(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Terrabacteria group: NE > Firmicutes: NE > Bacilli: NE > Bacillales: NE > Paenibacillaceae: NE > Thermobacillus: NE > Thermobacillus composti: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MVSLRIAKTENGYVQGLPAADPRITSFKGIPFAAPPVGENRWRAPQPAKN WDGVLKAYEFSPIPLQVRQEIDVNNIYTREWAVDPDIAMSEDCLYLNIWT PAKSPDEKLPVFVWYFGGGLQVGHASEMEFDGERIARRGIVVVTINYRLN VFGFLCHPEITAESPDAPANFGHLDQQFATQWVKRNIAAFGGDPDNITIG GQSAGGGSVLAQLTSPQNEGLFNKAIIMSGIFSPVYPGQRGPGVRETLAE AEQEGIKFFEYLGVSSLAEARKLDAVYIRDKMVEYKRFWGAVVDNKFCVG HPFERFLRNERWNVPILWGNTTDEFISRPQVGSIEELRQMAEGVFGEDAD RFLALCGADSGDLEQALKAASVNTIEYAIRVASRANAAAGAGTPLYYYKF DAEIPGWDNPGTFHSVDLWFFFETLAKCWRPFVGKHYDLARQMCNYWAHF IRSGDPNGPDSTGEDLPRWEPYTEEAPYAMVFGDKSEFVREQPGEMIRFL VEQYFKRK
The large amount of waste synthetic polyester plastics has complicated waste management and also endangering the environment due to improper littering. In this study, a novel carboxylesterase from Thermobacillus composti KWC4 (Tcca) was identified, heterologously expressed in Escherichia coli, purified and characterized with various plastic substrates. Irregular grooves were detected on polybutylene adipate terephthalate (PBAT) film by scanning electron microscopy (SEM) after Tcca treatment, and Tcca can also hydrolyze short-chain diester bis(hydroxyethyl) terephthalate (BHET). The optimal pH and temperature for Tcca were 7.0 and 40 C, respectively. In order to explore its catalytic mechanism and improve its potential for plastic hydrolysis, we modeled the protein structure of Tcca and compared it with its homologous structures, and we identified positions that might be crucial for the binding of substrates. We generated a variety of Tcca variants by mutating these key positions; the variant F325A exhibited a more than 1.4-fold improvement in PBAT hydrolytic activity, and E80A exhibited a more than 4.1-fold increase in BHET activity when compared to the wild type. Tcca and its variants demonstrated future applicability for the recycling of bioplastic waste containing a PBAT fraction.
