Cloning and characterization of a thermostable detergent‐compatible recombinant keratinase from Bacillus pumilus KS12

Abstract

Abstract.Functional expression of a keratinase from a potential feather-degrading bacterium, Bacillus pumilus KS12, was achieved in Escherichia coli using pEZZ18 vector. The enzyme was constitutively secreted at 37• C and 300 rpm after 18 H of incubation and was purified to homogeneity using diethylaminoethyl-Sepharose column. It was completely stable within the pH range of 7.0-10.0 with optima at pH 9.0, and temperature 30• C-90• C with optima at 70 • C. It had high thermostability with a t 1/2 of more than 4 H at 70• C, more than 2 H at 80• C, and 30 Min at 90• C. The enzyme was identified as a serine hydrolase as it was completely inhibited by 10 mM phenylmethylsulfonyl fluoride. It retained more than 90% relative activity in the presence of chelating agents such as ethylenediaminetetraacetic acid and 1,10-o-phenanthroline. It was also a thiol-activated enzyme with 3.32-and fourfold activation in the presence of 10 mM dithiothreitol and β-mercaptoethanol. The keratinase exhibited high detergent compatibility and oxidation stability with an eight-and fivefold enhancement in the presence of triton X-100 and saponin, respectively. It hydrolyzed a large array of protein substrates with a keratinolytic-caseinolytic ratio of more than 0.5. Amidolytic activity revealed its cleavage on phenylalanine → leucine → alanine-p-nitroanilides. In addition, electrospray ionization-mass spectrometry analysis of hydrolyzed insulin B-chain revealed cleavage between Leu 6 -Cys 7 , Cys 7 -Gly 8 , Tyr 16 -Leu

Zymogram Analysis of Alkaline Keratinase Produced by Nitrogen Fixing Bacillus pumilus ZED17 Exhibiting Multiprotease Enzyme Activities

“…Mazotto et al described that B. subtilis 1273 uses feather as a substrate for keratinase production (32). A 45-kDa keratinase obtained from B. pumilus KS12 was reported by Rajput et al (33).…”

Challenges and Opportunities in Identifying and Characterising Keratinases for Value-Added Peptide Production

“…In most cases the addition of denaturing and non-denaturing detergents resulted in an increase in activity (110-150%). However, the addition of the non-ionic detergents to the assay mixture with keratin as substrate of rK 27 had a dramatic effect on activity compared to the control without detergent [103]. Activities of 677% (Triton X-100), 242% (Tween 80), 461% (saponin) and 276% (cholate) were achieved.…”

“…Although many of the characterized enzymes have been produced by the native unmodified organism [94][95][96][97][98], several examples involve heterologous expression. Different organisms have been used for recombinant production, including yeast such as Komagataella Pastoris (Pichia Pastoris) [99] and bacteria such as Escherichia coli [100][101][102][103][104][105][106][107][108][109][110] and Streptomyces lividans [111]. Including the pre-pro-domains with the catalytic domain in heterologous systems have been shown to maintain enzyme activity and secretion [99,102,107,110] and inclusion of C-domains, when present, is important for substrate binding and recognition [105].…”

“…Keratin powder and solubilized keratin were generally obtained from commercial sources; however, the sources and preparation were not described. Pretreatments like autoclaving and milling [103,107], or treating with solvents at high temperatures [106], are known methods for keratin powder preparation from the literature. In the case of the rK 27 keratinase, the feather powder used in the assay was autoclaved and dried at 60 • C [103].…”

“…Pretreatments like autoclaving and milling [103,107], or treating with solvents at high temperatures [106], are known methods for keratin powder preparation from the literature. In the case of the rK 27 keratinase, the feather powder used in the assay was autoclaved and dried at 60 • C [103]. These preparation methods, as already described in Section 3, would compromise the keratin structure.…”