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This study is based upon a work funded by European Union Projects (grant numbers NMP4-SL-2009-229255-3D-Nanobiodevice, FP7-KBBE-2010-4-26537-Peroxicats and COST Action CM0701) and a Spanish National Project (Evofacel, BIO2010-19697) and by the Austrian Science Fund (FWF, P25148-B20). We thank Prof. S. Shleev from Malmo University (Sweden) for carrying out the measurements of the laccase activity in human plasma and blood. D. M. M. is grateful to the CSIC for a JAE fellowship.

Analysis of institutional authors

Mate, Diana M.Author

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Article

Functional expression of a blood tolerant laccase in Pichia pastoris

Publicated to:BMC BIOTECHNOLOGY. 13 (38): 38-12 - 2013-04-30 13(38), DOI: 10.1186/1472-6750-13-38

Authors: Mate, Diana M.; Gonzalez-Perez, David; Kittl, Roman; Ludwig, Roland; Alcalde, Miguel;

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Abstract

Background: Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory. Results: A blood-active laccase (ChU-B mutant) fused to the native/evolved a-factor prepro-leader was cloned under the control of two different promoters (P-AOX1 and P-GAP) and expressed in Pichia pastoris. The most active construct, which contained the P-AOX1 and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the a-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers. Conclusions: The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications.

Keywords

Blood toleranceBlueDirected evolutionFunctional expressionFungal laccasesGeneHalide inhibitionHigh-redox potential laccaseHydroxyl inhibitionMolecular-cloningPichia pastorisRecombinant proteinRedox potential laccaseSaccharomyces cerevisiaeSaccharomyces-cerevisiaeTrametes-trogiiYeast

Quality index

Bibliometric impact. Analysis of the contribution and dissemination channel

The work has been published in the journal BMC BIOTECHNOLOGY due to its progression and the good impact it has achieved in recent years, according to the agency Scopus (SJR), it has become a reference in its field. In the year of publication of the work, 2013, it was in position , thus managing to position itself as a Q1 (Primer Cuartil), in the category Biotechnology.

From a relative perspective, and based on the normalized impact indicator calculated from World Citations provided by WoS (ESI, Clarivate), it yields a value for the citation normalization relative to the expected citation rate of: 1. This indicates that, compared to works in the same discipline and in the same year of publication, it ranks as a work cited above average. (source consulted: ESI Nov 14, 2024)

This information is reinforced by other indicators of the same type, which, although dynamic over time and dependent on the set of average global citations at the time of their calculation, consistently position the work at some point among the top 50% most cited in its field:

  • Weighted Average of Normalized Impact by the Scopus agency: 1.18 (source consulted: FECYT Feb 2024)
  • Field Citation Ratio (FCR) from Dimensions: 2.33 (source consulted: Dimensions Jun 2025)

Specifically, and according to different indexing agencies, this work has accumulated citations as of 2025-06-07, the following number of citations:

  • WoS: 27
  • Scopus: 36
  • Europe PMC: 17
  • OpenCitations: 32

Impact and social visibility

From the perspective of influence or social adoption, and based on metrics associated with mentions and interactions provided by agencies specializing in calculating the so-called "Alternative or Social Metrics," we can highlight as of 2025-06-07:

  • The use, from an academic perspective evidenced by the Altmetric agency indicator referring to aggregations made by the personal bibliographic manager Mendeley, gives us a total of: 96.
  • The use of this contribution in bookmarks, code forks, additions to favorite lists for recurrent reading, as well as general views, indicates that someone is using the publication as a basis for their current work. This may be a notable indicator of future more formal and academic citations. This claim is supported by the result of the "Capture" indicator, which yields a total of: 96 (PlumX).

With a more dissemination-oriented intent and targeting more general audiences, we can observe other more global scores such as:

  • The Total Score from Altmetric: 3.

It is essential to present evidence supporting full alignment with institutional principles and guidelines on Open Science and the Conservation and Dissemination of Intellectual Heritage. A clear example of this is:

  • The work has been submitted to a journal whose editorial policy allows open Open Access publication.

Leadership analysis of institutional authors

This work has been carried out with international collaboration, specifically with researchers from: Austria.

There is a significant leadership presence as some of the institution’s authors appear as the first or last signer, detailed as follows: First Author (MATE MATE, DIANA) .