Unexpected destruction of chitosomal chitin synthetase by an endogenous protease during sucrose density gradient purification.
(74/258)
Because of their intrinsic low buoyant density, chitosomes can be separated from crude cell homogenates (1000 g or 35,000 g supernatants) of Mucor rouxii by isopycnic sedimentation in sucrose density gradients. To accelerate and simplify the isolation of chitosomes, we centrifuged the cell-free extracts at ultrahigh speed (in a fixed-angle rotor at forces up to 311,000 g Rav) and found that the duration of centrifugation was critical. Prolonged centrifugation at ultrahigh speed caused severe distortion of the chitin synthetase profile in the gradient as the peak of chitosomal chitin synthetase nearly disappeared. We traced the problem to a soluble protease(s) that moved into the chitosome band during protracted centrifugation and destroyed the chitin synthetase activity. The interfering protease was a soluble protein with a sedimentation coefficient of 4.6 S and a pH optimum of 7-7.5, and it was sensitive to PMSF (phenylmethylsulfonyl fluoride), indicating that it was a serine protease. Unlike other proteases, it destroyed chitin synthetase but did not activate the chitin synthetase zymogen. The interfering protease could be eliminated either by adding PMSF to the cells immediately after breakage or by removing the upper part of the sucrose gradient midway through the centrifugation of the cell-free extract and then completing the sedimentation with the 'decapitated' gradient. (+info)
A subunit of protein kinase a regulates growth and differentiation in the fungus Mucor circinelloides.
(75/258)
Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli.
(77/258)
Efficient glycosynthase mutant derived from Mucor hiemalis endo-beta-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan.
(78/258)
Immobilization and stability of lipase from Mucor racemosus NRRL 3631.
(80/258)
The lipase from Mucor racemosus NRRL 3631 was partially purified by fractional precipitation using 60% ammonium sulfate, which resulted in a 8.33-fold purification. The partially purified lipase was then immobilized using different immobilization techniques: physical adsorption, ionic binding, and entrapment. Entrapment in a 4% agar proved to be the most suitable technique (82% yield), as the immobilized lipase was more stable at acidic and alkaline pHs than the free enzyme, plus 100% of the original activity was retained owing to the thermal stability of the immobilized enzyme after heat treatment for 60 min at 45 degrees Celsius. The calculated half-lives (472.5, 433.12, and 268.5 min at 50, 55, and 60 degrees Celsius, respectively) and the activation energy (9.85 kcal/mol) for the immobilized enzyme were higher than those for the free enzyme. Under the selected conditions, the immobilized enzyme had a higher K(m) (11.11 mM) and lower V(max) (105.26 U/mg protein) when compared with the free enzyme (8.33 mM and 125.0 U/mg protein, respectively). The operational stability of the biocatalyst was tested for both the hydrolysis of triglycerides and esterification of fatty acids with glycerol. After 4 cycles, the immobilized lipase retained approximately 50% and 80% of its original activity in the hydrolysis and esterification reactions, respectively. (+info)