New Bacillus paralicheniformis strain with high proteolytic and keratinolytic activity

Abstract

Bacillus paralicheniformis T7, which exhibits high proteolytic and keratinolytic activities, was isolated from soil in Kazakhstan. Its secreted proteases were thermostable and alkaline, demonstrating maximum activity at 70 °C and pH 9.0. The proteases and keratinases of this strain were sensitive to Ni²⁺, Co²⁺, Mn²⁺, and Cd²⁺, with Cu²⁺, Co²⁺ and Cd²⁺ negatively affecting keratinolytic activity, and Fe³⁺ ions have a strong inhibitory effect on proteolytic and keratinolytic activity. Seven proteases were identified in the enzymatic extract of B. paralicheniformis T7: four from the serine peptidase family and three from the metallopeptidase family. The proteases hydrolyzed 1 mg of casein, hemoglobin, gelatin, ovalbumin, bovine serum albumin, or keratin within 15 s to 30 min. The high keratinolytic activity of this strain was confirmed through the degradation of chicken feathers, horns, hooves, wool, and cattle hide. Chicken feathers were hydrolyzed in 4 days, and the degrees of hydrolysis for cattle hide, wool, hoof, and horn after 7 days of cultivation were 97.2, 34.5, 29.6, and 3.6%, respectively. During submerged fermentation with feather medium in a laboratory bioreactor, the strain secreted enzymes with 249.20 ± 7.88 U/mL protease activity after 24 h. Thus, B. paralicheniformis T7 can be used to produce proteolytic and keratinolytic enzymes for application in processing proteinaceous raw materials and keratinous animal waste.

Keywords: Bacillus paralicheniformis T7; Keratinases; Proteases.

Fig. 1

Т7 strain on skim milk (a), gelatin (b) and feather (c) agar after 24 h incubation at 37 °C.

Fig. 2

Effect of temperature on proteolytic and keratinolytic activity of Bacillus paralicheniformis T7 enzymatic extract.

Fig. 3

Residual proteolytic activity of Bacillus paralicheniformis T7 enzymatic extract after 2 h incubation at 50 °С, 60 °С, and 70 °С.

Fig. 4

Effects pH on proteolytic and keratinolytic activity Bacillus paralicheniformis Т7 enzymatic extract.

Fig. 5

Zymogram with copolymerized casein (a), keratin (b), gelatin (c), and BSA (d) for the enzyme extract of B. paralicheniformis T7. Enzymatic extract (lane 1), enzymatic extract with PMSF (lane 2), enzymatic extract with EDTA (lane 3), enzymatic extract with Pepstatin A (lane 4), and enzymatic extract with E64 (lane 5). The raw zymograms are presented in the supplementary materials.

Fig. 6

Hydrolysis of the proteins by enzyme extract of Bacillus paralicheniformis T7: М – protein marker NEB cat.#P7719S; K – enzymatic extract; 1 – hemoglobin, 2 – hydrolyzed hemoglobin in 5 min, 3 – gelatin, 4 – hydrolyzed gelatin in 5 min, 5 – keratin, 6 – hydrolyzed keratin in 30 min, 7 – ovalbumin, 8 – hydrolyzed ovalbumin in 1 min; 9 – casein; 10 – hydrolyzed casein in 15 s; 11 – BSA; 12 – hydrolyzed BSA in 5 min. The raw gels are presented in the supplementary materials.

Fig. 7

Degradation of horn (a), hoof (b), hide (c), wool (d) and feather (e) samples using B. paralicheniformis T7 culture for 168 h. C - control sample after 168 h of incubation without addition of bacterial culture.

Fig. 8

Scanning electron microscopy image of a chicken feather after hydrolysis by B. licheniformis T7 strain, where (a) is a whole feather, (b) and (d) is a feather after 48 h of incubation, (c) is a feather after 72 h of incubation. Scale bars in (a), (d): 20 μm; in (b): 10 μm and in (c): 2 μm.

Fig. 9

Pilot scale production of proteolytic enzymes by B. paralicheniformis T7.