• Journal of agriculture & life science
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• v.46 no.1
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• pp.163-176
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• 2012

TTo increase productivity of a strong extracellular alkaline protease (BCAP), stable strains of Bacillus clausii I-52 carrying another copy of BCAP gene in the chromosome were developed. Integrative vector, pHPS9-fuBCAP carrying BCAP promoter, ribosome binding site, signal sequence and active protease gene was constructed and transferred into B. clausii I-52, and integration of the constructed plasmid into chromosome was identified by PCR. An investigation was carried out on BCAP production by B. clausii I-52 and transformant C5 showing the highest relative activity of alkaline protease using submerged fermentation. Maximum enzyme activity was produced when cells were grown under the submerged fermentation conditions at $37^{\circ}C$ for 48 h with an aeration rate of 1 vvm and agitation rate of 650 rpm in a optimized medium (soybean meal 2%, wheat flour 1%, sodium citrate 0.5%, $K_2HPO_4$ 0.4%, $Na_2HPO_4$ 0.1%, NaCl 0.4%, $MgSO_47H_2O$ 0.01%, $FeSO_47H_2O$ 0.05%, liquid maltose 2.5%, $Na_2CO_3$ 0.6%). A protease yield of approximately 134,670U/ml was achieved using an optimized media, which show an increase of approximately 1.6-fold compared to that of non-transformant (83,960 U/ml). When the stability of transformant C5 was examined, the integrated plasmid pHPS9-fuBCAP was detected in the transformant after cultivation for 8 days, suggesting that it maintained stably in the chromosomal DNA of transformant C5.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.6
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• pp.700-707
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• 2010

Effects of protease-resistant antimicrobial substances (PRA) produced by Lactobacillus plantarum and Leuconostoc citreum on rumen methanogenesis were examined using the in vitro continuous methane quantification system. Four different strains of lactic acid bacteria, i) Lactococcus lactis ATCC19435 (Control, non-antibacterial substances), ii) Lactococcus lactis NCIMB702054 (Nisin-Z), iii) Lactobacillus plantarum TUA1490L (PRA-1), and iv) Leuconostoc citreum JCM9698 (PRA-2) were individually cultured in GYEKP medium. An 80 ml aliquot of each supernatant was inoculated into phosphate-buffered rumen fluid. PRA-1 remarkably decreased cumulative methane production, though propionate, butyrate and ammonia N decreased. For PRA-2, there were no effects on $CH_4$ and $CO_2$ production and fermentation characteristics in mixed rumen cultures. The results suggested that PRA-1 reduced the number of methanogens or inhibited utilization of hydrogen in rumen fermentation.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.559-562
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• 2000

A halotolerant and extracellular pretense producing yeast was isolated from traditional Meju and identified as a strain of Hansenular sp. S-9 by investigation of its microbiological characteristics. The optimum pH, temperature and NaCl concentration for growth of Hansenular sp. S-9 were pH 7.5, $30^{\circ}C$ and 0.5M, respectively. The protease production from Hansenular sp. S-9 was maximized when it was grown on BD medium containing 1.0% beef extract, 1.0% glucose and 0.5M NaCl for 72 h at $30^{\circ}C$.

• Journal of Ginseng Research
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• v.6 no.2
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• pp.131-137
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• 1982

The effect of red ginseng residue on the several enzyme activities of the koji and alcohol fermentation were investigated. The koji showed maximum values of amylase and cellulase activity when it was prepared by 30% red ginseng residue and 70% wheat bran, and of protease activity when it was prepared by 40% red ginseng residue and 60% wheat bran-${\alpha}$ amylase activity of the koji during its fermentation was increased rapidly until 4 days and there after it was increased slowly, but ${\beta}$-amylase was rapidly increased after 3 days fermentation. During the preparation of the koji, the acidic, neutral protease and cellulase activities showed the maximum value after 3 days fermentation and the alkaline protease showed the maximum value within 4-6 days fermentation. On the otherhand, fermented broth, containing 6%(v/v) alcohol, could be obtained when the substrate was saccharified by the koji, based on 25% red ginseng residue and 75% wheat bran, prior to alcohol fermentation.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.331.2-331.2
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• 2002

Actinomycetes CS0707 has been isolated in soil sample from location in the Jeju province. Korea, and produces thermostable extracellular proteases. Actinomycetes CS0703 showed the highest protease activity at late exponential phase when grown in OSYM medium (oatmeal 2.0%, soybean meal 1 %, dried yeast 1 %, mannitol 1 %) at $48^{\circ}C$. Three forms of protease(Ta-1, TA-2 and Ta-3) were fractionated by Ultrogel AcA 54 column chromatography, and further purified through ammonium sulfate fractionation, ultramembrane filtration, and DEAE-sepharose CL-6B column chromatography. (omitted)

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.117-126
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• 2010

