793cc803f2451101115ca4f0142edb82 Salarich_Soldevila_Adria_Poster.pdf 372825234dd9e15805e79a8e26c18a0f9170eaee Salarich_Soldevila_Adria_Poster.pdf 948cbc809d2cb5b3360ee13368f865663466df10c88dfae3454df152f549ef83 Salarich_Soldevila_Adria_Poster.pdf Title: An in silico analysis of laccase thermostability determinants for the improvement of rational enzyme engineering - Keywords: DAHKyqty0A4,BACqCmSMERY,0 Author: Adrià Salarich Creator: Canva Producer: Canva CreationDate: Thu Jun 4 18:07:16 2026 CEST ModDate: Thu Jun 4 18:07:16 2026 CEST Custom Metadata: no Metadata Stream: yes Tagged: yes UserProperties: no Suspects: no Form: none JavaScript: no Pages: 1 Encrypted: no Page size: 2384.25 x 3370.5 pts (A0) Page rot: 0 File size: 4958601 bytes Optimized: no PDF version: 1.4 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AAAAAA+Calibri CID TrueType Identity-H yes yes yes 595 0 BAAAAA+Calibri-Bold CID TrueType Identity-H yes yes yes 596 0 CAAAAA+Calibri-Italic CID TrueType Identity-H yes yes yes 597 0 DAAAAA+Merriweather-Bold CID TrueType Identity-H yes yes yes 598 0 EAAAAA+JetBrainsMono-Regular CID TrueType Identity-H yes yes yes 599 0 FAAAAA+LiberationSans CID TrueType Identity-H yes yes yes 600 0 GAAAAA+LiberationSans-Bold CID TrueType Identity-H yes yes yes 601 0 HAAAAA+JetBrainsMono-Bold CID TrueType Identity-H yes yes yes 602 0 IAAAAA+Merriweather-BoldItalic CID TrueType Identity-H yes yes yes 603 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-07-10 04:03:51 CEST RepresentationInformation: Salarich_Soldevila_Adria_Poster.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-07-09 10:35:41 CEST Size: 4958601 Format: PDF Version: 1.4 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf Profile: Tagged PDF PDFMetadata: Objects: 829 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: false CenterWindow: false DisplayDocTitle: true NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Language: en-GB Outlines: Item: Title: An in silico analysis of laccase thermostability determinants for the improvement of rational enzyme engineering Destination: 1 Children: Item: Title: Adrià Salarich Soldevila - Degree in Microbiology Data Analysis Destination: 1 Item: Title: Introduction Destination: 1 Children: Item: Title: Laccases (EC 1.10.3.2) are enzymes capable of degrading a wide range of contaminants using oxygen, making them attractive tools in bioremediation. However, their low thermostability limits their large-scale application, and the molecular determinants of this property are not fully understood, hindering the rational design of more stable variants. This project compared thermostable and non-thermostable laccases through an MSA-based approach using statistical tests, Kullback-Leibler (KL) divergence and Shannon entropy (SE) to elucidate thermostability determinants that could guide rational enzyme engineering. The spore-coating laccase of Bacillus subtilis, CotA (PDB: 1GSK), served as the structural reference throughout. Destination: 1 Item: Title: Methodology Destination: 1 Children: Item: Title: Sequence collection: UniProt, NCBI, BRENDA. Classification in thermostable (stable at >50ºC) and non-thermostable (generic) Destination: 1 Item: Title: Clustering: CD-HIT (generic 90%, thermostable 95%) Destination: 1 Item: Title: MSA construction and trimming: MAFFT + trimAl Destination: 1 Item: Title: Relative solvent accessibility (RSA) classification: FreeSASA + ChimeraX, 20% RSA threshold → buried < 20% RSA | SA > 20% RSA Destination: 1 Item: Title: PSSM construction and extraction of amino acid (AA) frequencies in buried and solvent-accessible positions: Goalign, using PDB numbering as reference Destination: 1 Item: Title: Analyses: AA group enrichment (χ²), physicochemical properties (T-test + Mann-Whitney U), KL divergence and SE + structural interpretation Destination: 1 Item: Title: Collection Destination: 1 Item: Title: CD-Hit Destination: 1 Item: Title: MAFFT + trimAl Destination: 1 Item: