Actinomycete strain, Streptomyces sp. strain NEAE-94, has been tested for its cholesterol oxidase activity with a plate-based method, the formation of the pink areas around the colonies indicated the presence of cholesterol oxidase activity (Fig. 1). The process of cholesterol-oxidation is the oxidation of cholesterol with the use of cholesterol oxidase to 4-cholesten-3-one and hydrogen peroxide. The hydrogen peroxide generated by cholesterol oxidation was then combined with 4-aminoantipyrine and phenol by peroxidase to produce quinoneimine coloration (Supplementary Figure 1). The promising strain was identified based on morphological, cultural, physiological and chemotaxonomic characteristics, in addition to 16S rRNA sequence.
Cultural characteristics of Streptomyces sp. strain NEAE-94
The isolate showed well growth on four different media, including oatmeal agar, inorganic salt-starch agar, tyrosine agar and yeast malt extract agar. Weak growth was observed on glycerol asparagines agar and peptone-yeast extract iron agar (Supplementary Table 1). Aerial mycelium color was whitish yellow on yeast extract-malt extract agar (Fig. 2A); yellow on oatmeal agar, tyrosine agar and inorganic salt-starch agar (Fig. 2B), while is faint yellow on peptone-yeast extract iron agar and glycerol asparagines agar. However, the substrate mycelium develop a yellow color on oatmeal agar, inorganic salt-starch agar, yeast extract -malt extract agar; brownish orange color on tyrosine agar media. Whereas, a faint orange color was developed on both glycerol asparagine agar and peptone-yeast extract iron agar media. A yellow diffusible pigment was produced in inorganic salt-starch agar, yeast extract-malt extract agar, tyrosine agar and glycerol asparagine agar; a faint yellow pigment was produced in oatmeal agar. The diffusible pigment was not pH indicator. No pigments were produced in peptone-yeast extract iron agar.
Physiological properties of Streptomyces sp. strain NEAE-94
The physiological properties of Streptomyces sp. strain NEAE-94 are listed in Table 1. It utilized trehalose, D(+)xylose, D(+)mannose, rhamnose, D(+)galactose, raffinose, D(-)fructose, D(+)glucose, L-arabinose, sucrose, maltose and cellulose, but could not utilized ribose as the sole carbon source. Streptomyces sp. strain NEAE-94 has reduced nitrate to nitrite. Coagulation and peptonization of milk were positive. Production of protease, α–amylase (Fig. 2C), cellulase, gelatinase and asparaginase was positive. On the other hand, production of uricase, lecithinase and chitosanase was negative. The optimal growth temperature was 30oC and the optimal pH was 7.0. Streptomyces sp. strain NEAE-94 grew in the presence of NaCl up to 5 % (w/v). Streptomyces sp. strain NEAE-94 showed positive antimicrobial activities against Staphylococcus aureus, E. coli, Bacillus subtilis and pseudomonas aeruginosa, but no activities were shown against Candida albicans, Rhizoctonia solani, Aspergillus niger, Fusarium oxysporum, Alternaria solani, Sacchromyces cerevisiae, Bipolaris oryzae or Klebsiella pneumonia. Streptomyces sp. strain NEAE-94 did not produce melanoid pigments in tyrosine agar, peptone-yeast extract iron agar or tryptone-yeast extract broth.
Morphological features of Streptomyces sp. strain NEAE-94
Morphological characteristics of Streptomyces sp. strain NEAE-94 was observed by scanning electron micrograph after incubation on medium of starch nitrate agar medium at 30°C for 14 days. Microscopic observation of Streptomyces sp. strain NEAE-94 showed rectiflexibiles spores chains (Fig. 3). In general, chains of mature spores are long. Spore shape is elongated (0.593– 0.754 x 0.995–1.341 μm), irregular and the spore surface is smooth (Fig. 3).
