Isolation of bacteria from local soil sample
The soil samples were collected from Hazaribagh tannery industry, Dhaka and local poultry farms of Savar, Dhaka. Then bacteria were isolated by serial dilution and spread plate technique. The keratin agar (KA) plates were incubated at 37 °C for 5–6 days. The bacterial isolates were further sub-cultured to obtain pure culture. The modified KA medium contains (g/l): NaCl (0.5 g), KH2PO4 (0.7 g), K2HPO4 (1.4 g), MgSO4·7H20 (0.1 g), Keratin (ceratin) (10 g) and agar (15 g) with pH 7.5 (Agrahari and Wadhwa 2010).
Screening the keratinase positive bacteria
Keratinase positive bacteria were cultured on skim milk agar and feather meal agar plates. The composition of skim milk agar medium was (g/l): casein 5.0 g, glucose 1.0 g, skim milk powder 3.0 g, yeast extract 2.5 g and agar 15.0 g as solidifying agent. The pH was adjusted to 7.5. Bacterial isolates were inoculated onto plates and incubated at 37 °C for 48 h. The clear zone forming isolates were selected as keratinase producer. 10% trichloroacetic acid (TCA) was flooded on the milk agar plate to observe the zone clearly (Saran et al. 2007).
Selected keratinase positive bacteria were further confirmed by using feather meal powder in the medium instead of keratin. The isolates those produce clear zones on both media were considered as keratinase producers.
Identification of keratinase positive bacteria
Morphological and a range of biochemical tests were performed in order to identify the isolate (Accession Number KY593174; Strain Number CGMCC 1.16131). The isolate was identified based on morphological, biochemical characteristics as described in the Bergey’s manual of systematic bacteriology and by 16S rRNA gene sequencing.
Primers set used to amplify 16S rRNA sequence were 27f (5′-AGA GTT TGA TCC TGG CTG AG-3′) and 1492r (5′-GGC TAC CTT GTT ACG ACT T-3′) as forward and reverse primer-respectively in a PCR thermal cycler (ICycler 170-8740, USA). The thermal cycling program was initial denaturation at 95 °C for 5 min, followed by 30 cycles, denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 90 s and the final extension at 72 °C for 10 min. The amplified DNA was visualized by gel electrophoresis (Additional file 1: Figure S1). The 16S rDNA sequence was analyzed and compared with other deposited sequences in the Genbank via the online programme BLAST (http://www.ncbi.nlm.nih.gov/). Neighbor-joining phylogenetic tree was constructed based on the 16S rRNA sequences using MEGA6 (Tamura et al. 2013).
Bacterial inoculums preparation
50 ml of nutrient broth (13 g/l at pH 7.4 ± 0.2) was prepared and sterilized in an autoclave (CL-40 M, Japan) at 15 lbs/in.2 pressure, 121 °C for 20 min. After cooling the media at room temperature, freshly grown single colony was transferred aseptically to it and incubated at 37 °C overnight at 150 rpm in a rotary shaking incubator (Stuart SI 500, UK).
Enzyme production in shake flask cultures
The keratinase enzyme production was carried out in the basal medium by using shaking flask. The composition of the media was (g/l): feather meal powder (10 g), NH4Cl (1 g), NaCl (1 g), K2HPO4 (0.6 g), KH2PO4 (0.8 g), MgCl2·6H2O (0.48 g) and yeast extract (0.2 g) with pH 7.5 (Rajesh et al. 2014). 2.5 ml of the bacterial inoculums was added in 50 ml of medium and cultured on a rotary shaking incubator (Stuart SI 500, UK) at 150 rpm and 37 °C for 72 h. After incubation, fermented broth was centrifuged at 5000 rpm for 20 min at 4 °C. The cell free supernatant was collected and used for the assay of keratinase activity.
Enzyme assay
The enzyme activity was determined by keratin digestion method using 1% keratin in 0.05 M Tris–HCl buffer (pH 8.0) as substrate according to Cai et al. (2008). The reaction mixtures contain 1.75 ml substrate solution and 0.25 ml crude enzyme solution. Then mixture was incubated at 40 °C in water bath for 10 min and reaction was terminated by adding 2.0 ml 0.4-M trichloroacetic acid (TCA). The control also was made by incubating the enzyme solution with 2.0 ml TCA without addition of keratin. The mixture was then centrifuged at 3825 rpm for 30 min and absorbance was measured at 280 nm by spectrophotometer (Jenway 6305, USA) against the control. One unit (U/ml) of enzyme activity is defined an increase of absorbance of 0.01 at 280 nm (A280) (Gradišar et al. 2005) per minute under the assay condition calculated by the following equation.
$$ {\text{U}} = 4\times n \times A 2 80/\left( {0.0 1\times 10} \right) $$
(1)
where n is the dilution factor; 4 is the final reaction volume (ml); 10 is the incubation time (min).
Optimization of cultural conditions for keratinase production
The production of keratinase by bacterial inoculums was studied by considering the media components and culture conditions. All the experiments were carried out in triplicate and the mean values were presented.
Effects of substrates on keratinase production
Various substrates such as keratin, casein, peptone, skim milk powder and feather meal powder were used (10 g/l) as main nutrient sources separately for the production of keratinase. Fermentation was carried out with 5% inoculums at 37 °C for 72 h at 150 rpm. The pH and volume of the media were 7.5 and 50 ml respectively.
Effects of organic and inorganic nitrogen sources on keratinase production
The keratinase production by the isolated bacterium strain was also optimized by supplementing different organic and inorganic nitrogen sources individually. The organic nitrogen sources such as tryptone, peptone, beef extract, gelatin and yeast extract were used as 0.02% concentration as well as NH4NO3, KNO3, NH4H2PO4, NH4SO4 and NH4Cl were used 0.1% concentration as inorganic nitrogen sources in the media.
Effects of temperature and pH on keratinase production
To determine the suitable temperature for enzyme production, the culture media were incubated from 28 to 52 °C (28, 33, 37, 42, 47, 52 °C) temperature and for determination of optimum pH for keratinase production experiments were carried out from 5 to 10 (5, 6, 7, 7.5, 8, 9, 10) separately. The pH was adjusted by using 0.1 N HCl or 0.1 N NaOH. Fermentation was carried out for 72 h as stated earlier.
Effects of incubation period and inoculum volume on keratinase production
The effect of incubation period on keratinase production was examined by carrying out fermentation up to 24, 48, 72 and 96 h separately. Different inoculums volumes (2, 3, 4, 5, 6, and 7%) in media were also used to determine the appropriate inoculum volumes require for maximum production of enzyme.
Effects of agitation speed on keratinase production
The effect of agitation speed for keratinase production was studied at 120, 140, 150, 160 and 180 rpm independently. The assay was conducted after 72 h.
Partial characterization of crude keratinase activity
The optimum pH for enzyme activity were determined by incubating the reaction mixture at various pH range from 5 to 10 at shaking water bath for 10 min at 40 °C using 0.05 M Tris–HCl buffer and the effect of temperature on enzyme activity were checked by incubating the reaction mixture at temperatures from 30 to 80 °C (30, 40, 50, 60, 70, 80 °C) by using same buffer for 10 min and assayed for enzyme activity.
Keratinase production at optimized condition
After standardization of all production parameters, enzyme production was carried out at optimum condition and compared with initial production rate.