Structure and function of yeast mannoprotein degrading enzymes

酵母甘露糖蛋白降解酶的结构和功能

• 批准号: 11660069
• 负责人: NAKAJIMA Tasuku
• 金额: $ 2.3万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660069/
• 关键词: Mannoprotein; α-1, 2-mannosidase; Yeast cell wall; α-mannanase gene; Bacterial mannosidase; 1,6-α-マンナナーゼ

To Clarify the structure of yeast mannoprotein degrading enzymes, at first, a gene (aman2) encoding 1, 2-α-D-mannosidase from Bacillus sp.M-90 was cloned. The putative aman2 was 5928 base pairs long and encoded a mature 1, 2-α-D-mannosidase protein of 1939 amino acids and a signal peptide of 37 amino acids. The deduced amino acid sequence exhibits no similarity to other 1, 2-α-D-mannosidases identified in various organisms. The recombinant protein was verified by immunoblotting with anti-1, 2-α-D-mannosidease antibodies and showed hydrolytic activity to 1, 2-α-D-mannobise and yeast mannan. Secondly, a gene (aman6) encoding endo-1, 6-α-mannanase, a yeast mannan backbone degrading enzyme from Bacilles circulans was cloned and the gene was expressed in Escherichis coli. The putative aman6 wa 1767 base pares long and encodes a mature 1, 6-α-mannanase protein of 589 amino acids and a signal peptide of 36 amino acids. The purified mature 1, 6-α-D-mannanase from E.coli transformant showed 61-kDa protein, and N-terminal amino acid sequense and other general properties of the recombinant enzyme were identical to those of 1, 6-α-D-mannanase from Bacillus circulans TN-31.A data base serch indicates both aman2 and aman6 were novel genes.

为了阐明酵母甘露糖蛋白降解酶的结构，首先克隆了芽孢杆菌M-90编码1，2-α-D-甘露糖苷酶的基因aman2。该基因全长5928个碱基，编码1，2-α-D-甘露糖苷酶成熟蛋白，编码37个氨基酸的信号肽。推导的氨基酸序列与其他在各种生物中发现的1，2-α-D-甘露糖苷酶没有相似之处。用抗1，2-α-D-甘露糖苷酶抗体进行免疫印迹验证，表明重组蛋白对1，2-α-D-甘露糖和酵母甘露聚糖具有一定的水解性。其次，克隆了环状芽孢杆菌的酵母甘露聚糖酶内切-1，6-α-甘露聚糖酶基因(Aman6)，并在大肠杆菌中进行了表达。编码1，6-α-甘露聚糖酶成熟蛋白，编码589个氨基酸和36个氨基酸组成的信号肽。从大肠杆菌转化子中纯化的成熟1，6-α-D-甘露聚糖酶含有61 kDa的蛋白质，其N末端氨基酸序列等一般性质与环状芽孢杆菌TN-31中的1，6-α-D-甘露聚糖酶完全相同。

项目成果

前田浩 他: "Alkaline-Resistance Model of Subtilisin ALPI, a Novel Alkaline Subtilisin."Journal of Biochemistry. (印刷中). (2001)

Hiroshi Maeda 等人：“枯草杆菌蛋白酶 ALPI 的碱性抗性模型，一种新型碱性枯草杆菌蛋白酶”。生物化学杂志（2001 年出版）。

Y.Maruyama and T.Nakajima: "The aman6 gene encoding a yeast mannnan backbone degrading 1, 6-a-D-mannanase in Bacillus circulans : cloning, sequence analysis, and expression."Biosci.Biotecnol.Biochem.. 64. 2018-2020 (2000)

Y.Maruyama 和 T.Nakajima：“编码酵母甘露聚糖主链的 aman6 基因可降解环状芽孢杆菌中的 1, 6-a-D-甘露聚糖酶：克隆、序列分析和表达。”Biosci.Biotecnol.Biochem.. 64. 2018-2020

E.Ichishima, N.Taya, M.Ikeguchi, Y.Chiba, M.Nakamura, C.Kawabata, T.Inoue, K.Takahasi, T.Minetoki, K.Ozeki, C.Kumagai, K.Gomi, T.Yoshoda and T.Nakajima: "Molecular and enzymatic properties of recombinant 1, 2-a-mannosodase from Aspergillus saitoi overexpr

E.Ichishima、N.Taya、M.Ikeguchi、Y.Chiba、M.Nakamura、C.Kawabata、T.Inoue、K.Takahasi、T.Minetoki、K.Ozeki、C.Kumagai、K.Gomi、T.

前田浩 他: "Identification of N-terminal Autodigestion Target Site in Subitilisin ALPI."Biosci.Biotechnol.Biochem.(note). (印刷中). (2001)

Hiroshi Maeda 等人：“Subitilisin ALPI 中 N 末端自消化靶位点的识别”。Biosci.Biotechnol.Biochem.（注）（出版中）。

鈴木哉子 他: "Characterization of Recombinant Yeast Exo-β-1,3-Glucanase (Exglp) Expressed in Escherichia coli Cells."Biosci.Biotechnol. Biochem.. (印刷中). (2001)

Yako Suzuki 等人：“大肠杆菌细胞中表达的重组酵母外切-β-1,3-葡聚糖酶 (Exglp) 的表征”。Biosci.Biochem.（出版中）。