Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Characterization of Homocysteine ${\gamma}$-Lyase from Submerged and Solid Cultures of Aspergillus fumigatus ASH (JX006238)

  • El-Sayed, Ashraf S. (Microbiology Department, Faculty of Science, Zagazig University) ;
  • Khalaf, Salwa A. (Microbiology Department, Faculty of Science, Zagazig University) ;
  • Aziz, Hani A. (Microbiology Department, Faculty of Science, Zagazig University)
  • Received : 2012.08.28
  • Accepted : 2012.12.19
  • Published : 2013.04.28
Download PDF
⟨ Previous Next ⟩

Abstract

Among 25 isolates, Aspergillus fumigatus ASH (JX006238) was identified as a potent producer of homocysteine ${\gamma}$-lyase. The nutritional requirements to maximize the enzyme yield were optimized under submerged (SF) and solid-state fermentation (SSF) conditions, resulting in a 5.2- and 2.3-fold increase, respectively, after the last purification step. The enzyme exhibited a single homogenous band of 50 kDa on SDS-PAGE, along with an optimum pH of 7.8 and pH stability range of 6.5 to 7.8. It also showed a pI of 5.0, as detected by pH precipitation with no glycosyl residues. The highest enzyme activity was obtained at $37-40^{\circ}C$, with a $T_m$ value of $70.1^{\circ}C$. The enzyme showed clear catalytic and thermal stability below $40^{\circ}C$, with $T_{1/2}$ values of 18.1, 9.9, 5.9, 3.3, and 1.9 h at $30^{\circ}C$, $35^{\circ}C$, $40^{\circ}C$, $50^{\circ}C$, and $60^{\circ}C$, respectively. Additionally, the enzyme $K_r$ values were 0.002, 0.054, 0.097, 0.184, and 0.341 $S^{-1}$ at $30^{\circ}C$, $35^{\circ}C$, $40^{\circ}C$, $50^{\circ}C$, and $60^{\circ}C$, respectively. The enzyme displayed a strong affinity to homocysteine, followed by methionine and cysteine when compared with non-S amino acids, confirming its potency against homocysteinuria-related diseases, and as an anti-cardiovascular agent and a specific biosensor for homocysteinuria. The enzyme showed its maximum affinity for homocysteine ($K_m$ 2.46 mM, $K_{cat}\;1.39{\times}10^{-3}\;s^{-1}$), methionine ($K_m$ 4.1 mM, $K_{cat}\;0.97{\times}10^{-3}\;s^{-1}$), and cysteine ($K_m$ 4.9 m M, $K_{cat}\;0.77{\times}10^{-3}\;s^{-1}$). The enzyme was also strongly inhibited by hydroxylamine and DDT, confirming its pyridoxal 5'-phosphate (PLP) identity, yet not inhibited by EDTA. In vivo, using Swiss Albino mice, the enzyme showed no detectable negative effects on platelet aggregation, the RBC number, aspartate aminotransferase, alanine aminotransferase, or creatinine titer when compared with negative controls.

