2018 Volume 24 Issue 4 Pages 669-676
This study aimed to characterize the crude enzyme from Hericium erinaceum (H. erinaceum) as a substitute source of rennet and assess its proteolytic behavior on bovine caseins. The crude enzyme from H. erinaceum was active in the pH range from 3.7 to 6.5 and was inactivated completely by heating at 45 °C for 15 min. The use of specific inhibitors revealed that the crude enzyme from H. erinaceum contains an aspartic protease with an optimum temperature of 30–35 °C. The addition of CaCl2 enhanced the milk-clotting activity of the enzyme. A comparison of SDS-PAGE and kinetic properties of the crude enzyme from H. erinaceum with chymosin on bovine caseins showed that αs- and β-caseins were preferentially degraded relative to κ-casein.
The results indicate that crude enzyme from H. erinaceum can be used as an effective coagulant in cheesemaking.
During cheese production, rennet coagulation of milk is a crucial step. Calf rennet, which contains chymosin (EC.3.4.23.4) as the main enzyme component, has traditionally been used for the manufacture of cheese with proper texture and good flavor. The reaction is initiated by the chymosin in most rennet, which specifically cleaves the Phe105-Met106 bond of κ-casein, prior to the release of the C-terminal portion of κ-casein, referred to as caseinomacropeptide (CMP). The removal of CMP from the surface results in the destabilization of casein micelles, and then the formation of a gel network through hydrophobic interactions.
The worldwide increase of cheese production and consumption has resulted in an inadequate supply of animal rennet for many years. Thus, other sources of coagulant enzymes have been sought as rennet substitutes of microbial, animal, and plant origins. A microbial source of rennet would be practical because of its stable availability and low cost. However, except for fermentation-produced chymosin, most rennet substitutes contain various proteases, which exhibit high proteolytic activity and low specificity, resulting in reduced yield in the manufacture of cheese and a bitter taste during cheese ripening (Corredig and Salvatore, 2016). Plant proteases are extensively used in the production of raw sheep and goat milk cheese from Spain and Portugal. The specificity of caseinolytic activity from Moringa oleifera flowers indicated that hydrolysis of κ-casein started after 30 min of incubation, while αs- and β-caseins were degraded after 60 min (Pontual et al., 2012). In the case of a partially purified enzyme from Solanum dubium seeds, β- and κ-caseins were rapidly degraded to lower molecular weight peptides (Mohamed Ahmed et al., 2010). Extract of artichoke flowers (Cynara scolymus, L.) and their purified proteinases (cynarases A and B) hydrolyzed αs-, β- and κ-caseins (Chazarra et al., 2007). Some properties of milk-clotting enzyme produced by mushrooms have been investigated and compared with those of calf rennet and other microbial rennets. Kobayashi et al. (1983) reported that the two milk-clotting enzymes from Irpex lacteus were the same as commercial microbial rennets obtained from Mucor pusillus and Mucor miehei because there was no degradation of αs- and β-caseins. An extracellular metalloprotease from the edible mushroom Termitomyces clypeatus MTCC5091 preferentially hydrolyzed κ-casein, whereas the degradation of αs- and β-caseins proceeded slowly (Majumder et al., 2015).
Nakamura et al. (2014) reported that the crude enzyme obtained from the edible mushroom Hericium erinaceum (H. erinaceum) had high milk-clotting activity and low protease activity. We found that the crude enzyme from H. erinaceum clotted pasteurized milk as well as UHT milk. Interestingly, CMP was not detected in the obtained whey solution (Sato et al., 2016).
This work reports the characterization of the crude enzyme from H. erinaceum. Further, the enzymatic hydrolysis of bovine casein was investigated to clarify the milk-clotting mechanism.
Chemicals and reagents The αs-casein, β-casein, κ-casein, protease inhibitors (PMSF, iodoacetamide, 1,10-phenanthroline monohydrate, aprotinin, and pepstatin A) were purchased from Sigma-Aldrich Co., St. Louis, MO, USA. CHY-MAX Powder-Extra (Christian Hansen A/S, Hoersholm Denmark) was used as a chymosin for comparison. All the other chemicals were of analytical grade.
