The catalytic activity for ginkgolic acid biodegradation, homology modeling and molecular dynamic simulation of salicylic acid decarboxylase
Graphical abstract
Introduction
As a living fossil, Ginkgo biloba is one of the oldest species of tree, which has existed on the earth for two hundred million years (Major, 1967). Ginkgo biloba mainly grows in extratropical, warm and subtropical zones, and it’s estimated that 70% of this plant is distributed in China (Son and Kim, 1998). As a traditional medicine and food materials, Ginkgo biloba seeds have been used for several thousand years. In recent decades, the medical and health properties of extracts from Ginkgo biloba leaves have attracted considerable attention (Kobus-Cisowska et al., 2014; Gu et al., 2015; Ribeiro et al., 2016; Yan et al., 2015). Additionally, Ginkgo biloba seeds can be used as food or food ingredient to make desserts, glazed fruit, beverages and tipple (Deng et al., 2011). However, the existence of the toxic ginkgolic acids (2-hydroxy-6-alkyl/alkenyl benzoic acid, mainly include C13:0, C15:0, C15:1, C17:1 and C17:2) (Yang et al., 2002) has become a main safety concern for consumption of the products derived from Ginkgo biloba (Baron-Ruppert and Luepke, 2001; Liu and Zeng, 2009). Ginkgo acids have the potential to induce sensitization and mutagenicity (Koch et al., 2000), and strong cytotoxicity (Hecker et al., 2002). They can cause allergic reaction, gene mutation and nerve damage, which leads to nausea, heartburn, allergic shock, allergic purpura, exfoliative dermatitis, gastrointestinal mucosal allergy, convulsion, paralysis and other adverse reactions. There are some reports about death cases because excessive consumption of Ginkgo biloba seeds (Miyazaki et al., 2010; Fujisawa et al., 2002). Therefore, ginkgolic acids in the ingredients derived from Ginkgo biloba seeds and leaves must be removed or the content should be controlled under 5 mg kg−1 according to the European Pharmacopoeia and United States Pharmacopoeia (Wang et al., 2015).
Supercritical CO2 extraction (Liao and Ruiming, 2011), selective adsorption (Li et al., 2014) and chromatography technique (Beek and Wintermans, 2001) are usually utilized for removing the ginkgolic acids from Ginkgo biloba. However, the methods are very costly and inefficient for treatment of the large amount of Ginkgo biloba materials. Recently, biological degradation of the food toxins has been intensively investigated (Karlovsky, 1999; Alberts et al., 2006; Friedman and Rasooly, 2013). Compared with the non-biological methods, the biodegradation methods for food toxins are safer, cheaper and low requirement for expensive equipment.
Kirimura et al. (2010) previously reported that salicylic acid carboxylase (SDC EC 4.1.1.91) can decarboxylate salicylic acid to phenol (Kirimura et al., 2010). Given that ginkgolic acids have the common skeleton of salicylic acid, it was supposed that SDC may also possess the biodegradation activity for ginkgolic acids and it was found from our experiments that SDC can decarboxylate ginkgolic acid C15:1 (Scheme 1). On this basis, the structure of SDC was built by homology modeling. The interaction of SDC with the substrate of ginkgolic acid C15:1, was analyzed by molecular docking and molecular dynamics.
Section snippets
Bacterial strains, plasmids, and cultivation
The oligonucleotide encoding of salicylic acid carboxylase (SDC) gene sequence (Genbank No. DM040453) was synthesized by GENEWIZ, Inc. (Beijing, China). Plasmid pET21a(+) (Tianjin University of Science and Technology Culture Collection Center, Tianjin, China) was used in this study to construct pET21a(+)-sdc recombinant plasmid. E. coli BL21 (DE3) (Tianjin University of Science and Technology Culture Collection Center, Tianjin, China) was used as a host system for expression of recombinant
Cloning, transformation and expression of SDC in E. coli BL21
Kirimura et al. (2010) reported that a kind of enzyme named as salicylic acid decarboxylase in Trichosporon moniliiforme WU-0401 can catalyze Kolbe-Schmitt reaction reversibly at room temperature. It not only decarboxylates salicylic acid to phenol, but also produces salicylic acid using phenol as substrate. Salicylic acid was the sole product of the carboxylation and no meta-hydroxy benzoic acid or ortho-hydroxy benzoic acid was found. They obtained the purified salicylic acid decarboxylase
Conclusion
In conclusion, the application of SDC in decarboxylation of ginkgolic acid was investigated. The results showed that the disrupted cell suspension of recombined pET21a(+)-sdc/E.coli could decrease the ginkgolic acid by 39.02 ± 0.13%, indicated that SDC could be used to degrade ginkgolic acid. In order to understand the interaction of SDC with substrates of salicylic acid and ginkgolic acid, the 3D structure of SDC was constructed by homology modeling using the crystal structure of
Conflict of interest
The authors declare no competing financial interest.
Acknowledgement
This work was supported by National Natural Science Foundation of China (Grant No. 21272171).
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