• Title/Summary/Keyword: protease production

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Enhancement of human serum albumin production in Saccharomyces cerevisiae

  • Seo, Dong-Sik;Choi, Byung-Hyun;Kang, Whan-Koo;Kim, In-Ho;Hong, Eock-Kee
    • 한국생물공학회:학술대회논문집
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    • 2001.11a
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    • pp.767-770
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    • 2001
  • Experiments were accomplished to reduce the extent of proteolysis by simply controlling the culture conditions instead of the gene manipulation techniques. L-arginine and L-lysine were chosen as a protease inhibitor analogue With the assumption that they might act as the potential inhibitors against proteases involved in the rHSA proteolysis. The addition of arginine and lysine resulted in a considerable positive effect on the secreted rHSA production level.

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Functional Characterization of Antagonistic Fluorescent Pseudomonads Associated with Rhizospheric Soil of Rice (Oryza sativa L.)

  • Ayyadurai, N.;Naik, P. Ravindra;Sakthivel, N.
    • Journal of Microbiology and Biotechnology
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    • v.17 no.6
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    • pp.919-927
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    • 2007
  • Antagonistic fluorescent pseudomonads isolated from rhizospheric soil of rice were characterized by 16S rRNA amplicon and fatty acid methyl ester (FAME) analyses. Antagonistic isolates were grown in the fermentation media, and production of antibiotics was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Production of fungal cell-wall-degrading enzymes such as protease, cellulase, pectinase, and chitinase was determined. Dendrogram based on the major and differentiating fatty acids resulted into 5 clusters, viz., cluster I (P. pseudoalcaligenes group), cluster II (P. plecoglossicida group), cluster III (P. fluorescens group), cluster IV (P. aeruginosa group), and cluster V (P. putida group). Characteristic presence of high relative proportions of cyclopropane (17:0 CYCLO w7c) was observed in antagonistic bacteria. Data revealed biodiversity among antagonistic fluorescent pseudomonads associated with the rice rhizosphere. Results presented in this study will help to identify the antagonistic isolates and to determine their mechanisms that mediate antagonism against fungal pathogens of rice.

Production and Characteristics of Enzymatically Hydrolyzed Soy Sauce by the Treatment Using Proteases (단백질 분해효소로 원료 처리하여 제조한 효소분해 간장의 특성)

  • 채희정;인만진;김민홍
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.26 no.5
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    • pp.784-787
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    • 1997
  • Enzymatically hydrolyzed soy sauce(eHSS) was prepared by the treatment of defatted soy flake using two types of proteases, followed by maillard reaction and formulation with some ingredients. The eHSS was mixed with fermented soy sauce(FSS) to make enzymatically hydrolyzed mixed soy sauce(eHMSS). The properties and sensory characteristics were evaluated and compared with commercially available soy sauces. The control of salt and total nitrogen contents in eHSS and eHMSS was easy, and the production of soy sauce of low salt and high protein was possible. However, the free amino acid content of eHSS was lower than FSS. due to lower degree of hydrolysis. In sensory evaluation, the eHSS have no loss taste and overall acceptance than FSS. Consequently, the eHSS and eHMSS have the potential for use with FSS to produce high quality soy sauce of low salt and high protein contents.

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Functional Properties of Soybean Curd Residue Fermented by Bacillus sp. (Bacillus firmus NA-1 균주와 Bacillus subtilis G7-D 균주를 이용한 발효비지의 기능성)

  • Oh, Soo-Myung;Kim, Chan-Shick;Lee, Sam-Pin
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.35 no.1
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    • pp.115-120
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    • 2006
  • To convert the soybean curd residue (SCR) to functional food ingredient, alkaline fermentation of SCR was performed by Bacillus firmus NA-1 and Bacillus subtilis GT-D for 22 hr at $42^{\circ}C$. The micronized full-fat soy flour (MFS) was fortified to reduce the moisture content as well as to supply protein source. The mucilage and flavor productions in the fermented SCR were enhanced by the fortification of $20\%$ MFS. The peptide production from the SCR fermented with B. subtilis GT-D substantially increased when judged by the detectable amount of tyrosine $(480\;mg\%)$. The production of fibrinolytic enzyme was increased by the fermentation for 22 hr, indicating the relative activity of $62\%$ (B. firmus NA-1) and $47\%$ (B. subtilis GT-D), respectively. The SCR fermented by B. firmus NA-1 and B. subtilis GT-D indicated the consistency of $1.95\;Pa{\cdot}s^n\;and\;0.21\;Pa{\cdot}s^n$, respectively. After freeze-drying, the protease activity (615 unit/g) and a-amylase activity (180 unit/g) were obtained from SCR fermented by Bacillus firmus NA-1 and Bacillus subtilis GT-D, respectively.

