盐水球菌Salinicoccus ventosaetal B2-3-5的代谢产物分析及其抑制酪氨酸酶活性的机制

Analysis of metabolites from Salinicoccus ventosaetal B2-3-5 and their inhibitory mechanism of tyrosinase activity Application and Regulation in Xinjiang Key Laboratory Project (XJTSWZ-2018-01)

摘要:

[背景] 酪氨酸酶是黑色素合成过程中的关键酶，也是引起人体色素障碍性疾病和产生果蔬酶促褐变的主要原因。目前，酪氨酸酶抑制剂的开发已引起广泛关注，但一些酪氨酸酶抑制剂如熊果苷、曲酸等均存在一定的安全隐患。微生物资源丰富且具有许多优点，从微生物中寻找特异性强、高效的酪氨酸酶抑制剂已成为该领域研究的热点。[目的] 通过测定分离自新疆乌鲁木齐达坂城盐湖的盐水球菌Salinicoccus ventosaetal B2-3-5和B6-1-4代谢物提取物对酪氨酸酶活性的影响，比较2株菌发酵过程中代谢物的差异，了解所筛选菌株B2-3-5抑制酪氨酸酶活性的机制。[方法] 以曲酸为阳性对照分别测定B2-3-5和B6-1-4这2个菌株发酵产生的代谢物提取物对蘑菇酪氨酸酶的抑制活性；应用LC-MS代谢组学方法检测2株菌在相同条件下产生的所有代谢物质；采用单变量、多元变量、正交偏最小二乘判别分析（Orthogonal Partial Least Squares-Discrimination Analysis，OPLS-DA）法识别差异代谢物；利用层次聚类分析（Hierarchial Cluster Analysis，HCA）法对识别的差异物进行聚类分析；通过Kyoto Encyclopedia of Genes and Genomes （KEGG）代谢通路对比法分析这些差异代谢物主要参与的代谢途径。[结果] 菌株B2-3-5代谢物提取物对蘑菇酪氨酸酶二酚酶活性的抑制率为67%，其IC₅₀为0.277 mg/mL，同属菌株B6-1-4代谢物提取物则对酪氨酸酶无抑制活性。采用代谢组学的检测方法从2株菌的代谢物中筛选出63个差异代谢物，其中氨基酸类化合物、维生素类化合物和羧酸类化合物的种类及相对含量均是B2-3-5菌株明显高于B6-1-4菌株。通过代谢途径分析发现这些差异代谢物主要参与15个代谢通路，其中维生素B6生物合成通路的影响较为显著。[结论] 推测B2-3-5菌株可能是通过增加一些氨基酸类、维生素类及羧酸类等小分子化合物的含量来抑制酪氨酸酶活性。维生素B6代谢途径的上调也表明菌体细胞可通过产生维生素B6与酪氨酸酶中的必需氨基作用或清除酶催化循环过程中产生的活性氧自由基（reactive oxygen species，ROS）来抑制酪氨酸酶活性。

关键词:盐水球菌属;代谢产物;酪氨酸酶抑制剂;维生素B6

Abstract:

[Background] Tyrosinase is the key enzyme of melanin biosynthesis, which easily causes the pigment disorders and enzymatic browning of fruits and vegetables. At present, the development on tyrosinase inhibitor has obtained more and more attention. However some tyrosinase inhibitors have certain potential safety hazards, such as arbutin and kojic acid. Microorgansims are rich in resources and have many advantages. It has become a spot to search for tyrosinase inhibitors with strong specificity and high efficiency from microorganisms. [Objective] We investigated the effects of metabolite extracts of Salinicoccus ventosaetal strains B2-3-5 and B6-1-4 isolated from dabancheng Salt Lake of Urumqi in Xinjiang on tyrosinase activity. And also compared metabolic differences between the two strains during the fermentation. The aim of this study was to understand the inhibitory mechanism of tyrosinase activity of the strain B2-3-5. [Methods] Taken kojic acid as positive control, we determined the inhibitory effects of metabolite extracts produced by the two strains B2-3-5 and B6-1-4 on mushroom tyrosinase, respectively. We detected all the metabolites of the two strains under the same fermentation conditions by liquid chromatograph-mass spectrometer. We identified metabolic differences with univariate and multivariate analysis, Orthogonal Partial Least Squares-Discrimination Analysis (OPLS-DA) method. We carried out cluster analysis on the identified differential metabolites by Hierarchial Cluster Analysis (HCA) method and searched the differential metabolic pathways through the databases kyoto encyclopedia of genes and genomes (KEGG). [Results] These findings indicated that the inhibitory effects of metabolite extracts of the strain B2-3-5 on the diphenolase activity of mushroom tyrosinase was 67% and its IC₅₀ was estimated as 0.277 mg/mL, while the metabolite extracts of the strain B6-1-4 among the same genus had no inhibitory activity. We obtained 63 differential metabolites from the two strains by metabolomics methods. The types and relative contents of amino acids, vitamins and carboxylic acids were significantly higher in B2-3-5 than those in B6-1-4. The results of metabolic pathway analysis showed that these differential metabolites were mainly participated in 15 metabolic pathways, among which vitamin B6 biosynthesis pathway had the highest correlation with differential metabolites and fermentation process. [Conclusion] We speculated that the strain B2-3-5 might increase the contents of some small molecular compounds such as amino acids, vitamins and carboxylic acids, etc. to inhibit the tyrosinase activity. The upregulation of vitamin B6 biosynthesis pathway also suggested that the strain had the inhibitory activity of tyrosinase through producing vitamin B6 to interact with the essential amino group in tyrosinase or scavenging the reactive oxygen species (ROS) during the enzymatic catalytic process.

Key words:Salinicoccus;metabolites;tyrosinase inhibitor;vitamin B6