• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• Journal of Food Hygiene and Safety
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• v.22 no.3
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• pp.151-158
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• 2007

Among 68 strains of lactic acid bacteria (LAB) isolated from Dongchimi, a strain K11 was selected due to its bactericidal activity against Escherichia coli O157 The strain K11 was identified as Lactobacillus plantarum, based on physiological and biochemical characteristics. In the late exponential phase, La. plantarum K11 showed maximum bacteriocin activity (12,800 BU/mL) and maintained until the early stationary phase. The bacteriocin activity was completely inactivated by all the proteolytic enzymes such as pepsin, protease, proteinase K, papain, chymotrypsin, and trypsin, but the activity was not affected by catalase, a-amylase, lysozyme, and lipase, suggesting proteinaceous nature of the bacteriocin. Additionally, this activity was not affected in the pH range from 3.0 to 9.0 and under storage conditions like 30 days at -20,4, or $25^{\circ}C$. Although the bacteriocin activity was absolutely lost after 15 min treatment at 121, it was relatively stable at $70^{\circ}C$ for 60 min or $100^{\circ}C$ for 30 min. The activity was disappeared by treatment with acetone, benzene, ethanol, or methanol, but it was not affected by treatment with chloroform or hexane. The antibacterial activity of the bacteriocin was good against some LAB including Lactobacillus spp., Enterococcus spp., and Streptococcus spp., but not against food-borne pathogens such as Bacillus spp., Listeria spp., and Staphylococcus spp. as well as yeasts and molds. Especially, some intestinal bacteria such as Enterobacter aerogenes and E. coli were significantly affected by the bacteriocin of La, plantarum K11. Furthermore, the addition of 640 BU/mL resulted in the complete clearance of E. coli O157 after 10 hr.

• Journal of the Korean Society of Food Science and Nutrition
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• v.35 no.5
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• pp.588-593
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• 2006

This study was intended to observe possibility of which radiation technique can be used for oligopeptide production from pigskin collagen to reduce environmental pollution in processing and simplify the processing steps. Raw pigskin was ground using chopper, and then defatted in acetone cooled at $-20^{\circ}C$ freezer. Defatted dried pigskin was irradiated at 20, 40, 60, 100, 150, 200, 250, and 300 kGy using Co-60 gamma rays irradiator. With irradiation doses, the amount of soluble proteins increased, and the viscosity and turbidity of soluble proteins decreased, which could be clue of that irradiation degrade high molecular proteins directly. pH of soluble proteins from defatted pigskin increased in the sample above 150 kGy, and low molecular weight components (below 24 kDa) in SDS-PAGE increased. From gel permeation chromatography of the hydrolysates of pigskin irradiated at 300 kGy showed the major peak of 9,000, 8,200, 860, and 170 Da.

• Korean Journal of Food Science and Technology
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• v.29 no.1
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• pp.77-81
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• 1997

In order to develop a new type of natural seasoning material combining fish meat with seaweed, a processing method of the mixture of enzyme treated mackerel meat and Laminaria powder was studied. Mackerel meat previously boiled and deboned was treated with proteolytic enzyme to enhance taste of meat by proper hydrolysis. The enzyme-treated meat was dried at $100{\pm}2^{\circ}C$ for 4 hrs, and finally mixed with kelp power, moistened in advance, plus binding agents (0.02% calcium carbonate) to aid the formation of pellets by extrusion. Boiled mackerel meat of enzyme treated (0.03% Protease-A) at $50^{\circ}C$ for 90 min was adequate to result an increase in 6 times of total free amino acid content and about 10% increase of taste-enhancing amino acids such as glutamic acid, glycine, arginine, lysine.

• Korean Journal of Food Science and Technology
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• v.26 no.1
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• pp.81-87
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• 1994

Two different trypsin inhibitors, TRTI-1 and TRTI-2, were purified to near homogenity from Trichosanthes kirilowii root, by $0{\sim}90%$ saturated ammonium sulfate salting out, DEAE-Sephacel ion exchange chromatography, Sephadex G-50 gel filtration chromatography and trypsin-affinity chromatography. The molecular weight of TRTI-1 and TRTI-2 were estimated to be about 5,000 Da and 24,000 Da, respectively, by gel filtration and must be monomer and homodimer since they contain 4,000 Da and 10,000 Da each on SDS-polyacrylamide gel electrophoresis. TRTI-1 was stable after heating for at least 2 hr at $100^{\circ}C$ but TRTI-2 was completely inactivated after heating for 10 min at $90^{\circ}C$. When Bz-dl-Arg-pNA was used as a substrate of TPCK-treated trypsin, half-maximal inhibitions of TRTI-1 and TRTI-2 were observed at $0.8\;{\mu}M$ and 6\;${\mu}M$, repectively. Both TRTI-1 and TRTI-2 inhibited the hydrolysis of trypsin competitively and Km values were $0.97\;{\mu}M$ and $0.63\;{\mu}M$, respectively. Both TRTI-1 and TRTI-2 specifically inhibited trypsin but they did not inhibit other proteases tested, chymotrypsin, papain, elastase, collagenase, thermolysin, Nagarase, pepsin, and thrombin.

