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.

Scheme1.Schematicdiagram of laccase catalysis. Generalrepresentation of laccase-catalyzedredox cycles for substrate oxidation, with 2,6-dimethoxyphenol (2,6-DMP) as am odel substrate (SUB).

The fls gene encoding fervidolysin, a keratin-degrading proteolytic enzyme from the thermophilic bacterium Fervidobacterium pennivorans, was isolated using degenerate primers combined with Southern hybridization and inverse polymerase chain reaction. Further sequence characterization demonstrated that the 2.1-kb fls gene encoded a 699-amino-acid preproenzyme showing high homology with the subtilisin family of the serine proteases. It was cloned into a pET9d vector, without its signal sequence, and expressed in Escherichia coli. The heterologously produced fervidolysin was purified by heat incubation followed by ion exchange chromatography and emerged in the soluble fraction as three distinct protein bands, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal-sequence analysis of these bands and their comparison with that determined from biochemically purified keratinase and its predicted protein sequence, identified them as a 73-kDa fervidolysin precursor, a 58-kDa mature fervidolysin, and a 14-kDa fervidolysin propeptide. Using site-directed mutagenesis, the active-site histidine residue at position 79 was replaced by an alanine residue. The resulting fervidolysin showed a single protein band corresponding in size to the 73-kDa fervidolysin precursor, indicating that its proteolytic cleavage resulted from an autoproteolytic process. Knowledge-based modeling experiments showed a distinctive binding region for subtilases, in which binding of the propeptide could take place prior to autoproteolysis. Assays using keratin and other proteinaceous substrates did not display fervidolysin activity, perhaps because of the tight binding of the propeptide in the substrate-binding site, where it could then function as an inhibitor.

The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86^0.29 m for intrinsic fluorescence and 4.90^0.31 m for far-UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 m guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 m in the first dilution step. Particular attention has been paid to the role of Ca 2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca 2+ ions with a K d of 0.080^0.010 mm and a Hill coefficient H of 1.00^0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride-induced unfolding and the DG H 2 O D increased from 6.83^0.43 to 8.42^0.55 kcal´mol 21 . The refolding of the pullulanase, on the other hand, was not affected by Ca 2+ .Keywords: pullulanase; archaea; thermostable proteins; stability; calcium binding.A number of thermophilic, extreme thermophilic and hyperthermophilic microorganisms belonging to bacteria and archaea have been found to be capable of degrading starch and other biopolymers such as hemicellulose, cellulose and proteins [1,2]. In addition to the industrial relevance of heatstable amylolytic enzymes, the existence of these enzymes in all kingdoms of life provides the possibility of comparative studies on (thermo)stability and folding of proteins from mesophilic and thermophilic microorganisms.Pyrococcus woesei, a hyperthermophilic archaeon of the order Thermococcales grows optimally at 100±103 8C on a complex medium under strictly anaerobic conditions [3]. This archaeon is equipped with a polysaccharide-degrading enzyme system which is composed of an a-amylase [4], a pullulanase [5] and an a-glucosidase [6].The pullulanase from P. woesei belongs to pullulanase of type II (also named amylopullulanase) and it hydrolyzes the a-1,6 glycosidic linkages in pullulan and branched oligosaccharides as well as a-1,4 glycosidic linkages in linear and branched polysaccharides such as amylose and amylopectin. Many pullulanases of type II have been purified and characterized from anaerobic thermophilic bacteria and archaea [7±14]. On the other hand, type I pullulanases which attack specifically a-1,6 glycosidic linkages have been generally found in mesophilic, aerobic microorganisms [1,2,15]. Only recently a heat-stable pullulanase of type I has been detected in the newly isolated thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 [16].The pullulanase from P. woesei is a monomeric enzyme with a molecular mass of 90 kDa. Its gene has been cloned and expressed in Escherichia coli. The enzyme is opti...

The stability of the hexameric glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus at low pH values has been studied by activity assay, spectroscopic methods, size-exclusion chromatography and ultracentrifugation analysis. The enzyme is exceptionally stable and at pH 2.0 its hexameric assembly is preserved despite the changes observed in its tertiary structure. Below pH 1.7 dissociation into monomers starts and is accompanied by a progressive loss of tertiary interactions. Dissociation interniediate(s) were not detectable. At pH 2.0 the addition of NaCl causes the same structural changes observed upon further addition of protons. The monomeric state of the enzyme at pH 1.0 shows a significant content of native secondary structure and can be unfolded by guanidinium chloride. The role of electrostatic interactions in the high stability of the enzyme structure at low pH values is discussed.Keywords: acid denaturation; glutamate dehydrogenase ; hyperthermophile; oligomeric protein; unfolding.The stability of proteins at different pH values depends on the electrostatic forces originating from the change in the net charge and charge distribution upon addition of protons [I]. In particular the acid unfolding is generated by the repulsive forces resulting from the protonation of the amino acid residues. However, several proteins do not unfold completely at low pH, but adopt different conformational state(s) with respect to the native enzyme 121. These alternatively folded conformational state(s) are soluble and can be characterized spectroscopically without the interference due to the presence of denaturants such as guanidinium chloride (GdmC1) or urea. The mechanism of pH-induced denaturation has been studied in detail for several monomeric proteins and the different intermediate and unfolded states formed at low pH have been characterized at variable ionic strength [2, 31. For oligomeric proteins the stability at low pH has been studied in detail in the case of dimers, trimers and pentamers [4-91 but, to our knowledge, the effect of proton addition on more complex polymeric enzymes has not been reported. The importance of this type of study lies in the possibility of finding intermediate(s) during the enzyme disassembly and reconstitution. In turn, characterization of these intermediates may help in elucidating the minimum state of folding required for a correct subunit association and the changes induced by polymer assembly on the subunit tertiary structure.The study of the changes of structure and function induced by pH is particularly interesting for proteins characterized by a high resistance towards chemical and physical denaturants, as hydrogenase; V,, elution volume.those extracted from hyperthermophilic microorganisms whose adaptation to extreme environmental conditions has gained considerable interest in biotechnology [lo, 1 I]. The enzymatic machinery of these microorganisms is adapted to play its function at temperatures around 100 "C 1121. The hyperthermophilic Pyrococcus furios...