Isolation and Characteristics of Bacteria Showing Biocontrol and Biofertilizing Activities (생물방제 및 생물비료 활성을 가지는 세균의 분리 및 특성)

  • Jung, Ho-Il;Kim, Keun-Ki;Park, Hyean-Cheal;Lee, Sang-Mong;Kim, Yong-Gyun;Kim, Hong-Sung;Lee, Cnung-Yeol;Son, Hong-Joo
    • Journal of Life Science
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    • v.17 no.12
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    • pp.1682-1688
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    • 2007
  • To develop multifunctional microbial inoculant, microorganisms with antagonistic activity and biofertilizing activity were screened. Pantoea agglomerans and Bacillus megaterium from our laboratory culture collection, and strain MF12 from soil near poultry farm in Miryang were selected. On the basis of morphological, physiological studies and 16S rDNA sequence analysis, isolate MF12 was identified as the Bacillus pumilis. Three strains were studied for insoluble phosphate solubilization, indole-3-acetic acid (IAA) and siderophore production, ammonification ability, hydrolytic enzyme production and antifungal activity against phytopathogenic fungi. P. agglomerans did not produce any visible clear zone on agar plate containing 0.5% $Ca_3(PO_4){_2}$ as a sole phosphorus source. However, this strain could solubilize insoluble phosphate in liquid medium. All strains produced IAA ranged from $3{\sim}639{\mu}g/ml$ depending on culture time and had ammonification ability. Among three strains, only P. agglomerans produced siderophore. P. agglomerans produced pectinase and lipase, B. megaterium produced amylase, protease and lipase while B. pumilis produced protease and lipase. P. agglomerans showed antifungal activities against phytopathogenic fungi, Fusarium oxysporum and Colletotrichum gloeosporioides. B. pumilis showed antifungal activities against Botrytis cinerea, Sclerotinia sclerotiorum and Phythium ultimum.

A Fibrinolytic Enzyme from the Medicinal Mushroom Cordyceps militaris

  • Kim Jae-Sung;Sapkota Kumar;Park Se-Eun;Choi Bong-Suk;Kim Seung;Hiep Nguyen Thi;Kim Chun-Sung;Choi Han-Seok;Kim Myung-Kon;Chun Hong-Sung;Park Yeal;Kim Sung-Jun
    • Journal of Microbiology
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    • v.44 no.6
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    • pp.622-631
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    • 2006
  • In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and $37^{\circ}C$, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin $\alpha$-chain followed by the $\gamma$-$\gamma$ chains. It also hydrolyzed the $\beta$-chain, but more slowly. The A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

Characterization of Transgenic Lettuce (Lactuca sativa L.) Using a BL1 Gene Encoding Bromelain Isolated from Pneapple (제주산 파인애플 유래 Bromelain관련 유전자 (BL1)를 이용반 형질전환 상추의 특성)

  • Jung, Yu-Jin;Kim, Gi-Hyun;Choi, Jang-Sun;Lee, Soon-Youl;Nou, Il-Sup;Park, Jin-Heui;Kang, Kwon-Kyoo
    • Journal of Plant Biotechnology
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    • v.33 no.1
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    • pp.27-32
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    • 2006
  • To clarify the roles of bromelain in plants, we isolated BL1 gene encoding bromelain from pineapple stem tissues and sequenced. The full length cDNA is 933 bp and encodes a polypeptide of 311 amino acid residues. The cDNA is most similar 94% at the amino acid level to bromelain previously isolated from pineapple (BAA21929). Explants of Lactuca sativa were co-cultivated with Agrobacterium tume-faciences LBA 4404 strains containing nptII and BL1 gene for transformation. Through initial selection of regenerated explants by culturing on a kanamycin and carbenicillin containing MS medium, multiple shoots were obtained after 2 months of culture. For a complementary step of selection, putative transgenic shoots were transferred to 1/2 Ms basal medium supplemented with 100 mg/L kanamycin and 500 mg/L carbenicillin. The selected shoots were obtained T1 generation seeds with emasculation, and tested with PCR analysis using 35S promoter and BL1 specific primers whether BL1 gene was introduced to genome of the plants. These results confirmed that produced the specific PCR bands in the putative transgenic lines. Additionally the Northern blot and endo protease activity showed that transcripts of BL1 gene were detected in transgenic lines. Theses results suggest that BL1 gene be successfully integrated and transcripted in the transgenic lettuce plants.

