**4.4. Characteristics of the protease as a soybean milk curdling enzyme**

Optimum pH, temperature, and stability of the enzyme are presented in Figs. 6a and 6b. Optimum pH was assayed at pH 4.0–8.0 using 50 mM phosphate-citric buffer or phosphate-

New Cheese-Like Food Production from Soy Milk — Utility of Soy Milk Curdling Yeast http://dx.doi.org/10.5772/60848 91

cence was measured using a 485-nm excitation wave and a 535-nm emission wave. The proteolytic activity was decided for one unit expressed equal amount of trypsin (1 ng⋅mL–1)

After ion chromatography, fractions containing the highest level of activity were pooled and reprecipitated using 80% saturation of ammonium sulfate. After redissolving the precipitate, gel filtration chromatography (10 mm × 350 mm, P-100 gel; Bio-Rad Laboratories Inc., CA, USA) was carried out. Then the molecular weight of the enzyme was analyzed using also the chromatography as various molecular weight standards (myosin, 200 kDa; serum albumin,

The results are portrayed in Figs. 4a and 4b. One main curdling activity peak was identified using ion-exchange chromatography. The peak (fraction no. 49) agreed with protein and activity. Furthermore, curdling activity agreed with the same fractions presenting protease

As a result, the larger peak of proteolysis activity was found around fraction number 25, and a small peak was found at fraction number 49. The fraction of the proteolysis enzyme around number 25 was not representative of curdling activity. It is considered that the former fractions are attributable to intense proteolysis enzymes and that the latter fractions are attributable to

After reprecipitation, the sample was analyzed using gel filtration chromatography. A peak was found at fraction numbers 11–14. Their fractions agreed with soybean milk curdling activity, proteolytic activity, and protein. This result on their chromatograms demonstrates that protease and soybean milk curdling enzyme have some mutual relation of activity.

After purification, the enzyme protein band was approximately 45 kDa (Fig. 5a), which agrees with data of other proteases. The protease molecular weight was measured using gel filtration chromatography (Fig. 5b). The molecular mass is about 45 kDa. The protease was inferred. The soy milk curdling enzyme has proteolytic activity. Results suggest that the soy milk curdling enzyme was a proteolysis enzyme. Many researchers have reported protease produced by yeasts as *Candida albicans* [30, 31, 32], *Candida humicola* [33], and *Saccharomyces cerevisiae* [34]. Extracellular proteases produced by yeasts as *Candida* spp. are 42–45 kDa [35,

By contrast, few reports describe intracellular protease producing *Saccharomyces cerevisiae*, although many intracellular proteases in the vacuole or other organelles are known to be related to proteinase A, which is 42 k Da [34]. The molecular weight of curdling soy protein enzyme protease agreed with protease produced by other yeast as Ascomycota. However, the *Mucor* sp. enzyme, which curdles bovine milk, produced a 49-kDa protease [37], which is larger

Optimum pH, temperature, and stability of the enzyme are presented in Figs. 6a and 6b. Optimum pH was assayed at pH 4.0–8.0 using 50 mM phosphate-citric buffer or phosphate-

**4.4. Characteristics of the protease as a soybean milk curdling enzyme**

doing proteolysis of FTC solution.

90 Food Production and Industry

activity (fraction no. 49).

soybean milk-curdling enzymes.

32]; those by bacteria are 21 kDa [36].

than those produced by these yeasts.

66.2 kDa; ovalbumin, 45.0 kDa; trypsin inhibitor, 21.5 kDa).

Fig. 4a. Ion-exchange chromatography of soybean milk curdling. **enzyme.** Fig. 4b. Gel filtration chromatography of **Figure 4.** (a). Ion-exchange chromatography of soybean milk curdling. **enzyme.** (b). Gel filtration chromatography of soybean milk curdling. **enzyme**.

NaOH. For optimum pH of the enzyme assaying, 0.1 mL of a fluorescein isothiocyanatelabeled casein (FTC) solutions, which dissolved in each phosphate buffer (50 mM, pH 4.0–8.0), and 20 μL of enzyme solution were reacted at 40°C for 60 min. For optimum temperature of the enzyme assaying, FTC solution at pH 7.5 (0.1 mL) and 20 μL of enzyme solution were reacted at 15°C–70°C for 60 min to find optimum temperature. After reaction, fluorescence was measured using a 485-nm excitation wave and a 535-nm emission wave.

excitation wave and a 535-nm emission wave.

15C–70C for 60 min to find optimum temperature. After reaction, fluorescence was measured using a 485-nm **Figure 5.** (a). Photograph of SDS-PAGE of soybean curding enzyme. (b). Measurement of molecular weight.

