Department of Post Graduate Studies and Research in Microbiology, Gulbarga
University, Kalaburagi, Karnataka, India.

Corresponding author email: drvandanarathods@gmail.com

Article Publishing History

INTRODUCTION

The insoluble keratin which is highly stable is present in fur, feathers, beak, horns, nails, and hair of living animals (Onifade et al. 1998; Pandian et al. 2012). The length of Keratin fibre depends on their water-containing complex hydrates, which increases their length by 10-12% (Bhuyar et al. 2018). According to the sulfur content, hair, nail, hoof and also feathers are grouped as hard keratins where as  callus and skin belong to soft keratins. Hard keratins from chicken feathers obtained from feather industries have high level of disulphide bonds, cross linked structures which are resistant and insoluble, having hydrophobic interactions, (Annapurna et al. 1996). Chicken feathers constitute 5-7% of keratin producing a large number of poultry wastes which decomposes slowly causing massive environmental concerns, (Ningthoujam et al. 2016; Almahasheer et al. 2022).

Feather keratin characteristically attributes to the presence of various amino acids (Kumar et al. 2011; Gupta and Singh 2014). Physical and chemical methods of chicken feather degradation consumes high energy, damaging the products (Zoccola et al. 2012; Lee et al. 2016; Holkar et al. 2016). Hence, an alternative method is suggested for converting keratinous wastes into economic, reusable, eco-friendly products by using such enzymes produced from vast microbial sources. Such enzymes are produced by various keratinolytic microorganisms. Keratinous substrates can be hydrolyzed by keratin degrading enzymes which have potential applications. Keratinases are produced by fungi, bacteria and actinomycetes (Onifade et al. 1998; Gupta and Ramnani 2006; Bhuyar et al. 2018; Almahasheer et al. 2022). Feather degradation by the enzyme keratinase produced by B. subtilis, B. cereus, and B. licheniformis has been reported (Thys et al. 2004; Almahasheer et al. 2022).

Keratinase are used in several biotechnological processes, such as removing hair and feather for the production of feather meal, clearing the obstructions in the sewage systems and dehairing process in the leather industry (Gupta et al. 2002). Other uses include production of biogas, biodegradable films, glues, prion hydrolysis, wastewater processing, recovery of silver from X-ray films, drug delivery in medicine cosmetics and additives (Patinvoh et al. 2016; Almahasheer et al. 2022).

This work highlights the characterization and purification of the enzyme keratinase from Aneurinibacillus aneurinilyticus isolated from an endophytic xerophyte (Opuntia–ficus indica). Several studies have been done in the past on keratinase production, purification and characterization by bacteria. However, this paper is one-of-its-kind that has isolated endophytic bacteria from xerophytes in drought regions. These enzymes from such bacteria can tolerate high temperatures and pH which can be used in various biotechnological industries. The keratin degrading protein i.e., keratinases has diversified application in green technology. Therefore, there is a search for an efficient bacterial strain by the researchers (Almahashree et al. 2022). The keratin from poultry waste is degraded by the purified enzyme keratinases from B. licheniformis dcs1, has the efficiency to enhance the nutritional quality of the waste keratin from poultry, (Liaqat et al 2022).

MATERIAL AND METHODS

Bacteria was grown in 1000 ml production media under submerged fermentation and fermenting broth at 10,000 rpm at 4⁰C for 10 mins and it was centrifuged. Obtained supernatant was considered as a crude enzyme for purification purposes and at each step, enzyme activity was estimated. The crude enzyme from A. aneurinilyticus was partially purified further, ammonium sulphate precipitation was used at different saturation levels (10-80%) as per the standard chart with continuous stirring overnight under cold conditions to precipitate the desired protein.

Collected precipitate was centrifuged at 4⁰C for 10 min., further protein pellet was dissolved in 20 mmol/L Tris-HCl buffer (pH8.0). Folin-Phenol reagent along with bovine serum albumin was used as standard for protein estimation. The protein sample was loaded into the activated dialysis membrane and sealed at both ends. The dialysis 150 membrane bag was suspended in 500mL (50 mM, pH 7.0) phosphate buffer was used. Experiment was carried out with four changes in the buffer (Kamble et al. 2020).

