Kinetics of improved productivity of β-galactosidase by a cycloheximide-resistant mutant of Kluyveromyces marxianus

National Institute for Biotechnology and Genetic Engineering
Biotechnology Letters (Impact Factor: 1.59). 04/2004; 26(9):741-746. DOI: 10.1023/B:BILE.0000024089.52462.98


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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    • "It was also significantly lower than the values on the thermostabilized glucose isomerase system (160–235 kJ/mol) (Converti and Dominguez 2001). The entropy value of thermal inactivation of enzyme formation by mutant cultures was also very low (and had negative symbol) in SSF and was comparable with that for product formation by the thermo-tolerant yeast (Rajoka et al. 2004) but lower than for a thermo-stabilized reaction (Chen and Stites 2001). This suggested that there was negligible disorder up to 45°C in SSF. "
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    ABSTRACT: The aim of the present investigation was to comparatively evaluate the behaviour of A. niger and its derepressed mutant in production of α-galactosidase in submerged (SmF) and solid state fermentation (SSF) using basal Vogel’s medium or corn steep liquor as nitrogen source and observe the response of latter source under both cultural techniques under different temperature regimes, and determine if SSF can be exploited in a wide range of temperature expected to vary in this fermentation system. All studies were performed in both systems under pre-optimized cultural conditions. Higher melting temperature and negative values of entropy of activation in SSF indicated that the genetic system of both organisms was thermodynamically resistant in the presence of corn steep liquor but sensitive to inactivation in the presence of Vogel’s nitrogen sources in submerged fermentation. This was reflected as the organisms demanded higher magnitudes of energy for product formation in the presence of ammonium salts. Studies on influence of corn steep liquor revealed that it had stabilizing effect too in both fermentation systems but the mutant strain was more stable in both fermentation systems. Because of these properties, the mutant organism may be exploited for bulk production of α-galactosidase in SSF under condition where temperature may fluctuate during fermentation.
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    ABSTRACT: Seven strains of the genus Kluyveromyces were screened for β-galactosidase activity and Kluyveromyces marxianus ATCC 16045 was selected as the best enzyme producer for culture medium optimization. The production of β-galactosidase by submerged cultivation was evaluated using a factorial design and response surface methodology. The culture medium containing whey and parboiled rice effluent was formulated to maximize the production of β-galactosidase. The effects of the initial pH and the concentrations of whey lactose, peptone, (NH4)2SO4, yeast extract, and parboiled rice effluent on enzyme production were studied using a 2IV6-2 fractional design. A CCRD (24 trials plus axial and central points) was used for the four variables selected from the fractional design (lactose, peptone, (NH4)2SO4 and yeast extract), with β-galactosidase activity as the response. The optimum conditions established for production were a whey (lactose) concentration of 120g/L, a yeast extract concentration of 5g/L, a peptone concentration of 15g/L, a (NH4)2SO4 concentration of 15g/L, a parboiled rice effluent concentration of 30g/L, and a pH value of 4.0. Under these conditions, the highest enzymatic activity of 10.4U/mL was measured, being 9.5–9.7 as the values predicted by the proposed model, showing an enzymatic activity increase of 30% using alternative sources of lactose and nitrogen for β-galactosidase production. KeywordsExperimental design–Galactosidase–Parboiled rice effluent–RSM–Whey
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    ABSTRACT: Industrial byproducts namely canola meal, rice bran, sunflower meal, and wheat straw were used as substrates for endo-xylanase production by Humicola lanuginosemutant TH1 through solid substrate fermentation. The enzyme was secreted extracellularly by both wild and mutant cultures. Rice bran supported the maximum production of endo-xylanase followed by wheat straw, canola meal and sunflower meal. The highest activity was achieved after 72 h of culture and the highest yields from the above substrates were 842, 840, 610 and 608 IU per g substrate consumed respectively. The highest productivity (281 IU flask−1 h−1 corresponding to 5620 l−1 h-1) of endo-xylanase by the mutant of H. lanuginosa was 1.6-fold more than that produced by the parental organism in solid-state fermentation of rice bran at 45 °C. Maximum specific activity (180 IU mg−1 protein) and substrate consumption rates were significantly more than those reported by previous researchers on Humicola sp. The mutant possessed markedly low accompanying cellulase activity. Thermodynamic studies revealed that the mutant required significantly lower activation energy for enzyme production and higher for thermal inactivation which signified that the endogenous metabolic machinery of mutant cells exerted more protection against thermal inactivation during product formation than that needed by its parental cultures.
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