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42번째 alanine 잔기의 proline 치환에 의한 보리 α-amylase isozyme 2의 대장균 내 발현 증가 및 기질특이성 변화
최승호(Seung-Ho Choi),장명운(Myoung-Uoon Jang),이홍균(Hong-Gyun Lee),Birte Svensson,김태집(Tae-Jip Kim) 한국식품과학회 2010 한국식품과학회지 Vol.42 No.2
보리 맥아에는 2종의 α-amylase isozyme(AMY1, AMY2)이 존재하며, 이들 효소는 80% 이상의 높은 아미노산 서열 상동성을 보이지만, calcium 의존성 등 효소의 작용특성은 서로 매우 다르다. 따라서 본 연구에서는 AMY2의 활성부위 중 2번째 β→α loop에 존재하는 42번째 alanine 잔기를 saturation mutagenesis를 이용하여 다양한 아미노산으로 치환하고, 전분 분해활성이 증가한 돌연변이를 선발하였다. 결과적으로 alanine이 proline으로 치환된 AMY2-A42P의 경우에서만 발현도가 2배 증가하는 것을 확인하였으며, 특히 정제 과정에서의 회수율 또한 4배 증가하므로 향후 효소의 생산 및 활용에 유리할 것으로 판단하였다. 이 돌연변이 효소의 calcium 의존성 및 pH 안정성 등은 AMY2와 유사한 것으로 나타났으나, 각종 전분에 대한 기질특이성은 AMY1과 AMY2의 중간적인 특성으로 변화되었다. 결국 42번째 아미노산 잔기의 proline 치환에 의해 상대적으로 발현율이 높고 기질특이성이 변화된 AMY2 유사효소의 생산이 가능하였으며, 향후 이를 이용하여 분자진화기술 등 최신 효소공학적 방법론을 적용한 다양한 연구가 가능할 것으로 기대한다. Although barley α-amylase isozyme 1 (AMY1) and 2 (AMY2) share up to 80% of amino acid sequence identity, their enzymatic properties differ remarkably. In this study, the 42<SUP>nd</SUP> alanine residue of AMY2 was replaced with another random amino acid via saturation mutagenesis. Eight out of 370 recombinant E. coli cells showing enhanced starch-hydrolyzing activity were characterized as possessing the same proline residue instead of alanine. Even though the specific activity of AMY2-A42P is reduced to 81% of wild-type, its expression level and purification yield were enhanced by approximately 2 and 4 times that of AMY2, respectively. Characterization of its enzymatic properties confirmed that AMY2-A42P is similar to that of wild-type. However, its specificity to starch substrates is likely to be intermediate between AMY1 and AMY2.
Maxillomandibular arch width differences at estimated centers of resistance
Yun-Jin Koo,Sung-Hwan Choi,Byeong-Tak Keum,Hyung-Seog Yu,Chung-Ju Hwang,Birte Melsen,Kee-Joon Lee 대한치과교정학회 2017 대한치과교정학회지 Vol.47 No.3
Objective: To evaluate the differences in maxillomandibular transverse measurements at either the crown or the estimated center of resistance (CR), and to compare values between normal occlusion and Class III malocclusion groups. Methods: Dental casts and computed tomography (CT) data from 30 individuals with normal occlusion and 30 with skeletal Class III malocclusions were evaluated. Using the casts, dental arch widths (DAWs) were measured from the cusp tips, and basal arch widths (BAWs-cast) were measured as the distance between the points at the mucogingival junction adjacent to the respective cusp tips. The BAWs determined from CT (BAWs-CT) images were measured from the estimated CRs of the teeth. Results: None of the DAW measurements or maxillomandibular DAW differences showed statistically significant intergroup differences. In contrast, the maxillary BAWs-CT and BAWs-cast were lesser in the Class III malocclusion group than in the normal occlusion group. The mandibular BAWs-CT were significantly greater in the Class III malocclusion group than in the normal occlusion group. Moreover, the maxillomandibular BAW differences on both CT and cast showed significant intergroup differences in all transverse measurements. Conclusions: The maxillomandibular DAW differences showed no significant intergroup differences. In contrast, the maxillomandibular BAW differences on both CT and cast showed significant intergroup differences in all transverse measurements. The maxillomandibular BAW differences at the estimated CRs, measured using CT or casts, can reveal underlying transverse maxillary basal arch deficiencies in patients with skeletal Class III malocclusions.
Stö,ber, Gerald,Ben-Shachar, Dorit,Cardon, M.,Falkai, Peter,Fonteh, Alfred N.,Gawlik, Micha,Glenthoj, Birte Y.,Grü,nblatt, Edna,Jablensky, Assen,Kim, Yong-Ku,Kornhuber, Johannes,McNeil, Thomas Informa UK (TaylorFrancis) 2009 The world journal of biological psychiatry Vol.10 No.2
<P>Objective. The phenotypic complexity, together with the multifarious nature of the so-called 'schizophrenic psychoses', limits our ability to form a simple and logical biologically based hypothesis for the disease group. Biological markers are defined as biochemical, physiological or anatomical traits that are specific to particular conditions. An important aim of biomarker discovery is the detection of disease correlates that can be used as diagnostic tools. Method. A selective review of the WFSBP Task Force on Biological Markers in schizophrenia is provided from the central nervous system to phenotypes, functional brain systems, chromosomal loci with potential genetic markers to the peripheral systems. Results. A number of biological measures have been proposed to be correlated with schizophrenia. At present, not a single biological trait in schizophrenia is available which achieves sufficient specificity, selectivity and is based on causal pathology and predictive validity to be recommended as diagnostic marker. Conclusions. With the emergence of new technologies and rigorous phenotypic subclassification the identification of genetic bases and assessment of dynamic disease related alterations will hopefully come to a new stage in the complex field of psychiatric research.</P>
Pichia pastoris에서 발현된 보리 알파아밀라제 Chimera 효소들의 특성
김태집,육정빈,최승호,장명운,Kim, Tae-Jip,Yuk, Jeong-Bin,Choi, Seung-Ho,Jang, Myoung-Uoon,Svensson, Birte 한국미생물학회 2010 미생물학회지 Vol.46 No.1
보리 맥아로부터 발견된 서로 다른 알파아밀라제 동질효소(AMY1, AMY2)는 80%에 달하는 높은 아미노산 서열의 상동성을 보이지만, 두 효소의 특성은 서로 달라 AMY1 효소는 낮은 농도의 칼슘 조건에서 최대 활성을 보이는 반면, AMY2 효소는 높은 칼슘이온 농도에서 높은 활성을 나타낸다. 또한 BASI (Barley ${\alpha}$-Amylase/Subtilisin Inhibitor) 단백질은 AMY2 효소만을 특이적으로 저해한다. 따라서 본 연구에서는 AMY1과 AMY2 효소의 유전자를 I, II, III의 세 부위로 나눈 후, 제한효소 처리에 의해 일부 부위를 상호 치환한 4종의 chimera 효소를 추가로 제조하고, Pichia pastoris 균주에서 대량 발현하였다. 이들 효소의 특성을 비교한 결과, 제 I 부위만이 상호치환된 AMY211 및 AMY122 효소의 경우, AMY1과 AMY2의 중간적 칼슘 의존성을 나타내었으며, BASI에 의한 저해효과는 AMY2의 제 I, II 부위를 포함하는 AMY221 효소에서만 관찰되었다. 따라서 보리 아밀라제의 제 I 부위 및 제 II 부위에 존재하는 아미노산 잔기들이 칼슘 의존성 및 BASI와의 결합에 중요한 역할을 담당하는 반면 제 III 부위는 이들 효소의 활성 차이에 영향을 미치지 않음을 확인하였다. Two different ${\alpha}$-amylase isozymes (AMY1 and AMY2) found in barley malt share up to 80% of amino acid sequence identity with each other, but their enzymatic properties differ remarkably. AMY1 shows the highest activity at low concentration of calcium ion, while AMY2 is highly active at high calcium concentration. Meanwhile, BASI (Barley ${\alpha}$-Amylase/Subtilisin Inhibitor) protein specifically inhibits only AMY2. In the present study, three separate regions in AMY genes (I, II, and III) were assigned on the basis of restriction enzyme sites and four kinds of chimeric amylases have been obtained by swapping a part of regions with each other. Each chimera gene was successfully over-expressed in Pichia pastoris. From the results of enzymatic characterization, both AMY211 and AMY122 showed the mixed or intermediate type of calcium-dependent activity between AMY1 and 2. Meanwhile, only AMY221 chimera could be significantly inhibited by BASI protein. As a result, it can be proposed that some amino acid residues in the region I and II, except region III, of barley ${\alpha}$-amylases play very important roles in calcium-dependency and interaction with BASI.