Expression of an alpha-galactosidase from Saccharomyces cerevisiae in Aspergillus awamori and Aspergillus oryzae.
A gene encoding alpha-galactosidase activity was isolated by polymerase chain reaction (PCR) from Saccharomyces cerevisiae NCYC686 and separately placed under the control of transcriptional elements regulating alpha-amylase expression in Aspergillus oryzae and glucoamylase expression in A. awamori. Following transformation of both A. oryzae and A. awamori with their respective expression vectors, induction of heterologous alpha-galactosidase from positively selected clones was effected through the addition of soluble starch (10% wt/vol) to the growth medium. Upon induction in A. oryzae, a transcriptional instability resulted in degradation of mRNA encoding heterologous alpha-galactosidase, thus preventing expression of the active enzyme. The use of a gene fusion strategy in A. awamori overcame this instability and resulted in stable expression of S. cerevisiae alpha-galactosidase. Subsequent to initial (shake flask) experiments, a series of scale-up and optimisation studies led to heterologous expression of the recombinant enzyme in batch fermentation at 51 U mg(-1) total extracellular protein. This was higher than previously published works, which reported extracellular levels of heterologous alpha-galactosidase up to 38 U mg(-1) total protein. Analysis of crude extracts of the fermentation medium revealed significant differences between the activity parameters reported previously in the literature for this enzyme and those observed here. The recombinant enzyme exhibited thermostability properties not previously reported for S. cerevisiae alpha-galactosidase, a trait which would make it suitable for use in processes requiring high temperatures.
Murphy
R A
RA
Alltech Ireland, Dunboyne, Meath, Ireland.
Power
R F G
RF
D001230:Aspergillus / Q000235:genetics*; D001236:Aspergillus oryzae / Q000235:genetics*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D000519:alpha-Galactosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D000519:alpha-Galactosidase