ORCID Profile
0000-0002-3390-9638
Current Organisations
Universität Leipzig Institut für Biologie
,
istituto di scienze e tecnologie chimiche-CNR
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Publications
Novel Transaminase and Laccase from Streptomyces spp. Using Combined Identification Approaches
Publisher: MDPI AG
Date: 29-07-2021
Abstract: Three Streptomyces sp. strains with a multitude of target enzymatic activities confirmed by functional screening, namely BV129, BV286 and BV333, were subjected to genome sequencing aiming at the annotation of genes of interest, in-depth bioinformatics characterization and functional expression of the biocatalysts. A whole-genome shotgun sequencing followed by de novo genome assembly and annotation was performed revealing genomes of 6.4, 9.4 and 7.3 Mbp, respectively. Functional annotation of the proteins of interest resulted in between 2047 and 2763 putative targets. Among the various enzymatic activities that the three Streptomyces strains demonstrated to produce by functional screening, we focused our attention on transaminases (TAs) and laccases due to their high biocatalytic potential. Bioinformatics search allowed the identification of a putative TA from Streptomyces sp. BV333 as a potentially novel broad substrate scope TA and a putative laccase from Streptomyces sp. BV286 as potentially novel blue multicopper oxidase. The two sequences were cloned and overexpressed in Escherichia coli and the two novel enzymes, transaminase Sbv333-TA and laccase Sbv286-LAC, were characterized. Interestingly, both enzymes resulted to be exceptionally thermostable, Sbv333-TA showing a melting temperature (TM = 85 °C) only slightly lower compared to the TM of the most thermostable transaminases described to date (87–88 °C) and Sbv286-LAC being even thermoactivated at temperature °C. Moreover, Sbv333-TA showed a broad substrate scope and remarkably demonstrated to be active in the transamination of β-ketoesters, which are rarely accepted by currently known TAs. On the other hand, Sbv286-LAC showed an improved activity in the presence of the cosolvent acetonitrile. Overall, it was shown that a combination of approaches from standard microbiological and biochemical screens to genome sequencing and analysis is required to afford novel and functional biocatalysts.
Unraveling Energy and Dynamics Determinants to Interpret Protein Functional Plasticity: The Limonene-1,2-epoxide-hydrolase Case Study
Publisher: American Chemical Society (ACS)
Date: 29-03-2017
Abstract: The balance between structural stability and functional plasticity in proteins that share common three-dimensional folds is the key factor that drives protein evolvability. The ability to distinguish the parts of homologous proteins that underlie common structural organization patterns from the parts acting as regulatory modules that can sustain modifications in response to evolutionary pressure may provide fundamental insights for understanding sequence-structure-dynamics relationships. In applicative terms, this would help develop rational protein design methods. Herein, we apply recently developed computational methods, validated by experimental tests, to address these questions in a set of homologous enzymes representative of the limonene-1,2-epoxide-hydrolase family (LEH) characterized by different stabilities, namely Rhodococcus erythropolis LEH (Re-LEH), Tomks-LEH, CH55-LEH, and the two thermostable Re-LEH variants Re-LEH-F1b and Re-LEH-P. Our results show that these enzymes, despite significant sequence variations, exploit a few highly conserved stabilization determinants to guarantee structural stability linked to biological functionality. Multiple sequence analysis shows that these key elements are also shared by a larger set of LEHs structural homologues, despite very low sequence identity and functional ersity. In this framework, stabilizing elements that we hypothesize to have an accessory role are characterized by a lower degree of sequence identity and higher mutability. We suggest that our approach can be successfully used to pinpoint the distinctive energy fingerprint of a class of proteins as well as to locate those modulators whose modification could be exploited to tune protein stability and dynamic properties.
Characterization of Four Bifunctional Plant IAM/PAM-Amidohydrolases Capable of Contributing to Auxin Biosynthesis
Publisher: MDPI AG
Date: 07-08-2014
Related Organisations
Universidad Politécnica De Madrid - Campus De Montegancedo
Location: Spain
Universidad Politécnica De Madrid Escuela Técnica Superior De Ingenieros Industriales
Location: Spain
Istituto Di Chimica Del Riconoscimento Molecolare Consiglio Nazionale Delle Ricerche
Location: Italy
Related Funding Activities
No related grants have been discovered for Erica Elisa Ferrandi.