展示南极假丝酵母脂肪酶B的毕赤酵母全细胞催化合成短链芳香酯

Synthesis of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in non-aqueous phase

摘要:

展示酶的酵母细胞作为全细胞催化剂, 既具有固定化酶的优点, 又有制备简单、成本较低的特点。本研究将细胞表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B, CALB)的重组毕赤酵母用于非水相中催化合成短链芳香酯, 通过滴定和气相色谱的方法测定底物酸的转化率, 从底物的碳链长度、醇的结构、酵母冻干粉的添加量、底物浓度及底物的酸醇摩尔比等方面考察了展示CALB的毕赤酵母全细胞催化合成短链芳香酯的特性。研究结果表明: 该全细胞催化剂可催化C10以下的酸和醇直接酯化合成多种短链芳香酯, 酸的转化率达到90%以上; 其中己酸和乙醇为酶的最适底物; 酵母冻干粉的添加量20 g/L(306.0 U/g-dry cell)、己酸浓度0.8 mol/L、酸醇摩尔比1:1.1是合成己酸乙酯的最佳条件。在此条件下反应1.5 h, 己酸的转化率达到97.3%。在现有的关于脂肪酶非水相催化合成短链芳香酯的报道中, 该全细胞催化剂显示出较好的底物耐受性以及较高的催化反应速率。因此, 展示CALB的毕赤酵母全细胞催化剂在合成短链芳香酯方面具有较大的商业化应用潜能。

Abstract:

An enzyme-displaying yeast as a whole-cell biocatalyst seemed an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, we tried to use a recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) to catalyze the synthesis of short chain flavor esters in n-heptane. We studi some major influential factors of esterification reactions, such as carbon chain length of the substrates, alcohol structure, enzyme concentration, substrates concentration, molar ratio of the substrates. The acid conversions were determined by titration and gas chromatography analysis. About ten kinds of esters were synthesized successfully, and the acid conversions of eight esters reached as high as 90% after reaction for 6 h. The result also indicated that ethanol and hexanoic acid were the most suitable substrates for this whole-cell catalyst. Under the optimal reaction conditions (the amount of lipase 20 g/L (306.0 U/g-dry cell), hexanoic acid concentration 0.8 mol/L, the molar ratio of hexanoic acid to ethanol 1:1.1), hexanoic acid conversion reached 97.3% after reaction for 1.5 h. To our knowledge, the CALB-displaying P. pastoris whole-cell biocatalyst showed good tolerance for high substrates concentration and exhibited high reaction rate on esterification of short chain flavor esters among the present enzyme/cell reported. Thus, CALB-displaying P. pastoris whole-cell biocatalyst was promising in commercial application for flavor esters synthesis in non-aqueous phase.