Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Heterologous Expression and Characterization of a Thermostable α-L-Rhamnosidase from Thermoclostridium stercorarium subsp. thermolacticum DSM 2910 and Its Application in the Biotransformation of Rutin

  • Lin Ge (Department of Medical Science and Technology, Suzhou Chien-Shiung Institute of Technology) ;
  • Yingying Liu (Department of Medical Science and Technology, Suzhou Chien-Shiung Institute of Technology) ;
  • Fangming Zhou (Department of Medical Science and Technology, Suzhou Chien-Shiung Institute of Technology) ;
  • Lingling Zhan (Department of Medical Science and Technology, Suzhou Chien-Shiung Institute of Technology) ;
  • Linguo Zhao (Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University)
  • Received : 2023.05.28
  • Accepted : 2023.07.14
  • Published : 2023.11.28
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Abstract

An α-L-rhamnosidase gene from Thermoclostridium. stercorarium subsp. thermolacticum DSM 2910 (TstRhaA) was cloned and expressed. The maximum TstRhaA activity of the protein reached 25.2 U/ml, and the molecular mass was approximately 106.6 kDa. The protein was purified 8.0-fold by Ni-TED affinity with an overall recovery of 16.6% and a specific activity of 187.9 U/mg. TstRhaA activity was the highest at 65℃ and pH 6.5. In addition, it exhibited excellent thermal stability, better pH stability, good tolerance to low concentrations of organic reagents, and high catalytic activity for p-nitrophenyl-α-L-rhamnopyranoside (pNPR). Substrate specificity studies showed that TstRhaA exhibited a high specific activity for rutin. At 60℃, pH 6.5, and 0.3 U/ml enzyme dosage, 60 g/l rutin was converted to 45.55 g/l isoquercitrin within 150 min. The molar conversion rate of rutin and the yield of isoquercitrin were 99.8% and 12.22 g/l/h, respectively. The results suggested that TstRhaA could be used for mass production of isoquercitrin.

Keywords

Acknowledgement

This work was supported by the General Project of Natural Science Research of Jiangsu Province Higher Education Institutions (Grant No. 21KJB220015), and the Science and Technology Programme of Taicang (Grant No. TC2020JC17), "Two Funds" Project of Suzhou Chien-Shiung Institute of Technology (Grant No. JXLJ202208) as well as the start-up funds for doctoral research of Suzhou Chien-Shiung Institute of Technology.

