Characterization of Mucor pusillus rennin expressed in Pichia pastoris: enzymic, spectroscopic and calorimetric studies.
The aspartic proteinase gene of Mucor pusillus rennin expressed in Pichia pastoris was characterized in terms of structural and conformational stability induced by temperature. This enzyme is 12% glycosylated, with a similar specific activity to the native fungal enzyme. The secondary structure determined by CD is mainly due to beta-sheet structures with an important contribution of aromatic components. The calorimetric studies were carried out in the temperature range in which the enzyme is most stable. The enzyme undergoes an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. Between pH 3.0 and 7.0, only one endotherm characterized the thermal denaturation of enzyme. At pH 5.0, the most stable condition found, the denaturation can be fitted to the two-state irreversible model. Thus the kinetic constant and activation parameters of the denaturation process could be obtained. Upon reaching pH 7.5, the denaturation is characterized by two endotherms. This evidence indicates the complex tridimensional structure of this enzyme. Finally, taking into account the conservative tertiary structure of the aspartic proteinase family we comment on our results with reference to the crystallographic structure of M. pusillus pepsin [Newman, Watson, Roychowdhury, Badasso, Cleasby, Wood, Tickle and Blundell (1993) J. Mol. Biol. 221, 1295-1309].
Beldarraín
A
A
Processes and Systems Evaluation Division, Production Plant, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba.
Acosta
N
N
Montesinos
R
R
Mata
M
M
Cremata
J
J
D001709:Biotechnology; D002152:Calorimetry, Differential Scanning; D012085:Chymosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D015870:Gene Expression; D005800:Genes, Fungal; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009089:Mucor / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
D011994:Recombinant Proteins; D012085:Chymosin