Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors

Miksch, Gerhard; Kleist, Sofia; Friehs, Karl; Flaschel, Erwin

doi:10.1007/s00253-002-1071-z

Cited by 47 publications

(30 citation statements)

References 33 publications

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“…Antibody Production-Anti-AppA rabbit polyclonal antisera was generated by using the overexpression plasmid pPhyt190 (18). E. coli BL21 strains harboring pPhyt190 were grown overnight in 2 liters of TB medium (12 g of casein peptone, 24 g of yeast extract, 4 g of glycerol, and 20 g of NaCl in 1 liter of deionized water) with 1 mM isopropyl 1-thio-␤-D-galactopyranoside and 40 g/ml kanamycin at 37°C in an Erlenmeyer flask.…”

Section: Bacterial Strains and Growthmentioning

confidence: 99%

The Nonconsecutive Disulfide Bond of Escherichia coli Phytase (AppA) Renders It Dependent on the Protein-disulfide Isomerase, DsbC

Berkmen¹,

Boyd

Beckwith

2005

Journal of Biological Chemistry

120

119

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The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.Disulfide bonds between cysteine residues make an important contribution to the folding pathway and stability of many proteins. Early in vitro studies on protein folding showed that the protein RNase when denatured and its disulfide bonds reduced could refold into an active enzyme with the correct disulfide bonds formed (1). Although these results indicated that the information necessary for the proper folding of proteins was intrinsic to the amino acid sequence, the rate of disulfide bond formation in these experiments was much slower than the rates observed in living cells, and the yield of active enzyme was low. The low yields of properly folded enzyme were attributed to the formation of incorrect disulfide bonds in many of the assembled protein molecules. Thus, the in vitro studies, although remarkably successful, were unable to come close to replicating the rapid kinetics and the accuracy of in vivo disulfide bond formation. To explain this inaccuracy observed in biochemical experiments, Anfinsen and co-workers

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Section: Bacterial Strains and Growthmentioning

confidence: 99%

The Nonconsecutive Disulfide Bond of Escherichia coli Phytase (AppA) Renders It Dependent on the Protein-disulfide Isomerase, DsbC

Berkmen¹,

Boyd

Beckwith

2005

Journal of Biological Chemistry

120

119

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“…Residual phytase activities of wild-type Ymphytase and M1 variant after being incubated at 55°C and 58°C for 20 min (b). Melting temperature (T m ) of wild-type (Wt) and M1 variant as determined using thermal shift assay (c) mg; Miksch et al 2002). A further example is the Yersinia homolog phytase from Yersinia frederiksenii, which has compared to other Yersinia phytases a ∼fourfold reduced specific activity (500 U/mg) when expressed in E. coli (Fu et al 2009).…”

Section: Discussionmentioning

confidence: 99%

Directed evolution of a highly active Yersinia mollaretii phytase

Shivange

Serwe

Dennig

et al. 2011

Appl Microbiol Biotechnol

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Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073 U/mg which is ∼10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ∼8,400 clones) a phytase variant with ∼20% improved thermostability (58°C for 20 min; residual activity wild type ∼34%; variant ∼53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993 U/mg).

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“…The cultivation details (bacterial strain, culture medium and cultivation of bacteria), on-line and off-line methods, and experimental data have been published previously (Miksch et al 2002;Kleist et al 2003;Arndt et al 2004). Short description of the considered here cultivation process is given below.…”

Section: Methodsmentioning

confidence: 99%

Multiple model approach to modelling of Escherichia coli fed-batch cultivation extracellular production of bacterial phytase

Roeva¹,

Pencheva²,

Tzonkov³

et al. 2007

Electron. J. Biotechnol.

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Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors

Cited by 47 publications

References 33 publications

The Nonconsecutive Disulfide Bond of Escherichia coli Phytase (AppA) Renders It Dependent on the Protein-disulfide Isomerase, DsbC

The Nonconsecutive Disulfide Bond of Escherichia coli Phytase (AppA) Renders It Dependent on the Protein-disulfide Isomerase, DsbC

Directed evolution of a highly active Yersinia mollaretii phytase

Multiple model approach to modelling of Escherichia coli fed-batch cultivation extracellular production of bacterial phytase

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