DOI QR코드

DOI QR Code

Characterization of Alpha Amylase Producing Thielaviopsis ethacetica and Its Raw Starch Hydrolyzing Ability on Different Agricultural Substrates

  • Dissanayaka, Dissanayaka M.S. (Department of Biotechnology, Faulty of Agriculture and Plantation Management, Wayamba University of Sri Lanka) ;
  • De Silva, Sembukuttige N.T. (Department of Nano Science Technology, Faculty of Technology, Wayamba University of Sri Lanka) ;
  • Attanayaka, D.P.S.T.G. (Department of Biotechnology, Faulty of Agriculture and Plantation Management, Wayamba University of Sri Lanka) ;
  • Kurera, Mihidukulasuriya J.M.S. (Department of Biotechnology, Faulty of Agriculture and Plantation Management, Wayamba University of Sri Lanka) ;
  • Fernando, Charakrawarthige A.N. (Department of Nano Science Technology, Faculty of Technology, Wayamba University of Sri Lanka)
  • Received : 2018.12.05
  • Accepted : 2019.04.12
  • Published : 2019.09.28

Abstract

The present study reports the morphological and molecular characterization of the fungal strain, CMSS06 and evaluates its raw starch hydrolyzing ability in four different agricultural substrates (rice bran, banana peel, cassava tubers, and coconut water). The potential use of each agricultural substrate to replace the expensive fermentation media was evaluated with six different fermentation media: rice bran (RB), banana peel (BP), cassava starch (CS), cassava in coconut water (CSCW), cassava in modified coconut water (CMCW), and pure Coconut water (CW). The fungal strain CMSS06 was identified as Thielaviopsis ethacetica by the analysis of the ITS sequences. The T. ethacetica alpha amylase enzyme exhibited maximum alpha amylase activity at 72 h, pH 7.0, and $40^{\circ}C$ on soluble starch. This species resulted in the highest enzyme activity (mU/ml) of 26.06, 10.89, 58.82, 14.2, and 54.67 with the RB, BP, CS, CSCW, and CMCW fermentation media, respectively. The results indicate that CS can be used as a carbon substrate and CMCW can be used to accelerate the fermentation by T. ethacetica. The enzyme was partially purified by 40-60% ammonium sulphate fraction, and it showed total enzyme activity, total protein content, specific activity, purification fold, and a recovery of 2400 mU, 30 mg, 80 mU/mg, 2.7, and 71.1%, respectively. The molecular mass of the T. ethacetica alpha amylase was estimated on SDS-PAGE, and two bands around 50 kDa and 70 kDa were identified. The present study implies that T. ethacetica can produce alpha amylase, and it can be used to hydrolyze raw starch during the fermentation processes.

Keywords

Thielaviopsis ethacetica;molecular characterization;alpha amylase;raw starch;cassava

Acknowledgement

Supported by : National Science Foundation in Sri Lanka, Wayamba University of Sri Lanka

