**1. Introduction**

Keratinase (EC 3.4.99.11) is a type of protease enzyme that started to gain interest due to its broad application in industry. They are commonly extracellular inducible enzymes secreted by various microorganism in the medium containing keratin showing high substrate specificity toward keratin [1]. They are widely used in most of the biotechnological processing industry, mainly in feed formulation, nitrogen fertilizer, leather processing, and pharmaceutical industry [2–4].

In food and feed supplements, keratinase-treated feather is increasingly seen as a viable source of dietary protein, as the enzyme-treated final product preserved good nutritional value. Keratinases are expected to develop a significant total global demand comparable to other commercial proteases. Diverse class of keratinase have been isolated from various microbial populations, such as bacteria [5,6] actinomycetes [7,8], and fungi [9,10]. However, among bacteria, keratinase from *Bacillus* genera has been widely reported as keratinase from this genera and appears to be the most promising keratinase producer for commercial application [11,12]. In general, the reasons why *Bacillus* spp. are preferred in bioremediation and industrial biotechnology are due to their generally regarded as safe (GRAS) property and the capacity of selected *Bacillus* strains to produce and secrete large quantities (20–25 g/L) of extracellular enzymes [13]. This is also the reason as to why an increase in the number of reports on the isolation of keratin-degrading *Bacillus* spp. is on the rise.

Numerous commercial keratinases are from *Bacillus* spp., such as Versazyme from *B. licheniformis PWD-1* (Odetallah et al. 2005); Prionzyme (Genencor) and Cibenza DP100 ™, both also from *B. licheniformis* PWD-1; Esperase and Savinase (Novozymes A/S), both from *Bacillus* spp.; and Alcalse (Novozymes A/S) from *B. licheniformis* [14]. We have been approached by a small feather-processing company interested in feather-degrading technology with the main target in producing keratinase at ambient temperature (25 to 32 ◦C) without utilizing additional nitrogen sources and heating process to lower the cost. There are many *Bacillus* spp. keratin-degrading bacteria reported in the literature, but most require additional nitrogen sources, such as yeas<sup>t</sup> extract, peptone, soybean meal, ammonium ions, and soy flour [4,13,15–25], that may elevate the cost. Scouring through the literature shows that only two *Bacillus* spp. keratin-degrading bacteria fits the bill with chicken feather as the sources of carbon and nitrogen, but both required elevated temperatures 37 ◦C [26] and 50 ◦C [27] for optimum activity. In light of the current Covid-19 pandemic, where imported products have grea<sup>t</sup> di fficulties in being available, sometimes months at a time, this mean that local sources need to be developed.

The objectives of this work are to optimize keratinase production using feather as the only source of carbon and nitrogen and to numerically select the best conditions to maximize keratinase activity under ambient temperature and maximum feather concentration as a substrate. In this work, we report the optimization via response surface method (RSM) followed by a numerical optimization of a *Bacillus* sp. keratin-degrading bacterium having optimum ambient temperature for growth with chicken feather as the sole carbon and nitrogen sources.