Microbial preparation and enzyme production The strain of H. erinaceum (MAFF435060) was obtained from the National Institute of Agrobiological Sciences (NIAS) in Tsukuba, Japan. Crude enzyme produced by H. erinaceum was prepared from the cultured wheat bran medium as described previously (Nakamura et al., 2014, Sato et al., 2016). The medium, containing 100 g of wheat bran and 120 mL of distilled water (approximately 60 % moisture), was sterilized at 115 °C for 20 min. Enzyme production was carried out by cultivating the mycelia of H. erinaceum in the media at 25 °C for 14 days. Next, the crude enzyme from H. erinaceum was prepared by adding 1000 mL of 0.05 M McIlvaine buffer (pH 6.0) to the cultivated material and mixing gently, and the preparation was left overnight at 4 °C. The material was filtered and centrifuged at 10,000 × g for 15 min at 7 °C. The supernatant was used for further study.
Milk-clotting activity assay Milk-clotting activity (MCA) was measured according to the method of Arima et al. (1970) with some modifications. The substrate was prepared as follows. Commercial milk pasteurized at 66 °C for 30 min was obtained from Takanashi Milk Co., Ltd., in Iwate, Japan and defatted by centrifugation at 4320 × g for 30 min at 10 °C. The substrate was prepared by dissolving CaCl2 into the defatted milk to 10 mM. The substrate (2.0 mL, pH 6.6) was preincubated for 5 min at 30 °C, and 40 µL of the enzyme solution was added. The time necessary for the formation of curd fragments was recorded as milk-clotting time. MCA was expressed in Soxhlet units (SU). One unit of MCA was defined as the volume of milk coagulated per volume of the enzyme in 40 min.
MCA (SU)=2400/t*S/E
Where t=clotting time (sec), S=volume of reaction mixture (mL), and E=volume of enzyme (mL).
Effects of pH, temperature and protease inhibitors on MCA The optimum pH for the activity of the crude enzyme from H. erinaceum was determined by assaying MCA in the pH range 5.50–9.35, by adjusting the pH of the substrate with 0.1 M HCl or 0.1 M NaOH. To determine the pH stability, the crude enzyme from H. erinaceum was previously mixed (1:1, v/v) with 0.1 M citrate-HCl buffer (pH 3.0 to 5.5), 0.1 M sodium phosphate buffer (pH 6.0 to 7.5), and 0.1 M Tris-HCl buffer (pH 8.0 to 9.0) at 10 °C for 24 hr. Determination of milk-clotting activity was conducted at 30 °C as described in the above section.
The optimum temperature for the activity of the crude enzyme from H. erinaceum was determined by assaying MCA at 15, 25, 30, 35, 40, 45, 50 and 60 °C. Thermal stability was analyzed by incubation for 15, 30, 45 and 60 min at pH 6.0 and at different temperatures (20, 30, 35, 40 and 45 °C). Residual activity after each incubation time was measured as described above.
To study the inhibition of MCA, the crude enzyme from H. erinaceum was pre-incubated with inhibitors such as PMSF (10 mM), aprotinin (5 U/mL), EDTA (10 mM), o-phenanthroline (10 mM), pepstatin (50 and 150 µM), and iodoacetamide (10 mM). The mixtures were incubated at room temperature for 60 min and residual MCA was measured. MCA obtained without inhibitors was taken to be 100 %.
Effects of CaCl2 and NaCl The effects of various concentrations (0 to 50 mM) of CaCl2 and NaCl in the substrate on MCA of the crude enzyme were determined. The maximum activity obtained was taken to be 100 %.
SDS-polyacrylamide gel electrophoresis Hydrolyses of αs-, β- and κ-caseins by the crude enzyme from H. erinaceum and chymosin were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 5–20 % (w/v) polyacrylamide gradient gels (Perfect NT Gel M; DRC Co., Ltd., Tama, Japan) using the method of Laemmli (1970), under reduced conditions. Electrophoresis was performed at a constant 20 mA in 25 mM Tris-glycine buffer (pH 8.8) containing 0.1 % SDS. Samples were prepared in 62.5 mM Tris-HCl buffer (pH 6.8) containing 2 % SDS, 20 % glycerol, 5 % 2-mercaptoethanol and 0.005 % bromophenol blue (BPB). After electrophoresis, the gels were stained with Coomassie Brilliant Blue G-250 (Quick-CBB; Wako Pure Chemical Industries, Ltd., Osaka, Japan).
Protein determination The protein content was determined using the Pierce™ BCA Protein Assay (Thermo Scientific, Rockford, IL, USA) with bovine serum albumin as the standard.
