Advertisement

Marine Biotechnology

, Volume 17, Issue 4, pp 479–492 | Cite as

Chondroitin Lyase from a Marine Arthrobacter sp. MAT3885 for the Production of Chondroitin Sulfate Disaccharides

  • Varsha Kale
  • Ólafur FriðjónssonEmail author
  • Jón Óskar Jónsson
  • Hörður G. Kristinsson
  • Sesselja Ómarsdóttir
  • Guðmundur Ó. Hreggviðsson
Original Article

Abstract

Chondroitin sulfate (CS) saccharides from cartilage tissues have potential application in medicine or as dietary supplements due to their therapeutic bioactivities. Studies have shown that depolymerized CS saccharides may display enhanced bioactivity. The objective of this study was to isolate a CS-degrading enzyme for an efficient production of CS oligo- or disaccharides. CS-degrading bacteria from marine environments were enriched using in situ artificial support colonization containing CS from shark cartilage as substrate. Subsequently, an Arthrobacter species (strain MAT3885) efficiently degrading CS was isolated from a CS enrichment culture. The genomic DNA from strain MAT3885 was pyro-sequenced by using the 454 FLX sequencing technology. Following assembly and annotation, an orf, annotated as family 8 polysaccharide lyase genes, was identified, encoding an amino acid sequence with a similarity to CS lyases according to NCBI blastX. The gene, designated choA1, was cloned in Escherichia coli and expressed downstream of and in frame with the E. coli malE gene for obtaining a high yield of soluble recombinant protein. Applying a dual-tag system (MalE-Smt3-ChoA1), the MalE domain was separated from ChoA1 with proteolytic cleavage using Ulp1 protease. ChoA1 was defined as an AC-type enzyme as it degraded chondroitin sulfate A, C, and hyaluronic acid. The optimum activity of the enzyme was at pH 5.5–7.5 and 40 °C, running a 10-min reaction. The native enzyme was estimated to be a monomer. As the recombinant chondroitin sulfate lyase (designated as ChoA1R) degraded chondroitin sulfate efficiently compared to a benchmark enzyme, it may be used for the production of chondroitin sulfate disaccharides for the food industry or health-promoting products.

Keywords

Chondroitin sulfate Chondroitin sulfate lyase Marine Arthrobacter sp. 

Notes

Acknowledgments

This work was supported by a grant from the Technology Development Fund and the AVS R&D Fund of the Ministry of Fisheries and Agriculture in Iceland and the European Union, projects 265992 (Amylomics) and 311932 (SeaBioTech). We thank Solveig Olafsdottir, Brynjar Örn Ellertsson and Dr. Björn Viðar Aðalbjörnsson for technical assistance, Dr. Josef Altenbuchner for providing the expression vectors and Dr. Bryndis Björnsdottir for critical reading of the manuscript.

