Medium optimization for keratinase production in hair substrate by a new Bacillus subtilis KD-N2 using response surface methodology

Abstract

Culture medium for keratinase production from hair substrate by a new Bacillus subtilis strain, KD-N2, was optimized. Effects of culture conditions on keratinase production were tested, and optimal results were obtained with 10% inocula (v/v), 16 g/L hair substrate, an initial pH value of 6.5 and a culture volume of 20 mL. Several carbon sources (sucrose, cornflour) and nitrogen sources (yeast extract, tryptone and peptone) had positive effects on keratinase production, with sucrose giving optimal results. To improve keratinase yield, statistically based experimental designs were applied to optimize the culture medium. Fractional factorial design (FFD) experiments showed that MgSO4 and K2HPO4 were the most significant factors affecting keratinase production. Further central composite design (CCD) experiments indicated that the optimal MgSO4 and K2HPO4 concentrations were 0.91 and 2.38 g/L, respectively. Using an optimized fermentation medium (g/L: NaCl 1.0, CaCl2 0.05, KH2PO4 0.7, sucrose 3, MgSO4 0.91, K2HPO4 2.38), keratinase activity increased to 125 U/mL, an approximate 1.7-fold increase over the previous activity (75 U/mL). Human hair was degraded during the submerged cultivation.

Optimization of Physico-Chemical Parameters for Hyper Keratinase Production from a Newly Isolated Aspergillus sp. DHE7 using Chicken Feather as Substrate- Management of Biowaste

“…5, among all the carbon sources tested, sucrose proved to be the best for keratinase production yielding 226+4.6 U/mL followed by arabinose (198+2.5 U/mL) and glucose (195+3.1 U/mL), while maltose, raffinose and galactose negatively modulated keratinase production. This result is congruent with that reported by Cai and Zheng (2009) for the production of keratinase by Bacillus subtilis KD-N2. Similarly, Malviya et al (1992) and El-Naghy et al (1998) reported maximum keratinase production from Chrysosporium queenslandicum and C. georgiae, respectively, in the presence of keratin, which was further stimulated by the addition of glucose.…”

“…All fungal strains were quantitatively screened for keratinolytic activity in submerged fermentation conditions using chicken feathers as substrate in the basal medium. Results in Table 1, shown that the highest keratinase production was observed with isolate DHE7 (168±4.7 U/mL) followed by DHE15 (154±2.8 U/mL), DHE12 (127±2.7 U/mL) and DHE13 (123±3.6 U/mL), which are much higher than data reported by Cai and Zheng (2009) and Mazotto et al (2013) for extracellular keratinase production by Bacillus subtilis KD-N2 (75 U/mL) and A. niger 9D40 (21.3 U/mL), respectively. Therefore, this promising result justified the selection of strain DHE 7 for the following experimental studies.…”

“…They reported that higher feathers concentrations (30-50 g/L) resulted in repression of keratinase production. In contrast, Cai et al (2008) reported that 10 g/L of feather was the optimal concentration for the production of keratinase by Bacillus subtilis.…”

“…Therefore, because of the growing interest of microbial keratinases in different industrial sectors, it is essential to search for new keratinolyic microbes that produce novel keratinases with prospective industrial applications. However, the production of keratinase by microorganisms and biodegradation of keratin, vary considerably according to species, culture conditions and keratin substrates as reported by Cai and Zheng (2009).…”

A novel extracellular keratinase from Exiguobacterium sp. Dg1: enzyme production and dehairing application

“…Based on this fi nding, we could use substrate as much as 10 g/L to 25 g/L. This result was similar with B. sub• lis KD-N2 that produced highest kera" nase ac" vity at 16 g/L human hair as a substrate [20] and Bacillus sp. produced the highest kera" nase ac" vity in 2% feather meal as a substrate [21].…”