Kinetics of improved productivity of β-galactosidase by a cycloheximide-resistant mutant of Kluyveromyces marxianus
ABSTRACT The maximum volumetric productivity of -galactosidase by a Kluyveromyces marxianus mutant, grown on lactose/corn steep liquor medium for 3d, was 150IUl–1h–1 which is twice that of the parent organism. During product formation, mutated cells provided more resistance against thermal inactivation.
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ABSTRACT: In 1988 McCusker and Haber generated a series of mutants which are resistant to the minimum inhibitory concentration of the protein synthesis inhibitor cycloheximide. These cycloheximide-resistant, temperature-sensitive (crl) mutants, in addition, exhibited other pleiotropic phenotypes, e.g., incorrect response to starvation, hypersensitivity against amino acid analogues, and other protein synthesis inhibitors. Temperature sensitivity of one of these mutants, crl3-2, had been found to be suppressed by a mutation, SCL1-1, which resided in an alpha-type subunit of the 20S proteasome. We cloned the CRL3 gene by complementation and found CRL3 to be identical to the SUG1/CIM3 gene coding for a subunit of the 19S cap complex of the 26S proteasome. Another mutation, crl21, revealed to be allelic with the 20S proteasomal gene PRE3. crl3-2 and crl21 mutant cells show significant defects in proteasome-dependent proteolysis, whereas the SCL1-1 suppressor mutation causes partial restoration of crl3-2-induced proteolytic defects. Notably, cycloheximide resistance was also detected for other proteolytically deficient proteasome mutants (pre1-1, pre2-1, pre3-1, pre4-1). Moreover, proteasomal genes were found within genomic sequences of 9 of 13 chromosomal loci to which crl mutations had been mapped. We therefore assume that most if not all crl mutations reside in the proteasome and that phenotypes found are a result of defective protein degradation.Molecular Biology of the Cell 01/1998; 8(12):2487-99. · 4.60 Impact Factor
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ABSTRACT: The influence of enzyme immobilization on the main thermodynamic quantities of glucose isomerization to fructose and vice versa was studied in batch tests by using a commercial immobilized glucose isomerase. The values calculated for the immobilized system showed, when compared with those reported for the native enzyme, higher equilibrium constants at T > 70°C and simultaneous increases in both standard enthalpy and entropy changes of reaction. Activation enthalpy and entropy changes of both forward and reverse enzyme-catalyzed reactions were calculated from the initial glucose isomerase activity evaluated at different temperatures by the Briggs-Haldane model. The activation enthalpy of the forward reaction was about 24% less than that of the native enzyme. Enzyme inactivation tests performed at different temperatures and sugar equilibrium concentrations confirmed the existence of the so-called substrate protection phenomenon which resulted in a 29–36% reduction in the decay constant. A slight increase in the activation free enthalpy of the inactivation reaction was also shown.Enzyme and Microbial Technology. 01/1997;
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ABSTRACT: Lactose-intolerance is manifested in 50 % of the world's population. This can be reme-diated by removing lactose from the diet or converting it into glucose and galactose with b-galactosidase (EC 220.127.116.11). In this work, batch production of this enzyme in the presence of lactose, galactose, cellobiose, xylose, arabinose, sucrose and glucose was investigated using Kluyveromyces marxianus in shake flask culture studies. Substrate type and tempera-ture were the independent variables that directly regulated the specific growth and b-ga-lactosidase production rates. Lactose (2 %) supported the maximum specific product yield (Y P/X), followed by galactose, sucrose, cellobiose, xylose, arabinose and glucose. Its synthe-sis was regulated by an induction and a growth-dependent repression mechanism. The optimum temperature for the production was found to be 35–37 °C. The highest volume-tric productivity of enzyme (80.0 IU/L/h) occurred on lactose-corn steep liquor medium. This was significantly higher than the calculated values reported in the literature. Ther-modynamic studies revealed that the cells provided a defence mechanism against thermal inactivation. The enzyme was stable at 60 °C and pH=5.0–7.0, and it may find application in commercial lactose hydrolysis.01/2003; 152346631:547-455.