References

  1. Aguiar JA, Lima CR, Berto AGA, Michelacci YM (2003) An improved methodology to produce Flavobacterium heparinum chondroitinases, important instruments for diagnosis of diseases. Biotechnol Appl Biochem 37:115–127PubMedCrossRefGoogle Scholar
  2. Ajisaka K, Agawa S, Nagumo S, Kurato K, Yokoyama T, Arai K, Miyazaki T (2009) Evaluation and comparison of the antioxidative potency of various carbohydrates using different methods. J Agric Food Chem 57:3102–3107PubMedCrossRefGoogle Scholar
  3. Albertini R, De Luca G, Passi A, Moratti R, Abuja PM (1999) Chondroitin-4-sulfate protects high-density lipoprotein against copper-dependent oxidation. Arch Biochem Biophys 365:143–149PubMedCrossRefGoogle Scholar
  4. Aziz R, Bartels D, Best A, Dejongh M, Disz T, Edwards R, Formsma K, Gerdes S, Glass E, Kubal M, Meyer F, Olsen G, Olson R, Osterman A, Overbeek R, Mcneil L, Paarmann D, Paczian T, Parrello B, Pusch G, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST server: rapid annotations using subsystems technology. BMC Genomics 9:75PubMedCentralPubMedCrossRefGoogle Scholar
  5. Baici A, Horler D, Moser B, Hofer HO, Fehr K, Wagenhauser FJ (1992) Analysis of glycosaminoglycans in human serum after oral-administration of chondroitin sulfate. Rheumatol Int 12:81–88PubMedCrossRefGoogle Scholar
  6. Bakalash S, Rolls A, Lider O, Schwartz M (2007) Chondroitin sulfate-derived disaccharide protects retinal cells from elevated intraocular pressure in aged and immunocompromised rats. Invest Ophthalmol Vis Sci 48:1181–1190PubMedCrossRefGoogle Scholar
  7. Bali JP, Cousse H, Neuzil E (2001) Biochemical basis of the pharmacologic action of chondroitin sulfates on the osteoarticular system. Semin Arthritis Rheum 31:58–68PubMedCrossRefGoogle Scholar
  8. Black GW, Elmabrouk ZH, Vincent F, Zhang M, Smith NL, Turkenburg JP, Charnock SJ, Taylor EJ (2011) Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations. Proteins 79:965–974PubMedCrossRefGoogle Scholar
  9. Bradbury EJ, Moon LD, Popat RJ, King VR, Bennett GS, Patel PN, Fawcett JW, Mcmahon SB (2002) Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416:636–640PubMedCrossRefGoogle Scholar
  10. Campo GM, Avenoso A, Campo S, D’ascola A, Traina P, Sama D, Calatroni A (2008) NF-kB and caspases are involved in the hyaluronan and chondroitin-4-sulphate-exerted antioxidant effect in fibroblast cultures exposed to oxidative stress. J Appl Toxicol 28:509–517PubMedCrossRefGoogle Scholar
  11. Caterson B, Mahmoodian F, Sorrell JM, Hardingham TE, Bayliss MT, Carney SL, Ratcliffe A, Muir H (1990) Modulation of native chondroitin sulfate structure in tissue-development and in disease. J Cell Sci 97:411–417PubMedGoogle Scholar
  12. Conte A, Volpi N, Palmieri L, Bahous I, Ronca G (1995) Biochemical and pharmacokinetic aspects of oral treatment with chondroitin sulfate. Arzneimittel-Forschung 45–2:918–925Google Scholar
  13. Cygler M, Fethiere J, Eggimann B (1999) Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes. J Mol Biol 288:635–647PubMedCrossRefGoogle Scholar
  14. Cygler M, Lunin VV, Li YG, Linhardt RJ, Miyazono H, Kyogashima M, Kaneko T, Bell AW (2004) High-resolution crystal structure of Arthrobacter aurescens chondroitin AC lyase: an enzyme–substrate complex defines the catalytic mechanism. J Mol Biol 337:367–386PubMedCrossRefGoogle Scholar
  15. Cygler M, Shaya D, Hahn BS, Bjerkan TM, Kim WS, Park NY, Sim JS, Kim YS (2008) Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid. Glycobiology 18:270–277PubMedGoogle Scholar