The dialysate obtained from the above step was concentrated using (DEAE-cellulose), chromatographic anion coloumn (1.5X30cm). Sodium chloride with different concentrations (pH-8.0) in 20mmol/L Tris HCl buffer was used to elute the sample at 6 ml/min flow rate for collecting the fractions (Cheng-Gang Cai et al. 2008). The concentrated enzyme was equilibrated with 100mM Tris-HCl buffer (pH8.0) and Sephadex G-100 (50cm X 1.7 Cm) to prepare for gel filtration. The eluted pure protein fractions were characterized at the rate of 1ml/min of the fraction collected using same buffer. 10% separating acryl amide gel and 5% stacking gel having 0.1% SDS were used to visualize protein bands through Coomassei Brilliant Blue R-250. The protein bands were then destained with acetic acid and methanol. Pure enzymes were used for characterization (Lammli 1970; Cheng-Gang Cai et al. 2008).

Bovine Serum Albumin 66KDa, Lysozyme 14.3kDa, Phosphorylase 97kDa, Ovalbumin 45kDa and Bovine Carbonic Anhydrase 31 kDa, were used as standard protein markers (Eun-Jan Jeong et al. 2010). Zymogram analysis was performed by using 2% keratin as substrate copolymerized with stacking gel of 4% and 10% resolving gel, the sample buffer and purified proteins together were loaded to polymerized gel. Tris-HCl buffer 0.01M having a pH of 9.0 Triton X-100 (v/v) 2.0% were subjected to electrophoresis, for about half an hour. To remove Triton X-100 the gel was washed with D. H₂O and incubated at 37⁰C for 30 min.

After incubating at pH-9.0 and 37⁰C for 30mins in Tris-Cl, stained with Coomassie Brilliant Blue Dye R-250 for half an hour then de-stained, clear colourless zone was detected and considered as enzyme activity (Vermelho et al. 2009). For the characterization of purified keratinase, various physicochemical factors like temperature, pH, metal ions, enzyme inhibitors along with substrate were studied. Enzyme activity was noted using 0.1M acetate buffer (pH3-5), sodium phosphate buffer 0.1M (pH 6.0-8.0), glycine sodium hydroxide buffer (pH 9.0-12.0) with 0.1M Tris HCl buffer (pH 7.0-9.0) and temperature 10-80⁰C were taken to study the effect of pH and temperature on enzyme activity. The residual activities were measured by studying the effect of temperature and pH on enzyme stability by incubating the enzyme solution at pH between 3.0-12.0 and temperature between 10-80⁰C for 60 min (Murthy et al. 2019).

The effect of enzyme inhibitors such as Ethylene Diamine Tetra Acetic Acid (EDTA), Phenyl Methyl Sulfonyl Fluoride (PMSF), Dithiothreitol (DTT), Diisopropyl Fluoro Phosphate (DFP), Sodium Dodecyl Sulfate (SDS), β-mercaptoethanol, Triton X-114, Triton X-100, Tween-60, at 5mM (1%) along with Purified keratinase were pre-incubated in 100mM Glycine /NaOH buffer for 1h. The enzyme without inhibitor served as control and was taken as 100% activity (Ghasemi et al. 2012). Along with the effect of metal ions such as Cu²⁺, Co²⁺, Hg²⁺, Na²⁺, K⁺, Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺(1mM each) then incubating the purified fraction to determine the effect of metal ions.

Different substrates such as haemoglobin, bovine serum albumin, fibrin, gelatin, keratin, and casein (1% w/v) were used for determining the enzyme Substrate specificity. The enzyme was incubated for10 min in each of the substrates. The degree of substrate hydrolysis was analyzed by protease activity using standard assay conditions (Gupta and Singh 2014).

For measuring the enzyme activity various concentrations of keratin (1mM-10mM) dissolved in 0.05 M glycine NaOH buffer of pH 9.0 and incubation time of 30 min. under standard assay conditions were used for determining the Kinetic parameters Km, Vmax of purified keratinase by ploting Line weaver-Burk plot (1934).

RESULTS AND DISCUSSION

Enzyme mass production: Further the crude enzyme was used for purification steps.

