Summary
A new thermophilic Bacillus strain 3183 (ATCC 49341) was isolated from hot-spring sediments. The organism grew on pullulan as a carbon source and showed optimum pH and temperature at pH 5.5 and 62° C, respectively, for growth. The strain reduced nitrate to nitrite both aerobically and anaerobically. It produced extracellular thermostable pullulanase and saccharidase activities which degraded pullulan and starch into maltotriose, maltose, and glucose. Medium growth conditions for pullulanase production were optimized. The optimum pH and temperature for pullulanase activity were at pH 6.0 and 75° C, respectively. The enzyme was stable at pH 5.5-7.0 and temperature up to 70° C in the absence of substrate. The K m for pullulan at pH 6.0 and 75° C was 0.4 mg/ml. The pullulanase activity was stimulated and stabilized by Ca2+. It was inhibited by ethylenediaminetetraacetate (EDTA), beta and gamma-cyclodextrins but not by alpha-cyclodextrin and reagents that inhibit essential enzyme SH-groups.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abdullah M, French D (1970) Substrate specificity of pullulanase. Arch Biochem Biophys 137:483–493
Adams KR, Priest FG (1977) Extracellular pullulanase synthesis in Bacillus macerans. FEMS Microbiol Lett 1:269–273
Antranikian G, Herzberg C, Gottschalk G (1987) Production of thermostable alpha-amylase, pullulanase and alpha-glucosidase in continuous culture by a new Clostridium isolate. Appl Environ Microbiol 53:1668–1673
Bernfeld H (1955) Amylases alpha- and beta-. Methods Enzymol 1:149–150
Coleman RD, Yang S-S, McAlister MP (1987) Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis. J Bacteriol 169:4302–4307
Cowan ST, Steel KJ (eds) (1974) Manual for the identification of medical bacteria. Cambridge University Press, Cambridge, UK
Fogarty WM (1983) Microbial amylases. In: Fogarty WM (ed) Microbial enzymes and biotechnology. Applied Science Publishers, London, pp 1–92
Hyun HH, Zeikus JG (1985) General biochemical characterization of thermostable pullulanase and glucoamylase from Clostridium thermohydrosulfuricum. Appl Environ Microbiol 49:1168–1173
Imanaka T, Kuriki T (1989) Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan. J Bacteriol 171:369–374
Jensen B, Norman BE (1984) Bacillus acidopullulyticus pullulanase application and regulatory aspects in the food industry. Process Biochem 19:129–134
Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 19:265–275
Melasniemi H (1987) Characterization of alpha-amylase and pullulanase activities of Clostridium thermohydrosulfuricum. Biochem J 246:193–197
Nakamura N, Watanabe K, Horikoshi K (1975) Purification and some properties of alkaline pullulanase from a strain of Bacillus no. 202–1, an alkaline microorganism. Biochem Biophys Acta 397:188–193
Nakamura N, Sashihara N, Nagayama H, Horikoshi K (1987) Production of a thermostable pullulanase by a Thermus sp. J Jpn Soc Starch Sci 34:38–44
Nikolov ZL, Meagher MM, Reilly PJ (1985) High-performance liquid chromatography of trisaccharides on amine-bonded silica columns. J Chromatogr 321:393–399
Norman BE (1979) The application of polysaccharide degrading enzymes in the starch industry. In: Berkeley RCW, Gooday GW, Ellwood DC (eds) Microbiol polysaccharides and polysaccharidases. Academic Press, New York, pp 339–376
Plant AR, Morgan HW, Daniel RM (1986) A highly stable pullulanase from Thermus aquaticus YT-1. Enzyme Microb Technol 8:668–672
Plant AR, Clemens RM, Daniel RM, Morgan HW (1987) Purification and preliminary characterization of an extracellular pullulanase from Thermoanaerobium Tok6-B1. Appl Microbiol Biotechnol 26:427–433
Saha BC, Zeikus JG (1987) Biotechnology of maltose syrup production. Process Biochem 22:78–82
Saha BC, Zeikus JG (1989) Novel highly thermostable pullulanase from thermophiles. Trends Biotechnol 7:234–239
Saha BC, Mathupala SP, Zeikus JG (1988) Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum. Biochem J 252:343–348
Saha BC, Shen G-J, Srivastava KC, Lecureux LW, Zeikus JG (1989) New thermostable alpha-amylase-like pullulanase from thermophilic Bacillus sp. 3183. Enzyme Microb Technol 11:760–764
Sneath PHA, Mair NS, Sharpe EM, Holt JG (eds) (1986) Bergey's manual of systematic bacteriology, vol 2. Williams and Wilkins, Baltimore, pp 1130–1139
Suzuki Y, Chishiro M (1983) Production of extracellular thermostable pullulanase by an amylolytic obligatory thermophilic soil bacterium Bacillus stearothermophilus KP 1064. Appl Microbiol Biotechnol 17:24–29
Suzuki Y, Imai T (1985) Bacillus stearothermophilus KP 1064 pullulan hydrolase. Its assignment to a unique type of maltogenic alpha-amylase but neither to pullulanase nor isopullulanase. Appl Microbiol Biotechnol 21:20–26
Takasaki Y (1987) Pullulanase-amylase complex enzyme from Bacillus subtilis. Agric Biol Chem 51:9–16
Zeikus JG, Ben-Bassat A, Hegge PW (1980) Microbiology of methanogenesis in thermal, volcanic environments. J Bacteriol 143:432–440
Author information
Authors and Affiliations
Additional information
Offprint requests to: B. C. Saha
Rights and permissions
About this article
Cite this article
Shen, GJ., Srivastava, K.C., Saha, B.C. et al. Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183. Appl Microbiol Biotechnol 33, 340–344 (1990). https://doi.org/10.1007/BF00164533
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00164533