Bacillus sp. BE101 was isolated from soil and used to obtain the cyclodextrin glycosyltransferase(E.C.3.2.1.19 CGTase). This enzyme has a high activity for producing cyclodextrins. Cyclodextrin glycosyltransferase was purified by acetone precipitation, DEAE-cellulose chromatography, and sephadex G-150 chromatography. The molecular weight of the enxyme was calculated 73,000 by SDS-PAGE. The optimum conditions for CGTase activity were pH 6.0-6.5 and temperature 45℃. CGTase from Bacillus sp. BE101 synthesized mainly β-cyclodextrin from soluble starch; composition of α-, β-, $\gamma$- cyclodextrin was 1:7:2. An increase in cyclodextrin production by organic solvents such as acetone, propanol, and ethanol was observed. The maximum increase was observed at 10%(v/v) ethanol, and the enhancement of production yield was about 10%. The cause of increase in production yield of cyclodextrins by ethanol was due to the increase in hydrosis of starch by CGTase in the presence of ethanol.
A novel enzymatic process for the production of cyclodextrins from unliquefied starch was develpoed. Cyclodextrins were produced from unliquefied corn starch in an attrition bioreactor without liquefaction. The effect of milling media size and material on the performance of the attrition bioreactor was investigated, and operational parameters such as agitation speed, volume of milling media, ratio of enzyme to starch, and starch concentration were optimized. Optimum conditions were milling media glass beads of 3㎜ diameter, agitation speed 300 rpm, volume of milling media 30%(v/v), and ratio of enzyme to starch 500units/g starch. The production yield of cyclodextrins reached 35%(w/w) (53g/L) from 15%(w/v) unliquefied corn starch at 24h under optimized conditions.
Cyclodextrins were produced from unliquefied corn starch in the presence of water-miscible organic solvents using cyclodextrin glycosyltransferase (CGTase) in an attrition bioreactor. The production yield was significantly increased by tertiary butanol or isopropanol, and maximum enhancement was observed to be about 40% by 5%(v/v) tertiary butanol. Increase in the production of cyclodextrins by organic solvents seems to be due to the fact that organic solvents decreased the product inhibition of CGTase by forming an inclusion complex with cyclodextrins.
Cyclodextrins inhibited the activity of CGTase. In order to increase the production yield by reducing product inhibition and reusing the CGTase, an ultrafiltration membrane bioreactor system was employed in the production of cyclodextrins from milled corn starch. In a batch operation with ultrafiltration, the conversion yield was increased 57% when compard to that without ultrafiltration. Operating conditions for the continuous production of cyclodextrins in the membrane bioreactor were optimized by taking into consideration the filtraion rate and the conversion yield as follows: initial starch concentration, 7%(w/v); starch feeding rate, 240mg/h; CGTase loading, 350units/gram starch. When cyclodextrins were continuously produced under optimized conditions, amount of CGTase required to produce one gram of cyclodextrins was calculated to be about 340units, while in the case of conventional batch operation, about 1410units of CGTase was needed.
토양에서 분리한 Bacillus sp. BE101 균주를 이용하여 균체외로 배출되는 사이클로덱스트린 합성효소인 사이클로덱스트린 클루코실트랜스퍼라아제(Cyclodextrin glycosyltransferase)를 얻었다. 이 효소를 acetone 침전법, DEAE-cellulose chromatography 및 sephadex G-150 chromatography 법을 이용하여 분리하였으며 분리한 효소의 분자량은 73,000 이며, 최적 pH 및 온도는 6.0-6.5와 45℃ 이었다. 이 효소에 의해 가용성 전분으로부터 생산되는 α-, β-, $\gamma$-cyclodextrin 비율은 1:7:2 이었고, 이 효소는 cyclodextrins에 의해 저해받았다. 가용성 전분으로부터 사이클로덱스트린을 생산시 생산수율을 높이기 위하여 유기용매를 첨가하여 반응시킨 결과 에탄올(ethanol)을 반응액에 10%(v/v) 첨가시 생산 수율이 100% 증가하였고, 생산수율 증가원인은 에탄올 존재시 효소의 전분분해활성이 증가하였기 때문으로 조사되었다.
분쇄마찰반응기(attrition bioreactor)를 이용하여 액화과정없이 생전분으로부터 사이클로텍스트린을 생산할 수 있었다. 분쇄마찰반응기에서 생산조건을 검토한 결과 그램 전분당 효소를 500units 첨가하고 지름 3㎜ 유리구슬을 반응액에 30%(v/v) 첨가하여 300rpm으로 교반시 최대생산량을 보였다. 위와 같은 조건에서 15%(w/v) 생전분을 이용하여 생산한 결과 24 시간만에 53g/l 의 사이클로텍스트린을 얻을 수 있었으며 이때의 수율은 35% 이었다. 분쇄마찰반응기에서 사이클로텍스트린을 생산시 반응액을 HPLC를 이용하여 분석한 결과 포도당(glucose), 맥아당(maltose), 말토트리오스 (maltotriose) 등은 검출되지 않았으며 에너지 소비도 기존의 공정에 비하여 25% 이었다.
분쇄마찰반응기에서 생전분으로부터 사이클로텍스트린을 생산시 생산수율을 증가시키기 위하여 유기용매를 첨가하여 반응시킨결과 tertiary butanol을 5% 첨가 하거나, isopropanol을 10% 첨가시 생산수율이 40% 증가하였다. 생산수율 증가원인을 조사한 결과 사이클로덱스트린 합성효소인 사이클로텍스트린 글루코실트랜스퍼라아제의 산물저해현상(product inhibition)을 감소시키기 때문인 것으로 밝혀졌다.
사이클로텍스트린 글루코실트랜스퍼라아제의 사이클로텍스트린에 의한 산물 저해현상을 효과적으로 감소시키고 효소를 재사용하기 위하여 한외여과 막반응기(ultrafiltration membrane bioreactor)을 이용하여 사이클로텍스트린 생산을 수행 하였다. 한외여과막 반응기에서의 생산조건은 초기기질농도 7%, 기질첨가량 240㎎/h, 효소첨가량 350units/그램 전분 이었다. 한외여과 막반응기에서 산물저해현상을 효과적으로 제거하면서 사이클로텍스트린을 연속생산한 결과 1g의 사이클로텍스트린을 얻기 위하여 소모된 효소량이 340units 이었고 분쇄마찰반응기에서는 1410units이므로 보다 효과적으로 사이클로텍스트린을 생산할 수 있었다.