16S rRNA gene sequence analysis and phylogenetic analysis
The obtained 16S rRNA sequence of Streptomyces sp. strain NEAE-94 was determined which gave an almost complete sequence with 1536 bp and further subjected to the BLAST search of the GenBank database and the results showed homologies with other relevant sequences of many species belonging to the Streptomyces genus. The phylogenetic tree (Fig. 4) showed that Streptomyces sp. strain NEAE-94 shared gene similarity of 99.38% to that of Streptomyces anulatus strain BZ10-24, query cover 94% (GenBank accession no. KC493992.1); 99.59% to that of Streptomyces parvus strain 3151, query cover 94% (GenBank accession no. EF063462.1); 99.38% to that of Streptomyces flavofuscus strain NRRL B-2594, query cover 94% (GenBank accession no. EF178690.1) and 99.19% to that of Streptomyces fimicarius strain BWL-H1, query cover 95% (GenBank accession no. MG197994.1).
The whole morphological and physiological properties of Streptomyces sp. strain NEAE-94 and its closest phylogenetic neighbors of the genus Streptomyces which showed significant similarities are shown in Table 1. The strain (Streptomyces anulatus) has been deposited in the Culture Collection Ain Shams University (CCASU). The culture collection accession number CCASU 20202 is assigned to the deposited strain.
Screening of significant factors for production of cholesterol oxidase using Plackett–Burman design
The design matrix used for screening of the significant factors for production of cholesterol oxidase and the appropriate responses are shown in Table 2. The mycelial growth of Streptomyces anulatus strain NEAE-94 during cholesterol oxidase production in shake flask in submerged fermentation is shown in Fig. 2D. The results showed broad variability of the cholesterol oxidase activity (0.87 to 11.03 U/mL) reflecting the significance of the medium optimization for enhanced cholesterol oxidase production. The highest production of cholesterol oxidase (11.03 U/mL) was achieved in the run no. 18 using 50 mL medium/250 mL conical flask consists of (g/L): Glucose 5; starch 10; cholesterol 3; yeast extract 4; peptone 4; (NH4)2SO4 4; FeSO4.7H2O 0.01; MgSO4.7H2O 0.5; NaCl 0.5; K2HPO4 1 and pH 7; inoculum size was 4 % (v/v) and incubated for 5 day at 37°C using an agitation speed of 150 rpm. In the run no. 4, the lowest production of cholesterol oxidase was obtained (0.87 U/mL) using 50 mL medium/250 mL conical flask consists of (g/L): Glucose 5; starch 10; cholesterol 1; yeast extract 1; peptone 1; (NH4)2SO4 4; FeSO4.7H2O 0.05; MgSO4.7H2O 0.1; NaCl 1; K2HPO4 1 and pH 9; inoculum size was 4 % (v/v) and incubated for 7 day at 37°C using an agitation speed of 100 rpm.
Table 3 shows the statistical analysis of the results of the Plackett–Burman design. Fig. 5A shows the main effect of the individual independent factors on the cholesterol oxidase production. Fig. 5A revealed that, temperature, agitation speed, pH, starch, cholesterol, peptone, yeast extract, ammonium sulphate and K2HPO4 positively affect cholesterol oxidase production, whereas the remaining factors named incubation time, glucose, inoculum size, MgSO4, NaCl and FeSO4 negatively affect cholesterol oxidase production. The data revealed that, starch (G), peptone (K) and ammonium sulphate (L) with higher P-values (0.9271, 0.9573, 0.9370; respectively), lower effects (0.09, 0.05 and 0.07; respectively) and lower contribution % (0.04, 0.02 and 0.04; respectively) are insignificant factors. The Pareto chart shows absolute effects values and illustrates the significance order of the factors that influence cholesterol oxidase production. The Pareto chart shows a reference line, any absolute effect value extending past this reference line is highly essential (Fig. 5B). Fig. 5C displays a normal probability plot of the residuals. Fig. 5D shows the plot of the predicted cholesterol oxidase production versus actual values.