Purification of crude enzyme: Salt precipitation, dialysis, Gel filtration, sephadex G-100, DEAE cellulose were used for keratinase Purification from A. aneurinilyticus. Over all it was observed that the specific activity was 203.87U/mg, yield of 25.43% with a fold increase of 20.84 (Table-1). Keratinases from P. vulgaris EMB-14 showed purification folds of 14.46 with specific activity of 74.74 U/mg and 8.27% yield as reported by (Dada et al. 2020). A 10-fold purification with 3.46% yield and specific activity of 297.5U/mg with recovery of 45% was and keratinase with 7.5-fold increases in a specific activity with 45% recovery was reported in the previous studies (Ire et al. 2017).

Keratinase with a 7.5-fold increase in a specific activity with 45% recovery (Adina et al. 2021). A similar result was observed with a single band at 26KDa both in zymogram and SDS PAGE with B. licheniformis H62 (Kazzaz et al. 2015). B. subtilis SCK6 showed 30.95KDa and B. amyloliquefaciens strain TCCC 11319 with 28KDa (Tian et al. 2019). Zhang et al. (2016) Han et al.  (2012) also reported a nearer mol. wt. of 25KDa and 33KDa of keratinase from P. aeruginosa C11. The reports of various authors producing keratinase from Bacillus sps. correlate with our results (Tian et al. 2019; Adina et al. 2021).

Keratinase from A. aneurinilyticus and its molecular weight was determined by comparing the electrophoretic mobility of the marker proteins. Standard protein markers used were 97KDa (phosphorylase), 66KDa (BSA), 45KDa (Ovalbumin), and 31KDa (Bovine carbonic anhydrase) and 14.33KDa (Lysozyme). The mol. wt. of the purified enzyme was 34KDa when SDS PAGE was done (Fig.1a). Zymogram analysis showed a clear colourless zone against a dark blue background (Fig.1b). Similar results were observed with Kazzaz et al. (2015) revealing a single band at 26KDa both in zymogram and SDS PAGE with B. licheniformis H62.

B. subtilis SCK6 showed 30.95KDa and B. amyloliquefaciens strain TCCC 11319 with 28KDa (Tian et al. 2019, Zhang et al. (2016) and Han et al. (2012) also reported almost nearer mol. wt. of 25KDa and 33KDa of keratinase from P. aeruginosa C-11. Liaqat et al. (2022) also purified and characterized keratinase from B. licheniformis dcs1 from poultry waste processing and the keratinase which correlates our results, keratinase with mol. wt. of 34KDa.as reported by (Yong et al. 2020). Thus, showing similar reports of keratinase production with almost similar mol.Wt. from Bacillus sps (Yong et al. 2020; Liaqat et al. 2022).

Table 1. Steps of keratinase Purification from A. aneurinilyticus VRCS-4

Steps of Purification	Activity (U/ml)	Protein (mg)	Specific activity (U/mg)	Fold of Purification	(%) Yield
Crude culture fluid	38478	3931	9.78	1.0	100
(NH4)2SO4 Precipitation (70%)	27863	2453	11.35	1.16	74.41
DEAE cellulose	11602	143	81.13	8.29	30.15
CM Sephadex G-100 Gel filtration	9786	48	203.87	20.84	25.43

Figure.1 .a: SDS PAGE of keratinase (M: Marker protein), (1: Keratinase).

Figure.1 .b: Keratinase Zymogram

Characterization of Purified Enzyme: Maximum activity of purified keratinase enzyme was at 8.0 to10.0 pH, while moderate activity was between 5.0-11.0. Relative keratinase activity which was maximum (100%) was at pH 9.0 (Fig.2). To check the enzyme stability from A.  aneurinilyticus it was pre incubated between the temperatures of 20-80⁰C. It was observed that the enzyme was stable at 50⁰C. Further, increase in the temperature to 80⁰C, the relative activity was reduced to 40% (Fig.3).by increasing the temperature to 80⁰C, revealing that the purified keratinase showed the optimum activity at pH9.0and temperature 50⁰C (Kumar et al. 2021).

Similar results of optimum pH and temperature with B. megaterium were reported by Saibabu et al. (2012). Using the organism Chrysosporium indicum as reported by Kumar et al. (2021) it also showed maximum keratinase activity at temperature 50⁰C and pH-10. Yong et al. (2020) reported optimal temperature, pH 55⁰C and 10.0 by B. subtilis S1-4. When these results of keratinase production were compared with all the researchers it was observed that the pH ranged between 8.0 -9.0 and the temperature between 50-60⁰C (Kumar et al. 2021).

