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首页> 外文期刊>Arabian Journal for Science and Engineering. Section A, Sciences >Purification and Characterization of a Membrane-Unbound Highly Thermostable Metalloprotease from Aeromonas Caviae
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Purification and Characterization of a Membrane-Unbound Highly Thermostable Metalloprotease from Aeromonas Caviae

机译：从Aeromonas Caviae纯化和表征膜 - 未凝固高稳压金属蛋白酶

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摘要

The main objective of the current study was to purify and characterize an alkaline thermostable metalloprotease from Aeromonas caviae. The enzyme was subjected to a two-step purification scheme using ammonium sulfate [(NH_4)_2SO_4] fractionation and ion-exchange chromatography on a DEAE-sepharose fast flow column. The fraction which precipitated with 40-60% (w/v) of [(NH_4)_2SO_4] exhibited the highest enzyme activity. Anion-exchange chromatography resulted in approximately 51-fold purification, with a yield of 64.7%. The enzyme was successfully purified to homogeneity, and this was confirmed on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE).Asingle band with an approximate molecular mass of ∼42 kDa was observed. The maximum enzymatic activity was observed at pH 8 with azocasein as the substrate, and the enzyme was stable in the pH range of 6-10 after 5 h of incubation. The purified enzyme was active in the temperature range between 50 and 60 °C and showed 80% loss in enzymatic activity upon heating at 70 °C. The K_m and Vmax values for the purified protease, with azocasein as substrate under optimal reaction conditions (pH 8 and 50 °C) were 0.74 and 122.4 mg/ml, respectively. The protease was investigated for its application for the degradation of chicken feathers. The high enzymatic activity, pH, and thermal stability of AU04 proteasemake it an industrially important enzyme.
机译：目前研究的主要目的是纯化和表征来自Aeromonas Caviae的碱性恒温金属蛋白酶。使用硫酸铵[（NH_4）_2SO_4]分馏和离子交换色谱法在DEAE-Sepharose快速流动柱上进行两步纯化方案进行两步纯化方案。用40-60％（w / v）的[（NH_4）_2SO_4]沉淀的级分具有最高的酶活性。阴离子交换色谱法导致约51倍的纯化，产率为64.7％。将酶成功纯化为均匀性，并在十二烷基硫酸钠 - 聚丙烯酰胺凝胶（SDS-PAGE）上证实。观察到〜42kDa的近似分子量的脉冲带。用偶氮酶在pH8的pH 8中观察到最大酶活性，作为底物，在孵育5小时后，酶在6-10的pH范围内稳定。纯化的酶在50至60℃的温度范围内活性，在70℃下加热时在酶活性中显示80％的损失。纯化蛋白酶的K_M和VMAX值，在最佳反应条件（pH8和50℃）下作为基质的氮杂氨酰均为0.74和122.4mg / ml。研究了蛋白酶以施用鸡羽的降解。 Au04蛋白酶的高酶活性，pH和热稳定性是工业上重要的酶。

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