• Title/Summary/Keyword: Protease production

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Optimization of Culture Conditions of Bacillus pumilus JB-1 for Chungkook-jang Fermentation in Soybean Boiling-Waste Liquor Medium (대두 열수 침출액을 이용한 청국장 발효균주 Bacillus pumilus JB-1의 배양 최적화)

  • Kwon, Ha-Young;Ryn, Hee-Young;Kwon, Chong-Suk;Lee, Sang-Han;Sohn, Ho-Yong
    • Microbiology and Biotechnology Letters
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    • v.35 no.4
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    • pp.304-309
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    • 2007
  • Soybean is useful source of protein, especially in Asia. But soybean needs heat inactivation or fermentation process before consumption, since it contains the toxic lectin and various protease inhibitors. Therefore, production of soybean boiling-waste liquor (SBWL) as a byproduct is inevitable. In this study, the chemical composition of SBWL and the optimization of culture conditions for Bacillus pumilus JB-1, a selected strain for functional chungkuk-jang fermentation, using SBWL were investigated. The SBWL contains 88% water, 9.5% free sugar, 1.6% crude protein, 0.3% crude fat, 0.1% crude fiber and 2.1% ash, respectively. The contents of total polyphenol, total flavonoids and free amino acid in SBWL were 55%, 76%, and 30% of those of raw soybean, respectively. Culture conditions for B. pumilus JB-1 in SBWL were optimized. The 1/10-diluted, 0.1 % of $(NH_4)_2SO_4$ added SBWL without pH adjustment and carbon source addition was cultured at $37^{\circ}C$ for 48 h with agitation (120 rpm). The 0.5% of inoculation was enough. The large scale fermentation in 5-L jar fermentor showed that the SBWL is a good resource for production of chungkuk-jang starter and functional ingredients.

Production of Phenyl Lactic Acid (PLA) by Lactic Acid Bacteria and its Antifungal Effect

  • Song, June-Seob;Jang, Joo-Yeon;Han, Chang-Hoon;Yoon, Min-Ho
    • Korean Journal of Soil Science and Fertilizer
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    • v.48 no.2
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    • pp.125-131
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    • 2015
  • Phenyllactic acid (PLA) which is known as antimicrobial compound can be synthesized through the reduction of phenylpyruvic acid (PPA) by lactate dehydrogenase (LDH) of lactic acid bacteria (LAB). LAB producing PLA was isolated from Korea Kimchi and identified to Lactobacillus plantarum SJ21 by 16 rRNA gene sequence analysis. Cell-free supernatant (CFS) from L. plantarum SJ21 was assessed for both the capability to produce the antimicrobial compound PLA and the antifungal activity against four fungal pathogens (Rhizoctonia solani, Aspergillus oryzae, Botrytis cinerea, and Collectotricum aculatum). PLA concentration was investigated to be 3.23mM in CFS when L. plantarum SJ21 was grown in MRS broth containing 5mM PPA for 16 h. PLA production also could be promoted by the supplement of PPA and phenylalanine in MRS broth, but inhibited by the supplement of 4-hydroxyphenylpyruvic acid and tyrosine as precursors. Antifungal activity demonstrated that all fungal pathogens were sensitive to 5% CFS (v/v) of L. plantarum SJ21 with average growth inhibitions ranging from 27.32% to 69.05% (p<0.005), in which R. solani was the most sensitive to 69.05% and followed by B. cinerea, C. aculatum, and A. oryzae. The minimum inhibitory concentration (MIC) for commercial PLA was also investigated to show the same trend in the range from $0.35mg\;mL^{-1}$ (2.11 mM) to $0.7mg\;mL^{-1}$ (4.21 mM) at pH 4.0. The inhibition ability of CFS against the pathogens was not affected by heating or protease treatment. However, pH modification in CFS to 6.5 caused an extreme reduction in their antifungal activity. These results may indicate that antifungal activities in CFS were caused by acidic compounds like PLA or organic acids rather than proteins or peptides molecules.

