The protease was produced extracellularly in submerged fermentation by the yeast Rhodotorula oryzicola using different sources of nitrogen and maximum activity (6.54 × 10⁻³ U/mg) was obtained in medium containing 2% casein (w/v). Purification of the protease by gel filtration chromatography resulted in a 3.07-fold increase of specific protease activity. The optimal pH and temperature for enzyme activity were 6.51 and 63.04 °C, respectively. Incubation in the presence of some salts enhanced enzyme activity, which peaked under 0.01 M BaCl₂. The enzyme retained about 90% of enzymatic activity at temperatures 50–60 °C. The commercially available enzyme carriers evaluated, silica gel, Celite 545, and chitosan effectively immobilized the protease. The enzyme immobilized in Celite 545 retained 73.53% of the initial activity after 15 reuse cycles. These results are quite promising for large-scale production and immobilization of protease from R. oryzicola, as the high operational stability of the immobilized enzyme lowers production costs in biotechnological applications that require high enzymatic activity and stability under high temperatures.

中文翻译：

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米氏红酵母蛋白酶的生产、表征和固定化

蛋白酶是由酵母红酵母使用不同的氮源在深层发酵中产生的，并且在含有 2% 酪蛋白 (w/v) 的培养基中获得最大活性 (6.54 × 10 ^-3 U/mg)。通过凝胶过滤层析纯化蛋白酶导致比蛋白酶活性增加 3.07 倍。酶活性的最佳 pH 值和温度分别为 6.51 和 63.04 °C。在某些盐的存在下孵育可增强酶活性，在 0.01 M BaCl ₂下达到峰值. 该酶在 50–60 °C 的温度下保留了约 90% 的酶活性。评估的市售酶载体、硅胶、Celite 545 和壳聚糖有效地固定了蛋白酶。固定在硅藻土 545 中的酶在 15 次重复使用循环后保留了初始活性的 73.53%。这些结果对于米曲霉蛋白酶的大规模生产和固定化非常有希望，因为固定化酶的高操作稳定性降低了生物技术应用中需要高酶活性和高温稳定性的生产成本。