*2.4. Optimization of 7β-OH-DHEA Production by Absidia Coerulea CICC 41050* 2.4.1. Influence of Different Cosolvents

Figure 5A showed that, compared with the control group, using ethyl acetate, acetone, and ethanol as cosolvents can increase the transformation rate; when DMSO and chloroform were used as cosolvents, the transformation rate decreased. Therefore, acetone was selected as the best cosolvent. It can be seen from the results in Figure 5B that when the acetone concentration was 2%, the transformation rate was the highest. If the concentration of acetone was too low, the substrate could not be completely dissolved. However, with the increase in acetone concentration, acetone will have a toxic effect on fungi, inhibiting their growth and the activity of hydroxylase, thus affecting the conversion rate of substrate. Therefore, 2% acetone was selected as the cosolvent for the subsequent experiment. *Catalysts* **2022**, *12*, x FOR PEER REVIEW 6 of 13

**Figure 5.** The effect of cosolvent type(A) and concentration(B) on transformation rate. A, volume of cosolvent 1 mL(2%, V:V), concentration of DHEA 1 g/L, pH 6.5, 28 °C, 220 r/min. \**P* < 0.05, \*\**P* < 0.01. B, concentration of DHEA 1 g/L. **Figure 5.** The effect of cosolvent type (**A**) and concentration (**B**) on transformation rate. (**A**), volume of cosolvent 1 mL (2%, V:V), concentration of DHEA 1 g/L, pH 6.5, 28 ◦C, 220 r/min. \* *p* < 0.05, \*\* *p* < 0.01. (**B**), concentration of DHEA 1 g/L.

It can be seen from the results in Figure 6 A that the type of carbon source has a great impact on the transformation rate of 7β-OH-DHEA. When sucrose is the carbon source,

The Impact of the alternative to yeast extract, nitrogen sources (NH4NO3, (NH4)<sup>2</sup> SO4, peptone, yeast extract powder, and beef extract) on the improvement of 7β-hydroxylation catalyzed by *Absidia coerulea* CICC 41050 was investigated (Figure 6 C). Replacement of yeast extract with peptone provided an 8–10% higher 7β-OH-DHEA yield (up to 37%). The results showed that peptone as a nitrogen source was superior to the original nitrogen source (yeast extract) of the transformation medium (section 3.2). The effect of peptone at various concentrations on the transformation of DHEA by *Absidia coerulea* CICC 41050 was evaluated. The highest 7β-hydroxylase activity towards DHEA was reached at pep-

When studying the influence of pH of the transformation medium on DHEA conversion, it was shown that pH 6.5 provided the highest yield of 7β-OH-DHEA, while higher

in the transformation medium (section 3.2). The production of 7β-OH-DHEA by *Absidia coerulea* CICC 41050 also depends on the concentration of sucrose. The concentration of sucrose (40 g/L) provides the highest yield of 7β-OH-DHEA, which is higher than that of

30 g/L in the original transformation medium (section 3.2) (Figure 6 B).

acidic or alkaline pH negatively affected the transformation rate.

2.4.2. Effect of Key Nutrient Components and pH

tone content (15 g/L) (Figure 6 D).

#### 2.4.2. Effect of Key Nutrient Components and pH The results showed that peptone as a nitrogen source was superior to the original nitrogen

2.4.2. Effect of Key Nutrient Components and pH

0.01. B, concentration of DHEA 1 g/L.

*Catalysts* **2022**, *12*, x FOR PEER REVIEW 6 of 13

It can be seen from the results in Figure 6A that the type of carbon source has a great impact on the transformation rate of 7β-OH-DHEA. When sucrose is the carbon source, the transformation rate is the highest, which is consistent with the type of carbon source in the transformation medium (Section 3.2). The production of 7β-OH-DHEA by *Absidia coerulea* CICC 41050 also depends on the concentration of sucrose. The concentration of sucrose (40 g/L) provides the highest yield of 7β-OH-DHEA, which is higher than that of 30 g/L in the original transformation medium (Section 3.2) (Figure 6B). source (yeast extract) of the transformation medium (section 3.2). The effect of peptone at various concentrations on the transformation of DHEA by *Absidia coerulea* CICC 41050 was evaluated. The highest 7β-hydroxylase activity towards DHEA was reached at peptone content (15 g/L) (Figure 6 D). When studying the influence of pH of the transformation medium on DHEA conversion, it was shown that pH 6.5 provided the highest yield of 7β-OH-DHEA, while higher acidic or alkaline pH negatively affected the transformation rate.

