Fig. 2: Enzyme identification through sequence similarity networks (SSN) and enzyme determination by in vitro biodegradation performance and in silico computational examination.

a Circular genomic map of Chryseobacterium sp. PET-29. Functional proteins were determined by the BLAST software against the COG database, and enrichment analysis of molecular function, cellular components, and biological process was identified using the GO database. b The sequence similarity networks (SSN) analysis of PET hydrolase in Bacillus subtilis PET-86 and Chryseobacterium sp. PET-29. Each protein is represented by a node. The proteins within the same functional family are connected by the edge to form a cluster. The nodes representing BsEst and ChryBHETase are labeled with an arrow. c Sequence identity results of BsEst and ChryBHETase with other reported enzymes. Biodegradation performance of d BsEst and e ChryBHETase upon incubation on BHET. Reaction condition: 5 mM BHET at 30 °C in pH 7.5 buffer for 24 h. Error bars correspond to the standard deviation (s.d.) of three measurements (n = 3). f 3D structures of BsEst (left) and ChryBHETase (right) predicted by AlphaFold2. The sphere model represents the catalytic triad. S189-E310-H399 of BsEst; S194-E310-H379 of ChryBHETase. The black dashed line represents the substrate binding cleft (SBC). g The surface electrostatic potential distribution of BsEst (left) and ChryBHETase (right). The positive charge is shown in blue, and the negative charge is shown in red. h RMSF of BsEst and ChryBHETase residues determined from the last 40 ns of MD simulation in water and BHET systems. Data plotted from the average of three independent MD runs (n = 3). i The number of BHET/water molecules at the substrate binding site. The dotted line represents the quartiles of the data, and the middle-dotted line represents the median. Data plotted from the last 40 ns of three independent MD runs (n = 243).