References

  1. Randez-Gil F, Prieto JA, Murcia A, Sanz P. 1995. Construction of baker's yeast strains that secrete Aspergillus oryzae alpha-amylase and their use in bread making. J. Cereal Sci. 21: 185-193. https://doi.org/10.1016/0733-5210(95)90034-9
  2. Couto SR, Sanroman MA. 2006. Application of solid-state fermentation to food industry- A review. J. Food Eng. 76 : 291-302. https://doi.org/10.1016/j.jfoodeng.2005.05.022
  3. Nielsen JE, Borchert TV. 2000. Protein engineering of bacterial ${\alpha}$-amylases. Biochimi. Biophys. Acta 1543: 253-274. https://doi.org/10.1016/S0167-4838(00)00240-5
  4. Bruinenberg PM, Hulst AC, Faber A, Voogd RH. 1996. A process for surface sizing or coating of paper. European Patent Application EP0, 690,170 A1.
  5. Haq I, Ali S, Javed MM, Hameed U, Saleem A, Adnan F, et al. 2010. Production of alpha amylase from a randomly induced mutant strain of Bacillus amyloliquefaciens and its application as a desizer in textile industry. Pak. J. Bot. 42: 473-484.
  6. Singh S, Singh S, Bali V, Sharma L, Mangla J. 2014. Production of fungal amylases using cheap, readily available agriresidues, for potential application in textile industry. Biomed. Res. Int. 2014: 215748.
  7. Arikan B. 2008. Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15. Bioresour. Technol. 99: 3071-3076. https://doi.org/10.1016/j.biortech.2007.06.019
  8. Sharma V, Rausch KD, Graeber JV, Schmidt SJ, Buriak P, Tumbleson ME, et al. 2010. Effect of resistant starch on hydrolysis and fermentation of corn starch for ethanol. Appl. Biochem. Biotechnol. 160: 800-811. https://doi.org/10.1007/s12010-009-8651-7
  9. Nguyen CN, Le TM, Chu-Ky S. 2014. Pilot scale simultaneous saccharification and fermentation at very high gravity of cassava flour for ethanol production. Ind. Crops Prod. 56: 160-165. https://doi.org/10.1016/j.indcrop.2014.02.004
  10. Ivanova VN, Dobreva EP, Emanuilova EI. 1993. Purification and characterization of a thermostable ${\alpha}$-amylase from Bacillus licheniformis. J. Biotechnol. 28: 277-289. https://doi.org/10.1016/0168-1656(93)90176-N
  11. Takasaki Y, Furutani S, Hayashi S, Imada K. 1994. Acid-stable and thermostable ${\alpha}$-amylase from Bacillus licheniformis. J. Ferment. Bioeng. 77: 94-96. https://doi.org/10.1016/0922-338X(94)90216-X
  12. Shaw A, Bott R, Day AG. 1999. Protein engineering of alpha-amylase for low pH performance. Curr. Opin. Biotechnol. 10: 349-352. https://doi.org/10.1016/S0958-1669(99)80063-9
  13. Sajedi RH, Naderi-Manesh H, Khajeh K, Ahmadvand R, Ranjbar B, Asoodeh A, et al. 2005. A Ca-independent ${\alpha}$-amylase that is active and stable at low pH from the Bacillus sp. KR 8104. Enzyme Microb. Technol. 36: 666-671. https://doi.org/10.1016/j.enzmictec.2004.11.003
  14. Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R. 2000. Advances in microbial amylases. Biotechnol. Appl. Biochem. 31: 135-152. https://doi.org/10.1042/BA19990073
  15. Abe J, Bergmann FW, Obata K, Hizukuri S. 1988. Production of the raw-starch digesting amylase of Aspergillus sp. K-27. Appl. Microbiol. Biotechnol. 27: 447-450. https://doi.org/10.1007/BF00451611
  16. Okolo BN, Ire FS, Ezeogu LI, Anyanwu CU, Odibo FJ. 2001. Purification and some properties of a novel raw starch-digesting amylase from Aspergillus carbonarius. J. Sci. Food Agric. 81: 329-336. https://doi.org/10.1002/1097-0010(200102)81:3<329::AID-JSFA815>3.0.CO;2-3
