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Tiến hóa có hướng của lipase mã hóa di truyền được cố định cho sản xuất hiệu quả biodiesel từ dầu ăn thải
Tóm tắt
Chúng tôi gần đây đã phát triển một phương pháp cố định mã hóa di truyền trong một bước, dựa trên sự kết hợp của một enzyme mục tiêu với protein tự tinh thể hóa Cry3Aa, tiếp theo là sản xuất và tách tinh thể fusion trực tiếp từ Bacillus thuringiensis. Sử dụng phương pháp này, lipase A của Bacillus subtilis đã được kết hợp gen với Cry3Aa để sản xuất một xúc tác Cry3Aa–lipA có khả năng chuyển đổi dầu dừa thành biodiesel một cách dễ dàng qua 10 chu kỳ phản ứng. Ở đây, chúng tôi nghiên cứu sự kết hợp của một lipase khác với Cry3Aa với mục tiêu tạo ra một xúc tác phù hợp cho việc chuyển đổi dầu ăn thải thành biodiesel. Việc kết hợp gen của lipase Proteus mirabilis (PML) với Cry3Aa đã cho phép sản xuất các tinh thể lipase cố định (Cry3Aa–PML) trực tiếp trong các tế bào vi khuẩn. Tuy nhiên, sự kết hợp này đã dẫn đến sự mất hoạt tính của PML, do đó chúng tôi đã thực hiện tiến hóa có hướng trên Cry3Aa–PML trong trạng thái cố định của nó in vivo, tận dụng tính chất cố định mã hóa di truyền của nó. Chiến lược mới này đã cho phép lựa chọn một đột biến PML cố định với hiệu suất xúc tác cao hơn 4.3 lần và độ ổn định được cải thiện. Xúc tác Cry3Aa–PML đã cải tiến có thể được sử dụng để xúc tác chuyển đổi dầu ăn thải thành biodiesel ít nhất trong 15 chu kỳ với sự mất mát tối thiểu về hiệu suất chuyển đổi. Tính chất mã hóa di truyền của nền tảng cố định Cry3Aa-fusion của chúng tôi cho phép thực hiện cả tiến hóa có hướng và sàng lọc các enzyme cố định trực tiếp in vivo. Công trình này là ví dụ đầu tiên về việc sử dụng tiến hóa có hướng để tối ưu hóa một enzyme trong trạng thái cố định của nó, cho phép xác định một đột biến mà sẽ khó thấy khi sàng lọc dạng hòa tan của nó. Chúng tôi chứng minh rằng xúc tác Cry3Aa–PML thu được phù hợp cho việc chuyển đổi có thể tái sử dụng dầu ăn thải thành biodiesel.
Từ khóa
#Tiến hóa có hướng; lipase mã hóa di truyền; dầu ăn thải; biodiesel; cố định enzymeTài liệu tham khảo
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