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2015 | 14 | 1 |

Tytuł artykułu

Studies on the production of alkaline alpha - amylase from Bacillus Subtilis CB-18

Warianty tytułu

Języki publikacji

EN

Abstrakty

EN
Background. Amylases are among the main enzymes used in food and other industries. They hydrolyse starch molecules into polymers composing glucose units. Amylases have potential applications in a number of industrial processes including foods and pharmaceutical industries. Alkaline a-amylase has the potential of hydrolysing starch under alkaline pH and is useful in the starch and textile industries and as an ingredient of detergents. Amylases are produced from plants, however, microbial production processes have dominated applications in the industries. Optimization of microbial production processes can result in improved enzyme yields. Material and methods. Amylase activity was assayed by incubating the enzyme solution (0.5 ml) with 1% soluble starch (0.5 ml) in 0.1 M Tris/HCl buffer (pH 8.5). After 30 minutes, the reaction was stopped by the addition of 4 mL of 3,5-dinitrosalicylic acid (DNS) reagent then heated for 10 min in boiling water bath and cooled in a refrigerator. Absorbance readings were used to estimate the units of enzyme activity from glucose standard curve. Hydrolysed native starches from cassava, rice, com, coco yam, maize and potato and soluble starch were adjusted to pH 8.5 prior to incubation with crude enzyme solution. Reducing sugars produced were therefore determined. The effect of pH on enzyme activity of the alkaline a-amylase was determined by using buffer solutions of different pH (potassium phosphate buffer, 6.0-7.0; Tris-HCl buffer 7.5 to 9.0 and carbonate/bicarbonate buffer, pH 9.5-11) for enzyme assay. The pH stability profile of the enzyme was determined by incubating 0.5 ml of a-amylase enzyme in 0.1 M Tris/HCl buffer (pH 8.5) and 0.5 ml of 1% (w/v) soluble starch (Merck) in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h in various buffers. The effect of temperature on enzyme activity was studied by incubating 0.5 mL of the enzyme solution contained in the test tube and 0.5 mL of 1% soluble starch (Merck) solution prepared in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h at various temperatures (25, 30, 35, 40, 45, 50, 55 and 60°C) in a thermo static water bath. The reactions were stopped by adding DNS reagent. The enzyme activity was therefore determined. Thermal stability was studied by incubating 0.5 ml of enzyme solution in 0.1 M Tris/HCl buffer (pH 8.5) and 0.5 ml of 1% (w/v) soluble starch (Merck) in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h at various temperatures (20, 30, 40, 50, 60 and 70°C) for 60 min. Results. The enzyme displayed optimal activity at pH 8.0 at which it produced maximum specific activity of 34.3 units/mg protein. Maximum stability was at pH 8.0 to 9.0. Maximum activity was observed at temperature of 50°C while thermo stability of the enzyme was observed at 40-50°C. The enzyme displayed a wide range of activities on starch and caused the release of 5.86,4.75,5.98,3.44,3.96, 8.84 mg/mL reducing sugar from cassava, potato, cocoyam, com, rice and soluble starch respectively. Conclusion. This investigation reports some biochemical characterization of alkaline a-amylase from Bacillus subtilis CB-18. The substrate specificities of this enzyme on various starches suggested that the alkaline a-amylase enzyme had combined activities on raw and soluble starch.

Słowa kluczowe

Wydawca

-

Rocznik

Tom

14

Numer

1

Opis fizyczny

p.71-75,fig.,ref.

Twórcy

autor
  • Industrial Microbiology and Biotechnology Laboratory, Department of Microbiology, University of Nigeria, Nsukka, Nigeria
autor
  • Industrial Microbiology and Biotechnology Laboratory, Department of Microbiology, University of Nigeria, Nsukka, Nigeria

