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Dihydroxy-acid Dehydratase Involved in the Biosynthesis of the Branched-Chain Amino acids, Isoleucine and Valine, from the archaeon Sulfolobus solfataricus

  • Kim, Seong-Hun;Lee, Sun-Bok
    • 한국생물공학회:학술대회논문집
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    • 2005.04a
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    • pp.327-333
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    • 2005
  • Dihydroxy-acid dehydratase (DHAD, 2,3-dihydroxy-acid hydrolyase, EC 4.2.1.9) is one of the key enzymes involved in the biosynthetic pathway of the branched chain amino acid isoleucine and valine. Although the enzyme have been purified and characterized in various mesophiles including bacteria and eukarya, the biochemical properties of DHAD has bee not yet reported from hyperthermophilic archaea. In this study, we cloned, expressed, and purified a DHAD homologue from the thermoacidophilic archaeon Sulfolobus solfataricus P2, which grows optimally at $80\;^{\circ}C$ and pH 3, in E. coli. Characterization of the recombinant S. solfataricus DHAD (rSso_DHAD) revealed that it is the dimeric protein with a subunit molecular weight of 64,000 Da in native structure. rDHAD showed the highest activity toward 2,3-dihydroxyisovaleric acid among 17 aldonic acid substrates Interestingly, this enzyme also displayed 50 % activities toward some pentonic acids and hexonic acids when compared with the activity of this enzyme to the natural substrate. Moreover, rSso_DHAD indicated relatively higher activity toward D-gluconate than any other hexonic acids tested in substrates. $K_m$ and $V_{max}$ values of rSso_DHAD were calculated as $0.54\;{\pm}\;0.04\;mM$ toward 2,3dihydroxyisovalerate and $2.42\;{\pm}\;0.19\;mM$ toward D-gluconate, and as $21.6\;{\pm}\;0.4\;U/mg$ toward 2,3-dihydroxyisovalerate and $13.8\;{\pm}\;0.4\;U/mg$ toward D-gluconate, respectively. In the study for biochemical properties, the enzyme shows maximal activity between $70^{\circ}C$ and $80^{\circ}C$, and the pH range of pH 7.5 to 8.5. The half life time at $80^{\circ}C$ was 30 min. A divalent metal ion, $Mn^{2+}$, was only powerful activators, whereas other metal ions made the enzyme activity reduced. $Hg^{2+}$, organic mercury, and EDTA also strongly inhibited enzyme activities. Particularly, the rSso_DHAD activity was very stable under aerobic condition although the counterparts reported from mesophiles had been deactivated by oxygen.

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Studies on Physiological Reactions of Soybean Cultivars Tolerant and Susceptible to Rust (Phskopsora pachyrhizi Syd.) (대두 녹병에 대한 내병성 및 이병성 품종의 생리적 반응)

  • 신두철
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.31 no.4
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    • pp.440-446
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    • 1986
  • The physiological reaction of two soybean (Glycine max (L.) Merrill) cultivars tolerant and susceptible to rust (Phakopsora pachyrhizi Syd.) was studied at the AVRDC in Taiwan, ROC. The rust epidemic on the susceptible cultivar began earlier and progressed more rapidly than on the tolerant cultivar. The defoliation by rust infection increased rapidly after the latter half of pod filling. The reduction of LA! by rust in the susceptible cultivar occurred earlier than in the tolerant cultivar. The differences in the chlorophyll content between the rust-free and rust-infected plants was 2.04% in the tolerant, and 16.43% in the susceptible cultivar. The shoot dry weight increased at each growth stage in the fungicide protected plots, but decreased in the non-fungicide protected plots after the R6 growth stage onward and the tendency to decrease was more severe in the susceptible than in the tolerant cultivar. The pod and seed dry weight of the suscep-tible cultivar in the fungicide-protected plot increased dramatically from the R6 growth stage, but in the non-fungicide plot, there was almost no increase in pod and seed dry weight from R6 growth stage, due to rust. The number of empty pods and imperfect grains were increased by rust infection, but the protein content was not afftected. There were reductions of oil content, seed length, seed width, seed thickness, pod thickness, number of pods and seeds, 100 seed weight, and yield due to rust infection. The yield losses by rust infection were 22.3%in the tolerant and 68.7% in the susceptible cultivar.