Effect of Combined Use of Anti-microbial Materials on Storage of Low Salted Kochujang (저식염 고추장 저장시 항균물질 혼합첨가의 영향)

  • Han, Sun-Mi;Kim, Dong-Han
    • Applied Biological Chemistry
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    • v.51 no.4
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    • pp.281-287
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    • 2008
  • Effect of combined use of anti-microbial materials, such as alcohol, mustard and chitosan, or pasteurization on the quality of low salted kochujang was investigated during storage at $30^{\circ}C$ for 12 weeks. Activity of amylase decreased during storage, with lower activity in pasteurized kochujang than the other groups. Acidic protease activity increased during storage, but neutral protease activity decreased after 4 weeks. Viable cells of yeast increased during storage, but bacterial counts decreased gradually and did not show any remarkable difference among the test groups. Hunter a-values decreased as storage time increased, whereas L- and b-values decreased after 4 weeks and the degree of increase in total color difference (${\Delta}E$) was low in the supplementary ingredients added kochujang. The moisture contents and water activities decreased during storage with being lower in supplementary ingredients added groups. Titratable acidity of kochujang was decreased after 4 weeks of storage with the highest in combination of the supplementary ingredients added group. Oxidation-reduction potential was low in the supplementary ingredients added kochujang. Total sugar and reducing sugar contents of kochujang decreased during storage, with the highest contents in the supplementary ingredients added group. Ethanol content of kochujang increased during storage, whereas ethanol production was reduced in ethanol added one. Amino-nitrogen and ammonia-nitrogen contents decreased during storage with being lower in kochujang prepared with supplementary ingredients. Therefore, supplementary ingredients added kochujang would be effective for extending shelf-life of kochujang.

Production and Separation of Anti-hypertensive Peptide during Chunggugjang Fermentation with Bacillus subtilis CH-1023 (청국장 발효과정 중 항고혈압성 peptide의 생산 및 분리)

  • Cha, Woen-Suep;Bok, Su-Kyung;Kim, Myoung-Uk;Chun, Sung-Sook;Choi, Ung-Kyu;Cho, Young-Je
    • Applied Biological Chemistry
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    • v.43 no.4
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    • pp.247-252
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    • 2000
  • As functionality investigation of Korean traditional soybean fermentation foods, an antihypertensive peptide was separated during Chunggugjang fermentation by Bacillus subtilis CH-1023 and investigated inhibitory effect against angiotensin converting enzyme. After incubation at $20^{\circ}C,\;30^{\circ}C,\;40^{\circ}C,\;50^{\circ}C,\;60^{\circ}C$ for the $0{\sim}72$ hrs, protein content, protease activity and angiotensin converting enzyme inhibitory rate were determined. The protein content and protease activity were increased and reached maximum at 60 hrs fermentation with $40^{\circ}C$ and decreased after the 60 hrs fermentation. The optimum condition for antihypertensive peptide from Chunggugjang was appeared for 60 hrs at $40^{\circ}C$. Crude extract of Chunggugjang was partially purified by Amicon YM-3 membrane filtration and Sephadex G-10, G-25 gel filtration. The purified peptide showed inhibitory rate of 94.3% with 0.5 mg peptide content. The most prominent amino acid composition of the peptide from Chunggugjang was alanine, followed by phenylalanine, histidine.

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Characteristics and Action Pattern of Pretense from Bacillus subtilis CCKS-111 in Korean Traditional Soy Sauce (한국재래간장으로 부터 분리한 Bacillus subtilis CCKS-111이 생성하는 Protease의 특성 및 작용양상)

  • Choi, Cheong;Choi, Kwang-Soo;Cho, Young-Je;Lim, Sung-il;Kim, Sung;Son, Jun-Ho;Lee, Hee-Duck;Kim, Young-Hwal
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.25 no.6
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    • pp.915-921
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    • 1996
  • An alkaline pretense Producing microorganism was isolated from Korean traditional soy sauce and identified as Bacillus subtilis CCKS-111. The optimum culture condition of Bacillus subtilis CCKS-111 for the production of alkaline pretense was as follow: 2% soluble starch, 0.2% peptone, 0.1% (NB$_4$)$_2$S$_2$O$_{8}$ , 0.2% MgSO$_4$, pH 7.0, 35$^{\circ}C$ and 24hrs. The optimum pH and temperature for the enzyme activity of alkaline pretense producing Bacillus subtilis CCKS-111 were pH 9.0 and 5$0^{\circ}C$, respectively. The enzyme was relatively stable at pH 6.0~11.0 and at temperature below 4$0^{\circ}C$. The activity of the enzyme was inhibited by $K^{+}$ and Hg$^{2+}$, whereas Cu$^{2+}$ exhibited rather activating effects on the enzyme activity. Ethylenediaminetetraacetic acid and phenylmethanesulfonyl fluoride inhibited the enzyme activity. This indicates that this is serine pretense which requires metal ion group for the enzyme activity. Km value was 2.313$\times$10$^{-4}$ M/L, V$_{max}$ value was 39.216$\mu\textrm{g}$/min. This enzyme hydrolyzed casein more rapidly than the hemoglobin.lobin.

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