Properties of chitosanase produced from Bacillus subtilis EG1 and Bacillus wiedmannii DAM2 = Bacillus subtilis EG1 및 Bacillus wiedmannii DAM2에서 생성된 키토산아제의 특성

다국어 초록 (Multilingual Abstract)

Natural biopolymers such as chitin and chitosan have attracted considerable attention in various industries because of their advantageous properties. Chitosan, which is obtained by deacetylation of chitin, is composed of N- acetyl-D-glucosamine and D-glucosamine. It exhibits excellent characteristics, including biodegradability, antibacterial activity, and non- toxicity, which make it widely applicable in medicine, cosmetics, food, and agriculture. In particular, chitosan oligomers, produced through the hydrolysis of chitosan, have low molecular weights, high water solubility, and strong physiological activities, such as antibacterial, antitumor, antioxidant, and plant growth -promoting effects, resulting in high industrial potential. Conventional chemical methods for producing chitosan oligomers have several limitations, including harsh reaction conditions and environmental pollution. Therefore, enzymatic approaches using chitosanase, a chitosan-degrading enzyme, have gained attention as an alternative. Chitosanase hydrolyzes the β-glycosidic bonds of chitosan under mild reaction conditions with high specificity. Therefore, this study aimed to produce and characterize chitosan oligomers using chitosanases produced by two bacterial strains, Bacillus subtilis EG1 and Bacillus wiedmannii DAM2. Bacteria exhibiting chitosanase activity were first isolated and identified. The carbon and nitrogen source sources were then optimized to maximize enzyme activity. Following optimization, chitosanase activity increased 54.4-fold and 6.7-sfold in B. subtilis EG1 and B. wiedmannii DAM2, respectively. The basic characteristics of the two strains were subsequently investigated, and enzyme activity bands were analyzed by electrophoresis. The chitosan degradation patterns were examined using thin- layer chromatography, confirming the breakdown of chitosan into monosaccharides and hexasaccharides. Chitosan oligomers were further quantified by high-performance liquid chromatography, with total yields of 2.5% for EG1 and 9% for DAM2 at the optimal temperature and reaction time. In addition, the average molecular weight of chitosan was determined by gel permeation chromatography, and the optimal temperatures for chitosan oligomer production were identified as 60°C for EG1 and 50°C for DAM2. Collectively, our results confirmed that both B. subtilis EG1 and B. wiedmannii DAM2 produce active chitosanase enzymes capable of generating chitosan oligomers. These findings provide fundamental data supporting the industrial application of chitosanases derived from Bacillus spp. and for the large-scale production of high- value-added chitosan oligomers.

국문 초록 (Abstract)

