Tạo dòng gen mã hóa enzyme AA9 Polysaccharide Monooxygenase AN3860 trên vector pEX2B hướng tới biểu hiện trên Aspergillus oryzae

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Thông tin xuất bản: Tạp chí Khoa học và Công nghệ - Đại học Nguyễn Tất Thành, 2020

Mô tả vật lý: tr.5

Bộ sưu tập: Báo, Tạp chí

ID: 315964

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Polysaccharide monooxygenases (PMO) là enzyme xúc tác phản ứng oxi hóa thủy giải các chuỗi carbohydrate có cấu trúc khó phá vỡ như cellulose, tinh bột và chitin. Trong lĩnh vực năng lượng sinh học, sự chuyển đổi nguồn sinh khối lignocellulose dồi dào thành đường đơn cho quá trình lên men bằng enzyme li giải cellulase có thể tăng hiệu suất đáng kể khi bổ sung enzyme nhóm AA9 PMO. Những enzyme này được tìm thấy với một số lượng khá lớn trong các loại nấm sinh trưởng sử dụng nguồn sinh khối làm cơ chất. Aspergillus nidulans thuộc nhóm nấm sợi sở hữu một số gen mã hóa cho PMO, tuy nhiên chỉ có AN3860 được biểu hiện ở mức cao khi nuôi trong môi trường bổ sung sợi rơm lúa mì làm nguồn cacbon duy nhất. Phân tích tin học trình tự gen cho thấy AN3860 thuộc loại 3 AA9 PMO có thể oxi hóa linh động cả hai vị trí C1 và C4 của liên kết glycosidic. Do đó, AN3860 có tiềm năng cải thiện được hoạt tính li giải cellulose nhưng cho đến nay vẫn chưa được mô tả. Trong nghiên cứu này, trình bày cách tiếp cận AN3860 bằng phương pháp tạo dòng biểu hiện tái tổ hợp trong Aspergillus oryzae AUT1-PlD. Để thuận lợi cho quá trình tinh sạch AN3860, đuôi CBM20 được gắn vào đầu C của gen mục tiêu. Quá trình tối ưu biểu hiện và nghiên cứu thử nghiệm hoạt tính sẽ được thực hiện tiếp theo.Polysaccharide monooxygenases (PMOs) catalyze oxidative degradation of recalcitrant carbohydrate chains in cellulose, starch, and chitin. In biofuel industry, the conversion of rich lignocellulose source to fermentable sugars by hydrolytic cellulases can be synergistically boosted by cellulose-active PMOs (AA9 PMOs) found in a vast number of fungi that grow on biomass. Aspergillus nidulans, a filamentous fungus, possess a dozen of PMO-encoding genes, but only the AN3860 is expressed at a high level when cultured with wheat straw as the sole carbon source. Bioinformatic analysis indicates that AN3860 belongs to type 3 AA9 PMO subfamily that is capable of hydroxylating both C1 and C4 of the glycosidic linkages. Therefore, AN3860 may be a potential enzyme to improve cellulose hydrolysis efficiency, which has not been characterized. In this study, we describe the AN3860 cloning into Aspergillus oryzae AUT1-PlD. To facilitate the purification of AN3860, we added a CBM20 tag to its C-terminal. The recombinant vector was designed and constructed successfully. Simultaneously, we have obtained the clone of A. oryzae carrying the target gene by the ATMT method. Further expression optimization and characterization of AN3860 by both activity assays and spectroscopic techniques are underway.

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