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Cloning, Expression, Purification, and Properties of an Endoglucanase Gene (Glycosyl Hydrolase Family 12) from Aspergillus niger VTCC-F021 in Pichia pastoris

  • Pham, Thi Hoa;Quyen, Dinh Thi;Nghiem, Ngoc Minh;Vu, Thu Doan
    • Journal of Microbiology and Biotechnology
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    • v.21 no.10
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    • pp.1012-1020
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    • 2011
  • A gene coding for an endoglucanase (EglA), of the glycosyl hydrolase family 12 and derived from Aspergillus niger VTCC-F021, was cloned and sequenced. The cDNA sequence, 717 bp, and its putative endoglucanase, a 238 aa protein with a predicted molecular mass of 26 kDa and a pI of 4.35, exhibited 98.3-98.7% and 98.3-98.6% identities, respectively, with cDNA sequences and their corresponding endoglucanases from Aspergillus niger strains from the GenBank. The cDNA was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 1.59 U/ml culture supernatant, after 72 h of growth in a YP medium induced with 1% (v/v) of methanol. The molecular mass of the purified EglA, determined by SDS-PAGE, was 33 kDa, with a specific activity of 100.16 and 19.91 U/mg toward 1% (w/v) of ${\beta}$-glucan and CMC, respectively. Optimal enzymatic activity was noted at a temperature of $55^{\circ}C$ and a pH of 5. The recombinant EglA (rEglA) was stable over a temperature range of $30-37^{\circ}C$ and at pH range of 3.5-4.5. Metal ions, detergents, and solvents tested indicated a slightly inhibitory effect on rEglA activity. Kinetic constants ($K_m$, $V_{max}$, $k_{cat}$, and $k_{cat}/K_m$) determined for rEglA with ${\beta}$-glucan as a substrate were 4.04 mg/ml, 102.04 U/mg, 2,040.82 $min^{-1}$, and 505.05, whereas they were 10.17 mg/ml, 28.99 U/mg, 571.71 $min^{-1}$, and 57.01 with CMC as a substrate, respectively. The results thus indicate that the rEglA obtained in this study is highly specific toward ${\beta}$-glucan. The biochemical properties of rEglA make it highly valuable for downstream biotechnological applications, including potential use as a feed enzyme.

Commercial Production of Seed Garlic by Tissue Culture Technique (조잭배양에 의한 씨마늘의 상업적 생산)

  • Nam, Sang-Il;Park, Ju-Hyun;Choi, Jong-In;Kwon, Ki-Seok;Uhm, Jeong-Sik
    • Proceedings of the Korean Society of Plant Biotechnology Conference
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    • 2002.04b
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    • pp.33-40
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    • 2002
  • We, Tong Yang Moolsan Co. Ltd. (TYM) set up the mass-production system for virus-free seed garlic via tissue culture technique. TYM's tissue culture technique is called as 'Multiple shoot propagation technique'. This technique can lead mass propagation of genetically homogeneous seed garlic in a short period because of its highly proliferation ,ate of in vitro shoots $(15^{10}/year)$. TYM researchers applied the technique to some selected garlic cultivars with superior characteristics and carried out Held test of productivity in the inside and outside of the country for several years. According to the yearly results of Held test with virus-free seed garlic, we ascertained that virus-free seed garlic can produce the highly yield increase (max. above 50%) and also can enhance the product quality. Consequently, we estimated that TYM's seed garlic will contribute to farmers with increase of income and can elevate the national position of garlic market in the world for its competitive power of technical and production cost.

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Optimum Monitoring Parameters for the Safety of Mechanical Seals (미캐니컬 씰의 안전운용 감시를 위한 최적 계측인자)

  • Soon-Jae Lim;Man-Yong Choi
    • Journal of the Korean Society of Safety
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    • v.12 no.4
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    • pp.214-219
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    • 1997
  • The mechanical seals, which are installed in rotating machines like pump and compressor, are generally used as sealing devices in the many fields of industries. The failure of mechanical seals such as leakage, crack, breakage, fast and severe wear, excessive torque, and squeaking results in big problems. To identify abnormal phenomena on mechanical seals and to propose the proper monitoring parameter for the failure of mechanical seals, sliding wear experiments were conducted. Acoustic emission, torque, and temperature were measured during experiments. Optical microstructure was observed for the wear processing after every 10 minute sliding at rotation speed of 1750 rpm and scanning electron microscopy was also observed. Except for the initial part of every experiment, the variation of acoustic emission was well coincided with torque variation during the experiments. This study concludes that acoustic emission and torque are proper monitoring parameters for the failure of mechanical seals. The intensity of acoustic emission signals is measured in root mean square voltage. Temperature of sealing face will be used as a parallel parameter for increasing the reliability of monitoring system.