References

  1. Cho WK, Lee MM, Ma JY. 2022. Antiviral effect of isoquercitrin against influenza A viral infection via modulating hemagglutinin and neuraminidase. Int. J. Mol. Sci. 23: 13112.
  2. Kolesarova A, Michalcova K, Roychoudhury S, Baldovska S, Tvrda E, Vasicek J, et al. 2021. Antioxidative effect of dietary flavonoid isoquercitrin on human ovarian granulosa cells HGL5 in vitro. Physiol. Res. 70: 745-754. https://doi.org/10.33549/physiolres.934692
  3. Park J. 2020. Anti-anaphylactic activity of isoquercitrin (Quercetin-3-O-beta-d-Glucose) in the cardiovascular system of animals. Biomedicines 8: 139.
  4. Wu P, Liu S, Su J, Chen J, Li L, Zhang R, et al. 2017. Apoptosis triggered by isoquercitrin in bladder cancer cells by activating the AMPK-activated protein kinase pathway. Food Funct. 8: 3707-3722. https://doi.org/10.1039/C7FO00778G
  5. Gasparotto Junior A, Gasparotto FM, Lourenco EL, Crestani S, Stefanello ME, Salvador MJ, et al. 2011. Antihypertensive effects of isoquercitrin and extracts from Tropaeolum majus L.: evidence for the inhibition of angiotensin converting enzyme. J. Ethnopharmacol. 134: 363-372. https://doi.org/10.1016/j.jep.2010.12.026
  6. Wang J, Sun GX, Yu L, Wu FA, Guo XJ. 2013. Enhancement of the selective enzymatic biotransformation of rutin to isoquercitrin using an ionic liquid as a co-solvent. Bioresour. Technol. 128: 156-163. https://doi.org/10.1016/j.biortech.2012.10.098
  7. Shimada Y, Dewa Y, Ichimura R, Suzuki T, Mizukami S, Hayashi SM, et al. 2010. Antioxidant enzymatically modified isoquercitrin suppresses the development of liver preneoplastic lesions in rats induced by beta-naphthoflavone. Toxicology 268: 213-218. https://doi.org/10.1016/j.tox.2009.12.019
  8. Gasparotto Junior A, Prando TB, Leme Tdos S, Gasparotto FM, Lourenco EL, Rattmann YD, et al. 2012. Mechanisms underlying the diuretic effects of Tropaeolum majus L. extracts and its main component isoquercitrin. J. Ethnopharmacol. 141: 501-509. https://doi.org/10.1016/j.jep.2012.03.018
  9. Jiang P, Burczynski F, Campbell C, Pierce G, Austria JA, Briggs CJ. 2007. Rutin and flavonoid contents in three buckwheat species Fagopyrum esculentum, F. tataricum, and F. homotropicum and their protective effects against lipid peroxidation. Food Res. Int. 40: 356-364. https://doi.org/10.1016/j.foodres.2006.10.009
  10. Wang D, Zheng P, Chen P, Wu D. 2021. Immobilization of alpha-L-rhamnosidase on a magnetic metal-organic framework to effectively improve its reusability in the hydrolysis of rutin. Bioresour. Technol. 323: 124611.
  11. Li LJ, Liu XQ, Du XP, Wu L, Jiang ZD, Ni H, et al. 2020. Preparation of isoquercitrin by biotransformation of rutin using α-L-rhamnosidase from Aspergillus niger JMU-TS528 and HSCCC purification. Prep. Biochem. Biotechnol. 50: 1-9. https://doi.org/10.1080/10826068.2019.1655763
  12. Shin KC, Seo MJ, Oh DK, Choi MN, Kim DW, Kim YS, et al. 2019. Cloning and characterization of alpha-L-rhamnosidase from Chloroflexus aurantiacus and its application in the production of isoquercitrin from rutin. Biotechnol. Lett. 41: 419-426. https://doi.org/10.1007/s10529-019-02648-8
  13. Zhang R, Zhang BL, Xie T, Li GC, Tuo Y, Xiang YT. 2015. Biotransformation of rutin to isoquercitrin using recombinant α-L-rhamnosidase from Bifidobacterium breve. Biotechnol. Lett. 37: 1257-1264. https://doi.org/10.1007/s10529-015-1792-6
  14. Lou H, Liu X, Liu S, Chen Q. 2022. Purification and characterization of a novel alpha-L-rhamnosidase from Papiliotrema laurentii ZJU-L07 and its application in production of icariin from epimedin C. J. Fungi 8: 644.
