Expression of a Bacillus alpha-amylase gene in yeast.
A recombinant plasmid, pSR11.3, containing the alpha-amylase gene (AMY) of Bacillus amyloliquefaciens was characterized and expressed in Bacillus subtilis. A 2.3 kilobase BamHI-BglII fragment carrying AMY was cloned into pBR322 (pEL322) and in both orientations into a multi-copy Escherichia coli-yeast shuttle vector YEp13 (pAM13) and expressed in E. coli HB101 and various Saccharomyces stains. We report on the successful secretion of an active bacterial enzyme in yeast without using yeast promoter and secretory signals. Enzyme production in B. subtilis 1A297(pSR11.3), E. coli HB101(pEL322) and Saccharomyces JM2773-15B(pAM13) transformants was measured as 125, 22 and 123 U/ml, respectively. The molecular weight of the purified alpha-amylase secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B-(pAM13) was estimated to be 55 kDa. The pH and temperature optima for the alpha-amylase activities of the transformants were 6.5 to 8.0 and 50 to 65 degrees C, respectively. Amylose hydrolysis profiles of the alpha-amylases secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) indicate effective meso-thermostable hydrolytic enzymes with maltotriose and maltose, respectively, as major end products.
Pretorius
I S
IS
Department of Microbiology, University of Stellenbosch, South Africa.
Laing
E
E
Pretorius
G H
GH
Marmur
J
J
D001412:Bacillus subtilis / Q000235:genetics*; D003001:Cloning, Molecular; D003470:Culture Media; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014162:Transfection; D000516:alpha-Amylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D003470:Culture Media; D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes; D000516:alpha-Amylases