• 제목/요약/키워드: Production and Purification

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Molecular cloning, purification, expression, and characterization of β-1, 4-endoglucanase gene (Cel5A) from Eubacterium cellulosolvens sp. isolated from Holstein steers' rumen

  • Park, Tansol;Seo, Seongwon;Shin, Teaksoon;Cho, Byung-Wook;Cho, Seongkeun;Kim, Byeongwoo;Lee, Seyoung;Ha, Jong K.;Seo, Jakyeom
    • Asian-Australasian Journal of Animal Sciences
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    • 제31권4호
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    • pp.607-615
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    • 2018
  • Objective: This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated microorganism. Methods: To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens) Ce2 (Accession number: AB163733). The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21. Results: The maximum activity of recombinant Cel5A (rCel5A) was observed at $50^{\circ}C$ and pH 4.0. The enzyme was constant at the temperature range of $20^{\circ}C$ to $40^{\circ}C$ but also, at the pH range of 3 to 9. The metal ions including $Ca^{2+}$, $K^+$, $Ni^{2+}$,$Mg^{2+}$, and $Fe^{2+}$ increased the endoglucanase activity but the addition of $Mn^{2+}$, $Cu^{2+}$, and $Zn^{2+}$ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and $45.66{\mu}mol/min/mg$. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was $96.69(s^{-1})$ and 6.88 (mL/mg/s), respectively. Conclusion: Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.

Effect of Amino Acids and Organic Nitrogen Sources on Cyclosporin A Fermentation by Tolypocladium inflatum (Tolypocladium inflatum을 이용한 Cyclosporin A 발효에서 아미노산과 유기질소원의 영향)

  • Kim, Jeong-Keun;Lee, Byung-Kyu;Chang, Seog-Won;Park, Yong-Deok;Rho, Yong-Taek
    • Microbiology and Biotechnology Letters
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    • 제37권2호
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    • pp.140-146
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    • 2009
  • Cyclosporin, an immunosuppressant, is a representative group of biologically active secondary metabolites produced by the fungus Tolypocladium inflatum. The amount and ratio of cyclosporin derivatives in the culture broth are an important factors for the production of cyclosporin A and the purification in the industrial process. Therefore, we studied the effect of amino acids and complex organic nitrogen sources using Tolypocladium inflatum mutants on the productivity of cyclosporin A and the ratio of cyclosporin derivatives. Overproducing mutant YHC-004 having seven times higher productivity than mother strain's could be obtained through the artificial mutation by UV irradiation. The concentration and kind of organic nitrogens and amino acids shows the profound effect on the productivity of cyclosporin A and ratio of cyclosporin derivatives. As a result, it was possible to raise the productivity and the ratio of cyclosporin A up to 3,430 mg/L and 93% respectively, but on the other hand the other cyclosporin derivatives decreased less than 2% in the culture broth.

Production of Red-spotted Grouper Nervous Necrosis Virus (RGNNV) Capsid Protein Using Saccharomyces cerevisiae Surface Display (Saccharomyces cerevisiae 표면 발현을 이용한 붉바리 신경괴사 바이러스 외피단백질의 생산)

  • Park, Mirye;Suh, Sung-Suk;Hwang, Jinik;Kim, Donggiun;Park, Jongbum;Chung, Young-Jae;Lee, Taek-Kyun
    • Journal of Life Science
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    • 제24권9호
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    • pp.995-1000
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    • 2014
  • The studies of marine viruses in terms of viral isolation and detection have been limited due to the high mutation rate and genetic diversity of marine viruses. Of the modern methods currently used to detect marine viruses, serological methods based on enzyme-linked immunosorbent assay (ELISA) are the most common. They depend largely on the quality of the antibodies and on highly purified suitable antigens. Recently, a new experimental system for using viral capsid protein as an antigen has been developed using the yeast surface display (YSD) technique. In the present study, the capsid protein gene of the red-spotted grouper nervous necrosis virus (RGNNV) was expressed and purified via YSD and HA-tagging systems, respectively. Two regions of the RGNNV capsid protein gene, RGNNV1 and RGNNV2, were individually synthesized and subcloned into a yeast expression vector, pCTCON. The expressions of each RGNNV capsid protein in the Saccharomyces cerevisiae strain EBY100 were indirectly detected by flow cytometry with fluorescently labeled antibodies, while recognizing the C-terminal c-myc tags encoded by the display vector. The expressed RGNNV capsid proteins were isolated from the yeast surface through the cleavage of the disulfide bond between the Aga1 and Aga2 proteins after ${\beta}$-mercaptoethanol treatment, and they were directly detected by Western blot using anti-HA antibody. These results indicated that YSD and HA-tagging systems could be applicable to the expressions and purification of recombinant RGNNV capsid proteins.

