Strains, plasmids, and media
The original keratinase gene kerBp was mined and expressed in our previous studies (Su et al. 2017). B. subtilis WB600, Escherichia.coli JM109, and the vector pMA5 used in this experiment were all preserved in our laboratory.
The seed medium composition for E.coli and B. subtilis (g/L): tryptone 10, yeast extract 5, NaCl: 10. The fermentation medium composition for B. subtilis (g/L): glycerin 5, yeast extract 24, tryptone 12, K2HPO4 12.54, KH2PO4 2.31. The initial pH of the medium was natural pH. The optimized medium for B. subtilis (g/L): glucose 10, soyabean 18, soybean cake power 60, K2HPO4 12.54, KH2PO4 2.31.
Error-prone PCR and expression of keratinase
The gene kerBp was amplified with upstream primer (5′-CGG GAT CCA TGT GCG TTA AAA AGA AAA ATG TTA TGA CAA G-3′) and downstream primer (5′-GCA CGC GTT TAA TTT GAT GCT GCT TGC ACA TTA ATC-3′). The plasmid pMA5 was extracted and double-digested with the restriction enzyme Mlu I and Bam H. I. A randomly mutation library was constructed according to Zhang et al. (Zhang and Zhang 2011) with modifications by error-prone PCR reaction (5 mM MgCl2, 0.2 mM MnCl2, 0.2 mM dATP, 0.2 mM dGTP, 1 mM dCTP, 1 mM dTTP, 0.05 U/μL polymerase, and 0.4 mM each of the primers). The error-prone PCR was conducted by using the NEB Taq DNA polymerase (95 °C denaturation, 3 min; 29 cycles of 95 °C denaturation, 30 s; 57 °C annealing, 30 s; and 72 °C extension, 1.5 min, followed by 72 °C extension for 5 min). The error-prone PCR products were gel-purified and connected to the plasmid pMA5, which were tramsformed into E. coli JM109 competent cells. Plasmids obtained were finally expressed in B. subtilis WB600 and the strains with large transparent circles were selected directly. The mutant strains were cultured at 37 °C in 250-mL flasks containing 30 mL TB medium (50 µg/mL Kanr) for 60 h and the supernatant was collected by centrifuging at 4 °C and 8000g for 20 min for keratinase activity measurement.
Enzymatic properties of mutant keratinase
The optimum temperature of keratinase was determined by measuring enzyme activity at different temperatures (40, 45, 50, 55, 60, 65 and 70 °C). To evaluate thermal stability, the properly diluted keratinase solution was treated at series temperatures for 30 min and cooled on ice. The residual keratinase activity was determined at 50 °C according to the standard enzyme activity method, and the enzyme activity of the untreated enzyme solution was taken as the control. The optimum pH of keratinase was examined with the keratin substrate and enzyme solution appropriately diluted in series pH buffer (pH 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0).
Homology modeling of keratinase KerBp
The 3D structure of keratinase KerBp was homologous modeled with the crystal structure of subtilisin NAT (3VYN) from Bacillus subtilis (76% sequence similarity), subtilisin BPN (1TO2) from Bacillus amyloliquefaciens (76% sequence similarity), and subtilisin DY (1BH6) from Bacillus licheniformis (71% sequence similarity) as templates using Discovery Studio. Ramanchandran plot and profile-3D evaluation model were used to verify the rationality of the protein structure.
Scale-up production of keratinase in 7-L fermenter
Single colony of Bacillus subtilis with transparent circle was selected on LB-milk solid plate and inoculated with LB liquid medium for 12 h at 37 °C. 1 mL of the above seed solution was inoculated in 50 mL LB medium and cultured to OD600 0.6–0.8. The secondary seeds were transferred to a 7-L fermenter (Ependorff) with 5% inoculation for high-density fermentation, and kanamycin sulfate was added to 50 μg/mL finally. The speed of fermenter was set as 500 rpm and the temperature was 37 °C. 50% glacial acetic acid or 50% ammonia water was used to adjust the pH value around 7.5. The dissolved oxygen concentration at the initial stage of fermrntation is maintained at 20–30% by adjusting the speed. During the fermentation process, samples were taken periodically to detect bacterial concentration and enzyme activity. When a sudden increase in dissolved oxygen was detected, 50% glucose was added at a flow acceleration rate of 39.53 mL/h.
