Abstract
Eight constructions involving the Bacillus subtilis α-amylase gene (amyE), a mouse pancreatic α-amylase cDNA (AMY2) and an Aspergillus awamori glucoamylase cDNA (glaA) were prepared: three fusion genes, involving one α-amylase and the glucoamylase, two double-cassette plasmids (expressing one or other α-amylase and the glucoamylase) and three single-cassette plasmids, expressing the individual coding sequences. Following transformation of each plasmid into Saccharomyces cerevisiae, a plate test revealed that the largest starch hydrolysis halo was produced by the strain bearing the B. subtilis α-amylase/glucoamylase fusion (BsAAase/GAase), and the smallest halo by the one expressing the mouse pancreatic α-amylase/glucoamylase fusion (MAAase/GAase). When assayed for enzymatic activity in liquid medium, the strains bearing the fusion and the double-cassette plasmids involving B. subtilis α-amylase and the glucoamylase exhibited both enzymic activities. Moreover, the BsAAase/GAase hybrid was able to adsorb and digest raw starch. The MAAse/GAase fusion protein was found to exhibit only α-amylase activity. Finally, the capacity to grow on soluble and corn starch was tested in liquid medium for the strains bearing plasmids coding for the fusion proteins and the separate enzymes. The strain carrying the double-cassette BsAAase + GAase, which produced one of the smallest hydrolysis haloes in the place test, showed the best performance, not only in digesting soluble and corn starch but also in using all of the hydrolysis products for growth. The transformant bearing the BsAAase/GAase fusion was able to grow on soluble starch, but not on corn starch.
Similar content being viewed by others
References
Astolfi-filho S, Galembeck EV, Faria JB, Frascino ACGS (1986) Stable yeast transformant that secretes functional α-amylase encoded by cloned mouse pancreatic cDNA. Biotechnology 4:311–315
Astolfi-filho S Moraes LMP Azevedo MO Schenberg ACG, Oliver SG (1990) Cloning of an A. awamori glucoamylase cDNA. In: Limonta M, Gaoilondo J (eds) Abstracts of the 2nd Latin-American Congress of Biotechnology. Soc Iberolatinamerica de Biotec, Habana, Cuba. S192
Bernfeld P (1955) Amylases α and β. Methods Enzymol 1:149–158
Bradford MM (1976) A rapid and sensitive methods for the quantification of microgram quantities of protein utilising the principal of protein-dye binding. Anal Biochem 72:248–254
Chen DC, Yang BC, Kuo TT (1992) One-step transformation of yeast in stationary phase. Curr Genet 21:83–84
Coombs J (1984) Sugar-cane as an energy crop. Biotechnol Genet Eng Rev 1:311–345
De Mot R (1990) Conversion of starch by yeasts. In: Verchtert T, De Mot R (eds) Yeast biotechnology and biocatalysis. Dekker, Amsterdam, pp 163–222
Hacking AJ (1987) Economic aspects of biotechnology. Camb Stud Biotechnol 3
Harris EMS, Aleshin AE, Firsoy LM, Honzatko RB (1993) Refined structure for the complex of 1-deoxynojirimycin with glucoamylase from Aspergillus awamori var X100 to 24 Å resolution. Biochemistry 32:1618–1626
Hill J, Donald KAIG, Griffiths DE (1991) DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res 19:5791
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382
Ludwig JR II, Oliver SG, McLaughlin CS (1977) The effect of amino acids on growth and phosphate metabolism in a phototropic yeast strain. Biochem Biophys Res Commun 79: 16–23
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Marzola DL, Bartholomew DP (1979) Photosynthetic pathway and biomass and energy production. Science 205: 555–559
Mcpherson MJ, Quirke P, Taylor GR (1991) PCR: a practical approach. IRL, Oxford
Merlotti Wulff MC (1992) PhD Thesis, University of Manchester
Moraes LMP (1993) PhD Thesis, University of Manchester
Moraes LMP, Carmago SS, Azevedo MO, Lima BD, Schenburg ACG, Oliver SG, Astolfi-filho S (1991) Clonagem moleculare expressao em Saccharomyces cerevisiae do cDNA la glucoamilase de Aspergillus awamori sob o controle do promotor PGK. Anais da 17a Reuniao Annal de Genetica de Microorganismos, Soc. Bras. Genet., Brasilia DF, Brasil, B19.
Nunberg JH, Meade JH, Cole G, Lawyer FC, McCabe P, Schweickart V, Tal R, Wittman VP, Flatgaard JE, Innis MA (1984) Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori. Mol Cell Biol 4:2306–2315
Ribeiro dos Santos MGG (1988) MSc Thesis, Universidade de São Paulo, Brasil
Rosillo-Calle F, Hall DO, Arora AL, Carioca JOB (1992) Bioethanol production: economic and social considerations in failure and success. In: Da Silva EJ, Ratledge C, Sasson A (eds) Biotechnology: economic and social aspects. Cambridge University Press, Cambridge London pp 23–54
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Scheidtmann KH (1989) Immunological detection of proteins of known sequence: In: Creighton TE (ed) Protein structure: a practical approach. IRL, Oxford, pp 93–143
Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Shibuya I, Tamura G, Shima H, Ishikawa T, Hara S (1992) Construction of an α-amylase/glucoamylase fusion gene and its expression in Saccharomyces cerevisiae. Biosci Biotechnol Biochem 56:884–889
Souza MB (1986) Clonagem moleculaar e expressão de gene de α-amilase de Bacillus subtilis. MSc Thesis, Universidade de Brasília, Brasil
Stateva LI, Oliver SG, Trueman LJ, Venkov PV (1991) Cloning and characterisation of a gene which determines osmotic stability in Saccharomyces cerevisiae Mol Cell Biol 11:4235–4243
Stewart GG, Russell I (1987) Biochemical and genetic control of sugar and carbohydrate metabolism in yeasts In: Berry DR, Russell I, Stewart GG ed Yeast Biotechnology. Allen and Unwin, London, pp 277–310
Steyn AJC, Pretorius IS (1990) Expression and secretion of amylolytic enzymes by Saccharomyces cerevisiae. Acta Varia 5:76–126
Thomsen KK (1983) Mouse α-amylase synthesized by Saccharomyces cerevisiae is released in the culture medium Carlsberg Res Commun 48:545–555
Tosi M, Bovey R, Astolfi S, Bodary F, Meisler M, Wellauer PK (1984) Multiple non-allelic genes encoding pancreatic alpha amylase of mouse are expressed in a strain-specific fashion. EMBO J 3:2809–2816
Valência FF (1990) Sequenciamento do gene de α-amylase de Bacillus subtilis. MSc Thesis, Universidade de Brasília, Brasil
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
de Moraes, L.M.P., Astolfi-filho, S. & Oliver, S.G. Development of yeast strains for the efficient utilisation of starch: evaluation of constructs that express α-amylase and glucoamylase separately or as bifunctional fusion proteins. Appl Microbiol Biotechnol 43, 1067–1076 (1995). https://doi.org/10.1007/BF00166927
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00166927