# Microbial and Enzymatic Biodegradation of Plastic Waste for a Circular Economy
## 2 Microbial and Enzyme-Based Degradation of Plastic
### Enzymatic Degradation of Polymers
#### 2.3.1 Cutinase-Catalysed Depolymerisation of Plastics  
Cutinases enzymes can break down the ester bonds present in polyesters, especially polyesters like PET [103; 104; 106]. Cutinases have been identified in various organisms, including fungi and bacteria. The enzymatic depolymerisation reported by [PERSON] et al. using the cellulolytic actinomycete _Thermobifida fusca_ isolated from soil resulted in up to 50% degradation of PET at 55 \({}^{\circ}\)C in three weeks. After this work, many cutinases from various _Thermobifida_ sp. were isolated which could degrade plastics [105]. Alongside this work, engineered cutinases were developed and employed to degrade polyester-based plastics. For example, cutinases from _Thermobifida cellulosilytica_ DSM44535 and  
Figure 4: Role of insect gut microbiota in plastic degradation, with released enzymes facilitating depolymerisation [81; 84].  
Thermobifida fusca DSM44342 were successfully cloned and expressed in _E. coli_ BL21. These enzymes from _Thermobifida_, specifically ThcCut1, exhibit favorable kinetic parameters for soluble substrates and release MHT and terephthalatic acid from PET. Comparative homology modelling suggests that His-209, located in the active site, is crucial for hydrolysis activity [107]. _Thermobifida fusca_ cutinase (TfC) effectively degrades PET films pretreated with _S. paunii_ JWG-G1, showing up to 91.4% degradation within 23 days. The pretreatment with _S. paunii_ JWG-G1 likely enhanced the accessibility of the PET substrate, making it more susceptible to the enzyme. This combination of microbial pretreatment and enzymatic degradation resulted in substantial degradation of PET films, suggesting the benefit of a synergetic approach for efficient PET degradation [108].  
The cutinases (FoCut5a) from _Fusarium oxysporum_ and _Thermobifida alla_ AHK119 fungus, expressed in _E. coli_ BL21, show optimal catalytic activity on synthetic esters at 40 \({}^{\circ}\)C and a pH of 8.0 [109]. Their successful expression in _E. coli_ BL21 is an advantage which allows for high yield production using a well-established and cost-effective microbial expression system. The _Fusarium oxysporum_ enzyme represents a promising approach for hydrolysing PET-like substrates and other synthetic polymers [110]. A purified lipase from the yeast _Cryptococcus_ sp. showed sequence homology with proteins in the cutinase family rather than the lipase family, and this enzyme demonstrated effective degradation of the high molecular weight polymer PLA, as well as other biodegradable polymers including PBS, PCL, and PHB [95]. [PERSON] et al. demonstrated the potential of an extracellular cutinase-like enzyme from the biofilm forming-bacterium _Steotrophonomas maltophilis_ PRS8 in PET breakdown [111]. This enzyme's stability and activity were maintained during various environmental changes (including temperature, pH level, substrate concentration, and the presence of inhibitors or activators), and it was effective in the hydrolysis of PET. The depolymerisation process generated terephthalatic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHT), and bis(2-hydroxyethyl) terephthalate (BIE) from PET flakes, indicating its potential for managing PET waste and recovering value-added products [111].  
Cutinases could be a promising tool in combating plastic pollution and recovering various plastics monomers. Mutation and engineering of these enzymes may enhance their functional activity and stability over a wider range of pH levels and temperatures. However, it is important to note that while cutinases are effective in the degradation of polyester, a single enzyme is not sufficient for all types of polymers. Multi-enzyme systems are needed to target a wide range of plastics. This aspect needs further exploration and research [112].