Citrobacter diversus-derived keratinases and their potential application as detergent-compatible cloth-cleaning agents

Abstract

Currently, poultry farming is one of the sectors that have a significant impact on the global economy. In recent years, there has been an increase in the production of broilers, inflicting this segment of the industry to generate tons of keratin due to huge disposal of chicken feathers. This points to the need to degrade these chicken feathers, as they have emerged as a major threat to the environment. Thus, in this study we aimed to identify keratinases that are produced by the bacterium Citrobacter diversus and further investigate the biochemical characteristics of these keratin-degrading enzymes. In a submerged medium, the bacterium was capable of degrading chicken feathers almost completely after 36 h of fermentation. We found a maximum caseinolytic activity at pH 9–10.5 and 50–55 °C, and keratinolytic activity at pH 8.5–9.5 and 50 °C. Thus, given its stability at higher temperatures, upon incubation of this enzyme extract for 1 h at 50 °C, it showed approximately 50% of the keratinolytic and 100% of the caseinolytic activity. Further, under pH stability for 48 h at 4 °C, the enzyme extract maintained greater residual activity in the pH range 6–8. Caseinolytic activity was inhibited by EDTA and PMSF, whereas the keratinolytic activity was inhibited only by EDTA. Additionally, due to its alkaline activity and detergent compatibility, this enzyme extract could improve washing performance when added to a commercial detergent formulation. Using application tests, we could demonstrate a potential use of this bacterial enzyme extract as an additive in detergents to remove egg stains from cloth.

Introduction

Poultry farming is one of the industrial sectors that have a significant impact on the global economy, and it generates tons of keratin as the recalcitrant waste [1]. According to a report by the Department of Agriculture of the United States of America, in the last few years there has been an increase in the production of broilers, with an estimated value that has reached up to 103 million tons in 2020, to which Brazil has contributed 13 million tons of chicken per year [2].

The feathers constitute about 5–10% of the weight of a chicken and are comprised of approximately 90% of keratin [1]. In this scenario, a large amount of keratinous material (in the form of feathers) is expected to be disposed off each year in the environment. This reveals the importance of adding commercial value to this waste.

Keratin is a natural and insoluble protein that is generally present in the epidermal layer and in specialized structures, such as hair, nails, feathers, and animal horns and hooves [3, 4]. Based on the folding of their polypeptide chains, keratins can be classified into α-keratins, whose secondary structure is predominantly comprised of α-helix chains, which is mainly present in skin epidermis; and β-keratins, whose secondary structure is predominantly comprised of β-sheets, which is present in structures such as feathers, beaks, and claws of birds [4–6].

Industrially, keratin residues are principally derived from the meat industry, such as pig hooves, cattle, and especially chicken feathers. For this reason, keratinolytic enzymes have attracted biotechnological interest as an alternative for dealing with the disposal of chicken feathers [7].

By definition, keratinases are proteolytic enzymes (peptidases or proteases) that are capable of cleaving peptide bonds in keratin [8]. Most studies on keratinases have mainly identified and characterized the alkaline monomeric serine peptidases [9–18] and less frequently, the alkaline monomeric metallopeptidases [19–23]. Some studies have also described aspartic keratinases isolated from yeasts [24, 25] and dimeric keratinases [13].

Upon feather degradation, the keratin hydrolysates can be readily used as biofertilizers and animal feed supplements, and possess biological activity such as antioxidant and antimicrobial [26]. Additionally, keratinases also have potential industrial applications, such as additives in detergent formulations [7, 27]. The biochemical action of these enzymes on various proteins and their stability at alkaline pH makes them an important component of formulations that can be readily used for removing protein stains from cloth.

Thus, in this study, we first aimed to investigate the ability of the bacterium Citrobacter diversus to degrade chicken feathers, followed by biochemical characterization of the keratinases produced by this bacterium, in order to adapt its use in solving the problem of keratin disposal in the environment and application as an additive in detergents.

Our results demonstrated that the bacterium C. diversus is potentially capable of degrading chicken feathers. Additionally, in the application test, when the enzymatic extract of C. diversus was added in the composition of the detergent powder, we could demonstrate a potential use of this bacterial enzyme extract as an additive in detergents to remove egg stains from cloth.