Figure 2: Influence of pH on keratinase activity

Figure 3: Influence of temperature on keratinase activity

Keratinase activity on metal ions is presented in (Fig. 4). The metal ions, studied were Mg²⁺, Ca²⁺, K+, Fe²⁺, Zn²⁺, Mn²⁺, Na²⁺, Cu²⁺, Hg²⁺ and Co²⁺ at 1mM concentration. The percent residual activity was maximum with Mg²⁺, Ca²⁺, K+, Fe²⁺, Zn²⁺and Mn²⁺ while Cu²⁺, Co²⁺, and Hg²⁺ showed inhibitory action against keratinase from A. aneurinilyticus. Keratinase activity was inhibited by Hg²⁺, which signifies that Hg^2+, might reduce the enzyme activity by binding to the SH-group present at the active site or Hg²⁺ may bind to the carboxylic group and also tryptophan residue to decrease the enzyme activity (Saibu et al. 2013; Ul–Haq 2020). While in case with A. aneurinilyticus some cations stimulated the keratinase activity. Saibabu et al. (2013) reported that the enzyme inhibition with Hg²⁺ is not due to the thiol group but is due to the tryptophan residue interaction or may be the carboxyl group present in the amino acid of that particular enzyme. The stimulatory effect of Fe²⁺, Ca²⁺and Mn²⁺ at 1mM increases the keratinase activity by acting as co-factors (Ul–Haq 2020).

Suntornsuk et al. (2005) reported that Ca²⁺ a divalent cat ions enhanced the enzyme activity of B. licheniformis FK-14, indicating the enzyme to be typical for serine protease. To maintain the enzyme structural confirmation or to stabilize the substrate binding and to form enzyme complex probably these metal ions may be acting as ion bridge. Keratinase from A. aneurinilyticus is highly activated by Mg²⁺, Ca²⁺ and Mn²⁺ proving that these metal ions confer for protection of enzyme against denaturation by heat performing a vital function in the maintenance of its active conformation (Kumar and Takagi 1999). Dada (2020) expressed that keratinase largely from gram-positive bacteria are mostly serine proteases as they possess two Ca^{2 +} active sites thereby enhancing the enzymatic activity. The role of Ca^{2 +} associated with the stability of the activated forms of the keratinases agrees with our results (Dada 2020; Ul–Haq 2020).

Figure 4: Effect of metal ions on keratinase activity

Keratinase was stimulated by EDTA, DTT, DFP while the most potent inhibitor was PMSF (Fig.5). When the surfactants results were compared the keratinase activity was inhibited (19.92%) to the maximum with β-mercaptoethanol and stimulated by tween-60 (84.11%), tritonX-100 (82.01%), tritonX-114 (58.65%) and SDS (32.46%) (Fig.6). Inhibition of the enzyme activity in presence of PMSF indicates the keratin belongs to serine group. This inhibition is due to covalent binding of PMSF to the residual serine and in activating it, there by blocking the sulphonate releasing hydrolytic sides for keratinolytic attack (Suntornsuk et al. 2005). The active site of protease was blocked by PMSF by sulfonating the essential serine residue, there by resulting in complete inhibition of protease activity (Jaouadi et al. 2013). However,reducing agents able to break the disulphide bonds in the substrate keratin releasing different hydrolytic sites for the keratinolytic attack (Dada 2020; Ul–Haq 2020).

Moreover 70% of reduction in enzyme activity was observed with β-mercaptoethanol. Xian et al. (2016) also presented 70% of enzyme inhibition with β-mercaptoethanol, while it was 55% with DTT. More than 50% of residual activity was with other nonionic surfactants. We evaluated the impact of inhibitors and surfactants on keratinase activity by A. aneurunilyticus. Enzyme activity was inhibited by PMSF, β-mercaptoethanol and stimulated by EDTA, while amongst the surfactant used is again stimulated by Tween-60 and anionic surfactant such as SDS inhibited them. Our results correlate with Bose et al. (2014) where PMSF completely inhibited the enzyme activity while nonionic and anionic were marginal stable. Stability of the keratinase enzyme from B. subtilis k-5 with SDS and Tween-80 while activity was inhibited by β-mercaptoethanol was reported by Singh et al. (2014). Rai and Mukharjee (2009) stated that the stable alkaline protease in presence of surfactants is highly desirable for industrial application (Ul–Haq 2020).

