Engineering of the zinc-binding domain of an esterase from Clostridium botulinum towards increased activity on polyesters

Biundo, Antonino; Steinkellner, Georg; Gruber, Karl; Spreitzhofer, Theresa; Ribitsch, Doris; Guebitz, Georg M.

doi:10.1039/c7cy00168a

Cited by 16 publications

(15 citation statements)

References 54 publications

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“…The hydrolysis product (BG) present in the supernatant was determined by HPLC according to Biundo et al. with slight modifications . The samples were eluted from the column by using a nonlinear gradient with a constant flow rate of 0.85 mL min −1 .…”

Section: Methodsmentioning

confidence: 99%

Towards Sustainable High‐Performance Thermoplastics: Synthesis, Characterization, and Enzymatic Hydrolysis of Bisguaiacol‐Based Polyesters

et al. 2018

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The utilization of wood-derived building blocks (xylochemicals) to replace fossil-based precursors is an attractive research subject of modern polymer science. Here, we demonstrate that bisguaiacol (BG), a lignin-derived bisphenol analogue, can be used to prepare biobased polyesters with remarkable thermal properties. BG was treated with different activated diacids to investigate the effect of co-monomer structures on the physical properties of the products. Namely, derivatives of adipic acid, succinic acid, and 2,5-furandicarboxylic acid were used. Moreover, a terephthalic acid derivative was used for comparison purposes. The products were characterized by H NMR spectroscopy, attenuated total reflectance FTIR spectroscopy, gel-permeation chromatography, thermogravimetric analysis, and differential scanning calorimetry to assess their structural and thermal properties in detail. The polymers showed glass-transition temperatures ranging up to 160 °C and thermal stabilities in excess of 300 °C. Furthermore, the susceptibility of the polyester to enzymatic hydrolysis was investigated to assess the potential for further surface functionalization and/or recycling and biodegradation. Indeed, hydrolysis with two different enzymes from the bacteria Thermobifida cellulosilytica led to the release of monomers, as quantified by HPLC. The results of this study indicate that our new polyesters represent promising renewable and biodegradable alternatives to petroleum-based polyesters currently employed in the plastics industry, specifically for applications in which high-temperature stability is essential to ensure overall system integrity.

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Section: Methodsmentioning

confidence: 99%

Towards Sustainable High‐Performance Thermoplastics: Synthesis, Characterization, and Enzymatic Hydrolysis of Bisguaiacol‐Based Polyesters

et al. 2018

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“…In contrast, the truncated version of the enzyme, del71Cbotu_EstA, released some Ta and MHET from PET. Substitutions of the zinc-binding domain residues on the other hand did not improve PET hydrolysis when compared to the wild-type enzyme despite the fact that such mutations had previously been described to increase activity on other polyesters 29 (Fig. 5b ).…”

Section: Characterization Of Variantsmentioning

confidence: 78%

“…1 ), which was previously solved at a resolution of 1.2 Å, (PDB-Code 5AH1) 18 , we hypothesized that concomitant modification of the Zn-binding and N-terminal domain could improve the activity on the synthetic polyester PET. Individual modification in these regions has previously been shown to increase the activity of Cbotu_EstA on synthetic polyesters 28 , 29 . Substitution of amino acids present in the so-called entrance to the zinc ion cavity has an influence on the lid opening during interfacial activation in members of the I.5 lipase family such as the lipase from Geobacillus stearothermophilus (BTL2) 23 .…”

Section: Resultsmentioning

confidence: 95%

“…DNA and protein sequences were analyzed by CLC Main Workbench, version 7.0.3 (Qiagen, Netherlands). Site-directed mutagenesis (SDM) of the mutants of Cbotu_EstA carrying mutations for the residues present on the zinc-binding domain was accomplished using the QuickChange multi site-directed mutagenesis kit (Stratagene) with plasmids previously prepared for each specific substitution 29 and megaprimers for the deletion of the 71 amino acids at the N-terminus of each variant. The following megaprimers were designed so as to anneal on the vector (underlined) and on the desired gene sequence: 5′- TTG TTT AAC TTT AAG AAG GAG ATA TAC ATA GCA TTA TTG GTG GTA ACA ACT ATC CGA TTG-3′ (forward primer) and 5′-CAA TCG GAT AGT TGT TAC CAC CAA TAA TGC TAT GTA TAT CTC CTT CTT AAA GTT AAA CAA -3′ (reverse primer).…”

