从黑曲霉固态发酵产物中纯化α-L-鼠李糖苷酶及酶法制备普鲁宁

Purification of α-L-rhamnosidase from the Solid-state Fermentation Product of Aspergillus niger and Its Application in Enzymatic Production of Prunin

摘要:

本文研究了从黑曲霉固态发酵产物中纯化α-L-鼠李糖苷酶，并探究利用该酶转化柚皮苷制备普鲁宁。用黑曲霉固态发酵柚皮产生α-L-鼠李糖苷酶，通过40%~80%硫酸铵沉淀、疏水层析、亲和层析和凝胶过滤层析，从黑曲霉固态发酵产物中分离纯化得到了1种α-L-鼠李糖苷酶；该酶为单体分子量约为160 kDa；它由二硫键连接的两个肽段构成，其中有一个大肽段分子量约为130 kDa。其水解柚皮苷的最适温度为50~60 ℃，最适pH 4.0~5.0，米氏常数和最大酶反应速度分别为0.24 μmol/mL和312.5 U/mL。用该酶转化柚皮苷制备普鲁宁的最适反应时间为60~90 min，柚皮苷转化率达98%以上。转化产物中普鲁宁的含量在95%以上，普鲁宁的分解产物柚皮素的含量小于5.0%。用从黑曲霉固态发酵产物中纯化的α-L-鼠李糖苷酶制备普鲁宁具有热稳定性好、底物亲和力强、转化率高和副产物少等优点，为开发酶法制备普鲁宁的工艺提供了重要参考。

Abstract:

In this study, α-L-rhamnosidase was purified from the product of solid-state fermentation of pomelo peel powder by Aspergillus niger and used to convert naringin to prunin. α-L-rhamnosidase was isolated and purified by precipitation using ammonium sulfate at concentrations ranging from 40% to 80%, hydrophobic interaction chromatography, affinity chromatography, and gel filtration chromatography. The enzyme was found to be a monomer with molecular weight of approximately 160 kDa and contained two polypeptides linked by a disulfide bond. The larger polypeptide had a molecular weight of 130 kDa. Naringin was hydrolyzed to prunin using this enzyme, and the optimal temperature and pH, Km, and Vmax were 50 ℃~60 ℃, 4.0~5.0, 0.24 μmol/mL, and 312.5 U/mL, respectively. The optimal hydrolysis time was 60~90 min and a conversion rate above 98% was achieved. The content of Prunin was accounted for more than 95% in the final product, while that of the byproduct naringenin was less than 5%. The approach of using α-L-rhamnosidase purified from Aspergillus niger solid-state fermentation products to prepare prunin shows advantages such as good thermostability, strong substrate affinity, high conversion rate, and fewer by-products, thus providing an important basis for the enzymatic production of prunin.