Wastewater treatment from a science faculty during the COVID-19 pandemic by using ammonium-oxidising and heterotrophic bacteria.

During and after the pandemic caused by the SARS-CoV-2 virus, the use of personal care products and disinfectants increased in universities worldwide. Among these, quaternary ammonium-based products stand out; these compounds and their intermediates caused substantial changes in the chemical composition of the wastewater produced by these institutions. For this reason, improvements and environmentally sustainable biological alternatives were introduced in the existing treatment systems so that these institutions could continue their research and teaching activities. For this reason, the objective of this study was to develop an improved culture medium to cultivate ammonium oxidising bacteria (AOB) to increase the biomass and use them in the treatment of wastewater produced in a faculty of sciences in Bogotá, D.C., Colombia. A Plackett Burman Experimental Design (PBED) and growth curves served for oligotrophic culture medium, and production conditions improved for the AOB. Finally, these bacteria were used with total heterotrophic bacteria (THB) for wastewater treatment in a pilot plant. Modification of base ammonium broth and culture conditions (6607 mg L-1 of (NH4)2SO4, 84 mg L-1 CaCO3, 40 mg L-1 MgSO4·7H2O, 40 mg L-1 CaCl2·2H2O and 200 mg L-1 KH2PO4, 10% (w/v) inoculum, no copper addition, pH 7.0 ± 0.2, 200 r.p.m., 30 days) favoured the growth of Nitrosomonas europea, Nitrosococcus oceani, and Nitrosospira multiformis with values of 8.23 ± 1.9, 7.56 ± 0.7 and 4.2 ± 0.4 Log10 CFU mL-1, respectively. NO2- production was 0.396 ± 0.0264, 0.247 ± 0.013 and 0.185 ± 0.003 mg L-1 for Nitrosomonas europea, Nitrosococcus oceani and Nitrosospira multiformis. After the 5-day wastewater treatment (WW) by co-inoculating the three studied bacteria in the wastewater (with their self-microorganisms), the concentrations of AOB and THB were 5.92 and 9.3 Log10 CFU mL-1, respectively. These values were related to the oxidative decrease of Chemical Oxygen Demand (COD), (39.5 mg L-1), Ammonium ion (NH4+), (6.5 mg L-1) Nitrite (NO2-), (2.0 mg L-1) and Nitrate (NO3-), (1.5 mg L-1), respectively in the five days of treatment. It was concluded, with the improvement of a culture medium and production conditions for three AOB through biotechnological strategies at the laboratory scale, being a promising alternative to bio-augment of the biomass of the studied bacteria under controlled conditions that allow the aerobic removal of COD and nitrogen cycle intermediates present in the studied wastewater. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03961-4.

Use of biochar and a post-coagulation effluent as an adsorbent of malachite green, beneficial bacteria carrier, and seedling substrate for plants belonging to the poaceae family.

Wastewater treatment plants produce solid and semi-solid sludge, which treatment minimises secondary environmental pollution because of wastewater treatment and obtaining new bioproducts. For this reason, in this paper, the co-pyrolysis of biogenic biomasses recovered from a biological reactor with immobilised fungal and bacterial biomass and a tertiary reactor with Chlorella sp. used for dye-contaminated wastewater treatment was carried out. Biogenic biomasses mixed with pine bark allowed the production and characterisation of two types of biochar. The raw material and biochar were on the "in vitro" germination of Lolium sp. seeds, followed by adsorption studies for malachite green (MG) dye using the raw material and the biochar. Results showed that using 60 mg L-1 of a cationic coagulant at pH 6.5 allowed for the recovery of more than 90% of the microalgae after 50 min of processing. Two biochar resulted: BC300, at pH 5.08 ± 0.08 and BC500, at pH 6.78 ± 0.01. The raw material and both biochars were co-inoculated with growth-promoting bacteria; their viabilities ranged from 1.7 × 106 ± 1.0 × 101 to 7.5 × 108 ± 6.0 × 102 CFU g-1 for total heterotrophic, nitrogen-fixing and phosphate-solubilising bacteria. Re-use tests on Lolium sp. seed germination showed that with the post-coagulation effluent, the germination was 100%, while with the biochar, with and without beneficial bacteria, the germination was 98 and 99%, respectively. Finally, BC500 adsorbed the highest percentage of malachite green at pH 4.0, obtaining qecal values of 0.5249 mg g-1 (R2: 0.9875) with the pseudo-second-order model. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03766-x.