Section: Methodsmentioning

confidence: 99%

“…Freshly transformed E. coli BL21-Gold(DE3) cells were incubated and induced for protein expression as previously reported 29 . Purification was performed by IMAC as previously reported 21 .…”

Section: Methodsmentioning

confidence: 99%

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Synergistic effect of mutagenesis and truncation to improve a polyesterase from Clostridium botulinum for polyester hydrolysis

Biundo

Reich

Ribitsch

et al. 2018

Sci Rep

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The activity of the esterase (Cbotu_EstA) from Clostridium botulinum on the polyester poly(ethylene terephthalate) (PET) was improved by concomitant engineering of two different domains. On the one hand, the zinc-binding domain present in Cbotu_EstA was subjected to site-directed mutagenesis. On the other hand, a specific domain consisting of 71 amino acids at the N-terminus of the enzyme was deleted. Interestingly, a combination of substitution of residues present in the zinc-binding domain (e.g. S199A) synergistically increased the activity of the enzyme on PET seven fold when combined to the truncation of 71 amino acids at the N-terminus of the enzyme only. Overall, when compared to the native enzyme, the combination of truncation and substitutions in the zinc-binding domain lead to a 50-fold activity improvement. Moreover, analysis of the kinetic parameters of the Cbotu_EstA variants indicated a clear shift of activity from water soluble (i.e. para-nitrophenyl butyrate) to insoluble polymeric substrates. These results evidently show that the interaction with non-natural polymeric substrates provides targets for enzyme engineering.

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Insights into polyester plastic biodegradation by carboxyl ester hydrolases

Gricajeva

Nadda

Gudiukaitė

2021

J of Chemical Tech & Biotech

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Environmental pollution caused by polyesters has become a major ecological safety concern that needs to be managed urgently. One way to resolve this problem is giving the spotlight to current emerging research of microbial biocatalysts. During the last two decades many researchers have reported the ability to break down and modify natural and synthetic polyesters using different microbial carboxyl ester hydrolases (lipases, esterases, cutinases, PETases, etc.) also called polyesterases, and contribution of these enzymes towards the reduction of plastic levels in the future. Continuous search of such lipolytic biocatalysts and their improvement via protein engineering strategies results in beneficial findings making the use of polyesterases in the biodegradation of plastics increasingly more realistic. The present review provides a comprehensive insight into the structural properties enabling microbial lipolytic-type carboxyl ester hydrolases to effectively catalyze the cleavage of ester linkages in different polyester plastics. Moreover, the management of extensively used polyester plastics using different lipolytic enzymes as an innovative eco-friendly solution is presented in this report. Furthermore, improvement of polyesterases via protein engineering for the development of more effective and suitable polyester-degrading lipolytic biocatalysts is summarized in this review as well.

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Engineering of the zinc-binding domain of an esterase from Clostridium botulinum towards increased activity on polyesters

Abstract: Variants of the zinc-binding domain of Clostridium botulinum EstA (Cbotu_EstA) release more building blocks (Ta and BTa) from the aromatic/aliphatic copolyester poly(butylene adipate-co-terephthalate) (PBAT).

Cited by 16 publications

References 54 publications

Towards Sustainable High‐Performance Thermoplastics: Synthesis, Characterization, and Enzymatic Hydrolysis of Bisguaiacol‐Based Polyesters

Towards Sustainable High‐Performance Thermoplastics: Synthesis, Characterization, and Enzymatic Hydrolysis of Bisguaiacol‐Based Polyesters

Synergistic effect of mutagenesis and truncation to improve a polyesterase from Clostridium botulinum for polyester hydrolysis

Insights into polyester plastic biodegradation by carboxyl ester hydrolases

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