Cloning, heterologous expression, and characterization of Thielavia terrestris glucoamylase.
Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/ cellular biology is poorly understood. Only a few genes have been cloned from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae alpha-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 micromol/(min x mg) with maltose as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60 degrees C. The enzyme was stable at 60 degrees C for 30 min. The Km and kcat of the enzyme for maltotriose were determined at various pHs and temperatures. At 20 degrees C and pH 4.0, the enzyme had a Km of 0.33 +/- 0.07 mM and a kcat of (5.5 +/- 0.5) x 103 min(-1) for maltotriose. The temperature dependence of kcat/Km indicated an activation free energy of 2.8 kJ/mol across the range of 20-70 degrees C. Overall, the enzyme derived from the thermophilic fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi.
Rey
Michael W
MW
Novozymes Biotech, 1445 Drew Avenue, Davis, CA 95616, USA.
Brown
Kimberly M
KM
Golightly
Elizabeth J
EJ
Fuglsang
Claus C
CC
Nielsen
Bjarne R
BR
Hendriksen
Hanne V
HV
Butterworth
Amy
A
Xu
Feng
F
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics*; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004795:Enzyme Stability; D015966:Gene Expression Regulation, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D013696:Temperature; D000516:alpha-Amylases / Q000378:metabolism
D003470:Culture Media; D011134:Polysaccharides; D011994:Recombinant Proteins; C008315:maltodextrin; D000516:alpha-Amylases; D005087:Glucan 1,4-alpha-Glucosidase