Non-domestic wastewater treatment with fungal/bacterial consortium followed by Chlorella sp., and thermal conversion of the generated sludge.

Liquid waste from biological stains is considered non-domestic wastewater difficult to treat, generating high environmental impact. Therefore, the objective of this work was to carry out secondary and tertiary treatment of these effluents at a pilot scale, using a fungal/bacterial consortium followed by Chorella sp., for 15 days. In addition, to obtain an adsorbent material for Malachite Green dye removal, sludge generated in the plant and pine bark co-pyrolysis was performed. For microalgae isolation and selection of the Chlorophyceae class, Chlorococcales order, and Chorella sp. genus Winogradsky columns were employed. After 15 days of pilot plant treatment, removal percentages of 91 ± 2%, 90 ± 4% and 17 ± 2% were obtained for Colour Units, Chemical Oxygen Demand and Nitrates, respectively. Two types of class II biochar (BC500 and BC700) and one of class III (BC300) were produced. The highest value for Fixed carbon (FC) was obtained at 300 °C (27.3 ± 3%), decreasing as the temperature increased by 25.9 ± 5% and 24.8 ± 2%, for BC500 and BC700, respectively. Biochar yield was 62.1 ± 3%, 46.3 ± 4% and 31.6 ± 3% for BC300, BC500 and BC700, respectively. Finally, BC500 and BC700 biochar efficiently adsorbed Malachite Green obtaining qe values of 0.290 ± 0.032, 0.281 ± 0.015, 0.186 ± 0.009 and 0.191 ± 0.012 mg g-1 at pH values of 4.0 and 8.0 ± 0.2, respectively. Pseudo-second order model demonstrated a chemical adsorption took place, which was influenced by pH. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02780-1.

Phosphate-solubilizing Pseudomonas sp., and Serratia sp., co-culture for Allium cepa L. growth promotion.

Different genus of bacteria has been reported with the capacity to solubilize phosphorus from phosphate rock (PR). Pseudomonas sp., (A18) and Serratia sp., (C7) isolated from soils at the "Departamento de Boyacá" Colombia, where Allium cepa is cultivated. Bacteria were cultured in MT11B media and evaluated as a bio-fertilizer for A. cepa germination and growth during two months at greenhouse scale. Pseudomonas sp., and Serratia sp., cultured at 30 °C, 48 h in SMRS1 agar modified with PR, (as an inorganic source of phosphorus), presented a phosphate solubilization index (SI) of 2.1 ± 0.2 and 2.0 ± 0.3 mm, respectively. During interaction assays no inhibition halos were observed, demonstrating there was no antagonism between them. In MT11B media growth curve (12 h) demonstrated that co-culture can grow in the presence of PR and glucose concentrations 7.5-fold, lower than in SMRS1 media and brewer's yeast hydrolysate; producing phosphatase enzymes with a volumetric activity of 1.3 ± 0.03 PU at 6 h of culture and 0.8 ± 0.04 PU at 12 h. Moreover, co-culture released soluble phosphorus at a rate of 58.1 ± 0.28 mg L-1 at 8 h and 88.1 ± 0.32 mg L-1 at 12 h. After five days of evaluation it was observed that germination percentage was greater than 90 % of total evaluated seeds, when placing them in contact with the co-culture in a concentration of 1 × 108 CFU mL-1. Furthermore, it was demonstrated that co-culture application (10 mL per experimental unit to complete 160 mL in two months) at 8.0 Log10 CFU mL-1 twice a week for two months increased A. cepa total dry weight (69 ± 13 mg) compared with total dry weight (38 ± 5.0 mg) obtained with the control with water.

Tertiary treatment (Chlorella sp.) of a mixed effluent from two secondary treatments (immobilized recombinant P. pastori and rPOXA 1B concentrate) of coloured laboratory wastewater (CLWW).

