초록

○ 연구결과
가. 개량된 chitinase 제작
나. Bacilllus 형질전환주 제작 및 활용
다. chitin deacetylase 생산 능력이 우수한 균주의 선발 및 특성 구명
라. 항균활성이 우수한 키토올리고당 및 chitinase의 조제 과정 설정
마. 길항세균, chitinase 및 키토올리고당을 이용한 식물 병 방제 효과 증진 체계 확립

Abstract ▼

I. Improvement for chitinase properties and cloning of chitin deacetylase
1. DNA shuffling and domain shuffling
A total of 5,500 transformants were obtained. By primary screening, 9 candidates out of 35 positive clones were screened for further study. One clone with high chitinase activity (Ch

I. Improvement for chitinase properties and cloning of chitin deacetylase
1. DNA shuffling and domain shuffling
A total of 5,500 transformants were obtained. By primary screening, 9 candidates out of 35 positive clones were screened for further study. One clone with high chitinase activity (Chi2-389) and one clone with thermostable chitinase activity(Chi2-33) were obtained. The alkali-resistant chitinase was similar to the wild type chitinase. Mutant Chi2-389 showed higher specific activity than the wild type chitinase by 16%. The optimum temperature of the mutant Chi2-33 was 50℃ and was similar to that of the wild type. However, the thermostability of the Chi2-33 was higher than that of the wild type by 18%. The mutants Chi2-33 and Chi2-389 showed no change in the molecular weight of the enzyme, suggesting no internal cleavage or modification of terminal regions of the enzyme.
Domain-shuffled mutants were obtained by substituting ChBD with CBD and XBD. Most of the enzyme existed in the internal region of the cells. Chi40, a mutant lacking ChBD, showed only slight enzyme activity. This phenomenon seemed to be the result of the differences of CBD and ChBD in their affinity towards colloidal chitin. The enzyme with CBD and ChBD in series showed higher affinity towards the substrate than the enzymes with CBD or ChBD. XBD was less effective than CBD.
2. Consturction of cDNA library and isolation of chitin deacetylase clone
The molecular weight of the mRNAs obtained from Rhizopus microsorus var. oligosporus were 0.5 ~ 2.5 kb. A primary cDNA library of 870,000 was constructed by introducing cDNA synthesized from the mRNA into ZAP Expression vector. Based on the sequence of the chitin deacetylase gene of Rhizopus oryzae, three probes of ca. 30 bp were prepared. When hybridization was preformed with the probes, no positive clones could be obtained. A total of 25 positive plaques which hybridized to the antibody were obtained using a immunoscreening kit.
Of the 25 positive plaques, 2 chitin deacetylase-producing plaques were screened. The plaques with chitin deacetylase activity were introduced into E. coli XLOLR. Chitin deacetylase activity was detected in the periplasm and cytoplasm of the cells, but not in the culture supernatant of the culture broth.
The cDNA in RCDA-1 with high chitin deacetylase activity had an ORF of 1,308 bp encoding a protein consisted of 435 amino acids. The molecular weight of the protein was expected to be 48,000 Da.
Considering the molecular weight, 60 kDa, of the protein determined by activity staining of the protein, the protein is thought to be a glycoprotein. The similarity of the protein to other fungal chitin deacetylases were less than 69%. The enzyme had a conserved amino acid sequence in the middle region of the protein.
3. Introduction of the chitin deacetylase gene into Bacillus sp. and its expression
Vector pWB705 was used for the introduction of the gene into Bacillus sp. The vector was partially digested with EcoRI and the chitin deacetylase cDNA was ligated with the vector that had been cut at the interior of SacB SP. The competent cells of Bacillus subtilis WB700 and WB800 was transformed with the ligated DNA. The culture supernatant of the transformant B. subtilis WB800 (pRDA83) showed an enzyme activities of 0.45 U/㎖ which is 6 times higher activity than that of the Rhizopus microsorus var. oligosporus.
B. subtilis WB800 (pRDA83) was grown for 12 h and the culture
supernatant was electrophoresed. The activity staining of the gel revealed an active band of 60 kDa. The result indicated that the chitin deacetylase gene of Rhizopus microsorus var. oligosporus was expressed in the transformant.
II. System for improvement of control efficacy against plant diseases using chitinase, chitin- and chito-oligosaccharides
1. Selection of chitin deacetylase producing strain and its enzymatic characteristics
Rhizopus microsporus var. oligosporus (KCTC 6969) was selected as a chitin deacetylase producing strain from 60 strains. This strain showed best growth on 25 C, and enzyme producing ability was better in PDB than in YPG. The enzyme production reached at peak after 5 days incubation when solution of 105 spore/ml was inoculated, and after 3 days incubation when solution of 105 spore/ml was inoculated. The enzyme was not induced by chitin and chitosan. This strain grew slowly on the chitin agar plates and did not produce chitinase This strain produced two chitin deacetylases with molecular weight of 60-kDa and 45-kDa. The enzyme activity was best in 60 C and pH 7.0.