30 g/L in the original transformation medium (section 3.2) (Figure 6 B).

**Figure 5.** The effect of cosolvent type(A) and concentration(B) on transformation rate. A, volume of cosolvent 1 mL(2%, V:V), concentration of DHEA 1 g/L, pH 6.5, 28 °C, 220 r/min. \**P* < 0.05, \*\**P* <

It can be seen from the results in Figure 6 A that the type of carbon source has a great impact on the transformation rate of 7β-OH-DHEA. When sucrose is the carbon source, the transformation rate is the highest, which is consistent with the type of carbon source in the transformation medium (section 3.2). The production of 7β-OH-DHEA by *Absidia coerulea* CICC 41050 also depends on the concentration of sucrose. The concentration of sucrose (40 g/L) provides the highest yield of 7β-OH-DHEA, which is higher than that of

The Impact of the alternative to yeast extract, nitrogen sources (NH4NO3, (NH4)<sup>2</sup> SO4, peptone, yeast extract powder, and beef extract) on the improvement of 7β-hydroxylation catalyzed by *Absidia coerulea* CICC 41050 was investigated (Figure 6 C). Replacement of yeast extract with peptone provided an 8–10% higher 7β-OH-DHEA yield (up to 37%).

**Figure 6.** The effect of carbon source (**A**,**B**), nitrogen source(**C**,**D**) and initial pH (**E**) on transformation rate. A&B, DHEA 1 g/L, acetone 2%, yeast extract 10 g/L, pH=6.5, A, 48 h B,24 h. \*\**P* < 0.01. C&D, 1: NH<sup>4</sup> NO3, 2:(NH4)<sup>2</sup> SO4, 3: peptone, 4: yeast extract, 5: yeast extract powder, 6: beef extract, DHEA 1 g/L, acetone 2%, sucrose 30 g/L, pH=6.5, C, 48 h D,24 h. \**P* < 0.05. E, DHEA 1 g/L, acetone 2%, yeast extract 10 g/L, sucrose 30 g/L. **Figure 6.** The effect of carbon source (**A**,**B**), nitrogen source (**C**,**D**) and initial pH (**E**) on transformation rate. (**A**,**B**), DHEA 1 g/L, acetone 2%, yeast extract 10 g/L, pH = 6.5, (**A**), 48 h (**B**), 24 h. \*\* *p* < 0.01. (**C**,**D**), 1: NH<sup>4</sup> NO<sup>3</sup> , 2: (NH<sup>4</sup> )<sup>2</sup> SO<sup>4</sup> , 3: peptone, 4: yeast extract, 5: yeast extract powder, 6: beef extract, DHEA 1 g/L, acetone 2%, sucrose 30 g/L, pH = 6.5, (**C**), 48 h (**D**), 24 h. \* *p* < 0.05. (**E**), DHEA 1 g/L, acetone 2%, yeast extract 10 g/L, sucrose 30 g/L.

According to the results of the single factor experiment, a three-factor and three-level orthogonal experiment were designed to explore the best medium composition (Table 2). The orthogonal experiment results are shown in Table 3. **Table 2.** Orthogonal experiment factors and levels assignment for medium composition. **Factor Level A/ sucrose**(**g/L**) **B/peptone**(**g/L**) **C/ initial pH** The Impact of the alternative to yeast extract, nitrogen sources (NH<sup>4</sup> NO3, (NH4)<sup>2</sup> SO4, peptone, yeast extract powder, and beef extract) on the improvement of 7β-hydroxylation catalyzed by *Absidia coerulea* CICC 41050 was investigated (Figure 6C). Replacement of yeast extract with peptone provided an 8–10% higher 7β-OH-DHEA yield (up to 37%). The results showed that peptone as a nitrogen source was superior to the original nitrogen source (yeast extract) of the transformation medium (Section 3.2). The effect of peptone at various concentrations on the transformation of DHEA by *Absidia coerulea* CICC 41050 was

