Dennis J. Díaz‐Rincón

Pontificia Universidad Javeriana

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Alexander Rodríguez‐López

Hospital de Clínicas "José de San Martín"

Carlos Javier Alméciga-Díaz

Pontificia Universidad Javeriana

Raúl A. Poutou‐Piñales

Pontificia Universidad Javeriana

Claudia Cuervo

Pontificia Universidad Javeriana

Edwin D. Morales-Álvarez

University of Caldas

Balkys Quevedo-Hidalgo

Pontificia Universidad Javeriana

Aura M. Pedroza-Rodríguez

Pontificia Universidad Javeriana

ÁJ

Ángela J. Espejo

Pontificia Universidad Javeriana

Claudia M. Rivera-Hoyos

Pontificia Universidad Javeriana

ÁM

Ángela M. Cardozo-Bernal

Pontificia Universidad Javeriana

Cooperative Institutions

Pontificia Universidad Javeriana

University of Quindío

Universidad Nacional de Colombia

Hospital Universitario San Ignacio

Universidad de Antioquia

University of Applied and Environmental Sciences

Instituto de Investigación Biomédica de Málaga

Universidad de Málaga

Universidade de São Paulo

CasaLuker (Colombia)

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Plackett-Burman Design for rGILCC1 Laccase Activity Enhancement in Pichia pastoris: Concentrated Enzyme Kinetic Characterization

Enzyme Research (2017)

Edwin D. Morales-Álvarez Claudia M. Rivera-Hoyos Ángela M. Cardozo-Bernal Raúl A. Poutou‐Piñales Aura M. Pedroza-Rodríguez

Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZαA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 ± 0.39 UL-1 at 168 h of culture in a 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50°C and retained a residual enzymatic activity greater than 70% at 60°C and 50% at 70°C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity greater than 55% was obtained at pH 10.0 ± 0.2. Furthermore, calculated apparent enzyme kinetic parameters were a Vmax of 6.87 × 10-5 mM s-1, with an apparent Km of 5.36 × 10-2 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges.

ABTS

Plackett–Burman design

Enzyme Kinetics

10.1155/2017/5947581

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Citations (9)

Media improvement for 10 L bioreactor production of rPOXA 1B laccase by P. pastoris

3 Biotech (2019)

Leidy D. Ardila-Leal Diego A. Albarracín-Pardo Claudia M. Rivera-Hoyos Edwin D. Morales-Álvarez Raúl A. Poutou‐Piñales

ABTS

Plackett–Burman design

10.1007/s13205-019-1979-y

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Citations (11)

Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

Enzyme Research (2017)

Dennis J. Díaz‐Rincón Ivonne Duque Erika Osorio Alexander Rodríguez‐López Ángela J. Espejo

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus . Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L −1 . The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and V max of 23.1 µ M min −1 ) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.

Enzyme Kinetics

10.1155/2017/6980565

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Citations (8)

Production and characterization of a human lysosomal recombinant iduronate‐2‐sulfatase produced inPichia pastoris

Biotechnology and Applied Biochemistry (2018)

Natalia Pimentel Alexander Rodríguez‐López Sergio Díaz Juan Camilo Losada Dennis J. Díaz‐Rincón

Abstract Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an X‐linked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronate‐2‐sulfatase (IDS). Currently, MPS II patients are mainly treated with enzyme replacement therapy (ERT) using recombinant enzymes produced in mammalian cells. As an alternative, several studies have shown the production of active and therapeutic forms of lysosomal proteins in microorganisms. In this paper, we report the production and characterization of a recombinant IDS produced in the yeast Pichia pastoris (prIDS). We evaluated the effect of culture conditions and gene sequence optimization on prIDS production. The results showed that the highest production of prIDS was obtained at oxygen‐limited conditions using a codon‐optimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mg −1 H −1 and an apparent molecular mass of about 90 kDa. The highest stability was achieved at pH 6.0, and prIDS also showed high stability in human serum. Noteworthy, the enzyme was taken up by culture cells in a dose‐dependent manner through mannose receptors, which allowed the delivery of the enzyme to the lysosome. In summary, these results show the potential of Pichia pastoris as a host to produce an IDS intended for a MPS II ERT.

Pichia

Hunter syndrome

Mucopolysaccharidosis type II

Lysosomal storage disease

10.1002/bab.1660

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Citations (21)