食用油生物制造高效特異性脂酶的創制

廖祥儒 喻曉蔚

摘 要：該研究利用酶的分子改造與高效制備技術，自主創制并篩選獲得食用油制造用高效底物專一性脂酶總計11種，其中3種脂肪酶具有高1，3-位置選擇性以及酯化能力，4種脂肪酶具有高轉酯化活力及高熱穩定性，1種磷脂酶具有專一性磷脂-2-酰基水解作用，適用于油脂酶法脫膠。代表性成果如下：（1）創制了適用于油脂改性的1，3-位置選擇性脂肪酶，通過共表達伴侶蛋白、基因劑量調控以及氮源發酵調控將酶活提高到11719U/mL。構建了一系列解酯耶氏酵母Lip2基因工程菌，發酵活力達到30 800 U/mL，為目前國際報道最高。（2）創制了熱穩定性酯交換特異性脂肪酶，該研究采用限定區域內的隨機突變和結構熱點位置的飽和突變的策略構建基因突變庫，創造一些全新的酶反應，大大擴展酶催化的應用范圍。（3）創制了油脂脫膠用磷脂酶，該酶對甘油三酯沒有降解作用，與國外的酶制劑相比，專一性強，對水解條件和原料的要求寬松；適應性強，使用溫度范圍較寬，成本低；利用多拷貝質粒構建的鏈霉菌磷脂酶A2高產重組菌株的活力達到3 000 U/mL，對現有的酶法脫膠工藝進行了改進，使之更加簡便和節能。

關鍵詞：酯酶 脂肪酶 食用油

Abstract：In this study，by the technology of molecular evolution and preparation， totally 11 kinds of esterase and lipases for edible oil production were created，of which three kinds of lipase showed high 1，3-position selectivity，and esterification capabilities， four kinds of lipase exhibited high transesterification activity with high thermal stability， one kind of specific phospholipase for enzymatic degumming could hydrolysis phospholipids at sn-2 position. Representative results are as follows：（1）A 1，3-positions selective lipase used for oil modification was created. Through coexpression of chaperone， gene dosage optimization and fermentation the lipase activity reached 1 1719 U/mL. Construct a series of engineered yeast strains for production of Yarrowia Lip2 and the fermentation activity reached 3 0800 U/mL， which was the highest ever reported.（2）A series of thermal stable lipases and esterases were created with high transesterification specificity.This study constructed gene library by adopting random mutations within the defined protein region and hotspot of the structure，which created some new enzyme properties and greatly expanded the scope of application.（3）A phospholipase for oil degumming was created which showed no degradation activity towards triglyceride.This enzyme was also not stringent to reaction conditions and raw material. Taking advantage of multi-copy plasmid technology， Streptomyces phospholipase A2 activity reached 3 000 U/mL. The enzymatic degumming process has been improved to make it more convenient and efficient.

Key Words：Esterases；Lipases；Edible Oil

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