Energy Big Data Pre-processing System for Energy New Industries (에너지신산업을 위한 에너지 빅데이터 전처리 시스템)

  • Yang, Soo-Young;Kim, Yo-Han;Kim, Sang-Hyun;Kim, Won-Jung
    • The Journal of the Korea institute of electronic communication sciences
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    • 제16권5호
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    • pp.851-858
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    • 2021
  • Due to the increase in renewable energy and distributed resources, not only traditional data but also various energy-related data are being generated in the new energy industry. In other words, there are various renewable energy facilities and power generation data, system operation data, metering and rate-related data, as well as weather and energy efficiency data necessary for new services and analysis. Energy big data processing technology can systematically analyze and diagnose data generated in the first half of the power production and consumption infrastructure, including distributed resources, systems, and AMI. Through this, it will be a technology that supports the creation of new businesses in convergence between the ICT industry and the energy industry. To this end, research on the data analysis system, such as itemized characteristic analysis of the collected data, correlation sampling, categorization of each feature, and element definition, is needed. In addition, research on data purification technology for data loss and abnormal state processing should be conducted. In addition, it is necessary to develop and structure NIFI, Spark, and HDFS systems so that energy data can be stored and managed in real time. In this study, the overall energy data processing technology and system for various power transactions as described above were proposed.

Trend Analysis for River Water Quality Change Before and After the Prohibition of Ocean Dumping of Livestock Manure (가축분뇨 해양투기 금지 전후 하천 수질 변동 경향성 분석)

  • Choi, Hyeon-Mi;Cho, Yong-Chul;Lee, Bomi;Ryu, In-Gu;Kim, Sang-hun;Shin, Dongseok;Yu, Soonju
    • Journal of Korean Society on Water Environment
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    • 제37권2호
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    • pp.75-91
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    • 2021
  • Ocean dumping manure was prohibited in Korea until the end of 2012. Undumped manure was treated by resourcification, purification, and public process in a facility. But poor storage of untreated manure and overproduced compost can pollute river water. In this study, linear regression, Mann-Kendall, Seasonal Mann-Kendall, LOWESS trend, and Spearman's correlation analysis were conducted for investigating the changes in the water quality of Bukhan and Imjin river before and after the prohibition of ocean dumping. In 2017, the Imjin Riv er basin had more than 5.7 times liv estock breeding heads than the Bukhan Riv er basin and more than 3.5 times livestock manure. The areas of land, farmland, and nutrient balance were also higher in the Imjin River basin. Since the two basins have different environments, it is expected that the characteristics of changes in water quality due to the increase in livestock manure generation and treatment methods will be different when comparing the water quality trends before and after the ban. The result of the trend analysis revealed a decrease in EC before the ban but an increase afterward. T-N and BOD of Imjin river increased slightly before the ban but demonstrated great enhancement thereafter. Other sites and T-P exhibited no trend. The correlation analysis result showed that the increase in the number of livestock rearing was correlated with the increase in T-P of Bukhan River and BOD of Imjin River. The increase in livestock manure production was not correlated with the Imjin River basin, while a correlation was observed with the Bukhan River with an increase in T-N and EC and the decrease in T-P.