Hydrolysis of bovine caseins Commercial bovine αs-, β- and κ-caseins were individually dissolved in 10 mM sodium phosphate buffer (pH 6.0) to a final concentration of 2 mg/mL. The crude enzyme from H. erinaceum was added to the αs-, β- and κ-casein solutions at a ratio of 0.0025:2 (v/v), and the reaction was allowed to proceed at 30 °C for 15, 30, 45 and 60 min. Aliquots were then taken at each time point and heated at 80 °C for 15 min to stop the reaction. Each sample was analyzed by SDS-PAGE. Chymosin was used to compare with the hydrolysis of casein substrates.
Determination of kinetic parameters Bovine αs-, β- and κ-caseins were individually dissolved in 10 mM sodium phosphate buffer (pH 6.0). The reaction was initiated by adding the crude enzyme from H. erinaceum (at a ratio of 1:20, v/v) into αs- and β-casein solutions diluted at concentrations from 0.1 to 5.0 mg/mL, and κ-casein solutions from 1.0 to 20 mg/mL. The reaction was incubated at 30 °C for 300 sec, and then terminated immediately by mixing (1:1, v/v) with 0.4 M trichloroacetic acid (TCA). After being left at 40 °C for 20 min, TCA-insoluble protein was removed by centrifugation at 7133 × g for 15 min. Protein content in the supernatant was measured in quadruplicate. Kinetic parameters of chymosin to κ-casein were also determined for comparison. The kinetic parameters, apparent Michaelis constant (Km), apparent catalytic turnover number (kcat), and apparent proteolytic coefficient (kcat/Km) were calculated from the Lineweaver-Burk plot. The molecular weights of αs-, β- and κ-caseins were taken to be 23.5, 24.0, and 19.0 kDa, respectively. The total protein concentrations ([E]0) of chymosin and the crude enzyme from H. erinaceum were 0.60 and 2.57 mg/mL, respectively.
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Effects of temperature and pH on the crude enzyme MCA MCA increased with increasing temperature in the range of 15 to 30 °C, and the optimum temperature was from 30 to 35 °C, followed by a decline until 60 °C (Figure 1). This result differs from that reported for calf rennet, which is in the range of 42 to 45 °C (Fox, 1969). The thermal behavior of the crude enzyme from H. erinaceum could be applicable for cheese manufacturing, as milk-clotting is usually performed at temperatures < 40 °C. The heat stability of the crude enzyme from H. erinaceum is shown in Figure 2. The enzyme was active even after 60-min incubation at 20 °C. However, heat treatment of the crude enzyme at 40 °C lead to a decrease in MCA (26 % at 15 min, and 8 % at 60 min). The crude enzyme was completely inactivated after 15 min at 45 °C. The crude enzyme from H. erinaceum was thermostable up to 35 °C. Compared with milk-clotting enzymes from other sources, MCA of the crude enzyme from H. erinaceum appeared to decrease dramatically at > 40 °C. For example, the milk-clotting protease from the thermophilic fungus Thermomucor indicae-seudaticae N31 remained stable up to 45 °C for 1 hr, and lost activity after 60 °C (Merheb-Dini et al., 2010). The thermostable proteases from Balanites aegyptiaca fruit pulp (Beka et al., 2014) showed complete loss of activity after 2 hr at 70 °C. The proteases from Mucor pusillus (Nouani et al., 2009) and Rhizomucor miehei (Preetha and Boopathy, 1997) showed almost complete loss of MCA after 30 min at 65 °C.
Effects of temperature on the milk-clotting activity (MCA) of the crude enzyme from H. erinaceum (pH 6.0).
Values presented are the means of three determinants. Error bars represent standard deviations.
Thermal stability of the crude enzyme from H. erinaceum at 20 °C (○), 30 °C (△), 35 °C (◊), 40 °C (●), and 45 °C (▲).
Values presented are the means of three determinants. Error bars represent standard deviations.
The effect of milk pH on the MCA of the crude enzyme from H. erinaceum is shown in Figure 3. MCA decreased with increasing pH. At alkaline pH, the activity was completely lost. The effect of pH on the MCA of the crude enzyme from H. erinaceum is shown in Figure 4. The enzyme was stable at a relatively wide range of pH 3.7 to 6.5, with maximum stability at pH 4.1. At pH > 6.9, the enzyme activity was completely lost, which indicated that the crude enzyme from H. erinaceum was stable under acidic conditions.
Effect of pH on the MCA of the crude enzyme from H. erinaceum at 30 °C.
Values presented are the means of three determinants. Error bars represent standard deviations.
