New Stereospecific Synthesis of Bioactive Compounds Using Lipase as Catalyst in Organic Solvent

有机溶剂中脂肪酶催化新型立体定向合成生物活性化合物

• 批准号: 04556014
• 负责人: ODA Junichi
• 金额: $ 10.18万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 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/grant/KAKENHI-PROJECT-04556014/
• 关键词: lipase; stereospecific synthesis; organic solvent; cyanohydrin; transesterification; lipase activator; 活性化因子; 大量発現; 封入体; リパーゼ活性化因子; シャペロン; Catalytic triad; 融合タンパク質; 動的速度論的光学分割; 光学活性シアノヒドリン類; 固定化リパーゼ

Using lipases as catalyst in organic solvent, optically pure cyanohydrins in (s) -configuration were stereoselectively synthesized by transesterification reaction in almost 100% chemical yeild. When disaccharide such as trehalose or sucrose was added at the immobilization of lipases, the carbohydrate added keeps lipase in optimal hydration state and increases the maximal lipase activity. The addition of carbohydrate also increases the durability of the enzyme in organic solvent. Lipase was inhibited by organophosphorus esterase inhibitors in organic solvent, but not inhibited in aqueous solvent. This indicates that the lipase catalyzes the transesterification reaction as a typical serine esterase in organic solvent. Lipase and its activator genes from Pseudomonas aeruginosa TE3285 were cloned and expressed in Escherichia coli. The product lipase was not secreted, but stored in the cells as inactive inclusion bodies. The activator protein was expressed and purified. The lipase activity were recovered by refolding the inclusion bodies under the presence of the activator protein. They are reactivated through a 1 : 1 complex without ATP.

在有机溶剂中，以脂肪酶为催化剂，通过酯交换反应，立体选择性地合成了光学纯的（S）-构型氰醇，化学产率几乎为100%。在固定化脂肪酶时加入海藻糖或蔗糖等二糖，可使脂肪酶保持最佳水合状态，提高最大酶活。碳水化合物的加入也增加了酶在有机溶剂中的耐久性。有机磷酯酶抑制剂在有机溶剂中对脂肪酶有抑制作用，但在水溶剂中对脂肪酶无抑制作用。这表明该脂肪酶在有机溶剂中作为典型的丝氨酸酯酶催化酯交换反应。从铜绿假单胞菌TE 3285中克隆了脂肪酶及其激活因子基因，并在大肠杆菌中表达。产物脂肪酶不被分泌，而是以无活性的包涵体形式储存在细胞中。表达并纯化激活蛋白。包涵体在激活蛋白的存在下复性，酶活恢复。它们在没有ATP的情况下通过1：1复合物重新激活。

项目成果

Nakatani, T.: "Chemical inactivation of lipase in organic solvent: A lipase from Pseudomonas aeruginosa TE3285 is more like a typial serine enzyme in an organic solvent than aqueous media" Biosci. Biotech. Biochem. 56. 1118-1123 (1992)

Nakatani, T.：“有机溶剂中脂肪酶的化学失活：来自铜绿假单胞菌 TE3285 的脂肪酶在有机溶剂中比在水介质中更像是典型的丝氨酸酶”Biosci。

Nakai, K.: "Kinetic resolution of racemic α-aminonitriles Via stereo-selective N-acetylation catalyzed by lipase in organic solvent" Bull. Inst Chen Res. Kyoto Unn.70. 333-337 (1992)

Nakai，K.：“在有机溶剂中通过脂肪酶催化的立体选择性 N-乙酰化进行外消旋 α-氨基三酯的动力学拆分”Bull。Inst Chen Res.70 (1992)。

M.Chihara-Siomi: "Purification,Molecular Cloning,and Expression of Lipase from Pseudomonas aeruginosa" Arch.Biochem.Biophys.296. 505-513 (1992)

M.Chihara-Siomi：“铜绿假单胞菌脂肪酶的纯化、分子克隆和表达”Arch.Biochem.Biophys.296。

Ohshima-Hirayama, N.: "Lipase from Pseudomonas aeruginosa-Production in Escherichia coli and activation in vitro with a protein from the downstream gene" Eur. J. Biochem.215. 239-246 (1993)

Ohshima-Hirayama，N.：“来自铜绿假单胞菌的脂肪酶 - 在大肠杆菌中生产并用来自下游基因的蛋白质进行体外激活”Eur。

西岡孝明: "Pseudomonas aeruginosa TE3285 リパーゼ活性化遺伝子(lipB)産物の機能〓." 生物工学. 72. 194-196 (1994)

Takaaki Nishioka：“铜绿假单胞菌 TE3285 脂肪酶激活基因 (lipB) 产物的功能。生物技术”72. 194-196 (1994)。