• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1944-1953
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• 2015

A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80℃ in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50℃ and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni²⁺, Mn²⁺, and Cu²⁺ to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The K_m and V_max values were estimated to be 35.7 mg/ml, and 5 × 10⁴ U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.

• Journal of Dairy Science and Biotechnology
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• v.31 no.1
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• pp.9-20
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• 2013

The study was conducted to evaluate the feasibility of Mozzarella cheese analogue generation by using a mixture of soy milk and raw milk and to compare the quality of the resultant cheese with that of Mozzarella cheese manufactured using the traditional method. The mixtures showed increase in protein and decrease in lactose and SNF in a dose-dependent manner with the addition of soy milk. The Mozzarella cheese analogue had lower total solids content than the control cheese product, while the fat content was similar between both. The analogue cheese had lower ash content than the traditionally prepared cheese; the content was proportional to the amount of soy milk in the mixture. Higher soy milk quantities within mixtures also resulted in proportionally higher levels of fat content within analogue cheese. Water-soluble nitrogen content was lower in the analogue cheese than in the control cheese. While the WSN level increased in the control cheese, it was almost constant in the analogue cheese. The control cheese had much higher actual and predicted yield than the analogue cheese, while the analogue cheese had a higher stability level. The control cheese had a higher transfer rate than the analogue cheese, with the exception of lactose. Electrophoresis analysis showed bands for Mozzarella cheese analogues that were present in addition to the normal ${\alpha}$-casein and ${\beta}$-casein bands. Physical characteristic analysis showed that hardness was affected by the addition of soy milk to cheese, while cohesiveness and brittleness were affected by the addition of raw milk, and elasticity was barely affected by milk composition. The meltability of the control cheese was higher than that of the analogue cheese and increased during 30 days of storage at $4^{\circ}C$. Browning, oiling-off, and stretching characteristics were almost identical between the 2 types of cheeses. Sensory evaluation findings showed that the control cheese had much better body texture, appearance, and flavor than the analogue cheese.

• Applied Biological Chemistry
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• v.35 no.4
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• pp.237-241
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• 1992

The protease from Halomonas sp. ES 10 was purified by methanol precipitation, gel filtration on Sephadex G-150 and G-200, and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The specific activity of purified enzyme was 1,014 units/mg protein, and the yield of the total activity from the culture filtrate was 7%. The optimal temperature and pH for the enzyme activity were $35^{\circ}C$, and pH 11.0, respectively. And the enzyme was stable in the range of $pH\;7.5{\sim}11.0$. The residual activity of the enzyme was 70%, when the enzyme was incubated at $50^{\circ}C$ for 40 min. The Km value of the enzyme was 7.4 mg/ml to milk casein. $Li^+$, $Ca^{2+}$, SDS and Tween 80 were appeared to activators, whereas $Hg^{2+}$ and EDTA to inhibitors. The addition of DFP and PMSF showed the relative enzyme activities of 63% and 107%, respectively, suggesting that the enzyme may not belong to serine type protease. When the alkaline protease was treated with 0.5 M and 1 M NaCl, the relative enzyme activities were 95% and 65%, respectively. This enzyme showed 20% and 15% higher enzyme activity than that of Aspergillus oryzae (Sigma Chemical Company product, P4755) in the presence of 0.5 M and 1 M NaCl.

• Journal of Animal Science and Technology
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• v.62 no.2
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• pp.263-275
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• 2020

Studies on promoting milk protein yield by supplementation of amino acids have been globally conducted. Nevertheless, there is a lack of knowledge of what pathways affected by individual amino acid in mammary epithelial cells that produce milk in practice. Phenylalanine (PHE) and valine (VAL) are essential amino acids for dairy cows, however, researches on mammary cell levels are still lacking. Thus, the aim of this study was conducted to evaluate the effects of PHE and VAL on milk protein synthesis-related and energy-mediated cellular signaling in vitro using immortalized bovine mammary epithelial (MAC-T) cells. To investigate the effects of PHE and VAL, the following concentrations were added to treatment medium: 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. The addition of PHE or VAL did not adversely affect cell viability compared to control group. The concentrations of cultured medium reached its maximum at 0.9 mM PHE and 0.6 mM VAL (p < 0.05). Therefore, aforementioned 2 treatments were analyzed for proteomics. Glucose transporter 1 and mammalian target of rapamycin mRNA expression levels were up-regulated by PHE (166% and 138%, respectively) (p < 0.05). Meanwhile, sodium-dependent neutral amino acids transporter type 2 (ASCT2) and β-casein were up-regulated by VAL (173% in ASCT2, 238% in and 218% in β-casein) (p < 0.05). A total of 134, 142, and 133 proteins were detected in control group, PHE treated group, and VAL treated group, respectively. Among significantly fold-changed proteins, proteins involved in translation initiation or energy metabolism were detected, however, expressed differentially between PHE and VAL. Thus, pathway analysis showed different stimulatory effects on energy metabolism and transcriptional pathways. Collectively, these results showed different stimulatory effects of PHE and VAL on protein synthesis-related and energy-mediated cellular signaling in MAC-T cells.

