Abstract

Bacillus sp. DL-1 was isolated from the deep sea of the Western Pacific Ocean and behaved very good resistance to NaCl. The extracellular proteases of Bacillus sp. DL-1 were found to exhibit excellent enantioselectivity for the kinetic resolution of (±)-1-phenylethyl acetate. To improve the stability of the enzyme, the immobilized extracellular proteases were preparated by using 60 g kieselguhr and 1-L crude fermentation broth containing extracellular proteases of Bacillus sp. DL-1, shaking at 25 °C, 200 r/min for 10 h. Every gram of kieselguhr adsorbed 25.7 mg extracellular protease and the enzymatic activity recovery was 79.86%. The immobilized proteases preserved about 35.8% of its activity after 6 repeated uses, which were also used as biocatalyst to asymmetrically hydrolyse (±)-1-phenylethyl acetate for the preparation of (R)-1-phenylethanol and (S)-1-phenylethyl acetate with high optical purities. The effects of pH, temperature, enzyme concentration, substrate concentration, reaction time and additives (metal ions/surfactants) on the resolution were investigated by single factor experiments. Under the optimal reaction conditions (10 mM (±)-1-phenylethyl acetate, 40 mg/mL immobilized extracellular proteases, pH 7.5 (Tris-HCl), 5% (v/v) methanol and 45 °C for 2 h), (R)-1-phenylethanol was generated with the e.e._p being > 97%, and the yield being 53%, respectively. Analogously, under the optimal reaction conditions (10-mM (±)-1-phenylethyl acetate, 360 mg/mL immobilized extracellular proteases, pH 6.0 (PB), 5% (v/v) DMSO and 35 °C for 1.5 h), (S)-1-phenylethyl acetate was generated with the e.e._s being over 99% and the yield being 79%, respectively. Compared with the extracellular proteases from Bacillus sp. DL-2, the immobilized extracellular proteases from Bacillus sp. DL-1 exhibited higher hydrolytic activity and could asymmetrically hydrolyse (±)-1-phenylethyl acetate by using higher substrate concentrations, shorter reaction times to obtain higher yields. Notably, the extracellular proteases of Bacillus sp. DL-1 were demonstrated to behave the same enantio-preference as those of most other reported esterases/lipases. Proteases from deep-sea Bacillus sp. DL-1 are promising biocatalysts for the synthesis of valuable chiral chemicals.

摘要

芽孢杆菌 DL-1 分离自西太平洋深海，对 NaCl 有很好的抗性。芽孢杆菌属的细胞外蛋白酶。发现 DL-1 对 (±)-1-苯基乙酸乙酯的动力学拆分表现出优异的对映选择性。为了提高酶的稳定性，使用 60 g 硅藻土和 1 L 含有芽孢杆菌胞外蛋白酶的粗发酵液制备固定化胞外蛋白酶。sp。DL-1，25°C，200 r/min 振荡 10 h。每克硅藻土吸附胞外蛋白酶25.7mg，酶活回收率为79.86%。固定化蛋白酶经过 6 次重复使用后保留了约 35.8% 的活性，还可作为生物催化剂不对称水解 (±)-1-苯乙酯用于制备 ( R )-1-苯乙醇和 ( S )-1-具有高光学纯度的醋酸苯乙酯。通过单因素实验研究了pH、温度、酶浓度、底物浓度、反应时间和添加剂（金属离子/表面活性剂）对分辨率的影响。在最佳反应条件下（10 mM (±)-1-苯基乙酸乙酯，40 mg/mL 固定化胞外蛋白酶，pH 7.5 (Tris-HCl)，5% ( v/ v ) 甲醇和45°C 2 h)，分别生成( R )-1-苯基乙醇，ee _p > 97%，收率分别为53%。类似地，在最佳反应条件下（10-mM (±)-1-苯基乙酸乙酯，360 mg/mL 固定化胞外蛋白酶，pH 6.0 (PB)，5% ( v / v ) DMSO 和 35 °C 1.5 小时） , 生成( S )-1-苯基乙酸乙酯, ee _s大于99%, 收率79%。与来自芽孢杆菌属的细胞外蛋白酶相比。DL-2，来自芽孢杆菌的固定化胞外蛋白酶sp。DL-1表现出更高的水解活性，并且可以通过使用更高的底物浓度、更短的反应时间来不对称地水解(±)-1-苯乙酯以获得更高的产率。值得注意的是，芽孢杆菌属的细胞外蛋白酶。DL-1 表现出与大多数其他报道的酯酶/脂肪酶相同的对映体偏好。来自深海芽孢杆菌的蛋白酶。DL-1 是用于合成有价值的手性化学品的有前途的生物催化剂。