University of Turku, Department of Biochemistry, Turku, Finland.
Saint Petersburg State Chemical Pharmaceutical University, Department of Biotechnology, Saint Petersburg, Russia.
Sci Rep. 2019 Aug 14;9(1):11850. doi: 10.1038/s41598-019-48132-1.
Cholesterol oxidases are important enzymes with a wide range of applications from basic research to industry. In this study, we have discovered and described the first cell-associated cholesterol oxidase, ChoD, from Streptomyces lavendulae YAKB-15. This strain is a naturally high producer of ChoD, but only produces ChoD in a complex medium containing whole yeast cells. For characterization of ChoD, we acquired a draft genome sequence of S. lavendulae YAKB-15 and identified a gene product containing a flavin adenine dinucleotide binding motif, which could be responsible for the ChoD activity. The enzymatic activity was confirmed in vitro with histidine tagged ChoD produced in Escherichia coli TOP10, which lead to the determination of basic kinetic parameters with K 15.9 µM and k 10.4/s. The optimum temperature and pH was 65 °C and 5, respectively. In order to increase the efficiency of production, we then expressed the cholesterol oxidase, choD, gene heterologously in Streptomyces lividans TK24 and Streptomyces albus J1074 using two different expression systems. In S. albus J1074, the ChoD activity was comparable to the wild type S. lavendulae YAKB-15, but importantly allowed production of ChoD without the presence of yeast cells.
胆固醇氧化酶是一类重要的酶，在基础研究和工业领域都有广泛的应用。在本研究中，我们发现并描述了来自玫瑰色链霉菌 YAKB-15 的首个细胞相关胆固醇氧化酶 ChoD。该菌株是 ChoD 的天然高产生菌，但仅在含有完整酵母细胞的复杂培养基中产生 ChoD。为了对 ChoD 进行表征，我们获得了玫瑰色链霉菌 YAKB-15 的草图基因组序列，并鉴定出一个含有黄素腺嘌呤二核苷酸结合基序的基因产物，该基序可能与 ChoD 活性有关。通过在大肠杆菌 TOP10 中表达带有组氨酸标签的 ChoD，我们在体外证实了该酶的活性，这导致了确定基本动力学参数 K 15.9 μM 和 k 10.4/s。最适温度和 pH 值分别为 65°C 和 5。为了提高生产效率，我们随后在链霉菌变铅青链霉菌 TK24 和白色链霉菌 J1074 中通过两种不同的表达系统异源表达胆固醇氧化酶 choD 基因。在白色链霉菌 J1074 中，ChoD 活性与野生型玫瑰色链霉菌 YAKB-15 相当，但重要的是，它允许在没有酵母细胞存在的情况下生产 ChoD。
