(546f) Side-Chain Cleavage of Phytosterols By Mycobacterium sp. in a Biphasic Ionic Liquid-Aqueous System

Androst-4-ene-3,17-dione (AD) is an important steroid intermediate which can be used for synthesis of various hormone drugs. Phytosterols, that are abundant in oil residues and papermaking waste liquor, are preferred raw materials for production of AD. However, low substrate solubility and product inhibition are two major problems existed in phytosterols biotransformation. Recently, biphasic systems are considered to be efficient strategies to solve above problems, in which the hydrophobic phase act as a â??reservoirâ? for substrate dissolving and in-situ product removal (ISPR).

Ionic liquids (ILs), with advantages of tunable physical and chemical properties, negligible vapor pressure, and non-flammability, are promising alternatives to be applied in biphasic systems. In this work, nine kinds of water-immiscible ILs were used in phytosterols side-chain cleavage by growing cells of Mycobacterium sp. MB 3683. The solubilities of phytosterols and AD measured in these ILs were greatly higher than those in water, and the logarithm of distribution coefficients of AD between ILs and water were all above 2.0, suggesting their potential for ISPR. Specifically, [PrMIM][PF₆] was determined as a suitable candidate to establish a biphasic IL-aqueous system for phytosterols biotransformation to AD, and the effects of inoculation, IL addition time, and phase ratio were studied in detail. The results indicated that IL preferred to be added to fermentation medium when the cells were in stationary phase due to the toxicity of IL on cell membrane, and a small quantity of IL was appropriate because of its superior dissolving property. Under optimal conditions, i.e. inoculum size of 12% with 48 h age, IL addition at 84 h, and phase ratio of 20:1 (v/v, aqueous/IL), AD production achieved 2.23 g L^-1 after 5 days biotransformation with substrate concentration of 5 g L^-1 in a scaled-up biphasic system of 200 mL. Moreover, IL recovery was attained to be above 90%, and [PrMIM][PF₆] can be sequentially used for five batches with stable conversion in AD production.

Owing to increasing environmental constraints from organic solvents, biphasic IL-aqueous systems which presented efficient conversion over monophasic aqueous system at high feeding concentration, are potential in microbial side-chain cleavage of phytosterols and up-scale AD production.