  15. Ferreira-Lazarte A, Plaza-Vinuesa L, de Las Rivas B, Villamiel M, Munoz R, Moreno FJ. 2021. Production of α-rhamnosidases from Lactobacillus plantarum WCFS1 and their role in deglycosylation of dietary flavonoids naringin and rutin. Int. J. Biol. Macromol. 193: 1093-1102. https://doi.org/10.1016/j.ijbiomac.2021.11.053
  16. Singh P, Sahota PP, Singh RK. 2015. Evaluation and characterization of new α-L-rhamnosidase-producing yeast strains. J. Gen. Appl. Microbiol. 61: 149-156. https://doi.org/10.2323/jgam.61.149
  17. Yadav V, Yadav PK, Yadav S, Yadav KDS. 2010. α-L-rhamnosidase: a review. Process Biochem. 45: 1226-1235. https://doi.org/10.1016/j.procbio.2010.05.025
  18. Wu T, Pei J, Ge L, Wang Z, Ding G, Xiao W, et al. 2018. Characterization of a α-l-rhamnosidase from Bacteroides thetaiotaomicron with high catalytic efficiency of epimedin C. Bioorg. Chem. 81: 461-467. https://doi.org/10.1016/j.bioorg.2018.08.004
  19. Ge L, Xie J, Wu T, Zhang S, Zhao L, Ding G, et al. 2017. Purification and characterisation of a novel α-L-rhamnosidase exhibiting transglycosylating activity from Aspergillus oryzae. Int. J. Food Sci. Technol. 52: 2596-2603. https://doi.org/10.1111/ijfs.13546
  20. De Winter K, Simcikova D, Schalck B, Weignerova L, Pelantova H, Soetaert W, et al. 2013. Chemoenzymatic synthesis of α-l-rhamnosides using recombinant α-l-rhamnosidase from Aspergillus terreus. Bioresour. Technol. 147: 640-644. https://doi.org/10.1016/j.biortech.2013.08.083
  21. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. 2009. The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37: D233-D238. https://doi.org/10.1093/nar/gkn663
  22. de Araujo MEMB, Moreira Franco YE, Alberto TG, Sobreiro MA, Conrado MA, Priolli DG, et al. 2013. Enzymatic de-glycosylation of rutin improves its antioxidant and antiproliferative activities. Food Chem. 141: 266-273. https://doi.org/10.1016/j.foodchem.2013.02.127
  23. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  24. Ge L, Chen A, Pei J, Zhao L, Fang X, Ding G, et al. 2017. Enhancing the thermostability of α-L-rhamnosidase from Aspergillus terreus and the enzymatic conversion of rutin to isoquercitrin by adding sorbitol. BMC Biotechnol. 17: 21.
  25. Li B, Ji Y, Li Y, Ding G. 2018. Characterization of a glycoside hydrolase family 78 alpha-l-rhamnosidase from Bacteroides thetaiotaomicron VPI-5482 and identification of functional residues. 3 Biotech 8: 120.
  26. Manzanares P, de Graaff LH, Visser J. 1997. Purification and characterization of an α-L-rhamnosidase from Aspergillus niger. FEMS Microbiol. Lett. 157: 279-283. https://doi.org/10.1016/S0378-1097(97)00487-4
  27. Ge L, Li D, Wu T, Zhao L, Ding G, Wang Z, et al. 2018. B-factor-saturation mutagenesis as a strategy to increase the thermostability of alpha-L-rhamnosidase from Aspergillus terreus. J. Biotechnol. 275: 17-23. https://doi.org/10.1016/j.jbiotec.2018.03.013
  28. Birgisson H, Hreggvidsson GO, Fridjonsson OH, Mort A, Kristjansson JK, Mattiasson B. 2004. Two new thermostable α-L-rhamnosidases from a novel thermophilic bacterium. Enzyme Microb. Technol. 34: 561-571. https://doi.org/10.1016/j.enzmictec.2003.12.012
  29. Xie J, Zhao J, Zhang N, Xu H, Yang J, Ye J, et al. 2022. Efficient production of isoquercitin, icariin and icariside II by a novel thermostable alpha-l-rhamnosidase PodoRha from Paenibacillus odorifer with high alpha-1, 6-/alpha-1, 2- glycoside specificity. Enzyme Microb. Technol. 158: 110039.
  30. De Lise F, Mensitieri F, Tarallo V, Ventimiglia N, Vinciguerra R, Tramice A, et al. 2016. RHA-P: Isolation, expression and characterization of a bacterial α- l -rhamnosidase from Novosphingobium sp. PP1Y. J. Mol. Catal. B: Enzymatic. 134: 136-147. https://doi.org/10.1016/j.molcatb.2016.10.002
  31. Robert X, Gouet P. 2014. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42: W320-324. https://doi.org/10.1093/nar/gku316