Purification and Biochemical Characterization of a Detergent Stable α-amylase from Pseudomonas stutzeri AS22

다국어 초록 (Multilingual Abstract)

This study reports the purification and biochemical characterization of a novel maltotetraose-forming-α-amylase from Pseudomonas stutzeri AS22, designated PSA. The P. stutzeri α-amylase (PSA) was purified from the culture supernatant to homogeneity by Sepharose mono Q anion exchange chromatography, ultrafiltration and Sephadex G-100 gel filtration, with a 37.32-fold increase in specific activity, and 31% recovery. PSA showed a molecular weight of approximately 57 kDa by SDS-PAGE.
The N-terminal amino acid sequence of the first 7 amino acids was DQAGKSP. This enzyme exhibited maximum activity at pH 8.0 and 55oC, performed stably over a broad range of pH 5.0 ~ 12.0, but rapidly lost activity above 50oC. Both potato starch and Ca2+ ions have a protective effect on the thermal stability of PSA. The enzyme activity was inhibited by Hg2+, Mn2+, Cd2+, Cu2+, and Co2+, and enhanced by Ba2+. PSA belonged to the EDTA-sensitive α-amylase. The purified enzyme showed high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), and oxidizing agents, such as sodium per borate and H2O2. In addition, PSA showed excellent compatibility with a wide range of commercial solid and liquid detergents at 30oC, suggesting potential application in the detergent industry. Maltotetraose was the specific end product obtained after hydrolysis of starch by the enzyme for an extended period of time, and was not further degraded.

참고문헌 (Reference)

1 Sivaramakrishnan, S., "α-Amylases from microbial sources-An overview on recent developments" 44 : 173-184, 2006

2 Jane ek, Š., "α-Amylases and approaches leading to their enhanced stability" 304 : 1-3, 1992

3 Soleimani, M., "α-Amylase immobilization on the silica nanoparticles for cleaning performance towards starch soils in laundry detergents" 74 : 1-5, 2012

4 Miller, G. L., "Use of dinitrosalycilic acid reagent for determination of reducing sugars" 31 : 426-428, 1959

5 Volkin, D. B., "Thermal destruction processes in proteins involving cysteine" 89 : 2945-2950, 1989

6 Kikani, B. A., "The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium, Anoxybacillus beppuensis TSSC-1" 47 : 1791-1798, 2012

7 Tanaka, A., "Secondary calcium-binding parameter of Bacillus amyloliquefaciens α-amylase obtained from inhibition kinetics" 96 : 262-267, 2003

8 Malabendu, J., "Salt-independent thermophilic α-amylase from Bacillus megaterium VUMB109: An efficacy testing for preparation of maltooligosaccharides" 41 : 386-391, 2013

9 Sakano, Y., "Purification of a maltotetraose-forming exo-amylase of Pseudomonas stutzeri: Two-forms of the amylase and their enzymatic properties" 47 : 1761-1768, 1983

10 Sakano, Y., "Purification and properties of an exo-α-amylase from Pseudomonas stutzeri" 46 : 639-646, 1982

11 Nakada, T., "Purification and characterization of two forms of maltotetraose-forming amylase from Pseudomonas stutzeri" 54 : 737-743, 1990

12 Murakami, S., "Purification and characterization of five alkaline, thermotolerant, and maltotetraose- producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli" 43 : 321-328, 2008

13 Michelin, M., "Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii" 345 : 2348-2353, 2010

14 Kim, T. U., "Purification and characterization of a maltotetraose-forming alkaline α-amylase from an alkalophilic Bacillus strain GM8901" 61 : 3105-3112, 1995

15 Van der Maarel, M. J. E. C., "Properties and applications of starch converting enzymes of the α-amylase family" 94 : 137-155, 2002

16 Kimura, T., "Production of amylase and pullulanase by an alkalopsychrotrophic Micrococcus sp" 53 : 2963-2968, 1989

17 Hatada, Y., "Oxidatively stable maltopentaose-producing α- amylase from a deep-sea Bacillus isolate, and mechanism of its oxidative stability validated by site-directed mutagenesis" 39 : 1333-1340, 2006

18 Zhou, J., "Nucleotide sequence of the maltotetraohydrolase gene from Pseudomonas saccharophila" 255 : 37-41, 1989

19 Hagihara, H., "Novel α-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38" 67 : 1744-1750, 2001

20 Zhang, J., "Molecular cloning and expression of an extracellular α-amylase gene from an Antarctic deep sea psychrotolerant Pseudomonas stutzeri strain 7193" 27 : 841-850, 2011

21 Gupta, R., "Microbial α-amylase: A biotechnological perspective" 38 : 1599-1616, 2003

22 Vallee, B. L., "Metal content of α-amylases of various origins" 234 : 2901-2905, 1959

23 Robyt, J. F., "Isolation, purification and characterization of a maltotetraose-producing amylase from Pseudomonas stuzeri" 145 : 105-114, 1971

24 Gangadharan, D., "Immobilized bacterial α-amylase for effective hydrolysis of raw and soluble starch" 42 : 436-442, 2009

25 Owino, V. O, "Fortified complementary foods with or without alpha amylase treatment increase hemoglobin but do not reduce breast milk intake of 9-mo-old Zambian infants" 86 : 1094-1103, 2007

26 Fogarty, W. M., "Extracellular maltotetraose-forming amylase of Pseudomonas sp. IMD 353" 16 : 473-478, 1994

27 Miller, J. H., "Experiments in Molecular Genetics" Cold spring Harbor Laboratory 431-435, 1972

28 Morishita, Y., "Crystal structure of a maltotetraoseforming exo-amylase from Pseudomonas stutzeri" 267 : 661-672, 1997

29 Suvd, D., "Crystal structure of Bacillus Stearothermophilus α-amylase: Possible factors determining the thermostability" 129 : 461-468, 2001

30 Fujita, M., "Cloning and nucleotide sequence of the gene (amyP) for maltotetraose-forming amylase from Pseudomonas stutzeri MO-19" 171 : 1333-1339, 1989

31 Laemmli, U. K, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4" 227 : 680-685, 1970

32 Kobayashi, H., "Characterization of α-maltotetraohydrolase produced by Pseudomonas sp. MS300 isolated from the deepest site of Mariana trench" 16 : 109-116, 2000

33 Mehta, M., "Biochemical and molecular characterization of recombinant acidic and thermostable raw-starch hydrolyzing α-amylase from an extreme thermophile Geobacillus thermoleovorans" 85 : 229-238, 2013

34 Sneath, P. H. A., "Bergey’s Manual of Systematic Bacteriology. 9th ed." Williams and Wilkins 1063-1065, 1065

35 Takasaki, Y., "An amylase producing maltotetraose and maltopentaose from Bacillus circulans" 47 : 2193-2199, 1983

36 Aiyer, P. V., "Amylases and their applications" 4 : 1525-1529, 2005

37 Heinen, W., "Amylase activity and stability at high and low temperature depending on calcium and other divalent cations" 26 : 77-89, 1976

38 Pandey, A., "Advances in microbial amylases" 31 : 135-152, 2000

39 Bradford, M., "A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding" 72 : 248-254, 1976

40 Sajedi, R. H., "A calcium independent α-amylase that is active and stable at low pH from the Bacillus sp. KR-8104" 36 : 666-671, 2005