> 1 30 10 5.5 2 40 15 6.5

**No. <sup>A</sup> <sup>B</sup> <sup>C</sup> Transformation rate**(**%**)

 1 1 1 41.75 1 2 2 40.48 1 3 3 37.92 2 1 2 39.80 2 2 3 32.02 2 3 1 28.75 3 1 3 23.58 3 2 1 39.29 3 3 2 46.57

**Factor**

**Table 3.** Orthogonal experimental design and results for medium composition.

K1 40.05 35.04 36.60 K2 33.52 37.26 42.28 evaluated. The highest 7β-hydroxylase activity towards DHEA was reached at peptone content (15 g/L) (Figure 6D).

When studying the influence of pH of the transformation medium on DHEA conversion, it was shown that pH 6.5 provided the highest yield of 7β-OH-DHEA, while higher acidic or alkaline pH negatively affected the transformation rate.

According to the results of the single factor experiment, a three-factor and three-level orthogonal experiment were designed to explore the best medium composition (Table 2). The orthogonal experiment results are shown in Table 3.

**Table 2.** Orthogonal experiment factors and levels assignment for medium composition.


**Table 3.** Orthogonal experimental design and results for medium composition.


Table 3 showed that the order of influence of the three factors on the transformation rate is C > A > B. Through range analysis, the optimal combination of the three factors is C<sup>2</sup> A<sup>1</sup> B3: initial pH 6.5, sucrose 30 g/L, and peptone 20 g/L. However, the compositionoptimized medium is not in Table 3, and verification experiments are required. Three parallel experiments were carried out. It was defined that an initial pH 6.5, sucrose 30 g/L, and peptone 20 g/L provided the maximum production (50.48%) of 7β-OH-DHEA by *Absidia coerulea* CICC 41050.

#### 2.4.3. Effect of Biotransformation Conditions

Figure 7A shows that when the inoculum is less than 12%, the transformation rate increases with the increase of the inoculum, and when the inoculum is more than 12%, the transformation rate decreases. The production of 7β-OH-DHEA by *Absidia coerulea* CICC 41050 also depended on medium volume (Figure 7B). The transformation rate is highest when the medium volume is 60 mL in a 250 mL Erlenmeyer flask. When the volume of the medium is too large, the ventilation and dissolved oxygen in the medium are poor. The transformation rate reached its highest when the substrate was added for 48 h and became extremely low after 96 h (Figure 7C). It is speculated that the nutrients in the medium were consumed and the enzyme activity decreased. The effect of different concentrations of substrate (DHEA, 0.5–8.0 g/L) in the transformation medium was estimated. Figure 7D shows that 1 g/L DHEA can provide the highest transformation rate of 7β-OH-DHEA, while at more than 1 g/L, the transformation rate is declining; greater than 6 g/L, the transformation rate is very low, and the substrate is almost completely converted. The

reason may be that the concentration of cosolvent increases with the increase in substrate concentration, and the toxicity of cosolvent inhibits the growth of fungi, thus negatively affecting DHEA conversion. reason may be that the concentration of cosolvent increases with the increase in substrate concentration, and the toxicity of cosolvent inhibits the growth of fungi, thus negatively affecting DHEA conversion.

*Catalysts* **2022**, *12*, x FOR PEER REVIEW 8 of 13

*sidia coerulea* CICC 41050.

2.4.3. Effect of Biotransformation Conditions

K3 36.48 37.75 31.17 R 6.53 2.70 11.11

Table 3 showed that the order of influence of the three factors on the transformation rate is C > A > B. Through range analysis, the optimal combination of the three factors is C2 A1 B3: initial pH 6.5, sucrose 30 g/L, and peptone 20 g/L. However, the compositionoptimized medium is not in Table 3, and verification experiments are required. Three parallel experiments were carried out. It was defined that an initial pH 6.5, sucrose 30 g/L, and peptone 20 g/L provided the maximum production (50.48%) of 7β-OH-DHEA by *Ab-*

