• 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.

• The Korean Journal of Food And Nutrition
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• v.16 no.4
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• pp.388-396
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• 2003

The freeze dried young antler residue was extracted by proteases and hydrochloric acid(HCl). The young antler was extracted by water at 50$^{\circ}C$ and the residue was reacted by proteases for 5 hours at 50$^{\circ}C$. The extraction rate of its residue was 32.8%(absorbance 3.61 at 280nm) of bacteria protease, 23.8%(absorbance 0.69) of papain, and 31.2% (absorbance 2.96) of pepsin. The young antler was extracted by boiling water and the residue was reacted by proteases for 5 hours at 50$^{\circ}C$. The extraction rate of its residue was 45.0%(absorbance 3.61) of bacteria protease, 30.4%(absorbance 0.33) of papain, and 51.2% (absorbance 2.77) of pepsin. The result of HPLC analysis reveals that in 50$^{\circ}C$ water extract and boiling water extract, all high molecular peak was reduced under MW 1,000 by proteases. The result from the extract of young antler residue reacted by HCl for 5 hours at 50$^{\circ}C$ shows that its extraction rate was 45% (absorbance 0.78) in concentration of 0.1N HCl, 61% (absorbance 1.82) in 0.2N, 81% (absorbance 2.29) in 0.4N, and 82.0% (absorbance 3.28) in 2.0N. The result of HPLC analysis also reveals that in the extract by 0.8N HCl, the peak of about MW 70,000 accounted for 78% in total. Protein content of the extract by 0.8N HCl was 8.2%, and content of amino acid was 81.6%, ash was 1.3%, and mineral contents were 0.1 % of Ca, 2.3% of P, 0.8 % of Mg, 3.4% of Na, 0.002% of F by dry base.

• KSBB Journal
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• v.13 no.1
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• pp.114-118
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• 1998

The effects of fibron solubilization conditions on molecular weight distribution of enzymatically-hydrolyzed silk peptides were investigated. The weight-averaged molecular weights of silk proteins prepared by solubilization with calcium chloride, ethylenediamine and sulfuric acid were 41600, 3308, and 1268 dalton, respectively. Silk peptides in the average molecular weight range of 600-1200 dalton were obtained by protease treatment from solubilized silk fibroin. After the acid hydrolysis of silk protein using hydrochloric acid for 24 hr, silk protein was hydrolyzed to peptides whose average molecular weight and free amino acid content were 145 dalton and 80%, respectively. It was possible to control molecular weight distribution of silk peptides by the combination of solubilization and hydrolysis methods. Among the various treatment methods, acid solubilization followed by protease treatment had an advantage of molecular weight control for the peptide production.

• Journal of the Korean Society of Food Culture
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• v.6 no.1
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• pp.61-69
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• 1991

Brewing method and quality of 10 sample of traditional Dungge-Jang in Kyungsang-Do area were investigated. In order to improve the taste of Dungge-Jang, some amount of boiled bean was added in the Dungge-Jang at early stage of fermentation. The level of amino nitrogen turned out to be low while that of water soluble protein and salt soluble protein was high. Glutamic acid, aspartic acid and proline were the major amino acid in water and salt soluble protein in traditional Dungge-Jang in Kyungsang-Do area. The content of total sugar and free reducing sugar were found to be considerably high, and among the free sugar, glucose was the highest$(2.16{\sim}4.02\;mg/ml)$, followed by maltose and maltotriose. Activities of acid protease and liquefying amylase were $0.13{\sim}1.36$ unit per milliliter and $10.18{\sim}15.19D^{40o}_{30}$ respectively. Result of sensory evaluation showed that the good Dungge-Jang turned out to have well harmonized taste of flavor, sweetness and sourness while the color looked slightly dark and yellow.

