• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1077-1086
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• 2001

The sprA and sprB gene encoding chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB) and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from Streptomyces griseus ATCC10137 and overexpressed in Streptomyces lividans TK24 as a heterologous host. The chymotrypsin activity of tole culture broth measured with the artificial chromogenic substrate , N-succinyl-ala-ala-pro-phe-p-nitroanilide, was 10, 14 and 14 units/mg in the transformants haboring the sprA, sprB and sprD genes, respectively. The growth of S. lividans reached the maximum cell mass after 4 days of culture, yet SGPA and SGPD production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The trypsin activity of the culture broth measured with the artificial chromogenic substrate , N-${\alpha}$-benzoyl-DL- arginine-p-nitroanilide , was 16 units/mg and SGT production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The introduction of the sprA gene into S, lividans TK24 triggered the biosynthesis of pigmented antibiotics, actinorhodin and undecylprodigiosin, and induced significant morphological changes in the colonies in Benedict, R2YE, and R1R2 media. In addition, the introduction of the sprT gene also induced morphological changes in the colony shape without affecting the antibiotic production, thereby implying that certain proteases would appear to play very important and specific roles in secondary-metabolites formation and morphological differentiation in Streptomyces.

• 한국식품영양과학회지
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• 제31권2호
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• pp.211-215
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• 2002

전통 메주의 산업화를 위해서는 먼저 제조공정이 확립되어야 하며 이를 위해서는 적합한 종균이 개발되어야 한다. 재래식 메주로부터 Rhizopus stolonifer, Rhizopus oryzae 및 Absidia corymbifera를 분리하여 균주 유래 protease의 특성을 조사하여 장류산업에 있어 기초자료로 이용하고자 한다. 세 균주를 각각 배양한 밀기울 배지에서의 protease 최적 생산조건은 모두 3$0^{\circ}C$, 4일간이었으며 초기 pH에 의한 영향은 없었다. 3$0^{\circ}C$에서 4일간 각 균주를 배양하고 증류수로 조효소액을 추출하여 효소작용을 위한 최적 pH와 온도를 조사한 결과, 각각 pH 6.0, 5$0^{\circ}C$였고 pH 4.0~7.0의 범위인 약산성 영역과 4$0^{\circ}C$이하에서 안정하였다. 된장의 염도 범위를 고려해서 16% 이내의 영도에 의한 영향을 조사한 결과, 염농도는 효소활성에 거의 영향을 미치지 않았다. Rhizopus stolonifer, Rhizopus oryzae 및 Absidia corymbifera로부터 유래된 각 효소액의 Hammastein casein 기질에 대한 결합력과 가수분해속도를 비교한 결과 $K_{m}$ 은 각각 3.3$\times$$10^{-4}$ , 0.75$\times$$10^{-4}$ , 1.3$\times$$10^{-4}$ 로 상호간에 큰 차이가 없었으며, $V_{max}$도 17.2$\mu\textrm{g}$/min, 9.4 $\mu\textrm{g}$/min, 7.8 $\mu\textrm{g}$/min으로 유사하였다.

• Journal of Animal Science and Technology
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• 제54권2호
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• pp.133-139
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• 2012

Bacillus 균주가 생산하는 효소를 식품과 동물사료 첨가제로 이용하기 위해서 맥아로부터 단백질 분해능력과 전분 분해능력이 우수한 균주를 분리 동정하여 $Bacillus$ $amyloliquefaciens$ CNL-90으로 명명하였다. 분리된 $B.$ $amyloliquefaciens$ CNL-90이 생산하는 ${\alpha}$-amylase의 안정성은 pH는 7, 온도는 $40^{\circ}C$, protease의 경우는 pH가 7, 온도는 $50^{\circ}C$에서 안정했다. $B.$ $amyloliquefaciens$ CNL-90을 밀기울에 접종하여 고체 배양한 결과 ${\alpha}$-amylases의 효소활성은 6일 배양 후 290,000 unit/kg이었고, protease의 효소활성은 310,000 unit/kg으로 나타났다. 밀기울에 고체 배양한 생균수는 배양 6일 후에는 $2.2{\times}10^9$ CFU/g으로 높았다. 사료 요구율 개선은 $B.$ $amyloliquefaciens$ CNL-90 배양액 0.2% 첨가구가 대조구에 비해서 일당 증체량은 6.66% 높았고, 사료 요구율은 0.05% 효과가 있었다. 이러한 결과는 $B.$ $amyloliquefaciens$ CNL-90이 생산하는 단백질 분해효소와 전분 분해효소는 식품산업 및 가축사료 첨가제 산업에 응용할 수 있는 가능성을 확인하였다.