Figure 5: Effect of enzyme inhibitors on keratinase activity

Figure 6: Effect of surfactant on enzyme activity

Keratinases from A. aneurunilyticus showed broad substrate specificity since it could hydrolyze keratin substrate and casein (85.47%), BSA (69.04%), gelatin (59.72%), haemoglobin (48.21%) and the least were fibrin (36.43%) (Table-2).

Moridshashi et al. (2020) reported maximum relative activity with feather keratin then was casein, and the least hydrolysed were keratin azure, gelatine and BSA. Prakash et al. (2010) reported high activity towards casein, followed by keratin, using B. halodurans PPKS-2.  Gang et al. (2008) also revealed maximum keratinase activity by using casein, BSA, Feather meal and feather keratin with the organism B. subtilis KD-N2. When the relative activity of different substrates was compared it was observed that the substrates which were having more disulphide bonds could be easily hydrolysed than the substrates having less disulphide bonds, suggesting the solubility depends on the high percentage of disulphide bonds in the substrates (Moridshashi et al. 2020).

Different concentrations of the substrate keratin (1mM to10mM) were used to determine the Km and Vmax of purified keratinase. Km and Vmax values were determined by LB plot and were found to be 3mM-Km and 249µmol/mL/min Vmax respectively (Fig.7). Lower values of Km suggest high affinity towards the substrates indicating that the enzyme-substrate complex is tightly held before the substrate is converted to the product thus indicating that keratinase enzyme from A. aneurinilyticus has great affinity towards its substrate keratin. 1/S on the X-axis, 1/V on the Y-axis, a double reciprocal plot gave a straight line suggesting that our enzyme obeys the Michaelis Menton equation. Km being independent of enzyme concentration shows the characteristics of enzyme under defined temperature and pH condition (Moridshashi et al. 2020).

Singh (2014) and Srinivasan (2008) also reported a low Km value of 0.01µg/ml/min and Vmax of 1176mg/ml with B. subtillis K-1. Dada (2020) also reported Km and Vmax kinetic constants of 25.60mM and 74.46U/ml respectively, with B. licheniformis K-51. Hydrolysis efficiency of 7mg/ml for Km and 384.6U/mg of Vmax was observed when casein was the substrate, while it was 7.2mg/ml and 103mg/ml respectively when the substrate was keratin azure with Laeeyella sacchari (Dada et al. 2020). Moridshashi et al. (2020) also reported a low Km value of 8.74mg/ml and Vmax of 59.04U/ml/min with feather meal substrate using B. zhangzhouensis. The above-said report correlates with our data (Dada et al. 2020).

Table 2. Specificity of keratinase in the presence of different substrate

Substrates	Enzyme activity (U/ml)	Relative activity (%)
Bovine serum albumin	252.5	69.04
Casein	312.0	85.47
Gelatin	218.0	59.72
Fibrin	136.0	36.43
Keratin	365.0	100.00
Haemoglobin	176.0	48.21

Figure 7: Line Weaver-Burk plot (1/[S] V/S 1/ [V]) Km and Vmax

CONCLUSION

The findings of the present study showed that proteases of microbial origin are interesting, compared to plants or animal sources, as these enzymes from microbial origin possess all the features desired for biotechnological applications. Consequently, keratin can be transformed by keratinolytic microorganisms such as A. aneuinilyticus with low molecular weight of (34KDa). Generally, the molecular weight of bacterial keratinases varied between species and combination of various peptidases is required for keratin degradation. Thus, keratinase with low mol. wt. can be used in various biotechnological processes. Our studies of keratinase production from xerophytic endophytes will unravel the complex mechanism of keratinolysis and stability of the enzymes at high pH and temperatures which can be employed in various industries.

Ethics approval and consent to participate: Not applicable.

Funding:  No funding.

Conflict of interests: Authors declare no conflict of interests to disclose.

ACKNOWLEDGEMENTS

The work was carried out  in the Department of Microbiology, Gulbarga University, Kalaburgi, Karnataka, India, which is  greatly acknowledged
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