Materials and methods

Bacterial strains, growth conditions, and submerged fermentation

The bacterium C. diversus was obtained from the collection of microorganisms of the Laboratory of Biochemistry and Applied Microbiology, Unesp, São José do Rio Preto/SP.

An initial culture of C. diversus was set up in a slant tube containing enriched medium with the following composition: glucose (0.2%), NaCl (0.1%), tryptone (1%), yeast extract (0.5%), and agar (1.5%), and incubated for 2 days at 30 °C. After 2 days of growth, a pre-inoculum was initiated in a submerged medium that was similar to the previous medium (without agar), in order to obtain a bacterial cell suspension. Then, 0.3 mg of harvested bacterial cells was inoculated in the fermentation medium that was comprised of NaCl (0.05%), KH₂PO₄ (0.07%), K₂HPO₄ (0.15%), MgSO₄ (0.01%), and 0.5% chicken feather, pH 7.2. The final volume of this fermentation medium was adjusted to 50 mL and it was placed in a 250 mL Erlenmeyer flask. The cells were allowed to grow in a controlled growth chamber for 36 h at 30 °C and 120 rpm.

Degradation of chicken feathers was evaluated by quantifying the final dry biomass of the substrate in relationship with the increase in the turbidity of the medium, measured at 600 nm and the increase in absorbance of the medium, measured at 280 nm.

Proteolytic activity assay

The keratinolytic and caseinolytic activity of the enzyme extract obtained from C. diversus was measured using the substrates keratin azure (0.5%) and bovine casein (1%), respectively.

For assessing the keratinolytic activity, 0.4 mL of the enzyme extract was incubated with 1.2 mL of 0.5% keratin azure diluted in 50 mM Tris-HCl buffer, pH 8, and the reaction was maintained at 40 °C for 5 h. Subsequently, the enzymatic reaction was stopped by adding 1 mL of 10% trichloroacetic acid (TCA). Parallelly, the blank reaction tubes were prepared by adding 10% TCA to the reaction mixture before adding the substrate. Then, the reaction tubes and the blank tubes were centrifuged at 10,000×g for 10 min at 25 °C, and the supernatant was collected and the enzymatic activity measured spectrophotometrically at 595 nm wavelength.

For assessing the caseinolytic activity, 0.1 mL of the enzyme extract was incubated with 0.5 mL of 1% casein diluted in 50 mM Tris-HCl buffer, pH 8, and the reaction was maintained at 40 °C for 20 min. Subsequently, the enzymatic reaction was stopped by adding 0.3 mL of TCA. Blank tubes were prepared by adding 10% TCA to the reaction mixture before adding the substrate. Then, the reaction tubes and the blank tubes were centrifuged at 10,000×g for 10 min at 25 °C, and the supernatant was collected and the enzymatic activity measured spectrophotometrically at 280 nm wavelength.

One unit of keratinolytic activity (U/mL/h) or caseinolytic activity (U/mL/min) was defined as the amount of enzyme required to increase the absorbance of the reaction mixture by a measure of 0.01 at 595 nm wavelength for keratin azure [18], or at 280 nm for casein [28], respectively.

Functional biochemical characterization of the crude extract

Analysis of the effect of pH and temperature on activity and stability of peptidases

These experiments were carried out using the substrates casein and keratin azure. The optimum pH for maximum enzymatic activity was determined by performing the reaction at 45 °C using the following buffer mixtures: acetate (pH 5.0), MES (pH 5.5; 6.0 and 6.5), HEPES (pH 7.0 and 7.5), POPSO (pH 8.0 and 8.5), glycine (pH 9.0 and 9.5), and CAPS (pH 10.0, 10.5 and 11.0), all of them adjusted to 0.1 M final concentration. Further, the effect of temperature on the enzymatic activity was evaluated by performing the reaction at different temperatures ranging from 40 °C to 65 °C.

To assess the thermal stability, the enzyme extract was incubated for 1 h at different temperatures ranging from 40 °C to 60 °C (the sample incubated at 4 °C was used as 100% activity). Further, the stability of the enzyme extract at different pH conditions (ranging from 5.0 to 11) was determined by incubating the fermentation extract for 48 h at 4 °C (the peak activity was used as 100%). In both the cases, the enzymatic reactions were carried out using the optimum pH and temperature for predetermined activity.