Industrial development has increased wastewater (WW) volume; generating contamination and disturbing ecosystems, because of breeching disposal parameters. In this work, Coloured Laboratory Wastewater (CLWW), (1500.00 colour units, CU) was separately submitted to two secondary treatments. For the first one CLWW was treated for three cycles C1, C2 and C3 with P. pastoris X33/pGAPZαA-LaccPost-Stop producing rPOXA 1B laccase, immobilized in calcium alginate beads. For the second-one, rPOXA 1B enzyme concentrate was used (three processes: P1, P2, and P3). Both treatments were carried out in a 15 L reactor with 10 L effective work volume (EWV) with 72 h hydraulic retention time. C1, C2, and C3 effluents were flocculated and filtered through quartzite sand, while P1, P2, and P3 effluents were only filtered through quartzite sand. The mixture of secondary effluents was submitted to a tertiary treatment with Chlorella sp. For C1, C2, C3, P1, P2, and P3, CU removal was of 99.16, 99.58, 99.53, 96.72, 97.05 and 96.47%, respectively. Discharge parameters, total organic carbon (TOC), inorganic carbon (IC), chemical oxygen demand (COD) and biological oxygen demand (BOD5) decreased, although they reached different final values. After the tertiary treatment (144 h) effluent discharge parameters were reduced to 34 ± 4 CU, TOC to 6.6 ± 0.9 mg L-1 and COD to 155 ± 4 mg L-1. It was demonstrated that secondary treatments (immobilized recombined cells or recombinant enzyme concentrate) combined with Chlorella sp., (tertiary treatment) attained a considerable removal of discharge parameters, demonstrating a promissory alternative for CLWW sequential treatment.

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

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, 20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] 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.

Detoxification of pulping black liquor with Pleurotus ostreatus or recombinant Pichia pastoris followed by CuO/TiO2/visible photocatalysis.

Cellulose-pulping requires chemicals such as Cl2, ClO2, H2O2, and O2. The black liquor (BL) generated exhibits a high chemical oxygen demand (COD), five-day biochemical oxygen demand (BOD5), and chlorophenol content, along with an augmented colour and increased pH. BL is often discharged into water bodies, where it has a negative impact on the environment. Towards that end, laccases are of great interest for bioremediation, since they can degrade aromatic and non-aromatic compounds while reducing O2 to water instead of H2O2. As such, we evaluated Pleurotus ostreatus and Pichia pastoris (which produces rPOXA 1B laccase) in the treatment of synthetic BL (SBL) in an "in vitro" modified Kraft process followed by CuO/TiO2/visible light photocatalysis. Treating SBL with P. ostreatus viable biomass (VB) followed by CuO/TiO2/visible light photocatalysis resulted in 80.3% COD removal and 70.6% decolourisation. Toxic compounds such as 2-methylphenol, 4-methylphenol, and 2-methoxyphenol were eliminated. Post-treated SBL exhibited low phytotoxicity, as evidenced by a Lactuca sativa L seed germination index (GI) > 50%. Likewise, SBL treatment with P. pastoris followed by VB/CuO/TiO2/visible light photocatalysis resulted in 63.7% COD removal and 46% decolourisation. Moreover, this treatment resulted in the elimination of most unwanted compounds, with the exception of 4-chlorophenol. The Lactuca sativa L seed GI of the post-treated SBL was 40%, indicating moderate phytotoxicity.

Malachite Green and Crystal Violet Decolorization by Ganoderma lucidum and Pleurotus ostreatus Supernatant and by rGlLCC1 and rPOXA 1B Concentrates: Molecular Docking Analysis.

Laccases catalyze the oxidation of various aromatic organic compounds concomitantly with molecular oxygen reduction to water. Triphenylmethane dyes are synthetic compounds widely used in diverse industries. Their removal from effluents is difficult, due to their high degree of structural complexity; hence, their high concentration in effluents cause a negative impact on the environment. In the present work, molecular docking was used to evaluate interactions between rGlLCC1 or rPOXA 1B enzymes with Crystal Violet (CV) or Malachite Green (MG) dyes. In addition, removal tests of the two dyes were performed. Van der Waals interactions were obtained for only the CV dye for both GlLCC1 and POXA 1B enzymes. Nevertheless, in the GlLCC1 model, two π-π interactions were observed. For the MG dye only, Van der Waals interactions were obtained. Moreover, amino acid composition interacting in each model with each dye was similar. It is important to highlight that by molecular docking, none of the estimated ligand configurations generated hydrogen bonds. Thus, explaining the difficulty to degrade CV and MG. Regarding CV, maximum decolorization percentage was 23.6 ± 1.0% using Ganoderma lucidum supernatant and 5.0 ± 0.5% with Pleurotus ostreatus supernatant. When using recombinant laccase enzyme concentrates, decolorization percentages were 9.9 ± 0.1 and 7.5 ± 1.0% for rGlLCC1 and rPOXA 1B, respectively. On the other hand, for the MG dye, maximum decolorization percentages were 52.1 ± 5.1 and 2.3 ± 0.2% using G. lucidum and P. ostreatus concentrates, respectively. Whereas with recombinant laccase enzymatic concentrates, values of 9.4 ± 0.8% were obtained, with rGlLCC1, and 2.1 ± 0.1% when using rPOXA 1B. These findings represent an important step in bioremediation processes improvement and efficiency of industry-generated products, using environmentally friendly alternatives.