  17. Matsubara T, Ben Ammar Y, Anindyawati T, Yamamoto S, Ito K, Iizuka M, et al. 2004. Degradation of raw starch granules by ${\alpha}$-amylase purified from culture of Aspergillus awamori KT-11. J. Biochem. Mol. Bol. 37: 422-428.
  18. Vijayaraghavan P, Remya CS, Prakash Vincent SG. 2011. Production of ${\alpha}$-amylase by Rhizopus microspores using agriculture byproducts in solid state fermentation. Res. J. Microbiol. 6: 366-375. https://doi.org/10.3923/jm.2011.366.375
  19. Buranakarl L, Cheng-Ying F, Ito K, Isaki K. 1985. Production of molecular hydrogen by photosynthetic bacteria with raw starch. Agric. Biol. Chem. 49: 3339-3341.
  20. Primarini D, Ohta Y. 2000. Some enzyme properties of raw starch digesting amylases from Streptomyces sp. No. 4. Starch 52: 28-32. https://doi.org/10.1002/(SICI)1521-379X(200001)52:1<28::AID-STAR28>3.0.CO;2-J
  21. Kar S, Datta TK, Ray RC. 2010. Optimization of thermostable ${\alpha}$-amylase production by Streptomyces erumpens MTCC 7317 in solid-state fermentation using cassava fibrous residue. Braz. Arch Biol. Technol. 53: 301-309. https://doi.org/10.1590/S1516-89132010000200008
  22. Liu XD, Xu YA. 2008. Novel raw starch digesting ${\alpha}$-amylase from a newly isolated Bacillus sp. YX-1: Purification and characterization. Bioresour. Technol. 99: 4315-4320. https://doi.org/10.1016/j.biortech.2007.08.040
  23. Gangadharan D, Madhavan Nampoothiri K, Sivaramakrishnan S, Pandey A. 2009. Biochemical characterization of raw-starchdigesting alpha amylase purified from Bacillus amyloliquefaciens. Appl. Biochem. Biotechnol. 158: 653-662. https://doi.org/10.1007/s12010-008-8347-4
  24. Soni SK, Goyal N, Gupta JK, Soni R. 2012. Enhanced production of ${\alpha}$-amylase from Bacillus subtilis subsp. spizizenii in solid state fermentation by response surface methodology and its evaluation in the hydrolysis of raw potato starch. Starch 64: 64-77. https://doi.org/10.1002/star.201100119
  25. Abd-Elhalem BT, El-Sawy E, Gamal RF, Abou-Taleb KA. 2015. Production of amylase from Bacillus amyloliquefaciens under submerged fermentation using some agro industrial byproducts. Ann. Agric. Sci. 60: 193-202. https://doi.org/10.1016/j.aoas.2015.06.001
  26. Krishna C, Chandrasekaran M. 1996. Banana waste as substrate for ${\alpha}$-amylase production by Bacillus subtilis (CBTK 106) under solid-state fermentation. Appl. Microbiol. Biotechnol. 46: 106-111. https://doi.org/10.1007/s002530050790
  27. Balkan B, Ertan F. 2007. Production of alpha-amylase from Penicillium chrysogenum under solid-state fermentation by using some agricultural by-products. Food Technol. Biotechnol. 45: 439-442.
  28. Abd-Elhalem BT, El-Sawy M, Gamal RF, Abou-Taleb KA. 2015. Production of amylases from Bacillus amyloliquefaciens under submerged fermentation using some agro-industrial by-products. Ann. Agric. Sci. 60: 193-202. https://doi.org/10.1016/j.aoas.2015.06.001
  29. De Silva SNT, Attanayaka DPSTG, Nirosha SF, Aththanayaka AMWS. 2009. Isolation of raw starch hydrolyzing fungi and purification of ${\alpha}$-Amylase from Geotrichum candidum CMSS06. J. Natl. Sci. Found. 37: 93-98.
  30. Zivkovic S, Stojanovic S, Ivanovic Z, Trkulja N, Dolovac N, Aleksic G, et al. 2010. Morphological and molecular identification of Colletotrichum acutatum from Tomato Fruit. Pestic. Phytomed (Belgrade) 25: 231-239. https://doi.org/10.2298/PIF1003231Z
  31. Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes application to the identification of mycorrhizae and rusts. Mol. Ecol. 2: 113-118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x