키틴, 키토산과 같은 천연 바이오폴리머는 다양한 분야에서의 장점으로 산업에서 주목받고 있다. 키틴을 탈아세틸화하여 얻을 수 있는 키토산은 N-acetyl-Dglucosamine과 D-glucosamine으로 구성되어 있다. 이러한 키토산은 생분해성, 항균성, 무독성 등의 뛰어난 특성을 가지고 있어 의약, 화장품, 식품, 농업 등의 다양한 분야에 광범위하게 활용될 수 있다. 특히 키토산을 가수분해하여 얻어지는 키토산 올리고머의 경우, 분자량이 작고 수용성이 높으며 항균, 항종양, 항산화 및식물 생장 촉진 등의 높은 생리활성을 가지고 있어 산업에서 활용도가 매우 높다. 키토산 올리고머를 생산하는 기존의 화학적 방법은 높은 반응 조건과 환경오염 등의 단점을 가지고 있다. 따라서 이에 대한 대안으로 키토산 분해 효소인chitosanase를 사용하는 효소적 방법이 주목받고 있다. Chitosanase는 낮은 반응 조건과 높은 특이성으로 키토산의 b-glycoside 결합을 가수분해할 수 있다. 따라서본 연구는 Bacillus subtilis EG1, Bacillus wiedmannii DAM2 두 균주가 생산하는chitosanase를 사용하여 키토산 올리고머를 생산하고 그 특성을 규명하고자 한다. 그에 대한 방법으로 먼저, chitosanase 활성을 보이는 박테리아를 분리 및 동정하였다. 이후, 탄소원 질소원 조건을 최적화하여 효소 활성을 극대화하였다. 최적화를 통해 chitosanase 활성은 B. subtilis EG1와 B. wiedmannii DAM2 균주에서 각각 54.4배 및 6.7배까지 증가하였다. 또한 두 균주의 기본적인 특성 및 전기영동을통해 두 균주의 효소 활성 밴드를 측정하였다. 최종적으로, Thin-Layer-Chromatography(TLC)를 이용하여 키토산 올리고머 분해 패턴 분석한 결과, 키토산이 1당과 6당으로 분해되는 것을 확인하였다. 이후 High-Performance Liquid Chromatography (HPLC)를 이용하여 키토산 올리고머를 정량 한 결과, 최적 온도 및 시간에서 총 수율이 EG1은 2.5%, DAM2는 9%로 확인되었다. 그리고 Gel Permeation Chromatography (GPC)를 이용하여 키토산의 평균 분자량을 측정한 결과,두 균주의 키토산 올리고머 생성의 최적 온도는 각각 60도와 50도임을 확인하였다. 따라서 본 연구는 B. subtilis EG1, B. wiedmannii DAM2 두 균주 모두 chitosanase 효소를 생산하고, 그 효소를 이용하여 키토산 올리고머를 생산할 수 있음을 확인하였다. 이러한 결과는 Bacillus sp. 균주 유래 chitosanase의 산업적 응용과 고부가가치 키토산 올리고머의 대량 생산을 위한 기초 데이터를 제공할 것이다.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 2. MATERIALS AND METHODS 10
• 2.1. Chitosan preparation 10
• 2.2. Preparation of chitosan medium 10
• 2.3. Isolation and identification of chitosanase-producing microorganisms · 10
• 2.4. Basic biochemical characteristics of EG1 and DAM2 strains 11
• 2.4.1. API test 11
• 2.4.2. Cell growth, protein content, chitosanase activity, and chitinase activity 12
• 2.4.3. Evaluation of cellulase, protease, and lipase activities in media 13
• 2.4.4. Enzyme activity staining on SDS-PAGE 14
• 2.5. Optimization of chitosanase activity 16
• 2.5.1. Chitosanase activity by chitosan substrate concentration 16
• 2.5.2. Chitosanase activity by carbon source 16
• 2.5.3. Chitosanase activity by nitrogen source 17
• 2.6. Confirmation of chitosan oligomer production by strains EG1 and DAM2 18
• 2.6.1. Chitosan degradation pattern and qualitative analysis 18
• 2.6.2. Molecular weight analysis of chitosan 19
• 2.6.3. Chitosan oligomer analysis 19
• 2.6.4. Calculation of chitosan oligomer yield 20
• 2.7. Biological control characteristics 20
• 2.7.1. Antifungal activity of two strains against plant pathogenic fungi 21
• 2.7.2. Antifungal activity of supernatants from two strains against plant pathogenic fungi 21
• 2.7.3. Antifungal activity of two strains and chitosan reactants against plant pathogenic fungi 21
• 3. RESULTS AND DISCUSSION 23
• 3.1. Isolation and identification of chitosanase-producing microorganisms · 23
• 3.2. Basic biochemical characteristics of EG1 and DAM2 strains 25
• 3.2.1. API test 25
• 3.2.2. Cell growth, protein content, chitosanase activity, and chitinase activity 28
• 3.3. Optimal activity of chitosanases by substrate, carbon source, and nitrogen source 31
• 3.3.1. Chitosanase activity by chitosan substrate concentration 31
• 3.3.2. Chitosanase activity by carbon source type 32
• 3.3.3. Chitosanase activity by nitrogen source 35
• 3.4. Basic biochemical characteristics of EG1 and DAM2 strains in the optimized medium 36
• 3.5. Evaluation of cellulase, lipase, and protease activities in media 40
• 3.6. SDS-PAGE 42
• 3.7. Confirmation of chitosan oligomer production by EG1 and DAM2 44
• 3.7.1. Investigation of chitosan degradation patterns by temperature on TLC 44
• 3.7.2. GPC analysis of chitosan polymer by temperature 45
• 3.7.3. Analysis of chitosan degradation over time on TLC 49
• 3.7.4. GPC analysis of chitosan polymer degradation over time 51
• 3.7.5. HPLC analysis of chitosan oligomers over time 57
• 3.7.6. Calculation of chitosan oligomer yield 64
• 3.8. Biological control characteristics 66
• 3.8.1. Investigation of the antifungal activity of strains EG1 and DAM2 against plant pathogenic fungi using a dual culture assay 66
• 3.8.2. Antifungal activity of supernatants from strains EG1 and DAM2 against plant pathogenic fungi using a dual culture assay 67
• 3.8.3. Antifungal activity of EG1 and DAM2 strains and chitosan reactants against plant pathogenic fungi using a dual culture assay 69
• 4. CONCLUSIONS 72
• REFERENCES 73
• ABSTRACT IN KOREAN 82
• RESEARCH PERFORMANCE 84