Author
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Zahnley, James |
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Smith, Michael |
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Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 2/24/2004 Publication Date: 8/12/2004 Citation: Zahnley, J.C., Smith, M.R. 2005. Production of amylase by Arthrobacter psychrolactophilus. J. Ind. Microbiol. Biotechnol. 32:277-283. Interpretive Summary: A major cost of converting starch to high-value products is the energy required for heating steps in the process. Use of enzymes that break down starch at lower temperature would make starch processing more competitive by reducing fuel costs. Other potential low-temperature enzyme applications include removing starch from heat-labile high-value food components. Bacteria that grow at low temperatures are potential sources of cold-active enzymes for starch hydrolysis, but few such enzymes have been described. Culture conditions for production of these enzymes by a marine bacterium grown at 10C were developed. Crude enzyme was most active under mild conditions (20-25C and pH 6-8). The major enzyme, alpha-amylase, cuts the starch molecules into fragments that can be broken down by other enzymes. The activity has an unusually low temperature optimum and, unlike most amylases, the activities are located in the cells. Once enzymes are purified from the concentrated cell extracts, they can be be utilized in protein engineering to generate new enzymes having both increased stability and retention of activity at low temperatures. Technical Abstract: We investigated production of starch-degrading enzyme activity by the marine psychrophile Colwellia psychroerythraea (ATCC 27364) in culture media with or without added carbohydrate. Inclusion of starch and buffer in the culture medium markedly affected the level and stability of the activity, with 0.5% soluble starch and 100 mM HEPES (pH 7.0) in Difco Marine Broth 2216 yielding the highest levels of amylolytic activity. Whole cultures assayed with soluble starch as substrate showed optimal activity at pH 6-8 and 20-25C. Few amylolytic enzymes described to date have such a low temperature optimum. Most of the activity was found in the cell pellet, but it could be released by re-suspending the cells at lower ionic strength of 0.2 (osmotic shock). The solubilized activity was stabilized by extraction at pH 7-8 in the presence of calcium ions and glycerol. Chromatography on anion exchange or hydrophobic interaction columns suggested that adsorption of the extracted proteins depends more on ionic than on hydrophobic interactions. Possible use of these amylolytic enzymes in low-temperature processes to remove starch from high-value thermolabile constituents of food plants will most likely depend on increasing expression levels by molecular genetic means. |
