Polymerase chain reaction(PCR) was conducted to obtain the gene encoding glucose oxidase(GOD) from Aspergillus niger(ATCC 2110) and the DNA sequence determined was coincided with published GOD sequence from A. niger. Recombinant transforming vector containing GOD and hygromycin B(hyg.B) resistant gene(hph) was constructed and used for further transformation of A. niger ATCC 2110. Selectivity of hyg.B against A. niger differed depending on which media were used i.e., nutrient-rich media such as potato dextrose agar(PDA) and complete medium(CM) showed only 50% growth inhibition at 400 m ml of hyg.B while the minimal media inhibited mycelial growth completely at 200um ml of hyg.B. Twenty to sixty putative transformants were isolated from the hyg.B-containing minimal top agar, transferred successively onto alternating selective and nonselective media for a mitotic stability of hyg.B resistance and, then, single-spored. Among the stable transformants, the transformant(GOD1-6) grown by flask culture showed the considerable increase of extracellular GOD activity, which was estimated to the degree of 50% - 100% comparing to that of wild type. Transformation of tGOD1-6 was resulted from integration of the vectors into heterologous as well as homologous regions of the A. niger genome. Southern blot analysis revealed that there were two independent integrations of vector into fungal genome and one into the GOD gene due to homologous recombination. In addition, GOD activity and sodium gluconate production when tGOD1-6 was fed-batch fermented were enhanced 11 fold and 2.25 fold, respectively, compared to that of the wild type.
목차
서론 재료 및 방법 사용균주 및 plasmid A. niger로부터 포도당 산화효소의 클로닝 재조합 유전자의 제조 A. niger에 재조합 유전자의 도입 Gluconic acid 발효 포도당 산화효소를 이용한 sodium gluconate의 제조 결과 및 고찰 포도당산화효소 유전자(God)의 cloning A. niger의 형질전환체의 선발 및 확보 Southern analysis Batch-fermentation Fed-batch culture 요약 감사 참고문헌
한국생물공학회 [The Korean Society for Biotechnology and Bioengineering]
설립연도
1984
분야
공학>생물공학
소개
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