• The Korean Journal of Food And Nutrition
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• v.17 no.2
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• pp.147-155
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• 2004

Freeze dried antler, heat dried antler, antler were extracted through processing step by water, protease and hydrochloric acid(HCl). Extraction rate of freeze dried antler at 50$^{\circ}C$ by water was 9.01％(8.82, absorbance at 280 nm), that of heat dried antler was 9.01％(4.45, absorbance at 280 nm), and that of antler was 1.10％(0.31, absorbance at 280 nm), respectively. Extraction rate of freeze dried antler by bacterial protease was 16.89％(4.50, absorbance at 280 nm), and that of heat dried antler was 17.29％(5.62, absorbance at 280 nm), and that of antler was 18.22％(0.64, absorbance at 280 nm), respectively. Extraction rate of freeze dried antler by 0.8N HCl was 72.25％(4.60, absorbance at 280 nm), that of heat dried antler was 71.14％(4.70 absorbance at 280 nm), and that of antler was 79.82％ (2.80, absorbance at 280 nm), respectively. Extraction rate of freeze dried antler through three processing steps was 98.15％, that of heat dried antler was 97.35％, that of antler was 99.14％, respectively. The result of analysis by HPLC shows that high molecular pe which appears in young antler and antler extraction was changed into a small molecular peak of about 1,000 by the reaction of protease, and protein of about MW 70,000 was extracted from their remaining residue by 0.8N HCl. The above result shows that water extraction and protease extraction in the freeze dried young antler, protease extraction and HCl extraction in dried young antler, and HCl extraction in antler are most effective.

• Applied Biological Chemistry
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• v.11
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• pp.1-27
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• 1969

More than thirty kinds of sea food pickles have been eaten in Korea. Out of these salted yellow tail pickle, salted clam pickle, salted oyster pickle, and salted cuttlefish pickle were employed for the analysis of their components, identification of main fermenting microbes, and determination of enzyme characteristics concerned. Also studied was the effect of enzymic action of microbes, which are concerned with the fermenting of pickles, on the production of flavorous 5'-mononucleotides and amino acids. The results are summarized as follows: 1. Microflora observed in the pickles are: (a) Total count of viable cells after 1-2 months of pickling was found to be $10^7$ and that after 6 months decreased to $10^4$. (b) Microbial occurence in the early stage of pickling was observed to be 10-20% Micrococcus spp., 10-20% Brevibacterium spp., 0-30% Sarcina spp., 20-30% Leuconostoc spp., ca 30% Bacillus spp., 0-10% Pseudomonas spp., 0-10% Flavobacterium spp., and 0-20% yeast. (c) Following the early stage of pickling, mainly halophilic bacteria such as Bacillus subtilis, Leuconostoc mesenteroides, Pediococcus halophilus and Sarcina litoralis, were found to exhibit an effect on the fermentation of pickle and their enzyme activities were in direct concern in fermentation of pickles. (d) Among the bacteria participating in the fermentation, Sarcina litoralis 8-14 and 8-16 strains were in need of high nutritional requirement and the former was grown only in the presence of purine, pyrimidine and cystine and the latter purine, pyrimidine and glutamic acid. 2. Enzyme characteristics studied in relation to the raw materials and the concerned microbes isolated are as follows: (a) A small amount of protease was found in the raw materials and 30-60% decrease in protease activity was demonstrated at 7% salt concentration. (b) Protease activity of halophilic bacteria, Bacillus subtilis 7-6, 11-1, 3-6 and 9-4 strains, in the complete media decreased by 10-30% at the 7% salt concentration and that of Sarcina litoralis 8-14 and 8-16 strains decreased by 10-20%. (c) Proteins in the raw materials were found to be hydrolyzed to yield free amino acids by protease in the fermenting microbes. (d) No accumulation of flavorous 5'-mononucleotides was demonstrated because RNA-depolymerase in the raw materials and the pickles tended to decompose RNA into nucleoside and phosphoric acid. (e) The enzyme produced in Bacillus subtilis 3-6 strain isolated from the salted clam pickles, was ascertained to be 5'-phosphodiesterase because of its ability to decompose RNA and thus accumulating 5'-mononucleotide. (f) It was demonstrated that the activity of phosphodiesterase in Bacillus subtilis 3-6 strain was enhanced by some components in the corn steep liquor and salted clam pickle. The enzyme activity was found to decrease by 10-30% and 40-60% at the salt concentration of 10% and 20%, respectively. 3. Quantitative data for free amino acids in the pickles are as follows: (a) Amounts of acidic amino acids such as glutamic and aspartic acids in salted clam pickle, were observed to be 2-10 times other pickles and it is considered that the abundance in these amino acids may contribute significantly to the specific flavor of this food. (b) Large amounts of basic amino acids such as arginine and histidine were found to occur in salted yellow tail pickle. (c) It is much interesting that in the salted cuttlefish pickle the contents of sulfur-containing amino acids were exceedingly high compared with those of others: cystine was found to be 17-130 times and methionine, 7-19 times. (d) In the salted oyster pickle a high content of some essential amino acids such as lysine, threonine, isoleucine and leucine, was demonstrated and a specific flavor of the pickle was ascribed to the sweet amino acids. Contents of alanine and glycine in the salted oyster pickle were 4 and 3-14 times as much as those of the others respectively. 4. Analytical data for 5'-mononucleotides in the pickles are as follows: (a) 5'-Adenylic acid and 3'-adenylic acid were found in large amounts in the salted yellow tail pickle and 5'-inosinic acid in lesser amount. (b) 5'-Adenylic acid, especially 3'-adenylic acid predominated in amount in the salted oyster pickle over that in the other pickles. (c) The salted cuttlefish pickle was found to contain only 5'-adenylic acid and 3'-adenylic acid. It has become evident from the above fact that clam and the invertebrate lack of adenylic deaminase and contain high content of adenylic acid. Thus, they were demonstrated to be the AMP-type. (d) 5'-Inosinic acid was contained in the salted yellow tail pickle in a significant concentration, and it might be considered to be IMP-type. 5. Comparative data for flavor with regard to the flavorous amino acids and the contents of 5'-mononucleotides are: (a) A specific flavor of salted yellow tail pickle was ascribed to the abundance in glutamic acid and aspartic acid, and to the existence of a small amount of flavorous 5'-inosinic acid. The combined effect of these components was belived to exhibit a synergistic action in producing a specific fiavor to the pickle. (b) A specific flavor of salted clam pickle has been demonstrated to be attributable to the richness in glutamic acid and aspartic acid rather than to that of 5'-mononucleotides.

• Journal of the Korean Society of Food Science and Nutrition
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• v.17 no.3
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• pp.242-248
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• 1988

Plasteins were synthesized from a peptic sardine protein hydrolysate by pepsin, ${\alpha}-chymotrypsin$ pretense(from Aspergillus saitoi) and papain under the optimum conditions of previous paper. L -glutamic acid diethylester and L-leucine ethylester also were incorporated into plastein during the plastein reaction by papain. General composition, yield, molecular weight and amino acid composition were measured. The protein, ash and lipid rontent of plasteins were $81.1{\sim}88.2%$, $1.9{\sim}7.6%$ and $0.3{\sim}0.8%$, respectively. The yield of plasteins were pretense plastein 52.3%, papain plastein 44.2%, pepsin plnstein 43.6%, ${\alpha}-chymotrypsin$ plastein 43.2%. Leu -papain plastein 33. 2% and Glu - papain plastein 29.0%. The glutamic acid and leucine content in Glu -papain plastein and Leu -papain plastein were 39.0%, 37.5%, respectively. While the contents in the papain plastein were 14.3%, 7.1%, respectively. The amino acid composition of plasteins were similar to that of peptic sardine protein hydrolysate. The major molecular weight of the peptic hydrolysnte estimated by gelfilteration were 1,800 and 285, and those of plasteins were 26,000 and 9,100 for ${\alpha}-chymotrypsin$, 23,000, 10,000 and 4,300 for pepsin, 18,000 for pretense, 13,000 for papain, 29,000 for Leu -papain plastein and 19,000 for Glu -papain plastein.

