80c8159febbf36ec8f3f0578031d010e pmc_9844320.pdf 0d3960cccf1b0f8241e98a4f7f4837248ecd4694 pmc_9844320.pdf a50dfbdb0dffca41e87e28ba709203733db56cedb749d8ff90de3a9d5548bed8 pmc_9844320.pdf Title: Novel CaLB-like Lipase Found Using ProspectBIO, a Software for Genome-Based Bioprospection Subject: Enzymes have been highly demanded in diverse applications such as in the food, pharmaceutical, and industrial fuel sectors. Thus, in silico bioprospecting emerges as an efficient strategy for discovering new enzyme candidates. A new program called ProspectBIO was developed for this purpose as it can find non-annotated sequences by searching for homologs of a model enzyme directly in genomes. Here we describe the ProspectBIO software methodology and the experimental validation by prospecting for novel lipases by sequence homology to Candida antarctica lipase B (CaLB) and conserved motifs. As expected, we observed that the new bioprospecting software could find more sequences (1672) than a conventional similarity-based search in a protein database (733). Additionally, the absence of patent protection was introduced as a criterion resulting in the final selection of a putative lipase-encoding gene from Ustilago hordei (UhL). Expression of UhL in Pichia pastoris resulted in the production of an enzyme with activity towards a tributyrin substrate. The recombinant enzyme activity levels were 4-fold improved when lowering the temperature and increasing methanol concentrations during the induction phase in shake-flask cultures. Protein sequence alignment and structural modeling showed that the recombinant enzyme has high similarity and capability of adjustment to the structure of CaLB. However, amino acid substitutions identified in the active pocket entrance may be responsible for the differences in the substrate specificities of the two enzymes. Thus, the ProspectBIO software allowed the finding of a new promising lipase for biotechnological application without the need for laborious and expensive conventional bioprospecting experimental steps. Keywords: bioprospection; CaLB; protein; functional characterization Author: Gabriela C. Brêda, Priscila E. Faria, Yuri S. Rodrigues, Priscila B. Pinheiro, Maria Clara R. Nucci, Pau Ferrer, Denise M. G. Freire, Rodrigo V. Almeida and Rafael D. Mesquita Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jan 6 10:53:46 2023 CET ModDate: Fri Jan 6 10:56:32 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1692848 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CDFLWE+VnURWPalladioL Type 1 Custom yes yes no 10 0 VYUJDZ+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 QJSEMF+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 DOQVBU+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 DTGNSD+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 72 0 VIWIOF+CMSY10 Type 1 Builtin yes yes no 79 0 FPSBGA+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 91 0 FPSBGA+PalatinoLinotype,Bold CID TrueType Identity-H yes yes yes 97 0 FPVLLZ+PalatinoLinotype CID TrueType Identity-H yes yes yes 103 0 FPSBGA+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 119 0 FPSBGA+PalatinoLinotype,Bold CID TrueType Identity-H yes yes yes 125 0 FPVLLZ+PalatinoLinotype CID TrueType Identity-H yes yes yes 131 0 FPSBGA+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 149 0 FPSBGA+PalatinoLinotype,Bold CID TrueType Identity-H yes yes yes 155 0 FPVLLZ+PalatinoLinotype CID TrueType Identity-H yes yes yes 161 0 FPSBGA+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 177 0 FPSBGA+PalatinoLinotype,Bold CID TrueType Identity-H yes yes yes 183 0 FPVLLZ+PalatinoLinotype CID TrueType Identity-H yes yes yes 189 0 FPSBGA+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 205 0 FPSBGA+PalatinoLinotype,Bold CID TrueType Identity-H yes yes yes 211 0 FPVLLZ+PalatinoLinotype CID TrueType Identity-H yes yes yes 217 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-20 02:29:33 CEST RepresentationInformation: pmc_9844320.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-01-26 02:12:16 CET Size: 1692848 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 449 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Bioinformatics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Experimental Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Children: Item: Title: Bioprospecting Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: ProspectBIO Software Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Prospecting a Novel CaLB-like Lipase Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: UhL Three-Dimensional Modeling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Experimental Validation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: UhL Expression in Pichia pastoris Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: UhL Characteristics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Substrate Specificity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Patents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Info: Title: Novel CaLB-like Lipase Found Using ProspectBIO, a Software for Genome-Based Bioprospection Author: Gabriela C. Brêda, Priscila E. Faria, Yuri S. Rodrigues, Priscila B. Pinheiro, Maria Clara R. Nucci, Pau Ferrer, Denise M. G. Freire, Rodrigo V. Almeida and Rafael D. Mesquita Subject: Enzymes have been highly demanded in diverse applications such as in the food, pharmaceutical, and industrial fuel sectors. Thus, in silico bioprospecting emerges as an efficient strategy for discovering new enzyme candidates. A new program called ProspectBIO was developed for this purpose as it can find non-annotated sequences by searching for homologs of a model enzyme directly in genomes. Here we describe the ProspectBIO software methodology and the experimental validation by prospecting for novel lipases by sequence homology to Candida antarctica lipase B (CaLB) and conserved motifs. As expected, we observed that the new bioprospecting software could find more sequences (1672) than a conventional similarity-based search in a protein database (733). Additionally, the absence of patent protection was introduced as a criterion resulting in the final selection of a putative lipase-encoding gene from Ustilago hordei (UhL). Expression of UhL in Pichia pastoris resulted in the production of an enzyme with activity towards a tributyrin substrate. The recombinant enzyme activity levels were 4-fold improved when lowering the temperature and increasing methanol concentrations during the induction phase in shake-flask cultures. Protein sequence alignment and structural modeling showed that the recombinant enzyme has high similarity and capability of adjustment to the structure of CaLB. However, amino acid substitutions identified in the active pocket entrance may be responsible for the differences in the substrate specificities of the two enzymes. Thus, the ProspectBIO software allowed the finding of a new promising lipase for biotechnological application without the need for laborious and expensive conventional bioprospecting experimental steps. 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