Development of new food materials using specific catalitical activities of microbial enzymes

利用微生物酶的特定催化活性开发新食品材料

• 批准号: 07556023
• 负责人: KUMAGAI Hidehiko
• 金额: $ 6.98万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556023/
• 关键词: fucosylglucose; bifidus factor; gamma-glutamyltranspeptidase; glutathione; gentiobiose; gamma-glutamyltransferase; Bifidobacterium; beta-glucodidase; γグルタミルトランスペピチダーゼ; ゲンチオビオース; γ-グルタミリトランスペプチダーゼ; γ-グルタミル-DOPA; γ-グルタミル-リジン; フコシグルコース; b-D-グルコ

1.(1) gamma-Glutamyl-DOPA,gamma-glutamyl-Gln were synthesized using gamma-glutamyltrans-peptidase (GGT) from E.coli.(2) Mechanism of low temperature (20ﾟC) induction of E.coli GGT was studied. It was found to be regulated at transcriptional level.(3) X-ray crystallographical study of GGT was performed and three dimensional structure of the main chain was obtained.(4) Catabolism pathway of glutathione in E.coli cell was elucidated.2.(1)beta-Glucosidase gene was cloned from Bifidobacterium breve. It was overexpressed in E.coli and purified to electrophoretic homogeneity and crystallized.(2) This enzyme was found to catalyze the reverse reaction of hydrolysis reaction. This enzymatic activity was used to synthesize gentiobiose and fucosyl (beta1-6) glucose by serial columns of immobilized recombinant beta-glucosidase and activated charcoal. Various strains of Bifidobacteria, Lactobacillus and other intestinal bacteria were tested if they can catabolize thus synthesized fucosyl (beta1-6) glucose. We found that 8 out of 9 Bifidobacteria could cataboliza this disaccharide but no other bacteria could.(3) alpha-Galactosidase of Bifidobacterium was found to be an inducible enzyme. We isolated this enzyme and identified its characters. Its gene was cloned and the enzyme was overxpressed in E.coli.

1. (1)用大肠杆菌的γ-谷氨酰转肽酶（GGT）合成γ-谷氨酰-多巴、γ-谷氨酰-谷氨酰胺。(2)对大肠杆菌GGT的低温诱导机制进行了研究。发现它在转录水平上受到调控。(3)对GGT进行了X射线晶体学研究，得到了其主链的三维结构。(4)阐明了谷胱甘肽在大肠杆菌细胞中的催化途径.（1）从短双歧杆菌中克隆了β-葡萄糖苷酶基因。在大肠杆菌中过表达，纯化至电泳均一并结晶。(2)发现该酶催化水解反应的逆反应。通过固定化重组β-葡萄糖苷酶和活性炭的串联柱，将这种酶活性用于合成龙胆二糖和岩藻糖基（β 1 -6）葡萄糖。测试双歧杆菌、乳杆菌和其它肠道细菌的各种菌株是否可以分解代谢如此合成的岩藻糖基（β 1 -6）葡萄糖。我们发现，9个双歧杆菌中有8个能分解这种二糖，但没有其他细菌可以。(3)发现双歧杆菌的α-半乳糖苷酶是一种诱导酶。我们分离了该酶并鉴定了其性质。克隆了其基因，并在大肠杆菌中过量表达该酶。

项目成果

布浦 直樹: "ビフィズス菌の酵素でビフィズス因子を作る" 化学と生物. 35(6). 451-457 (1997)

Naoki Nunoura：“使用双歧杆菌酶创建双歧因子”，化学与生物学 35(6) (1997)。

熊谷英彦: "微生物が作る酵素 食品分野への応用" Bulletion of the Research Institute for Food Science,Kyoto University. 60. 112-113 (1997)

熊谷秀彦：“微生物产生的酶：在食品领域的应用”京都大学食品科学研究所公告 60. 112-113 (1997)。

Wataru Hashimoto: "Low temperature inducible γ-glutamyltranspeptidase of Eschericha coli K-12" Biosci.Biotech.Biochem.61(1). 34-39 (1997)

Wataru Hashimoto：“大肠杆菌 K-12 的低温诱导型 γ-谷氨酰转肽酶”Biosci.Biotech.Biochem.61(1) (1997)。

Ping Xu: "Characterization of a lactate oxidate from a strain of gram negative becterium from soil." Applied Biochemistry and Biotechnology. 56(3). 277-288 (1996)

Ping Xu：“土壤革兰氏阴性菌菌株乳酸氧化物的表征。”

Naoki Nunoura: "Purification and characterization of β-D-glucosidase (β-D-fucosidase) from Bifidobacterium breveclb acclimated to cellobiose." Bioscience Biotechnology and Biochemistry. 60 (2). 188-193 (1996)

Naoki Nunoura：“来自适应纤维二糖的短双歧杆菌的 β-D-葡萄糖苷酶（β-D-岩藻糖苷酶）的纯化和表征。”生物科学生物技术和生物化学 60 (2)。