A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis

Ren, Xiaodong; Yu, Dawei; Yu, Lei; Gao, Gui; Han, Si‐ping; Feng, Yan

doi:10.1016/j.jbiotec.2007.01.038

Cited by 36 publications

(20 citation statements)

References 17 publications

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“…Indeed, permeabilization and lysis of E. coli cells can be achieved upon exposure to increased temperatures, which enables the use of a whole cell bioconversion [18]. Furthermore, endogenous E. coli enzymes are typically inactivated under these conditions, which considerably reduces contaminating activities.…”

Section: Introductionmentioning

confidence: 78%

See 1 more Smart Citation

Enzymatic production of β‐D‐glucose‐1‐phosphate from trehalose

Borght¹,

Desmet²,

Soetaert³

2010

Biotechnology Journal

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beta-D-Glucose-1-phosphate (beta Glc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of beta Glc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60 C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, beta Glc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what ist observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline beta Glc1P is now available from the cheap substrates trehalose and inorganic phosphate

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Section: Introductionmentioning

confidence: 78%

“…Thermolysis of E. coli to release a recombinant thermostable esterase was previously described by Ren et al [18]. The host cells are lysed, and E. coli proteins are denatured while the thermostable enzyme remains active.…”

Section: Production Of the Biocatalystsmentioning

confidence: 99%

Enzymatic production of β‐D‐glucose‐1‐phosphate from trehalose

Borght¹,

Desmet²,

Soetaert³

2010

Biotechnology Journal

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“…The membrane structure of E. coli cells is partially or entirely disrupted at high temperatures, and thus thermophilic enzymes, which are produced as soluble proteins, leak out of the cells (6)(7)(8)(9). Although the heat-induced leakage of thermophilic enzymes results in better accessibility between the enzymes and substrates, it limits the applicability of thermophilic whole-cell catalysts to continuous and repeated-batch reaction systems.…”

mentioning

confidence: 99%

Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst

Ninh

Honda

Yokohigashi

et al. 2013

Appl Environ Microbiol

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The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.T hermophilic enzymes are promising tools for biotransformation owing to their high operational stability, cosolvent compatibility, and low risk of contamination (1-3). The direct use of recombinant mesophiles (e.g., Escherichia coli) having heterologous thermophilic enzymes at high temperatures results in the denaturation of indigenous enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. Honda et al. have demonstrated that the rational combination of these thermophilic whole-cell catalysts enables the construction of in vitro artificial biosynthetic pathways for the production of value-added chemicals (4). Recently, Ye et al. have successfully constructed a chimeric Embden-Meyerhof pathway with the balanced consumption and regeneration of ATP and ADP, using nine recombinant E. coli strains, each of which overproduces a thermophilic glycolytic enzyme (5).The membrane structure of E. coli cells is partially or entirely disrupted at high temperatures, and thus thermophilic enzymes, which are produced as soluble proteins, leak out of the cells (6-9). Although the heat-induced leakage of thermophilic enzymes results in better accessibility between the enzymes and substrates, it limits the applicability of thermophilic whole-cell catalysts to continuous and repeated-batch reaction systems. This limitation prevents us from exploiting the most advantageous feature of thermophilic biocatalysts, namely, their excellent stability. To overcome this limitation, one potential strategy is the integration of thermophilic enzymes to the membrane struct...

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“…Currently, in the field of biotechnology, sonoporation has a broad range of applications, including its use for inhibition of food-borne pathogens in processed food (Forghani et al, 2013;Koolman et al, 2014), disruption of various kinds of cells (Ren et al, 2007;Sudar et al, 2013;Zhou et al, 2014), and assistance in bioactive component extraction (Kwun et al, 2009;Liu et al, 2011). The present study demonstrated that sonoporation can immediately and effectively homogenize liquid-cultured mycelial pellets of G. lucidum, and highly uniform mycelia can be obtained.…”

Section: Discussionmentioning

confidence: 53%

Sonoporation of Ganoderma lucidum Mycelium for High Biomass and Exopolysaccharide Productivity in Submerged Culture

Liu¹,

Zhang²,

Kuang³

et al. 2016

IJAB

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Ganoderma lucidum, known as Lingzhi in China and Reishi in Japan, is a famous medicinal mushroom that is commonly produced by solid-state fermentation. Recently, submerged culture has been considered as a promising alternative for production of G. lucidum. Herein, to provide a theoretical base for preparation of a homogenous inoculum for G. lucidum submerged culture we investigated the effect of sonoporation on G. lucidum mycelium. Submerged culture was also evaluated using the sonoporated mycelium. Microscopic investigation indicated that the mycelial pellets (0.55 g, dry weight) contained in 60 mL solution were almost completely disappeared within 20 s by sonoporation (20 kHz and 125 w). The homogenate obtained after sonoporation had a 3.75×106 cfu/mL cell concentration. The sonoporation caused rapid leakage of intracellular protein that increased linearly with time. The metabolic activity of the cells decreased rapidly after sonoporation. Sonoporation also negatively affected laccase activity. When the homogenate was used at 4.76% (v/v) inoculum level, the cultures produced biomass and exopolysaccharide of 15.75 and 0.54 g/L, respectively; these values were 129.26 and 45.95% higher than that of the control culture using a pellet-type inoculum without sonoporation. Optimal inoculum size was 5.88-6.97% (v/v). We conclude that sonoporation is an effective method for homogenization of G. lucidum mycelium, and sonoporated mycelium can improve productivity significantly in G. lucidum submerged culture and requires lower levels of starting inoculum.

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A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis

Cited by 36 publications

References 17 publications

Enzymatic production of β‐D‐glucose‐1‐phosphate from trehalose

Enzymatic production of β‐D‐glucose‐1‐phosphate from trehalose

Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst

Sonoporation of Ganoderma lucidum Mycelium for High Biomass and Exopolysaccharide Productivity in Submerged Culture

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