Efficient, non-killing extraction of β-d-fructofuranosidase (an exo-inulase) from Kluyveromyces fragilis at high cell density

Lam, Kwok-Wai; GrootWassink, J.W.D.

doi:10.1016/s0141-0229(85)80010-7

Cited by 12 publications

(7 citation statements)

References 10 publications

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“…For this reason, washing of cells before incubation in the enzyme release buffer was omitted. Buffer solutions with other sulfhydryl compounds such as cysteine (13) yielded 60 to 80% of the amount of solubilized enzyme released by the combined activity of 2-mercaptoethanol and dithiothreitol.…”

Section: Resultsmentioning

confidence: 99%

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556

Rouwenhorst

Visser

Baan

et al. 1988

Appl Environ Microbiol

121

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From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45°C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight-' at D = 0.1 h-l to 2 U mg of cell dry weight1 l at D = 0.8 h-'. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/ repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.

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Section: Resultsmentioning

confidence: 99%

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556

Rouwenhorst

Visser

Baan

et al. 1988

Appl Environ Microbiol

121

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“…Similarly, the inulinase of yeast is in part associated with the cell wall, but, in contrast to the invertase of Saccharomyces, much more of the enzyme is actually secreted into the culture fluid. When grown under conditions which derepress enzyme synthesis, the yeast K. marxianus secretes over 50% of its enzyme into the culture fluid and 35% can be released from the cell wall by sulfhydryls (23,32). In contrast to the biochemistry of the invertase of Saccharomyces, very little is known about the biochemistry of inulinase.…”

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confidence: 99%

Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556

Rouwenhorst

Hensing

Verbakel

et al. 1990

Appl Environ Microbiol

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In the yeast Kluyveromyces marxianus two forms of inulinase were present, namely, an inulinase secreted into the culture fluid and an inulinase retained in the cell wall. Both forms were purified and analyzed by denaturing and nondenaturing polyacrylamide gel electrophoresis. With the use of endo-ID-N-acetyl-glucosaminidase H, it was established that the enzyme retained in the cell wall and the enzyme secreted into the culture fluid have similar subunits consisting of a 64-kDa polypeptide with varying amounts of carbohydrate (26 to 37% of the molecular mass). The two forms of inulinase differed in size because of their differences in subunit aggregation. The enzyme present in the culture fluid was a dimer, and the enzyme retained in the cell wall was a tetramer. The differences in oligomerization did not affect the apparent Km values towards the substrates sucrose and raffinose. These findings support the hypothesis that the retention of glycoproteins in the yeast cell wall may be caused by a permeability barrier towards larger glycoproteins. The amino-terminal end of inulinase was determined and compared with the amino terminus of the closely related invertase. The kinetic and structural evidence indicates that in yeasts two distinct ,3-fructosidases exist, namely, invertase and inulinase.

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“…The cysteine treatment gave almost 80% recovery of the enzyme with four fold purification. Lam and Grootwassink reported complete recovery of inulase from Kluyveromyces fragilis after 30 min treatment with 8 mmol L −1 cysteine solution at pH 7–8. Kidby and Davies reported 60% invertase release from Saccharomyces fragilis at 10 mmol L −1 aqueous mercaptoethanol solution at 30 °C.…”

Section: Resultsmentioning

confidence: 99%

Mathematical model for reactive recovery of invertase by chemical permeabilization of baker's yeast

Koli

Subhedar

Pamidipati

et al. 2015

J. Chem. Technol. Biotechnol.

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BACKGROUND Cell permeabilization techniques allow selective release of an enzyme and specific reactions without breaking the cells. A mathematical model has been developed for selective permeabilization of Saccharomyces cerevisiae to recover invertase at high purity. The effect of thiols on the cell walls of S. cerevisiae was studied under different conditions of pH, cysteine concentration, speed of agitation and solid suspension density. RESULT The rate of release of invertase was a function of pH and cysteine (or thiolate ion) concentration. The maximum recovery of invertase was obtained at pH 10.0, 10 mmol dm−3 cysteine concentration and 15% (w/v) yeast loading. CONCLUSION Thiols are capable of permeabilizing the cell walls of S. cerevisiae and releasing the invertase under basic conditions. The insignificant effect of agitation shows that the external mass transfer resistance does not play any role in the release of invertase. © 2015 Society of Chemical Industry

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Efficient, non-killing extraction of β-d-fructofuranosidase (an exo-inulase) from Kluyveromyces fragilis at high cell density

Cited by 12 publications

References 10 publications

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556

Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556

Mathematical model for reactive recovery of invertase by chemical permeabilization of baker's yeast

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