曲霉属α-l-鼠李糖苷酶的挖掘、酶学性质表征与应用

Mining, enzymatic characterization, and application of α-l-rhamnosidase from Aspergillus sp.

摘要:

异槲皮素是一种黄酮类化合物，具有抗氧化、抗炎和免疫调节等多种生理活性。然而，由于其含量很低，传统的提取方法难以大规模制备。【目的】α-l-鼠李糖苷酶可特异性水解天然糖苷的末端l-鼠李糖残基。本研究筛选可高效且特异性转化芦丁生产异槲皮素的菌株，并从中挖掘新型α-l-鼠李糖苷酶且应用于异槲皮素的生产，为后续规模化生产异槲皮素提供新元件。【方法】通过芦丁唯一碳源的选择培养法筛选和鉴定可特异性水解芦丁为异槲皮素的菌株；利用转录组分析，获得高效且特异性强的α-l-鼠李糖苷酶，通过结构模拟确定其结构域组成，并对其酶学性质和底物特异性进行研究；通过毕赤酵母异源表达，在5 L发酵罐中对其水解效果进行验证。【结果】通过结构模拟确定AfRhase具有5个结构域，包括1个α-结构域(结构域A)和4个β-结构域(结构域N、结构域E、结构域F和结构域C)。以芦丁为底物，重组酶AfRhase的最适温度和最适pH分别为55 ℃和4.5。重组酶AfRhase底物特异性研究表明，其具有广泛的底物特异性，可以水解芦丁、橙皮苷、柚皮苷和朝藿定C的鼠李糖基。通过5 L发酵罐体系的水解芦丁生产异槲皮素的放大验证，其可将120 g芦丁粗品(纯度70%)水解生成61 g异槲皮素，摩尔转化率为95.4%，生产效率为2.0 mmol/(L·h)。【结论】本研究成功从曲霉(Aspergillussp.) XT-1中挖掘到一种能够高效且特异性水解芦丁生成异槲皮素的α-l-鼠李糖苷酶，在毕赤酵母中异源表达，对该酶进行结构域分析和酶学性质研究，测试底物特异性，并进行5 L发酵罐的水解效果验证。综上所述，本研究拓宽了真菌来源的α-l-鼠李糖苷酶用于黄酮类化合物芦丁生物转化法的功能研究，也为异槲皮素的工业化生产奠定了基础。

Abstract:

Isoquercetin is a flavonoid with antioxidant, anti-inflammatory, and immunomodulatory activities. However, the low content in plants poses a challenge to the large-scale production of isoquercetin by the extraction method. [Objective] α-l-rhamnosidase can specifically hydrolyze the terminal l-rhamnose residues of natural glycosides. In this study, we screened the strains capable of efficiently and specifically transforming rutin to produce isoquercetin with rutin as the sole carbon source and applied the α-l-rhamnosidase to the production of isoquercetin, aiming to provide new elements for the large-scale production of isoquercetin. [Methods] The selective culture medium with rutin as the sole carbon source was used to screen and identify the strains that can specifically hydrolyze rutin into isoquercetin. The transcriptome analysis was carried out to obtain highly efficient and specific α-l-rhamnosidase, the domain composition of which was determined by structural simulation. The enzymatic properties and substrate specificity of the α-l-rhamnosidase were studied. Furthermore, the hydrolysis effect of the enzyme heterologously expressed in Pichia pastoris in a 5 L fermenter was determined. [Results] AfRhase had five domains, including one α-domain (domain A) and four β-domains (domains N, E, F, and C). With rutin as the substrate, the recombinant enzyme AfRhase showcased the best performance at 55 ℃ and pH 4.5. AfRhase had a wide range of substrates including rutin, hesperidin, naringin, and epimedin C. In a 5 L fermenter for scaled-up production of isoquercetin, P. pastoris expressing AfRhase generated 61 g isoquercetin by hydrolyzing 120 g crude rutin (purity of 70%), with the molar conversion rate of 95.4% and production efficiency of 2.0 mmol/(L·h). [Conclusion] This study for the first time discovered a highly efficient and specific α-l-rhamnosidase from Aspergillus sp. XT-1 for the production of isoquercetin from rutin and heterologously expressed this enzyme in P. pastoris. The domain composition, enzymatic properties, substrate specificity, and hydrolysis efficiency in a 5 L fermenter of this enzyme were determined. In conclusion, this study broadened the function of a fungus-derived α-l-rhamnosidase for the transformation of rutin and laid a foundation for the industrial production of isoquercetin.