Molecular Characterization of a Novel Family VIII Esterase from &lt;i&gt;Burkholderia multivorans&lt;/i&gt; UWC10

Rashamuse, Konanani; Burton, Stephanie G.; Stafford, William; Cowan, Don A.

doi:10.1159/000103610

Cited by 13 publications

(7 citation statements)

References 59 publications

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“…In native PAGE, the molecular mass of active PsEstA was found to be substantially greater than 200 kDa, implying that PsEstA is in an oligomeric conformation. A similar behavior indicating an oligomeric state of PsEstA was also observed in gel filtration chromatography ( Figure 4E), which has been reported for other family VIII esterases [38][39][40]. The mass spectrometric analysis showed a major peak (m/z) at 47.7 kDa with all additional amino acids including the His-tag ( Figure 4F).…”

Section: Characterization Of Psestasupporting

confidence: 84%

Molecular Characterization of a Novel Family VIII Esterase with β-Lactamase Activity (PsEstA) from Paenibacillus sp.

et al. 2019

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Molecular information about family VIII esterases, which have similarities with class C β-lactamases and penicillin-binding proteins, remains largely unknown. In this study, a novel family VIII esterase with β-lactamase activity (PsEstA) from Paenibacillus sp. was characterized using several biochemical and biophysical methods. PsEstA was effective on a broad range of substrates including tertiary butyl acetate, glyceryl tributyrate, glucose pentaacetate, olive oil, and p-nitrophenyl esters. Additionally, PsEstA hydrolyzed nitrocefin, cefotaxime, and 7-aminocephalosporanic acid. Interestingly, two forms of immobilized PsEstA (CLEAs-PsEstA and mCLEAs-PsEstA) showed high recycling property and enhanced stability, but hybrid nanoflowers (hNFs) of PsEstA require improvement. This study provides a molecular understanding of substrate specificities, catalytic regulation, and immobilization of PsEstA, which can be efficiently used in biotechnological applications.

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Section: Characterization Of Psestasupporting

confidence: 84%

Molecular Characterization of a Novel Family VIII Esterase with β-Lactamase Activity (PsEstA) from Paenibacillus sp.

et al. 2019

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“…The resultant gene library was screened for esterase activity on LB agar plates containing 100 μg ml −1 of ampicillin, 1% (v/v) tributyrin and 0·5% (w/v) gum arabic. Recombinant esterase positive clones were identified by a zone of clearance around the colony margins (Rashamuse et al. 2007a).…”

Section: Methodsmentioning

confidence: 99%

Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library

Rashamuse¹,

Ronneburg²,

Hennessy³

et al. 2009

Journal of Applied Microbiology

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Aims: The aim of this study was to demonstrate the application of environmental sample pre‐enrichment to access novel carboxylesterases from environmental genomes, along with subsequent heterologous expression and characterization of the discovered enzyme(s). Methods and Results: A positive recombinant clone (UVCL29), conferring an esterase phenotype was identified from a shotgun gene library. The complete sequence of the 3·0 kb DNA insert from the pUVCL29 recombinant plasmid was obtained using primer‐walking strategies. Nucleotide sequence analysis revealed a complete 945 bp open reading frame (ORF1). Translational analysis of the ORF1 showed a protein of 314 amino acids (named EstAM) with a predicted molecular weight of 34 kDa. EstAM’s primary structure showed a classical (–G–D–S–A–G–) motif, corresponding with the generally conserved (G–x–S–x–G) esterase signature motif. Identity searches indicated that EstAM has high sequence similarity with esterases from family IV. EstAM was successfully expressed in Escherichia coli in a biologically active form. Partial purification was achieved using a one‐step Pro‐PurTM IMAC column. Biochemical characterization revealed that EstAM has a temperature optimum of 40°C. Conclusion: Based on its substrate profile, EstAM was classified as a carboxylesterase because of its preference for short p‐nitrophenyl ester substrates. Significance and Impact of the Study: This study is a demonstration of the successful application of environmental sample pre‐enrichment technology in accessing novel esterases from a mining environment.

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“…For routine enzyme assay, esterase activity was determined as essentially described by [17]. Protein concentrations were determined by the method of [18], using bovine serum albumin (BSA, Sigma-Aldrich) as a standard.…”

Section: E Protein and Enzyme Activity Assaysmentioning

confidence: 99%

Molecular Cloning, Over-expression, Kinetic and Structural Properties of Purified Recombinant Family VII Carboxyl esterases

2019

SETWM-19, ACBES-19, EEHSS-19 Nov. 18-19, 2019 Johannesburg (South Africa)

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Carboxyl esterases (EC 3.1.1.1) are α/β hydrolases which act on acylglycerols to liberate fatty acids and glycerol. They are amongst the most sort-after biocatalysts due to the potential for application in various industries i.e. food, dairy, pharmaceutical, detergents, textile, biodiesel, and cosmetic industries, a factor that drives the continued identification and kinetic characterization for these enzymes. The carboxyl esterase (CEST) genes from Geobacillus kaustophilus (Est179), B. licheniformis (Est96) and B. pumilus (Est95) were expressed in Escherichia coli. For the purified CESTs, temperature optima assays showed that Est95 and 96 were optimally active at 45 o C while the optimum activity for Est179 was recorded between 45 o C and 55 o C. Furthermore, Est95 and 96 retained more than 95% of activity at temperature ranges between 25-40 ° C while, Est179 retained about 80-100% activity between 25-60 o C. The CESTS were also subjected to substrate specificity assays which revealed that the enzymes showed high affinity for p-nitrophenyl butyrate (ρNP-C4) as demonstrated by the low K m obtained in comparison to other test substrates. However, the ratio for the specificity constants (k cat /K M) revealed ρNP-C3 (propionate) was a preferred substrate for Est95, while ρNP-C2 (acetate) and ρNP-C4 were preferred substrates for Est96 and Est179, respectively. Circular Dichroism (CD) spectroscopy-assisted secondary structure predictions recorded spectra that were consistent with a α-helical content for all three proteins. Intrinsic fluorescence spectroscopy-based comparative analysis of protein tertiary structure revealed a difference in the conformation for the 3 proteins, a possible explanation for the differences in the kinetic properties. Therefore, this study constitutes a report on three family VII CESTs that are optimally active at near-neutral to alkaline pH (6.5-9), mesotable to moderately thermostable and with a preference for short-chain acyl esters. The outcomes of this study forms the basis for future experiments aimed at evaluating CESTs properties for potential application in specific industries.

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Molecular Characterization of a Novel Family VIII Esterase from <i>Burkholderia multivorans</i> UWC10

Cited by 13 publications

References 59 publications

Molecular Characterization of a Novel Family VIII Esterase with β-Lactamase Activity (PsEstA) from Paenibacillus sp.

Molecular Characterization of a Novel Family VIII Esterase with β-Lactamase Activity (PsEstA) from Paenibacillus sp.

Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library

Molecular Cloning, Over-expression, Kinetic and Structural Properties of Purified Recombinant Family VII Carboxyl esterases

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