immobilization of pichia pastoris cells containing alcohol oxidase activity
نویسندگان
چکیده
background and objectives : the attempts were made to describe the development of a whole cell immobilization of p. pastoris by entrapping the cells in polyacrylamide gel beads. the alcohol oxidase activity of the whole cell pichia pastoris was evaluated in comparison with yeast biomass production. materials and methods : methylotrophic yeast p. pastoris was obtained from collection of standard microorganisms, department of bacterial vaccines, pasteur institute of iran (csmpi). stock culture was maintained on ypd agar plates. alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. the cells were harvested by centrifugation then permeabilized. finally the cells were immobilized in polyacrylamide gel beads. the activity of alcohol oxidase was determined by method of tane et al. results : at the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell p. pastoris reached the highest level. in comparison, the alcohol oxidase activity was measured in an immobilized p. pastoris when entrapped in polyacrylamide gel beads. the alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. the cells were permeabilized by cetyltrimethylammonium bromide (ctab) and immobilized. ctab was also found to increase the gel permeability. alcohol oxidase activity of immobilized cells was then quantitated by abts/pod spectrophotometric method at od420. there was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. by addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. conclusion : immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media.
منابع مشابه
Immobilization of Pichia pastoris cells containing alcohol oxidase activity
BACKGROUND AND OBJECTIVES The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. MATERIALS AND METHODS Methylotrophic yeast P. pastoris was obtained from Collection of Standard M...
متن کاملCrystal Structure of Alcohol Oxidase from Pichia pastoris
FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta...
متن کاملStructure of Alcohol Oxidase from Pichia pastoris by Cryo-Electron Microscopy
The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the...
متن کاملNon-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris.
The growth of Pichia pastoris in a mixture of either glycerol or glucose and methanol follows a diauxic growth, with C1 utilizing enzymes being repressed. Therefore, these carbon sources can not be used as a mixture with methanol to simultaneously grow P. pastoris and induce C1 utilizing enzymes, especially in a shake flask cultures of AOX-deficient P. pastoris. Among the alternative carbon sou...
متن کاملPeroxisomal Targeting, Import, and Assembly of Alcohol Oxidase in Pichia pastoris
Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal pro...
متن کاملمنابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
iranian journal of microbiologyجلد ۳، شماره ۴، صفحات ۲۱۰-۲۱۵
میزبانی شده توسط پلتفرم ابری doprax.com
copyright © 2015-2023