Expression of the Aspergillus aculeatus endo-beta-1,4-mannanase encoding gene (man1) in Saccharomyces cerevisiae and characterization of the recombinant enzyme.
The endo-beta-1,4-mannanase encoding gene man1 of Aspergillus aculeatus MRC11624 was amplified from mRNA by polymerase chain reaction using sequence-specific primers designed from the published sequence of man1 from A. aculeatus KSM510. The amplified fragment was cloned and expressed in Saccharomyces cerevisiae under the gene regulation of the alcohol dehydrogenase (ADH2(PT)) and phosphoglycerate kinase (PGK1(PT)) promoters and terminators, respectively. The man1 gene product was designated Man5A. Subsequently, the FUR1 gene of the recombinant yeast strains was disrupted to create autoselective strains: S. cerevisiae Man5ADH2 and S. cerevisiae Man5PGK1. The strains secreted 521 nkat/ml and 379 nkat/ml of active Man5A after 96 h of growth in a complex medium. These levels were equivalent to 118 and 86 mg/l of Man5A protein produced, respectively. The properties of the native and recombinant Man5A were investigated and found to be similar. The apparent molecular mass of the recombinant enzyme was 50 kDa compared to 45 kDa of the native enzyme due to glycosylation. The determined K(m) and V(max) values were 0.3 mg/ml and 82 micromol/min/mg for the recombinant and 0.15 mg/ml and 180 micromol/min/mg for the native Man5A, respectively. The maximum pH and thermal stability were observed within the range of pH 4-6 and 50 degrees C and below. The pH and temperature optima and stability were relatively similar for recombinant and native Man5A. Hydrolysis of an unbranched beta-1,4-linked mannan polymer released mannose, mannobiose, and mannotriose as the main products.
Setati
M E
ME
Department of Microbiology, University of Stellenbosch, Stellenbosch, 7600, South Africa.
Ademark
P
P
van Zyl
W H
WH
Hahn-Hägerdal
B
B
Stålbrand
H
H
D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008361:Mannosidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013816:Thermodynamics
D011994:Recombinant Proteins; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase