• Journal of Life Science
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• v.22 no.4
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• pp.552-558
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• 2012

Severe acute respiratory syndrome (SARS) is a severe respiratory infectious disease caused by a novel human coronavirus, SARS-CoV. The 3CL protease is a key enzyme in the proteolytic processing of replicase polyprotein precursors, pp1a and pp1ab, which mediate all the functions required for viral genomic replication and transcription. Therefore, this enzyme is a target for the development of chemotherapeutic agents against SARS. A large quantity of active SARS-3CL protease is required for development of anti-SARS agents. Here we have constructed overexpression vector for the production of the SARS-3CL protease. The gene encoding SARS-3CL protease was amplified using polymerase chain reaction and cloned into the pET29a expression vector, resulting in pET29a/SARS-3CLP. Recombinant SARS-3CL protease was successfully synthesized by the dialysis mode of the cell-free protein expression system, and purified by three-step fast protein liquid chromatography using HighQ and MonoP column chromatographies and Sephacryl S-300 gel filtration. In addition, the produced SARS-3CL protease was found to be an active mature form. This study provides efficient methods not only for the development of anti-SARS materials from natural sources, but also for the study of basic properties of the SARS-3CL protease.

• KSBB Journal
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• v.22 no.4
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• pp.185-190
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• 2007

Productivity of secreted recombinant protein depends largely on its stability in the extracellular environment with protease. Most hGM-CSF produced by transgenic tobacco cell cultures and secreted to the medium was confirmed to be rapidly degraded by protease in medium. To increase the productivity, therefore, various protein stabilizers such as gelling agents such as carrageenan and alginate, polymers, polyols, and amino acids have been tested. The stability of hGM-CSF in spent medium without cells was improved by the presence of gelling agents. However, the reason for the enhanced production by the addition of gelling agents may be due to the increased expression level and permeability rather than stability. The addition of DMSO inhibited the cell growth, but improved specific yield. The others were not effective for stability as well as hGM-CSF production.

• Journal of Life Science
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• v.10 no.2
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• pp.157-163
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• 2000

One hundred eight strains of Pseudomonas aeruginosa isolated from the patients (sputum, urine, burn skin, stool and blood) of Pusan National University hospital were tested for exonezyme production, antimicrobial susceptibility and serotyping. The results obtained were as follow: In exonezyme production test, 50 strains (46.30%) produced both protease and elastase. Thirty three strains (30.55%) did not produce any exoenzyme, 18 strains (16.67%) produced only protease and 7(6.48%) stains only produced elastase. As the result of antimicrobial susceptibility by the disc diffusion method, most strains were resistant to sulfamethoxazole (96.30%). But the resistant rate against gentamicin and ticarcillin were 47.23% and 46.30% respectively. The resistant rate to other antibiotics were less than 40%. All strains could be serologically typed. Most strains were identified as type Ⅲ: among them, 51 strains were belonged to serotype E. The correlation of serotype and exoenzyme production was not found.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.1952-1960
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• 2017

Recently, soybean isoflavone aglycones (i.e., daidzein and genistein) and ${\gamma}-aminobutyric$ acid (GABA) have begun to receive considerable consumer attention owing to their potential as nutraceuticals. To produce these ingredients, multiple microorganisms and their enzymes are commonly used for catalysis in the nutraceutical industry. In this work, we introduce a novel fermentation process that uses whole-cell biocatalysis to accelerate GABA and isoflavone aglycone production in doenjang (a traditional Korean soybean paste). Microbial enzymes transform soybean isoflavone glycosides (i.e., daidzin and genistin) and monosodium glutamate into soybean isoflavone aglycones and GABA. Lactobacillus brevis GABA 100 and Aspergillus oryzae KACC 40250 significantly reduced the production time with the aid of a protease. The resulting levels of GABA and daidzein were higher, and genistein production resembled the levels in traditional doenjang fermented for over a year. Concentrations of GABA, daidzein, and genistein were measured as 7,162, 60, and $59{\mu}g/g$, respectively on the seventh day of fermentation. Our results demonstrate that the administration of whole-cell L. brevis GABA 100 and A. oryzae KACC 40250 paired with a protease treatment is an effective method to accelerate GABA, daidzein, and genistein production in doenjang.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.87-92
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• 2005

