Alam Amany A, Goda Doaa A, Soliman Nadia A, Abdel-Meguid Dina I, El-Sharouny Ebaa E, Sabry Soraya A
Botany and Microbiology Department, Faculty of Science, Alexandria, Egypt.
Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City),New Borg El-Arab City, Universities and Research Institutes Zone, P.O. 21934, Alexandria, Egypt.
J Genet Eng Biotechnol. 2022 Nov 10;20(1):156. doi: 10.1186/s43141-022-00433-1.
Cholesterol oxidases (CHOs) have attracted enormous attention because of their wide biotechnological potential. The present study explores the production of CHOs by Streptomyces sp. AN. Evaluation of culture conditions affecting enzyme production, medium optimization and released metabolite characteristics were also investigated.
The current work reports the isolation of 37 colonies (bacteria/actinobacteria) with different morphotypes from different soil/water samples. The isolate-coded AN was selected for its high potency for CHO production. Morphological characteristics and the obtained partial sequence of 16srRNA of AN showed 99.38% identity to Streptomyces sp. strain P12-37. Factors affecting CHO production were evaluated using Plackett-Burman (PB) and Box-Behnken (BB) statistical designs to find out the optimum level of the most effective variables, namely, pH, starch, NHNO and FeSO.7HO with a predicted activity of 6.56 U/mL. According to this optimization, the following medium composition was considered to be optimum (g/L): cholesterol 1, starch 6, MgSO.7HO 0.1, CaCl 0.01, FeSO.7HO 0.1, NHNO 23.97, yeast extract (YE) 0.2, KHPO 0.01, KHPO 0.1, NaCl 0.01, Tween 20 0.01, pH 6.36 and incubation temperature (30 °C) for 9 days. Spectophotometric analysis for released metabolites against cholesterol (standard) via Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) was carried out. FTIR spectrum showed the appearance of new absorption peaks at 1644 and 1725cm; this confirmed the presence of the Keto group (C=O) stretch bond. Besides, fermentation caused changes in thermal properties such as melting temperature peak (99.26; 148.77 °C), heat flow (- 8; - 3.6 Mw/mg), capacity (- 924.69; - 209.77 mJ) and heat enthalpy (- 385.29; 69.83 J/g) by comparison to the standard cholesterol as recognized through DSC thermogram. These changes are attributed to the action of the CHO enzyme and the release of keto derivatives of cholesterol with different properties.
Streptomyces sp. AN was endowed with the capability to produce CHO. Enzyme maximization was followed using a statistical experimental approach, leading to a 2.6-fold increase in the overall activity compared to the basal condition. CHO catalyzed the oxidation of cholesterol; this was verified by the appearance of a new keto group (C=O) peak at 1644 and 1725 cm observed by FTIR spectroscopic analysis. Also, DSC thermogram demonstrates the alteration of cholesterol triggered by CHO.
胆固醇氧化酶（CHOs）因其广泛的生物技术潜力而备受关注。本研究探索了链霉菌属菌株AN产胆固醇氧化酶的情况。同时还研究了影响酶产量的培养条件评估、培养基优化及释放代谢产物的特性。
目前的工作报道了从不同土壤/水样中分离出37个具有不同形态型的菌落（细菌/放线菌）。分离株编码为AN，因其产胆固醇氧化酶的高效力而被选中。AN的形态特征和获得的16srRNA部分序列显示与链霉菌属菌株P12 - 37的同一性为99.38%。使用Plackett - Burman（PB）和Box - Behnken（BB）统计设计评估影响胆固醇氧化酶产量的因素，以找出最有效变量（即pH、淀粉、NH₄NO₃和FeSO₄·7H₂O）的最佳水平，预测活性为6.56 U/mL。根据此优化，以下培养基组成被认为是最佳的（g/L）：胆固醇1、淀粉6、MgSO₄·7H₂O 0.1、CaCl₂ 0.01、FeSO₄·7H₂O 0.1、NH₄NO₃ 23.97、酵母提取物（YE）0.2、KH₂PO₄ 0.01、K₂HPO₄ 0.1、NaCl 0.01、吐温20 0.01、pH 6.36，在30℃下培养9天。通过傅里叶变换红外光谱（FTIR）和差示扫描量热法（DSC）对释放的代谢产物与胆固醇（标准品）进行分光光度分析。FTIR光谱显示在1644和1725cm处出现新的吸收峰；这证实了酮基（C = O）伸缩键的存在。此外，与通过DSC热谱图识别的标准胆固醇相比，发酵导致热性质发生变化，如熔点峰（99.26；148.77℃）、热流（-8；-3.6 Mw/mg）、热容（-924.69；-209.77 mJ）和热焓（-385.29；69.83 J/g）。这些变化归因于胆固醇氧化酶的作用以及具有不同性质的胆固醇酮衍生物的释放。
链霉菌属菌株AN具有产胆固醇氧化酶的能力。采用统计实验方法使酶产量最大化，与基础条件相比，总体活性提高了2.6倍。胆固醇氧化酶催化胆固醇氧化；通过FTIR光谱分析在1644和1725 cm处观察到新的酮基（C = O）峰，证实了这一点。此外，DSC热谱图证明了胆固醇氧化酶引发的胆固醇变化。
