earticle

영어

Leuconostoc mesenteroides NRRL B-1149 produces various glucosyltransferases for the synthesis of dextran, levan and glucose-1-phosphate using sucrose as a substrate. A sucrose phosphorylase (1149SPase) was purified from L. mesenteroides NRRL B-1149 culture by using hollow fiber filtration (30 kDa cut off), Toyopearl DEAE 650 M column chromatography and following two times of DEAE-Sepharose column chromatographies. The specific activity of the purified 1149SPase was 25.7 (U/㎎) with 16% yield. The 1149SPase showed a molecular size of 56 kDa on denatured 10% SDS-PAGE. The N-terminal amino acid sequence of the enzyme was MEIQNKAM. The optimum pH and temperature of this enzyme were 6.2～6.5 and 37℃, respectively. It had an apparent Km of 6.0 mM and Kcat of 1.62/s for sucrose. 1149SPase
crystal was formed by hanging drop diffusion technique using 20 mM calcium chloride dihydrate, 100 mM sodium acetate trihydrate pH 4.6 and 30% 2-methyl-2,4-pentanediol as vaporizing and reservation solution. The 1149SPase catalyzes transferring of glucose from isomaltose or sucrose to salicin and salicyl alcohol by disproportionation reaction or acceptor reaction and synthesized two acceptor products, respectively.

한국어

Leuconostoc mesenteroides NRRL B-1149로부터 sucrose를 기질로 하여 효소 반응기에 phosphate를 첨가하면 fructose와 G-1-P를 합성하는 sucrose phosphorylase을 정제하였다. 배양액을 hollow fiber filtration (30 kDa cut off)을 이용하여 농축하고 Toyopearl DEAE 650 M column과 두 번의 DEAE-Sepharose column chromatography를 통해 16% yield를 갖는 정제된 1149SPase 25.7 (U/㎎)을 얻었다. 정제한 1149SPase는 분자량이 56 kDa (10%, poly acrylamide gel)이었고, 37℃와 pH 6.2～6.5에서 효소 최적 활성을 보였다. 또한, sucrose를 기질로 한 Km과 Kcat 값은 각각 6.0 mM과 1.62/s로 계산 되었고, N-terminal아미노산 서열은 MEIQNKAM이였다. 1149SPase의 결정은 20mM calcium chloride dihydrate, 100 mM sodium acetate trihydrate pH 4.6, 30% 2-methyl-2,4-pentanediol의 혼합 용액을 저장액과 건조액으로 사용한 hanging drop diffusion방법으로 얻을 수 있었다. Salicin과 salicin alcohol을 isomaltose를 이용한 disproportionation 반응과 sucrose을 이용한 수용체 반응으로 당전이 시킨 화합물을 합성하였다. 이러한 1149SPase의 disproportionation반응과 수용체 반응 특성은 여러 신소재 개발에 활용 가능하겠다.