High-yield production of cycloamylose from starch using combined treatment of debranching enzyme and 4-a-glucanotransferase : Debranching 효소와 4-a-glucanotransferase 복합처리를 통한 전분으로부터의 고효율 cycloamylose 생산

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서울대학교 대학원
학위논문 (석사)-- 서울대학교 대학원 : 바이오시스템·소재학부(바이오시스템공학), 2012. 2. 김용노.
Cycloamylose is better than the conventional cyclodextrin as a hosting material. In this study, starch from various sources of rice and sweet potato was debranched with isoamylase and coupling with 4-a-glucanotransferase to increase the yield of cycloamyloses. Different concentrations of 1.0, 2.5, 5.0 and 7.5 U/g of isoamylase at 50 o C for 0 to 12 hr adding to the Goami rice starch in the different reaction media: 50 mM Tris-HCl, 100 mM sodium acetate, and 20 % demetyl sulfoxide (DMSO) in Tris-HCl; pH = 4.5 were compared. The debranched starches were treated with 1 U/mg of TATG and 40 U/g of b-amylase.
In buffer solution, reducing power increased about 4 times when the reaction time was 4 hr, and reducing power of samples treated in 20% DMSO considerably lower than that of samples in a solution without DMSO. Optimum reaction time of 5 U/g isoamylase could be determined as 4 hr. With 5 U/g of isoamylase, the apparent amylose content increased to 62.43 % when the treatment time was 4 hr. This was the highest apparent amylose content. 1NMR indicated that the proportion of a -1,6 was 6 % initially and decreased to about 2 % after 6 hr treatment which the most rapid change occurred within initial 4 hr of treatment. There was no different in the peak after 8 hr of treatment; and the average molecular weight of 4.17 x 104 Da with elution time of 50 min was observed. However, the sample treated in 20% DMSO showed very slow change during the reaction time.
The amount of linear maltooligosaccharide decreased at the same time cyclic glucan peaks newly appeared and the purified cycloamylose was obtained which ranged from DP23 to DP40 in MALDI-TOF analysis. The production of cycloamylose of starch with and without debranching process showed the significant different in intensity of the peaks at the same regions. The maximum yield of cycloamylose was observed at 6 hr treatment time. Without debranching step the maximum yield values ranged between 6 and 7 %, whereas the maximum yield with debranching step ranged from 31 % to 49.69 %, indicating the significance of debranching step in the cycloamylose production in rice starches. The maximum yields of cycloamylose of sweet potatoes without debranching step were between 33 % and 42 %, and with isoamylase significantly increased the maximum yield (45 % to 60 %). Sweet potato starches contained high amylose content than those of rice starches. Therefore, the increasing of cycloamylose was proportionate to amylose content. The chromatogram of HPEAC and MALDI-TOF indicated that the final products of cyclic glucans of cycloamyloses were produced. The highest yields of DP were observed at 25 and 26. In conclusion, adding the debranching step to the rice starch increased the production of cycloamylose by increasing the amylose content.
Cycoamylse는 포접복합체 형성에 있어 cyclodextrin에 비해 여러 장점을 가지고 있다. 본 연구에서는 다양한 품종의 전분에 대해 isoamylase와 4-α-glucanotransferase의 복합처리를 통해 cycloamylose의 수율을 증가시키기 위한 조건을 구명하였다. 고아미 전분에 isoamylase의 다양한 농도 (1.0, 2.5, 5.0 and 7.5 U/g )와 시간(0 to 12 hr), 용매 (50 mM Tris-HCl, 100 mM sodium acetate, and 20% demetyl sulfoxide (DMSO) in Tris-HCl;)를 처리하여 비교한 결과 isoamylse를 모든 용매에서 4시간 이상 처리했을 때 환원당이 최대값 부근으로 증가하였고, 겉보기 아밀로스 함량은5U/g 농도로 4시간 처리하였을 경우 가장 높았으며, 이 때 최종 분자량은 약 104 Da로 수렴하였다. 쌀전분에서 isoamylose처리 후 4-α-glucanotransferase 를 6시간 처리하였을 때 cycloamylose 의 수율은 6-7%에서 50%로 증가하였다. 고구마전분은isoamylase를 처리하지 않을 경우 cycloamylose 생산 수율은 33-42%였으며, isoamylase를 처리하였을 경우 45-60%로 증가하였다. 생산된 cycloamylose는 HPAEC와 MALDI-TOF MS를 이용하여 중합도와 분자량 분포를 측정한 결과 cyclic glucan의 이론적인 값과 일치하는 물질을 생산하였으며 cycloamylose 표준물질의 분자량 분포와 유사한 물질을 생산하였다. 가장 높은 수율을 가진
cycloamylose의 중합도는 25와 26으로 나타났다. 아밀로스 함량이 다른 쌀전분과 고구마 전분을 이용하여 cycloamylose 수율을 분석한 결과 전분의 아밀로스 함량과 cycloamylose 수율간의 상관관계가 있음을 확인하였다. 이 연구를 통해 환형 아밀로스 제조에 적합한 전분의 선택과 더불어 debranching 효소와 4-α-glucanotransferase의 복합 처리를 통해 전분으로부터 보다 높은 수율의 cycloamylose 제조가 가능함을 밝힐 수 있었다.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Biosystems and Biomaterials Science and Engineering (바이오시스템·소재학부)Theses (Master's Degree_바이오시스템·소재학부)
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