Isolation of Serratia marcescens CK-3 against phytopathogenic fungi and its enzymatic properties (식물(植物) 병원류(病源惟) 사상균(絲狀菌)에 길항력(拮抗力)을 갖는 Serratia marcescens CK-3의 분리(分離) 및 효소적(酵素的) 성질(性質))

  • Kim, Yeong-Yil;Rhee, Young-Hwan;Kim, Kwang-Sik;Park, Hwa-Sung;Chun, Woo-Bock;Lee, Jae-Wha;Kim, Jong-Hyun
    • Applied Biological Chemistry
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    • v.34 no.1
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    • pp.54-60
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    • 1991
  • Serratia marcescens CK-3, decomposing chitin which is a mar component of cell wall in phyitopathogenic fungi, was isolated from the continuous cropping rhizosphere of pepper and cucumber and its enzymatic property was examined. S. marcescens CK-3 was found tn have an tagonistic effects against, Fusarium axysporum and Rhizoctonia solani and to have complex enzyme system such as chitinase, laminarinase, and proteinase. The preferable composition of the medium for production of chitinase was fond and was as follows : colloidal chitin 1.5%, tryptone 0.5%, glucose 1.0%, peptone 0.2%, $MgSO_4{\cdot}7H_2O\;0.1%,\;K_2HPO_4\;0.1%,\;and\;NaCl\;0.1%$(w/v), pH 6.8. The maximum enzyme production was observed after culture of 72 hours at $30^{\circ}C$ using a medium containing the above chemical composition. The optimal pH and temperature for in vitro activity of chitinase from S. marcescens CK-3 were pH 7.5 and $50^{\circ}C$, respectively. The enzyme activity in-creased by metal ions such as$Ag^+$ and $Mn^{++}$.

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Purification and Characterization of Bacillus subtilis JS-17 Collagenase. (Bacillus subtilis JS-17이 생산하는 Collagenase의 정제 및 특성)

  • Lim Kyoung-Suk;Son Shung-Hui;Kang Ho Young;Jun Hong-Ki
    • Journal of Life Science
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    • v.15 no.4 s.71
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    • pp.657-663
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    • 2005
  • Collagenases are generally defined as enzymes that are capable of degrading the polypeptide backbone of native collagen under conditions that do not denature the protein. An extracellular collagenase-producing bacterial strain was isolated from kimchi and identified to be Bacillus subtilis JS-17 through morphological, cultural, biochemical characteristics and 16S rDNA sequence analysis. Optimum culture condition of Bacillus subtilis JS-17 for the production of collagenase was $1.5\%$ fructose, $1\%$ yeast extract, $0.5\%\;K_2HPO_4,\;0.4\%\;KH_2PO_4,\;0.01\%\;MgSO_4\cdot7H_2O,\;0.01\%\; MnSO_4\cdot4H_2O,\;,0.1\%$ citrate and $0.1\%\;CaCl_2$. The production of collagenase was optimal at $30^{\circ}C$ for 72 hr. A collagenase was isolated from the culture filtrate of Bacillus subtilis JS-17. The enzyme was purified using Amberlite IRA-900 column chromatography, Sephacryl S-300 HR column chromatography and DEAE-Sephadex A-50 column chromatography The purified collagenase has an specific activity 192.1 units/mg. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PACE. The purified collagenase has $100\%$ activity up to $55^{\circ}C$.

