Exoproduction and characterization of a detergent-stable alkaline keratinase from Arthrobacter sp. KFS-1

Highlights

• Arthrobacter sp. KFS-1 produced extracellular keratinase in basal salt medium.

• Process conditions optimization enhanced extracellular keratinase secretion.

• The enzyme was optimally active at pH 8.0 and 60 °C; with considerable pH and temperature stability.

• The enzyme sensitivity to protease inhibitors suggested a metallo-class of protease.

• The enzyme showed high compatibility with chemical agents and laundry detergents.

Abstract

Arthrobacter sp. KFS-1 previously isolated from a dump site was used to produce keratinase in basal medium. The physico-chemical conditions were optimized to enhance the keratinase production, and biochemical properties of the enzyme were also evaluated. Arthrobacter sp. KFS-1 optimally produced keratinase in a basal medium that contained 1.0 g/L xylose, 2.5–5.0 g/L chicken feather; with initial pH, incubation temperature and agitation speed of 6.0, 30 °C and 200 rpm, respectively. Maximum keratinase activity of 1559.09 ± 29.57 U/mL was achieved at 96 h of fermentation; while optimal thiol concentration of 665.13 ± 38.73 μM was obtained at 144 h. Furthermore, the enzyme was optimally active at pH 8.0 and 60 °C. The enzyme activity was inhibited by ethylene diamine tetraacetic acid and 1,10-phenanthroline, but not affected by phenylmethylsulfonyl floride. In addition, the crude enzyme retained 55%, 63%, 80%, 81% and 90% of the original activity after respective pretreatment with some commercial detergents (Maq, Omo, Surf, Sunlight and Ariel). Moreso, the enzyme showed remarkable stability in the presence of reducing agents, surfactants, and organic solvents. Arthrobacter sp. KFS-1 significantly produced keratinase which exhibited excellent stability in presence of chemical agents and commercial laundry detergents; hence, suggesting its industrial application potentials especially in detergent formulation.

Introduction

Keratinases (EC 3.4.21/24/99) are a group of hydrolytic enzymes that effectively participate in bioconversion of soluble and insoluble proteinacous substrates into amino acid and peptide fragments [1]. According to the sensitivity to protease inhibitors and nature of catalytic sites, keratinases predominantly belong to the serine- and metallo-class of proteases [2,3]. Keratinases have the potentials to revolutionise, and broaden the application of proteases due to their robustness, stability and catalytic tendencies as may be embodied in the bioconversion of recalcitrant keratinous biomass into functional products [4]. Perhaps, soon, keratinases would be a significant player in the proteolytic enzyme market.

Catalytic stability and tenacity for the dismemberment of keratinous biomass is a peculiar characteristic of keratinases [5]. However, the enzyme battery thought to present an effective and efficient disintegration of keratinous materials would include the synergy of keratinolytic protease and disulfide bond reductase-like protein [6]. The genetic complexity of keratinase producing microbes may have provided for the robust enzymes systems that mediate the complete breakdown of biomass composed of keratinous materials [4]. Some groups of bacterial and fungal species have been implicated in keratinase production [6,7]. Among the fungal species, keratinase production was predominantly reported within the confines of the pathogenic dermatophytes; consequently, the commercialisation prospects were restrained because of safety issues [7]. Therefore, the discovery of novel keratinases from non-pathogenic bacteria has increased over the past decades and has been on upward trajectory after the isolation and characteriation of feather degrading Bacillus licheniformis PWD-1 [8].

Microbial keratinase production is inducible as keratinous biomass in the fermentation media, in the absence of any other carbon and nitrogen sources, compels microbial species to express the keratinase genes [[9], [10], [11]]. The features of inducible enzyme production process offers the advantage not seen with the constitutive enzyme production systems. The keratinase production through the inducible-enzyme process offers the dual benefits of yielding extracellular keratinase and biomass degradation products that may have economic viability. The import of agro-waste keratinous biomass in the media constitution for the production of keratinases is another important advantage as upstream production processes would be highly cost effective [12,13].

Keratinase has profound prospects in sectors such as the feedstock, leather, cosmetic and detergent [14,15]. However, the prospects needs translation into contemporary applications, which has been hampered by factors including the insufficiency in application diversity of the commercially available keratinase [3]. The use of agro-industry waste keratinous biomass would open new vistas in keratinase production and application. Therefore, microbial exploration for novel keratinases must remain topical, and as such, the study reported herein was set out to optimize some process variables that enhanced extracellular keratinase production by Arthrobacter sp. KFS-1. The biochemical properties of the keratinase produced were likewise evaluated.