In this work, we statistically improved culture media for rPOXA 1B laccase production, expressed in Pichia pastoris containing pGAPZαA-LaccPost-Stop construct and assayed at 10 L bioreactor production scale (6 L effective work volume). The concentrated enzyme was evaluated for temperature and pH stability and kinetic parameter, characterized by monitoring oxidation of different ABTS [2,] substrate concentrations. Plackett-Burman experimental design (PBED) implementation improved previous work results by 3.05-fold, obtaining a laccase activity of 1373.72 ± 0.37 U L −1 at 168 h of culture in a 500 mL shake flask. In contrast, one factor experimental design (OFED) applied after PBED improved by threefold the previous study, additionally increasing the C/N ratio. Employing OFED media at 10 L bioreactor scale was capable of producing 3159.93 ± 498.90 U L −1 at 192 h, representing a 2.4-fold increase. rPOXA 1B concentrate remained stable between 10 and 50 °C and retained over 70% residual enzymatic activity at 60 °C and 50% at 70 °C. Concerning pH stability, the enzyme was stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity (60%) was obtained at pH 10.0 ± 0.2. Furthermore, the apparent kinetic parameters were V max of 3.163 × 10 −2 mM min −1 and K m of 1.716 mM. Collectively, regarding enzyme stability our data provide possibilities for applications involving a wide range of pH and temperatures. Keywords Plackett-Burman experimental design • One-factor experimental design • Pichia pastoris • Recombinant laccase • Enzyme stability • Enzyme kinetics Abbreviations MCOs Multicopper oxidases ABTS 2, 20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) PBL Pulping black liquor LDPE Low density polyethylene pAOX1 Alcohol oxidase promoter pGAP Glyceraldehyde-3-phosphate dehydrogenase promoter POXA 1B Laccase from P. ostreatus rPOXA 1B Recombinant laccase from P. ostreatus V max Maximum reaction rate K M Michaelis constant YPG Yeast extract, peptone and glucose culture media MCB Master cell bank YPG-Z Yeast extract, peptone, glucose-zeocin culture media EWV Effective work volume PBED Plackett-Burman experimental design OFED One-factor experimental design v.v.m. Air volume per media volume µ x Specific growth rate T d Duplication time r.p.m. Revolutions per minute Y (x/s) Biomass/substrate yield Y (p/s) Enzyme/substrate yield P (x) Biomass productivity P (p) Enzyme productivity SD Standard deviation GFP Green fluorescent protein

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 H 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.

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.

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 Vmax of 23.1 µM 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.