4-乙烯基愈创木酚(4-vinylguaiacol,4-VG)在制药、化妆品和食品工业中应用广泛,具有重要的商业价值,目前主要通过化学法合成,不环保且有副产物。通过酚酸脱羧酶(phenolic acid decarboxylase,PAD)催化阿魏酸(ferulic acid,FA)实现非氧化脱羧的生物法合成4-VG被认为是一种具有前景的方法,然而,FA和4-VG的抑制效应导致转化率低、产物浓度低、无重复利用,限制了该方法的实际应用。该研究将萎缩芽孢杆菌(Bacillus atrophaeus)来源的酚酸脱羧酶(Bapad)展示于酿酒酵母表面(SD-Bapad),并同步在毕赤酵母中表达,用于比较酶学特性及其对高浓度底物、产物和有机溶剂环境的耐受性。随后,采用双相反应体系在5L生物反应器中应用SD-Bapad进行4-VG的生产。结果显示,在55 ℃下孵育1 h后SD-Bapad仍保留54.7%相对酶活性,在pH 3和pH 10下孵育1 h仍保留65%以上相对酶活性,而游离酶几乎失活,且其在高浓度底物、产物及有机溶剂条件下表现出更强的耐受性。此外,SD-Bapad在有机/水两相体系中重复使用6次后仍保持63%的酶活性。最终,在5L反应器中利用SD-Bapad作为全细胞催化剂,将FA高效转化为4-VG,在20 h内获得约为157.4 g/L(1 047.9 mmol/L),产率约为7.9 g/(L·h),摩尔转化率约为84.8%。研究结果表明,酿酒酵母表面展示技术可显著提升Bapad的稳定性和重复使用性,为工业化生物法生产4-VG提供了新途径和潜在解决方案。
4-Vinylguaiacol (4-VG) is widely used in the pharmaceutical, cosmetic and food industries and has important commercial value.At present, it is mainly synthesised by chemical methods, which are not environmentally friendly and have by-products.The biological synthesis of 4-VG by non-oxidative decarboxylation of ferulic acid (FA) catalyzed by phenolic acid decarboxylase (PAD) is considered to be a promising method.However, the inhibition effect of FA and 4-VG leads to a low conversion rate, low product concentration, and no reusability, which limits the practical application of this method.In this study, the phenolic acid decarboxylase (Bapad) from Bacillus atrophaeus was displayed on the surface of S.cerevisiae (SD-Bapad) and expressed in Pichia pastoris to compare its enzymatic properties and tolerance to high concentrations of substrates, products and organic solvents.Subsequently, a two-phase reaction system was used to produce 4-VG in a 5 L bioreactor using SD-Bapad.Results showed that SD-Bapad retained 54.7% of the relative enzyme activity after incubation at 55 ℃ for 1 h, and retained more than 65% of the relative enzyme activity after incubation at pH 3 and 10 for 1 h, while the free enzyme was almost inactivated, and showed stronger tolerance under high concentration of substrates, products and organic solvents.In addition, SD-Bapad still maintained 63% enzyme activity after repeated use in the organic / water two-phase system 6 times.Finally, FA was efficiently converted to 4-VG in a 5 L reactor using SD-Bapad as a whole-cell catalyst.Efficient conversion of FA to 4-VG yielded approximately 157.4 g/L (1 047.9 mmol/L) within 20 hours, the yield was about 7.9 g/(L/h), and the molar conversion rate was about 84.8%.Results showed that the surface display technology of S.cerevisiae could significantly improve the stability and reusability of Bapad, which provided a new way and potential solution for industrial biological production of 4-VG.
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