Analysis of the effect of inhibitors on peptidase activity

To understand the mechanism of action of the peptidases present in the enzyme extract, we used the following inhibitors: E-64, phenyl methyl sulfonyl fluoride (PMSF), and ethylene-diaminotetraacetic acid (EDTA), at final concentrations of 5 mM. The enzymatic reactions were then carried out at the optimum pH and temperature for predetermined activity.

Testing of washing performance and stability of the enzyme extract and its compatibility with commercial detergent

The fermentation extract was evaluated for its stability upon addition to the formulation of a commercial detergent powder (Soft/Brazil), and its washing performance in removing chicken egg stains from cloth.

Initially, the total unit of proteolytic enzymes per gram of detergent was quantified. In addition, part of this detergent powder was autoclaved to denature the proteolytic enzymes. Next, similar proportion of the C. diversus enzymes (as present in the active detergent) was then added to this autoclaved extract.

Hereafter, multiple white cotton fabrics (size, 2 × 2 cm²) were spotted with 100 μL of homogenized egg. The cotton fabrics were incubated at 60 °C until the stains were dried completely. Washing experiments were performed in falcon flasks (50 mL) using 40 mL of washing mixture that was agitated at 120 rpm in an incubator shaker for 60 min at 30 °C. Later, the cotton fabrics were rinsed and incubated at 45 °C until they were dried completely. Comparative washing tests were carried out using active detergent, inactive detergent (denatured enzymes), inactive detergent supplemented with C. diversus enzyme, a mixture of enzyme extract and buffer solution (detergent pH), and water [29].

Stability tests were performed using the enzyme mixture with inactive detergent and upon incubation (in liquid form) for 24 h at room temperature. Caseinolytic assay was performed to quantify the proteolytic activity during this period.

Statistical analyses

The experimental data were analyzed using the GraphPad Prism software version 5.0 (GraphPad Software, San Diego, CA, USA). Statistical variance was estimated using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. When p values was < 0.05, the differences were considered statistically significant.

Results

Degradation of chicken feathers in submerged culture medium

Using the submerged fermentation method, we observed that C. diversus was potentially capable of degrading the chicken feathers to the maximum extent (~ 85% feather degradation) at the 36-h time point. We next determined the level of degradation of the chicken feathers by quantifying the final dry biomass in relationship with the increase in the turbidity of the medium, measured at 600 nm and the increase in absorbance of the medium, measured at 280 nm (Fig. 1). Clearly, the loss of dry biomass upon fermentation was correlated with the increase in the turbidity of the medium that resulted from the degradation of the chicken feathers and also with the increase in microbial growth over time. Further, the microbial growth led to an increase in the pH of the medium.

Fig. 1.

Growth of C. diversus in submerged medium containing 0.5% chicken feather, initial pH 7.2. During culture, samples were collected at different time points (4 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 60 h) and evaluated for OD at 280 nm and 600 nm, final pH and feather degradation

Thus, after the 36-h time point, the enzymatic extract was removed and used for further biochemical characterization.

Effect of pH and temperature on enzyme activity and stability

The proteolytic enzymes present in the fermentation extract were evaluated for both the caseinolytic and keratinolytic activity. As shown in Fig. 2a, b, the maximum caseinolytic activity was observed at pH 9–10.5 (p = 0.6) and at a temperature range of 50–55 °C (p = 0.12), whereas the highest keratinolytic activity was observed at pH 8.5–9.5 (p = 0.14) and 50 °C.

Fig. 2.

Effect of (a) pH and (b) temperature on caseinolytic and keratinolytic activity. Reactions were performed using 1% casein and 0.5% keratin azure as substrates, respectively

For both the substrates, proteolytic activity was significant at alkaline pH. For instance, by comparing the two activities, the casein hydrolysis was more active at pH above 9.5, with approximately 80% activity at pH 11, whereas the keratinolytic activity was completely lost at pH 10.5–11. A similar behavior in the activity of the enzyme extract was observed due to the effect of temperature, as the caseinolytic activity was less sensitive than the keratinolytic activity at 60 °C. Specifically, the keratinolytic activity was not observed at this temperature, whereas 60% of caseinolytic activity was still maintained.

