원문정보
Optimization for Production of Exo-β-1,3-glucanase (Laminarinase) from Aspergillus oryzae in Saccharomyces cerevisiae
초록
영어
In this study, a EXGA gene code for exo-β-1,3- glucanase from Aspergillus oryzae was overexpressed and secretory produced in Saccharomyces cerevisiae. To overexpress the β-1,3-glucanase, pGInu-exgA and pAInu-exgA plasmids having GAL10 and ADH1 promoter, respectively, and exoinulinase signal sequence (Inu s.s) were constructed and introduced in S. cerevisiae SEY2102 and 2805. The recombinant β-1,3- glucanase was successfully expressed and secreted into the medium and the β-1,3-glucanase activity in 2102/pGInu-exgA and 2102/pAInu-exgA strain were 5.01 unit/mL and 4.09 unit/mL, respectively. In the 2805/pGInu-exgA and 2805/pAInu-exgA strain, the β-1,3-glucanase activity showed 3.23 unit/mL and 3.22 unit/mL, respectively. Secretory efficiency in each strain reached 95% to 98%. Subsequently, the recombinant β-1,3-glucanase was used for ethanol production. Ethanol productivity in 2102/pAInu-exgA strain was 0.83 g/L when pre-treated Laminaria japonica which has initial reducing sugar of 1.4 g/L was used as substrate. It is assumed that the polysaccharides of Laminaria japonica was effectively saccharified by recombinant β-1,3-glucanase, resulting in increase of ethanol productivity. These results suggested that recombinant β-1,3- glucanase was efficiently overexpressed and secreted in S. cerevisiae SEY2102 as host strain by using ADH1 promoter-Inu s.s system.
목차
1. 서론
2. 재료 및 방법
2.1. 사용균주 및 plasmids
2.2. 재조합 plasmid 구축 및 형질전환
2.3. 배지 조성 및 배양조건
2.4. 균체 농도 및 Plasmid 안정성
2.5. MUG plate assay
2.6. 균체분획 및 β-1,3-glucanase 활성 측정과 분비효율
2.7. SDS-PAGE에 의한 단백질 확인
2.8. 에탄올 생산성 측정
3. 결과 및 고찰
3.1. β-1,3-glucanase 발현 재조합 plasmid의 구축
3.2. 효모 형질전환체 선별 및 MUG assay
3.3. β-1,3-glucanase 과발현 및 분비생산
3.4. β-1,3-glucanase 단백질 확인 및 에탄올 생산성
4. 결론
감사
References