Discovery of Polyesterases from Moss-Associated Microorganisms

Abstract

The growing pollution of the environment with plastic debris is a global threat which urgently requires biotechnological solutions. Enzymatic recycling not only prevents pollution but also would allow recovery of valuable building blocks. Therefore, we explored the existence of microbial polyesterases in microbial communities associated with the Sphagnum magellanicum moss, a key species within unexploited bog ecosystems. This resulted in the identification of six novel esterases, which were isolated, cloned, and heterologously expressed in Escherichia coli. The esterases were found to hydrolyze the copolyester poly(butylene adipate-co-butylene terephthalate) (PBAT) and the oligomeric model substrate bis[4-(benzoyloxy)butyl] terephthalate (BaBTaBBa). Two promising polyesterase candidates, EstB3 and EstC7, which clustered in family VIII of bacterial lipolytic enzymes, were purified and characterized using the soluble esterase substrate p-nitrophenyl butyrate (K m values of 46.5 and 3.4 M, temperature optima of 48°C and 50°C, and pH optima of 7.0 and 8.5, respectively). In particular, EstC7 showed outstanding activity and a strong preference for hydrolysis of the aromatic ester bond in PBAT. Our study highlights the potential of plant-associated microbiomes from extreme natural ecosystems as a source for novel hydrolytic enzymes hydrolyzing polymeric compounds.IMPORTANCE In this study, we describe the discovery and analysis of new enzymes from microbial communities associated with plants (moss). The recovered enzymes show the ability to hydrolyze not only common esterase substrates but also the synthetic polyester poly(butylene adipate-co-butylene terephthalate), which is a common material employed in biodegradable plastics. The widespread use of such synthetic polyesters in industry and society requires the development of new sustainable technological solutions for their recycling. The discovered enzymes have the potential to be used as catalysts for selective recovery of valuable building blocks from this material.

Shotgun proteomics reveals putative polyesterases in the secretome of the rock-inhabiting fungus Knufia chersonesos

“…The monomer Ta represented the most abundant compound detected at all experimental conditions and was proven not to be metabolised by the organism. This suggests the involvement of the fungus extracellular enzymes in the cleavage of aromatic ester bonds present in PBAT chains 17,45 , which are reportedly less prone to hydrolysis than the ester bond with aliphatic monomer (adipic acid) as also described for P. pseudoalcaligenes polyesterase PpEst 15 . Previous studies on single enzyme hydrolysis process of milled PBAT reported that BTaB and BTa were more abundant than the monomer, as well as that an increase of Ta concentration is given by higher pH than that selected in the current study 7 .…”

“…bation with PBAT, the supernatants were diluted with ice-cold methanol (1:1 v/v) in order to precipitate the enzymes and acidified to pH 3.0 with 1 M HCl. The samples were centrifuged at 18.213 x g (5427 R, Eppendorf) for 15 min at 4 °C, then filtered through 0.45 μm nylon syringe filters and thereafter analysed by a HPLC-DAD system consisting of a 1290 Infinity II LC (Agilent Technologies), coupled with a reversed phase column C18 (Poroshell 120 EC-C18 2,7 µm 3.0 × 150 mm), at a flow rate of 0.4 mL/min 15,19 . The PBAT hydrolysis products were separated using a nonlinear gradient according to Quartinello et al 2 with some modifications (i.e.…”

“…Microbial activities against PBAT have been shown in bacteria and to a lesser extent in fungi from soil [13][14][15][16][17] , by esterolytic hydrolases 7,15,18,19 . Typically, the investigation of PBAT degrading enzymes involves functional screenings and protein isolation through chromatographic purification 14,16,20,21 .…”

Unravelling native plant resistomes – The Sphagnum microbiome harbours versatile and novel antimicrobial resistance genes

“…The 3.6 Gbps metagenomic fosmid library was established in E. coli EPI300 pCC2FOS (Epicentre, Wisconsin, USA), incorporating ~40 kb metagenomic DNA from the Alpine peat bog moss S. magellanicum as previously described 55 . The generated 90 000 metagenomic clones were pooled by resuspending them in LB medium supplied with 20% glycerol for long term storage at −70°C.…”

Agar plate‐based screening methods for the identification of polyester hydrolysis by Pseudomonas species

“…However, lipolytic enzymes clustering within other families (Arpigny and Jaeger, ), e.g. family VIII (β‐lactamase like), were likewise associated with polyesterase activity very recently (Biundo et al ., ; Müller et al ., ; Hajighasemi et al ., ). Besides biodegradation of artificial polyesters like PET, cutinases/polyesterases are discussed for different biotechnological applications, e.g.…”