Effect of pH on the stability of the crude enzyme from H. erinaceum at 30 °C.
Values presented are the means of three determinants. Error bars represent standard deviations.
The loss of MCA at > 40 °C and under alkaline conditions may be due to irreversible conformation changes of the protein or proteolysis by the other proteases coexisting in the crude enzyme from H. erinaceum.
Effect of protease inhibitors on MCA Protease inhibitors were used to identify the group at the active site of the enzyme. Inhibition studies on MCA were performed on the crude enzyme from H. erinaceum. Table 1 summarizes the susceptibility of the crude enzyme from H. erinaceum to serine-protease inhibitors (PMSF and aprotinin), cysteine-protease inhibitor (iodoacetamide), metalloprotease inhibitors (EDTA and o-phenanthroline) and aspartic protease inhibitor (pepstatin A). The crude enzyme from H. erinaceum retained MCA in the presence of PMSF, aprotinin, EDTA and iodoacetamide. However, pepstatin A, which is a tight binding inhibitor specific to aspartic proteases, produced a strong inhibition of 61.5 % and 68.1 % at 50 µM and 150 µM pepstatin A, respectively. These results suggest the presence of an aspartate residue at the active site as well as chymosin and pepsin.
| Inhibitor class | Inhibitor | Concentration | Residual MCA (%) |
|---|---|---|---|
| None (control) | None | - | 100 |
| Serine protease | PMSF | 10 mM | 85.0 |
| Aprotinin | 5U/mL | 82.9 | |
| Cystein protease | Iodoacetamide | 10 mM | 90.6 |
| Metalloprotease | EDTA | 10 mM | 108.2 |
| o-Phenanthroline | 10 mM | 83.0 | |
| Aspartic protease | Pepstatin | 150 µM | 31.9 |
| 50 µM | 38.5 |
The activity of the enzyme without any inhibitor was taken as control and recorded as 100 %. All tests were performed in triplicate, and the results are presented as mean values.
Effects of CaCl2 and NaCl concentration on the crude enzyme MCA The presence of calcium and sodium ions is an important factor in cheese processing and product quality. The effects of CaCl2 and NaCl concentrations on the activity of the crude enzyme from H. erinaceum were evaluated. As shown in Figure 5, the addition of CaCl2 significantly increased MCA. In the range of 0–30 mM CaCl2, the activity increased with the increasing concentration of calcium ion. Application of 30 mM CaCl2 resulted in maximum activity. MCA of the crude enzyme from H. erinaceum was CaCl2-dependent, similar to that reported for S. dubium (Ahmed et al., 2010) and Withania coagulans seeds (Naz et al., 2009), Bromelia hieronymi fruits (Bruno et al., 2010), C. scolymus flowers (Chazarra et al., 2007), and ginger (Huang et al., 2011). It is well known that calcium is essential during milk-clotting, creating isoelectric conditions and acting as a bridge between casein micelles.
Effects of NaCl and CaC2 concentration on the MCA of the crude enzyme from H. erinaceum at 30 °C.
Values presented are the means of three determinants. Error bars represent standard deviations.
NaCl had no effect on the activity of the crude enzyme from H. erinaceum, even at 50 mM. Most cheeses are salted by mixing dry salt with the drained curd or by immersion of the pressed cheeses in brine. Salt influences cheese ripening through its effects on water activity, control of microbial growth and various enzyme activities in cheese; and therefore reduces the moisture content of cheese and improves the flavor, texture and color of its cheese (Fox et al., 2015). The lower level of sensitivity to NaCl of the crude enzyme from H. erinaceum makes it beneficial in cheese making.
Casein hydrolysis Hydrolysis of casein components (αs-, β- and κ-caseins) with the crude enzyme from H. erinaceum and with chymosin during incubation was analyzed by SDS-PAGE. Chymosin mainly hydrolyzed κ-casein, as expected, into a low molecular weight product, likely para-κ-casein. The αs- and β-caseins were not remarkably degraded by chymosin. In contrast, αs-, β- and κ-caseins seemed to be susceptible to the action of the crude enzyme from H. erinaceum. Degradation of αs-casein gradually occurred after 60 min of incubation (Figure 6a). Hydrolysis of β-casein by the crude enzyme from H. erinaceum generated several polypeptides with lower molecular weights of between 10 and 20 kDa. Decrease in the intensity of the β-casein band due to hydrolysis by the crude enzyme from H. erinaceum was accompanied by an increase in the intensity of these polypeptides (Figure 6b). κ-Casein was also degraded by the crude enzyme from H. erinaceum into a small product with a molecular weight of approximately 13 kDa (Figure 6c).