Effect of Solidago altissima L. Extract on Forage Crop Germination

  • Ho-Jun Gam;Yosep Kang;Eun-Jung Park;Ki-Yong Kim;Sang-Mo Kang;In-Jung Lee
    • Proceedings of the Korean Society of Crop Science Conference
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    • 한국작물학회 2022년도 추계학술대회
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    • pp.114-114
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    • 2022
  • There are 28 families and 166 species of exotic weeds on agricultural land and among these, 23 families and 80 species of exotic weeds occur on pastures. Among them, the Solidago altissima is a perennial weed belonging to the asteraceae family and it is an exotic weed that spreads to the surrounding area using methods such as high seed production, vegetative propagation using underground rhizomes and allelochemical. Accordingly, in 2009, the Ministry of Environment designated it as an ecosystem-disrupting species. This study was conducted to obtain basic data about the effects of S.altissima derived allelochemicals on forage crops. The root of S.altissima was separated, dried in the shade and then pulverized to prepare an root powder. Powder was repeatedly extracted with methanol for 3 days and concentrated under reduced pressure to obtain an root methanol extract. Dissolve the extract in distilled water, dispense it in a separate-funnel and proceed with liquid-liquid extraction by adding equal amounts of n-haxane (Hex), chloroform (CHCI3), ethyl acetate (EtoAC), and butanol (BuOH) in order of increasing polarity. A seed-bioassay was performed using fractions for each solvent, followed by separation and purification by silica gel column chromatography. As a result of the fraction germination test for each solvent, the IC50 values using the fresh weight of each fraction were 898.3 mg L-1, 676.3 mg L-1, 1160 mg L-1 and 1360 mg L-1. CA, CB, and CC fractions were obtained through primary silica gel column chromatography that used CHCI3 fraction. As a result of seed-bioassay using each fraction, the IC50 values for the fresh weight of each fraction was 537.3 mg L-1, 1280 mg L-1 and 1947 mg L-1. Based on this, 5 fractions were obtained as a result of secondary silica gel column chromatography using the CA fraction. A seed-bioassay was performed, as a result, the lowest IC50 value was calculated as 226.7 mg L-1 in the CAE fraction. Based on this, the fraction was analyzed by GC-MS. The results of this study can be used as basic research data on the effects of weeds on forage crops and allelochemicals secreted from S. altissima.

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Melamine Concentration in Han River Basin and the in GAC Column Breakthrough Curve Model (한강수계 내 Melamine 농도검출과 GAC처리에서의 파과모델링)

  • Lee, Sang-Jung;Lee, Jai-Yeop;Han, Ihn-Sup
    • Journal of Korean Society of Environmental Engineers
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    • 제33권10호
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    • pp.717-722
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    • 2011
  • Currently, melamine is being used variously in our lives such as resins, flame retardants, adhesive, laminate etc. And understandably sewer of stream of wastewater containing Melamine has also increased. GHS (Globally Harmonized System of Classification and Labelling of Chemicals) of EU safety guidelines says that it can cause cancer. Still, study on toxicity of Melamine is going on. In this research, melamine contamination level of the Han River and River Basin was analyzed by HPLC/UV. And the experiments of GAC adsorption were conducted and the model was studied. We collected the 3 same samples at the suburbs of Paldang Dam located in the relative upstream in Han River and Ttukseom amusement park, the downstream region and collected samples equally at the Hongreung stream, Wangsuk stream, Cheonggye stream among streams flowing into Han River and then measured Melamine concentration after purification. As a result, melamine was not detected at the suburbs of Paldang Dam and it was detected at Ttukseom amusement park, the downstream of it, in the concentration of $0.312{\mu}g/L$. The Wangsuk stream with $0.578{\mu}g/L$ highest Cheonggye stream and Hongreung stream was detected with each $0.197{\mu}g/L$ and $0.325{\mu}g/L$. Although the concentration was low in general, melamine detection could be checked at most point. In 1970, the world capacity of Melamine was estimated at 200,000 ton, with current production estimated to be 1,400,000 ton. Melamine of Han River and rivers flowing into Han River is present at low concentration but pollution will increase in the future due to increase of use. Depending on the size of activated carbon in the experiment were slightly different. But the breakthrough model is almost identical.

Self-purification Capacity of Eutrophic Buk Bay by DO mass Balance (부영양화된 북만의 용존산소 수지에 의한 자정능력)