Bibliografia

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  • Boyer, E. W, Ingle, M. B. (1972). Extracellular alkaline amylase from Bacillus species. J. Bacteriol., 110, 992-1000.
  • Burhan, A., Nisa, U., Khan, G., Gokhan, C., Ashabil, A., Osman, G. (2003). Enzymatic properties of a novel thermostable, thermophilic alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6. Process Biochem., 38, 1397-1403.
  • Dastager, G. S., Agasar, D., Pandey, A. (2009). Production and partial purification of a-amylase from a novel isolate Streptomyces gulbargensis. J. Ind. Microbiol. Bio- technol, 36, 189-194.
  • Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K., Chau- han, B. (2003). Microbial a-amylases: a biotechnological perspective. Process Biochem., 381, 599-1616.
  • Hagihara, H., Igarashi, K., Hayashi, Y, Endo, K., Ikawa- -Kitayama, K., Ozaki, K., Kawai, S., Ito, S. (2001). Novel alpha amylase that is highly resistant to chelating reagents and chemical oxidants from alkaliphilic Bacillus isolate KSM-K38. Appl. Environ. Microbiol., 67, 1744-1750.
  • Horikoshi, K., 1971. Production of alkaline enzymes by alkalophilic microorganisms 11. Alkaline amylase produced by Bacillus No A-40-2. Agric. Biol. Chem., 35, 1783-1791.
  • Igarashi, K., Hatada, Y. Hagihara, H., Saeki, K, Takaiwa, M., Uemura, T. (1998). Enzymatic properties of a novel liquefying a-amylase from an alkaliphilic Bacillus isolate and entire nucleotide and amino acid sequence. Appl. Environ. Microbiol., 64, 3282-3289.
  • Kim, T. U., Gu, B. G., Jeong, J. Y., Byun, S. M., Shin, Y. C. (1996). Purification and characterization of a maltotet- rose - forming alkaline a-amylase from an alkalophilic Bacillus strain GM8901. Appl. Environ. Microbiol., 61, 3105-3112.
  • Lin, L. L., Chyau, C. C., Hsu, W. H. (1996). Production and properties of a raw starch - degrading amylase from the thermophilic and alkaliphilic Bacillus sp. TS-23. Bio- technol. Appl. Biochem., 28, 61-68.
  • Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. (1951). Protein measurement with folin-phenol reagent. J. Biochem., 193, 265-275.
  • Mamo, G., Gessesse, A. (1999). Purification and characterization of two raw - starch digesting thermostable a-amylases from a thermophilic Bacillus. Enz. Microbiol. Technol., 25, 433-438.
  • Miller, G. L. (1959). Use of dinitrosalicic acid reagent for determination of reducing sugar. Anal. Chem., 31, 426-428.
  • Murakami, S., Nishimato, H., Toyama, Y., Shimamoto, E., Takenaka, S., Kaulpiboon, J., Prousoontorn, M., Lim- paseni, T., Pongsawasdi, P., Aoki, K. (2007). Purification and characterization of two alkaline, thermotolerant alphamylases from Bacillus halodurans 38C-2-I and expression of the cloned gene in Escherichia coli. Biosc. Biotechnol. Biochem., 71 (10), 2393-2401.
  • Nwokoro, O., Odiase, A. O. (2012). Influence of media composition on the production of alkaline a-amylase from Bacillus subtilis CB-18. Acta Sci. Pol. Technol. Aliment, 11(3), 231-238.
  • Obi, S. K. C, Odibo, J. F. C. (1984). Some properties of a highly thermostable a-amylase from Thermoactinomy- ces sp. Can. J. Microbiol, 30, 780-785.
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  • Shanmugapriya, S, Kiran, G. S, Selvin, J, Gandhimathi, R, Baskar, T. B, Manilal, A, Sujith, S. (2009). Optimization, production and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halotacterium salinarum MMD047. Biotechnol. Bioproc. Eng, 14, 67-75.
  • Van der Maarel, M. J. E. C, Van der Veen, B, Uitdehaag, J. C. M, Leemhuis, H, Dijkhuizen, L. (2002). Properties and applications of starch-converting enzymes of the a-amylase family. J. Bacteriol, 94, 137-155.
  • Yang, H, Liu, L, Li, J, Du, G, Chen, J. (2011). Heterologous expression, biochemical characterization and overproduction of alkaline a-amylase from Bacillus al- calophilus in Bacillus subtilis. Microbial. Cell Fact, 10, 77-85.
  • Zhao, J, Lan, X, Su, J, Sun, L, Rahman, E. (2008). Isolation and identification of an alkaliphilic Bacillus flexus XJU-3 and analysis of its alkaline amylase. Wei Sheng Wu Xue Bao, 48(6), 750-756.

Typ dokumentu

Bibliografia

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