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Streptomyces lividans에 재조합된 α - 1 , 3 glucanase 의 특성
윤정원,--,한홍근 대한구강보건학회 1995 大韓口腔保健學會誌 Vol.19 No.4
The α-1,3 glucanase gene from Streptomyces sp. SW403 was cloned into Streptomyces lividans 1326JI with plasmid pIJ702 for high and stable production of α-1,3 glucanase. Genomic DNA from Streptomyces sp. SW403 was partially digested with the restriction enzyme Sau3A I and ligated into Bgl B -digested pIJ702 for the transformation into Streptomyces lividans 1326JI. This transfromant encoded α-1,3 glucanase and was named Streptomyces lividans T1027. The enzyme was partially purfied by 30-70% (NH4)2S04 precipitation. The cloned enzyme activity was higher and more stable than the enzyme from Streptomyces sp. SW403. The optimum pH and temperature of the wild and cloned enzyme were pH 6.5 and 37℃, respectively. The activity of cloned enzyme was more stable than wild enzyme at 60℃ below, respectively.
Streptomyces sp. Y9343이 生産하는 齒面細菌膜 分解酵素의 精製와 特性
김성주,한홍근,윤정원 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.1
치면세균막을 제거하거나 형성을 억제하여 치아우식증 예방제를 개발할 목적으로 α-1,3 glucanase를 분비하는 새로운 균주로 부터 분리 정제하여 그 특성을 조사하였다. 불용성 glucan을 유일탄소원으로 하는 agar plate를 제조하여 토양으로 부터 α-1,3 glucanase 분비균주를 탐색한 결과 Streptomyces sp. Y9343을 얻었다. 액체배양시 최적 효소생산조건은 탄소원으로 1% soluble starch와 indcer로 0.5% insoluble glucan을 첨가하였을 때 가장 효율적이었다. α-1,3 glucanase는 황산 암모늄 염석, DEAE-CEllulose 이온교환 크로마토그래피, Sephadex G-75 겔 여과 등에 의하여 32.1배까지 정제되었고 수율은 0.53%이었으며, 이 때의 활성도는 7840.9 U/mg protein이었다. 정제된 α-1,3 glucanase를 SDS-PAGE로 분석한 결과, 단일체임을 확인하였으며, 이 때 분자량은 22,500이었다. 효소의 최적 pH는 6.5이었다. 효소의 최저온도는 37℃이었고, 열에 대한 안정성은 70℃ 이상에서 40%~60%의 효소활성이 상실함을 보였다. Detergent의 영향은 SDS에 의해 83%, Tween 20에 의해서는 약 27% 정도의 활성저해를 받았다. 효소활성의 금속이온에 의한 영향은 Co^2+, Mn^2_+에 의해 각각 81.8, 69.7%의 활성의 증가를 보였고 이들의 최적농도는 10mM이었으며, 반면에 Hg^2+에 의해서는 93.9%의 효소활성의 저해를 나타내었다. 또한 초기속도(30분 이내)에 금속이온에 의한 영향이 큰 것으로 나타났다. α-1,3 glucanase의 불용성 glucan에 대한 K_m 값은 2.50mM이었고, V_max는 0.0431 mM/min이었다. α-1,3 glucanase의 기질특이성을 조사한 결과, 반응 30분 후 IG와 soluble starch에는 각각73, 100%의 높은 분해력을 보였으며, raw starch, dextran T-10에 대해서는 낮은 분해력을 보였다. 한편, 인조치면세균막을 S. mutans로부터 시험관 벽에 제조한 후, α-1,3 glucanase를 처리한 결과 2시간 이내에 완전히 분해 제거되는 것을 알 수 있어 강력한 치아우식예방제로 개발될 수 있음을 보여 주었다. Streptococcus mutans has been implicated as primary causative agents of dental caries by insoluble glucan (IG) in human and experimental animals. An attempt was made to search for the α-1,3 glucanase that degrades IG produced by S. mutans. α-1,3 glucanase was detected in the culture supernatant of microorganisms, which are isolated from soils on agar medium containing IG as a sole carbon source. This Streptomyces sp. hydrolysed IG produced by immobilized S. mutans and was named as Y9343. This enzyme required α-1,3 glucan (IG) as an inducer. The optimum conditions for enzyme production were studied. The enzyme was purified by 30~70% (NH_4)_2SO_4 precipitation, anion exchange chromatography on DEAE-cellulose and gel filration on Sepadex G-75. The purified enzyme has a specific activity of 7840.0 U/mg protein giving 32.1-fold purification and final yield of 0.53%. The molecular weight was estimated to be about 22.5 kDa by SDS-PAGE. The optimum pH and temperature for enzyme reactiorr were 6.5 and 37℃, respectively and the enzyme was relatively stable at the temperature below 60℃. The activity of purified enzyme was enhanced by adding Co^2+, Mn^2+, and Mg^2+ into the medium, whereas inhiited by adding Hg^2+, Zn^2+ and SDS. The K_m and V_max value of α-1,3 glucanase for IG were estimated to be 2.50 mM and 0.0431 mM/min, respectively. The thin layer chromatographic analysis of hydrolysates from IG with α-1,3 glucanase showed that glucose was the main product of reaction. This enzyme activity was about 14 times higher than marketing dextranase as preventive agent against artificial dental caries by S. mutans in TH medium including 5% sucrose after 30 minutes.