Pnh_Lac1 (Lac1) gene from the marine-derived fungus Peniophora sp. CBMAI 1063 was expressed in Pichia pastoris using the Pichia Toolkit system. Constitutive (pGAP, pPET9, pG1, pG6, and pADH2) and methanol-inducible (pAOX1, pDAS1, and pPMP20) promoters were assessed in combination with 21 different signal peptides and His-tag about efficiency in laccase production. Next, 3,200 variants were screened, different culture conditions were evaluated, and an investigation was performed in a bench-scale bioreactor for the best variant selected. The influence of promoters and signal peptides on Lac1 expression was demonstrated in the constitutive libraries. The change from pG6 to pGAP resulted in a 171-fold increase in production. Changing the alpha-mating factor peptide by the native signal peptide of the Lac1 gene decreased laccase production 22-fold. The promoters pGAP (constitutive library) and pAOX1 (inductive library) performed best. The association with the signal peptide αAmylase-αMFD was more efficient for both promoters. The constitutive expression of Lac1 had a 1.37-fold greater production compared to the inducible expression achieved by pAOX1 and was considered more suitable for laccase expression. Culturing the best producer variant pGAP_αA1 at pH 6 and 18 °C resulted in the best production rate in deep-well plates (90 U/L). Constitutive laccase production in a 2-L bioreactor resulted in a peak production of 178 U/L after 78 h. Pichia Toolkit was efficient in the selection of the best molecular regulation and secretion of Lac1. Our findings contribute to the development of marine biotechnology and will serve as the basis for Lac1 production optimization.

• Keywords
• Marine biotechnology, Multicopper oxidase, Synthetic biology,
• Publication type
• Journal Article MeSH

Low-expression levels remain a challenge in the quest to use the small laccase (rSLAC) as a viable catalyst. In this study, a recombinant Pichia pastoris strain (rSLAC-GAP-AOX) producing rSLAC under both AOX and GAP promoters (located in two different plasmids) was generated and cultivated in the presence of methanol and mixed feed (methanol:glycerol). Induction with methanol resulted in a maximum laccase activity of 1200 U/L for rSLAC-GAP-AOX which was approximately 2.4-fold higher than rSLAC-AOX and 5.1-fold higher than rSLAC-GAP. The addition of methanol:glycerol in a stoichiometric ratio of 9:1 consistently improved biomass and led to a 1.5-fold increase in rSLAC production as compared to induction with methanol alone. The rSLAC removed 95% of 5 mg/L ciprofloxacin (CIP) and 99% of 100 mg/L tetracycline (TC) in the presence of a mediator. Removal of TC resulted in complete elimination of antibacterial activity while up to 48% reduction in antibacterial activity was observed when CIP was removed. Overall, the present study highlights the effectiveness of a double promoter system in enhancing SLAC production.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Laccase * genetics   metabolism   MeSH
• Pichia * genetics   metabolism   MeSH
• Promoter Regions, Genetic MeSH
• Recombinant Proteins genetics   MeSH
• Saccharomycetales MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Laccase * MeSH
• Recombinant Proteins MeSH

Laccases are multi-copper oxidoreductases with broad biotechnological applications. Here, we report detailed biochemical characterization of purified recombinant laccases originating from Myceliophthora thermophila (MtL) and Trametes trogii (TtL). We identified optimal conditions for decolorization of commercial dyes and textile wastewater samples. We also tested the toxicity of decolorized wastewater samples using human peripheral blood mononuclear cells. MtL and TtL were expressed in Saccharomyces cerevisiae, and secreted enzymes were purified by consecutive hydrophobic and gel chromatography. The molecular masses of TtL (~ 65 kDa) and MtL (> 100 kDa) suggested glycosylation of the recombinant enzymes. Deglycosylation of MtL and TtL led to 25% and 10% decreases in activity, respectively. In a thermal stability assay, TtL retained 61% and MtL 86% of the initial activity at 40 °C. While TtL retained roughly 50% activity at 60 °C, MtL lost stability at temperatures higher than 40 °C. MtL and TtL preferred syringaldazine as a substrate, and the catalytic efficiencies for ABTS oxidation were 7.5 times lower than for syringaldazine oxidation. In the presence of the mediator HBT, purified TtL almost completely decolorized dyes within 30 min and substantially decolorized wastewater samples from a textile factory (up to 74%) within 20 h. However, products of TtL-catalyzed decolorization were more toxic than MtL-decolorized products, which were almost completely detoxified.

• Keywords
• Decolorization, Laccases, PBMCs, Textile dyes, Toxicity, Wastewater,
• Publication type
• Journal Article MeSH