 to produce alkaline extracellular lipase was isolated from oil-rich soil samples and identified as Aspergillus awamori.The growth conditions and nutritional factors for lipase production by strain HB-03 were optimized,and the maximum lipase production of (45.9±2.3) U/mL was obtained at 30 °C and pH 7.0 after 36 h using olive oil (1%) and sucrose (0.5%) as carbon sources and combination of peptone (2%),yeast extract (0.5%) and ammonium sulfate (0.1%) as nitrogen sources.The lipase was purified to homogeneity with 10.6-fold,18.84% yield and a specific activity of 1 862.2 U/mg using ammonium sulfate precipitation followed by SephadexG-75 gel filtration chromatography.The purified lipase with molecular mass of 68 ku was estimated by SDS-PAGE.The optimum pH and temperature for the purified lipase were found to be 8.5 and 40 °C,respectively.The lipase kept more than 80% of activity in pH 7.0-10.0 and temperatures up to 45 °C.The metal ions of Mn2+,Ba2+ significantly enhanced the lipase activity,whereas Cu2+,Fe3+ and Mg2+ strongly reduced the lipase activity.The Km and Vmax values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mmol/L and 60.6 mmol/(L-min),respectively.The results show that this novel lipase has potential industrial applications.
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)。
