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Extracellular expression of PETase in Bacillus subtilis

Abewe, Blandine LU (2025) KBTM01 20251
Biotechnology (MSc)
Biotechnology (M.Sc.Eng.)
Abstract
Polyethylene terephthalate (PET) is a widely used polyester worldwide, however its disposal has made a major contribution to climate change and other environmental issues. Therefore, sustainable approaches for PET depolymerization are essential for advancement of eco-friendly PET recycling. Enzymatic depolymerization of PET has attracted attention during the past decade.
In this study, to evaluate the potential of Bacillus subtilis (B. subtilis) K07 strain as a host for extracellular expression of PET-degrading enzyme LCC, two genetically engineered constructs (2 and 3) were designed based on initial construct 1 containing NBP350 promoter and AprE signal peptide. Construct 2 contained a modified signal peptide (WapA) whereas construct 3... (More)
Polyethylene terephthalate (PET) is a widely used polyester worldwide, however its disposal has made a major contribution to climate change and other environmental issues. Therefore, sustainable approaches for PET depolymerization are essential for advancement of eco-friendly PET recycling. Enzymatic depolymerization of PET has attracted attention during the past decade.
In this study, to evaluate the potential of Bacillus subtilis (B. subtilis) K07 strain as a host for extracellular expression of PET-degrading enzyme LCC, two genetically engineered constructs (2 and 3) were designed based on initial construct 1 containing NBP350 promoter and AprE signal peptide. Construct 2 contained a modified signal peptide (WapA) whereas construct 3 incorporated a modified promoter (paprE). The LCC enzymes produced using three constructs were tested for catalytic activity.
The enzymatic activity was confirmed by the hydrolysis of 40% crystalline PET into its monomers (TPA, MHET, and BHET) after 48 hours of incubation at 60°C and 600 rpm. The results showed that construct 1 released a total of 1.02*10-04 mol/L monomers, construct 2 released 1.12*10-04 mol/L, and construct 3 released 1.24*10-04 mol/L. These results may have been influenced by variations in LCC concentrations among the constructs. Future studies using standardized LCC enzyme concentrations will be essential to determine the precise effect of biobricks optimization and synthetic biology strategies on PET recycling efficiency. (Less)
Popular Abstract
Bacillus subtilis is a gram-positive bacterium that is generally recognized as safe (GRAS). Some of its characteristics made it an attractive candidate for biotechnological applications. Notably, it can uptake foreign DNA in the surrounding environment and secrete proteins outside the cell, making it useful for producing extracellular protein. Additionally, widely available and standardized methods for genetic manipulation, transformation and cultivation make it possible to engineer B. subtilis for the intended use.
PET is one of the most used plastics in the world known for its durability and cost-effectiveness. Apart from its good properties, its accumulation in the environment poses serious risks to human health, wildlife, and... (More)
Bacillus subtilis is a gram-positive bacterium that is generally recognized as safe (GRAS). Some of its characteristics made it an attractive candidate for biotechnological applications. Notably, it can uptake foreign DNA in the surrounding environment and secrete proteins outside the cell, making it useful for producing extracellular protein. Additionally, widely available and standardized methods for genetic manipulation, transformation and cultivation make it possible to engineer B. subtilis for the intended use.
PET is one of the most used plastics in the world known for its durability and cost-effectiveness. Apart from its good properties, its accumulation in the environment poses serious risks to human health, wildlife, and ecosystem due to its resistance to natural degradation. To address this issue, various strategies have been explored, including reducing plastic consumption, and PET recycling. The latter became a topic of interest to many researchers, with scientists exploring mechanical, chemical, and enzymatic methods to break down PET
To date, many PET- degrading enzymes have been discovered and improved through protein engineering approaches. Additionally, enzymatic degradation is considered a more sustainable alternative, as it avoids toxic chemicals and generate less waste. In this study, Bacillus subtilis was engineered to produce proteins that break PET into small building blocks that can be retrieved and reused, contributing to more efficient and environmentally friendly PET recycling. (Less)
Please use this url to cite or link to this publication:
author
Abewe, Blandine LU
supervisor
organization
course
KBTM01 20251
year
type
H2 - Master's Degree (Two Years)
subject
keywords
biotechnology, B.subutilis, LCC, hydrolysis, PET structure
language
English
id
9203910
date added to LUP
2025-06-23 12:56:28
date last changed
2025-06-23 12:56:28
@misc{9203910,
  abstract     = {{Polyethylene terephthalate (PET) is a widely used polyester worldwide, however its disposal has made a major contribution to climate change and other environmental issues. Therefore, sustainable approaches for PET depolymerization are essential for advancement of eco-friendly PET recycling. Enzymatic depolymerization of PET has attracted attention during the past decade.
In this study, to evaluate the potential of Bacillus subtilis (B. subtilis) K07 strain as a host for extracellular expression of PET-degrading enzyme LCC, two genetically engineered constructs (2 and 3) were designed based on initial construct 1 containing NBP350 promoter and AprE signal peptide. Construct 2 contained a modified signal peptide (WapA) whereas construct 3 incorporated a modified promoter (paprE). The LCC enzymes produced using three constructs were tested for catalytic activity.
The enzymatic activity was confirmed by the hydrolysis of 40% crystalline PET into its monomers (TPA, MHET, and BHET) after 48 hours of incubation at 60°C and 600 rpm. The results showed that construct 1 released a total of 1.02*10-04 mol/L monomers, construct 2 released 1.12*10-04 mol/L, and construct 3 released 1.24*10-04 mol/L. These results may have been influenced by variations in LCC concentrations among the constructs. Future studies using standardized LCC enzyme concentrations will be essential to determine the precise effect of biobricks optimization and synthetic biology strategies on PET recycling efficiency.}},
  author       = {{Abewe, Blandine}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Extracellular expression of PETase in Bacillus subtilis}},
  year         = {{2025}},
}