Kinetic analysis of cellulose synthase of Gluconacetobacter hansenii in whole cells and in purified form

John B. McManus, Liza Anne Wilson, Hui Yang, James D. Kubicki, Ming Tien

Research output: Contribution to journalArticle

Abstract

The Gram-negative bacterium, Gluconacetobacter hansenii, has been long studied and is a model for cellulose synthesis. It produces cellulose, using the enzyme AcsA-AcsB, of exceptionally high crystallinity in comparison to the cellulose of higher plants. We determined the rate of cellulose synthesis in whole cells measured as moles of glucose incorporated into cellulose per second per mole of enzyme. This was determined by quantifying the rate of cellulose synthesis (over a short time span, such that the enzyme concentration is not changing due to cell growth) and the amount of enzyme in the whole cell by quantitative western blotting. We found that the whole cell rate of 24 s−1 is much faster than the kcat, measured from steady-state kinetic analysis, of 1.7 s−1. Our whole cell rates are consistent with previous studies using microscopy. We postulate that the rationale for this difference is the presence of an alternative in vivo priming mechanism. This in turn can increase the rate of initiation, which we previously postulated to be the rate-limiting step in catalysis.

Original languageEnglish (US)
Pages (from-to)24-29
Number of pages6
JournalEnzyme and Microbial Technology
Volume119
DOIs
StatePublished - Dec 1 2018

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Gluconacetobacter
Cellulose
Kinetics
Enzymes
Cell growth
Gram-Negative Bacteria
Catalysis
Glucose
cellulose synthase
Microscopy
Bacteria
Microscopic examination
Western Blotting
Growth

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

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abstract = "The Gram-negative bacterium, Gluconacetobacter hansenii, has been long studied and is a model for cellulose synthesis. It produces cellulose, using the enzyme AcsA-AcsB, of exceptionally high crystallinity in comparison to the cellulose of higher plants. We determined the rate of cellulose synthesis in whole cells measured as moles of glucose incorporated into cellulose per second per mole of enzyme. This was determined by quantifying the rate of cellulose synthesis (over a short time span, such that the enzyme concentration is not changing due to cell growth) and the amount of enzyme in the whole cell by quantitative western blotting. We found that the whole cell rate of 24 s−1 is much faster than the kcat, measured from steady-state kinetic analysis, of 1.7 s−1. Our whole cell rates are consistent with previous studies using microscopy. We postulate that the rationale for this difference is the presence of an alternative in vivo priming mechanism. This in turn can increase the rate of initiation, which we previously postulated to be the rate-limiting step in catalysis.",
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Kinetic analysis of cellulose synthase of Gluconacetobacter hansenii in whole cells and in purified form. / McManus, John B.; Wilson, Liza Anne; Yang, Hui; Kubicki, James D.; Tien, Ming.

In: Enzyme and Microbial Technology, Vol. 119, 01.12.2018, p. 24-29.

Research output: Contribution to journalArticle

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