Gene_locus Report for: clobh-A51055

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.

The carboxylesterase from Clostridium botulinum (Cbotu_EstA) has been shown to hydrolyze the surface of the polyester poly.butylene adipate-co-terephthalate) (PBAT) releasing the monomeric building blocks. Cbotu_EstA contains a zinc ion, tetrahedrally coordinated by two histidine and two aspartic acid residues, which is buried inside an extra domain typical for members of the I. 5 lipase family. To elucidate the role of this extra domain with regard to polyester hydrolysis, variants of the zinc-binding domain were constructed and expressed in E. coli BL21-Gold.DE3). These enzyme variants were characterized with respect to their specific activity, kinetic parameters and thermostability on soluble substrates as well as on PBAT. All the variants exhibited a similar affinity towards the small substrate para-nitrophenyl butyrate (pNPB), with KM values between 0.4 and 1.2 mM, while the catalytic efficiency decreased approximately 1000-fold for the zinc-binding variants and 5-fold for the zinc cavity variants. Moreover, all four variants of the zinccoordination site (D130L, H150F, H156F, and D302L) showed a loss of thermostability. However, H156F and D302L revealed a drastic loss of thermostability compared to D130L and H150F. Nevertheless, compared to Cbotu_EstA, variants carrying substitutions of amino acids in the zinc-binding domain were able to release up to 10 times more soluble products from the polymeric substrate PBAT. The thermostability at 50 degrees C was increased in the case of F154Y and W274H, carrying more hydrophilic residues. These data clearly demonstrate the importance of different regions of the zinc-binding domain for the hydrolysis of polyesters like PBAT.

Clostridium botulinum is a heterogeneous Gram-positive species that comprises four genetically and physiologically distinct groups of bacteria that share the ability to produce botulinum neurotoxin, the most poisonous toxin known to man, and the causative agent of botulism, a severe disease of humans and animals. We report here the complete genome sequence of a representative of Group I (proteolytic) C. botulinum (strain Hall A, ATCC 3502). The genome consists of a chromosome (3,886,916 bp) and a plasmid (16,344 bp), which carry 3650 and 19 predicted genes, respectively. Consistent with the proteolytic phenotype of this strain, the genome harbors a large number of genes encoding secreted proteases and enzymes involved in uptake and metabolism of amino acids. The genome also reveals a hitherto unknown ability of C. botulinum to degrade chitin. There is a significant lack of recently acquired DNA, indicating a stable genomic content, in strong contrast to the fluid genome of Clostridium difficile, which can form longer-term relationships with its host. Overall, the genome indicates that C. botulinum is adapted to a saprophytic lifestyle both in soil and aquatic environments. This pathogen relies on its toxin to rapidly kill a wide range of prey species, and to gain access to nutrient sources, it releases a large number of extracellular enzymes to soften and destroy rotting or decayed tissues.

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.

The carboxylesterase from Clostridium botulinum (Cbotu_EstA) has been shown to hydrolyze the surface of the polyester poly.butylene adipate-co-terephthalate) (PBAT) releasing the monomeric building blocks. Cbotu_EstA contains a zinc ion, tetrahedrally coordinated by two histidine and two aspartic acid residues, which is buried inside an extra domain typical for members of the I. 5 lipase family. To elucidate the role of this extra domain with regard to polyester hydrolysis, variants of the zinc-binding domain were constructed and expressed in E. coli BL21-Gold.DE3). These enzyme variants were characterized with respect to their specific activity, kinetic parameters and thermostability on soluble substrates as well as on PBAT. All the variants exhibited a similar affinity towards the small substrate para-nitrophenyl butyrate (pNPB), with KM values between 0.4 and 1.2 mM, while the catalytic efficiency decreased approximately 1000-fold for the zinc-binding variants and 5-fold for the zinc cavity variants. Moreover, all four variants of the zinccoordination site (D130L, H150F, H156F, and D302L) showed a loss of thermostability. However, H156F and D302L revealed a drastic loss of thermostability compared to D130L and H150F. Nevertheless, compared to Cbotu_EstA, variants carrying substitutions of amino acids in the zinc-binding domain were able to release up to 10 times more soluble products from the polymeric substrate PBAT. The thermostability at 50 degrees C was increased in the case of F154Y and W274H, carrying more hydrophilic residues. These data clearly demonstrate the importance of different regions of the zinc-binding domain for the hydrolysis of polyesters like PBAT.

Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site. Biotechnol. Bioeng. 2016;113: 1024-1034. (c) 2015 Wiley Periodicals, Inc.

Clostridium botulinum is a heterogeneous Gram-positive species that comprises four genetically and physiologically distinct groups of bacteria that share the ability to produce botulinum neurotoxin, the most poisonous toxin known to man, and the causative agent of botulism, a severe disease of humans and animals. We report here the complete genome sequence of a representative of Group I (proteolytic) C. botulinum (strain Hall A, ATCC 3502). The genome consists of a chromosome (3,886,916 bp) and a plasmid (16,344 bp), which carry 3650 and 19 predicted genes, respectively. Consistent with the proteolytic phenotype of this strain, the genome harbors a large number of genes encoding secreted proteases and enzymes involved in uptake and metabolism of amino acids. The genome also reveals a hitherto unknown ability of C. botulinum to degrade chitin. There is a significant lack of recently acquired DNA, indicating a stable genomic content, in strong contrast to the fluid genome of Clostridium difficile, which can form longer-term relationships with its host. Overall, the genome indicates that C. botulinum is adapted to a saprophytic lifestyle both in soil and aquatic environments. This pathogen relies on its toxin to rapidly kill a wide range of prey species, and to gain access to nutrient sources, it releases a large number of extracellular enzymes to soften and destroy rotting or decayed tissues.