The values of the determination coefficient (R2 = 0.9996) and the adjusted determination coefficient (Adj. R2 = 0.9978) are very high. Cholesterol, agitation speed with a P-value of <0.0001 was determined to be the most significant factors, followed by the concentration of yeast extract, incubation time (0.0001) then glucose (0.0002) (Table 3). The P-value < 0.05 (0.0001) implies that the model terms are significant. The F-value of the model (573.21) means that it is significant. The first order polynomial equation representing the production of cholesterol oxidase in relation to the independent factors was obtained by neglecting the insignificant factors:
Y = 5.17+ 0.15 A− 0.88 B − 0.45 C + 1.01 D + 0.42 E − 0.78 F + 2.18 H + 0.95 K + 0.17 M− 0.41 N− 0.11 O− 0.19 P Equation (3)
Where Y is the production of cholesterol oxidase and A,B,C,D,E,F,H,K,M,N,O,P are temperature, the time of incubation, size of inoculum, speed of agitation, pH, concentration of glucose, concentration of cholesterol, concentration of yeast extract, concentration of K2HPO4, NaCl, MgSO4 and FeSO4; respectively.
For assessment of Plackett-Burman design precision, the following production medium was used for cholesterol oxidase production (g/L): FeSO4.7H2O 0.01; MgSO4.7H2O 0.5; NaCl 0.5; K2HPO4 1; yeast extract 4; cholesterol 3 and pH 7. The production medium was inoculated with inoculum size of 4% (v/v) and incubated at a temperature of 37°C in a shaker incubator at 150 rpm for 5 days. The production of cholesterol oxidase using the previous medium was 11.03 U/mL, which was increased 2.45 times compared to the enzyme activity obtained before application of Plackett-Burman design (4.51 U/mL).
Optimization of the selected significant variables by Box–Behnken design
Box–Behnken design was used to obtain the optimal levels of the most significant factors influencing cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 and to study the interaction effects among these factors. In the current study, fifteen experiments with various combinations of agitation speed, cholesterol concentration and yeast extract concentration were performed and the experimental and predicted cholesterol oxidase production and residuals for the fifteen trials are provided in Table 4.
Based on the variations in the agitation speed, cholesterol concentration and yeast extract concentration, the results showed variations in the cholesterol oxidase production. Cholesterol oxidase production ranged from 5.64-27.31 U/mL. The lowest cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 (5.64 U/mL) was achieved in the 13th run when the agitation speed was 100 rpm, cholesterol concentration was 4 g/L and yeast extract concentration was 3 g/L. The maximum value of cholesterol oxidase production was achieved in the 12th run with value of 27.31 U/mL, when agitation speed was 150 rpm, cholesterol concentration was 4 g/L and yeast extract concentration was 5 g/L.
The analysis of variance (ANOVA) for multiple regression analysis
Table 5 contains multiple regression analysis and ANOVA for the results of the Box–Behnken design. The ANOVA of the multiple regression analysis show the model to be highly significant, as can be seen from the low probability value (<0.0001) and the value of Fisher’s F-test (113.82) (Table 5). The current R2 and adjusted R2 values are 0.9951 and 0.9864; respectively. While, predicted R2 value is 0.9427. Accuracy and reliability of the model can be seen in the small percentage of the coefficient of variation value (CV=5.44%), mean value (15.16), adequate precision value (31.469), PRESS value (40.03) and standard deviation value (0.82) (Table 5).
The significance of each coefficient was defined in terms of both P and F values listed in Table 5. It can be seen from the P-values and F-values that the linear coefficients of cholesterol concentration, interaction between the agitation speed and cholesterol concentration; agitation speed and yeast extract concentration; cholesterol concentration and yeast extract concentration and quadratic effects of agitation speed, cholesterol concentration and yeast extract concentration are significant as it is evident from the F-values of 119.05, 7.80, 62.24, 7.80, 321.59, 79.45, 526.73; respectively, and P-values of 0.0001, 0.0383, 0.0005, 0.0383, <0.0001, 0.0003, <0.0001; respectively. On the other hand, P-values of the linear coefficients of agitation speed (X1) and yeast extract concentration (X3) indicate that they had nonsignificant effects on cholesterol oxidase production by the strain under study.
