Stepwise error‐prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic<i>Bacillus</i> sp

Melzer, Susanne; Sonnendecker, Christian; Föllner, C.; Zimmermann, Wolfgang

doi:10.1016/j.fob.2015.06.002

Cited by 18 publications

(12 citation statements)

References 74 publications

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“…In this technique, random fragmentation of genes in a library (e.g., by DNase I digestion) is followed by PCR-based reassembly of overlapping fragments with sufficient homology, which effectively recombines mutations within the gene library [ 71 ]. Examples of DNA shuffling in combination with epPCR are seen in protein engineering efforts that have introduced mutations to the CBM of cyclodextrin glucanotransferase from Bacillus sp., and the glycan-binding regions of N -oligosaccharyltransferase from Campylobacter jejuni resulting in increased specificity and efficiency of those enzymes [ 72 , 73 ].…”

Section: Mutagenesis Methods For Library Generationmentioning

confidence: 99%

Resources and Methods for Engineering “Designer” Glycan-Binding Proteins

Warkentin

Kwan

2021

Molecules

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This review provides information on available methods for engineering glycan-binding proteins (GBP). Glycans are involved in a variety of physiological functions and are found in all domains of life and viruses. Due to their wide range of functions, GBPs have been developed with diagnostic, therapeutic, and biotechnological applications. The development of GBPs has traditionally been hindered by a lack of available glycan targets and sensitive and selective protein scaffolds; however, recent advances in glycobiology have largely overcome these challenges. Here we provide information on how to approach the design of novel “designer” GBPs, starting from the protein scaffold to the mutagenesis methods, selection, and characterization of the GBPs.

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Section: Mutagenesis Methods For Library Generationmentioning

confidence: 99%

Resources and Methods for Engineering “Designer” Glycan-Binding Proteins

Warkentin

Kwan

2021

Molecules

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“…Synthesis reactions were performed in 1 mL total volume at 50 °C (GeoT at 60 °C), and 100 μL of aliquots was removed after 1, 4, 8, 16, and 24 h (with GeoT also after 48 and 72 h) and added to 100 μL 0.2 m acetic acid buffer, pH 4.5, followed by heat inactivation and glucoamylase treatment as previously described . Samples were diluted and analyzed by high‐pressure anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) as described previously . Elution was performed at −10 to 2 min with 8 m m NaNO 3 , followed by linear gradients to 14, 22, 36, 80, 200, 100, and 8 m m NaNO 3 at 10, 35, 45, 55 57, 58, and 62 min after injection.…”

Section: Methodsmentioning

confidence: 99%

Domain shuffling of cyclodextrin glucanotransferases for tailored product specificity and thermal stability

Sonnendecker

Zimmermann

2019

FEBS Open Bio

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Cyclodextrin glucanotransferases (CGTases) convert α‐1,4‐glucans to cyclic oligosaccharides (cyclodextrins, CD), which have found applications in the food and the pharmaceutical industries. In this study, we used two CGTases with different cyclization activities, product specificities, and pH and temperature optima to construct chimeric variants for the synthesis of large‐ring CD. We used (a) a synthetic thermostable CGTase mainly forming α‐ and β‐CD (CD6 and CD7) derived from Geobacillus stearothermophilus ET1/NO2 (GeoT), and (b) a CGTase with lower cyclization activity from the alkaliphilic Bacillus sp. G825‐6, which mainly synthesizes γ‐CD (CD8). The A1, B, A2, and CDE domains of the G825‐6 CGTase were replaced with corresponding GeoT CGTase domains by utilizing a megaprimer cloning approach. A comparison of the optimum temperature and pH, thermal stability, and CD products synthesized by the variants revealed that the B domain had a major impact on the cyclization activity, thermal stability, and product specificity of the constructed chimera. Complete suppression of the synthesis of CD6 was observed with the variants GeoT‐A1/B and GeoT‐A1/A2/CDE. The variant GeoT‐A1/A2/CDE showed the desired enzyme properties for large‐ring CD synthesis. Its melting temperature was 9 °C higher compared to the G825‐6 CGTase and it synthesized up to 3.3 g·L−1 CD9 to CD12, corresponding to a 1.8‐ and 2.3‐fold increase compared to GeoT and G825‐6 CGTase, respectively. In conclusion, GeoT‐A1/A2/CDE may be a candidate for the further development of CGTases specifically forming larger CD.

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“…Protein sequences from 30 CGTases were obtained from the National Center of Biotechnology Information (www.ncbi.nlm.nih.gov; accession numbers in Table S1) and compared to the CGTase G825-6 sequence [43] using a clustalW algorithm implemented in the homology software MEGA, version 6.06 [44]. Based on the X-ray structure of the CGTase from Bacillus clarkii (PDB:4JCM), obtained from the Protein Data Bank (PDB) (www.rcsb.org), structure homology modeling was performed using the SWISS-MODEL platform [45,46].…”

Section: Protein Sequence Alignments and Molecular Modelingmentioning

confidence: 99%

Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins

Sonnendecker

Zimmermann

2019

Catalysts

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Cyclodextrin glucanotransferases (CGTases) convert starch to cyclodextrins (CD) of various sizes. To engineer a CGTase for the synthesis of large-ring CD composed of 9 to 12 glucose units, a loop structure of the protein involved in substrate binding was targeted for semi-rational mutagenesis. Based on multiple protein alignments and protein structure information, a mutagenic megaprimer was designed to encode a partial randomization of eight amino acid residues within the loop region. The library obtained encoding amino acid sequences occurring in wild type CGTases in combination with a screening procedure yielded sequences displaying a changed CD product specificity. As a result, variants of the CGTase from the alkaliphilic Bacillus sp. G825-6 synthesizing mainly CD9 to CD12 could be obtained. When the mutagenesis experiment was performed with the CGTase G825-6 variant Y183R, the same loop alterations that increased the total CD synthesis activity resulted in lower activities of the variant enzymes created. In the presence of the amino acid residue R183, the synthesis of CD8 was suppressed and larger CD were obtained as the main products. The alterations not only affected the product specificity, but also influenced the thermal stability of some of the CGTase variants indicating the importance of the loop structure for the stability of the CGTase.

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Stepwise error‐prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilicBacillus sp

Abstract: Graphical abstract

Cited by 18 publications

References 74 publications

Resources and Methods for Engineering “Designer” Glycan-Binding Proteins

Resources and Methods for Engineering “Designer” Glycan-Binding Proteins

Domain shuffling of cyclodextrin glucanotransferases for tailored product specificity and thermal stability

Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins

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