Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases

Blažić, Marija; Kovačević, Gordana; Prodanović, Olivera; Ostafe, Raluca; Gavrović-Jankulović, Marija; Fischer, Rainer; Prodanović, Radivoje

doi:10.1016/j.pep.2013.03.014

Cited by 27 publications

(26 citation statements)

References 26 publications

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“…The obtained parental Aga2‐GOx and the three mutant Aga2‐GOx enzymes are denoted as WT, and M1, M2, and M3, respectively. It was previously reported (Blazic et al, ) that Aga2‐GOx fusion proteins produced using the surface display in S. cerevisiae exhibited a ~1.5‐fold decrease in catalytic turnover rate as compared with GOx lacking the Aga2 fusion domain. This prior work confirmed, however, that both an enhanced mutant and WT GOx were affected equally by fusion with Aga2, indicating that detection of an improved GOx variant as an Aga2‐fusion reflects the improvement in the properties of the enzyme and not an artifact of the Aga2 fusion domain.…”

Section: Resultsmentioning

confidence: 98%

“…We characterized the differences in thermal stability and glycosylation profiles of the mutant enzymes to account for their ability to outperform the WT under the assay conditions. Previously, a mutant Aga2‐GOx called B11 was purified and characterized (Blazic et al, ) with k cat = 80 s −1 , K M = 16 mM, and catalytic efficiency = 5.0. Our Aga2‐GOx mutant M1 reported here, by comparison, exhibits k cat = 136.1 s −1 , K M = 10.68 mM, and catalytic efficiency = 12.74.…”

Section: Resultsmentioning

confidence: 99%

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Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

Vanella

Nash

2019

Biotech & Bioengineering

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Directed evolution of oxidoreductases to improve their catalytic properties is being ardently pursued in the industrial, biotechnological, and biopharma sectors.Hampering this pursuit are current enzyme screening methods that are limited in terms of throughput, cost, time, and complexity. We present a directed evolution strategy that allows for large-scale one-pot screening of glucose oxidase (GOx) enzyme libraries in well-mixed homogeneous solution. We used GOx variants displayed on the outer cell wall of yeasts to initiate a cascade reaction with horseradish peroxidase (HRP), resulting in peroxidase-mediated phenol crosscoupling and encapsulation of individual cells in well-defined fluorescent alginate hydrogel shells within~10 min in mixed cell suspensions. Following application of denaturing stress to whole-cell GOx libraries, only cells displaying GOx variants with enhanced stability or catalytic activity were able to carry out the hydrogel encapsulation reaction. Fluorescence-activated cell sorting was then used to isolate the enhanced variants. We characterized three of the newly evolved Aspergillus niger GOx enzyme sequences and found up to~5-fold higher specific activity, enhanced thermal stability, and differentiable glycosylation patterns. By coupling intracellular gene expression with the rapid formation of an extracellular hydrogel capsule, our system improves high-throughput screening for directed evolution of H 2 O 2producing enzymes many folds. K E Y W O R D Scell encapsulation, directed evolution, glucose oxidase, hydrogels, yeast display

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

Vanella

Nash

2019

Biotech & Bioengineering

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“…The experiments were performed with Escherichia coli DH5α strain for transformation, kindly donated by Fraunhofer IME, Germany [19]. The exogenous recombinant DNA taken in experimental working was a chiA_pUC57 plasmid, which was purchased from Gene Script (#SD1176).…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Use of factorial design to optimize the efficiency of bacterial transformation

Menghiu¹,

Zbîrcea²,

Ostafe³

2019

Studia UBB Chemia

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A Plackett-Burman factorial design of experiments was created to optimize the protocols of preparation of E. coli DH5α competent cells and transformation of these cells by heat shock method using a chiA_pUC57 plasmid. The numerical parameters to be optimized were: the pH, the concentration of CaCl2, the cell concentration of the culture used for the preparation of the competent cells, the temperature of defrosting of the competent cells, the concentration of plasmid DNA. It was also considered a qualitative factor that might influence the transformation efficiency, namely the use of ultrasound in the heat shock step of transformation protocol. A design of experiments based on 26 experimental values was created. Analyzing this experimental setup by both, Plackett-Burman factorial design and surface response design, it was highlighted that the pH, the concentration of calcium chloride and the concentration of plasmid DNA have a significant influence on the transformation efficiency. The optimal conditions for the preparation and transformation of E. coli DH5α competent cells with chiA_pUC57 plasmid where when the pH of a 40 mM CaCl2 solution was 6, the competent stock cells were thawed slowly on ice and in the heat shock step the cells were subjected to ultrasounds treatment.

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“…In the case of Aga2-based yeast display systems, the cells typically are grown initially in glucose-or raffinose-containing medium to an optical density at 600 nm (OD600) of 2-5 units, then shifted to galactose-supplemented medium for an additional 48-72 h of cultivation (Puthenveetil et al, 2009;Chen et al, 2011;Han et al, 2011;Lee et al, 2011;Dang et al, 2012, Blazic et al, 2013. Alternatively, Andreu & Del Olmo (2013) demonstrated that a long (48-to72-h) incubation period with galactose was not required to achieve good display efficiency.…”

Section: Accepted Manuscriptmentioning

confidence: 99%

“…Therefore, 6-8 h of incubation was suitable for good levels of display efficiency. Blazic et al (2013) reported that a 12-h incubation in galactose was appropriate for optimizing display efficiency. These reports also showed that cell-surface display efficiency was dependent on the anchor protein and target protein/peptide.…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Cell surface engineering of industrial microorganisms for biorefining applications

Tanaka

Kondo

2015

Biotechnology Advances

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Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases

Cited by 27 publications

References 26 publications

Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

Use of factorial design to optimize the efficiency of bacterial transformation

Cell surface engineering of industrial microorganisms for biorefining applications

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