Pronase, a protease produced for commercial purpose by Streptomyces griseus, was composed of serine protease, alkaline protease, aminopeptidase and carboxypeptidase complex, and it has been widely used as anti-inflammatory drugs for human therapy. In this study, we developed a new integration vector, pHJ101 derived from pSET152, containing strong promoter, ermE, to overexpress a certain protease gene. Specific PCR primers for cloning of sprA (a gene for S. griseus protease A) and sprB (a gene for S. griseus protease B) genes were designed from the basis of nucleotide sequence in databases and amplified by PCR. Plasmid pHJ201 and pHJ202 were constructed by inserting of amplified each gene in a vector pHJ101. S. griseus HA and S. griseus HB were respectively obtained by conjugal process of a parent strain, S. griseus IFO 13350 with the recombinant Escherichia coli harboring plasmid pHJ201 or pHJ202. When protease activity was measured in flask cultivation, produced protease levels of S. griseus HA and S. griseus HB increased about 5.3 times and 5 times, respectively, more than that of parent strain. And, the constructed integrating plasmid pHJ101 was applicable for overexpression of a certain gene in Streptomyces sp.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.9
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• pp.1303-1313
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• 2017

Objective: This study evaluated the change and function of the pancreas, and small intestine in relation to growth performance of broilers on diets supplemented with raw soybean meal (RSBM) and protease. Samples of test ingredients and diets, after mixing and prior to being used were also assessed on contents of anti-nutritional factors. Methods: A $3{\times}3$ factorial study was used, with three levels of RSBM (commercial soybean meal [SBM] was replaced by RSBM at 0, 10%, or 20%) and protease (0.1, 0.2, or 0.3 g/kg). Each treatment was replicated six times with nine birds per replicate. Birds were housed in cages, in climate-controlled room and fed starter, grower and finisher diets. Results: Levels of trypsin inhibitors in the diets, containing varying levels of RSBM ranged between 1,730.5 and 9,913.2 trypsin inhibitor units/g DM. Neither RSBM nor protease supplementation in diets significantly affected (p>0.05) the body weight of broilers in the entire periods (0 to 35-d). Increasing the level of RSBM in diets increased the weight of the pancreas at d 10 (p<0.000), d 24 (p<0.001), and d 35 (p<0.05). Increasing levels of RSBM in the diets reduced the apparent ileal digestibility of crude protein (CP), and amino acid (AA) at d 24. Increasing level of RSBM in the diets decreased (p<0.01) pancreatic protein content, but this was increased (p<0.05) when protease was added to the diets (0 to 10-d). Increasing the level of protease improved the pancreatic digestive enzymes, including trypsin (p<0.05), chymotrypsin (p<0.01), and general proteolytic enzymes (p<0.05). Conclusion: The commercial SBM could be replaced at up to 20% by RSBM for broilers. Although protease supplementation slightly improved the digestive enzymes, and the ileal digestibilities of CP and AA, the CP and AA were negatively affected by increasing RSBM.

• The Korean Journal of Food And Nutrition
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• v.19 no.4
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• pp.496-501
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• 2006

This study was to isolate a bacterium producing a alkaline protease from mud flats of the west seaside of Korea and to investigate the biochemical analysis of the alkaline protease producing from the isolate. The isolate was named as Gelidibacter sp. HK-1 based on 16S rRNA sequence, Gram staining and the photograph of electron microsceope. Optimum temperature for growth and pretense production of the isolate was $25^{\circ}C$. Growth of the isolate was reached at stationary phase after 10hrs followed by inoculation. Maximum activity of protease produced from the isolate was shown after 14hrs. Optimum temperature and pH for the protease activity were $45^{\circ}C$ and pH 9, respectively. Molecular weight of the pretense was about 50KD and the partial amino acid sequence of the pretense was Ala-Try-Ala-Leu-Asn-Thr-Ser-Val-Thr-Glu-Thr-Phe-Ala-Lys. The partial amino acid sequences of the protease showed significant homology with a pretense produced from Streptomyces avermitilis.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.8
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• pp.1164-1173
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• 2006

