Engineered pro‐peptide enhances the catalytic activity of keratinase to improve the conversion ability of feather waste

Abstract

Keratinase is an attractive industrial enzyme that can specifically catalyze keratin waste to obtain value-added products. A challenge to the application of keratinase is improving catalytic capacity to achieve efficient hydrolysis. In this study, we effectively expressed the keratinase gene from Bacillus licheniformis BBE11-1 in Bacillus subtilis WB600 based on pro-peptide engineering. Partial deletion of the pro-peptide sequence and the substitution of amino acid at the pro-peptide cleavage site (P1) suggested that the "chaperone effect" and "cleavage efficiency" of the pro-peptide determine the activity of the mature enzyme. Subsequently, seven target sites that can increase the activity of the mature enzyme by 16%-66% were obtained through the multiple sequence alignment of pro-peptides and site-directed mutation. We further performed combinatorial mutations at six sites based on the design principle of three-codon saturation mutations and obtained mutant 2-D12 (236.8 KU/mg) with a mature enzyme activity of 186% of the original (127.6 KU/mg). Finally, continuous fermentation was carried out in a 5-L bioreactor for 22 h, and the activity of the 2-D12 mature enzyme was increased to 391.6 KU/mg. Most importantly, 2-D12 could degrade more than 90% of feather waste into amino acids and peptides within 12 h with the aid of sulfite.

Modifying the Substrate Specificity of Keratinase for Industrial Dehairing to Replace Lime-Sulfide

“…Identify the Dehairing Ability of Keratinase KerZ1 and Its Damage to Collagen. Given that KerZ1 has shown high keratinase activity (73710.22 U mL −1 , Figure 1A), 15 we selected sheepskin and cowhide with hair to test its dehairing ability and application potential. The results showed that KerZ1 had higher dehairing efficiency for sheepskin.…”

“…42 To explore the industrial production potential of the variant strain, fermentation test was carried out on the S100D/Y208V in 3-L bioreactor using a fed-batch strategy based on industrial media. 15 In view of past experience, excessively high glucose concentration will cause the accumulation of byproduct lactic acid, while less carbon source will inhibit cell growth and enzyme production. 43 Therefore, it was appropriate to pump glucose periodically at a constant speed.…”

“…The keratinase from Bacillus paralicheniformis MKU3 requires 24 h to completely remove wool . To this end, researchers have developed several over-expressed keratinases and their variants. , However, keratinase also exhibits hydrolytic activity on fibrin such as collagen and elastin due to poor substrate specificity . Leather damage caused by the hydrolytic activity of keratinase on collagen has become a new challenge .…”

Perspectives on Converting Keratin-Containing Wastes Into Biofertilizers for Sustainable Agriculture

“…It is shown that point mutation N122Y in keratinase increased the enzymatic activity with an approximately 5.6-fold (liu et al, 2013a ). As the N- and C-termini of a protease may play a regulatory role in the enzymatic activity, swapping these regions with similar domains of enzymes from other bacteria can also result in an enzyme with improved activity and stability (Fang et al, 2016 ; Peng et al, 2021 ).…”

Structure, Application, and Biochemistry of Microbial Keratinases

“…Other methods such as PCR-based methods and direct evolution will play a role in obtaining more potent keratinases ( Vidmar and Vodovnik, 2018 ). When the regulatory mechanism of a keratinase is understood, the modification on other regions of the keratinase can also improve its activity and stability ( Fang et al, 2016b ; Peng et al, 2021 ). In a study, the N- and C-terminal regions of KerSMD were replaced with those regions of a homogenous keratinase.…”