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Myriococcum albomyces 가 생산하는 Cellulase 에 관한 연구
정동효 한국농화학회 1971 Applied Biological Chemistry (Appl Biol Chem) Vol.14 No.1
As a study on the cellulase of Myriococcum albomyces the culture media for enzyme formation and properties of its crude preparation were investigated and the crude enzyme preparation was further fractionated. The results are summarized as follows: 1. Wheat bran solid culture produced stronger activities of cellulase than rice bran or defatted soy bean meal solid culture. 2. Shaking culture with wheat bran, rice bran or defatted soy bean meal produced higher cellulase activities than solid culture with the corresponding media. 3. The enzyme formation was higher at 45℃ than at 37℃ or 50℃ regardless of the kind of culture medium. 4. The formation of CMCase activity was more promoted by organic nitrogen source than inorganic nitrogen source. 5. The formation of cellulase activities were increased 1.5 to 3.0-fold by adding CMC, Avicel or cellulose powder as an inducer into 5% wheat bran basal medium. 6. Cellulase production using a tank culture procedure with addition of CMC or Avicel as an inducer was the highest at fifth day and thereafter decreased slightly. 7. The crude enzyme preparation showed pH optimum in 4.0 to 4.5, and pH stability in the range of 3.5 to 8.0. Optimum temperature for the activity was 65℃ which was higher than among other cellulases and it was stable at 60℃ for 120 minutes. 8. Dialyzed crude enzyme was activated by Ca^(++) and Mg^(++), but inhibited by Hg^(++), Cu^(++) and Ag^+. 9. Four different types of cellulase, i. e., fraction I, fraction II-a, fraction II-b, and fraction III were purified from the culture filtrate of Myriococcum albomyces through a sequence of ammonium sulfate fractionation, and elution chromatography on DEAE-Sephadex A-25, Amberlite CG-25 type 2 and hydroxyapatite columns. 10. These four cellulase fractions were showed to be homogenous by electrophoresis and ultracentrifugation and also gave a typical ultraviolet absorption spectrum of protein. 11. Four purified fraction showed different specificity toward substrates, fraction I has a stronger activity toward Avicel, cellulose powder, and gauze than that of other cellulase fractions. Fraction II-a had a powerful activity toward cellobiose but it was almost inactive agaisnt fibrous cellulose contrary to fraction I. On the contrary, the main component fraction II-b had a fairly higher activity on CMC and Avicel. Activity of fraction II-b toward cellobiose was about one-third of that of fraction II-a and activity on Avicel was lower than that of fraction I. Fraction III had a more powerful activity in decreasing viscosity of CMC. 12. Final hydrolysis products of fibrous cellulose by each fraction were cellobiose and glucose. Whereas oligosaccharides were predominant in the early stage of hydrolysis, prolonged reaction produced more glucose than cellobiose. Fraction I and fraction II-a acted synergically on Avicel. 13. Optimum pH for the activities of cellulase fraction I, fraction II-a, fraction II-b and fraction III were found to be 5.5, 5.0, 4.0 and 4.0∼4.5, respectively. These fractions were found to be stable in the range of pH 3.0∼7.5. 14. Optimum temperature for the activities of fraction I, fraction II-a, fraction II-b, and fraction III were 50℃, 55℃, 60℃ and 55℃, respectively. No less of activity was found by heating 120 minutes at 55℃ and fraction II-a was more stable than the others at 60℃. 15. Fraction I and fraction II-b were activated by Ca^(++) and Mg^(++) but inhibited by Hg^(++) and Ag^+.
정동효,정호권,박준희,심상국 中央大學校 遺傳工學硏究所 1988 遺傳工學硏究論集 Vol.1 No.1
본 연구는 lac'z gene의 promoter개발을 위하여 착수하였다. lac'z gene의 promoterⅠ과 Ⅱ를 효모의 염색체 Bam HI DNA 단편에서 발견하였다. PromoterⅠ의 크기의 2.5kb정도이고 β- galactosidase 활성은 124.6 U/㎎ protein이었으나 promoterⅡ의 크기와 효소활성은 4.0kb 와 168.8 U/㎎이었다. 형질전환체에서 YEp plasmid 안정성은 52.7%에서 67.4%정도였다. YEp plasmid에서 YIp plasmid를 구출할 수 있었고 이 YIp plasmid는 대장균에서나 효모에서도 발현되었고 특히 HIS5 gene이 promoter로서 가능함을 알 수 있었다. It was attempted to observe the development of promoter on lac'z gene. Two promoterⅠand Ⅱ of lac'z gene were isolated from chromosomal DNA Bam HI fragment of yeast. The size of the promoterⅠwas estimated to be 2.5kb and β- galactosidase activity was 124.6 U/㎎ protein but the size of the promoterⅡ was 4.0kb and its β- galactosidase activity was 168.8 U/㎎ protein respectively. The stability of the recombinant YEp plasmid in the transformant was from 52.7 to 67.4% at minimal medium. And YIp plasmid was constructed by YEp plasmid. This YIp plasmid both in E.coli and yeast. The HIS5 gene, coding gene of histidiolphosphate aminotransferase functioned as the promoter of YIp plasmid .