Cellulase from Aspergillus niger, (${\alpha}$-amylase from Bacillus sp. and protease from Bacillus globigii were used as enzyme sources in this study to examine how their respective substrates protect them in two kinds of simulated gastrointestinal tract digesting processes. Avian total digest tract simulation test showed that filter paper, Avicel and cellulose resulted in 7.7, 6.4 and 7.4 times more activity than of unprotected cellulose, respectively. Protease with addition of casein, gelatin or soybean protein showed no significant protection response. Starch protected amylase to be 2.5 times activity of the unprotected one. Monogastric animal total tract digestion simulation test showed that filter paper, Avicel and cellulose resulted in 5.9, 9.0 and 8.8 times activity of unprotected cellulase, respectively. Casein, gelatin and soybean protein resulted in 1.2, 1.3 and 2.0 times activity of unprotected protease, respectively. Starch did not protect amylase activity in monogastric animal total tract simulation. Protection of mixed enzymes by substrates in two animal total tract simulation tests showed that filter paper in combination with soybean protein resulted in 1.5 times activity of unprotected cellulose, but all substrates tested showed no significant protection effect to protease. Soybean protein and starch added at the same time protected the amylase activity to be two times of the unprotected one. Test of non-purified substrate protection in two animal total digest tract simulation showed that cellulase activity increased as BSA (bovine serum albumin) concentration increased, with the highest activity to be 1.3 times of unprotected enzyme. However, BSA showed no significant protection effect to protease. Amylase activity increased to 1.5 times as BSA added more than 1.5% (w/v). Cellulase activity increased to 1.5 times as soybean hull was added higher than 1.5%. Amylase had a significant protection response only when soybean hull added up to 2%. Protease activity was not protected by soybean hull to any significant extent.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.430-434
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• 2004

Three extracellular protease-deficient Bacillus subtilis strains were transformed with the plasmid pCK98 containing the endo-$\beta$-1,4-glucanase (Eng) gene of B. subtilis BSE616. The three transformants, B. subtilis DB104 (pCK98), WB600 (pCK98) and WB700 (pCK98), produced the same high level of enzyme activity and showed similar patterns of cell growth and enzyme production. When B. subtilis DB 104 (pCK98), a two-extracellular protease deficient strain, was cultured for 22 h, almost all the secreted enzyme was found to be in the completely cleaved form by both activity staining and Western blotting studies. B. subtilis WB600 (pCK98), a six-extracellular protease-deficient strain, produced a partially cleaved form in addition to the intact form of the enzyme, although the degree of internal cleavage of the enzyme was greatly reduced. With B. subtilis WB700 (pCK98), a seven-extracellular protease-deficient strain, almost all the enzyme was produced as the intact uncleaved form. This study illustrates that a role of the V pr protease is to degrade foreign proteins produced in B. subtilis and WB700 is a suitable expression system for producing the intact form of the Eng and other foreign proteins that may lose at least part of their efficacy due to internal proteolytic cleavage.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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• pp.1291-1300
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• 2018

Objective: This experiment was conducted to investigate the effects of dietary crude protein (CP) level and exogenous protease supplementation on growth performance, serum metabolites, carcass traits, small intestinal morphology and endogenous protease activity in broiler chickens reared under a tropical climate. Methods: A total of 480 day-old male broiler chicks were randomly assigned to eight dietary treatments in a $4{\times}2$ factorial arrangement. The main effects were CP level (21.0%, 19.7%, 18.5%, or 17.2% from 1 to 21 days and 19.0%, 17.9%, 16.7%, or 15.6% from 22 to 35 days) and protease enzyme supplementation (0 ppm or 500 ppm). All experimental diets were fortified with synthetic feed-grade lysine, methionine, threonine and tryptophan to provide the minimum amino acid recommended levels for Cobb 500. Results: Reducing dietary CP linearly reduced (p<0.05) growth performance, serum albumin, total protein, and carcass traits and increased (p<0.05) serum triglycerides and abdominal fat. There was no consistent effect of reducing dietary CP on morphological parameters of the intestine and on the pancreatic and intestinal endogenous protease activity (p>0.05). Protease supplementation improved (p<0.05) feed conversion ratio, body weight gain, carcass yield and intestinal absorptive surface area. Conclusion: Protease supplementation, as measured by growth performance, intestinal morphology and carcass yield, may alleviate the detrimental effects of low protein diets in broiler chickens.