Keywords

References

  1. Abdel-Azeem, A. M. 2010. The history, fungal biodiversity, conservation and future perspectives for mycology in Egypt. IMA Fungus 1: 123-142. https://doi.org/10.5598/imafungus.2010.01.02.04
  2. Benko, P. V., T. C. Wood, and I. H. Segel. 1967. Specificity and regulation of the methionine transport in filamentous fungi. Arch. Biochem. Biophys. 122: 783-804. https://doi.org/10.1016/0003-9861(67)90187-7
  3. Beruter, J., J.-P. Colombo, and C. Bachmann. 1978. Purification and properties of arginase from human liver and erythrocytes. Biochem. J. 175: 449-454.
  4. Caddick, M. X., D. Peters, and A. Platt. 1994. Nitrogen regulation in fungi. Antoine van Leeuwenhoek 65: 169-177. https://doi.org/10.1007/BF00871943
  5. Carmel, R. and D. W. Jacobsen. 2001. Homocysteine in Health and Disease. Cambridge University Press.
  6. De Bree, A., M. Verschuren, D. Kromhout, L. A. Kluijtmans, and H. J. Blom. 2002. Homocysteine determinants and the evidence to what extent homocysteine determines the risk of coronary heart disease. Pharmacol. Rev. 54: 599-618. https://doi.org/10.1124/pr.54.4.599
  7. Dias, B. and B. Weimer. 1998. Purification and characterization of L-methionine γ-lyase from Brevibacterium linens BL2. Appl. Environ. Microbiol. 64: 3327-3331.
  8. dos Passos, J. B., M. Vanhalewyn, and R. L. Brandao. 1992. Glucose induced activation of plasma membrane H(+)-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation, and the initiation of glycolysis. Biochem. Biophys. Acta 1136: 57-67. https://doi.org/10.1016/0167-4889(92)90085-P
  9. El-Sayed, A. S. A. 2010. Microbial L-methioninase, molecular characterization, and therapeutic applications. Appl. Microbiol. Biotechnol. 86: 445-467. https://doi.org/10.1007/s00253-009-2303-2
  10. El-Sayed, A. S. A. 2009. L-Methioninase production by Aspergillus flavipes under solid-state fermentation. J. Basic Microbiol. 49: 331-341. https://doi.org/10.1002/jobm.200800318
  11. El-Sayed, A. S. A. 2011. Purification and characterization of a new L-methioninase from Aspergillus flavipes under solid state fermentation. J. Microbiol. 49: 130-140. https://doi.org/10.1007/s12275-011-0259-2
  12. El-Sayed, A. S. A., A. A. Shindia, and Y. Zaher. 2012. L-Amino acid oxidase from filamentous fungi: Screening and Optimization. Ann. Microbiol. 62: 773-784. https://doi.org/10.1007/s13213-011-0318-2
  13. Faleev, N. G., M. V. Troitskaya, E. A. Paskonova, M. B. Saporovskaya, and V. M. Belikov. 1996. L-Methionine $\gamma$-lyase in Citrobacter intermedius cells. Stereo chemical requirements with respect to the thiol structure. Enzyme Microb. Technol. 19: 590-593. https://doi.org/10.1016/S0141-0229(96)00071-3
  14. Garraway, M. O. and R. C. Evans 1984. Fungal Nutrition and Physiology. Wiley-Interscience.
  15. Graham, I. M., L. E. Daly, H. M. Refsum, K. Robinson, L.E. Brattstrom, P. M. Ueland, et al. 1997. Plasma homocysteine as a risk factor for vascular disease. JAMA 277: 1775-1781. https://doi.org/10.1001/jama.1997.03540460039030
  16. Green, S. M., E. Eisenstein, P. McPhie, and P. Hensley. 1990. The purification and characterization of arginase from Saccharomyces cerevisiae. J. Biol. Chem. 265: 1601-1607.
  17. Han, Q., M. Lenz, Y. Tan, M. Xu, X. Sun, X. Tan, et al. 1998. High expression, purification and properties of recombinant homocysteine $\alpha$, $\gamma$-lyase. Protein Expr. Purif. 14: 267-274. https://doi.org/10.1006/prep.1998.0955
  18. Ito, S., T. Nakamura, and Y. Eguchi, 1976. Purification and characterization of methioninase from Pseudomonas putida. J. Biochem. 79: 1263-1272.
  19. Jennings, D. H. 1995. The Physiology of Fungal Nutrition, 1st Ed. Cambridge University Press, Cambridge
  20. Kebeish, R. M. and A. S. A. El-Sayed. 2012. Morphological and molecular characterization of L-methioninase-producing Aspergilli. Afr. J. Biotechnol. [In Press].
  21. Khalaf, S. A. and A. S. A. El-Sayed, 2009. L-Methioninase production by filamentous fungi: Screening and optimization under submerged conditions. Curr. Microbiol. 58: 219-226. https://doi.org/10.1007/s00284-008-9311-9
  22. Killham, K., N. D. Lindley, and M. Wainwright. 1981. Inorganic sulfur oxidation by Aureobasidium pullulans. Appl. Environ. Microbiol. 42: 629-631.
  23. Kudou, D., S. Misaki, M. Yamashita, T. Tamura, T. Takakura, T. Yoshioka, et al. 2007. Structure of the antitumor enzyme Lmethionine $\gamma$-lyase from Pseudomonas putida at 1.8 A resolution. J. Biochem. 141: 535-544. https://doi.org/10.1093/jb/mvm055
  24. Lockwood, B. and G. Coombs. 1991. Purification and characterization of methionine $\gamma$-lyase from Trichomonas vaginalis. Biochem. J. 279: 675-682.
  25. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.
  26. Merricks, D. L. and R. L. Salsbury. 1974. Involvement of vitamin B6 in the dethiomethylation of methionine by rumen microorganisms. J. Appl. Microbiol. 28: 106-111.
  27. Monreal, J. and E. Reese. 1969. The chitinase of Serratia marcescens. Can. J. Microbiol. 15: 689-696. https://doi.org/10.1139/m69-122
  28. Morris, M. S. 2003. Homocysteine and Alzheimer's disease. Lancet Neurol. 2: 425-428. https://doi.org/10.1016/S1474-4422(03)00438-1
