Table 1.

Enzymes developed through in silico-based engineering.

Enzyme	Development platform/Software used	Specific mutations incorporated into the enzyme	Type of PET used as a substrate for enzyme assay	In vitro/in vivo conversion efficiency	Ref.
FAST-PETase: functional, active, stable, and tolerant PETase)	MutCompute three-dimensional CNN (3DCNN) model. A structure-based machine learning algorithm	Three from ML algorithm prediction (N233K/R224Q/S121E) and two (D186H/R280A) from parental scaffold) Active in ambient temperature from 30 ℃ To 50 ℃	untreated, postconsumer-PET from 51 different thermoformed products (Marked thermostability and reactivity toward amorphous and less crystalline PET (1.2–11.7% crystallinity) at elevated temperatures (e.g., 50 ℃))	98.4% of TPA From digestion solution. Complete degradation of untreated, post-consumer-PET from 51 different thermoformed products in 1 week. Depolymerization of untreated, amorphous portions of a commercial water bottle and an entire thermally pretreated water bottle at 50 ºC	[54]
Dura-PETase	Greedy accumulated strategy for protein engineering, GRAPE. A systematic clustering analysis combined with greedy accumulation of beneficial mutations in a computationally derived. library	melting temperature increased by 31 °C	1. semicrystalline poly (ethylene terephthalate) (PET) films (30%)2. microplastics	1. 30% enhancement of PET degradation at mild temperatures (over 300-fold). 2. Complete biodegradation of 2 g/L microplastics 2. Complete biodegradation of 2 g/L microplastics	[52]
LCC-ICCG	M.Do. Binding analysis to 2- HE(MHET)3	F243I/D238C/S283C/Y127G	post-consumer colored-flake PET waste (PcW-PET)	90% of PET depolymerization into monomers over 10 h, Productivity is 16.7 g of TPA/L/h (200 g/kg of PET suspension, with an enzyme concentration of 3 mg/g of PET)	[56]
Thermo-PETase		IsPETaseS121E/D186H/R280A variant, have a stabilized β6-β7 connecting loop and extended subsite IIc, Thermostable IsPETase with Tm 56.8 ℃) that harbors three mutations, S121E, D186H, and R280A		Tm value increased by 8.81 °C, and PET degradation activity was enhanced by 14-fold at 40 °C compared with IsPETaseWT.	[49]
IsPETaseS121E/D186H/S242T/N246D	Structural bioinformatics-based protein engineering	integrating the S242 T and N246D mutations into the previously reported IsPETaseS121E/D186H/R208A variant		A 58-fold increase in activity compared with IsPETaseWT.	[50]
IsPETase W159H/F229Y variant	the mutation design tool, Premuse	two mutations (W159H and F229Y)	p-NPP, amorphous PET, and PET bottle	Its Tm and catalytic efficiency values (kcat/Km) increased by 10.4 °C and 2.0-fold using p-NPP as the substrate compared with the wild type. The degradation activity for amorphous PET was increased by almost 40-fold compared with the wild type at 40 °C in 24 h. biodegradation of PET bottles at a mean rate of 23.4 mgPET/h/mg enzyme.	[53]
L92F/Q94Y variant of PES-H1	Structural analyses and computational modeling using MD simulation		Amorphous Goodfellow PET (Gf-PET) films and pretreated postconsumer (‘real-world’) PET waste	2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently	[59]
Ple629 polyester hydrolase variant		D226A/S279A mutations improved activity and thermo-stability	PET nanoparticles	5.5-fold improved activity	[60]
Cut190*		S226P/R228S increased activity and higher thermostability. The mutant of the cutinases-like enzyme, Cut190, from Saccharomonospora viridis AHK190	Model substrate poly (butylene succinate-co-adipate).		[61]
IsPETase double mutant	Homology modeling and g-induced fit docking (IFD)	Two mutations are introduced at S238F/W159H to make the PETase-active site more like cutinase enzyme’s	polyethylene-2,5-furan dicarboxylate (PEF), PET coupons with an initial crystallinity of 14.8 ± 0.2%		[51]