Novel CaLB-like Lipase Found Using ProspectBIO, a Software for Genome-Based Bioprospection
Brêda, Gabriela C. (Universidade Federal do Rio de Janeiro. Departamento de Bioquímica)
Faria, Priscila E. (Universidade Federal do Rio de Janeiro. Departamento de Bioquímica)
Rodrigues, Yuri S. (Universidade Federal do Rio de Janeiro. Departamento de Bioquímica)
Pinheiro, Priscila B. (Universidade Federal do Rio de Janeiro. Departamento de Bioquímica)
Nucci, Maria Clara R. (Universidade Federal do Rio de Janeiro. Departamento de Bioquímica)
Ferrer, Pau (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Freire, Denise M. G. (Universidade Federal do Rio de Janeiro. Departamento de Bioquímica)
Volcan Almeida, Rodrigo (Universidade Federal do Rio de Janeiro. Departamento de Bioquímica)
Mesquita, Rafael D. (Universidade Federal do Rio de Janeiro. Departamento de Bioquímica)

Date:	2023
Abstract:	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.
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Bioprospection ; CaLB ; Protein ; Functional characterization
Published in:	BioTech, Vol. 12, Issue 1 (January 2023) , art. 6, ISSN 2673-6284

DOI: 10.3390/biotech12010006
PMID: 36648832

14 p, 1.6 MB

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