• 한국미생물·생명공학회지
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• 제17권4호
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• pp.358-364
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• 1989

토양으로부터 분리한 Streptomyces sp. YSA-130으로부터 활성이 좋은 결정화된 alkaline protease를 분리하였다. Alkaline protease 생산의 최적 배양조건은 2.0% soluble starch, 1.0% soytone, 0.3% $K_2$HPO$_4$, 0.02% MgSO$_4$.7$H_2O$, 0.8% $Na_2$CO$_3$ 3$0^{\circ}C$, pH 10.5에서 72 시간 배양하였을 때 였다. Alkaline protease이 정제는 (NH$_4$)$_2$SO$_4$. 분별침전, 투석, DEAE cellulose column chromatography, Sephadex G-75 gel filtration, crystallization으로 하였으며, 그 결과 비활성도 14,290unit/mg, 정제도 23.8 배였고, 수율은 20.0% 이었다. Alkaline protease의 반응 최적온도와 pH는 6$0^{\circ}C$ 와 11.5이었으며, 효소의 pH 안정성은 5.5-12.0에서 안정하였고, 온도 안정성은 5$0^{\circ}C$까지 안정하였으며, $Ca^{++}$ ion 첨가시 6$0^{\circ}C$까지 안정성이 증가하였다. Alkaline protease의 분자량은 30,000이었으며 금속이온, EDTA, 환원제는 활성에 영향이 없었고 DFP에 의해 저해되었다. 계면활성제에 저항성이 크고 $H_2O$$_2$에 대한 잔존활성은 60% 을 유지하였다.

• Applied Biological Chemistry
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• 제21권2호
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• pp.123-130
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• 1978

마늘가루 첨가(添加)가 국균(麴菌)의 각종(各種) 효소생산(酵素生産) 및 생육(生育)에 미치는 영향을 규명(糾明)할 목적으로 Asp. oryzae D(단모균(短毛菌))와 Asp. oryzae H(장모균(長毛菌)) 균주를 마늘가루를 함유하는 밀기울배지 및 Czapek-Dox액체배지에 배양하여 각종효소력(酵素力), 균체생성량(菌體生成量), pH, 적정산도(滴定酸度), 환원당 등(等)을 측정한 결과는 다음과 같다. 1, Asp. oryzae D균주의 경우 산성 protease는 마늘가루첨가농도 $0.5{\sim}2%$범위내에서, 중성 protease는 0.5%, alkali성(性) protease는 $2{\sim}6%$범위내에서 각각 대조구에비하여 활성이 높았다. 2, Asp. oryzae H균주의 경우 각(各) protease의 활성은 마늘가루첨가농도 $0.5{\sim}8%$범위내에서는 대조구와 큰차이가 없었으나 30%에서는 peak를 나타냈다. 3. Asp. oryzae H균주의 경우 마늘가루첨가는 $\alpha$ 및 glucoamylase생성을 증가시켰다. 4. 마늘가루첨가는 국균(麴菌)의 cellulase 생성을 저해(沮害)하였다. 5. 국균(麴菌)의 균체생성량(菌體生成量)은 마늘가루첨가농도 $0.5{\sim}6%$범위내에서 대조구에 비하여 증가하는 경향을 나타냈고 장모균(長毛菌)은 단모균(短毛菌)에 비해 증가현상이 현저하였다. 6. 마늘가루첨가농도가 증가함에 따라 Asp. oryzae D균주는 Czapek-Dox액체배양물의 pH를 저하(抵下)시키는 경향을 나타냈으나 Asp. oryzae H균주는 배양물의 pH를 상승시켰다. 7 마늘가루첨가농도가 증가함에 따라 Czapek-Dox 액체배양물의 정적산도(定滴酸度)는 대체로 증가하였다. 8. 마늘가루첨가농도가 10%이상인 밀기울고체(固體)배지 및 Czapek-Dox액체 배지에서는 국균(麴菌)의 생육은 거의 억제되었다. 9. Czapek-Dox액체배양시(時) 환원당의 이용율은 마늘첨가농도 $1{\sim}2%$범위에서 가장 양호하였다.

• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.222-228
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• 2001

Vibrio metschnikovii strain RH530 is a non-pathogenic, industrially-important alkaline protease producer which has been isolated from wastewater. In this paper, we report on the transformation of this strain by using the method of electroporation. A field strength of $7.5\;kVcm^{-1}$ and $25\;{\mu}F$, and using a 0.2-cm cuvette, appeared to be the optimal conditions for electroporation of the cells with the recombinant pSBCm plasmid carrying the vapK alkaline protease gene and the ColE1 replicon. Cells were subjected to osmotic shock in order to remove extracelluar DNase, and adding 200 mM of sucrose to electroporation buffer cells showed an increased transformation efficiency. Maximum efficiency of transformation was obtained at an early exponential growth phase. Using all of the conditions mentioned above, we routinely obtained a transformation efficiency of more than $10^4{({\mu}g\;plasmid\;DNA)}^{-1}$. The stability of the plasmid pSBCm in V. metschnikovii RH530 was 25% after 18h of growth (27 generations) in the medium without antibiotic selection. The insertion of the par locus to the pSBCm increased the stability of the plasmid up to 42% without selective pressure. The increase in plasmid stability was accompanied by the increase in the productivity of alkaline protease in the recombinant V. metschnikovii strain RH530. Determining optimal conditions for the transformation of the industrially-important, nonpathogenic Vibrio strain, and the improvement of plasmid stability by introducing the par locus into the high copy number plasmid vector, will allow the development of procedures involved in the genetic manipulation of this strain, particularly for its use in the production of industrial enzymes such as alkaline protease.