  29. Moubasher, A. H. 1993. Soil Fungi in Qatar and Other Arab Countries. Center of Scientific and Applied Research, University of Qatar, Qatar.
  30. Mrtinez-Cuesta, M. C., C. Pelaez, J. Eagles, M. J. Gasson, T. Requena, and S. B. Hanniffy. 2006. YtjE from Lactococcus lactis IL1403 is a C-S lyase with $\alpha$, $\gamma$-elimination activity towards methionine. Appl. Environ. Microbiol. 72: 4878-4884. https://doi.org/10.1128/AEM.00712-06
  31. Mudd, S. H., F. Skovby, H. L. Levy, K. D. Pettigerw, B. Wilcken, R. E. Pyeritz, et al. 1985. The natural history of homocysteinuria due to cystathionine $\beta$-synthase deficiency. Am. J. Hum. Genet. 37: 1-31.
  32. Nakayama, T., N. Esaki, H. Tanaka, and K. Soda. 1988. Chemical modifications of cysteine residues of L-methionine $\gamma$- lyase. Agric. Biol. Chem. 52: 177-183. https://doi.org/10.1271/bbb1961.52.177
  33. Pandey, A., C. R. Soccol, J. A. Rodriguez-Leon, and P. Nigam. 2001. Solid State Fermentation in Biotechnology. A siathech. Inc, New Delhi.
  34. Percudani, R. and A. Peracchi. 2003. Agenomic overview of pyridoxal-phosphate dependent enzymes. EMBO Rep. 4: 850-854. https://doi.org/10.1038/sj.embor.embor914
  35. Pratt, D. S. and M. M. Kaplan. 2000. Evaluation of abnormal liver enzyme results in asymptomatic patients. N. Engl. J. Med. 342: 1266-1271. https://doi.org/10.1056/NEJM200004273421707
  36. Raper, K. B. and D. I. Fennell. 1965. The Genus Aspergillus. The Williams and Wilkins Company, Baltimore.
  37. Refsum, H. 1998. Homocysteine and Cardiovascular disease. Annu. Rev. Med. 49: 31-62. https://doi.org/10.1146/annurev.med.49.1.31
  38. Ruiz-Herrera, J. and R. L. Starkey. 1969. Dissimilation of methionine by a demethiolase of Aspergillus species. J. Bacteriol. 99: 764-770.
  39. Ruiz-Herrera, J. and R. Starkey. 1970. Dissimilation of methionine by Achromobacter starkeyi. J. Bacteriol. 104: 1286-1293.
  40. Sato, D., W. Yamagata, S. Harada, and T. Nozaki. 2008. Kinetic characterization of methionine $\gamma$-lyase from the enteric protozoan parasite Entamoeba histolytica against physiological substrates and trifluoromethionine, a promising lead compound against amoebiasis. FEBS J. 275: 548-560. https://doi.org/10.1111/j.1742-4658.2007.06221.x
  41. Schuh, S., D. S. Rosenblatt, and B. A. Cooper. 1984. Homocysteinuria and megalo-blastic anemia responsive to vitamin B12 therapy. Nat. Eng. J. Med. 310: 686-690.
  42. Sharma, M., B. S. Chadha, M. Kaur, S. K. Ghatora, and H. S. Saini. 2007 Molecular characterization of multiple xylanase producing thermophilic/thermotolerant fungi isolated from composting materials. Lett. Appl. Microbiol. 46: 526-535.
  43. Skye, G. E. and H. Segel. 1970. Independent regulation of cysteine and cysteine transport in Penicillium chrysogenum. Arch. Biochem. Biophys. 138: 306-318. https://doi.org/10.1016/0003-9861(70)90311-5
  44. Stanger, O. 2004. The potential role of homocysteine in percutaneous coronary interventions (PCI): Review of current evidence and plausibility of action. Cell Mol. Biol. 50: 953-988.
  45. Sun, X., Z. Yang, S. Li, Y. Tan, N. Zhang, X. Wang, et al. 2003. In vivo efficiency of recombinant methioninase is enhanced by the combination of polyethylene glycol conjugation and pyridoxal 5-phosphate supplementation. Cancer Res. 63: 8377-8383.
  46. Suzuki, T., S. Akiyama, S. Fujimoto, M. Ishikawa, Y. Nakao, and H. Fukuda. 1976. The aconitase of yeasts. IV. Studies of iron and sulfur in yeast aconitase. J. Biochem. 80: 199-804.
  47. Takakura, T., T. Ito, S. Yagi, Y. Notsu, T. Itakura, T. Nakamura, et al. 2006. High-level expression and bulk crystallization of recombinant L-methionine $\gamma$-lyase, an anticancer agent. Appl. Microbiol. Biotechnol. 70: 183-192. https://doi.org/10.1007/s00253-005-0038-2
  48. Tanaka, H., N. Esaki, and K. Soda. 1977. Properties of Lmethionine $\gamma$-lyase from Pseudomonas ovalis. Biochemistry 16: 100-106. https://doi.org/10.1021/bi00620a016
  49. Tanaka, H., N. Esaki, T. Yamamoto, and K. Soda. 1976. Purification and properties of methioninase from Pseudomonas ovalis. FEBS Lett. 66: 307-311. https://doi.org/10.1016/0014-5793(76)80528-5
  50. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionsspecific gap penalties and weight matrix choice. Nucleic Acid Res. 22: 4673-4680. https://doi.org/10.1093/nar/22.22.4673
  51. Trinder, P. 1969. Determination of blood glucose using an oxidase-peroxidase system with non-carcinogenic chromogen. J. Clin. Pathol. 22: 158-161. https://doi.org/10.1136/jcp.22.2.158
  52. Wei, X. L., J. F. Wei, T. Li, L. Y. Qiao, Y. L. Liu, T. Huang, and S. H. He. 2007. Purification, characterization and potent lung lesion activity of an L-amino acid oxidase from Agkistrodon blomhoffii ussurensis snake venom. Toxicon 50: 1126-1139. https://doi.org/10.1016/j.toxicon.2007.07.022
  53. Yano, T., M. Ito, K. Tomita, K. Kumagai, and T. Tochikura. 1988. Purification and properties of glutaminase from Aspergillus oryzae. J. Ferment. Technol. 66: 137-143. https://doi.org/10.1016/0385-6380(88)90039-8
  54. Zhao, W.-F., X.-H. Ma, X.-M. Jia, Y. Ma, X. Li, K-P. Guo, et al. 2008. Isolation of a homocysteine $\gamma$-lyase producing bacterium and study of its enzyme production conditions. J. Appl. Microbiol. 104: 1042-1050. https://doi.org/10.1111/j.1365-2672.2007.03656.x