Feather degradation
The feather waste used for degradation was collected from a poultry farm (Wuxi, China). The feather degradation experiments were carried out in a 500-mL flask containing 50 mL enzyme solution and 10 g/L chicken feather waste. The prepared keratinase was applied to feather degradation, combining with papain, pepsin, trypsin, bromelain, flavor protease, neutral protease and alkaline protease. The optimal combination of enzymes was selected to degrade feathers and the degradation conditions of compound enzymes were optimized, including proportion of combined enzyme (The enzyme activity radio of trypsin and keratinase were 1:3, 1:2, 1:1, 2:1, 3:1, respectively), enzyme content (800, 1200, 1600, 2000, 2400, 2800 U/mL), temperature (30, 35, 40, 45, 50, 55 °C), pH (7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0), degradation time (8, 16, 24, 32, 40, 48 h) and sulfite content (0.1%, 0.5%, 1%, 1.5%, 2.0%, 2.5%).
Analytical methods
Keratinase assay
Add 100 μL 1% keratin substrate solution to 100 μL appropriately dissoluted keratinase solution, and then incubated at 50 °C for 20 min. Immediately after the reaction, add 200 μL 5%(W/V) TCA to stop the reaction. The control group was added with 200 μL TCA followed by 100 μL keratin substrate solution. Then, the processed samples were centrifuged at 12,000 rpm for 5 min. 200 μL supernatant was mixed with 1 mL 0.4 M Na2CO3 and 200 μL folinol solution, and placed in a 40 °C water bath for 20 min. The absorbance value was detected at 660 nm.
Feather degradation rate
The degradation products were filtered to leave the undegraded feathers, which then were washed with deionized water three times to completely remove the soluble substances and thallus, and dried at 65 °C for 24 h to constant weight. Degradation rate of feathers is defined as the change in dry weight before and after degradation.
Reducing power analysis of keratinase
Two different methods were used to evaluate the reducibility of keratinase. Reducing power was evaluated as the ability to reduce Fe3+ to Fe2+, using the potassium ferricyanide reduction method (Clerici et al. 2021). The diluted keratinase (300 μL) was mixed with 0.2 M PBS (300 μL) buffer solution (pH 6.6) and 300 μL 1% (W/V) potassium ferricyanate. After incubation at 50 °C for 20 min, 300 μL 10% (w/v) TCA was added to stop the reaction, and centrifuged at 4000 rpm for 10 min. Then 200 μL supernatant was mixed 800 μL 0.01% (W/V) ferric chloride solution and incubated at 30 °C for 10 min. Finally, the absorbance of the reaction system was detected at 700 nm. The value of the absorbance represents the level of reducing power.
2,2′-Azo-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical scavenging experiment is mainly based on Re et al. (1999). Add 10 μL of culture supernatant to 1 mL of ABTS radical working solution and messure the absorbance at 734 nm after 6 min. In the control group, 10 μL of distilled water was added to replace the culture supernatant. The ABTS radical scavenging is calculated as follows:
$${\text{Scavenging}}\,(\% ) = \left[ {{{\left( {{\text{Abs}}_{{{\text{control}}}} - {\text{Abs}}_{{{\text{sample}}}} } \right)} \mathord{\left/ {\vphantom {{\left( {{\text{Abs}}_{{{\text{control}}}} - {\text{Abs}}_{{{\text{sample}}}} } \right)} {{\text{Abs}}_{{{\text{control}}}} }}} \right. \kern-\nulldelimiterspace} {{\text{Abs}}_{{{\text{control}}}} }}} \right] \times 100.$$
Analysis of amino acids
The supernatant of degradation products was added with the same volume of TCA. After centrifugation for 30 min, the supernatant passed through a 0.2-μm membrane filter. The free amino acid composition was determined by high performance liquid chromatography (HPLC, Agilent 1260, Santa Clara, CA, USA) with o-phthalaldehyde-9-fluorovinyl methyl chloroformate (OPA-FMOC) pre-column derivatization. An Agilent spectroscopy system was used to calculate the concentration from the peak area obtained. The mobile phase is acetonitrile–methanol. The detector is VWD, the wavelength is 338 nm, and the flow rate is 1 mL/min. The chromatographic column is Hypersil ODS-2 (250 × 4.6 mm, 5 μm), the temperature is 40 °C, and the injection volume is 10 μL.
Analysis of soluble peptides
Based on the comparison of peak time and peak area, the molecular weight distribution of soluble peptides was determined by high performance liquid chromatography (Agilent 1260, USA). The samples were centrifuged at 8000 rpm for 5 min, and then the supernatant was collected and passed through a 0.2-μm membrane filter. The molecular weight distribution of soluble peptides was obtained by gradient elution on TSK gel G2000SWXL (7.8 × 300 mm) using phosphate buffer as mobile phase. The detector, wavelength and flow rate were VWD, 214 ηm and 0.8 mL/min, respectively (Peng et al. 2019).
Statistical analysis
All assays in this study were performed in triplicate. Data processing in this study was performed by using the mean standard deviation (± SD) and analyzed via GraphPad Prism 7 (San Diego, CA, USA).