• Fisheries and Aquatic Sciences
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• v.19 no.5
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• pp.25.1-25.8
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• 2016

The fractionation of serine protease inhibitor (SPI) from fish roe extracts was carried out using polyethylene glycol-4000 (PEG4000) precipitation. The protease inhibitory activity of extracts and PEG fractions from Alaska pollock (AP), bastard halibut (BH), skipjack tuna (ST), and yellowfin tuna (YT) roes were determined against target proteases. All of the roe extracts showed inhibitory activity toward bromelain (BR), chymotrypsin (CH), trypsin (TR), papain-EDTA (PED), and alcalase (AL) as target proteases. PEG fractions, which have positive inhibitory activity and high recovery (%), were the PEG1 fraction (0-5 %, w/v) against cysteine proteases (BR and PA) and the PEG4 fraction (20-40 %, w/v) against serine proteases (CH and TR). The strongest specific inhibitory activity toward CH and TR of PEG4 fractions was AP (9278 and 1170 U/mg) followed by ST (6687 and 2064 U/mg), YT (3951 and 1536 U/mg), and BH (538 and 98 U/mg). The inhibitory activity of serine protease in extracts and PEG fractions from fish roe was stronger than that of cysteine protease toward common casein substrate. Therefore, SPI is mainly distributed in fish roe and PEG fractionation effectively isolated the SPI from fish roes.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.624-631
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• 2007

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by tractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and Phenyl-Sepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63kDa protease (PRO63) at pH5.0. The optimum temperature for the two proteases was $65^{\circ}C$. The PRO33 had a $K_m$ value of 6.6mM and a $V_{max}$ value of $10.31{\mu}mol/l/min$, and PRO63 l7.6mM and $9.08{\mu}mol/l/min$, with casein as substrate. They were thermostable at $60^{\circ}C$. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at $70^{\circ}C$ for 1h. The thermal stability of the two enzymes was significantly enhanced by $Ca^{2+}$. The residual activity of PRO33 and PRO63 at $70^{\circ}C$ after 60min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing $Ca^{2+}$. These properties make them applicable for many biotechnological purposes.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.677-684
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enhanced by the increase of agitation speed. Two forms of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both proteases were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were $50^{\circ}C$ and $45^{\circ}C$, respectively. About 56% of the original protease BK7-2 activity remained after being treated at $50^{\circ}C$ for 30 min but protease BK7-1 was rapidly inactivated at above $25^{\circ}C$. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• Food Science of Animal Resources
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• v.44 no.2
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• pp.430-442
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• 2024

This research aimed to assess the effect of collagenolytic proteases from Bacillus subtilis B13 and Bacillus siamensis S6 for tenderizing goat meat during wet aging. Collagenolytic proteases B13 and S6 were prepared at 5 U/mL of collagenolytic activity before injecting into goat meat with 10% (v/w) of initial weight. The control sample was injected with distilled water and used as a negative control. The injected meats were placed in vacuum-sealed bags and wet aged at 4℃ for 0, 3, 5, 7, 14, and 21 days. Thereafter, total aerobic count and physicochemical quality were elucidated. Both enzyme-treated samples from B13 and S6 aged for 5 days showed an acceptable microbial quality with lower than 5.7 Log CFU/g. These conditions produced the tender meats by the reduction in shear force accounting for 30% for B13 and 26% for S6 as compared to the control. Moreover, the enzyme-treated samples showed lower values of hardness, gumminess, and chewiness, with higher springiness and trichloroacetic acid-soluble peptides than the control (p<0.05). The detrimental impact on cooking loss and lipid oxidation was not found. Enzyme-injected meat had a lower cooking loss than the control (p<0.05) with no significant difference in lipid oxidation (p>0.05). Notably, meats treated with B13 and S6 were lower in CIE L^* value as compared to the control (p<0.05) with no significant impact on CIE a^* and CIE b^* (p>0.05). These results suggested that these two collagenolytic proteases could enhance the quality of goat meat in terms of tenderness and reduce the aging time for meat tenderization.

• ALGAE
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• v.19 no.1
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• pp.59-68
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• 2004

Hizikia fusiformis hydroysates by five carbohydrases (Viscozyme, Celluclast, Termamyl and Ultraflo) and five proteases (Protamex, Kojizyme, Neutrase, Flavourzyme and Alcalase) were investigated for their extraction efficacy (yield and total total polyphenolic content) and antioxidative activity (DPPH radical and hydrogen peroxide scavenging activity). Termamyl and Ultraflo of the carbohydrases and Flavourzyme and Alcalase of proteases were selected by their high eficacy of extraction and antioxidative activity. Selected enzymes were used to investigate the optimum enzymatic reaction time and dosage (enzyme/substrate ratio) suitable for hydorolysis. Optimum reaction time for the enzymatic hydrolysis was 3 days and optimum dosage of hydrolysis was observed as 5%. Simultaneously, Ultraflo of the two carbohydrases and Alcalse of the two proteases were selected as the most effective enzymes. Combination of Ultraflo and Alcalase under optimum hydrolysis conditions could intensify the extraction efficacy of antioxidative materials form H. fusiformis. The hydrolysate obtained by combining the enzymes was separated into four different molecular weight fractions (<1kD, 1-10 kD, 10-30 kD and >30 kD) and recorded the polyphenolic content distribution and respective antioxidative ability. The fraction <1kD was identified as less effective and those fractions > 1kD indicated comparatively higher antioxidative activities related to their polyphenolic content.

