BioCatalyst Hub

Coordinators:

Prof. Marco Moracci (marco.moracci@unina.it)

The BioCatalyst Hub of the Department of Biology of the University of Naples Federico II is a technological platform designed to support the development, production, purification, and characterization of thermophilic and thermostable enzymes for industrial, agro-food, environmental, and pharmaceutical applications. The infrastructure provides a fully integrated workflow, from microbial cultivation to the validation of biocatalysts and the analysis of reaction products.

Core Activities

Expression and Protein Purification

The platform offers optimized strategies for the efficient expression of recombinant proteins, ensuring high yields and reproducibility. Proteins of interest can be purified with high resolution using the FPLC system, which supports affinity, ion exchange, and gel filtration chromatography. Downstream analysis includes gel imaging to assess protein purity and confirm successful expression and purification.

Structure–Function Studies

The core enables in-depth analysis of enzyme stability, conformational properties, and interactions with ligands. Differential scanning fluorimetry (DSF) and spectrofluorimetric approaches are employed to evaluate protein stability and ligand binding at nanomolar sensitivity. Biochemical characterization includes assessment of pH and temperature dependence as well as determination of kinetic parameters, providing a comprehensive understanding of protein functionality.

Reaction Product Characterization

The platform is equipped for advanced analysis of enzymatic reaction products. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) allows for the direct, high-sensitivity detection of mono- and oligosaccharides without the need for derivatization, reaching sensitivity in the nanomolar range. Complementary analyses are performed with HPLC systems, which enable broad-spectrum detection, quantification, and preparative separation of a wide variety of compounds using UV, fluorescence, and IR detectors.

Equipments

E. coli strains: Engineered strains for recombinant protein production: BL21(DE3), BL21(DE3) pG-KJE8, BL21(DE3) pGro7, BL21(DE3) pKJE7, BL21(DE3) pTf16, BL21(DE3) RIL, C43 (DE3), Origami (DE3), Lemo21 (DE3), RB791, ArctictExpress (DE3).

Incubators: Large-capacity incubators with precise temperature control (Temperature range 4–80 °C) and agitation for microbial growth.

FPLC Chromatographic Systems: High-resolution purification with affinity, ion exchange, and size-exclusion chromatography.

Gel Imager: Detection and analysis of proteins from SDS-PAGE and Western blot.

Plate Reader: Temperature-controlled system (Temperature range 4–70 °C) for enzymatic activity assays.

UV-Vis Spectrophotometer: Temperature-controlled measurements (Temperature range 4-90 °C) for cell growth monitoring, protein quantification, enzymatic activity assays.

Spectrofluorimeter: Analysis of protein stability, ligand binding and enzymatic activity with nanomolar sensitivity.

Real-Time PCR: Instrument used for Differential Scanning Fluorimetry to assess conformational stability.

HPAEC-PAD system: high sensitive detection of monosaccharides and oligosaccharides in the nanomolar range, without the need for derivatization, allowing detailed carbohydrate profiling and accurate quantification.

HPLC system: UV, fluorescence, and IR detection capabilities, it enables the identification, quantification, and preparative separation of a wide range of products

ADDITIONAL SERVICES

Cloning of the desired genes from (meta)genome into the appropriate vector.

Preparation and optimization of enzymatic cocktails for the degradation of desired complex substrates.

ACKNOWLEDGEMENTS

All the users and collaborators of the BioCatalyst Hub Core are obligated to acknowledge the core in publications: “The authors acknowledge the Enzymology for Discovery & Innovation Core at Department of Biology of University of Naples Federico II.

BioCat Mining

Coordinators:

Prof. Andrea Strazzulli (andrea.strazzulli@unina.it)

BioCat Mining provides high-performance computational resources for the in silico discovery of enzymes from (meta)genomic and (meta)transcriptomic datasets. It enables functional and taxonomic annotations as well as structural modeling to study folding and biomolecular interactions. Applications range from green chemistry to agro-industrial valorization and bioremediation, supporting innovative and sustainable solutions.

Core Activities

Functional and Taxonomic Annotation

The platform enables large-scale annotation of (meta)genomic and (meta)trascriptomic datasets, providing accurate classification and identification of putative enzymes of industrial interest.

Data Mining for Enzyme Discovery

Advanced computational pipelines support the identification of novel enzyme families and catalytic activities, accelerating the discovery of innovative biocatalysts for sustainable applications.

Structural Modeling and Folding Analysis

The infrastructure offers structural modeling of proteins to study folding, stability, and biomolecular interactions.

Equipments

Twin Professional Workstations optimized for advanced bioinformatics analysis.

Processing Power: Dual-processor architecture, 48 physical cores / 96 logical threads per unit.

Memory: 128 GB high-speed ECC RAM per workstation, for handling large biological datasets and molecular models.

Storage: ~15 TB per workstation for reference databases, raw sequences, and analysis outputs.

Applications: large-scale sequence alignment, functional/taxonomic annotation, machine learning–based data mining, protein structural modeling.

ACKNOWLEDGEMENTS

All the users and collaborators of the BioCat MiningCore are obligated to acknowledge the core in publications: “The authors acknowledge the Enzymology for Discovery & Innovation Core at Department of Biology of University of Naples Federico II.

