食用油生物制造高效特異性脂酶的創(chuàng)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(shù)，自主創(chuàng)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(zhuǎn)酯化活力及高熱穩(wěn)定性，1種磷脂酶具有專一性磷脂-2-?；庾饔?，適用于油脂酶法脫膠。代表性成果如下：（1）創(chuàng)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(dá)伴侶蛋白、基因劑量調(diào)控以及氮源發(fā)酵調(diào)控將酶活提高到11719U/mL。構(gòu)建了一系列解酯耶氏酵母Lip2基因工程菌，發(fā)酵活力達(dá)到30 800 U/mL，為目前國際報道最高。（2）創(chuàng)制了熱穩(wěn)定性酯交換特異性脂肪酶，該研究采用限定區(qū)域內(nèi)的隨機(jī)突變和結(jié)構(gòu)熱點位置的飽和突變的策略構(gòu)建基因突變庫，創(chuàng)造一些全新的酶反應(yīng)，大大擴(kuò)展酶催化的應(yīng)用范圍。（3）創(chuàng)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(qiáng)，對水解條件和原料的要求寬松；適應(yīng)性強(qiáng)，使用溫度范圍較寬，成本低；利用多拷貝質(zhì)粒構(gòu)建的鏈霉菌磷脂酶A2高產(chǎn)重組菌株的活力達(dá)到3 000 U/mL，對現(xiàn)有的酶法脫膠工藝進(jìn)行了改進(jìn)，使之更加簡便和節(jié)能。

關(guān)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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