• 제목/요약/키워드: S. griseus

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Heterologous Production of Streptokinase in Secretory Form in Streptomyces lividans and in Nonsecretory Form in Escherichia coli

  • Kim,, Mi-Ran;Choeng, Yong-Hoon;Chi, Won-Jae;Kang, Dae-Kyung;Hong, Soon-Kwang
    • Journal of Microbiology and Biotechnology
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    • 제20권1호
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    • pp.132-137
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    • 2010
  • The skc gene encoding streptokinase (SK) with a molecular mass of approximately 47.4 kDa was cloned from Streptococcus equisimilis ATCC 9542 and heterologously overexpressed in Streptomyces lividans TK24 and E. coli using various strong promoters. When the promoter for sprT [Streptomyces griseus trypsin (SGT)] was used in the host S. lividans TK24, a 47.4-kDa protein was detected along with a smaller hydrolyzed protein (44 kDa), suggesting that posttranslational hydrolysis had occurred as has been reported in other expression systems. The casein/plasminogen plate assay revealed that the plasmid construct containing the SGT signal peptide was superior to that containing the SK signal peptide in terms of SK production. Maximal production of SK was calculated to be about 0.25 unit/ml of culture broth, a value that was five times higher than that obtained with other expression systems using ermE and tipA promoters in the same host. When the skc gene was expressed in E. coli BL21(${\Delta}DE3$)pLys under the control of the T7 promoter, a relatively large amount of SK was expressed in soluble form without hydrolysis. SK activity in E. coli/pET28a-$T7_pSK_m$ was more than 2 units/ml of culture broth, even though about half of the expressed protein formed an inactive inclusion body.

패스틴®첨가가 단백질 분해율과 반추위 발효 및 영양소 소화율에 미치는 영향 (Effects of Passtein® Supplements on Protein Degradability, Ruminal Fermentation and Nutrient Digestibility)

  • 최유지;최낙진;박성호;송재용;엄재상;고종열;하종규
    • Journal of Animal Science and Technology
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    • 제44권5호
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    • pp.549-560
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    • 2002
  • 본 시험은 패스틴$^{(R)}$ 을 첨가하였을 때, in vitro 상에서 단백질 fraction과 분해율에 미치는 영향과, in vivo 상에서 반추위 성상, 미생물 군집, 암모니아태 질소 농도 및 영양소 소화율에 미치는 영향을 구명하고자 실시하였다. In vitro 실험에서는 1mm로 분쇄된 대두박을 기질로 하여 패스틴$^{(R)}$ ((주)은진인터내셔날)을 첨가하여 borate-phosphate buffer와 중성세제에서의 조단백질 분해율을 측정하였으며, exogenous enzyme (Streptomyces griseus 유래 protease)를 이용하여 39$^{\circ}C$에서 0, 2, 4, 8, 12, 48 시간동안 배양 후 조단백질 분해율을 측정하였다. 반추위 발효성상과 영양소 소화율은 반추위 fistula가 부착된 평균체중 300kg의 홀스타인 수소 4두를 이용하여 무첨가구, 패스틴$^{(R)}$ 첨가구의 두개 처리구에 2마리씩 4마리를 배치하여 측정하였다. Buffer Soluble Protein fraction은 패스틴$^{(R)}$ 첨가 수준별로 차이가 없었으나, 무첨가구에 비해 패스틴$^{(R)}$ 첨가구에서 감소하는 경향을 보였다. 단백질 분해율은 배양 0 시간대에서 4시간대까지는 처리구간 유의성이 없었지만, 12 h과 48 h에서는 패스틴$^{(R)}$ 첨가로 시험구에서 감소되었다. 용해 단백질 분해율 ‘a’는 패스틴$^{(R)}$ 시험구에서 경미하게 높은 수치를 나타내었지만, 소화 가능한 단백질 분해율 ‘a+b’는 패스틴$^{(R)}$ 시험구에서 낮은 경향을 보였다. 패스틴$^{(R)}$ 첨가로 pH와 $NH_3$- N 농도는 증가하는 경향이었으며 휘발성지방산, 미생물 수 및 enzyme activity는 감소하였고 영양소 소화율은 높았으나 유의적인 차이는 없었다.

