Howard P. Isermann Department of Chemical and Biological Engineering and the Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, United States.
Howard P. Isermann Department of Chemical and Biological Engineering and the Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, United States.
Bioresour Technol. 2019 Dec;294:122104. doi: 10.1016/j.biortech.2019.122104. Epub 2019 Sep 4.
A completely immobilized cell-free enzyme reaction system was used to convert ketoisovaleric acid to isobutanol, a desirable biofuel, with a molar yield of 43% and a titer of 2 g/L, which are comparable to high performing in vivo systems (e.g. 41% and 5.4 g/L, respectively, for Clostridium thermocellum). The approach utilizes, for the first time, a series of previously reported enzyme mutants that either overproduce the product or are more stable when compared with their wild type. The selected enzyme variants include keto-acid decarboxylase attached to a maltose binding protein, alcohol dehydrogenase, and formate dehydrogenase. These enzymes were screened for thermal, pH, and product stability to choose optima for this system which were pH 7.4 and 35 °C. This system is designed to address well-known limitations of in vivo systems such as low product concentrations due to product feedback inhibition, instability of cells, and lack of economic product recovery.
采用完全固定化的无细胞酶反应体系将酮异戊酸转化为理想的生物燃料异丁醇，摩尔产率为 43%，浓度为 2g/L，与高性能的体内系统相当（例如，产率分别为 41%和 5.4g/L，对于热纤梭菌）。该方法首次利用一系列先前报道的酶突变体，与野生型相比，这些酶突变体要么过度产生产物，要么更稳定。所选的酶变体包括与麦芽糖结合蛋白偶联的酮酸脱羧酶、醇脱氢酶和甲酸脱氢酶。对这些酶进行了热稳定性、pH 值和产物稳定性的筛选，以选择该体系的最佳条件为 pH 值 7.4 和 35°C。该系统旨在解决体内系统中众所周知的限制因素，如由于产物反馈抑制导致的产物浓度低、细胞不稳定和缺乏经济有效的产物回收等。
