Hannemann Frank, Virus Cornelia, Bernhardt Rita
FR 8.3--Biochemie, Universität des Saarlandes, Saarbrücken, Germany.
J Biotechnol. 2006 Jun 25;124(1):172-81. doi: 10.1016/j.jbiotec.2006.01.009. Epub 2006 Feb 28.
The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conversion assay as well as a CO difference spectroscopy assay, which enables the screening for enzyme variants with higher activity and stability.
巨大芽孢杆菌的15β-羟化酶（CYP106A2）是目前已分离和鉴定的少数细菌甾体羟化酶之一，可催化多种甾体的15β-羟化反应。该酶的活性可通过牛肾上腺的肾上腺铁氧还蛋白（Adx）和肾上腺铁氧还蛋白还原酶（AdR）来支持，为其提供甾体羟化活性所需的还原当量。通过共表达来自两个质粒的相应编码序列，在大肠杆菌中构建了这种三组分电子传递链，这两个质粒含有不同的选择标记和兼容的复制起点。第一个质粒上AdR和Adx的cDNA由核糖体结合序列隔开，还原酶位于铁氧还蛋白之前。用于CYP106A2表达的第二个质粒构建时具备分子进化方法所需的所有特征。转化后的细菌表现出可诱导的能力，在300 ml培养体积中，能以平均1 mM/d的速率有效地将11-脱氧皮质酮（DOC）转化为15β-DOC。甾体转化系统被缩小到微量滴定板形式，并开发了一种机器人设置用于基于荧光的转化测定以及CO差光谱测定，这使得能够筛选出具有更高活性和稳定性的酶变体。