Correction to: Malachite Green and Crystal Violet Decolorization by Ganoderma lucidum and Pleurotus ostreatus Supernatant and by rGlLCC1 and rPOXA 1B Concentrates: Molecular Docking Analysis.

The original version of this article unfortunately contained a mistake. The replacement image of Fig. 4 provided by the first corresponding author, Aura M. Pedroza-Rodríguez, is incorrect and that the originally submitted Fig. 4 should have been retained. The original article has been corrected.

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

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.

"In Silico" Characterization of 3-Phytase A and 3-Phytase B from Aspergillus niger.

Phytases are used for feeding monogastric animals, because they hydrolyze phytic acid generating inorganic phosphate. Aspergillus niger 3-phytase A (PDB: 3K4Q) and 3-phytase B (PDB: 1QFX) were characterized using bioinformatic tools. Results showed that both enzymes have highly conserved catalytic pockets, supporting their classification as histidine acid phosphatases. 2D structures consist of 43% alpha-helix, 12% beta-sheet, and 45% others and 38% alpha-helix, 12% beta-sheet, and 50% others, respectively, and pI 4.94 and 4.60, aliphatic index 72.25 and 70.26 and average hydrophobicity of -0,304 and -0.330, respectively, suggesting aqueous media interaction. Glycosylation and glycation sites allowed detecting zones that can affect folding and biological activity, suggesting fragmentation. Docking showed that H59 and H63 act as nucleophiles and that D339 and D319 are proton donor residues. MW of 3K4Q (48.84 kDa) and 1QFX (50.78 kDa) is similar; 1QFX forms homodimers which will originate homotetramers with several catalytic center accessible to the ligand. 3K4Q is less stable (instability index 45.41) than 1QFX (instability index 33.66), but the estimated lifespan for 3K4Q is superior. Van der Waals interactions generate hydrogen bonds between the active center and O2 or H of the phytic acid phosphate groups, providing greater stability to these temporal molecular interactions.

Partial removal and detoxification of Malachite Green and Crystal Violet from laboratory artificially contaminated water by Pleurotus ostreatus / Eliminación parcial y desintoxicación de Verde Malaquita y Cristal Violeta del laboratorio con agua contaminada artificialmente por Pleurotus ostreatus / Extração e desintoxicação de Verde Malaquita e Cristal Violeta de água contaminada artificialmente em laboratório por Pleurotus ostreatus

Abstract The triphenylmethane Malachite Green (MG) and Crystal Violet (CV) dyes are cationic dyes and mix with domestic wastewater when dumped; increasing, among others, the chemical and biological oxygen demand and can cause acute toxicity at different trophic levels. Promoting the removal (decolorization) of MG and CV, and laccase activity (54.8 ± 8.9 and 30.6 ± 2.9 UL-1 respectively) by using P. ostreatus viable biomass needed parameters such as pH (4.5 and 6.0), temperature (25 to 30 °C), stirring speed (120 rpm), percentage of inoculum (2% v/v), and dye concentration (20 and 10 mg L-1). In adsorption studies, it was showed that an acidic pH favors the adsorption of both dyes and the model of pseudo-second order describes best the phenomenon of adsorption. Finally, the germination index (GI), using Lactuca sativa seeds for the initial dyes solutions, was < 50%; demonstrating its high phytotoxic effect. When dye solutions were treated with viable biomass, the GI increased, leaving open the possibility to perform future research to determine if the aqueous solutions, post-treated with P. ostreatus, could be used in treatments that generate less toxic water which could be used in processes that do not require potable water.