Cited by

  1. Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH vol.82, pp.6, 2013, https://doi.org/10.1134/s0026261713060143
  2. Screening, morphological and molecular characterization of fungi producing cystathionine γ-lyase vol.66, pp.1, 2013, https://doi.org/10.1556/abiol.66.2015.1.10
  3. Purification, immobilization, and biochemical characterization ofL-arginine deiminase from thermophilicAspergillus fumigatusKJ434941: Anticancer activityin vitro vol.31, pp.2, 2013, https://doi.org/10.1002/btpr.2045
  4. Transcriptional and Proteomic Profiling of Aspergillus flavipes in Response to Sulfur Starvation vol.10, pp.12, 2013, https://doi.org/10.1371/journal.pone.0144304
  5. Genome editing approaches: manipulating of lovastatin and taxol synthesis of filamentous fungi by CRISPR/Cas9 system vol.101, pp.10, 2017, https://doi.org/10.1007/s00253-017-8263-z
  6. Purification and Characterization of Ornithine Decarboxylase from Aspergillus terreus; Kinetics of Inhibition by Various Inhibitors vol.24, pp.15, 2013, https://doi.org/10.3390/molecules24152756
  7. Purification and Characterization of Anabaena flos-aquae Phenylalanine Ammonia-Lyase as a Novel Approach for Myristicin Biotransformation vol.30, pp.4, 2013, https://doi.org/10.4014/jmb.1908.08009
  8. Exploiting the Biosynthetic Potency of Taxol from Fungal Endophytes of Conifers Plants; Genome Mining and Metabolic Manipulation vol.25, pp.13, 2013, https://doi.org/10.3390/molecules25133000
  9. Biochemical Properties of Tyrosinase from Aspergillus terreus and Penicillium copticola; Undecanoic Acid from Aspergillus flavus, an Endophyte of Moringa oleifera, Is a Novel Potent Tyrosinase Inhibit vol.26, pp.5, 2013, https://doi.org/10.3390/molecules26051309
  10. Biosynthesis and Anti-Mycotoxigenic Activity of Zingiber officinale Roscoe-Derived Metal Nanoparticles vol.26, pp.8, 2021, https://doi.org/10.3390/molecules26082290
  11. Microbial Tyrosinase: Biochemical, Molecular Properties and Pharmaceutical Applications vol.14, pp.3, 2013, https://doi.org/10.13005/bpj/2229
  12. Efficient biocontrol of Spodoptera littoralis by Aspergillus nidulans, an endophyte of Lantana camara vol.67, pp.4, 2013, https://doi.org/10.1080/09670874.2020.1771472
  13. Purification and Biochemical Characterization of Taxadiene Synthase from Bacillus koreensis and Stenotrophomonas maltophilia vol.89, pp.4, 2013, https://doi.org/10.3390/scipharm89040048