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Immunohistochemical Profile of Breast Cancer Patients at a Tertiary Care Hospital in New Delhi, India

  • Doval, Dinesh Chandra;Sharma, Anila;Sinha, Rupal;Kumar, Kapil;Dewan, Ajay Kumar;Chaturvedi, Harit;Batra, Ullas;Talwar, Vineet;Gupta, Sunil Kumar;Singh, Shailendra;Bhole, Vidula;Mehta, Anurag
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.12
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    • pp.4959-4964
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    • 2015
  • Background: To assess the immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor-2 (HER2) neu receptor in breast cancer and their associations with various clinicopathological characteristics. Materials and Methods: This is a retrospective analysis of women who presented with primary, unilateral breast cancer in the Department of Medical Oncology at Rajiv Gandhi Cancer Institute and Research Centre, Delhi, India during the period from January 2008 to December 2011. Data were retrieved from the medical records of the hospital including both early and locally advanced cancer cases. ER, PgR and HER2neu expression in these patients was assessed and triple negative patients were identified. Associations of triple negative and non-triple negative groups with clinicopathological characteristics were also evaluated. Results: A total of 1,284 women (mean age 52.1 years, 41.9% premenopausal) were included in the analysis. Hormone receptor positivity (ER and/or PgR) was seen in 63.4% patients, while 23.8% of tumors were triple negative. Only 23.0% were HER2 positive. Around 10.0% of tumors were both ER and HER2 positive. ER and PgR positivity was significantly associated with negative HER2 status (p-value <0.0001). Younger age, premenopausal status, higher tumor grade, lymph node negativity, advanced cancer stage, and type of tumor were strongly associated with triple negativity. Significantly, a smaller proportion of women had ductal carcinoma in situ in the triple negative group compared with the non-triple negative group (35.6% versus 60.8%, p-value<0.01). Conclusions: The present analysis is one of the largest studies from India. The majority of the Indian breast cancer patients seen in our hospital present with ER and PgR positive tumors. The triple negative patients tended to be younger, premenopausal, and were associated with higher tumor grades, negative lymph nodes status and lower frequency of ductal carcinoma in situ.

Myocardial Protection by Recombinant Soluble P-selectin Glyco-protein Ligand-1: Suppression of Neutrophil and Platelet Interaction Following Ischemia and Reperfusion

  • Ham, Sang-Soo;Jang, Yoon-Young;Song, Jin-Ho;Lee, Hyang-Mi;Kim, Kwang-Joon;Hong, Jun-Sik;Shin, Yong-Kyoo
    • The Korean Journal of Physiology and Pharmacology
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    • v.4 no.6
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    • pp.515-523
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    • 2000
  • Polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia/reperfusion (MI/R) injury. Moreover, platelets are also important blood cells that can aggravate myocardial ischemic injury. This study was designed to test the effects of PMNs and platelets separately and together in provoking cardiac dysfunction in isolated perfused rat hearts following ischemia and reperfusion. Additional control rat hearts were perfused with $75{\times}10^6$ PMNs, with $75{\times}10^6$ platelets, or with $75{\times}10^6\;PMNs+75{\times}10^6$ platelets over a five minute perfusion followed by a 75 min observation period. No significant reduction in coronary flow (CF), left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dt max) was observed at the end of the observation period in any non-ischemic group. Similarly, global ischemia (I) for 20 min followed by 45 minutes of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of blood cells. However, I/R hearts perfused with either PMNs or platelets alone exhibited decreases in these variables of $5{\sim}10%$ (p<0.05 from control). Furthermore, I/R hearts perfused with both PMNs and platelets exhibited decreases of 50 to 60% in all measurements of cardiac function (p<0.01). These dual cell perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity indicating a significant PMN infiltration, and enhanced P-selectin expression on the coronary microvascular endothelium. All cardiaodynamic effects as well as PMN accumulation and P-selectin expression were markedly attenuated by a recombinant soluble PSGL-1 which inhibits selectin mediated cell adhesion. These results provide evidence that platelets and PMNs act synergistically in provoking post-reperfusion cardiac dysfunction, and that this may be largely due to cell to cell interactions mediated by P-selectin. These results also demonstrate that a recombinant soluble PSGL-1 reduces myocardial reperfusion injury by platelet and PMNs interaction.

