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호열성 Geobacillus thermosacchalytycus가 생산하는 a-Cyclodextrin Glucanotransferase 의 분리정제와 당전이 반응 특성

Purification of a-Cyclodextrin Glucanotransferase Excreted from Themophilic Geobacillus thermosacchalytycusand Characterization of Transglycosylation Reaction of Glucosides.

이용현|이미숙|김태권|신현동
한국미생물·생명공학회 (The Korean Society For Microbiology And Biotechnology)|2004.03|한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)|제32권 제1호|pp. 29-36

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초록

토양으로부터 CGTase를 생산하는 새로운 호열성 세균을 분리하였으며, 형태 및 생리적 특성과 16S ribosomal DNA의 염기서열을 분석한 결과 Geobacillus thermosacchalytycus로 동정하였다. 호열성 G. thermosacchalytycus가 분비하는 CGTase를 한외여과, 소수성 Phenyl Sephadex CL-4B 크로마토그래피, 그리고 gel filtration의 순서로 분리 정제하여 정제도 28.2배, 정제수율 12.2%, 그리고 specific activity가 4952.5 units/mg인 CGTase를 얻을 수 있었다. 정제된 CGTase의 분자량은 69,000 dalton이었고, N-terminal amino sequence는 Asn-Leu-Asn-Lys-Val-Asn-Phe-Thr-Ser-Asp-Val-Val-Tyr-Gln-Ile이었다. 최적 pH 및 온도는 각각 pH 6와 50oC였고, pH 6~8과 60oC에서 장기간 보존할 수 있는 중성 pH의 내열성 효소임을 알 수 있었다. 내열성 CGTase의 cyclization과 coupling 반응의 특징을 검토한 결과 G. thermosacchalytycus가 생산하는 내열성 효소는 a-type CGTase이었고, cyclization 반응보다는 coupling 반응에 적합한 당전이성이 높은 효소임을 알 수 있었다. 당전이 반응에서 기질 특이성을 검토한 결과 당공여체로는 가용성 전분 그리고 당수용체로는 배당체인 stevioside에 대해 높은 기질 특이성을 보였다. 당전이 반응은 당수용체와 당공여체가 효소와 무작위로 결합하여 진행되는 random ternary complex mechanism으로 반응이 수행되었다.

Abstract

a-Cyclodextrin glucanotransferase excreted from a newly isolated themophilic Geobacillus thermosacchalytycus was purified through the ultrafiltraion, hydrophobic Sepharose CD-4B affinity chromatography, and gel filtration on Sephadex G-75, respectively. The molecular weight of the purified CGTase was 69 kDa and its N-terminal amino acid sequence was determined to be Asn-Leu-Asn-Lys-Val-Asn-Phe-Val-Ser-Asp-Val-Val-Val-Gln-Ile. The optimum pH and temperature were pH 6.0 and 60oC, respectively, and stably at the pH range of 6.0~8.0 and 60oC in the presence of Ca++. The excreted CGTase from the thermophilic G. thermosacchalytycus was a-type showing a high coupling activity for the transglycosylation on various glucosides. The coupling reaction was carried out according to the random ternary complex mechanism.

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