In enzyme stability test, when the fermentation extract was incubated at different pH values at 4 °C for 48 h, higher caseinolytic and keratinolytic activity (both above 60%) was observed at the pH range 6–8 (Fig. 3a). When incubated for 1 h at different temperatures, the keratinolytic activity of the extract was maintained at 80% at 40 °C, 60% at 45 °C, 45% at 50 °C, and 20% at 55 °C, and a total loss of activity was observed after incubation at 60 °C (Fig. 3b). In contrast, the caseinolytic activity was found to be more stable at temperatures up to 50 °C, as no loss of activity was observed when the extract was incubated for 1 h at 40 °C, 45 °C, and 50 °C.

Fig. 3.

Effect of (a) pH and (b) temperature on enzyme stability. Reactions were performed using 1% casein and 0.5% keratin azure as substrates

Effect of inhibitors on caseinolytic and keratinolytic activity

Using the proteolysis inhibition tests, we showed that the fermentation extract possesses two classes of peptidases (Fig. 4). Caseinolytic activity was inhibited by PMSF (90% inhibition) and EDTA (80% inhibition), whereas the keratinolytic activity was solely inhibited by EDTA (85% inhibition). E-64 and control sample are not variances significantly different (p = 0.2). This indicates that serine peptidase as well as metallopeptidase are present in the fermentative extract, as the keratinolysis activity is mainly dependent on a metal enzyme.

Fig. 4.

Effect of E-64, EDTA and PMSF (5 mM) inhibitors on caseinolytic and keratinolytic activity of the enzyme extract. Reactions were performed using 1% casein and 0.5% keratin azure as substrates, respectively

Washing performance in compatibility with commercial detergent and stability tests

Using the wash test that was performed to remove the egg stains from cloth, we could observe a better washing performance of the C. diversus enzyme extract when mixed with inactive detergent (Fig. 5d), in comparison with that for the other samples (Fig. 5). For instance, under the same washing conditions, the stain residues were still observed in the sample containing active detergent (Fig. 5b).

Fig. 5.

Washing performance of different detergent mixtures for removing the egg stains. a Washing with water, b Detergent with inactive enzyme, c detergent with active enzyme, d C. diversus enzyme + detergent with inactive enzyme, e C. diversus enzyme + CAPS buffer pH 10

In the stability test, the enzyme and detergent mixture was dissolved in water and incubated for up to 24 h at 25 °C (Fig. 6). The enzyme activity was maintained at around 90% for 2 h, and at 70% from 5 h to 24 h.

Fig. 6.

Evaluation of the stability of C. diversus enzyme in compatibility with commercial detergent (Soft/Brazil) at 25 °C. Enzyme and the detergent mixture was dissolved in water (incubation time: 1 h, 2 h, 5 h, and 24 h). Reactions were performed using 1% casein as a substrate

Discussion

In this study, we first determined the ability of the bacterium C. diversus to degrade chicken feathers in a submerged fermentation medium. As a result of microbial growth and metabolism of amino acids derived from protein hydrolysis, increases in turbidity and pH of the medium were observed, demonstrating the ability of this bacterium to efficiently degrade keratin [30]. The keratin degradation ability of C. diversus demonstrated in this study (~ 85% in 36 h) was higher than that of the bacterium Kocuria rhizophila strain p3–3, which was previously reported to degrade only 52% of chicken feathers in 96 h of fermentation in submerged culture [31]. In contrast, the keratin degradation ability of C. diversus was similar to that of the Alcaligenes sp. AQ05–001, which is reported to perform at similar levels in 36 h of fermentation [32].

The use of microbial enzymes for the degradation of keratinous residues, especially the chicken feather keratin, proves to be a valuable alternative to solve the problem of accumulation of this poultry by-product in the environment. Unlike acid hydrolysis, keratinases require milder conditions for hydrolysis with reduced impairment of the protein hydrolysate [7, 26].

Upon analyzing the effect of inhibitors, we found that the process of keratin degradation is dependent on metallopeptidase enzymes (EDTA—85% inhibition on keratinolytic activity). Additionally, serine peptidases could act in the process of auxiliary hydrolysis of fragments derived from partial keratin decomposition. Lange et al. [33] showed that keratin degradation depends on an arsenal of proteolytic enzymes, where both the endo- and exo-peptidases are important for complete hydrolysis of this protein. Furthermore, in general, keratinolysis depends on a sulfitolysis process that can be catalyzed by disulfide reductase, and thus EDTA can potentially compromise this reaction, and consequently, inhibit the degradation of keratin.

