Expression of a Bacillus phytase C gene in Pichia pastoris and properties of the recombinant enzyme.
The cloning and expression of a native gene encoding a Bacillus subtilis phytase using Pichia pastoris as the host is described. In addition, the influence of N-glycosylation on the biochemical properties of the B. subtilis phytase, the influence of pH on the thermostability of the recombinant and native B. subtilis phytases, and the resistance of both phytases to shrimp digestive enzymes and porcine trypsin are also described. After 48 h of methanol induction in shake flasks, a selected recombinant strain produced and secreted 0.82 U/ml (71 mg/liter) recombinant phytase. This phytase was N-glycosylated, had a molecular mass of 39 kDa after N-deglycosylation, exhibited activity within a pH range of 2.5 to 9 and at temperatures of 25 to 70 degrees C, had high residual activity (85% +/- 2%) after 10 min of heat treatment at 80 degrees C and pH 5.5 in the presence of 5 mM CaCl(2), and was resistant to shrimp digestive enzymes and porcine trypsin. Although the recombinant Bacillus phytase had pH and temperature activity profiles that were similar to those of the corresponding nonglycosylated native phytase, the thermal stabilities of the recombinant and native phytases were different, although both were calcium concentration and pH dependent.
Guerrero-Olazarán
Martha
M
Instituto de Biotecnología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León,San Nicolás de los Garza, Nuevo León, México. mguerrer58@yahoo.com.mx
Rodríguez-Blanco
Lilí
L
Carreon-Treviño
J Gerardo
JG
Gallegos-López
Juan A
JA
Viader-Salvadó
José M
JM
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002122:Calcium Chloride / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D003067:Coenzymes; D011994:Recombinant Proteins; D010832:6-Phytase; D014357:Trypsin; D002122:Calcium Chloride