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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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Cloning of an AA13 Polysaccharide Monooxygenase Gene from Neurospora crassa into the plasmid pEX2B / Ho Ta Giap, Ngo Nguyen Vu, Vu Van Van // Tạp chí Khoa học và Công nghệ - Đại học Nguyễn Tất Thành . - 2020. - p. 13-18. - ISSN: 2615-9015
Ho Chi Minh City : Nguyen Tat Thanh University, 2020
6 p.
Ký hiệu phân loại (DDC): 660
Polysaccharide monooxygenases (PMOs) cleave glycoside linkages in polysaccharides, which has great potential in manufacturing biofuel from polysaccharide biomasses. Therefore, heterologous production of PMOs is required for the development of their application. We aim to clone PMO genes into the plasmid pEX2B to express PMOs in Aspergillus oryzae, a filamentous fungus widely used for industrial enzyme production. Here we cloned the NCU08746 gene, an
AA13 PMO from Neurospora crassa, into pEX2B. We successfully designed primers and amplified the NCU08746 gene from the N. crassa genome. NCU08746 was then cloned into the pEX2B vector. The recombinant plasmid pEX2B-NCU08746 was subsequently transferred into A. tumefaciens AGL1 prior to being transferred into A. oryzae. This work provides the foundation for further study on PMOs expression in A. oryzae.
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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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Study on AA10 expression in E. coli / Vu Van Van, Ngo Thi Cam Nhung // Tạp chí Khoa học và Công nghệ - Đại học Nguyễn Tất Thành . - 2021. - tr. 01-05. - ISSN: 2615-9015
Tp. Hồ Chí Minh : Trường Đại học Nguyễn Tất Thành, 2021
5 tr.
Ký hiệu phân loại (DDC):
The GlcNAc-binding protein A (GbpA) has been known as a virulent factor of Vibrio vulnificus pathogen. Domain 1 of GbpA adhesion takes responsibility of binding both human intestine and the chitinous surface. The domain 1 structure is similar to a polysaccharide monooxygenase (PMO) AA10-type (PMO), which catalyzed oxidation toward the recalcitrant chitin polymer. The role of the VvPMO10 module in catalytic functions has not been fulfilled characterized. To aim of the VvPMO10 study, this protein was cloned to the pET22b system and transformed into the E. coli BL21 (DE3) strain. The recombinant enzyme was expressed at 37 0C with IPTG induced. Total protein was checked by SDS-PAGE method and stained using Coomassie blue solution. The target band showed a band of 20 kDa as expectation. Thus, the heterologous protein was expressed successfully in E. coli BL21 (DE3) strain and becomes the materials for future study.
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