• Korean Journal of Food Science and Technology
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• v.30 no.1
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• pp.82-89
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• 1998

This study was performed to elucidate the purification and characterization of pretease from saewoo-jeot, a Korean traditional salt-fermented shrimp product. The protease in saewoo-jeot (Acetes japonicus) were extracted, desalted through electrodialysis and purified by ammonium sulfate fractionation, Sephadex G-100 gel filtration and DEAE-cellulose column chromatography. Purified enzyme had specific activity of 8.4 unit/mg, yield of 14% and purification fold of 9.8. Purified enzyme was confirmed as single band protein by polyacrylamide gel electrophresis and the molecular weight was estimated to be about 24 kDa. The optimal pH and temperature for the enzyme activity were 8.0 and $40^{\circ}C$, respectively. The range of its stability to the pH and temperature were 7.0 to 10.0 and $30^{\circ}C\;to\;60^{\circ}C$, respectively. The activity of enzyme to synthetic substrate showed BAPNA and TAME. The enzyme was activated significantly by manganese ions, while inhibited by STI, TLCK. metals $(K^+,\;Li^+,\;Na^+,\;Ca^{++},\;Co^{++},\;Cu^{++},\;Mg^{++},\;Ba^{++},\;Hg^{++},\;Zn^{++},\;Fe^{+++})$. The Km value of the enzyme was $5.1{\times}10^{-7}\;M$ to hammersten casein. It's suggested that purified protease from saewoo-jeot seemed to be trypsin-like enzyme.

• Journal of Microbiology
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• v.41 no.3
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• pp.207-211
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• 2003

Extracellular protease, from Aeromonas hydrophila Ni 39, was purified 16.7-fold to electrophoretic homogeneity with an overall yield of 19.9％, through a purification procedure of acetone precipitation, and Q Sepharose and Sephacryl S-200 chromatographies. The isoelectric point of the enzyme was 6.0 and the molecular mass, as determined by Sephacryl S-200 HR chromatography, was found to be about 102 kDa. SDS/PAGE revealed that the enzyme consisted of two subunits, with molecular masses of 65.9 kDa. Under standard assay conditions, the apparent $K_{m}$ value of the enzyme toward casein was 0.32 mg/ml. About 90％ of the proteolytic activity remained after heating at 60$^{\circ}C$ for 30 min. The highest rate of azocasein hydrolysis for the enzyme was reached at 60$^{\circ}C$, and the optimum pH of the enzyme was 9.0. The enzyme was inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), by about 87.9％, but not by E64, EDTA, pepstatin or 1,10-phenanthroline. The enzyme activity was inhibited slightly by Ca$\^$2＋/, Mg$\^$2＋/ and Zn/supb 2＋/ ions.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.507-514
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• 1998

The strain K-54, the best producer of fibrinolytic enzyme, was isolated from Korean traditional food Chung Guk Jang and identified as Bacillus subtilis. Fibrinolytic enzyme was purified and characterized, and its molecular weight was determined. The fibrinolytic enzyme activity was increased about 66.9 times via purification with recovery yield of 10.1%. The optimum pH and temperature of this enzyme were 11 and $65^{\circ}C$. The enzyme was stable within a pH range 8-12 and unstable at 9$0^{\circ}C$. The molecular weight was estimated to be 29,000 dalton in the form of monomer with no other subunit. The isoelectric point was calculated 8.67. N-terminal sequence was identified Ala-Gly-Ser-Val-Pro-Try-Gly-Ser. Km value of the enzyme for $\alpha$-casein was calculated to be 0.31 (3.1 mg/$m\ell$). The enzyme activity highly inhibited by PMSF at 1 nM.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.10
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• pp.1372-1376
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• 2011