• Korean Journal of Food Science and Technology
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• v.34 no.2
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• pp.141-150
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• 2002

Owing to a need for simple extraction and purification for analysis of water soluble vitamins in food samples by RP-HPLC with UV-detector, the methods of bromelain and protease hydrolysis and $C_{18}$ Sep-Pak solid phase extraction were employed. The recoveries of standard water soluble vitamins by the bromelain and protease hydrolysis and $C_{18}$ Sep-Pak solid phase extraction were significantly high compared to AOAC methods in most of vitamins. The contents of pyridoxal determined with protest in the pork was similar, but in the bromelain hydrolysis and AOAC method, was high compared to the results of reference. The niacinamide, thiamin and riboflavin determined with bromelain and protease hydrolysis showed similar values to the results of references. In the potato, pyridoxamine was detected in the AOAC method, which was not detected in the bromelain and protease hydrolysis methods. Pyridoxal contents in the protease hydrolysis and AOAC methods were very similar to the results of references. The recoveries of fortified standard vitamins in food samples were significantly high and accurate compared to those of AOAC methods. The extraction and purification with $C_{18}$ Sep-Pak solid extractor might be considered superior method for the determination of water soluble vitamins in food samples.

• Journal of Applied Biological Chemistry
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• v.54 no.2
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• pp.135-142
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• 2011

A feather-degrading bacterium Elizabethkingia meningoseptica CS2-1 was isolated from compost in a chicken farm. Cultured on a basal medium containing 2% chicken feather, the bacterium showed 729.7 ${\mu}mol/mL$ of amino acid. Optimal culture conditions for feather degradation by E. meningoseptica CS2-1 were $25^{\circ}C$, pH 7.5, and 180 rpm. The optimal pH and temperature for protease activity were 8.0 and $40^{\circ}C$, respectively. The composition of an optimal medium for amino acid production was 0.05% NH4Cl, 0.05% NaCl, 0.03% $K_2HPO_4$, 0.03% $KH_2PO_4$, 0.01% $MgCl_2{\cdot}6H_2O$, 0.1% urea, and 2% chicken feather. Characteristics of amino acids extracted from the optimal medium under the optimal culture conditions of E. meningoseptica CS2-1 were analyzed. The total amino acid content of strain CS2-1 was 1063 ${\mu}mol/mL$, which was 46% higher compared to the basal condition (729.7 ${\mu}mol/mL$). The essential amino acid content in the total amino acid was 315.9 ${\mu}mol/mL$, which was 44% higher than that of the basal condition. Major amino acids were proline (14%), aspartic acid (12%), glutamic acid (11%), serine (10%), alanine (10%), glycine (9%), and tyrosine (7%) by strain CS2-1. These results suggest that strain CS2-1 can be used as a potential microbial resource for the production of amino acid using chicken feathers.

• Korean Journal of Food Science and Technology
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• v.23 no.4
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• pp.400-404
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• 1991

Protease was purified from Aspergillus niger propagated on katsuobushi. The optimal pH and temperature of the enzyme were 7.2 and $45^{\circ}C$, respectively. The enzyme was stable at $pH\;5{\sim}8$ and at below $40^{\circ}C$. Enzyme activity was promoted by $K^{-}\;and\;Fe^{2+}$, whereas it was inhibited by $Hg^{2},\;Zn^{2},\;Mn^{2}\;and\;Cd^{2}$. The acidic, basic and neutral amino acid compositions were found to be 22.63, 13.57 and 63.80%, respectively. The content of nonpolar, poler and sulfur-containing amino acids were 39.72, 20.03 and 9.53% respectively, and aspartic and glutamic acids were abundant. The molecular weight was 42,000 and isoelectric point was estimated pH 5.6.