A chitinase (CHT) and a protease (PRO) were purified from the culture supernatant of Serratia sp. TKU017, with shrimp shell as the sole carbon/nitrogen source. The molecular masses of CHT and PRO determined by SDS-PAGE were approximately 65 kDa and 53 kDa, respectively. CHT was inhibited by $Mn^{2+}$ and $Cu^{2+}$, and PRO was inhibited by most tested divalent metals and EDTA. The optimum pH, optimum temperature, pH stability, and thermal stability of CHT and PRO were pH 5, $50^{\circ}C$, pH 5-7, and <$50^{\circ}C$, and pH 9, $40^{\circ}C$, pH 5-11, and <$40^{\circ}C$, respectively. PRO retained 95% of its protease activity in the presence of 0.5 mM SDS. The result demonstrates that PRO is an SDS-resistant protease and probably has a rigid structure. The $4^{th}$-day supernatant showed the strongest antioxidant activity (70%, DPPH scavenging ability) and the highest total phenolic content ($196{\pm}6.2\;{\mu}g$ of gallic acid equiv./ml). Significant associations between the antioxidant potency and the total phenolic content, as well as between the antioxidant potency and free amino groups, were found for the supernatant. With this method, we have shown that shrimp shell wastes can be utilized and it is effective in the production of enzymes and antioxidants, facilitating its potential use in industrial applications and functional foods.

• Journal of Microbiology
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• v.39 no.4
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• pp.305-313
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• 2001

The spr D gene encoding Strptomyces griseus protease D(SGPD); a chymotrypsin-like proteae, was cloned from Strptomyces griseus IFO13350 and sequence. Most of the amino-acid sequence deduced from the nucleotide sequence is idential to that Strptomyces griseus IMRU3499 except that one amino acid has been deleted and Trp 369 has been substituted into Cys369 in the SGPD from S. griseus IFO13350 without affecting the protease activity. The spr D gene was overexpressed in Streptomyce liv-idans TK24 as a heterologous host. Various media with different compositions were also used to max-imize the productivity of SGPD inthe heterologous host. The SGPD productivity was best when the transformant S. lividans TK24 was cultivated in R2YE medium. The relative chymotrypsin activity of the culture broth measured with an artificial chromogenic substrate, N-scuccinyl-ala-ala-pro-phe-p-nitroanilide, was 16 units/ml. A high level of SGPD was also produced in YEME and SAAM medial but it was relatively lower that in R2YE medium and negligible amounts of SGPD were produced in GYE, GAE and Benedict media. The growth of S. lividans reacted the maximum level of cell mass at days 3 and 4 of the culture, but SGPD production started in the stationary phase of cell growth and kept increase in till the 10$^{th}$ day of culture in R2YE and YEME medium, but in GYE media the productivity reached maximum level at 8days of cultivation. The introduction of the spr D gene into S. lividans TK24 triggered biosyntheis of the pigmented antibiotic , actinorhodin, which implies some protease may paly a very improtant role in secondary-metabolite formation in sStreptomyces.

• Preventive Nutrition and Food Science
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• v.2 no.2
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• pp.109-120
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• 1997

In order to elucidate roles of lactic acid bacteria(LAB) for the antibiosis occurring in th fermenting environment of Kimchi, 2.052 strains of LAB were isolated from Kimchi. Fifty tow strains which showed antagonistic effect against 4 indicator strains were finally selected and investigated. Based upon responses to protease treatment, antibiosis of the 52 strains of LAB were classified into 3 types. Type A antibiosis resulted from action of antibiotic-like substances which were not affected by protease treatment and which had broad action spectra against even natural inhabitants of Kimchi. Type B antibiosis was due to bacteriocin-like substances which were very sensitive to treatment of protease and more effective against foreign bacteria than original inhabitant microflora. Type C antibiosis was owing to proteinaceous compounds which were activated or induced by the presence of protease and then exerted antibacterial activities. Therefore, lactic acid bacteria appeared to contribute to antibiosis of Kimchi by the concerted action of these three different types of antibacterial compounds. As one of model system for type B bacteriocin, the antagonistic compound produced by LAB31-9 as well as th producer strain itself was further charaacterized. Strain LAB31-9 was identified as L. casei. Bacteriocin produced by LAB31-9 was proteinaceous and stable over wide range of pH and to various solvents, but very labile to heat treatment. Its mode of action was bactericidal. Based upon these data, bacteriocin produced by LAB31-9 was named as 'caseicin K319'. Genetic determinant for the bacteriocin production of LAB31-9 was located in the chromosome.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.282-282
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• 2003

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.9
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• pp.1114-1121
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• 2017

Bacillus strains not producing harmful components were isolated from Korean traditional soybean products. Extracellular enzyme activities (amylase, protease, cellulase, and xylanase) of isolated Bacillus strains were measured, and Bacillus strains with high protease activity were selected. The selected 15 strains were identified as Bacillus amyloliquefaciens (10), Bacillus methylotrophicus (1), Bacillus velezensis (1), and Bacillus subtilis (3). Among them, B. subtilis JBG17019, B. amyloliquefaciens JBD17076, and B. amyloliquefaciens JBD17109 showed antimicrobial activities against food-borne microorganisms. The production abilities of glutamate, glutamine, and poly-${\gamma}$-glutamic acid (${\gamma}$-PGA) of the selected Bacillus strains were measured to analyze fermentation characteristics related to glutamic acid metabolism. The factor for multivariate was analyzed by the principal components analysis (PCA) method between fermentation characteristics and ${\gamma}$-PGA production. The three principal components were classified according to the PCA method: PC1 [enzyme activity (amylase, cellulase, and xylanase)], PC2 (${\gamma}$-PGA), and PC3 (protease, glutamate, and glutamine). As a result, B. amyloliquefaciens JBD17076 and B. subtilis JBG17019 strains were evaluated as having excellent enzyme activity and ${\gamma}$-PGA production.