Polyethylene terephthalate (PET) is the most important mass‐produced thermoplastic polyester used as a packaging material. Recently, thermophilic polyester hydrolases such as TfCut2 from Thermobifida fusca have emerged as promising biocatalysts for an eco‐friendly PET recycling process. In this study, postconsumer PET food packaging containers are treated with TfCut2 and show weight losses of more than 50% after 96 h of incubation at 70 °C. Differential scanning calorimetry analysis indicates that the high linear degradation rates observed in the first 72 h of incubation is due to the high hydrolysis susceptibility of the mobile amorphous fraction (MAF) of PET. The physical aging process of PET occurring at 70 °C is shown to gradually convert MAF to polymer microstructures with limited accessibility to enzymatic hydrolysis. Analysis of the chain‐length distribution of degraded PET by nuclear magnetic resonance spectroscopy reveals that MAF is rapidly hydrolyzed via a combinatorial exo‐ and endo‐type degradation mechanism whereas the remaining PET microstructures are slowly degraded only by endo‐type chain scission causing no detectable weight loss. Hence, efficient thermostable biocatalysts are required to overcome the competitive physical aging process for the complete degradation of postconsumer PET materials close to the glass transition temperature of PET.

BackgroundBacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M.ResultsHere we show that activity of the htrB promoter as induced by overproduction of AmyM was “noisy”, which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions.ConclusionExpression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement.