Spectroscopic and Kinetic Studies on the Active Site Structure and Substrate Specificities of Pullulanase.

普鲁兰酶活性位点结构和底物特异性的光谱和动力学研究。

• 批准号: 04660097
• 负责人: HIROMI Keitaro
• 金额: $ 0.7万
• 依托单位: Fukuyama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04660097/
• 关键词: pullulanase; cyclodextrins; active site of enzyme; プルラナーゼ; マルトオリゴ糖; オリゴ糖アリコール; 酵素反応速度論; 変異酵素

1) In the interaction of cyclodextrins (CDs) with pullulanase from Klebsiella pneumoniae, it has been known that beta-CD has the highest affinity compared with alpha- and gamma-CDs. Two lines of approach have strongly indicated that some side chain of the enzyme is included in only beta-CD ring. First, mono-tosylated beta-CD among the three CDs has the most decreased affinity. Second, adamantan carboxylate which is known to be included in the ring of beta-CD most remarkably decreased the binding affinity.2) Pullulanase from Klebsiella aerogenes has been found to have significantly different (better) K_m and k_<cat> values and also much stronger inhibition by cyclodextrins. Moreover, the shape of the difference fluorescence spectra caused by pullulan and cyclodextrins are significantly different from those of the enzyme from Klebsiella pneumoniae.3) Several kinds of site-directed mutagenesis of pullulanase from Klebsiella aerogenes were successfully done to determine the roles of amono acid residues importantly involved in the enzyme reaction.

1) 在环糊精 (CD) 与肺炎克雷伯菌支链淀粉酶的相互作用中，已知与 α-和 γ-CD 相比，β-CD 具有最高的亲和力。两种方法强烈表明酶的某些侧链仅包含在β-CD环中。首先，三种 CD 中单甲苯磺酰化的 β-CD 的亲和力下降幅度最大。其次，已知包含在β-CD环中的金刚烷羧酸盐最显着地降低了结合亲和力。2)已发现来自产气克雷伯氏菌的支链淀粉酶具有显着不同（更好）的K_m和k_<cat>值，并且环糊精的抑制作用也更强。而且，支链淀粉和环糊精引起的差异荧光光谱的形状与肺炎克雷伯菌酶的荧光光谱形状显着不同。3)成功地对产气克雷伯菌支链淀粉酶进行了多种定点诱变，以确定在酶反应中重要的氨基酸残基的作用。

项目成果

Hiroyuki Iwamoto et al.: "Comparison binding of the of β-cyclodextrin and α-and γ-cyclodextrins with pullulanase from Klebsiella pncumoniae as Studied by equilibrium and Kinetic fluo ro metry" J.Biochem. 116. 1264-1268 (1994)

Hiroyuki Iwamoto 等人：“通过平衡和动力学荧光测定法研究的 β-环糊精、α-和 γ-环糊精与支链淀粉酶的结合比较”J.Biochem. 116. 1264-1268 (1994)

M.Yamashita, T.Kinoshita, M.Ihara, T.Mikawa, Y.Murooka: "Random Mutagenesis of Pullulanase from Klebsiella aerogenes for Studies of the Structure and Function of the Enzyme" J.Biochem.116. 1233-1240 (1994)

M.Yamashita、T.Kinoshita、M.Ihara、T.Mikawa、Y.Murooka：“产气克雷伯菌普鲁兰酶的随机诱变，用于研究酶的结构和功能”J.Biochem.116。

松本 大 ら: "プルラナーゼの基質結合部位の同定"

Dai Matsumoto 等人：“普鲁兰酶底物结合位点的鉴定”

Mitsuo Yamashita et al.: "Random muutagenesis ot pullu lanase from Klebsiella aerognes for studies of the structure and furction of the exzyme" J.Biochem. 116. 1233-1240 (1994)

Mitsuo Yamashita 等人：“来自克雷伯菌的支链淀粉酶的随机诱变，用于研究酶的结构和功能”J.Biochem。

H.Iwamoto et al.: "Interaction between Pullulanase from Klebsiella pneumoniae and Cydodextrins" J.Biochemistry. 113. 93-96 (1993)

H.Iwamoto 等人：“肺炎克雷伯菌支链淀粉酶与环糊精之间的相互作用”J.Biochemistry。