고초균으로부터 대장균으로 cloning된 섬유소 분해 효소의 분리 및 특성

학위논문(석사) - 한국과학기술원 : 생물공학과, 1986.2, [ v, 42 p. ]Carboxymethyl cellulose degradating enzyme(CMCase) produced by Escherichia coli HB101 harboring plasmid pBS 2115 which contains Bacillus cellulase gene has been purified partially and characterized. The purpose of this study was of two fold; to obtain pure CMCase which is a component of the multiple system of microbial cellulases by producing with E. coli which had been free of cellulase genes until it was transformed by pBS 2115, and to find any alternative on the expression of the Bacillus cellulase gene in E. coli, if present. The E.coli HB101(pBS 2115) cells at midlogarithmic growth phase were harvested and the CMCase was extracted by applying osmotic shock to the cells. The curde enzyme preparation was purified by DEAE-Sephadex A-25 and hydroxyapatitic chromatography. Through the purification steps, the purity of the enzyme in the osmotic shock extract was increased 300 fold along with the yield reduction from 100 to 22\%. The molecular weight of this enzyme was estimated to be 34,000 in the SDS-polyacrylamide gel electrophoresis. The optimal activity of the enzyme for temperature and pH was at $65\,^\circ\!C$ and 6.0, respectively. The enzyme was stable at $55\,^\circ\!C$ for 1 hour. The substrate specificity of this enzyme was broad and hydrolyzed CMC, laminarin, pustulan, polymers of glucose linked by $\beta(1 \to 4),\; \beta(1 \to 3)$, and $\beta(1 \to 6)$ glycosidic bond. The enzyme was not inhibited by glucose or cellobiose. The enzyme liquefied CMC with sharp decrease in viscosity at early reaction time followed by a slow decrease, whereas the reducing ends were produced linearly. From these results it was concluded that the CMCase produced by the E. coli HB101 (pBS 2115) is endo-$\beta(1 \to 4)$ glucanase.한국과학기술원 : 생물공학과

대장균에서 종양괴사 인자의 대량 생산 및 비활성 단백질의 활성화

학위논문(박사) - 한국과학기술원 : 생물공학과, 1993.2, [ vii, 105 p. ]Overexpression of biologicall active lymphotoxin, i.e. tumor necrosis factor-$\beta$, was attempted using genetic engineering strates. The lympnotoxin gene contained in pLTl was transfered is pASI and constructed a new plasmid pALI. The signal sequence of precursor lymphotoxin gene in pALI was removed. With this pALIII, Escherichia coli M5248 was transformed and production of lymphotoxin was attempted. However, the E. coli transformant having pALIII did not produce lymphotoxin. The $P_L$ promoter of the lymphotoxin gene in pALIII was replaced with trc promoter and constructed prtcLT which produced lymphotoxin below 1\% of total cell proteins in E. coli. To increase the lymphotoxin production, the cDNA sequence of lymphotoxin gene was mutated by altering the translation initiation region using chemically synthesized oligonucleotides avoiding the local secondary structure of mRNA. The mutations were so designed to maintain the original amino acid sequence of lymphotoxin unchanged. The structures of mRNA of lymphotoxin were predicted in the translation initiation region based on the shorth range interaction of mRNA spanning 5`` terminal 130 nucleotides. The expressions of lymphotoxin were increased as the predicted local secondary structure of mRNA decreased. The elevation of cultivation temperature also increased the expression level of lymphotoxin. The amounts of mRNA were not significantly different when the cultivation temperature decreased to 32$^\circ$C or increased to $42^\circ$C. The maximal yield of expression level of lymphotoxin was 12\% of total cell proteins. Most of produced lymphotoxin in this system were biologically active form. Another trial of overproduction was performed by expressing the cDNA sequence of human lymphotoxin gene in T7 expression system. A production of lymphotoxin at the level of 16\% of total cell proteins was observed, but the production was in the form of insoltions by the criteria descrived in above section were resulted in ...한국과학기술원 : 생물공학과