Title: FreeSASA + Goalign Destination: 1 Item: Title: Results Destination: 1 Children: Item: Title: Amino acid group frequencies Destination: 1 Children: Item: Title: Amino acids were divided into functional categories Destination: 1 Children: Item: Title: AA Destination: 1 Item: Title: Comparison through chi squared (χ²) test Destination: 1 Item: Title: No significant results Destination: 1 Item: Title: Physicochemical properties Destination: 1 Children: Item: Title: Calculation of physicochemical index value at every position in the alignment Destination: 1 Item: Title: In thermostable: ↑ core hydrophobicity and ↓ surface hydrophobicity, but effects are small Destination: 1 Item: Title: Shannon entropy Destination: 1 Children: Item: Title: Shannon entropy was calculated at each common position Destination: 1 Item: Title: Mean SE was then compared between groups Destination: 1 Item: Title: Thermostable laccases consistently showed lower SE Destination: 1 Item: Title: Kullback-Leibler divergence Destination: 1 Children: Item: Title: KL divergence was calculated at each common position in the alignments to compare the amino acid frequency distributions between thermostable and generic laccases, separately for buried and solvent-accessible positions. Destination: 1 Item: Title: The positions compared were then ranked from highest to lowest KL divergence for downstream analysis of the most discriminating positions. Destination: 1 Item: Title: General patterns Destination: 1 Children: Item: Title: Most discriminating positions fall on loops and secondary structure termini Destination: 1 Item: Title: 6 proline residues among most discriminating positions Destination: 1 Item: Title: Overall rigidification strategy Destination: 1 Item: Title: Notable positions Destination: 1 Children: Item: Title: P44 - exposed loop (see Figure 1), strongest signal Destination: 1 Item: Title: P384 and Q378 - highly conserved β-hairpin in thermostable laccases (see Figure 1) Destination: 1 Item: Title: F123 , F127, V100 and K101 - potential stabilising hydrophobic cluster (see Figure 2) Destination: 1 Item: Title: Conclusions Destination: 1 Item: Title: Bibliography & Resources Destination: 1 Children: Item: Title: Expected global compositional differences are minor; thermostability is likely driven by local factors Destination: 1 Item: Title: Thermostable laccases show stronger positional conservation overall (lower mean SE) Destination: 1 Item: Title: Strong positional proline conservation at flexible positions in thermostable laccases Destination: 1 Item: Title: Identification of a hydrophobic cluster and a β-hairpin as potential targets for rational design Destination: 1 Item: Title: Overall, the findings suggest a stabilisation strategy based on the rigidification of flexible zones Destination: 1 Item: Title: In vitro experiments remain necessary to validate the findings Destination: 1 Item: Title: Find the full bibliography and project manuscript Destination: 1 Info: Title: An in silico analysis of laccase thermostability determinants for the improvement of rational enzyme engineering - Author: Adrià Salarich Keywords: DAHKyqty0A4,BACqCmSMERY,0 Creator: Canva Producer: Canva CreationDate: Thu Jun 04 18:07:16 CEST 2026 ModDate: Thu Jun 04 18:07:16 CEST 2026 ID: 0x87b734f953a1c2a4d61c64eb21436a8f4d37a1e68b15a85247a185d84a2029cd, 0x87b734f953a1c2a4d61c64eb21436a8f4d37a1e68b15a85247a185d84a2029cd Filters: FilterPipeline: FlateDecode XMP: An in silico analysis of laccase thermostability determinants for the improvement of rational enzyme engineering - An in silico analysis of laccase thermostability determinants for the improvement of rational enzyme engineering - Adrià Salarich Pages: Page: Sequence: 1 Checksum: 2eaa12a4 Type: CRC32 Checksum: 793cc803f2451101115ca4f0142edb82 Type: MD5 Checksum: 372825234dd9e15805e79a8e26c18a0f9170eaee Type: SHA-1 Checksum: 948cbc809d2cb5b3360ee13368f865663466df10c88dfae3454df152f549ef83 Type: SHA-256