Lactobacillus plantarum APsulloc 331261 Fermented Products as Potential Skin Microbial Modulation Cosmetic Ingredients (Lactobacillus plantarum APsulloc 331261 발효 용해물의 피부 미생물 조절 효과)

  • Kim, Hanbyul;Myoung, Kilsun;Lee, Hyun Gee;Choi, Eun-Jeong;Park, Taehun;An, Susun
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.46 no.1
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    • pp.23-29
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    • 2020
  • The skin is colonized by a large number of microorganisms with a stable composition of species. However, disease states of skin such as acne vulgaris, psoriasis, and atopic dermatitis have specific microbiome compositions that are different from those of healthy skin. The target modulation of the skin microbiome can be a potential treatment for these skin diseases. Quorum sensing (QS), a bacterial cell-cell communication system, can control the survival of bacteria and increase cell density. Also, QS affects the pathogenicity of bacteria such as biofilm formation and protease production. In this study, we confirmed anti-QS activity of Amorepacific patented ingredients, which are Lactobacillus ferment lysate (using Lactobacillus plantarum APsulloc 331261, KCCM 11179P) through bio-reporter bacterial strain Chromobacterium violaceum. The purple pigment production of C. violaceum controlled by QS was reduced 27.3% by adding 10 ㎍/mL of Lactobacillus ferment lysate (freeze dried). In addition, the Lactobacillus ferment lysate increased growth of Staphylococcus epidermidis 12% and decreased growth of Pseudomonas aeruginosa 38.5% and its biofilm formation 17.7% at a concentration of 10 ㎍/mL compared to the untreated control group. Moreover, S. epidermidis was co-cultured with the representative dermatological bacterium Staphylococcus aureus in the same genus, the growth of S. epidermidis was increased 134 % and the growth of S. aureus was decreased 13%. These results suggest that fermented lysate using Lactobacillus plantarum APsulloc 331261 may be useful as a cosmetic ingredient that can control the balance of skin microbiome.

Enhanced Tolerance of Chinese Cabbage Seedlings Mediated by Bacillus aryabhattai H26-2 and B. siamensis H30-3 against High Temperature Stress and Fungal Infections

  • Lee, Young Hee;Jang, Su Jeong;Han, Joon-Hee;Bae, Jin Su;Shin, Hyunsuk;Park, Hee Jin;Sang, Mee Kyung;Han, Song Hee;Kim, Kyoung Su;Han, Sang-Wook;Hong, Jeum Kyu
    • The Plant Pathology Journal
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    • v.34 no.6
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    • pp.555-566
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    • 2018
  • Two rhizobacteria Bacillus aryabhattai H26-2 and B. siamensis H30-3 were evaluated whether they are involved in stress tolerance against drought and high temperature as well as fungal infections in Chinese cabbage plants. Chinese cabbage seedlings cv. Ryeokgwang (spring cultivar) has shown better growth compared to cv. Buram-3-ho (autumn cultivar) under high temperature conditions in a greenhouse, whilst there was no difference in drought stress tolerance of the two cultivars. In vitro growth of B. aryabhattai H26-2 and B. siamensis H30-3 were differentially regulated under PEG 6000-induced drought stress at different growing temperatures (30, 40 and $50^{\circ}C$). Pretreatment with B. aryabhattai H26-2 and B. siamensis H30-3 enhanced the tolerance of Chinese cabbage seedlings to high temperature, but not to drought stress. It turns out that only B. siamensis H30-3 showed in vitro antifungal activities and in planta crop protection against two fungal pathogens Alternaria brassicicola and Colletotrichum higginsianum causing black spots and anthracnose on Chinese cabbage plants cv. Ryeokgwang, respectively. B. siamensis H30-3 brings several genes involved in production of cyclic lipopeptides in its genome and secreted hydrolytic enzymes like chitinase, protease and cellulase. B. siamensis H30-3 was found to produce siderophore, a high affinity iron-chelating compound. Expressions of BrChi1 and BrGST1 genes were up-regulated in Chinese cabbage leaves by B. siamensis H30-3. These findings suggest that integration of B. aryabhattai H26-2 and B. siamensis H30-3 in Chinese cabbage production system may increase productivity through improved plant growth under high temperature and crop protection against fungal pathogens.

Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean Red Ginseng

  • Jeonghui Moon;Younghun Jung;Seokoh Moon;Jaehyeon Hwang;Soomin Kim;Mi Soo Kim;Jeong Hyeon Yoon;Kyeongwon Kim;Youngseo Park;Jae Youl Cho;Dae-Hyuk Kweon
    • Journal of Ginseng Research
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    • v.47 no.1
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    • pp.123-132
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    • 2023
  • Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean Red Ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

Studies on the Mutation of Aspergillus niger (흑국균(黑麴菌)의 인공변이(人工變異)에 관(關)한 연구(硏究))

  • Park, Yoon-Joong;Sohn, Cheon-Bae
    • Korean Journal of Food Science and Technology
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    • v.14 no.1
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    • pp.72-79
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    • 1982
  • Several mutants were isolated from the parent strain of Aspergillus niger CF: the first mutant strain CF-11 was obtained by UV irradiation, and the second mutant strain CF-21 and CF-22 were from NTG (N-methyl-N'-nitroso-N-nitroso-guanidine) treatment on the CF-11. These mutants were characterized, and their enzyme and acid production on wheat bran Koji and wheat flour Koji were studied. Asp. niger CF-22 mutant appeared to be tan type which conidial heads were discolored. It's glucoamylase activity was inreased approximately two times and its ${\alpha}-amylase$ about 50 percent as compared with that of the parent strain of Asp. niger CF, when grown on wheat bran Koji under the optimal conditions. Asp. niger CF-21 mutant showed slower growth rate and poor sporulation than the wild type, although its conidial heads were not discolored. Approximately 4-fold increment in its acid production was observed as compared with the weld type. The activities of glucoamylase and ${\alpha}-amylase$ of the Asp. niger CF-22 and CF-21 mutants were higher than those of the wild type, but their protease activity was rather lower. The maximum production of glucoamylase by the Asp. niger CF-22 mutant was obtained after 2 to 3 days incubation on wheat bran at 30 to $35^{\circ}C;$ ${\alpha}-amylase$2 days incubation at 30 to $35^{\circ}C$. The maximal levels of acid production by the mutant CF-21 was appeared after 2 days incubation on wheat bran Koji, and after 3 days on wheat flour Koji at $30^{\circ}C$. Little differences in the levels of acid production were observed between on wheat bran and flour Koji.

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Predicting In Sacco Rumen Degradation Kinetics of Raw and Dry Roasted Faba Beans (Vicia faba) and Lupin Seeds (Lupinus albus) by Laboratory Techniques