The content of Ca in milk exceeds the typical saturation level of Ca salts, which is necessary for neonate growth. This calcium is distributed between the casein micelles in the colloidal and aqueous phases. Information on the properties of calcium activity in the aqueous phase is limited compared with that on the properties of bound or sequestrated calcium. The objectives of this study were to evaluate the changes in calcium activity in fresh milk using an ion-selective electrode and to assess the relationship between calcium activity and milk production in hot season. Milk samples collected from 10 cows at the National Agricultural Research Center for Kyushu Okinawa Region in June to October (Min-Max: 7.2-$35.2^{\circ}C$, 24.3-100% RH) were analyzed on total calcium concentrations and calcium activity. We observed that the rectal temperature of the cows increased according to elevation of ambient temperature but that the pH of the collected milk ($6.61{\pm}0.01$ (Mean${\pm}$SEM)) was not significantly influenced by rectal and ambient temperature. Total calcium concentrations and calcium activity in fresh milk decreased in July (Min-Max: 21.1-$33.5^{\circ}C$, 48.9-100.0% RH) compared with the values after August (Min-Max: 18.1-$35.0^{\circ}C$, 26.5-96.2% RH) (p<0.05); however, there was no significant correlation between the two parameters. The ratio of calcium activity to total calcium concentration decreased after August compared with the values in June and July (p<0.05). The calcium activity in fresh milk was positively correlated with milk yield (r = 0.45, p<0.01) and negatively correlated with milk lactose content (r = -0.53, p<0.01). These results suggest that the calcium activity in milk could be affected by ambient temperature and might be associated with milking production in hot season.

• Journal of Microbiology and Biotechnology
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• v.22 no.1
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• pp.58-68
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• 2012

An investigation was conducted on the enhancement of production and purification of an oxidant and SDS-stable alkaline protease (BHAP) secreted by an alkalophilic Bacillus horikoshii, which was screened from the body fluid of a unique Korean polychaeta (Periserrula leucophryna) living in the tidal mud flats of Kwangwha Island in the Korean West Sea. A prominent effect on BHAP production was obtained by adding 2% maltose, 1% sodium citrate, 0.8% NaCl, and 0.6% sodium carbonate to the culturing medium. The optimal medium for BHAP production contained (g/l) SBM, 15; casein, 10; $K_2HPO_4$, 2; $KH_2PO_4$, 2; maltose, 20; sodium citrate, 10; $MgSO_4$, 0.06; NaCl, 8; and $Na_2CO_3$, 6. A protease yield of approximately 56,000 U/ml was achieved using the optimized medium, which is an increase of approximately 5.5-fold compared with the previous optimization (10,050 U/ml). The BHAP was homogenously purified 34-fold with an overall recovery of 34% and a specific activity of 223,090 U/mg protein using adsorption with Diaion HPA75, hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose, and ion-exchange chromatography on a DEAE- and CM-Sepharose column. The purified BHAP was determined a homogeneous by SDS-PAGE, with an apparent molecular mass of 28 kDa, and it showed extreme stability towards organic solvents, SDS, and oxidizing agents. The $K_m$ and $k_{cat}$ values were 78.7 ${\mu}M$ and $217.4s^{-1}$ for N-succinyl-Ala-Ala-Pro-Phe-pNA at $37^{\circ}C$ and pH 9, respectively. The inhibition profile exhibited by PMSF suggested that the protease from B. horikoshii belongs to the family of serine proteases. The BHAP, which showed high stability against SDS and $H_2O_2$, has significance for industrial application, such as additives in detergent and feed industries.

• Applied Biological Chemistry
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• v.35 no.4
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• pp.300-307
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• 1992

Bromelains (EC 3.4.4.24) isolated from pineapple fruit and stem have been purified about 18 and 46-folds to homogenity in the same yield of 23%. Molecular weights of fruit and stem-bromelain were estimated to be 32.5 KDa and 37 KDa by Sephadex G-200, respectively. The enzymes were composed of one subunit. The fruit and stem-bromelain had their maximum activity at pH 8.0, $70^{\circ}C$ and at pH 7.0, $60^{\circ}C$. Especially the enzymes catalyzed hydrolysis of plant proteins such as ISP (Isolated soybean protein) and wheat gluten with high molecular activity compared to animal proteins. The enzymes were competitively inhibited by sulfhydryl reagent; $K_i$ values of fruit and stem-bromelain for pCMB(p-chloromercuribenzoate) were 0.18 mM and 0.10 mM. Activities of the enzymes inhibited by pCMB were reversibly restored with increasing concentration of cysteine.