Abstract
A Cellulomonas genomic fragment encoding extracellular amylase activity was isolated as a clone (ACs2) in Escherichia coli DH10B. The amylase was expressed in the absence of IPTG and in the presence of starch or maltose. This enzyme corresponded to the low mobility activity of Cellulomonas amylases as demonstrated on gel electrophoresis. Maltose, as well as lactose, xylose and xylan cross-induced the amylase of clone ACs2. Maltose-induced amylase was purified to homogeneity. ACs2-coded amylase is a 70kDa acidic protein, with a pH optimum of 7.0 at 45°C. This enzyme exhibited an endo mode of action, similar to the corresponding Cellulomonas enzyme.
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Alemohammed, S.J. & Pembroke, J.T. 1989 Mutants of Cellulomonas flavigena with altered cell associated amylase activity. Biotechnology Letters 11, 75
Alemohammed, S.J. & Pembroke, J.T. 1993 Molecular cloning and expression of the Cellulomonas flavigena cell-associated amylase gene in E. coli and C. flavigena. Biotechnology Letters 15, 219-222.
Bhalerao, J., Patki, A.H., Bhave, M., Khurana, I & Deobagkar, D.N. 1990 Molecular cloning and expression of a xylanase gene from Cellulomonas species into E. coli. Applied Microbiology and Biotechnology 34, 71-76.
Birnboim, H.C. & Doly, J. 1979 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7, 1513-1523.
Bradford, MM. 1976 A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry 72, 248-254.
Bucht, B & Eriksson, K.E. 1968 Extracellular enzyme system utilised by the rot fungus Stereum sanguinolentum for the breakdown of cellulose. 1. Studies on the enzyme production. Archives of Biochemistry and Biophysics 124, 135-141.
Chomczynski, P. & Quasba, P.K. 1984 Alkaline transfer of plasmid DNA to membrane. Biochemical Research Communications 122, 340-344.
Feinberg, A.P. & Vogelstein, B. 1984 A technique for radiolabelling restriction enzyme fragments to high specific activity. Analytical Biochemistry 137, 266-267.
Fogarty, W.M. 1983 Microbial Amylases. In Microbial Enzymes and Biotechnology; ed Fogarty, W.M. pp. 1-77. London: Applied Science Publishers.
Gilbert, H.J. & Hazelwood, G.P. 1993 Bacterial cellulases and xylanases. Journal of General Microbiology 139, 187-194.
Gokhale, D.V. & Deobagkar, D.N. 1989 Differential expression of xylanases and endoglucanases in the hybrid derived from inter-generic protoplast fusion between a Cellulomonas species and B. subtilis. Applied and Environmental Microbiology 55, 2675-2680.
Haggett, R.D., Gray, P.P. & Dunn, N.W. 1979 Crystalline cellulose degradation by a strain of Cellulomonas and its mutant derivatives. European Journal of Applied Microbiology 8, 183-190.
Han, Y.W. & Srinivasan, V.R. 1968 Isolation and characterization of a cellulose utilizing bacterium. Applied Microbiology 16, 1140-1145.
Kumar, N. 1995 Molecular cloning, purification and characterization of the amylases of Cellulomonas sp. NCIM 2353. PhD thesis, Department of Zoology, University of Poona, India.
Kumar, N.N. & Deobagkar, D.N. 1995 Presence of multiple amylases encoded by independent genes in Cellulomonas sp. NCIM 2353. Biotechnology Letters 17, 797-802.
Kumar, N.N. & Deobagkar, D.N. 1996 Multifunctional glucanases. Biotechnology Advances 14, 1-15.
Kumar, N.N., Bhide, A., Gokhale, D.V. & Deobagkar, D.N. 1995 Production of extracellular amylase by Cellulomonas sp. NCIM 2353. Biotechnology and Applied Biochemistry 22, 345-353.
Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680-685.
Lee, S.-P., Morikawa, M., Takagi, M. & Imanaka, T. 1994 Cloning of the aapT gene and characterization of its product, α-amylase-pullulanase (AapT), from thermophilic and alkalophilic Bacillus sp. strain XAL601. Applied and Environmental Microbiology, 60, 3764-3773.
McCarthy, J.F. & Pembroke, J.T. 1988 The amylase activity of Cellulomonas flavigena is cell associated and inducible. Biotechnology Letters 10, 285-288.
Meinke, A., Gilkes, N.R., Kilburn, D.G. Miller, R.C. Jr. & Warren, R.A.J. 1991 Multiple domains in endoglucanase B (Cen B) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides. Journal of Bacteriology 173, 7126-7135.
Rigby, P.W.J., Dieckmann, M., Rhodes, C. & Berg, P. Labelling DNA to high specific activity in vitro by nick translation with DNA polymerase I. 1977 Journal of Molecular Biology 113, 237-251.
Righetti, P. & Drysdale, J.W. 1971 Isoelectric focusing in polyacrylamide gel electrophoresis. Biochemica et Biophysica Acta 236, 17-23.
Saha, B.C. & Ueda, S. 1983 Raw starch adsorption and elution and digestion behaviour of glucoamylase of Rhizopus niveus. Journal of Fermentation Technology 61, 67-72.
Saito, H. & Miura, K-I. 1963 Preparation of transforming DNA by phenol treatment. Biochemica et Biophysica Acta 72, 619-629.
Telford, J.L., Kressmann, A., Koski, R.A., Grosschedl, R., Muller, F., Clarkson, S.G. & Birnsteil, M.L. 1979 Delimitation of a promoter for RNA polymerase III by means of a functional test. Proceedings of the National Academy of Sciences of the United states of America. 76, 2590-2594.
Towbin, H., Stahelin, T. & Gordan, J. 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76, 4350-4351.
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Kumar, N., Deobagkar, D. Expression and characterization of amylase encoded by a gene cloned from Cellulomonas sp. NCIM 2353. World Journal of Microbiology and Biotechnology 13, 491–496 (1997). https://doi.org/10.1023/A:1018548922051
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DOI: https://doi.org/10.1023/A:1018548922051