High Throughput Screening for New Fungal Polyester Hydrolyzing Enzymes.

Simone Weinberger,Doris Ribitsch,Joseph Strauss,Reinhard Beyer,Georg M Guebitz,Alessandro Pellis,Christoph Schüller

doi:10.3389/fmicb.2020.00554

Abstract

There is a strong need for novel and more efficient polyester hydrolyzing enzymes in order to enable the development of more environmentally friendly plastics recycling processes allowing the closure of the carbon cycle. In this work, a high throughput system on microplate scale was used to screen a high number of fungi for their ability to produce polyester-hydrolyzing enzymes. For induction of responsible enzymes, the fungi were cultivated in presence of aliphatic and aromatic polyesters [poly(1,4-butylene adipate co terephthalate) (PBAT), poly(lactic acid) (PLA) and poly(1,4-butylene succinate) (PBS)], and the esterase activity in the culture supernatants was compared to the culture supernatants of fungi grown without polymers. The results indicate that the esterase activity of the culture supernatants was induced in about 10% of the tested fungi when grown with polyesters in the medium, as indicated by increased activity (to >50 mU/mL) toward the small model substrate para-nitrophenylbutyrate (pNPB). Incubation of these 50 active culture supernatants with different polyesters (PBAT, PLA, PBS) led to hydrolysis of at least one of the polymers according to liquid chromatography-based quantification of the hydrolysis products terephthalic acid, lactic acid and succinic acid, respectively. Interestingly, the specificities for the investigated polyesters varied among the supernatants of the different fungi.

Highlights

Polymers and especially polyesters are components of materials with industrially interesting properties such as chemical resistance, low production costs and simple processability
Inoculation of fungi was performed on a Hamilton liquid handling robot (Hamilton Microlab STAR) while screening was conducted in a 2-stage process
Fungi were challenged with a polymer mix containing poly(butylene succinate) (PBS), poly(lactic acid) (PLA), and poly(butylene adipate-coterephthalate) (PBAT) and supernatants were tested for esterase activity

Summary

Introduction

Polymers and especially polyesters are components of materials with industrially interesting properties such as chemical resistance, low production costs and simple processability. High Throughput Screening for Polyesterases environment poses a major threat to natural environment since they are barely biodegradable and accumulate in the ecosystems. Increased awareness of this problem led to intensive research for environmentally friendly alternatives in the last decades. To obtain PLA, mainly starches and sugars are fermented to lactic acid, which is further processed to the polymer (Weng et al, 2013). The building blocks can be obtained via fermentative pathways from glucose or sucrose feedstock

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Results

Conclusion