  16. Dower WJ, Miller JF, Ragsdale CW (1988) High-efficiency transformation of Escherichia-coli by high-voltage electroporation. Nucleic Acids Res 16:6127–6145PubMedCentralPubMedCrossRefGoogle Scholar
  17. Du Souich P, García A, Vergés J, Montell E (2009) Immunomodulatory and anti-inflammatory effects of chondroitin sulphate. J Cell Mol Med 13:1451–1463PubMedCentralPubMedCrossRefGoogle Scholar
  18. Dumon-Seignovert L, Cariot G, Vuillard L (2004) The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3). Protein Expr Purif 37:203–206PubMedCrossRefGoogle Scholar
  19. Ebert S, Schoeberl T, Walczak Y, Stoecker K, Stempfl T, Moehle C, Weber BHF, Langmann T (2008) Chondroitin sulfate disaccharide stimulates microglia to adopt a novel regulatory phenotype. J Leukoc Biol 84:736–740PubMedCrossRefGoogle Scholar
  20. Ernst S, Langer R, Cooney CL, Sasisekharan R (1995) Enzymatic degradation of glycosaminogIycans. Crit Rev Biochem Mol 30:387–444CrossRefGoogle Scholar
  21. Gabay C, Medinger-Sadowski C, Gascon D, Kolo F, Finckh A (2011) Symptomatic effect of chondroitin sulfate 4&6 in hand osteoarthritis: the Finger Osteoarthritis Chondroitin Treatment Study (FACTS). Arthritis Rheum 63:3383–3391PubMedCrossRefGoogle Scholar
  22. Gonzalez RP, Soares FDD, Farias RF, Pessoa C, Leyva A, Viana GSD, Moraes MO (2001) Demonstration of inhibitory effect of oral shark cartilage on basic fibroblast growth factor-induced angiogenesis in the rabbit cornea. Biol Pharm Bull 24:151–154PubMedCrossRefGoogle Scholar
  23. Gu K, Linhardt RJ, Laliberte M, Zimmermann J (1995) Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum. Biochem J 312(Pt 2):569–577PubMedCentralPubMedGoogle Scholar
  24. Harris NG, Nogueira MS, Verley DR, Sutton RL (2013) Chondroitinase enhances cortical map plasticity and increases functionally active sprouting axons after brain injury. J Neurotrauma 30:1257–1269PubMedCentralPubMedCrossRefGoogle Scholar
  25. Hashemian S, Marschinke F, Af Bjerken S, Stromberg I (2014) Degradation of proteoglycans affects astrocytes and neurite formation in organotypic tissue cultures. Brain Res 1564:22–32PubMedCrossRefGoogle Scholar
  26. Hiyama K, Okada S (1975) Amino acid composition and physicochemical characterization of chondroitinase from Arthrobacter aurescens. J Biochem-Tokyo 78:1183–1190PubMedGoogle Scholar
  27. Hobel CFV, Marteinsson VT, Hauksdottir S, Fridjonsson O, Skirnisdottir S, Hreggvidsson GO, Kristjansson JK (2004) Use of low nutrient enrichments to access novel amylase genes in silent diversity of thermophiles. World J Microbiol Biotechnol 20:801–809CrossRefGoogle Scholar
  28. Hobel CFV, Marteinsson VT, Hreggvidsson GO, Kristjansson JK (2005) Investigation of the microbial ecology of intertidal hot springs by using diversity analysis of 16S rRNA and chitinase genes. Appl Environ Microbiol 71:2771–2776PubMedCentralPubMedCrossRefGoogle Scholar
  29. Hong SW, Kim BT, Shin HY, Kim WS, Lee KS, Kim YS, Kim DH (2002) Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ-15, a human intestinal anaerobic bacterium. Eur J Biochem 269:2934–2940PubMedCrossRefGoogle Scholar
  30. Ishiko T, Naitoh M, Kubota H, Yamawaki S, Ikeda M, Yoshikawa K, Fujita H, Yamaguchi H, Kurahashi Y, Suzuki S (2013) Chondroitinase injection improves keloid pathology by reorganizing the extracellular matrix with regenerated elastic fibers. J Dermatol 40:380–383PubMedCrossRefGoogle Scholar
  31. Ishizeki K, Hiraki Y, Kubo M, Nawa T (1997) Sequential synthesis of cartilage and bone marker proteins during transdifferentiation of mouse Meckel’s cartilage chondrocytes in vitro. Int J Dev Biol 41:83–89PubMedGoogle Scholar