The optimum pH for the protease activity as curdling was pH 7.5; the optimum temperature was 50°C. The optimum pH of a bovine milk curdling protease, *Mucor pusillus*, is pH 5.0. The optimum pH values of many commercially available proteases are pH 5.9–6.7. However, soymilk curdling activity decreases concomitantly with increasing alkalinity. The optimum pH for the protease activity as curdling was pH 7.5; the optimum temperature was 50C. The optimum pH of a bovine milk curdling protease, *Mucor pusillus*, is pH 5.0. The optimum pH values of many commercially available proteases are pH 5.9–6.7. However, soymilk curdling activity decreases concomitantly with increasing alkalinity.

Fig. 6a. Optimum pH of soybean curdling enzyme. Fig. 6b. Optimum temperature of soybean curdling enzyme. Park and coauthors [38] reported that the optimum pH of soybean milk curdling protease produced by *Bacillus* **Figure 6.** (a). Optimum pH of soybean curdling enzyme. (b). Optimum temperature of soybean curdling enzyme.

was pH 6.0. Regarding this enzyme, the optimum pH of the protease was pH 7.5. The optimum temperature of the protease was 50C. The enzyme also curdled soybean milk at pH 7.5 and 50C. Commercial soybean milks sold in

Effects of metal ions and inhibitors on protease are presented in Table 5. In fact, zinc, copper, and mercury all inhibit protease activity. The amino acid of the active site contains cysteine residue because of inhibition by mercury

Japan are pH 7.0–7.2. The pH range agrees with their optimum pH range.

Table 5. Effects of metal ion and inhibitor of the protease

Na<sup>+</sup> 1 mM 101.5 K<sup>+</sup> 1 mM 105.9 Mn2+ 1 mM 101.9 Mg2+ 1 mM 46.2

inhibitor Concentrations Relative activity (%)

Metal ion and

[39]. Furthermore, EGTA (10 mM) inhibited protease activity (62.0% of relative activity).

14

Park *et al.* [38] reported that the optimum pH of soybean milk curdling protease produced by *Bacillus* was pH 6.0. Regarding this enzyme, the optimum pH of the protease was pH 7.5. The optimum temperature of the protease was 50°C. The enzyme also curdled soybean milk at pH 7.5 and 50°C. Commercial soybean milks sold in Japan are pH 7.0–7.2. The pH range agrees with their optimum pH range.

Effects of metal ions and inhibitors on protease are presented in Table 5. In fact, zinc, copper, and mercury all inhibit protease activity. The amino acid of the active site contains cysteine residue because of inhibition by mercury [39]. Furthermore, EGTA (10 mM) inhibited protease activity (62.0% of relative activity).


**Table 5.** Effects of metal ion and inhibitor of the protease

The optimum pH for the protease activity as curdling was pH 7.5; the optimum temperature was 50°C. The optimum pH of a bovine milk curdling protease, *Mucor pusillus*, is pH 5.0. The optimum pH values of many commercially available proteases are pH 5.9–6.7. However,

The optimum pH for the protease activity as curdling was pH 7.5; the optimum temperature was 50C. The

temperature of the enzyme assaying, FTC solution at pH 7.5 (0.1 mL) and 20 L of enzyme solution were reacted at 15C–70C for 60 min to find optimum temperature. After reaction, fluorescence was measured using a 485-nm

**Figure 5.** (a). Photograph of SDS-PAGE of soybean curding enzyme. (b). Measurement of molecular weight.

0.0

0.0 20.0 40.0 60.0 80.0

**Temperature (oC)**

20.0

40.0

60.0

**Relative activity (%)**

Fig. 6a. Optimum pH of soybean curdling enzyme. Fig. 6b. Optimum temperature of soybean curdling enzyme. Park and coauthors [38] reported that the optimum pH of soybean milk curdling protease produced by *Bacillus* was pH 6.0. Regarding this enzyme, the optimum pH of the protease was pH 7.5. The optimum temperature of the protease was 50C. The enzyme also curdled soybean milk at pH 7.5 and 50C. Commercial soybean milks sold in

**Figure 6.** (a). Optimum pH of soybean curdling enzyme. (b). Optimum temperature of soybean curdling enzyme.

Effects of metal ions and inhibitors on protease are presented in Table 5. In fact, zinc, copper, and mercury all inhibit protease activity. The amino acid of the active site contains cysteine residue because of inhibition by mercury

80.0

100.0

14

soymilk curdling activity decreases concomitantly with increasing alkalinity.

