2.3.3. Numerical Optimization

As the company requested minimum costs to for the developed system, a numerical optimization was calculated so that the best conditions under the following criteria (Table 5)—minimum temperature, pH within range, minimum inoculum, maximum substrate (feather), and maximum keratinase activity—were obtained. Under the constraint criteria selected, ten solutions were obtained, and the best solution recommended was as follows.

**Table 5.** Numerical optimization for selected criteria for keratinase activity by *Bacillus* sp. strain UPMAGG-1.


For verification purposes, a series of validation experiment was conducted based on the conditions provided by CCD for optimum keratinase production (Table 6). Based on the provided solution, the highest keratinase activity obtained through solution 1 with a pH of 7.00, temperature 25.00, inoculums' size of 5.0 (v/v), feather concentration 4.97 (w/v) resulted in an activity of 56.218 U/mL with the desirability value of 0.968.

**Table 6.** Suggested value for each variable for optimum keratinase activity by *Bacillus* sp. strain UPMAGG-1.


*2.4. Amino Acid Profile of Hydrolysate of Bacillus sp. UPM-AAG1 Using High-Performance Liquid Chromatography (HPLC)*

Amino acid analysis profile of the keratinase from *Bacillus* sp. strain UPM-AAG1 (Figure 4) revealed the presence of 17 different amino acids, including essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine and valine, and non-essentials amino acids, like aspartic acid, glutamic, glycine, alanine, cysteine, tyrosine, arginine, serine, and proline, as evident from the HPLC chromatogram (Figure 5). The sequence is largely composed of phenylalanine (65.73 μmol/mL), isoleucine (24.04 μmol/mL), and lysine (20.14 μmol/mL) as essential amino acids and glutamine (32.48 μmol/mL), glycine (60.47 μmol/mL), and serine (158.42 μmol/mL) as a non-essential amino acid.

**Figure 4.** Amino acid profile of*Bacillus* sp. UPM-AAG1 hydrolysate. Error bars represent mean±standard deviation (n = 2).

**Figure 5.** High-performance liquid chromatography (HPLC) chromatogram of chicken feather degradation lysate by *Bacillus* sp. strain UPMAGG-1.
