Biosynthesis, purification, and biochemical properties of an alkaline-thermostable keratinase derived from Bacillus cereus J6

Determination of the factors and their levels: The impacts of the chicken feather inducer, nitrogen sources (peptone, yeast extract, (NH₄)₂SO₄, NH₄Cl, urea, and NaNO₃), carbon sources (glucose, lactose, maltose, sucrose, starch, and dextrin), and mineral salts (MgSO₄, ZnSO₄, CaCl₂, MnCl₂, FeSO₄ and FeSO₄) were investigated via the single-factor method.

Orthogonal experiments: The orthogonal method was used to determine the fermentation medium that is most suitable for Strain J6 to produce keratinase, providing support for increasing the yield of keratinase and the fermentation efficiency. Based on the above-mentioned experiments’ results, an appropriate orthogonal experimental array L₉(3⁴) was selected to obtain the optimal composition of the fermentation medium (Table 1).

Subsequently, a comprehensive validation process was implemented, involving three independent batches of experiments for the optimal fermentation medium derived from the orthogonal experiment. Each of these batches was also replicated three times.

The fermentation conditions of the strain were also optimized, including initial pH, fermentation temperature, inoculum size and rotary speed, by employing a single-factor optimization approach.

The activities of the enzyme were measured in the pH range of 6.0-12.0 at a buffer concentration of 20 mmol/L. And the residual activities of keratinase were detected after the enzyme was remained in the corresponding pH conditions for 60 min at 20°C. Moreover, the influence of temperatures in the range of 30-70°C on keratinase activity was measured to obtain its optimal temperature. Furthermore, the thermostability of the keratinase was measured by remaining the enzyme at the respective temperature for 60 min and subsequently monitoring its residual activity levels.

The impacts of various chemical agents on keratinase activity were investigated. Keratinase activities were assayed under the enzyme’s optimal pH and temperature conditions in the presence of multiple metal ions at 5 mmol/L, well-known protease inhibitors at 1 mmol/L, the reducing agent at 1 mmol/L, and surfactants at 1%. The enzyme’s activity without any chemicals served as the control.

Diverse substrates (wool, chicken feather, keratin, casein, Type-I collagen) were employed to test enzymatic activity of the keratinase at its optimal pH and temperature. Specifically, the soluble substrates were prepared at the concentration of 1%, then evaluate the keratinase’s activities by the keratinase assay method. For the insoluble substrates, the reaction system contained the suspended 0.05 g of substrates in 0.5 mL buffer solution, and 0.05 mL keratinase. The reaction time was 60 min. Afterward, add 1.0 mL of 10% trichloroacetic acid (TCA) solution to stop the reaction, and centrifuge (15,000 rpm, 10 min). Measure the absorbance of supernatant at 280 nm (OD280). For each reaction, use the reaction system with TCA added before the substrate as a control. The enzyme activity is defined as follows: in the above reaction system, a 0.01 increase in OD280 is considered as one unit.

To explore the kinetic properties of J6 keratinase, soluble keratin at concentrations ranging from 0.1% to 5% was used. The assays were performed under the enzyme’s optimal pH and temperature settings. Subsequently, based on the Michaelis-Menten Equation, J6 keratinase’s kinetic parameters (K_m and V_max) were calculated.

As depicted in Figure 2A, B. cereus J6 began to produce keratinase after 8 h of fermentation. Prior to 36 h, the enzyme activity exhibited a continuous increase trend. It peaked at 36 h with an enzyme activity value of 192 U/mL. Afterwards, the enzyme activity declined gradually. Therefore, it was determined that the optimal fermentation time was 36 h, which is shorter than the 60-h cultivation time of Pseudomonas sp. LM19, as previously reported by Mohamad et al. [16], and the 48-h cultivation time of Bacillus tropicus Gxun-17 reported by Shen et al. [7].

Figure 2.

Keratinase production by B. cereus J6. A. Enzyme production profile; B, Feather; C, Nitrogen sources; D, Urea; E, Carbon sources; F, Lactose; G, Mineral salts; F, MgSO4.

