Figure 1.
Phylogenetic tree based on 16S rRNA gene sequences within the radiation of the genus Bacillus.
The sequence of E. coli ATCC 11775T (X80725) was chosen arbitrarily as an outgroup. Bar, 0.02 nt substitutions per base. Numbers at nodes (>50%) indicate support for the internal branches within the tree obtained by bootstrap analysis (percentages of 100 bootstraps). NCBI accession numbers are presented in parentheses.
Figure 2.
Kinetic production and purification of KERUS.
(A) Time course of Br. brevis strain US575 cell growth (•) and KERUS production (○). Cell growth was monitored by measuring the OD at 600 nm. (B) Chromatography of the keratinase from Br. brevis US575 on Mono Q Sepharose. The column (2.6 cm×20 cm) was equilibrated with buffer C. Adsorbed material was eluted with a linear NaCl gradient (0 mM to 500 mM in buffer C) at a flow rate of 40 ml.h−1, and assayed for protein content at 280 nm (◊) and keratinase activity (♦) as described in Section 2. (B) SDS-PAGE of the purified keratinase. Lane 1, protein markers. Lane 2, purified KERUS (30 µg) obtained after Q Sepharose cation-exchange chromatography (fractions 26–34), (D) Zymogram activity staining of the purified keratinase, and (E) MALDI-TOF spectrum of 10 pmol purified KERUS from Br. brevis US575. The mass spectrum shows a series of multiply protonated molecular ions. The molecular mass of the enzyme was found to be 29121.11 Da.
Table 1.
Flow sheet for KERUS purification.
Table 2.
Effects of various inhibitors, reducing agents, and metal ions on KERUS stability.
Figure 3.
Effects of pH and temperature on the activity and stability of KERUS, NUE, and KOROPON.
Effects of pH on the activity (A) and stability (B) of KERUS, NUE, and KOROPON. The activity of the enzyme at optimum pH was taken as 100%. Buffer solutions used for pH activity and stability are presented in Section 2. Effects on the thermoactivity (C) and the thermostability of KERUS (D), NUE (E), and KOROPON (F). The enzyme was pre-incubated in the absence or presence of CaCl2 at various temperatures ranging from 30°C to 60°C. Residual protease activity was determined from 0 h to 72 h at 6 h intervals. The activity of the non-heated enzyme was taken as 100%. (G) Hydrolysis curves of chicken feather-meal proteins treated with various purified enzymes. The purified proteases used were: KERUS (♦), NUE (▪), and KOROPON (Δ). Each point represents the mean (n = 3) ± standard deviation.
Table 3.
Effect of substrate specificity on KERUS activity.
Table 4.
Kinetic parameters of purified keratinolytic proteases: KERUS, NUE, and KOROPON for the hydrolysis of protein, ester, and synthetic peptide substrates.
Figure 4.
Keratin(feather)-biodegradation by Br. brevis US575 and hide-dehairing function of KERUS.
(A) Feathers were incubated for 28 h at 37°C under shake culture condition with 3.5×107 cells ml−1 as an initial inoculum density for the US575 strain (right flask, t = 28 h) and with autoclaved inoculum as control (left flask, t = 0 h). (B) KERUS was incubated for 10 h at 37°C with feather, C = control. (C) KERUS was incubated for 10 h at 37°C with rabbit hair (R0: rabbit hair as control, t = 0 h; R10: rabbit hair treated, t = 10 h), (G0: goat hair as control, t = 0 h; G10: goat hair treated, t = 10 h), (B0: bovine hair as control, t = 0 h; B10: bovine hair treated, t = 10 h), (S0: sheep wool as control, t = 0 h; S10: sheep wool treated, t = 10 h).
Table 5.
Effect of keratinacious substrates on the keratinase KERUS of Br. brevis strain US575 after 28 h of incubation.
Figure 5.
Nucleotide and deduced amino acid sequences of the kerUS gene.
The kerUS consisted of 1149-bp encoding a polypeptide of 383 aa residues. The putative starting residues of the pre-protein (pre), pro-protein (pro), and mature protein (mature) are indicated. The nucleotide and predicted amino acids are numbered on the right and on the left. The inverted arrows indicate the position of the primers F-US and R-US. The catalytic center is indicated in bold and grey. The possible Shine-Dalgarno sequence and the transcriptional terminator sequences are bolded and underlined, and the putative −35 and −10 promoters are shown in bold. The black box indicates the N-terminal amino acid sequence of the purified KERUS.
Figure 6.
Amino acid sequence alignment of KERUS from Br. brevis strain US575 with other Bacillus keratinases.
The used keratinases are: the SAPB from B. pumilus strain CBS, the KERA1 from B. pumilus strain A1, and the KERK12 from B. pumilus strain KS12. The first amino acid of the mature keratinase, Ala, is counted as +1. The putative starting residues of the pre-peptide (pre), pro-peptide (pro), mature protease, and active site residues D32, H64, and S221 are indicated. X (highlighted character) shows amino acid changes in KERUS with regards to other serine keratinases.