(a) (b)

optimum pH of a bovine milk curdling protease, *Mucor pusillus*, is pH 5.0. The optimum pH values of many commercially available proteases are pH 5.9–6.7. However, soymilk curdling activity decreases concomitantly with

0

Metal ion and

4.0 5.0 6.0 7.0 8.0

**pH**

Japan are pH 7.0–7.2. The pH range agrees with their optimum pH range.

Table 5. Effects of metal ion and inhibitor of the protease

Na<sup>+</sup> 1 mM 101.5 K<sup>+</sup> 1 mM 105.9 Mn2+ 1 mM 101.9 Mg2+ 1 mM 46.2

inhibitor Concentrations Relative activity (%)

[39]. Furthermore, EGTA (10 mM) inhibited protease activity (62.0% of relative activity).

20

40

60

**Relativa activity (%)**

80

100

increasing alkalinity.

92 Food Production and Industry

excitation wave and a 535-nm emission wave.

The activity was not activated by metal ions, but it was inactivated by mercury. Soybean milkcurdling enzyme [38] was inhibited by zinc ions and mercury ions. These results agree with our data related to zinc and mercury. Its survival activity was 18% by mercury. The protease %%D %EDQG%DSSUR[LPDWHO\N'D>@

was not activated by metal ions, which indicates that the protease is not a metalloprotease: a metal-dependent enzyme. The amino acid of active site contains cysteine residue.

The mechanisms of curdling soybean milk protease were investigated. At first, The curdled soybean milk samples with added protease and without protease were treated with sample buffer solution. 7KH PHFKDQLVPV RI FXUGOLQJ VR\EHDQ PLON SURWHDVH ZHUH LQYHVWLJDWHG \$W ILUVW 7KH FXUGOHG VR\EHDQ PLON

Soybean milk was poured into a glass vessel (inner diameter 32 mm, height 45 mm). After 0.1 mL of enzyme solution adding to the soybean milk, or without enzyme 0.1 mL D.W., the mixtures were incubated at 40°C, and they were sampled sequentially; between 4- and 24-h. samples (0.01 mL) were added to 0.01 mL of sample buffer and then heated at 100°C for 3 min. Then samples (10 μl) were added in each well. The samples were electrophoresed on a 12.5% uniform gel at 20 mA. VDPSOHVZLWKDGGHGSURWHDVHDQGZLWKRXWSURWHDVHZHUHWUHDWHGZLWKVDPSOHEXIIHUVROXWLRQ 6R\EHDQPLONZDVSRXUHGLQWRDJODVVYHVVHOLQQHUGLDPHWHUPPKHLJKWPP\$IWHUP/RIHQ]\PH VROXWLRQDGGLQJWRWKHVR\EHDQPLONRUZLWKRXWHQ]\PHP/':WKHPL[WXUHVZHUHLQFXEDWHGDW&DQGWKH\ ZHUHVDPSOHGVHTXHQWLDOO\EHWZHHQDQGKVDPSOHVP/ZHUHDGGHGWRP/RIVDPSOHEXIIHUDQGWKHQ KHDWHGDWq&IRUPLQ7KHQVDPSOHVOZHUHDGGHGLQHDFKZHOO7KHVDPSOHVZHUHHOHFWURSKRUHVHGRQD

They were subsequently examined using SDS–PAGE (Fig. 7) of curdled soybeans. The left side lane shows the standard of protein size. The next lane (0 h.) shows soybean milk protein without reaction of protease. The other lanes show soybean milk protein decomposed for 4, 8, 12, 16, and 24 h. Lane 0 h shows the α′- and α-subunits of β-conglycinin (approximately 84– 73 kDa), the A3 acidic subunit (approximately 40 kDa), other acidic subunits as A4, A1a, A1b, and A2 (approximately 30–42 kDa) of glycinin, the β subunit (approximately 50 kDa), and basic subunits as B3, B1a, B1b, and B4 (approximately 20 kDa) [40, 41]. XQLIRUPJHODWP\$ 7KH\ZHUHVXEVHTXHQWO\H[DPLQHGXVLQJ6'6±3\$\*()LJRIFXUGOHGVR\EHDQV7KHOHIWVLGHODQHVKRZVWKH VWDQGDUGRISURWHLQVL]H7KHQH[WODQHKVKRZVVR\EHDQPLONSURWHLQZLWKRXWUHDFWLRQRISURWHDVH7KHRWKHUODQHV VKRZ VR\EHDQ PLON SURWHLQ GHFRPSRVHG IRU DQG K /DQH K VKRZV WKH Dc DQG DVXEXQLWV RI EFRQJO\FLQLQDSSUR[LPDWHO\±N'DWKH\$DFLGLFVXEXQLWDSSUR[LPDWHO\N'DRWKHUDFLGLFVXEXQLWVDV\$ \$D\$EDQG\$DSSUR[LPDWHO\±N'DRIJO\FLQLQWKHEVXEXQLWDSSUR[LPDWHO\N'DDQGEDVLFVXEXQLWVDV

)LJ'LJHVWLRQRIVR\SURWHLQGXULQJFXUGLQJE\VR\EHDQPLONFXUGOLQJHQ]\PH **Figure 7.** Digestion of soy protein during curding by soybean milk curdling enzyme.

