Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme.
Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of approximately 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K(M) was 42 mM, and V(max) was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.
de Almeida
Fabiana M
FM
Departamento de Biologia, Faculdade de Filosofia Ciências e Letras, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Bonini
Beatriz M
BM
Beton
Daniela
D
Jorge
João A
JA
Terenzi
Héctor F
HF
da Silva
Aline M
AM
D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014198:Trehalase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014198:Trehalase