Probing the crucial role of Leu31 and Thr33 of the Bacillus pumilus CBS alkaline protease in substrate recognition and enzymatic depilation of animal hide.

The sapB gene, encoding Bacillus pumilus CBS protease, and seven mutated genes (sapB-L31I, sapB-T33S, sapB-N99Y, sapB-L31I/T33S, sapB-L31I/N99Y, sapB-T33S/N99Y, and sapB-L31I/T33S/N99Y) were overexpressed in protease-deficient Bacillus subtilis DB430 and purified to homogeneity. SAPB-N99Y and rSAPB displayed the highest levels of keratinolytic activity, hydrolysis efficiency, and enzymatic depilation. Interestingly, and at the semi-industrial scale, rSAPB efficiently removed the hair of goat hides within a short time interval of 8 h, thus offering a promising opportunity for the attainment of a lime and sulphide-free depilation process. The efficacy of the process was supported by submitting depilated pelts and dyed crusts to scanning electron microscopic analysis, and the results showed well opened fibre bundles and no apparent damage to the collagen layer. The findings also revealed better physico-chemical properties and less effluent loads, which further confirmed the potential candidacy of the rSAPB enzyme for application in the leather industry to attain an ecofriendly process of animal hide depilation. More interestingly, the findings on the substrate specificity and kinetic properties of the enzyme using the synthetic peptide para-nitroanilide revealed strong preferences for an aliphatic amino-acid (valine) at position P1 for keratinases and an aromatic amino-acid (phenylalanine) at positions P1/P4 for subtilisins. Molecular modeling suggested the potential involvement of a Leu31 residue in a network of hydrophobic interactions, which could have shaped the S4 substrate binding site. The latter could be enlarged by mutating L31I, fitting more easily in position P4 than a phenylalanine residue. The molecular modeling of SAPB-T33S showed a potential S2 subside widening by a T33S mutation, thus suggesting its importance in substrate specificity

Flow sheet for KERUS purification.

a<p>The experiments were conducted three times and ± standard errors are reported.</p>b<p>One keratin unit is defined as an increase of 0.1 absorbance at 440 nm per minute, using keratin azure as a substrate under the experimental conditions used.</p>c<p>Amounts of protein were estimated by the method of Bradford <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076722#pone.0076722-Sambrook1" target="_blank">[21]</a>.</p

Effects of pH and temperature on the activity and stability of KERUS, NUE, and KOROPON.

<p>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.</p

Nucleotide and deduced amino acid sequences of the <i>kerUS</i> gene.

<p>The <i>kerUS</i> 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.</p

Phylogenetic tree based on 16S rRNA gene sequences within the radiation of the genus <i>Bacillus</i>.

<p>The sequence of <i>E. coli</i> ATCC 11775^T (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.</p

Kinetic parameters of purified keratinolytic proteases: KERUS, NUE, and KOROPON for the hydrolysis of protein, ester, and synthetic peptide substrates.

<p>Values represent the means of three replicates, and ± standard errors are reported.</p

Keratin(feather)-biodegradation by <i>Br. brevis</i> US575 and hide-dehairing function of KERUS.

<p>(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).</p

Effect of keratinacious substrates on the keratinase KERUS of <i>Br. brevis</i> strain US575 after 28 h of incubation.

<p>Values represent the means of three replicates, and ± standard errors are reported.</p

Kinetic production and purification of KERUS.

<p>(A) Time course of <i>Br. brevis</i> strain US575 cell growth (•) and KERUS production (○). Cell growth was monitored by measuring the OD at 600 nm. (B) Chromatography of the keratinase from <i>Br. brevis</i> 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 <i>Br. brevis</i> 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.</p

Effects of various inhibitors, reducing agents, and metal ions on KERUS stability.

a<p>Values represent the means of three replicates, and ± standard errors are reported.</p