  • Yu, P.;Egan, A.R.;Leury, B.J.
    • Asian-Australasian Journal of Animal Sciences
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    • v.13 no.10
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    • pp.1377-1387
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    • 2000
  • Two laboratory techniques: (1) an in vitro method with two procedures for measuring protein degradabilities and (2) an in vitro method with three procedures for measuring protein solubility, were investigated to determine which laboratory techniques could most accurately predict the quantity of rumen protein degradation kinetics of legume seeds after dry roasting under various conditions, in terms of (1) rumen protein disappearance ($D_j$, where j=0, 2, 4, 8, 12, 24 and 48 h incubation), (2) rumen protein effective degradability (EDCP), (3) the parameters describing rumen degradation characteristics (the soluble fraction: S, the potentially degradable fraction: D, undegradable fraction: U, lag time: T0 and the degradation rate: Kd) and (4) rumen bypass protein (BCP), which were determined by the method accepted internationally at present, in sacco nylon bag technique using the standardized Dutch method. Feeds evaluated were the raw and dry roasted whole faba (Vicia faba) beans (WFB) and whole lupin (Lupinus albus) seeds (WLS), each was dry roasted under various conditions (at 110, 130 or $150^{\circ}C$ for 15, 30 or 45 min). In vitro protein degradability ($D_1$_Auf and $D_{24}$_Auf) were determined using the modified Aufr re method by enzymatic hydrolysis for 1 h and 24 h using a protease extracted from Streptomyces griseus in a borate-phosphate buffer. In vitro protein solubility ($bf_1$_S, $bf_2$_S, $bf_3$_S) was measured in a borate-phosphate buffer with three different procedures. Results from laboratory techniques (in vitro) were correlated and linearly regressed with in sacco results. Of the three procedures of in vitro protein solubility evaluated, none of them could predict in sacco results with good precision. The highest Pearson correlation coefficient ($R^2$) was less than 0.50. Of two procedures of in vitro protein degradability studied, the $D_1$_Auf values were closely correlated with in sacco parameters: Kd, EDCP and %BCP with high R' values: 0.82, 0.85 and 0.85, respectively, and closely correlated with in sacco $D_j$ at 2, 4, 8 and 12 h rumen incubation with high $R^2$ values: 0.83, 0.91, 0.93 and 0.83, respectively. The $D_{24}$_Auf values could not predict in sacco results. The highest $R^2$ value was less then 0.40. These results indicated that in vitro protein solubility measured in borate-phosphate failed to identify differences in the rate and extent of protein degradation of legume seeds after dry roasting under various conditions and thus should not be used to predict rumen degradation, particularly for heat processed feedstuffs. But in vitro protein degradability using the modified Aufr re method by enzymatic hydrolysis for 1 h or possibly an intermediate time (>1 h and <24 h) is a promising laboratory procedure to detect effectiveness of dry roasting legume seeds on rumen protein degradation characteristics and could be used as a simple laboratory method to predict the rate and extent of protein degradation in the rumen in sacco with high accuracy. The equations to predict EDCP, Kd and BCP of dry roasted legume seeds (WLS and WFB) under various conditions are as follow: For both: EDCP (%)=-1.37+1.06*$D_1$_Auf ($R^2=0.85$, p<0.01). For both: Kd (%/h)=-21.81+0.49*$D_1$_Auf ($R^2=0.82$, p<0.01). For both: %BCP=103.37-1.07*$D_1$_Auf ($R^2=0.85$, p<0.01).

Biological Control of Fusarium Stalk Rot of Maize Using Bacillus spp. (Bacillus spp.를 이용한 옥수수 밑둥썩음병의 생물학적 방제)

  • Han, Joon-Hee;Park, Gi-Chang;Kim, Joon-Oh;Kim, Kyoung Su
    • Research in Plant Disease
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    • v.21 no.4
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    • pp.280-289
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    • 2015
  • Maize (Zea mays L.) is an economically important crop in worldwide. While the consumption of the maize is steadily increasing, the yield is decreasing due to continuous mono-cultivation and infection of soil-borne fungal pathogens such as Fusarium species. Recently, stalk rot disease in maize, caused by F. subglutinans and F. temperatum has been reported in Korea. In this study, we isolated bacterial isolates in rhizosphere soil of maize and subsequently tested for antagonistic activities against F. subglutinans and F. temperatum. A total of 1,357 bacterial strains were isolated from rhizosphere. Among them three bacterial isolates (GC02, GC07, GC08) were selected, based on antagonistic effects against Fusarium species. The isolates GC02 and GC07 were most efficient in inhibiting the mycelium growth of the pathogens. The three isolates GC02, GC07 and GC08 were identified as Bacillus methylotrophicus, B. amyloliquefaciens and B. thuringiensis using 16S rRNA sequence analysis, respectively. GC02 and GC07 bacterial suspensions were able to suppress over 80% conidial germination of the pathogens. GC02, GC07 and GC08 were capable of producing large quantities of protease enzymes, whereas the isolates GC07 and GC08 produced cellulase enzymes. The isolates GC02 and GC07 were more efficient in phosphate solubilization and siderophore production than GC08. Analysis of disease suppression revealed that GC07 was most effective in suppressing the disease development of stalk rot. It was also found that B. methylotrophicus GC02 and B. amyloliquefaciens GC07 have an ability to inhibit the growth of other plant pathogenic fungi. This study indicated B. methylotrophicus GC02 and B. amyloliquefaciens GC07 has potential for being used for the development of a biological control agent.