Green Separation Engineering Laboratory, School of Chemical and Biotechnology, SASTRA Deemed to be University, Thanjavur, Tamil Nadu, 613401, India.
Sci Rep. 2020 Aug 7;10(1):13356. doi: 10.1038/s41598-020-70414-2.
The present study report for the first time on the one-pot production and purification of fibrinolytic protease from Bacillus cereus by extractive fermentation using natural deep eutectic solvents (NADES). Cheese whey was chosen as a sustainable low-cost production alternative yielding a significantly high amount of protease (185.7 U/mg). Five natural deep eutectic solvents with menthol as hydrogen bond donor and sugar molecules as corresponding hydrogen bond acceptors were synthesized and their association was confirmed with H NMR. Thermophysical investigation of the synthetic NADES was accomplished as a function of temperature to define their extraction ability. Response surface methodology based optimization of concentration of NADES (77.5% w/w), NaSO (14% w/v) and cheese whey (1% w/w) were accomplished for extractive fermentation. Further, preparative purification using size exclusion chromatography was used to quantify the amount of enzyme obtained in the extraction phase (190 U/ml). On subsequent purification with an anion exchange column, the maximum purity fold (21.2) with enzyme activity (2,607.8 U/ml) was attained. The optimal pH (8.0), temperature (50 °C) were determined and the in-vitro fibrinolytic activity has been confirmed using a fibrin plate assay.
本研究首次报道了利用天然深共熔溶剂（NADES）进行提取发酵，从蜡样芽孢杆菌中一锅法生产和纯化纤溶蛋白酶。选择奶酪乳清作为可持续的低成本生产替代物，可显著提高蛋白酶的产量（185.7 U/mg）。合成了 5 种以薄荷醇为氢键供体、糖分子为氢键受体的天然深共熔溶剂，并通过 H NMR 对其缔合进行了确认。作为温度的函数，完成了对合成 NADES 的热物理性质研究，以确定其萃取能力。基于响应面法优化了 NADES（77.5% w/w）、NaSO（14% w/v）和奶酪乳清（1% w/w）的浓度，用于提取发酵。进一步使用分子筛层析法进行制备性纯化，以定量提取阶段获得的酶量（190 U/ml）。随后，用阴离子交换柱进行纯化，获得了最大的纯度倍数（21.2）和酶活（2,607.8 U/ml）。确定了最佳的 pH 值（8.0）和温度（50°C），并通过纤维蛋白平板测定法证实了体外纤溶活性。
