High keratinase and other types of hydrolase activity of the new strain of Bacillus paralicheniformis

Abstract

Keratinases, a subclass of proteases, are used to degrade keratin thereby forming peptones and free amino acids. Bacillus paralicheniformis strain T7 was isolated from soil and exhibited high keratinase, protease, collagenase, amylase, xylanase, lipase, and phosphatase activities. Keratinases of the strain showed maximum activity at 70°C and pH 9.0 as well as high thermal stability. A mass-spectrometric analysis identified seven peptidases with molecular masses of 26.8-154.8 kDa in the secretory proteome. These peptidases are members of S8 and S41 serine peptidase families and of M14, M42, and M55 metallopeptidase families. Additionally, α-amylase (55.2 kDa), alkaline phosphatase (59.8 kDa), and esterase (26.8 kDa) were detected. The strong keratinolytic properties of the strain were confirmed by degradation of chicken and goose feathers, which got completely hydrolyzed within 4 days. Submerged fermentation by strain B. paralicheniformis T7 was carried out in a pilot bioreactor, where the highest keratinase production was noted after 19 h of cultivation. After the fermentation, in the culture fluid, the keratinase activity toward keratin azure was 63.6 ± 5.8 U/mL. The protease activity against azocasein was 715.7 ± 40.2 U/mL. The possibility of obtaining enzyme preparations in liquid and powder form was demonstrated, and their comparative characteristics are given. In the concentrate, the keratinase, protease, α-amylase, phosphatase, and esterase/lipase activities were 2,656.7 ± 170.4, 29,886.7 ± 642.9, 176.1 ± 16.3, 23.9 ± 1.8, and 510.9 ± 12.2 U/mL, respectively. In the lyophilizate, these activities were 57,733.3 ± 8,911.4, 567,066.7 ± 4,822.2, 2,823.0 ± 266.8, 364.2 ± 74.8, and 17,618.0 ± 610.3 U/g, respectively. In the preparation obtained by air flow drying at 55°C, these activities were 53,466.7 ± 757.2, 585,333.3 ± 4,277.1, 2,395.8 ± 893.7, 416.7 ± 52.4, and 15,328.1 ± 528.6 U/g, respectively. The results show high potential of B. paralicheniformis strain T7 as a producer of keratinases and other enzymes for applications in agricultural raw materials and technologies for processing of keratin-containing animal waste.

Fig 1

The Т7 strain on skim milk (A), gelatin (B) and feather (C) agar after 24 h incubation at 37°C.

Fig 2

A: The impact of temperature on keratinase activity of the B. paralicheniformis T7 enzyme extract. B: The impact of temperature, at 50, 60, or 70°C, on the stability of the B. paralicheniformis T7 enzyme extract.

Fig 3

A: The impact of pH on keratinase activity in the B. paralicheniformis T7 enzyme extract. B: The impact of pH at 6.0, 8.0, and 10.0 on the stability of B. paralicheniformis T7 enzyme extract.

Fig 4

A zymogram with copolymerized keratin (A), casein (B), or gelatin (C) for the enzyme extract from B. paralicheniformis T7. Enzyme extract (lane 1), enzyme extract with PMSF (lane 2), enzyme extract with EDTA (lane 3), enzyme extract with Pepstatin A (lane 4), and enzyme extract with E64 (lane 5).

Fig 5. Hydrolysis of various proteins by the enzyme extract from B. paralicheniformis T7.

М: protein markers (NEB cat. # P7719S); 1: enzyme extract; 2: casein; 3: hydrolyzed casein in 15 s; 4: gelatin; 5: hydrolyzed gelatin in 5 min; 6: BSA; 7: hydrolyzed BSA in 5 min; 8: keratin; 9: hydrolyzed keratin in 30 min.

Fig 6

Degradation of goose (A) and chicken (B) feathers by B. paralicheniformis T7 culture for 96 h. Control: A control sample after 96 h of incubation without the addition of the bacterial culture.

Fig 7

An SEM image of a chicken feather after hydrolysis by B. paralicheniformis T7 strain, where (A) is an intact feather; (B–E) the feathers after 24, 48, 72, and 96 h of incubation, respectively; (F) the feathers after 24 h incubation (magnification 2000–4000×).

Fig 8. Pilot scale production of keratinases by B. paralicheniformis T7.