The development of functional enzymes capable of operating effectively in human physiological environments is a cornerstone of modern biotechnology, particularly in the design of implantable biosensors and biofuel cells. In this context, high-redox potential laccases (HRPLs) from basidiomycetes offer unique advantages due to their ability to catalyze oxygen reduction at high potentials. However, their natural susceptibility to inhibition by hydroxide ions and halides—abundant in blood—has limited their practical use. To overcome these barriers, we engineered a blood-tolerant laccase variant, ChU-B, through iterative directed evolution in Saccharomyces cerevisiae, and subsequently optimized its overexpression in Pichia pastoris for scalable production.
The ChU-B mutant was constructed by fusing the mature laccase domain of PM1 strain CECT 2971 to either native or evolved α-factor prepro-leader sequences. These constructs were cloned under two distinct promoters: PAOX1 (methanol-inducible) and PGAP (constitutive). Initial screening in shake-flask cultures identified the PAOX1-driven construct with the evolved prepro-leader as the most productive. This candidate was then subjected to small-scale fed-batch fermentation in a 500-mL bioreactor, achieving peak laccase activity of 990 ABTS-U/L after 151 hours of induction. Encouraged by these results, the process was scaled up to a 42-L bioreactor system using a 20-L fermentation volume. The final yield reached 43 mg/L of purified recombinant enzyme, marking a 5.4-fold increase over S. cerevisiae-based production (~8 mg/L).
Purification of the ChU-B laccase involved a three-step chromatographic protocol: ammonium sulfate precipitation, anion-exchange (Mono Q), and hydrophobic interaction chromatography (PHE sepharose). The resulting protein was homogeneous, as confirmed by SDS-PAGE and MALDI-TOF mass spectrometry. The molecular weight of the P. pastoris-produced enzyme was determined to be 62,541 Da, consistent with the presence of additional glycosylation compared to the S. cerevisiae version (60,310 Da). Glycosylation levels were estimated at 16% in P. pastoris versus 12% in S. cerevisiae, reflecting differences in post-translational processing between hosts.
Despite higher glycosylation, the enzyme retained full functionality. N-terminal sequencing revealed identical extra extensions (ETEAEF) in both expression systems, indicating incomplete cleavage of the α-factor pro-leader by STE13 protease—a phenomenon observed in both yeasts. This extension did not impair activity but may contribute to secretion efficiency. The pH-activity profile remained unchanged across hosts, with optimal activity at pH 4.0 (ABTS) and pH 5.0 (DMP), and significant residual activity at pH 7.4. Most notably, the ChU-B mutant exhibited exceptional resistance to halide inhibition: I50 values for Cl⁻ exceeded 1000 mM at acidic pH, and F⁻ inhibition was also substantially reduced.Bis(4-tert-butylphenyl)amine Cancer
Kinetic analysis demonstrated that Km values for ABTS and DMP were similar between the two hosts (~0.024 mM and ~0.26 mM, respectively), indicating no loss in substrate affinity.STAT3 Antibody manufacturer However, kcat values were consistently higher in the S.PMID:35086724 cerevisiae-expressed form, suggesting subtle differences in catalytic turnover possibly linked to glycosylation patterns or folding dynamics. Still, the overall catalytic efficiency remained sufficient for biomedical applications.
Functional validation in human plasma and whole blood confirmed the enzyme’s real-world utility. Oxygen consumption rates were measured using a Clark electrode, yielding values of 31 ± 7 min⁻¹ in blood and 27 ± 1 min⁻¹ in plasma—indicating robust activity in complex biological matrices without interference from serum proteins or cellular components. This performance underscores the enzyme’s suitability for integration into miniature biofuel cells and continuous monitoring devices.
In conclusion, the combination of directed evolution in S. cerevisiae followed by high-level expression in P. pastoris provides a scalable, efficient, and reliable platform for producing advanced biocatalysts. The ChU-B laccase exemplifies how rational engineering can transform a naturally sensitive enzyme into a stable, blood-compatible biocatalyst. Its successful production and characterization pave the way for future applications in implantable diagnostics, wearable sensors, and energy-harvesting devices operating within the human body.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com