  • CHOI Woo-Jeung;NA Gui-Hwan;CHUN Young-Yell;PARK Chung-Kil
    • Korean Journal of Fisheries and Aquatic Sciences
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    • 제24권1호
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    • pp.21-30
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    • 1991
  • In Summer, oxygen-deficient water masses were developed extensively in the closed eutrophic bays such as Chinhae Bay which results in mass mortality of marine organisms and severe decrease the production of the bay every year. Under the circumstances, this study was performed to investigate the oxygen depletion relating to eutrophication, and also to evaluate self-purfication capacity of Buk Bay by dissolved oxygen mass balance in 1988. The mean concentration of total inorganic nitrogen, phosphate phosphorus and chlorophyll-a were $11.06{\mu}g-at/l,\;0.80{\mu}g-at/l\;and\;1.11mg/m^3$ respectively, which were over eutrophication criteria. Oxygen-deficient water mass was formed in July with the minimum concentration of 2.08ml/l(mean) at the bottom of all stations and recovered slowly in August. The decay and reaeration coefficient calculated from dissloved oxygen sag curve were 0.222/day and 0.018ml/l/day, respectively. To maintain above 4ml/l of oxygen to prevent oxygen-deficient water mass, it is recommendable to supply as much as 0.856ml/l/day of dissolved oxygen or should be reduced the same mass loading of BOD from watershed by the construction of wastewater treatment plant.

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The properties of algal degradation and gas emission by thermophilic oxic process (고온호기발효장치를 이용한 조류 분해 및 가스 발생특성)

  • Kang, Changmin
    • Journal of the Korea Organic Resources Recycling Association
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    • 제7권2호
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    • pp.57-64
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    • 1999
  • The purpose of this study is to establish effective conditions for controlling $CH_4$, $N_2O$ emission from organic Waste / wastewater treatment processes. Continuous and batch experiments were conducted to treat the micro algae from polluted and eutrophicated lakes through the thermophilic oxic process. The microalgae used were mainly Microcystis sp.(collected from eutrophic lake) and Chlorella sp. (cultured in laboratory) Wasted cooking oil was added by aid-heating source. Physico-chemical components of sludges and microalgae were analyzed. In batch experiments, air supply was changed from 50ml/min to 150ml/min. The temperature. water content and drained water were affected by the air flow rate at initial stage. However, there was almost no influence of air flow rate on them in middle and last stages. At air flow rate of 100ml/min, the degradation rate of organic material was higher than that at other air flow rates. $CO_2$ concentration in exhaust was proportional to the strength of aeration, especially at initial stage when degradation was active. $CH_4$ with low concentration was detected only at starting stage when air diffusion was not enough. $N_2O$ production was not affected by variation of air supply. In continuous experiments no matter what the dewatering methods (with PAC and without PAC) and media (wood chip and reed chip) were changed, $N_2O$ was almost not affected by variation of injected air. Result showed that the reed chips using for lake purification could be used as media for thermophilic oxic process in lake and marshes area. $CO_2$ concentration was not so much affected by the change of dewatering methods and media types. $CH_4$ was not detected in the experimental period. So it can be shown that the thermophilic oxic process had been well operated in wide handling conditions regardless of media and dewatering methods.

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Characteristics of Microbial Pretense far Application to Abolished Protein Resource (폐단백자원에 이용하기 위한 미생물 Protease의 특성)

  • Chun, Sung-Sook;Cho, Young-Je;Sung, Tae-Soo;Son, Jun-Ho;Choi, Cheong
    • Applied Biological Chemistry
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    • 제41권1호
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    • pp.6-12
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    • 1998
  • To extract insoluble proteins and to improve functional properties of abolished proteins, a protease producing Aspergillus sp. MS-18 was isolated from soil. The enzyme was purified and its enzymological characteristics were investigated. It was found that production of protease reached to the maximum when the wheat brae medium containing, 3% arabinose, 0.5% polypepton, 0.1% $(NH_4)_2SO_4$ and 0.2% magnesium chloride was cultured for 3 days. Protease was purified 16.9 folds after ion exchange chromatography and gel filtration and the specific activity was 340.4 unit/mg. Purified enzyme was confirmed as a single band by the polyacrylamide gel electrophoresis. The molecular weight of protease was estimated to be 30,000. Crystalization form of purified protease was a stick shape with rounding edges. The optimum pH and temperature for the protease activity were 9.0 and $60^{\circ}C$, respectively. The enzyme was stable in pH 7.0-12.0 at $50^{\circ}C$. The activity of purified enzyme was inhibited by $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$ and $Pb^{2+}$, whereas it was activited by $Na^+$, $Mg^{2+}$ and $Mn^{2+}$. The activity of the protease was inhibited by the treatment with ethylenediaminetetraacetic acid and phenylmethane sulfonyl fluoride. The result suggests that the purified enzyme is a serine protease with metal ion at active site. Km and Vmax of purified protease were $29.33\;{\mu}mole/L$ and $5.13\;{\mu}g/min$, respectively.

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