戊糖乳杆菌单宁酶的性质及其水解石榴皮提取鞣花酸

Characterization of a tannase from Lactiplantibacillus pentosus and application of this enzyme in extraction of ellagic acid from pomegranate peel

摘要:

【背景】鞣花酸是一种天然多酚，具有抗癌、抗氧化、抗病毒和降血压等功能，在食品、饲料和制药等行业应用广泛，市场需求量逐年增加。单宁酶能水解鞣花酸单宁生成小分子鞣花酸，但现有的酶种少，转化率低、生产成本高。【目的】从戊糖乳杆菌(Lactiplantibacillus pentosus)中克隆一个新型单宁酶基因，在大肠杆菌(Escherichia coli)中表达，研究其相关酶学性质及从石榴皮中提取鞣花酸。【方法】以戊糖乳杆菌(L. pentosus)基因组DNA为模板，克隆一个单宁酶基因(LpeTanA)。构建重组表达载体pET32a-LpeTanA，并在大肠杆菌BL21(DE3)中表达。Ni-NTA亲和层析纯化后测定酶学性质及水解石榴皮提取鞣花酸。【结果】该酶与里昂葡萄球菌(Staphylococcus lugdunensis)单宁酶同源性最高，为48.50%。纯化后重组单宁酶分子质量为75 kDa，最适pH和温度分别为pH 7.0和25 ℃，在pH 6.0-8.0和40 ℃以下稳定。该酶对没食子酸甲酯具有最高比酶活力，为137.7 U/mg。LpeTanA以石榴皮粉为底物，于25 ℃水解24 h后鞣花酸提取率为11.5%，纯度达28.0%。【结论】通过对单宁酶LpeTanA的酶学性质研究，确定了LpeTanA对石榴皮的水解特性，为单宁酶的开发和水解石榴皮提取鞣花酸提供了参考依据。

Abstract:

[Background] Ellagic acid, a natural polyphenol, has anticancer, antioxidant, antiviral, and blood pressure-lowering effects. Because of the wide applications in food, feed, and pharmaceutical industries, the demand for ellagic acid has been growing year by year. Tannases can catalyze the conversion of ellagitannins into ellagic acid. However, few tannases are available, and the application of these enzymes in production is limited due to the low conversion rate and the high production cost. [Objective] To clone a tannase gene from Lactiplantibacillus pentosus and express in Escherichia coli, characterize the enzymatic properties of the expressed protein, and use the protein for the extraction of ellagic acid from pomegranate peel. [Methods] The tannase gene (LpeTanA) was cloned from the genomic DNA of L. pentosus. The recombinant plasmid pET32a-LpeTanA was constructed and expressed in E. coli BL21(DE3). The enzyme purified by Ni-NTA affinity chromatography was characterized and used for the extraction of ellagic acid from pomegranate peel. [Results] The enzyme shared the highest amino acid sequence identity of 48.50% with the tannase from Staphylococcus lugdunensis. The molecular weight of the purified LpeTanA was about 75 kDa. The optimal pH and temperature of LpeTanA were pH 7.0 and 25 ℃, respectively. The enzyme was stable up to 40 ℃ and within pH 6.0-8.0. It showed the highest specific activity (137.7 U/mg) towards methyl gallate. The extraction rate and purity of ellagic acid was 11.5% and 28.0%, respectively, after pomegranate peel power was hydrolyzed by LpeTanA at 25 ℃ for 24 h. [Conclusion] LpeTanA is characterized and used to hydrolyze pomegranate peel. The results provide reference for the development of tannases and the extraction of ellagic acid by tannases from pomegranate peel.