The production of galactooligosaccharide (GOS) by microorganisms and enzymes with transgalactosylation activity was studied. A number of microorganisms with the ability to produce GOS was screened and a fungal strain of Bullera singularis ATCC 24193 was selected as the best GOS-producing strain considering GOS yield and enzyme activity.
The cultural conditions of B. singularis were optimized for the production of GOS. Optimum temperature was 25℃, the pH was 6.0, and the inoculum size was over 5% (v/v). GOS production by the culture of B. singularis was shown to be proportional to cell growth. The production rate increased most sharply at an exponential growth phase. Through optimization of pH and initial lactose concentration, a maximum GOS% (defined as the ratio of GOS to total saccharides in the medium) of 92% was reached with 10% lactose at an initial pH 4.0. This value corresponds to 70 g-GOS/l medium. The cultural products were mainly of trisaccharides and tetrasaccharides as confirmed from the HPLC and TLC analysis. A new mechanism of GOS production was proposed based on the metabolism of the carbon source.
Enzymatic production of GOS was performed by partially purified -galactosidase. Ammonium sulfate precipitation and ultrafiltration methods were used to prepare the enzyme. A maximum yield of 40% GOS, corresponding to 120 g GOS/l, was reached from the 300 g/l lactose solution at 45 C and pH 3.7. The yield of GOS did not increase with initial lactose concentration and pH change. Reaction products were found to be comprised of a disaccharide and trisaccharides. The structure of the major product, a trisaccharide, was proposed to be ο-β-D-galactopyranosyl-(1-4)-o-β-D-galactopyranosyl-(1-4)-β-D-glucose (4'-galactosyl lactose).
GOS were continuously produced using lactose and immobilized -galactosidase in a packed bed reactor. Partially purified -galactosidase was immobilized onto a Chitopearl BCW 3510 bead (970 GU/g-resin) by simple adsorption method. Amounts of 55% oligosaccharides (sum of disaccharide and trisaccharides) were obtained sustainedly with a productivity of 4.4 g/(l-h) from the 100 g/l lactose solution during the 15 day operation.
During the enzymatic reactions the by-products, glucose and galactose, were found to inhibit GOS formation. To reduce the inhibition and to increase the yield of GOS, a mixed enzyme system was introduced by using glucose isomerase as a co-enzyme. The optimum ratio of glucose isomerase to β-galactosidase was in the range of 1.5 to 2 (GU/IGIU). At this ratio, a maximum yield of oligosaccharides reached to 64% after 112 hrs of reaction. Compared to the composition of GOS produced by the β-galactosidase only, the GOS syrup produced by the mixed enzyme system showed little difference with respect to sugar composition; i.e., more tetrasaccharides and fructose in the mixed system. Continuous operation by using the mixed enzyme system is successfully under way.
The present system proved to be adequate in extracting and immobilizing the enzyme and suitable for the commercial production of GOS.
갈락토실기 전이활성을 지니는 미생물과 효소에 의한 갈락토올리고당 생산에 관하여 연구하였다. 다양한 미생물의 분리 및 선별작업의 결과로 곰팡이의 일종인 Bullera singularis ATCC 24193을 갈락토올리고당 생산균주로 선정하였다.
B. singularis의 배양에 의한 생산의 경우 최적 배양조건은 25℃, pH 6.0, 접종농도는 5%(v/v) 이상 이었다. 미생물의 성장과 올리고당 생산은 정비례하였으며 지수증식기에서 올리고당 생산속도가 최고치를 나타내었다. 배지의 pH와 초기 유당농도를 조절하여 (pH 4.0, 100 g/l의 유당) 최고 92%의 순도로 70 g/l의 올리고당을 얻을 수 있었다. 생산된 올리고당은 고속액체 크로마토그래프와 박충 크로마토그래프의 결과로 부터 삼당과 사당이 대부분임을 알 수 있었다. 이 균주로 부터 효소를 부분정제한 후 이 효소에 의한 올리고당 생산에 관하여 연구하였다. 황산암모늄에 의한 침전과 한외여과법으로 고역가의 효소액을 얻을 수 있었으며, 이 효소를 이용하여 회분식 반응으로 300 g/l의 유당 용액으로 부터 45℃, pH 3.7 조건에서 120 g/l의 올리고당(삼당 이상)을 생산할 수 있었다. 올리고당의 생산수율은 초기 유당농도와 pH에 영향 받지 않았다. 생산된 올리고당은 이당류와 삼당이 대부분이었으며, 삼당은 4'-galactosyllactose로 밝혀졌다.
분리된 효소를 키토산 담체 (상품명 Chitopearl BCW 3510)에 고정화한 (900 GU/g-담체) 후 충진탑 반응기에 충진하여 15일간 55%의 수율로 올리고당을 연속생산하였다. 이때 생산성은 시간당 반응기 용적당 4.4g-올리고당 이었다.
올리고당 생산물의 부산물인 포도당의 저해작용을 줄이기 위하여 포도당 이성화효소를 이용한 혼합효소계 반응을 도입하였다. 최적조건 하에서 단일효소를 사용한 경우와 비교하여 올리고당 수율 및 유당의 전환율이 각각 10% 상승하였다. 구성당의 조성은 혼합효소계의 경우가 사당과 과당의 함량이 높게 나타났으며 상대적 감미도는 두 경우 공통적으로 40부근으로 유사하였다.