• Korean Journal of Food Science and Technology
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• v.5 no.2
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• pp.71-77
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• 1973

The results, which was analytically surveyed the free amino acids by the automatic amino acid analyzer adding the enzyme on the Korean cow's fore shank muscles, are as follows: 1. The content of free amino acids in the fore shank muscles, without addition of the enzyme orderly contains alanine, glutamic acid, lysine, glycine, histidine, leucine, threonine, arginine, cystine, serine, proline, isoleucine, phenylalanine, tyrosine, methionine, aspartic acid and valine. 2. In accordance with the addition of the enzyme, by 0.01%, 0.05% and 0.1% the nine free amino acids of glutamic acid, glycine, alanine, cystine, valine, isoleucine, leucine, lysine and arginine were continuosly increased. 3. Proline and histidine were decreased at the enzyme addition of 0.01% after showing the high content at the control, but the quantity of free amino acids was increased according to the increase of the quantity of the enzyme. 4. Aspartic acid, threonine, serine, methionine, tyrosine and phenylalanine were increased till the enzyme addition of 0.05% and remarkably decreased from 0.1%. 5. At cooking the meat, the quantity of the enzyme addition was most effective at 0.05% of meat weight.

• Korean Journal of Food Science and Technology
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• v.36 no.1
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• pp.152-157
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• 2004

Gluten was extracted from domestic wheat flour using UTH buffer (4 M urea in 0.1 M Tris-HCl, pH 8.6) and validated by SDS-PAGE analysis for production of wheat flour products with reduced gluten content.. Anti-gluten polyclonal antibody was made by administering extracted gluten fraction on animal model. Anti-gluten serum titer of extracted gluten fraction was evaluated by ELISA, and that of antibody titer according to administration period. Anti-gluten sera were used for ELISA and immunoblot analysis before and after hydrolysis of gluten fraction at optimal pH and temperature condition for each protease. Gluten fraction separated by SDS-PAGE showed several bands covering 75 to 10 kDa, in which anti-gluten sera were 25, 34, and 45 kDa. Enzyme hydrolysis of gluten fraction revealed protein band sizes to be lower than 15 kDa. Content of pretense from bovine pancreas (b.p. protease) for gluten hydrolysis was estimated as 1 mg in 10 mL gluten fraction extracted for 4 hr.

• Journal of the Korean Applied Science and Technology
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• v.34 no.3
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• pp.631-642
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• 2017

Protein hydrolysates derived from plants and animals having antioxidant, suppression of hypertension, immunodulatory, alleviation of pain, and antimicrobial activity has been known as playing important role like hormone. This study was performed to hydrolysis of Ogae egg white protein using the six proteases. The antioxidant activity of the produced peptides was analyzed. As a result, the maximum value of hydrolysis was protamex(46.3%), DPPH radical scavenging was bromelain(57.23%), hydroxy radical scavenging was alcalase(30.21%), superoxide radical scavenging was alcalase(58.07%), and $Fe^{2+}$ chelation ability was alcalase(72.06%). Furthermore, the antioxidant Inhibition concentration ($IC_{50}$) of peptides was evaluated for each enzyme. As a result, the maximum value of alcalase was $Fe^{2+}$ cheating ability($IC_{50}$, 1.24 mg/mL), bromelain was DPPH radical scavenging($IC_{50}$, 2.46 mg/mL), flavourzyme was $Fe^{2+}$ cheating ability($IC_{50}$, 1.25 mg/mL), neutrase was DPPH radical scavenging($IC_{50}$, 3.64 mg/mL), papain was DPPH radical scavenging ($IC_{50}$, 3.82 mg/mL) and protamex was DPPH radical scavenging($IC_{50}$, 1.93 mg/mL). Therefore, we expect that peptides produced from Ogae egg white protein using protease enzyme are useful as an antioxidant functional food ingredients.

• Korean journal of applied entomology
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• v.53 no.4
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• pp.331-338
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• 2014

Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus (CpBV). Our previous study indicated that a transient expression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. To directly demonstrate the protein function, this study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to determine its inhibitory activity against cysteine protease and to assess its physiological alteration in insect immune and development. The open reading frame of CpBV-CST1 encodes a polypeptide of 138 amino acids (${\approx}15kDa$). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was over-expressed by 0.5 mM IPTG for 4 h. In biological activity assay, the purified rCpBV-CST1 showed a significant inhibition against papain activity. It inhibited a cellular immune response of hemocyte nodule formation in the beet armyworm, Spodoptera exigua. Moreover, its oral administration retarded larval development of the diamondback moth, Plutella xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 may be applied to control insect pest populations.

• Journal of the Korean Applied Science and Technology
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• v.35 no.2
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• pp.367-377
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• 2018

Swimming crab(Ovalipes punctatus) is produced in Korea and utilized as semi-processed food at streamed cooked state. Recently, protein hydrolysates have been known as having function such as antioxidant, suppression of hypertension, immunodulatory, alleviation of pain, and antimicrobial activity. This research was investigated to find the functional antioxidant from crab hydrolysates. To fine optimal protease enzyme, alcalase, bromelain, flavourzyme, neutrase, papain, and protamex were selected to evaluate the DPPH radical scavenging activity and finally bromelain to show the best activity was selected. The molecular weight of bromelain hydrolysates were distributed with range from 500 to 3,200 Da and 7 different molecules or more. The amino acids related to antioxidant capacity was about 42.54%. The processes optimization study used was the response surface methodology. The ranges of processes were the reaction temperature of 40 to $60^{\circ}C$, pH 6 to 8, and enzyme concentration 1 to 3%(w/v). As a result, the optimization of process was determined at temperature of $55^{\circ}C$, pH of 6.5, and enzyme concentration of 3%(w/v). In these conditions, degree of hydrolysates were maximum 71.60%. Therefore, we expect that those products are useful as functional food ingredients.