• Korean Journal of Veterinary Research
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• v.55 no.3
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• pp.175-179
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• 2015

Proteases play important roles in parasite development and host parasite interactions. The protease of Kudoa spp. has been recognized as a key factor of severe proteolysis of fish muscle post-mortem; however, there is little information available regarding the protease of Kudoa (K.) septempunctata, which was recently identified as a cause of food poisoning in humans. The present study was conducted to isolate and characterize proteases to elucidate the type of protease contained in the parasite and determine the optimal pH for protease activity. We confirmed the cysteine protease and metalloprotease produced by K. septempunctata. While the cysteine protease showed optimal activity at pH 5 that decreased rapidly with increasing pH, the optimal activity of metalloprotease was pH 7, and it remained stable from pH 6 to pH 8. These results indicate that the pH of cysteine protease is not proper for fish muscle postmortem, and that metalloprotease can act in human intestines. Overall, the present study provides important information that improves our understanding of the role of protease physiology and the subsequent food poisoning caused by K. septempunctata.

• Korean Journal of Microbiology
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• v.27 no.3
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• pp.245-249
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• 1989

Extracellular proteases of Bacillus licheniformis were partially purified using ammonium sulfate fractionation and Sephadex G-75 gel filtration chromatography. The partial purification permited the weparation of two different protease activities, type I and type II. Protease type I is an enzyme with rather high protealytic activity toward dasein and was highly susceptible to organofluoride and EDTA inhibitions. It showed maximal proteolytic activity at pH 7.5 and was rapidly denatured at $71^{\circ}C$. Protease type II is a protease with relatively lower proteolytic activity than the type I. It was also inhibited by 10mM of EDTA and 1mM of PMSF by 30 min incubation. The enzyme showed maximal activity at pH 8.0 and was denatured relatively slowly at $71^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.5-14
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• 2006

Streptomycetes are Gram-positive microorganisms producing secondary metabolites through unique physiological differentiation [4]. The microbes show unusual morphological differentiation to form substrate mycelia, aerial mycelia, and arthrospores on solid medium [19]. Substrate mycelium growth is sustaining with sufficient nutrients in the culture medium. The concentration of a specific individual substrate in the culture environment is the most important extracellular factor allowing vegetative mycelia growth, where extracellular hydrolytic enzymes participate in the utilization of waterinsoluble substrates. However, with starvation of nutrients in the culture medium, the vegetative mycelia differentiate to aerial mycelia and spores. It has been considered that shiftdown of essential nutrients for mycelia growth is the most important factor triggering morphological and physiological differentiation in Streptomyces spp. Since proteineous macromolecule compounds are the major cellular components, these are faced to endogenously metabolize following a severe depletion of nitrogen source in culture nutrients (Fig. 1). Various proteases were identified of which production was specifically related with the phase of mycelium growth and also morphological differentiation. The involvement of proteases and protease inhibitor is reviewed as a factor explaining the mycelium differentiation in Streptomyces spp.

• Journal of Applied Biological Chemistry
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• v.49 no.4
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• pp.135-139
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• 2006

A alkalophilic strain, Bacillus licheniformis MH31 producing an alkaline protease was isolated from mine soil of Boryeong in Korea. Production of a high level of alkaline protease was achieved 42 h after incubation when the bacterium was grown at pH 9.0 and $35^{\circ}C$ in Horikoshi medium supplemented with 0.5%(w/v) starch and 1%(w/v) skim milk as carbon and nitrogen source, respectively. The molecular weight of partially purified enzyme was estimated to be 30 kDa by SDS-PAGE and its optimum pH was pH 10. The enzyme showed optimum temperature at $50^{\circ}C$, and was stable up to $60^{\circ}C$ after 1 h incubation. The protease was strongly inhibited by 1 mM of PMSF which was known well as strong inhibitor of serine proteases, but almost not inhibited by 5 mM of EDTA and 1,10-phenanthroline. When the protein hydrolysis products of 1% skim milk by partially purified protease was compared with available commercial proteases using HPLC analysis, most of hydrolysis products were detected below molecular weight of 10,000 and the hydrolysis ratio of purified enzyme was 24.8% lower than those(above 32%) of commercial proteases.

• Korean journal of food and cookery science
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• v.20 no.6 s.84
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• pp.658-666
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• 2004

In order to develop and commercialize high quality frozen soy yogurt, the effects of industrial proteases and commercial mixed cultures were examined on the functional properties and the sensory attributes of frozen soy yogurt. For quality improvement, soy protein isolates were primarily hydrolyzed by either Flavourzyme or Neurtrase, industrial Proteases, to reduce the beany flavor and increase the functional properties of the protein. The viable cell count of lactic acid bacteria was higher in the soy protein hydrolysates than whenuntreated. ABT-5 (L. acidophilus, Bifidobacterium lactis, and S. thermophilus) resulted in higher acid tolerance, bile acid tolerance and melt-down percent values than those with YC-X11 (Lactobacillus bulgaricus and Streptococci thermophilus). The overrun of frozen soy yogurt was improved by both Flavourzyme $(193.3\%)$ and Neurtrase $(156.7\%)$ treatments. With regard to thesensory characteristics, Flavourzyme improved the beany flavor, astringency taste, mouth feel and overall quality of frozen soy yogurts fermented with ABT-5. Further studies onproduct formulation will be needed to commercialize the frozen soy yogurt for the market.