EXPERIMENT-TEST

Construction of tyrosine standard curve

Tyrosine powder was dissolved in water to prepare standard tyrosine solutions ( 0, 20, 40, 60, 80 and 100 μg/m. 100 μL standard tyrosine solution with different concentrations and 500 μL 0.4 M Na₂CO₃ were added to a 1.5 mL centrifuge tube, and then 100 μL folinol reagent is added. Putted the mix into water bath and heated at 40℃ for 20 min. After reaction, took 200 μL of the reaction solution and measured the OD660 absorbance. Drew a standard curve with the concentration of tyrosine as the abscissa and the absorbance at OD660 as the ordinate. The obtained standard curve formula is y=0.0143+0.00529x, R2=0.9993.

Method for determining the activity of keratinase

Added 100 μL of the tested sample and 100 μL of 2% keratin into a 1.5 mL centrifuge tube and gently blending and incubated at 50℃ for 20 min. 200 μL 0.4 M TCA was added to stop the reaction.The mix was centrifuged at 14000 rpm for 2 min. 100 μL of supernatant, 500 μL 0.4 M Na₂CO₃ and 100 μL Folin are mixed in a new 1.5 mL centrifuge tube. The mixture was incubate at 40℃ for 20 min and the keratinolytic activity was measured at OD660. The 0-time reaction solution was used as blank control. The keratinase activity is calculated according to the tyrosine standard curve. One unit of keratinase activity was defined as the amount of enzyme needed to release 1 μg tyrosine from keratin per min.

Determining amino acid and soluble protein content in feather hydrolysate

The amino acid concentration was determined using ninhydrin reagent41 after the removal of protein with 20% TCA. Isoleucine powder was dissolved in water to prepare standard solutions at 0, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 and 350 μg/mL to draw the standard curve. The obtained standard curve formula is y=0.0067x-0.9642, R2=0.9944.For samples, 200 μL of supernatant was mixed with 50 μL 0.1 M phosphate buffer (pH 8.04) and 50 μL 2% ninhydrin reagent. The mixture was heated in a water bath at 90℃ for 30 min, followed by the addition of 950 μL distilled water. The absorbance was measured at 570 nm to quantify the amino acids present in the sample according to the prepared standard curve.

The concentration of soluble protein was quantified using a bicinchoninic acid analysis kit (Takara) with bovine serum albumin as the standard.

Employing weightlessness method to evaluate feather degradation rate

The feather degradation rate was evaluated using the weight loss method[1]. After feathers were degraded by SCUT-3 the fermented medium was centrifuged at 6000 rpm for 10 min. The precipitation was filtered through Whatman No. 1 filter paper and thoroughly washed with double-distilled water. The residue was dried at 65℃ for 2 d and then weighed to calculate the weight loss. Feather degradation was determined by calculating the weight loss after degradation divided by the weight of the feather before fermentation.

Statistical Analysis

We use t-test with 95% confidence interval to test the validity of each statement. The condition of t-test (or two-sample t test) was satisfied since our sample distribution can be considered as normal distribution.

Reference

[1] Zhen Fang, Juan Zhanga, Baihong Liu. et al. Cloning, heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1. Process Biochem.2014, 49, 647–654.