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Explora: Environment
and Resource Enzymatic degradation

United Nations Agenda 2030. Our proposed enzymatic serum albumin as the standard. The crude enzyme
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approach holds promise for application in decentralized concentration was measured at 10 mg/mL (20% purity). To
waste treatment facilities, bioreactors for plastic waste reduce the concentration for further assays, 10 mL of the
remediation, and as a pre-treatment step to accelerate extract was diluted with an equal volume of distilled water,
subsequent biodegradation. More importantly, such eco- resulting in a final protein concentration of 5 mg/mL,
friendly strategies can reduce the environmental footprint while maintaining the purity at 20%.
of incineration and landfilling, which aligns with circular
economy principles and global sustainability targets. 2.4. Incubation of PE samples
2.4.1. Isolation of LDPE film in the single-enzyme
2. Materials and methods system
2.1. Materials The pre-weighed LDPE films (0.5 × 0.5, 1 × 1, and 2 ×
Low-density PE (LDPE) samples (PWS) were collected 2 cm) were aseptically transferred into three pre-labeled
from dumpsites within Gidan Kwano village (Nigeria). 100 mL conical flasks, respectively. About 20 mL of the
Enzymes used were produced at the Step B Laboratory, prepared degrading enzyme was added to each conical
Centre for Genetic Engineering and Biotechnology, flask and thoroughly mixed with the LDPE samples. For
Federal University of Technology, Minna (FUTMinna), assessing the effect of different enzyme concentrations, the
Bosso Campus, Nigeria. The incubation media used prepared LDPE strips (1 × 1 cm each) were transferred into
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for the LDPE samples and all analytical chemicals were two pre-labeled 100 mL conical flasks, respectively. Then,
procured from the Department of Chemical Engineering, 100% pure Lip and a 50% diluted Lip solution were added
FUTMinna (Nigeria). separately to each flask and thoroughly mixed with the
LDPE samples. To minimize heat dissipation and preserve
2.2. Preparation of PE films a sanitized environment, a foil stopper was used to seal
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The LDPE samples were cut into 0.5 × 0.5, 1 × 1, and 2 × the conical flasks. The procedure was repeated for Lac.
2 cm strips and weighed using a digital weighing balance After 10 days of incubation, samples were collected, and
(MAB220, Wensar, India), as previously reported. 49,51 The the LDPE films were cleaned with ethanol and distilled
samples underwent a rigorous cleaning process, which water, then allowed to air-dry naturally. Following that,
involved immersion in a solution of 70% ethanol for the LDPE samples were evaluated to determine their
30 min, followed by washing with distilled water, and level of biodegradability. After 30 days, the process was
drying in an oven at 50°C for 20 min. terminated. The 30-day incubation period was selected to
allow sufficient time for measurable enzymatic degradation
2.3. Production of degrading enzymes of LDPE by Lac and Lip.
Ab initio, the medium was prepared through the following 2.4.2. Isolation of LDPE film in the two-enzyme system
steps. First, the Sabouraud Dextrose Agar was prepared by
dissolving 20 g of the powdered non-synthetic medium in The pre-weighed LDPE films (0.5 × 0.5 and 2 × 2 cm) were
two separate 2000 mL conical flasks labeled “Lipase” and transferred into two pre-labeled 100 mL conical flasks,
“Laccase,” respectively, each containing distilled water. respectively. Lip and Lac enzymes (20 mL) were added to
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For the Lip medium, 2 mL of olive oil (0.1% substrate each conical flask and thoroughly mixed with the LDPE
concentration) was added. For the Lac medium, 2 mL samples. For assessing the effect of different enzyme
of guaiacol (0.1% substrate concentration) was added. concentrations, the prepared LDPE strips (1 × 1 cm) were
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Both media were sterilized in an autoclave at 121°C for transferred into two pre-labeled 100 mL conical flasks,
3 h under a pressure of 1.5 N/m. After autoclaving, the respectively. The Lip-Lac enzyme mixture at 100% and
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medium was removed from the autoclave and allowed 50% concentrations was added to the two conical flasks,
to cool before inoculation. The 72-h-old broth culture of respectively. To minimize heat dissipation and preserve a
A. flavus (5 mL) was inoculated into 1 L of each medium sanitized environment, a foil stopper was used to seal the
and incubated for 5 days on a shaker incubator at ambient conical flasks. The solution was gently stirred to obtain
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or room temperature (25–28°C ± 2°C). After 5 days, a homogeneous mixture. The LDPE films were cleaned
crude enzymes were readily produced. The crude enzyme using ethanol and distilled water, then allowed to air-
solutions were centrifuged at 4000 rpm using a high-speed dry naturally. Biodegradation of the LDPE samples was
refrigerated centrifuge. The supernatants contained the analyzed after 10 days of incubation. Following that, the
crude enzymes, while the sediments consisted of fungal LDPE samples were evaluated to determine their level of
spores. The Bradford protein assay was used to quantify biodegradability. The process was terminated after 30 days,
the total protein content in the crude extract, using bovine in accordance with Yang et al. 56

Volume 2 Issue 3 (2025) 3 doi: 10.36922/EER025220042