GlycoEngineering Platform

Coordinators:

Prof. Marco Moracci (marco.moracci@unina.it)

Prof.  Andrea Strazzulli (andrea.strazzulli@unina.it)

GlycoEngineering is a technological platform dedicated to the engineering of biocatalysts for the production of high-value oligosaccharides. By integrating synthetic biology and enzyme engineering approaches, the infrastructure develops solutions that enable controlled, selective, and sustainable synthesis. with applications in pharmaceutical and nutraceutical sectors

Core Activities

Design and Engineering of Glycosidases

The platform supports the rational engineering of glycosidases into putative glycosynthases/thioglycoligases through site-directed mutagenesis, guided by structural modelling using high-performance workstations.

Expression and Protein Purification

Microbial expression systems allow the production at scales up to 10 L. Enzymes of interest are purified with high resolution using the ÄKTA Pure FPLC system, which enables affinity, ion exchange, and gel filtration chromatography.

Biochemical and Structural Analysis

Spectrophotometric and fluorimetric assays allow determination of enzymatic activity, catalytic efficiency, conformational stability, and enzyme–ligand interactions.

Reaction Product Characterization

The platform provides advanced tools for carbohydrate analysis. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) allows direct and quantitative detection of mono- and oligosaccharides at nanomolar concentrations. Complementary analyses with HPLC systems, equipped with UV, fluorescence, and IR detectors, enable quantitative and preparative separations of synthetic products.

Equipments

1. coli strains: Engineered strains for recombinant protein production: BL21(DE3), BL21(DE3) pG-KJE8, BL21(DE3) pGro7, BL21(DE3) pKJE7, BL21(DE3) pTf16, BL21(DE3) RIL, C43 (DE3), Origami (DE3), Lemo21 (DE3), RB791, ArctictExpress (DE3).

Incubators: Large-capacity incubators with precise temperature control (Temperature range 4–80 °C) and agitation for microbial growth.

FPLC Chromatographic Systems: High-resolution purification with affinity, ion exchange, and size-exclusion chromatography.

Gel Imager: Detection and analysis of proteins from SDS-PAGE and Western blot.

Plate Reader: Temperature-controlled system (Temperature range 4–70 °C) for enzymatic activity assays.

UV-Vis Spectrophotometer: Temperature-controlled measurements (Temperature range 4-90 °C) for cell growth monitoring, protein quantification, enzymatic activity assays.

Spectrofluorimeter: Analysis of protein stability, ligand binding and enzymatic activity with nanomolar sensitivity.

HPAEC-PAD system: high sensitive detection of monosaccharides and oligosaccharides in the nanomolar range, without the need for derivatization, allowing detailed carbohydrate profiling and accurate quantification.

HPLC system: UV, fluorescence, and IR detection capabilities, it enables the identification, quantification, and preparative separation of a wide range of products

ACKNOWLEDGEMENTS

All the users and collaborators of the GlycoEngineering Core are obligated to acknowledge the core in publications: “The authors acknowledge the Enzymology for Discovery & Innovation Core at Department of Biology of University of Naples Federico II.

Chaperone Screening Platform

Coordinators:

Prof. Andrea Strazzulli (andrea.strazzulli@unina.it)

Prof.  Roberta Iacono (roberta.iacono@unina.it)

Prof. Donato Giovannelli (donato.govannelli@unina.it)

The Chaperone Screening Platform (CSP) is a technological infrastructure dedicated to the engineering of biocatalysts for the development of robust and versatile enzymatic tools. These models are designed to mimic human pathological mutations responsible for rare genetic diseases, thereby enabling the screening of compounds with potential chaperone activity. The platform supports the identification of novel therapeutic strategies targeting to misfolded protein.

Core Activities

In Silico Design of Enzyme Mutants

High-performance workstations enable structural modelling and rational design of enzyme mutants reproducing human pathological variants.

Expression and Protein Purification

Mutant enzymes are produced through microbial recombinant expression. The purified proteins are obtained using the ÄKTA Pure FPLC system (Cytiva), which ensures high-resolution purification with affinity and ion exchange chromatography.

Biochemical and Structural Characterization

The core provides biochemical studies including evaluation of pH and temperature effects as well as determination of kinetic parameters. Protein stability can be monitored with high sensitivity through differential scanning fluorimetry (DSF) and spectrofluorimetric methods.

Chaperone Screening

The platform enables the systematic testing of small molecules, natural compounds, and chemical libraries to identify compounds with chaperone-like activity, measures temperature and pH stability and dissociation constant of the ligands.

E. coli strains: Engineered strains for recombinant protein production: BL21(DE3), BL21(DE3) pG-KJE8, BL21(DE3) pGro7, BL21(DE3) pKJE7, BL21(DE3) pTf16, BL21(DE3) RIL, C43 (DE3), Origami (DE3), Lemo21 (DE3), RB791, ArctictExpress (DE3).

Incubators: Large-capacity incubators with precise temperature control (Temperature range 4–80 °C) and agitation for microbial growth.

FPLC Chromatographic Systems: High-resolution purification with affinity, ion exchange, and size-exclusion chromatography.

Gel Imager: Detection and analysis of proteins from SDS-PAGE and Western blot.

Plate Reader: Temperature-controlled system (Temperature range 4–70 °C) for enzymatic activity assays.

UV-Vis Spectrophotometer: Temperature-controlled measurements (Temperature range 4-90 °C) for cell growth monitoring, protein quantification, enzymatic activity assays.

Spectrofluorimeter: Analysis of protein stability, ligand binding and enzymatic activity with nanomolar sensitivity.

Real-Time PCR: Instrument used for Differential Scanning Fluorimetry to assess conformational stability

ACKNOWLEDGEMENTS

All the users and collaborators of the Chaperone Screening Platform Core are obligated to acknowledge the core in publications: “The authors acknowledge the Enzymology for Discovery & Innovation Core at Department of Biology of University of Naples Federico II.