The enzyme-substrate interaction is very specific to each reaction complex [8]. Thus, upon interaction with the substrates keratin azure and casein, differences were observed in the peaks of enzymatic activity. The optimum pH and temperature were found to be lower for the keratinolytic activity (pH 8.5–9.5 and 50 °C) than for the caseinolytic activity (pH 9–10.5 and 50–55 °C). However, for both the processes, the maximum activity was obtained at alkaline pH. It is important to note that unlike that for casein, keratin degradation is a synergistic process that depends on enzymes that are capable of acting on keratin, through sulfitolysis and proteolysis [33]. This can also be the cause of differences in the peaks of enzyme activity for the two substrates.

Many studies have demonstrated keratinolytic activity at a pH similar to the one in this study. For example, Lv et al. [12] and Barman et al. [34] reported keratinases from Chryseobacterium L99 sp. nov. and Arthrobacter sp. NFH5 with substantial activity at pH 8, respectively. Zhang et al. [22, 35] also described a keratinase from Brevibacillus parabrevis CGMCC 10798 with optimal activity at pH 8 and great stability in the pH range 6–8 [22], and the keratinolytic activity of Gibberella intermedia CA3–1 with excellent performance at pH 9 and stability in the pH range 6–9 [35]. In addition to pH stability, the thermostability of the enzymes described here is significantly higher to that reported in Gibberella intermedia CA3–1, for which only 20% residual activity was observed after pre-incubation at 40 °C for 1 h. Other results with the same optimal activity range have been reported in Actinomyces, Bacillus, Chryseobacterium, Fervidobacterium, Micrococcus, Microsporum, Nocardiopsis, Pseudomonas, Stenotrophomonas, Streptomyces [36].

From the biotechnological perspective, peptidases with activities in the alkaline pH range are more favorable for detergent-based formulation studies. Due to their activity and stability at alkaline pH, combined with the broad spectrum of their action on different proteins, in recent years, there has been an increase in the demand of keratinolytic enzymes for application in the detergent industry [35].

In the application test, when the enzymatic extract of C. diversus was added in the composition of the detergent powder, excellent results were obtained in terms of washing performance with better removal of the egg stains compared with that for other samples, especially the active detergent. It is also notable that the washing performance of only the enzyme or only the detergent without the enzyme was much less efficient for removing the stains. This indicates that the combination of these compounds is very important to remove egg stains and to achieve better cloth-cleaning.

It is also important to note that, in addition to being able to remove stains, the enzyme must be active and stable in the detergent formulation. In this study, the enzyme was tolerant to the detergent mixture when dissolved in water, for up to 24 h. Stability results have not been reported extensively in other studies in which these proteolytic enzymes have been implemented in laundry detergent formulation. For example, Patil et al. [37], Ida et al. [29], and Rekik et al. [38] described the efficiency of the microbial peptidases mixed with laundry detergent in removing egg stains. However, in these studies, the enzyme stability in the detergent mixture had not been reported. Other reports have also been published without this information [39–43].

Based on the results obtained in this study, we conclude that there is potential for further research in order to improve the activity and stability of these proteolytic enzymes, and also their cloth-washing performance.

Conclusion

The problem of keratin degradation has resulted in increased attention to microorganisms that are capable of producing keratinases. Through the results presented in this study, we could demonstrate that the bacterium C. diversus is potentially capable of degrading chicken feathers, and thus can be used as one more alternative to solve the problem of accumulation of keratinous residues. With the worldwide trend of adapting sustainable technologies, the enzymatic hydrolysis of keratin by this bacterium opens up a futuristic opportunity for testing the use of the by-product/protein hydrolysate as a biofertilizer.

Another important feature that has been well-described in this study is the ability of this enzyme extract to be used as an additive in detergent formulation. The enzyme extract produced by C. diversus demonstrated compatibility with the formulation of commercial detergent and was stable in a liquid mixture (detergent dissolved in water), in addition to its excellent washing performance against the egg stains. Future tests to improve the application of these enzymes should be carried out in order to optimize the washing performance to remove stains from different proteins and to adapt the ideal dosages of enzyme in the detergent.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.