ABSTRACT
Polyethylene terephthalate (PET) is widely used for various industrial applications. However, owing to its extremely slow breakdown rate, PET accumulates as plastic trash, which negatively affects the environment and human health. Here, we report two novel PET hydrolases: PpPETase from Pseudomonas paralcaligenes MRCP1333, identified in human feces, and ScPETase from Streptomyces calvus DSM 41452. These two enzymes can decompose various PET materials, including semicrystalline PET powders (Cry-PET) and low-crystallinity PET films (gf-PET). By structure-guided engineering, two variants, PpPETaseY239R/F244G/Y250G and ScPETaseA212C/T249C/N195H/N243K were obtained that decompose Cry-PET 3.1- and 1.9-fold faster than their wild-type enzymes, respectively. The co-expression of ScPETase and mono-(2-hydroxyethyl) terephthalate hydrolase from Ideonella sakaiensis (IsMHETase) resulted in 1.4-fold more degradation than the single enzyme system. This engineered strain degraded Cry-PET and gf-PET by more than 40% and 6%, respectively, after 30 d. The concentrations of terephthalic acid (TPA) in the Cry-PET and gf-PET degradation products were 37.7% and 25.6%, respectively. The discovery of these two novel PET hydrolases provides opportunities to create more powerful biocatalysts for PET biodegradation.
Subject(s)
Feces , Hydrolases , Polyethylene Terephthalates , Streptomyces , Polyethylene Terephthalates/metabolism , Polyethylene Terephthalates/chemistry , Streptomyces/enzymology , Streptomyces/genetics , Hydrolases/metabolism , Hydrolases/genetics , Hydrolases/chemistry , Humans , Feces/microbiology , Pseudomonas/enzymology , Pseudomonas/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , BurkholderialesABSTRACT
Poly(ethylene terephthalate) (PET) and phthalate esters (PAEs) are used extensively as plastics and plasticizers. Enzymatic degradation of PET and PAEs has drawn great attention in recent years; however, evolution of PET- and PAE-degrading enzymes is still a big challenge, partly because of the lack of an effective screening method to detect phthalic acid (PA) and terephthalic acid (TPA), which are the main hydrolysis products of PAEs and PET. Here, by directed evolution of a promiscuous transcription factor, XylS from Pseudomonas putida, we created two novel variants, XylS-K38R-L224Q and XylS-W88C-L224Q, that are able to bind PA and TPA and activate the downstream expression of a fluorescent reporter protein. Based on these elements, whole-cell biosensors were constructed, which enabled the fluorimetric detection of as little as 10 µM PA or TPA. A PAE hydrolase, GoEst15, was preliminarily engineered using this new biosensor, yielding a mutant GoEst15-V3 whose activity toward dibutyl phthalate (DBP) and p-nitrophenyl butyrate was enhanced 2.0- and 2.5-fold, respectively. It was shown that 96.5% DBP (5 mM) was degraded by GoEst15-V3 in 60 min, while the wild-type enzyme degraded only 55% DBP. This study provides an effective screening tool for directed evolution of PAE-/PET-degrading enzymes.
Subject(s)
Phthalic Acids , Transcription Factors , Dibutyl Phthalate , Esters/metabolism , Phthalic Acids/metabolism , Transcription Factors/geneticsABSTRACT
Engineering of a promiscuous lactonase via semi-rational evolution gave a 1007-fold improvement in its catalytic activity in the degradation of triphenyl phosphate (TPHP). TPHP is a typical bulky organophosphate flame retardant (OPFR) and is widely used in industry. To the best of our knowledge, this is the first artificial enzyme capable of degrading OPFRs.
