含深共熔溶剂体系中晶胶微球固定化分枝杆菌生产雄烯二酮研究

Production of Androstenedione Using Immobilized Mycobacterium sp.via Cryogel Beads in Systems Containing Deep Eutectic Solvents

分枝杆菌全细胞催化的选择性植物甾醇侧链降解反应可用于生产甾体药物重要中间体雄烯二酮(AD),由于存在底物低水溶解度和产物抑制与降解等问题,该反应的转化效率较低。深共熔溶剂(DESs)是一种新型的绿色溶剂,可以作为助溶剂有效促进底物溶解。本研究在甜菜碱(Bet)与麦芽糖(Mal)或半乳糖(Gal)组成的2种DESs构成的转化体系中,以甲基丙烯酸羟乙酯和甲基丙烯酸正丁酯聚合的晶胶微球为基质,研究了固定化分枝杆菌细胞降解植物甾醇侧链生产AD的过程。结果表明:晶胶微球能够有效分散细胞,提高了细胞利用率,固定化后单位细胞AD产量提高了6.8倍。DESs可缓解AD降解和减少AD在晶胶微球内的累积,经过3批次半连续转化后,在含10%体积分数Bet/Mal体系中的单位细胞干重(DCW)的总AD产量可达401.4 mg/g,是缓冲液体系悬浮培养细胞的12倍。在含DESs体系中晶胶微球固定化分枝杆菌能促进植物甾醇降解从而提高AD产量,具有潜在的工业应用前景。

The whole-cell side-chain cleavage of phytosterols by Mycobacterium sp.is known as an environmentally friendly method for the production of androstenedione(AD),which is an important intermediate to produce steroids.However,AD yield was limited by factors such as low water solubility of phytosterols,product inhibition and degradation.Deep eutectic solvents(DESs)have been employed as a kind of popular green solvent with the ability to promote substrate dissolution and increase product yield.In this paper,the biotransformation of phytosterols by immobilized Mycobacterium sp.resting cells in systems containing DESs was investigated,and 3 batches of semi-continuous biotransformation were performed.First,two types of DESs,i.e.betaine/maltose and betaine/galactose,were prepared to construct solvent systems,and the cryogel beads polymerized with 2-hydroxyethyl methacrylate and butyl methacrylate were used as the carrier of immobilization.With better dispersions and utilization of cells,AD yield per unit of cells was improved to 6.8 times by immobilized cells,and high biotransformation efficiency was still maintained when the addition of immobilized cryogel beads was up to 180 g/L.The AD yield by immobilized cells was further enhanced by the introduction of DESs,which inhibited the AD degradation pathway and reduced the accumulation of AD in the interior of cryogel beads.Finally,high AD yield per unit of cells(401.4 mg/g-DCW)was achieved after 3 batches of semi-continuous biotransformations in the system with 10%Bet/Mal,which was 12 times of that by free resting cells in the buffer system.In summary,AD yield was significantly enhanced due to the improved cell utilization by immobilization of Mycobacterium sp.via cryogel beads combined with the promotion of DESs on biotransformation,which presented a potential industrial application prospect.