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Effect of Extract from Fermented Black Soybean (Glycine max var. Seoritae) on the Hair Damaged by Decolorization (서리태 발효추출물이 탈색을 통해 손상된 모발에 미치는 영향)

  • Jung, Heehoon;Shin, Min Kyu;Lee, Su Yel;Lee, Sang Rin;Kim, Moo Sung
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.43 no.2
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    • pp.149-155
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    • 2017
  • This study was carried out to investigate the hair protection effect of fermented black soybean extracts. The morphological characteristics, tensile strength and constitutional changes of the hair were analyzed and compared when the hair was chemically oxidized and then treated with fermented black soybean extract. As a result, treatment of oxidizing agent on virgin hair caused damage on the cuticle layer of the epidermis and decreased in tensile strength of hair from $14.32{\pm}0.83g/cm^2$ to $12.32{\pm}0.79g/cm^2$. FT-IR analysis showed the peaks at 1,077, 1,041, and $801cm^{-1}$ of the hair treated with oxidizing agent were increased compared to peak values of virgin hair, indicating that cystein in hair was decreased which is crucial to disulfide bond between keratin. On the other hand, when the damaged hair is treated with the fermented black soybean extract, cracks in the cuticle layer of the epidermis were filled, tensile strength was restored to $14.27{\pm}0.96g/cm^2$ and the ratio of oxidized cysteine in hair was decreased. These results suggest that the fermented black soybean extract is worthy of further investigation as a protective material for hair damaged by oxidizing agents.

A Study on the Distribution Characteristics of Nitrite Oxidizing Bacteria in Wastewater Nitrification Systems (폐수 질산화 시스템에서 아질산 산화 미생물의 분포 특성 연구)

  • Kim, Sun-Hee;Kim, Dong-Jin;Yoo, Ik-Keun;Cha, Gi-Cheol
    • Journal of Korean Society of Environmental Engineers
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    • v.28 no.10
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    • pp.1024-1030
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    • 2006
  • Genus Nitrospira and Nitrobacter species are the key nitrite-oxidizing bacteria(NOB) in nitrifying wastewater treatment plants. It has been hypothesized that genus Nitrospira are K-strategists(low $K_6$ value) that can exploit low amounts of nitrite more efficiently than Nitrobacter. In contrast, Nitrobacter species are r-strategists(high $V_{max}$) that can grow faster than Nitrospira. It has also been known that the availability of organic compounds and dissolved oxygen as well as nitrite affects the distribution of NOB. In this study, we determined the distribution and competition of NOB in wastewater nitrification systems where nitrite, organic compounds, and dissolved oxygen concentrations were compositively varied. For the purpose, several compounds of the laboratory-scale nitrificaiion bioreactor and full-scale $A_2O$ wastewater treatment plant and their distribution of NOB were analyzed and compared. The analysis showed that Nitrobacter was the dominant NOB in nitrification bioreactor where average nitrite was maintained at 5 mg-N/L with very low organic concentration in aerobic condition, whereas Nitrospira was the dominant NOB in full-scale $A_2O$ plant where nitrite was maintained very low and organic compounds were maintained relatively high in alternating aerobic-anoxic condition. The result indicates that nitrite concentration is more critical factor than organics and dissolved oxygen which determines the dominant NOB in nitrification system and it is confirmed that Nitrospira and Nitrobacter showed the characteristics of r-strategist and K-strategist, respectively.

Commercial Production of Seed Garlic by Tissue Culture Technique (조직배양에 의한 씨마늘의 상업적 생산)

  • Nam, Sang-Il;Park, Ju-Hyun;Choi, Jong-In;Kwon, Ki-Seok;Uhm, Jeong-Sik
    • Journal of Plant Biotechnology
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    • v.29 no.3
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    • pp.171-177
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    • 2002
  • We, Tong Yang Moolsan Co. Ltd. (TYM) set up the mass-production system for virus-free seed garlic via tissue culture technique. TYM's tissue culture technique is called as 'Multiple shoot propagation technique' This technique can lead mass propagation of genetically homogeneous seed garlic in a short period because of its highly proliferation rate of in vitro shoots (15/sup 10//year). TYM researchers applied the technique to some selected garlic cultivars with superior characteristics and carried out field test of productivity in the inside and outside of the country for several years. According to the yearly results of field test with virus-free seed garlic, we ascertained that virus-free seed garlic can produce the highly yield increase (max. above 50%) and also can enhance the product quality. Consequently, we estimated that TYM's seed garlic will contribute to farmers with increase of income and can elevate the national position of garlic market in the world for its competitive power of technical and production cost.