Figure 7A shows that when the inoculum is less than 12%, the transformation rate increases with the increase of the inoculum, and when the inoculum is more than 12%, the transformation rate decreases. The production of 7β-OH-DHEA by *Absidia coerulea* CICC 41050 also depended on medium volume (Figure 7B). The transformation rate is highest when the medium volume is 60 mL in a 250 mL Erlenmeyer flask. When the volume of the medium is too large, the ventilation and dissolved oxygen in the medium are poor. The transformation rate reached its highest when the substrate was added for 48 h and became extremely low after 96 h (Figure 7C). It is speculated that the nutrients in the medium were consumed and the enzyme activity decreased. The effect of different concentrations of substrate (DHEA, 0.5–8.0 g/L) in the transformation medium was estimated. Figure 7D shows that 1 g/L DHEA can provide the highest transformation rate of 7β-OH-DHEA, while at more than 1 g/L, the transformation rate is declining; greater than 6 g/L, the transformation rate is very low, and the substrate is almost completely converted. The

**Figure 7.** The effect of biotransformation conditions. (**A**–**D**), the composition-optimized medium: initial pH 6.5, sucrose 30 g/L, and peptone 20 g/L. **Figure 7.** The effect of biotransformation conditions. (**A**–**D**), the composition-optimized medium: initial pH 6.5, sucrose 30 g/L, and peptone 20 g/L.

According to the results of the single-factor experiment, an orthogonal experiment with four factors and three levels was designed to explore the optimal biotransformation conditions (Table 4). The orthogonal experiment results are shown in Table 5.



It can be seen from the results in Table 5 that the order of influence of the four factors in the orthogonal experiment of biotransformation conditions on the transformation rate is A > C > B > D. Through range analysis, the optimal combination of the four factors is A<sup>1</sup> C<sup>2</sup> B<sup>2</sup> D1: DHEA 1 g/L, medium volume 60 mL, biotransformation time 48 h, and inoculum 10%. As the optimal biotransformation conditions are not listed in Table 5, validation tests are required.

Three parallel experiments were carried out. It was defined that sucrose 30 g/L, peptone 20 g/L, corn steep liquor 10 g/L, K<sup>2</sup> HPO<sup>4</sup> 2 g/L, KH<sup>2</sup> PO<sup>4</sup> 1.6 g/L, MgSO<sup>4</sup> 0.5 g/L, FeSO<sup>4</sup> 0.05 g/L, pH 6.5, DHEA 1 g/L, medium volume 60 mL, biotransformation time 48 h, and inoculum 10% provided maximum production (69.61%) of 7β-OH-DHEA by *Absidia coerulea* CICC 41050. Compared with the highest transformation rate of 62.81% in the orthogonal test and the primary transformation rate of 27.23%, transformation rate was increased by 6.80% and 42.38% respectively.


**Table 5.** Orthogonal experimental design and results for biotransformation conditions.

#### **3. Materials and Methods**

#### *3.1. Chemicals*

Dehydroepiandrosterone (DHEA) was obtained from Hubei Gongtong Pharmaceutical Co., Ltd. (Xiangyang city, Hubei, China). Methanol and acetonitrile were purchased from Concord Technology Co., Ltd. (Tianjin, Tianjin, China). Yeast extract was purchased from HopeBio Co., Ltd. (Qingdao, Shandong, China). All other chemical reagents were purchased from Yuwang Chemical Co., Ltd. (Shenyang, Liaoning, China).

#### *3.2. Microorganism and Cultivation*

*Absidia coerulea* 41050 and *Gibberella sp.* 2498 were purchased from the China Center of Industrial Culture Collection (CICC).

Potato dextrose agar (PDA) is composed of potatoes (200 g), glucose (20 g), agar (20 g), and 1000 mL distilled water. Seed culture media (g/L) are composed of potato starch (45 g), yeast extract (3 g), corn steep liquor (10 g), CaCO<sup>3</sup> (3 g), MgSO<sup>4</sup> (0.5 g), and FeSO4 (0.05 g). Transformation media (g/L): sucrose (30 g), yeast extract (10 g), corn steep liquor (10 g), K<sup>2</sup> HPO<sup>4</sup> (2 g), KH<sup>2</sup> PO<sup>4</sup> (1.6 g), MgSO<sup>4</sup> (0.5 g), FeSO<sup>4</sup> (0.05 g), pH 6.5.