In general, the production of enzyme during microbial fermentation is regulated by the nutritional status of fermentation medium [2]. Therefore, the composition of medium and the corresponding levels of its components were determined. First, given that feather serving as the substrate/inducer as well as nitrogen/carbon source is usually important for the keratinase biosynthesis by feather-degrading microbial strains [17, 18], feather was chosen as a key factor. The experimental results demonstrated that at 10 g/L of feather powder, B. cereus J6 synthesized the maximal level of keratinase (Figure 2B), which is accordant with what was observed for Bacillus licheniformis PWD-1 [19]. When feathers exceeded 15 g/L, the increase in feather concentration led to a decrease in enzyme activity. This is possibly due to the fact that an excessive concentration of feathers affects the dissolved oxygen in the medium [7], thereby influencing the biosynthesis of keratinase by B. cereus J6. When investigating the effects of supplemented nitrogen sources on enzyme production, it was found that peptone and yeast powder had a negligible impact on the enzyme production by the strain, while yeast powder, ammonium chloride, and urea exhibited a certain promoting effect on enzyme production (Figure 2C). Among these nitrogen sources with promoting effects, urea had the most substantial promoting effect. And the addition of urea at 10 g/L had the most effective promoting effect on keratinase production by B. cereus J6 (Figure 2D). The result agrees with the finding regarding a remarkable increase in production of keratinase by Bacillus sp. with the addition of urea [20]. Furthermore, among the supplemented carbon sources tested, only lactose promoted the keratinase production of the strain (Figure 2G). Conversely, all other measured carbon sources inhibited the enzyme production. And lactose at 5 g/L most effectively promoted keratinase production (Figure 2F). Similarly, it has been reported that the addition of lactose promoted the yields of keratinases produced by Bacillus velezensis ZBE1, B. licheniformis PWD-1 and B. cereus [19, 21, 22]. Regarding inorganic salts, both Mg²⁺ and Ca²⁺ could evidently promote Strain J6 to produce keratinase with Mg²⁺ having the most significant promoting effect. In contrast, Fe²⁺, Zn²⁺ and Mn²⁺ exhibited inhibitory effects on enzyme production. And MgSO4 at concentration of 1 g/L most promoted keratinase production (Figure 2H). Shen et al. reported similar research showing that Mg²⁺ promoted the production of keratinase by Bacillus pumilus A1, B. cereus YQ15, and B. tropicus Gxun-17 [7, 14, 23].

The orthogonal experimental design method skillfully uses the orthogonal table’s “balanced dispersion and neat comparability” features. The experimental results are greatly scientifically reliable. So, it strongly supports optimizing experimental plans and finding optimal conditions, making research more efficient. Therefore, by employing the orthogonal design method, the four medium components (feather, lactose, urea, and MgSO₄) and their corresponding levels were determined for further systematic optimization to enhance the yield of keratinase. Based on the above experimental results, an appropriate orthogonal experimental array L₉(3⁴) was selected (Table 1). Subsequently, the orthogonal experiments were executed in strict accordance with the factor-level combinations delineated in the orthogonal table, wherein each experimental group was replicated three times. As shown in Table 2, in exploring the impacts of four factors on enzyme generation, magnesium sulfate was determined to possess the preponderant influence. Subsequently, feather meal, lactose, and urea manifested their respective degrees of influence in a descending sequence. The optimal combination of the four factors (A₂B₁C₁D₃) was chicken feather 10 g/L, lactose 2.5 g/L, urea 7.5 g/L, and MgSO₄ 1.1 g/L. When B. cereus J6 was fermented in the suitable medium, the enzyme activity reached 425 U/mL, 3.37 times that before optimization.

Apart from the components of fermentation medium, the fermentation conditions of the strain were also optimized, including initial pH, fermentation temperature, inoculum size and rotary speed employing a single-factor optimization approach. As shown in Figure 3, the best fermentation conditions were determined as follows: the initial pH of 8.0, the temperature of 37°C, the inoculum size of 6% (v/v), and the rotational speed of 220 rpm. The optimal cultural temperature differed from those of Bacillus sp. Nnolik-K1 (25°C), Chryseobacterium sp. RBT (45°C) due to the diverse characteristics of microbial producers [24, 25]. Researches have shown that an initial slightly alkaline pH condition, such as pH 8.0 for B. tropicus Gxun-17 and Pseudomonas sp. LM19, facilitates the biosynthesis of keratinase [7, 16]. Meanwhile, the result also suggests that the keratinase exhibits alkaliphilic characteristics, thereby highlighting its potential for applications within alkaline environments [26]. Under the optimal fermentation conditions, following 36-hour incubation, the keratinase activity of the strain B. cereus J6 attained 727 U/mL, 1.71 times the pre-optimization level.

Figure 3.

