Tối ưu điều kiện biểu hiện AA9 polysaccharide monooxygenases tái tổ hợp trong hệ thống Escherichia coli

0 Người đánh giá. Xếp hạng trung bình 0

Tác giả: Quỳnh Loan Lê, Thị Cẩm Nhung Ngô, Văn Vân Vũ

Ngôn ngữ: vie

Ký hiệu phân loại:

Thông tin xuất bản: Tạp chí Khoa học và Công nghệ - Đại học Nguyễn Tất Thành, 2022

Mô tả vật lý: tr.36

Bộ sưu tập: Metadata

ID: 335848

Thêm vào giỏ Liên kết toàn văn

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.Sinh khối lignocellulose là nguồn nguyên liệu dồi dào cho sản xuất nhiên liệu sinh học thế hệ thứ hai. Sự tham gia của enzyme Polysaccharide monooxygenases (PMO) trong phản ứng chuyển hóa sinh khối lignocellulose thành đường đơn đóng vai trò tăng cường hoạt động và nâng cao hiệu suất thủy phân của các enzyme hydrolase trên cơ chất lignocellulose. Enzyme AN3860 thu nhận từ chủng nấm Aspergillus nidulans thuộc nhóm AA9 PMO được dự đoán có thể xúc tác linh hoạt ở vị trí carbon C1 và C4 của liên kết β-glycosidic. Với tiềm năng là một enzyme có thể cải thiện khả năng thủy phân tinh thể cellulose, AN3860 đã được dòng hóa thành công trong hệ thống biểu hiện E. coli BL21 (DE3). Trong nghiên cứu này, chủng tái tổ hợp mang gen AN3860 được tối ưu quy trình nuôi cấy nhằm thu nhận lượng lớn protein mục tiêu đảm bảo thuận lợi cho quá trình tinh sạch và tiết kiệm chi phí sản xuất. Các kết quả tối ưu quy trình tạo dòng cho thấy trong tế bào E. coli tái tổ hợp tăng trưởng tốt trong môi trường TB (Terrific Broth) và lượng protein AN3860 thu được cao nhất với nồng độ chất cảm ứng IPTG là 0,05 mM kết hợp nhiệt độ cảm ứng ở 30 0C. Sinh khối tạo thành sau khi cảm ứng 6 giờ có thể đạt giá trị hơn 500 mg/L và lượng protein AN3860 được tạo ra chiếm khoảng (7-10) % lượng protein tổng số. Protein AN3860 tái tổ hợp tiếp tục được thu nhận ở quy mô lớn hơn và tiến hành tinh sạch qua cột sắc kí Ni-NTA cho các thí nghiệm phân tích hoạt tính, khối phổ trên các cơ chất lignocellulose trong tương lai.

1. Đọc trực tuyến

1. Đọc trực tuyến

Tạo bộ sưu tập với mã QR