Production and statistical optimization of cholesterol-oxidase generated by Streptomyces sp. AN strain

Amany A Alam; Doaa A Goda; Nadia A Soliman; Dina I Abdel-Meguid; Ebaa E El-Sharouny; Soraya A Sabry

doi:10.1186/s43141-022-00433-1

Production and statistical optimization of cholesterol-oxidase generated by Streptomyces sp. AN strain

J Genet Eng Biotechnol. 2022 Nov 10;20(1):156. doi: 10.1186/s43141-022-00433-1.

Authors

Amany A Alam¹, Doaa A Goda², Nadia A Soliman³, Dina I Abdel-Meguid¹, Ebaa E El-Sharouny¹, Soraya A Sabry¹

Affiliations

¹ Botany and Microbiology Department, Faculty of Science, Alexandria, Egypt.
² 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. doaa.rashid@yahoo.com.
³ 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.

Abstract

Background: 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.

Results: 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, NH₄NO₃ and FeSO₄.7H₂O 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, MgSO₄.7H₂O 0.1, CaCl₂ 0.01, FeSO₄.7H₂O 0.1, NH₄NO₃ 23.97, yeast extract (YE) 0.2, K₂HPO₄ 0.01, KH₂PO₄ 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^-1; 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.

Conclusion: 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^-1 observed by FTIR spectroscopic analysis. Also, DSC thermogram demonstrates the alteration of cholesterol triggered by CHO.

Keywords: Cholesterol oxidase; Experimental design; Optimization; Process; Streptomyces sp. AN.