**3. Factors affecting androstenedione production**

#### **3.1. Substrate solubility**

Several researchers have reported their work in developing a microbial biotransformation process with high yield of AD with wide industrial applications [9–11]. Therefore, it was important to understand the factors affecting the actual biotransformation process. The basic and the most reported research is on low solubility of phytosterols in the aqueous media. This leads to low mass transfer rate and low substrate availability for conversion of phytosterol to AD. Oxygen transfer rate was also reported to be another critical parameter in the biotransformation of phytosterol [12]. Along with oxygen transfer rate (OTR) the effect of by-products such as 1,4-HBC formed during the bioprocess was found to affect the amount of the products produced leading to a loss in yield by around 16.3 percent [12].

**Figure 3.** Enzymes involved in nucleus degradation of AD.

#### **3.2. Nucleus degradation**

The change in the steroidal nucleus of AD is one of the major obstacles reported in the microbial biotransformation of phytosterols. After formation of AD during microbial biotransformation, further degradation of AD takes place producing either ADD or 9-hydroxy androstenedione (9-OHAD) [7]. In a phenomenon which was coined as nucleus degradation by the researchers, two major enzymes have been implicated so far: 3-ketosteroid-1,2-dehydrogenase (KsdD) was found to be responsible for AD to ADD conversion and hence lead to low yield of AD. 3-ketosteroid-9-hydroxylase (Ksh), was observed to be responsible for AD to 9-OHAD conversion, leading to nucleus degradation and low yield of AD (**Figure 3**).

Xu et al. demonstrated that the enzymes involved in nucleus degradation of AD are temperature sensitive [7]. 3-ketosteroid-1,2-dehydrogenase (KsdD) and 3-ketosteroid-9-hydroxylase (Ksh) are enzymes sensitive at 37°. These enzymes break the B ring of steroidal nucleus which leads to degradation of the steroid. Hence it can be concluded that the microbial biotransformation of phytosterol is a temperature sensitive process.