  32. White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA gene for phylogenetics. pp. 315-322. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (ed.) PCR protocols: A guide to methods and applications. Academic Press, Inc, New York.
  33. Weir BS, Johnston PR, Damm U. 2012. The Colletotrichum gloeosporioides species complex. Stud. Mycol. 73: 115-180. https://doi.org/10.3114/sim0011
  34. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. Mega 5 molecular evolutionary genetics analysis. Mol. Biol. Evol. 28: 2731-2739. https://doi.org/10.1093/molbev/msr121
  35. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
  36. Edgar RC. 2004. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5: 133. https://doi.org/10.1186/1471-2105-5-133
  37. Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120. https://doi.org/10.1007/BF01731581
  38. Okolo BN, Ezeogu LI, Mba CN. 1995. Production of raw starch digesting amylase by Aspergillus niger grown on native starch sources. J. Sci. Food Agric. 69: 109-115. https://doi.org/10.1002/jsfa.2740690117
  39. Henry RJ, Chiamori N. 1960. Study of the saccharogenic method for the determination of serum and urine amylase. Clin. Chem. 6: 434-452.
  40. Bradford MM. 1976. A rapid and sensitive method for the quantization 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
  41. Wingfield PT. 2001. Protein precipitation using ammonium sulfate. Curr. Protoc. Protein Sci. Appendix 3F.
  42. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  43. Jansz ER, Pieris N, Jeyaraj EE, Abeyratne DJ. 1976. A process for improved manioc chips and flour. In: Industrial Microbiology Section-Ceylon Institute of Scientific and Industrial Research. pp. 3-5. 3rd ed. Industrial Technology Institute, Colombo.
  44. Mbenoun M, De Beer ZW, Wingfield MJ, Wingfield BD, Roux J. 2014. Reconsidering species boundaries in the Ceratocystis paradoxa complex, including a new species from oil palm and cacao in Cameroon. Mycologia 106: 757-784. https://doi.org/10.3852/13-298
  45. Mbenoun M, Wingfield MJ, Begoude Boyogueno AD. 2016. Diversity and pathogenicity of the Ceratocystidaceae associated with cacao agro forests in Cameroon. Plant Pathol. 65: 64-78. https://doi.org/10.1111/ppa.12400
  46. Wijeratnam RSW, Hewajulige IGN, Abeyratne N. 2005. Postharvest hot water treatment for the control of Thielaviopsis black rot of pineapple. Postharvest Biol. Technol. 36: 323-327. https://doi.org/10.1016/j.postharvbio.2005.01.003
  47. Kainuma K, Kobayashi S. 1987. Process for saccharification of starch using enzyme produced by fungus belonging to genus Chalara. Enzyme Microb. Technol. 121: 37-44.
  48. Monma M, Yamamoto Y, Kagei N, Kainuma K. 1989. Raw starch digestion by ${\alpha}$-amylase and glucoamylase from Chalara paradoxa. Starch 41: 382-385. https://doi.org/10.1002/star.19890411005
  49. Takaya T, Sugimato Y, Wako K, Fuwa H. 1979. Degradation of starch granules by alpha-amylase of Streptomyces praecox NA-273. Starch 31: 205-208. https://doi.org/10.1002/star.19790310607
  50. Fairbarn DA, Priest FG, Stark JR. 1986. Extracellular amylase synthesis by Streptomyces limosus. Enzyme Microb. Technol. 8: 89-92. https://doi.org/10.1016/0141-0229(86)90077-3
  51. Gupta A, Gupta VK, Modi DR, Yadav LP. 2008. Production and characterization of alpha amylase from Aspergillus niger. Biotechnology 7: 551-556. https://doi.org/10.3923/biotech.2008.551.556