2. Antifungal activity of chito-and chitin-oligosaccharides and chitinase against plant pathogens and their preparation.
The antifungal activity of chito- and chitin-oligosaccharides and chitinase was investigated with plant pathogens; Botrytis cinerea, Cladosporium sphaerospermum, Cladosporium tenuissimum, Alternaria panax, Colletotrichum gloeosporioides and Fusarium oxysporium. The antifungal activity against the plant pathogens was specifically high in chito-oligosaccharides as compared with chitin-oligosaccharides. The activity was also specifically high in pentamer and hexamer of chito-oligosaccharides. The hexamer showed higher antifungal activity against B. cinerea, C. sphaerospermum and C. tenuissimum with EC(50) values of 5ppm than A. panax, C. gloeosporioides, F. oxysporium with EC(50) values of 50ppm. The EC(50) values of chitinase were 20ppm for Cladosporium sphaerospermum and Cladosporium tenuissimum, 200ppm for B. cinerea and more than 2000ppm for F. oxysporium.
Degradation of crystal chitin by purified chitinase started to be detected after 1 hour incubation, and most pentamer and hexamer were detected after 6 hours incubation. The chitin deacetylase showed best activity in the hexamer as a substrate, and followed by order of pentamer and tetramer. However, the activity was very low in trimer and dimer. The activity in the glycol chitin was a half level as compared with that in pentamer, but the activity was very low in colloidal chitin and crystal chitin. These results suggest that it is better to convert chitin into chitin-oligomers with chitinase, and then to convert the chitin-oligomers into chito-oligomers with chitin deacetylase.
3. Large-scale production of antagonistic bacteria and chitinase, and stratigies for improvement of biocontrol of plant disease.
Previous experiments suggested that culture of chitinolytic bacteria in chitin media may lead to produce chitinase and chitin-oligomers as well as the bacteria, and also chito-oligomers by addition of chitin deacetylase. These culture solution may show better control efficacy against plant diseases. In this study, system for large-scale production of these culture solution was established and their control efficacy was investigated. In control of Lysobacter enzymogenensis and Chromobacterium sp. strain C-61 against powdery mildew of cucumber, the control efficacy was better in culture solution of two strains than in that of a single strain, in the culture solution than in suspension of bacteria cells alone. The control efficacy of the culture solution more increased by addition of chitin deacetylase. In the vinyl house experiment, control efficacy of the culture solution against powdery mildew was largely affected by environment of spray timing. The undiluted and 1/10 diluted solution of the combined culture solution showed high control value more than 90%, which were superior to commercial biocides. This culture solution also showed good control efficacy against leaf mold and powdery mildew of tomato, and root-knot nematode of cucumber. It is expected that this culture solution may be used by many farmers because it can be simply prepared with low cost.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 10
• CONTENTS ... 15
• 목차 ... 17
• 제1장 연구개발과제의 개요 ... 19
• 제1절 연구개발의 목적 및 범위 ... 19
• 제2절 연구개발의 필요성 ... 21
• 제2장 국내.외 기술개발 현황 ... 26
• 1) 유전자 변형에 의한 chitinase 활성 및 특성 변화 ... 26
• 2) chitin deacetylase 유전자의 클로닝 및 Bacillus sp.에 도입 및 발현 ... 28
• 3) 항미생물성 키틴유도체의 생산 ... 28
• 4) 키틴분해미생물 및 키틴유도체를 이용한 식물병 방제 ... 29
• 제3장 연구개발 수행 내용 및 결과 ... 31
• 제1절 효소활성 및 특성이 변화된 chitinase 유전자의 개발 및 chitin deacetylase 유전자의 클로닝 ... 31
• 1. 서 론 ... 31
• 2. 재료 및 방법 ... 32
• 3. 결과 및 고찰 ... 42
• 가. 효소활성 및 특성이 변화된 chitinase 유전자의 개발 ... 42
• 나. Chitin deacetylase 유전자의 클로닝 ... 56
• 다. Bacillus sp.에 chitin deacetylase 유전자 도입 및 발현 ... 64
• 4. 결과 요약 ... 66
• 제2절 Chitinase, 키틴 및 키토올리고당을 이용한 식물 병 방제효과 증진 시스템 확립 ... 68
• 1. 서 론 ... 68
• 2. 재료 및 방법 ... 70
• 3. 결과 및 고찰 ... 77
• 가. chitin deacetylase 생산 균주의 선발 및 효소 특성 ... 77
• 나. 키틴올리고당, 키토올리고당 및 Chitinase의 항균활성과 그들의 조제 ... 81
• 다. 길항세균, chitinase 및 키토올리고당의 대량 생산과 그들의 식물 병 방제 효과 증진 ... 91
• 4. 결과 요약 ... 106
• 제4장 목표달성도 및 관련분야에의 기여도 ... 109
• 제1절 1년 차 연구개발 목표, 목표의 달성도 및 기여도 ... 109
• 제2절 2년 차 연구개발 목표, 목표의 달성도 및 기여도 ... 111
• 제3절 3년 차 연구개발 목표, 목표의 달성도 및 기여도 ... 113
• 제5장 연구개발결과의 활용계획 ... 115
• 제1절 효소활성 및 특성이 변화된 chitinase 유전자의 개발 및 chitin deacetylase 유전자의 클로닝 ... 115
• 제2절 Chitinase, 키틴 및 키토올리고당을 이용한 식물 병 방제효과 증진 시스템 확립 ... 115
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 116
• 제7장 참고문헌 ... 117
• 끝페이지 ... 128