Resumen Los colorantes trifenilmetánicos Verde Malaquita (MG) y Crystal Violeta (CV) son catiónicos y al ser vertidos se mezclan con aguas residuales domésticas, incrementando, entre otros, la demanda química y biológica de oxígeno; pudiendo causar toxicidad aguda en diferentes niveles tróficos. En este estudio se encontró que los parámetros pH (4,5 y 6,0), temperatura (25 y 30 °C), velocidad de agitación (120 r.p.m.), porcentaje de inóculo (2 % v/v) y concentración de colorante (20 y 10 mgL-1), presentaron un efecto significativo (p < 0.05) para favorecer la remoción (decoloración) de MG y CV, así como la actividad lacasa (54,76 ± 8,91 y 30,59 ± 2,89 UL-1 respectivamente) al utilizar biomasa viable de P. ostreatus. En los estudios de adsorción se evidenció que pH ácidos favorecen la adsorción de ambos colorantes y que el modelo de Pseudo-segundo orden describe mejor el fenómeno de quimisorción. Finalmente los índices de germinación (IG) empleando semillas de Lactuca sativa, para los colorantes iniciales fueron < 50 %; demostrando su efecto fitotóxico elevado. Cuando las soluciones de colorantes fueron tratadas con biomasa viable, el IG aumentó, dejando abierta la puerta para la realización de investigaciones futuras con la intensión de determinar si las soluciones acuosas, postratadas con P ostreatus, pueden ser utilizadas en tratamientos que generen aguas menos tóxicas y que estas puedan ser empleadas en otros procesos que no requieran agua potable.

Resumen Os corantes de tipo trifenilmetano Verde Malaquita (VM) e Cristal Violeta (CV) são corantes catiônicos e se misturam com águas residuais domésticas quando descartadas; aumentando, entre outros, as demandas químicas e biológicas de oxigênio, podendo causar toxicidade aguda em diferentes níveis tróficos. Promoveu-se a remoção (descoloração) de VM e CV, e atividade da lacase (54.8 ± 8.9 e 30.6 ± 2.9 UL-1 respectivamente) utilizando como parâmetros necessários para a biomassa viável de P. ostreatus como pH (4,5 e 6,0), temperatura (25 a 30 °C), velocidade de agitação (120 RPM), porcentagem de inócuo (2 % v/v), e concentração de corante (20 e 10 mg L-1). Em estudos de absorção, se demonstrou que um pH mais ácido favorece a absorção de ambos corantes e o modelo de pseudo-segunda ordem descreve melhor o fenômeno da absorção. Finalmente, o índice de germinação (IG), utilizando sementes de Lactuca sativa para as soluções iniciais dos corantes, foi < 50 %; demonstrando assim seu alto efeito fitotóxico. Quando as soluções de corante foram tratadas com a biomassa viável, o IG aumentou, deixando em aberto a possibilidade de realizar futuras investigações para determinar se as soluções aquosas, tratadas com P. ostreatus, poderiam ser utilizadas em tratamentos que gerem águas menos tóxicas, que poderia ser utilizada em processos que não requerem água potável.

Bioinformatic Analysis of the Human Recombinant Iduronate 2-Sulfate Sulfatase.

Mucopolysaccharidosis type II is a human recessive disease linked to the X chromosome caused by deficiency of lysosomal enzyme Iduronate 2-Sulfate Sulfatase (IDS), which leads to accumulation of glycosaminoglycans in tissues and organs. The human enzyme has been expressed in Escherichia coli and Pichia pastoris in attempt to develop more successful expression systems that allow the production of recombinant IDS for Enzyme Replacement Therapy (ERT). However, the preservation of native signal peptide in the sequence has caused conflicts in processing and recognition in the past, which led to problems in expression and enzyme activity. With the main object being the improvement of the expression system, we eliminate the native signal peptide of human recombinant IDS. The resulting sequence showed two modified codons, thus, our study aimed to analyze computationally the nucleotide sequence of the IDSnh without signal peptide in order to determine the 3D structure and other biochemical properties to compare them with the native human IDS (IDSnh). Results showed that there are no significant differences between both molecules in spite of the two-codon modifications detected in the recombinant DNA sequence.

Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris.

Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.