Ruminal Protein Degradation Characteristics of Cell Mass from Lysine Production

  • Seo, S.;Kim, H.J.;Lee, S.Y.;Ha, Jong K.
    • Asian-Australasian Journal of Animal Sciences
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    • 제21권3호
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    • pp.364-370
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    • 2008
  • Chemical analysis and in vitro studies were conducted to investigate the nutritive value for ruminants of cell mass from lysine production (CMLP) which is a by-product of the lysine manufacturing process. Proximate analysis, protein fractionation, and in vitro protein degradation using protease from Streptomyces griseus and strained ruminal fluid were carried out to estimate ruminal protein degradability of CMLP with two reference feedstuffs-soybean meal (SBM) and fish meal (FM). Amino acid composition and pepsin-HCl degradability were also determined to evaluate postruminal availability. CMLP contained 67.8% crude protein with a major portion being soluble form (45.4% CP) which was composed of mainly ammonium nitrogen (81.8% soluble CP). The amount of nucleic acids was low (1.15% DM). The total amount of amino acids contained in CMLP was 40.60% DM, which was lower than SBM (47.69% DM) or FM (54.08% DM). CMLP was composed of mainly fraction A and fraction B2, while the protein fraction in SBM was mostly B2 and FM contained high proportions of B2 and B3 fractions. The proportion of B3 fraction, slowly degradable protein, in CP was the highest in fish meal (23.34%), followed by CMLP (7.68%) and SBM (1.46%). CMLP was degraded up to 51.40% at 18 h of incubation with Streptomyces protease, which was low compared to FM (55.23%) and SBM (83.01%). This may be due to the insoluble portion of CMLP protein being hardly degradable by the protease. The in vitro fermentation by strained ruminal fluid showed that the amount of soluble fraction was larger in CMLP (40.6%) than in SBM (17.8%). However, because the degradation rate constant of the potentially degradable fraction of CMLP (2.0%/h) was lower than that of SBM (5.8%/h), the effective ruminal protein degradability of CMLP (46.95%) was slightly lower than SBM (53.77%). Unavailable fraction in the rumen was higher in CMLP (34.0%) compared to SBM (8.8%). In vitro CP degradability of CMLP by pepsin was 80.37%, which was lower than SBM (94.42%) and FM (89.04%). The evaluation of protein degradability using different approaches indicated that soluble protein in CMLP may supply a large amount of ammonia in the rumen while insoluble protein can be by-passed from microbial attacks due to its low degradability. The results from this study suggest that CMLP can be used as a protein supplement to ruminants for supplying both non-protein nitrogen to rumen microbes and rumen undegradable protein to the host animal.

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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    • 제13권10호
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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).

2,2-Methylene bis (3,4,6-trichloroacetoxy benzene) 유도체에 관한 연구 제 1보 합성 및 항균성 (Studies on Derivatives of 2,2′-Methylene bis (3,4,6-trichloroacetoxy benzene) Part 1. Synthesis and Antimicrobial Sensitivity)

  • 유주현;김종호;사혜순;윤혜정;양융;김유삼
    • 한국미생물·생명공학회지
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    • 제1권1호
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    • pp.51-57
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    • 1973
  • (1) 2,2'-Methylene bis (3,4,6-trichloroacetoxy benzene)을 모체화합물로 하여 Mannich반응에 의해 11종의 새로운 화합물을 합성했다. (2) 합성화합물중 R위치에 (구조식 들어감) 기를 가진 유도체가 효모보다 세균에 대해 비교적 강한 항균력을 나타냈다.

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사료 내 조농비율에 따른 생균제의 첨가가 흑염소의 사료섭취량, 영양소 소화율 및 질소축적에 미치는 영향 (Effects of Dietary Probiotics on Feed Intakes, Nutrient Digestibility and Nitrogen Retention in Korean Black Goats Fed Two Diets Differing in Forage to Concentrate Ratios)

  • 황보순;조익환;송기준;이성훈
    • 한국유기농업학회지
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    • 제15권2호
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    • pp.195-205
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    • 2007
  • 본 연구는 흑염소의 보다 효율적인 사양관리 체계의 확립에 기여하기 위하여, 조사료와 농후사료의 비율을 각각 70:30과 30:70으로 달리 하여,이들 배합비율에 생균제를 첨가하였을 때, 흑염소의 사료 섭취량, 영양소 소화율 및 질소 축적율에 미치는 영향을 조사하기 위하여 실시하였다. 본 연구에 사용된 생균제는 Lactobacillus case, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae 및 Streptomyces griseus의 균종을 함유하는 혼합된 형태였고, 사료 내 첨가비율은 0.2%이었다. 12두의 흑염소(male)를 네 처리구로 나누어 처리구당 3두씩 완전 임의 배치하여 개별 대사케이지에 수용하였고, 실험기간은 21일간 지속되었다. 결과를 살펴보면 1일 두당 건물 섭취량은 조농비율 및 생균제의 첨가에 의하여 영향을 받지 않았다. 가소화 건물 섭취량은 사료 내 조사료 비율이 증가함에 따라 유의하게 감소하였으며(p<0.05), 생균제 첨가에 의한 효과는 나타나지 않았다. 대사체중당 건물 섭취량과 체중에 대한 건물 섭취비율은 건물 섭취량과 유사한 양상을 나타내어 조농비율 및 생균제 첨가에 의한 효과는 나타나지 않았고, 이에 따라 처리군 간에 유의한 차이가 나타나지 않았다. 영양소 소화율은 조사료 비율이 낮을수록 유의하게 높은 소화율을 나타내었고(p<0.05), 생균제 첨가에 의한 효과는 나타나지 않았다. 뇨를 통한 질소 배설은 조사료 비율이 낮을수록 유의하게 감소하였으나, 생균제 첨가 효과는 두 사료에서 공히 유의한 차이가 나타나지 않았다. 질소 축적은 조사료 비율이 증가함에 따라 감소하였고, 두 사료 내 생균제의 첨가는 질소 축적을 다소 증가시키는 경향을 나타내었다. 이상의 결과로부터, 사료 내 조농비율이 다른 두 사료에 생균제의 급여는 사료 섭취량, 영양소 소화율 및 질소 축적에 유의한 영향을 미치지 않았고, 흑염소에 대한 이들 변수는 생균제에 의한 결과라기보다는 조농비율에 의하여 지배적으로 나타나는 것으로 나타났다.

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