  32. Jerosch J (2011) Effects of glucosamine and chondroitin sulfate on cartilage metabolism in OA: outlook on other nutrient partners especially omega-3 fatty acids. Int J Rheumatol 2011:1–17CrossRefGoogle Scholar
  33. Ji D, Roman M, Zhou J, Hildreth J (2007) Determination of chondroitin sulfate content in raw materials and dietary supplements by high-performance liquid chromatography with ultraviolet detection after enzymatic hydrolysis: single-laboratory validation. J AOAC Int 90:659–669PubMedCentralPubMedGoogle Scholar
  34. Jobe KL, Odman-Ghazi SO, Whalen MM, Vercruysse KP (2003) Interleukin-12 release from macrophages by hyaluronan, chondroitin sulfate A and chondroitin sulfate C oligosaccharides. Immunol Lett 89:99–109PubMedCrossRefGoogle Scholar
  35. Kawaguchi Y, Sugiura N, Kimata K, Kimura M, Kakuta Y (2013) The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein. FEBS Lett 587:3943–3948PubMedCrossRefGoogle Scholar
  36. Kelly G (1998) The role of glucosamine sulfate and chondroitin sulfates in the treatment of degenerative joint disease. Altern Med Rev 3:27–39PubMedGoogle Scholar
  37. Konradsdottir M, Perttula M, Pere J, Viikari L, Kristjansson JK (1991) In situ enrichment of thermophilic acetate-utilizing bacteria. Syst Appl Microbiol 14:190–195CrossRefGoogle Scholar
  38. Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  39. Lee A, Langer R (1983) Shark cartilage contains inhibitors of tumor angiogenesis. Science 221:1185–1187PubMedCrossRefGoogle Scholar
  40. Linhardt RJ, Galliher PM, Cooney CL (1986) Polysaccharide lyases. Appl Biochem Biotechnol 12:135–176PubMedCrossRefGoogle Scholar
  41. Linn S, Chan T, Lipeski L, Salyers AA (1983) Isolation and characterization of 2 chondroitin lyases from Bacteroides thetaiotaomicron. J Bacteriol 156:859–866PubMedCentralPubMedGoogle Scholar
  42. Liu SG, Tao K, Long ZF, Gao Q, Tao Y, Jin H, Ran HY, Liu K (2005) Isolation of Serratia marcescens as a chondroitinase-producing bacterium and purification of a novel chondroitinase AC. Biotechnol Lett 27:489–493CrossRefGoogle Scholar
  43. Lovu M, Dumais G, Du Souich P (2008) Anti-inflammatory activity of chondroitin sulfate. Osteoarthr Cartil 16:S14–S18Google Scholar
  44. Lunin VV, Li YG, Linhardt RJ, Miyazono H, Kyogashima M, Kaneko T, Bell AW, Cygler M (2004) High-resolution crystal structure of Arthrobacter aurescens chondroitin AC lyase: an enzyme–substrate complex defines the catalytic mechanism. J Mol Biol 337:367–386PubMedCrossRefGoogle Scholar
  45. Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538PubMedCentralPubMedGoogle Scholar
  46. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  47. Morrison M (1977) Therapeutic application of C4S, appraisal of biological properties. Folia Angiol 25:225–232Google Scholar
  48. Motejadded H, Altenbuchner J (2009) Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing. Biotechnol Lett 31:543–549PubMedCrossRefGoogle Scholar
  49. Numakura M, Kusakabe N, Ishige K, Ohtake-Niimi S, Habuchi H, Habuchi O (2010) Preparation of chondroitin sulfate libraries containing disulfated disaccharide units and inhibition of thrombin by these chondroitin sulfates. Glycoconj J 27:479–489PubMedCrossRefGoogle Scholar
  50. Ooshima H, Sakata M, Harano Y (1983) Adsorption of cellulase from Trichoderma viride on cellulose. Biotechnol Bioeng 25:3103–3114PubMedCrossRefGoogle Scholar
  51. Pendleton JC, Shamblott MJ, Gary DS, Belegu V, Hurtado A, Malone ML, Mcdonald JW (2013) Chondroitin sulfate proteoglycans inhibit oligodendrocyte myelination through PTPsigma. Exp Neurol 247:113–121PubMedCrossRefGoogle Scholar