SDS-PAGE pattern of bovine αs-casein(a), β-casein(b), and κ-casein(c) hydrolyzed by the crude enzyme from H. erinaceum and chymosin.
(a) lane 1, molecular weight marker; lane 2 and 7, unhydrolyzed bovine αs-casein; lane 3–6, hydrolyzed bovine αs-casein by chymosin for 15, 30, 45, and 60 min, respectively. Lane 8–11, hydrolyzed bovine αs-casein by the crude enzyme from H. erinaceum for 15, 30, 45, and 60 min, respectively.
(b) lane 6, molecular weight marker; lane 1 and 7, unhydrolyzed bovine β-casein; lane 2–5, hydrolyzed bovine β-casein by chymosin for 15, 30, 45, and 60 min, respectively. Lane 8–11, hydrolyzed bovine β-casein by the crude enzyme from H. erinaceum for 15, 30, 45, and 60 min, respectively.
(c) lane 1 and 7, molecular weight marker; lane 2 and 8, unhydrolyzed bovine κ-casein; lane 3–6, hydrolyzed bovine κ-casein by chymosin for 15, 30, 45, and 60 min, respectively. Lane 9–12, hydrolyzed bovine κ-casein by the crude enzyme from H. erinaceum for 15, 30, 45, and 60 min, respectively.
Kinetic parameters Kinetic parameters of the crude enzyme from H. erinaceum on αs-, β- and κ-caseins were investigated and incorporated in Table 2. Results show that kcat of chymosin against κ-casein was almost 5-fold greater than that of the crude enzyme from H. erinaceum. However, chymosin had a similar apparent proteolytic coefficient (kcat/Km) to the crude enzyme from H. erinaceum, attributable to its smaller Km value.
| Enzyme | Substrate | Km (µM) | kcat (min−1) | kcat/Km (µM−1 -min−1) |
|---|---|---|---|---|
| Crude enzyme from H. erinaceum | αs-casein | 3.66 ± 0.10 | 0.40 ± 0.12 | 0.11 ± 0.03 |
| β-casein | 4.98 ± 0.24 | 0.41 ± 0.09 | 0.08 ± 0.01 | |
| κ-casein | 233.0 ± 21.6 | 2.43 ± 0.04 | 0.01 ± 0.001 | |
| Chymosin | κ-casein | 620.7 ± 127.0 | 13.04 ± 0.06 | 0.02 ± 0.004 |
Km: apparent Michaelis constant; kcat: apparent catalytic turnover number; and kcat/Km: apparent proteolytic coefficient.
In regards to the crude enzyme from H. erinaceum, the Km was 47 to 64 times higher for κ-casein (233 µM) than for αs-and β-caseins (3.66 and 4.98 µM, respectively), whereas the kcat/Km value of κ-casein was lower than that of αs- and β-caseins (Table 2). The results suggested that the hydrolyses of αs- and β-caseins with the crude enzyme from H. erinaceum showed a higher affinity and catalytic efficiency than κ-casein during the initial reaction. As we described previously (Sato et al., 2016), no CMP was detected in the whey treated with the crude enzyme from H. erinaceum by RP-HPLC analysis. From the above results, it can be presumed that the crude enzyme from H. erinaceum contained not only the milk-clotting enzyme, but also other proteases. As a result, the crude enzyme from H. erinaceum is likely to preferentially hydrolyze αs- and β-caseins over κ-casein, which might cause destabilization of the casein micelles, resulting in milk coagulation and the formation of cheese curds.
The effects on the taste and texture of cheese produced with the crude enzyme from H. erinaceum are unclear. Further investigations are needed to determine the proteolytic activities during cheese ripening.
The present study investigated the properties of the milk-clotting enzyme from H. erinaceum. The crude enzyme from H. erinaceum belongs to the aspartic proteases, with an optimum temperature of 30–35 °C. The crude enzyme remained stable in the pH range of 3.7–6.5. CaCl2 addition to the milk increased MCA, but NaCl had no effect. Moreover, αs- and β-caseins were more susceptible to proteolysis than κ-casein compared with chymosin. The crude enzyme from H. erinaceum is a potential new source of proteases applicable to cheese production.
Acknowledgements This work was supported by JSPS KAKENHI Grant Numbers 25560035 and 15K00780.