• 한국미생물·생명공학회지
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• 제26권5호
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• pp.450-455
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• 1998

Alkaline protease를 대량생산하는 Aspergillus oryzae를 만들기 위하여 A. oryzae의 alkaline pretense 유전자 alpA를 고발현시키는 plasmid pTAalp를 제조하고 이 plasmid로 A. oryzae M-2-3 균주를 형질전환시켰다. 16개의 형질전환체를 얻어 이들의 protease생산성을 skim milk 분해에 의한 halo 형성능에 의하여 확인하였다. 또한 protease 생산성이 증가한 형질전환체는 pTAalp가 multi-copy로 염색체 안에 integration되어 있음을 Southern blot에 의하여 확인하였고, 이들의 배양액을 polyacrylamide gel전기영동에 의하여 분석한 결과, 형질전환체 No. 14에서는 전체 분비단백질의 80-90%가 alkaline protease 임을 알 수 있었다. 간장원료 분해실험의 결과 No. 14에 의한 원료분해액은 간장 양조용 대조균에 의한 분해액보다 TN이 증가하였으며 원료분해율도 1.4-1.5배로 증가되었다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권12호
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• pp.1769-1774
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• 2001

Bacillus licheniformis strains L-25 and PWD-1 are two thermophilic feather-degrading bacteria. Despite isolated from different environmental conditions, they were both capable of breaking down chicken feathers and growing in a medium in which feather was the only source of carbon and nitrogen. A 1.46-kb keratinase gene (ker B) was isolated from strain L-25 by a polymerase chain reaction (PCR) using L-25 genomic DNA as templates. Sequencing results reveal that ker B shares great sequence identity with a previously published keratinase gene of B. licheniformis PWD-1 (ker A). Only two amino acids differences were found in the deduced amino acid sequence between the keratinases from L-25 and PWD-1. However several nucleotide changes were found upstream of the putative promoter region. Protease inhibition studies indicated that neutral protease activity accounted for approximate 25 to 30% of total extracellular proteolytic activity produced by strain L-25 in the feather medium. In contrast, no measurable neutral protease activity was produced by strain PWD-1 in the feather medium. When glucose (1%), a common catabolic repressor, was added into the feather medium, L-25 was still able to grow and produce keratinase. Strain PWD-1 produced no neutral protease activity and its growth was severely inhibited in the feather medium containing glucose. L-25 produced an enhanced level of keratinase in the feather medium in comparison with PWD-1.

• 한국식품영양과학회지
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• 제19권5호
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• pp.434-442
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• 1990

Aspergillus sp. CC-29 ws selected for its strong protease activity among various stains of molds found in soil. It was found that the production of alkaline protease reached to maximum when the wheat bran medium containing glucose as carbon source had been cultured for 4 days. Alkaline proteased was purified 36.10 fold from Aspergillus sp. CC-29 The purification procedures included ammonium sulfate fractunation gel filteration on Sepha-dex G-75 G-150 and DEAE-cellulose ion-exchange chromatography, The yield of the purified enzyme was 22.40% The purified enzyme was confirmed as a single band by the polyacryla-mide. When the purified enzyme was applied to SDS-PAGE the molecular weight was estima-ted 24000. The optimum pH for the enzyme activity was 9.0 and the optimum temperature was 4$0^{\circ}C$ The reaction of this enzyme followed typical Michaelis-Menten kinetics with the Km value of 2.10$\times$10-4M with the Vmax of 29.41 $\mu$g/min. The enzyme was reactively stable in alkalic condition and unstable by heat treatment. The activity of alkaline protease was increased by the addition of Ca2+ whereas it was inhibited by Hg2+ Zn2+ at concentration of 1$\times$10-3M.

• The Plant Pathology Journal
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• 제28권2호
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• pp.197-201
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• 2012

Potyviruses express their RNA genomes through the production of polyproteins that are processed in host cells by three virus-encoded proteases. Soybean plants produce large amounts of protease inhibitors during seed development and in response to wounding that could affect the activities of these proteases. The in vitro activities of two of the proteases of Soybean mosaic virus (SMV) and Tobacco vein mottling virus (TVMV) were compared in the rabbit reticulocyte lysate in vitro translation system using synthetic RNA transcripts. Transcripts produced from SMV and TVMV cDNAs that included the P1 and helper component-protease (HC-Pro) coding regions directed synthesis of protein products that were only partially processed. Unprocessed poly-proteins were not detected from transcripts that included all of the P1, HC-Pro, P3 and portions of the cylindrical inclusion protein coding regions of either virus. Addition of soybean trypsin inhibitor to in vitro translation reactions increased the accumulation of the unprocessed polyprotein from TVMV transcripts, but did not alter the patterns of proteins produced from SMV. These experiments suggest that SMV-and TVMV-encoded proteases are differentially sensitive to protease inhibitors.