7KHWZREDQGVVKRZQDVDcDQGDGLVDSSHDUHGJUDGXDOO\DIWHUWKHUHDFWLRQVKRZLQJWKHSURWHLQEDQGIURPFXUG PDNLQJWKHSURWHDVH,QWKHJO\FLQLQVXEXQLWVWKHEDQGRIWKH\$DFLGLFVXEXQLWGLVDSSHDUHGFRPSOHWHO\DIWHUK )XUWKHUPRUH\$\$D\$EDQG\$GLVDSSHDUHGWRDSDUWLDOGHJUHHVDPHDV\$DFLGLFVXEXQLW3HSWLGHVVPDOOHUWKDQ The two bands shown as α′ and α disappeared gradually after the reaction, showing the protein band from curd making the protease. In the glycinin subunits, the band of the A3 acidic subunit disappeared completely after 4 h. Furthermore, A4, A1a, A1b, and A2 disappeared to a partial

GHFRPSRVHG6R\EHDQSURWHLQEHFDPHORRVHO\FXUGOHGZLWKWKHDGGLWLRQRIRWKHUSURWHDVHVIURPPLFURRUJDQLVPVRU SODQWV7KHSURWHLQZDVGHFRPSRVHG7KHORZPROHFXODUZHLJKWSHSWLGHVLQFUHDVHGRQWKHSRO\DFU\ODPLGHJHO \*HQHUDOO\6JO\FLQLQZDVUHODWHGWRWKHIRUPDWLRQRIDVWLIIHUJHO)XUWKHUPRUH2QRHWDO>@UHSRUWHG

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degree same as A3 acidic subunit. Peptides smaller than 20 kDa were detected on the gel. The β-conglycinin as α, α′, and part of glycinin as A3 A4, A1b, and A<sup>2</sup> were decomposed. Soybean protein became loosely curdled with the addition of other proteases from microorganisms or plants. The protein was decomposed. The low-molecular-weight peptides increased on the polyacrylamide gel. Generally, 11S glycinin was related to the formation of a stiffer gel. Furthermore, Ono *et al.* [42] reported hydrophobic bonding and hydrogen bonding related to curdling *Tofu*. Utsumi *et al.* [43] reported that the basic subunit and β-subunit formed macro complexes by heating. The complexes were regarded as forming cores for *Tofu* coagulation. The complexes were reportedly wrapped in α-, α′-, and acidic subunits [42].

According to our data, after the curdling soy milk by enzyme, α- and α′-subunits cleaved by the protease easily, whereas basic and β-subunit remained. It is considered that surface proteins as α- and α′-subunits were decomposed easily. Some decomposed subunits such as α, α′, A3, acidic, and basic subunits are regarded as related to the curdling soybean milk.

The enzyme of mechanisms for proteolysis was searched that the enzyme had the peptidase activity as exotype proteolysis activity and protease as endotype proteolysis activity. The synthesis peptide substrates, Z-glutamyl-tyrosine, and casein, FTC, were reacted by the enzymes. Peptidase (carboxypeptidase) activity was determined by the increase in ninhydrin after hydrolysis of benzyloxycarbonyl-glutamyl-tyrosine (pH 8.0) at 40°C.

The results show that the enzyme had 0.14 U⋅mg–1 protein as peptidase activity and 0.55 U⋅mg–1 protein protease as endotype proteolysis activity (data not shown). Results also show that the soybean milk-curdling enzyme as a proteolysis enzyme had endotype proteolysis activity.

Jones *et al.* [34] reported proteolysis enzymes of three types in yeast classes: cytosolic protease, vacuolar proteases, and proteases located within the secretory pathway. They belong to aspartic type, serine, or metallo-type proteolysis enzyme cleaved substrates with endotype or exotype. Generally, metallo-type enzyme requires metal ions such as zinc. The optimum condition of aspartic protease is an acidic condition. The enzyme did not require ion metal. Its optimum pH was 7.5, which is weakly alkaline. It is therefore considered that the enzyme is a serine protease of one kind. These results agreed with serine protease from *Bacillus* sp. curdling soy protein [44]. For future studies, we will ascertain the amino acid sequence in a substrate cleaved by enzyme using synthesis substrate.