  52. Petursdottir SK, Kristjansson JK (1996) The relationship between physical and chemical conditions and low microbial diversity in the Blue Lagoon geothermal lake in Iceland. FEMS Microbiol Ecol 19:39–45CrossRefGoogle Scholar
  53. Ronca F, Palmieri L, Panicucci P, Ronca G (1998) Anti-inflammatory activity of chondroitin sulfate. Osteoarthr Cartil 6:14–21PubMedCrossRefGoogle Scholar
  54. Ruffell B, Poon GFT, Lee SSM, Brown KL, Tjew SL, Cooper J, Johnson P (2011) Differential use of chondroitin sulfate to regulate hyaluronan binding by receptor CD44 in inflammatory and interleukin 4-activated macrophages. J Biol Chem 286:19179–19190PubMedCentralPubMedCrossRefGoogle Scholar
  55. Salyers AA, Obrien M (1980) Cellular location of enzymes involved in chondroitin sulfate breakdown by Bacteroides-thetaiotaomicron. J Bacteriol 143:772–780PubMedCentralPubMedGoogle Scholar
  56. Sasisekharan R, Prabhakar V, Raman R, Capila I, Bosques CJ, Pojasek K (2005) Biochemical characterization of the chondroitinase ABC I active site. Biochem J 390:395–405PubMedCentralPubMedCrossRefGoogle Scholar
  57. Shim K-W, Kim D-H (2007) Cloning and expression of chondroitinase AC from Bacteroides stercoris HJ-15. Protein Expr Purif 58:222–228PubMedCrossRefGoogle Scholar
  58. Starkey ML, Bartus K, Barritt AW, Bradbury EJ (2012) Chondroitinase ABC promotes compensatory sprouting of the intact corticospinal tract and recovery of forelimb function following unilateral pyramidotomy in adult mice. Eur J Neurosci 36:3665–3678PubMedCrossRefGoogle Scholar
  59. Su HS, Tkalec AL, Fink D, Blain F, Zhang-Sun GY, Laliberte M, Bennett DC, Gu KF, Zimmermann JJF (2000) Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum. Appl Environ Microbiol 66:29–35PubMedCentralPubMedCrossRefGoogle Scholar
  60. Sugiura N, Setoyama Y, Chiba M, Kimata K, Watanabe H (2011) Baculovirus envelope protein ODV-E66 is a novel chondroitinase with distinct substrate specificity. J Biol Chem 286:29026–29034PubMedCentralPubMedCrossRefGoogle Scholar
  61. Sugiura N, Ikeda M, Shioiri T, Yoshimura M, Kobayashi M, Watanabe H (2013) Chondroitinase from baculovirus Bombyx mori nucleopolyhedrovirus and chondroitin sulfate from silkworm Bombyx mori. Glycobiology 23:1520–1530PubMedCrossRefGoogle Scholar
  62. Wegerer A, Sun T, Altenbuchner J (2008) Optimization of an E. coli l-rhamnose-inducible expression vector: test of various genetic module combinations. BMC Biotechnol 8:2PubMedCentralPubMedCrossRefGoogle Scholar
  63. Xie HX, Nie P, Chang MX, Liu Y, Yao WJ (2005) Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G4. Arch Microbiol 184:49–55PubMedCrossRefGoogle Scholar
  64. Yamada S, Sugahara K (2008) Potential therapeutic application of chondroitin sulfate/dermatan sulfate. Curr Drug Discov Technol 5:289–301PubMedCrossRefGoogle Scholar
  65. Zhou J, Nagarkatti PS, Zhong Y, Nagarkatti M (2009) Effect of chondroitin sulfate and its degraded disaccharide on the development of experimental autoimmune encephalomyelitis. J Neurochem 108:140Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Varsha Kale
    • 1
    • 2
  • Ólafur Friðjónsson
    • 1
    Email author
  • Jón Óskar Jónsson
    • 1
  • Hörður G. Kristinsson
    • 1
  • Sesselja Ómarsdóttir
    • 2
  • Guðmundur Ó. Hreggviðsson
    • 1
    • 3
  1. 1.MatísReykjavíkIceland
  2. 2.Faculty of Pharmaceutical SciencesUniversity of IcelandReykjavíkIceland
  3. 3.Department of BiologyUniversity of IcelandReykjavíkIceland

Personalised recommendations