Impact of cultural conditions on B. cereus J6 producing keratinase

As shown in Figure 5A, the optimum temperature for the keratinase was 55°C, which is consistent with that of the keratinases from Bacillus subtilis S1-4 and P. aeruginosa 4-3 [8, 30], and higher than that of B. amyloliquefaciens K11 keratinase (37°C) and P. aeruginosa SU-1 keratinase (30°C) [29, 31]. Notably, when the temperature was below 55°C, this enzyme retained more than 90% of its activity. This high level of residual activity indicated that the enzyme has remarkable thermal stability in this temperature range, which is a significant characteristic for its potential applications in various industries [32]. Moreover, the keratinase exhibited maximal activity at a pH value of 9.0, consistent with that of the keratinase of B. subtilis S1-4 and B. pacificus RSA27 keratinase [28, 30]. After being incubated for 60 min, exceeding 50% of the residual activity was retained within the pH range of 8.0 to 12.0 (Figure 5B). Considering that it had an optimal pH of 9.0 and was active in an alkaline environment, B. cereus J6 keratinase belongs to alkaline keratinase [33].

Figure 5.

Impact of temperature and pH

Enzymatic activities of the keratinase were assayed in the presence of metal ions, surfactants, reducing agent, and inhibitors (Table 4). Calcium ion, Mg²⁺, Zn²⁺ and Mn²⁺ at 5 mmol/L could evidently enhance the activity by 22.56%, 31.81%, 18.52% and 6.73% respectively. The promoting effects of Ca²⁺, Mg²⁺ and Mn²⁺ were in consistent with those of Bacillus maojavensis SA keratinase, B. pumilus AR57 and Ectobacillus sp. JY-23 keratinase [34-36]. Monovalent metal ions including Na⁺ and K⁺ did not show noticeable influence on keratinase activity. Additionally, Cu²⁺, Fe²⁺, Al³⁺, and Fe³⁺ at 5 mmol/L significantly inhibited the keratinase activity. Cu²⁺ and Fe²⁺ also remarkably reduced the activity of Bacillus zhangzhouensis keratinase [37]. Interestingly, the keratinase was observed to be stable with the most tested surfactants. Specifically, Tween 60, Tween80, Tween 20 and Triton X-100 not only failed to inhibit the enzyme activity but also enhanced it, with respective significant increases of 75.27%, 50.16%, 15.67%, and 6.06%. Only SDS inhibited the enzyme activity by 15.70%. The stability of B. cereus J6 keratinase with Tween 20 and Triton X-100 was similar to the keratinases derived from P. aeruginosa S-04 and B. cereus YQ15 [14, 26]. In addition, dithiothreitol (DTT) led to a 55.96% increase in keratinase activity, which is accordant with previous reports on the keratinases of Ectobacillus sp. JY-23 and B. zhangzhouensis [35, 37]. DTT, possessing reducing properties, facilitates breaking disulfide bonds in the structure of keratin [30]. Both ethylenediamine tetraacetic acid (EDTA) and phenylmethylsulfonyl fluride (PMSF), two well-known protease inhibitors, exerted nearly complete inhibitory effect on the activity of the purified keratinase. This finding implies that the enzyme contains serine in its catalytic active center and possesses catalytic sites that rely on metal ions for proper functionality [35]. Meanwhile, the enzyme might belong to the serine-metal protease family.

Understanding enzyme’s substrate specificity can potentially guide the development of more efficient bioprocessing methods that harness the unique properties of this enzyme for targeted substrate degradation. The degradation effects of the keratinase on various substrates were illustrated in Figure 6. This keratinase showed wide substrate specificity, and outstanding degradation abilities for casein, BSA, and soluble keratin. Additionally, it exhibited relatively high activity against type-I collagen. Conversely, when dealing with insoluble substrates like wool and feathers, the enzyme showed low activity levels. This distinct substrate-specific activity profile of the keratinase not only offers profound insights into its catalytic characteristics but also has far-reaching implications for exploring its potential applications in relevant industries such as management and bioconversion of keratinous waste.

Figure 6.

The enzymatic activity of the keratinase towards various substrates

The kinetic parameters of the purified B. cereus J6 keratinase were studied by employing soluble keratin as the substrate. A linear fit was performed using the double-reciprocal plot, specifically the Lineweaver-Burke plot (Figure 7). The results of these calculations revealed that the purified J6 keratinase’s values of K_m and V_max were determined as 8.33 mg/mL and 478.47 U/mg/min, respectively. The K_m value is slightly higher than that of the keratinase from P. aeruginosa S-04 (7.62 mg/mL), and is comparable to that of B. pumilus keratinase (8.74 mg/mL) [26, 38].

Figure 7.

The kinetics of the purified keratinase