Development of Reaction Element for Nonaquous Bioprocess

对铜绿假单胞菌有机耐溶剂蛋白酶PST-01进行了纯化和鉴定。PST-01蛋白酶在各种有机溶剂存在下都是稳定的。利用PST-01蛋白酶在含有机溶剂的单相水溶液中合成多肽。在60%(v/v) DMSO存在下，肽的初始反应速率和平衡产率分别比无有机溶剂时高5.1倍和7.0倍。具有亲水性胍基和疏水性硬脂酰基的两亲性聚合物颗粒是固定化delemar根霉脂肪酶的最佳颗粒。用两亲性聚合物颗粒制备的固定化脂肪酶的水解活性比以往用Dowex MWA-1、多孔玻璃微珠和Sepharose 4B制备的固定化脂肪酶高150 ~ 8700倍。将脂肪酶固定在两亲性聚合物颗粒上，提高了脂肪酶的稳定性和酯交换活性。用两亲性聚合物颗粒制备的固定化脂肪酶的比酯交换活性是冻干脂肪酶的93.4倍。用合成的表面活性剂制备表面活性剂包被蛋白酶。表面活性剂包被的蛋白酶在有机溶剂中的肽合成活性和转化活性均高于粉末状蛋白酶。表面活性剂包被的蛋白酶活性是粉末状蛋白酶的260倍。

An organic solvent-tolerant protease (PST-01 protease) from Pseudomonas aeruginosa PST-01 was purified and characterized. The PST-01 protease was stable in the presence of various organic solvents. Peptide syntheses were performed in monophasic aqueous solution containing an organic solvent using PST-01 protease. The initial reaction rate and the equilibrium yield of the peptide in the presence of 60%(v/v) DMSO were 5.1 times and 7.0 times higher than that in the absence of an organic solvent, respectively.Amphiphilic polymer particles which have hydrophilic guanidino groups and hydrophobic stearoyl groups were the best particles for the immobilization of Rhizopus delemar lipase. The hydrolytic activity of the immobilized lipase prepared with the amphiphilic polymer particles was 150-8700 times higher than those of the immobilized lipases prepared by previous investigators using Dowex MWA-1, porous glass beads, and Sepharose 4B. The stability and the transesterification activity of lipase were both enhanced by immobilizing lipase on the amphiphilic polymer particles. The specific transesterification activity of the immobilized lipase prepared with the amphiphilic polymer particles was 93.4 times higher than that of the lyophilized lipase.Surfactant-coated protease was prepared with a synthesized surfactant. The peptide synthetic activity and transeste rification activity of the surfactant-coated protease in organic solvents were higher than that of the powder protease. The activity of the surfactant-coated protease was 260 times higher that of the powder protease.