Production of surfactant-stable keratinase from Bacillus cereus YQ15 and its application as detergent additive

Abstract

Background: With the growing concern for the environment, there are trends that bio-utilization of keratinous waste by keratinases could ease the heavy burden of keratinous waste from the poultry processing and leather industry. Especially surfactant-stable keratinases are beneficial for the detergent industry. Therefore, the production of keratinase by Bacillus cereus YQ15 was improved; the characterization and use of keratinase in detergent were also studied.

Results: A novel alkaline keratinase-producing bacterium YQ15 was isolated from feather keratin-rich soil and was identified as Bacillus cereus. Based on the improvement of medium components and culture conditions, the maximum keratinase activity (925 U/mL) was obtained after 36 h of cultivation under conditions of 35 °C and 160 rpm. Moreover, it was observed that the optimal reacting temperature and pH of the keratinase are 60 °C and 10.0, respectively; the activity was severely inhibited by PMSF and EDTA. On the contrary, the keratinase showed remarkable stability in the existence of the various surfactants, including SDS, Tween 20, Tween 60, Tween 80, and Triton X-100. Especially, 5% of Tween 20 and Tween 60 increased the activity by 100% and 60%, respectively. Furtherly, the keratinase revealed high efficiency in removing blood stains.

Conclusion: The excellent compatibility with commercial detergents and the high washing efficiency of removing blood stains suggested its suitability for potential application as a bio-detergent additive.

Keywords: Bacillus cereus; Detergent compatibility; Keratinase; Production.

Fig. 1

Morphological graphs of B. cereus YQ15. a, the colony on skimmed milk agar medium; b, the bacterial image taken by TEM

Fig. 2

Neighbor-joining phylogenetic tree of the strain YQ15 based on the 16S rDNA gene sequences (Bootstrap values were based on 1000 replicates). The bar represents 0.005 substitutions per site. The 16S rDNA sequence of B. subtilis NZ 104 (MK184278) was used as the outgroup. The related bacterial accession numbers from NCBI database were showed in parentheses

Fig. 3

Effects of medium components on keratinase production by B. cereus YQ15 with an initial pH of 7.2 after cultivation at 37 °C and 200 rpm for 36 h. a, Keratinous inducers (10 g/L); b, Feather; c, Carbon sources (10 g/L); d, Soluble starch; e, Nitrogen sources (2 g/L); f, Peptone; g, Mineral salts (2 g/L); h, MgSO₄

Fig. 4

Effect of culturing conditions on the keratinase production by B. cereus YQ15 using the optimal medium. a, pH; b, Temperature; c, Inoculum density; d, Rotate speed

Fig. 5

Effects of pH and temperature on the keratinase activity. a, the optimal pH and pH stability of keratinase; b, the optimal temperature and thermal stability of keratinase. Black square and hollow circle indicated the enzyme activity and stability, respectively

Fig. 6

Compatibility and stability of the B. cereus YQ15 keratinase with commercial detergents after incubation at 50 °C for 30 min. The keratinase activity in absence of detergent was set as 100%. Different letters at the same concentration indicate significant differences (p ≤ 0.05)

Fig. 7

Substrate specificity of the B. cereus YQ15 keratinase

Fig. 8

Washing performance of B. cereus keratinase. a, the stain treated with 100 mL tap water; b, the stain treated with 100 mL detergent solution (0.7 mg/mL) for 20 min; c, the stain treated with 100 mL of crude enzyme solution (400 U/mL); d, the stain treated with 100 mL detergent and enzyme solution (0.7 mg/mL inactive commercial detergent combining with 200 U/mL keratinase) for 20 min