The fungi were routinely maintained on PDA slants. To obtain first-generation mycelium, the spore suspension from one agar slant (1 week old) was inoculated aerobically in 50 mL of seed culture media on a rotary shaker (200 rpm) at 28 ◦C, for 48 h in Erlenmeyer flasks (250 mL). Then 5 mL of seed culture were inoculated into the transformation medium (50 mL in 250 mL Erlenmeyer flask) and cultured at 28 ◦C, 200 rpm for 5 days. Substrate controls were set without inoculating the fungi into the media and strain controls were set without adding the substrate into the media, with all other conditions remaining the same.

#### *3.3. Sample Preparation*

The cultivation broth was centrifuged (3000 r/min, 10 min) to obtain mycelia and transformation solution after 5 days. The mycelium and the transformation solution, with an equal volume of ethyl acetate, were extracted. After three extractions, the extraction solution was combined, evaporated under reduced pressure on the rotary evaporator, and then redissolved with 5 mL of methanol. Added an appropriate amount of anhydrous magnesium sulfate to dry and obtained the transformation sample for detection.

#### *3.4. Thin Layer Chromatography (TLC)*

The concentrated extract was analyzed by TLC. TLC on silica gel 60 F254 (25 aluminum sheets 20 × 20 cm; Merck, New York, NY, USA) with a solvent mixture of CHCl3-CH<sup>3</sup> OH (10:1, *v*/*v*) was applied to separate the metabolites and stained by spraying the plates with H<sup>2</sup> SO4/CH<sup>3</sup> CH<sup>2</sup> OH mixture (1:9, *v*/*v*). A UV light at 254 nm was used to visualize them.

#### *3.5. HPLC Detection*

A 0.1 mL of conversion product containing dehydroepiandrosterone was diluted five times with methanol and then filtrated by 0.45 µM organic membrane to obtain the sample solution. HPLC analysis was performed on a WondaSil C18 Superb column (5 µm, 4.6 mm × 250 mm, Shimadzu, Kyoto, Japan) with a methanol/water mixture (62:38, *v*/*v*), as mobile phase was at 30 ◦C with UV absorbance detection of 206 nm. Flow rate: 0.8 mL/min; injection volume: 10 µL.

#### *3.6. Isolation and Identification of Major Metabolite*

Isolation of the target metabolite was performed by semi-preparative HPLC. HPLC analysis was performed on a SinoChrom ODS-BP column (5 µm, 10 mm × 250 mm, Elite, China) with an acetonitrile/water mixture (30:70, *v*/*v*) as mobile phase at 36 ◦C with UV absorbance detection of 206 nm. Flow rate: 3.7 mL/min; injection volume: 100 µL.

Purified metabolites were identified by ESI-MS and NMR analysis under standard conditions. <sup>1</sup> H, <sup>13</sup> C NMR spectra were taken using a Brüker AVANCE III 400 instrument (Bruker Biospin AG, Fallanden, Switzerland). <sup>1</sup> H NMR spectra were recorded in CDCl<sup>3</sup> and DMSO-d<sup>6</sup> using tetramethylsilane (TMS) as an internal standard. Mass spectra were taken in ESI mode on an Agilent 1200 LC-MS (Agilent, Santa Clara, CA, USA).

#### *3.7. Establishment of Standard Curve and Calculation of Transformation Rate*

The 7*β*-OH-DHEA samples were dissolved in methanol and prepared into solutions with different concentration gradients (0.05, 0.10, 0.15, 0.20, 0.25 mg/mL). After filtering, carry out HPLC detection. Draw a standard curve with the concentration of DHEA as the abscissa and the peak area as the ordinate. The transformation rate is calculated as follows:

$$\text{Transformation rate} = \frac{A \times M\_b}{B \times M\_a} \times 100\% \tag{1}$$

where *A* is the quantity of product (g), and *B* is the quantity of substrate (g). *Ma* and *Mb* are the relative molecular weight of the product (7β-OH-DHEA, 304.41) and the relative molecular weight of the substrate (DHEA, 288.43), respectively (Figure S10).