  52. Varalakshmi KN, Kumudini BS, Nandini BN, Solomon J, Suhas R, Mahesh B, et al. 2009. Production and characterization of alpha amylse from Aspergillus niger JGI 2 isolated in Bangalore. Pol. J. Microbiol. 58: 29-36.
  53. Famotemi AC, Lawal AK, Esan AO, Salihu I, Tijani AA, dike EN. 2015. Amylase production from Aspergillus flavus using millet pomace by solid state fermentation (SSF). Int. J. Adv. Res. Biol. Sci. 2: 66-72.
  54. Goyal N, Gupta JK, Soni SK. 2005. A novel raw starch digesting thermostable ${\alpha}$-amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch. Enzyme Microb. Technol. 37: 723-734. https://doi.org/10.1016/j.enzmictec.2005.04.017
  55. Ishigami H. 1987. Raw starch-digesting amylase from Chalara paradoxa. J. Jpn. Soc. Starch Sci. 34: 66-74. https://doi.org/10.5458/jag1972.34.66
  56. Rao BSN. 2000. Nutritive value of rice bran. Nutr. Foundation Ind. Bull. 21: 5-7. http://www.nutritionfoundationofindia.org/pdfs/BulletinArticle/Pages_from_nfi_10_00_2.pdf
  57. Serin B, Akcan N, Uyar F. 2012. Production and optimization of ${\alpha}$-amylase from Bacillus circulans ATCC 4516 with solid state fermentation. J. Biol. Chem. 40: 393-400.
  58. Puri S, Arora M, Sarao L. 2013. Production and optimization of amylase and glucoamylase using Aspergillus oryzae under solid state fermentation. Int. J. Res. Pure Appl. Microbiol. 3: 83-88.
  59. Kaur H, Arora M, Bhatia S, Alam MS. 2016. Solid state fermentation of deoiled rice bran for production of fungal enzyme-A review. Int. J. Appl. Pure Sci. Agric. 2: 143-157.
  60. Anhwange BA, Ugye TJ, Nyiaatagher TD. 2009. Chemical composition of Musa sepientum (Banana) peels. Electron J. Environ. Agric. Food Chem. 8: 437-442.
  61. Emaga TH, Bindelle J, Agneesens R, Buldgen A, Wathelet B, Paquot M. 2011. Ripening influences banana and plantain peels composition and energy content. Trop. Anim. Health Prod. 43: 171-177. https://doi.org/10.1007/s11250-010-9671-6
  62. Zhang P, Whistler RL, Bemiller JN, Hamaker BR. 2005. Banana starch: Production, physicochemical properties, and digestibility- A review. Carbohydr. Polym. 59: 443-458. https://doi.org/10.1016/j.carbpol.2004.10.014
  63. Soares CA, Peroni-Okita FHG, Cardoso MB, Shitakubo R, Lajolo FM, Cordenunsi BR. 2011. Plantain and banana starches: Granule structural characteristics explain the differences in their starch degradation patterns. J. Agric. Food Chem. 59: 6672-6681. https://doi.org/10.1021/jf201590h
  64. Teixeira EM, Curvelo AAS, Corrêa AC, Marconcini JM, Glenn GM, Mattoso LHC. 2012. Properties of thermoplastic starch from cassava bagasse and cassava starch and their blends with poly (lactic acid). Ind. Crops Prod. 37: 61-68. https://doi.org/10.1016/j.indcrop.2011.11.036
  65. Swain MR, Ray RC. 2007. Alpha-amylase production by Bacillus subtilis CM3 in solid state fermentation using cassava fibrous residue. J. Basic Microbiol. 47: 417-425. https://doi.org/10.1002/jobm.200710132
  66. Franco CML, Do Rio Preto SJ, Ciacco CF. 1992. Factors that affect the enzymatic degradation of natural starch granules-effect of the size of the granules. Starch 44: 422-426. https://doi.org/10.1002/star.19920441106
  67. Rocha TDS, Carneiro APDA, Franco CML. 2010. Effect of enzymatic hydrolysis on some physicochemical properties of root and tuber granular starches. Ciencia e Tecnol Aliment. 30: 544-551. https://doi.org/10.1590/S0101-20612010000200039