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Cloning of AA9 Polysaccharide Monooxygenase gene AN3860 into pEX2B for expression in Aspergillus oryzae / Nhung Ngo Thi Cam, Van Vu Van // Tạp chí Khoa học và Công nghệ - Đại học Nguyễn Tất Thành . - 2020. - p. 5-10. - ISSN: 2615-9015
Ho Chi Minh City : Nguyen Tat Thanh University, 2020
6 p.
Ký hiệu phân loại (DDC): 660
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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Optimization of culture conditions to express AA9 Polysaccharide monooxygenases AN3860 in Escherichia coli / Ngo Thi Cam Nhung, Le Quynh Loan, Vu Van Van // Tạp chí Khoa học và Công nghệ - Đại học Nguyễn Tất Thành . - 2022. - tr. 36-43. - ISSN: 2615-9015
Ký hiệu phân loại (DDC): 615
Lignocellulose biomass is a copious source for second generation biomaterial production. The participant of Polysaccharide monooxygenases enzyme (PMO) in the reactions which convert lignocellulose biomass into monosaccharides enhances the activity and improve the efficiency of hydrolysis of hydrolase enzymes on lignocellulose substrate. Enzyme AN3860, obtained from Aspergillus nidulans strain belonging to AA9 PMO, is expected to catalyze flexibly at C1 and C4 carbon positions of β-glycosidic linkage. As an enzyme with high potential of improving cellulose crystals hydrolysis capacity, AN3860 was successfully cloned into the expression system of E. coli BL21 (DE3) strain. In this study, the culture process of recombinant strain with AN3680 gene is optimized to increase the target proteins yield, thus ensure the outcome of purification process, and save production cost. The results demonstrate that the E. coli recombinant strains grow sufficiently in TB (Terrific Broth) culture media and the highest yield of AN3680 protein achieved when the concentration of Isopropyl β-D-1- thiogalactopyranoside (IPTG) is 0.05 mM and the temperature of the reaction is 30 0C at 150 rpm. After 6 hours of induction, the biomass reaches 500 mg/L and the yield of AN3860 account for (7-10) % total protein generated. The recombinant AN3860 protein is later harvested on larger scale and purified by Ni-NTA column chromatography method for analysis of bioactivities on lignocellulose substrates in the future.
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