Identification of cholesterol-assimilating actinomycetes strain and application of statistical modeling approaches for improvement of cholesterol oxidase production by Streptomyces anulatus strain NEAE-94

Abstract

Background: Cholesterol oxidase biosensors have been used to determine the level of cholesterol in different serum and food samples. Due to a wide range of industrial and clinical applications of microbial cholesterol oxidase, isolation and identification of a new microbial source (s) of cholesterol oxidase are very important.

Results: The local isolate Streptomyces sp. strain NEAE-94 is a promising source of cholesterol oxidase. It was identified based on cultural, morphological and physiological characteristics; in addition to the 16S rRNA sequence. The sequencing product had been deposited in the GenBank database under the accession number KC354803. Cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 in shake flasks was optimized using surface response methodology. The different process parameters were first screened using a Plackett-Burman design and the parameters with significant effects on the production of cholesterol oxidase were identified. Out of the 15 factors screened, agitation speed, cholesterol and yeast extract concentrations had the most significant positive effects on the production of cholesterol oxidase. The optimal levels of these variables and the effects of their mutual interactions on cholesterol oxidase production were determined using Box-Behnken design. Cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 was 11.03, 27.31 U/mL after Plackett-Burman Design and Box-Behnken design; respectively, with a fold of increase of 6.06 times compared to the production before applying the Plackett-Burman design (4.51 U/mL).

Conclusions: Maximum cholesterol oxidase activity was obtained at the following fermentation conditions: g/L (cholesterol 4, yeast extract 5, NaCl 0.5, K₂HPO₄ 1, FeSO₄.7H₂O 0.01, MgSO₄.7H₂O 0.5), pH 7, inoculum size 4% (v/v), temperature 37°C, agitation speed of 150 rpm, medium volume 50 mL and incubation time 5 days.

Keywords: 16S rRNA; Box Behnken design; Identification; Plackett-Burman design; Streptomyces sp. strain NEAE-94.

Fig. 1

Cholesterol oxidase production of Streptomyces sp. NEAE-94 detected by plate assay method

Fig. 2

The colored photograph of Streptomyces sp. NEAE-94 aerial mycelium after growth on yeast extract -malt extract agar (a) and inorganic salt-starch agar media (b) at 30°C for 7 days, (c) plate assay showing starch hydrolysis by Streptomyces sp. strain NEAE-94 and (d) cholesterol oxidase production in submerged fermentation

Fig. 3

Spore-chain and spore-surface morphology of Streptomyces sp. strain NEAE-94 under scanning electron microscope at magnification of 4000X-20,000X

Fig. 4

Phylogenetic tree obtained by Neighbour-joining method showing the relationship between Streptomyces sp. strain NEAE-94 and other related species of Streptomyces based on the 16S rRNA sequences

Fig. 5

a The main effects of the variables, b The Pareto chart shows the order of significance of each variable, c The normal probability plot of the residuals, d Correlation between the experimented and predicted values for cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 determined by the first-order polynomial equation

Fig. 6

a-c Three-dimensional response surface plots showing the effect of agitation speed, cholesterol, yeast extract concentration and their mutual effects on the cholesterol oxidase production