The quadratic model of Box–Behnken design used for cholesterol oxidase production by Streptomyces anulatus strain NEAE-94, with a non-significant lack of fit (F-value 1.51 and P-value = 0.4219) and a very low P-value< 0.0001 was shown in the fit summary results (Supplementary Table 2). The largest adjusted and predicted R2 of 0.9864 and 0.9427 and the lowest standard deviation (0.82) was reported in the summary statistics of the quadratic model.
The optimum levels of agitation speed, cholesterol concentration and yeast extract concentration giving the maximum cholesterol oxidase production was evaluated by a second-order polynomial equation. Cholesterol oxidase production can be predicted by applying the following second-order regression equation in terms of the independent variables:
Y = 26.55+0.04X1+3.18X2 +0.35X3 +1.15X1 X2 -3.25X1X3 +1.15X2X3 -7.69X12 -3.82X22-9.84X32 Equation (4)
Where Y is the cholesterol oxidase production, X1 is the coded value of agitation speed, X2 is the coded value of cholesterol concentration and X3 is the coded value of yeast extract concentration.
Three dimensional (3D) surface and Contour plots
To understand the interaction among the three factors (X1 - X3) and the optimum level of each factor required for the maximum cholesterol oxidase production, the 3D curves and its corresponding contour plots were generated by plotting the cholesterol oxidase production on the Z axis versus two factors are allowed to vary and the third variable is fixed at its zero level (shown in Fig. 6A–C). Fig. 6A represents the cholesterol oxidase production as the simultaneous effect of agitation speed (X1), cholesterol concentration (X2) while yeast extract was kept at the central point (5 g/L). The cholesterol oxidase activity increases gradually by increasing cholesterol concentration and agitation speed till reach its optimum, but further increase in both cholesterol concentration and agitation speed leads to decrease in cholesterol oxidase activity. By solving the equation (4), the highest cholesterol oxidase production of 27.21 U/mL could be reached using 5 g/L yeast extract at the optimal predicted levels of agitation speed and cholesterol concentration of 150 rpm and 4.8 g/L; respectively.
The three-dimensional surface and contour plots in Fig. 6B illustrates cholesterol oxidase production as a function of agitation speed (X1) and yeast extract concentration (X3) while cholesterol concentration (X2) was fixed at the central point (4 g/L). Fig. 6B indicates that low agitation speed (X1) results in lower cholesterol oxidase production and with increasing the agitation speed, the cholesterol oxidase production increases beyond 150 rpm after which cholesterol oxidase production was reduced. Lower and higher concentrations of yeast extract (X3) results in lower cholesterol oxidase production, and the maximum cholesterol oxidase production, obviously obtained at the central level of the yeast extract concentration. By analysis of Fig. 6B and solving the equation (4), the maximum predicted cholesterol oxidase production of 26.55 U/mL could be reached using 4 g/L cholesterol at the optimal predicted levels of agitation speed (150 rpm) and yeast extract concentration (5 g/L).
Fig. 6C shows cholesterol oxidase production as influenced by cholesterol concentration (X2) and the concentration of yeast extract (X3) by maintaining the agitation speed at the central point (150 rpm). With an increased concentrations of both cholesterol and yeast extract, cholesterol oxidase production by the selected strain (Streptomyces anulatus strain NEAE-94) was improved and the maximum cholesterol oxidase production was obtained at the middle levels of two factors, and further increase of cholesterol concentration or yeast extract concentration decreases cholesterol oxidase activity. By analysis of Fig. 6C and solving the equation (4), the maximal predicted cholesterol oxidase production of 27.21 U/mL could be reached using agitation speed (150 rpm) and the optimal predicted levels of 4.8 g/L cholesterol and yeast extract concentration (5 g/L).
Verification of the model
Using the optimal levels of the process variables as obtained using Box–Behnken design, the experimental cholesterol oxidase production was verified and compared with the predicted value of cholesterol oxidase production (26.55 U/mL). The maximum experimental cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 was 27.31 U/mL. The verification revealed a high degree of model accuracy (97.21%).