Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides.

In Saccharomyces kluyveri, dihydropyrimidinase (DHPaseSK) is involved in the pyrimidine degradation pathway, which includes the reversible ring cleavage between nitrogen 3 and carbon 4 of 5,6-dihydrouracil. In this study, DPHaseSK was successfully cloned and expressed in E. coli BL-21 Gold (DE3) with and without affinity tags. Thereby, the Strep-tag enabled fastest purification and highest specific activity (9.5 ± 0.5 U/mg). The biochemically characterized DHPaseSK_Strep had similar kinetic parameters (Kcat/Km) on 5,6-dihydrouracil (DHU) and para-nitroacetanilide respectively, with 7,229 and 4060 M-1 s-1. The hydrolytic ability of DHPaseSK_Strep to polyamides (PA) was tested on PA consisting of monomers with different chain length (PA-6, PA-6,6, PA-4,6, PA-4,10 and PA-12). According to LC-MS/TOF analysis, DHPaseSK_Strep showed a preference for films containing the shorter chain monomers (e.g., PA-4,6). In contrast, an amidase from Nocardia farcinica (NFpolyA) showed some preference for PA consisting of longer chain monomers. In conclusion, in this work DHPaseSK_Strep was demonstrated to be able to cleave amide bonds in synthetic polymers, which can be an important basis for development of functionalization and recycling processes for polyamide containing materials.

Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters.

Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in Thermoanaerobacter thermohydrosulfuricus lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met-142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in significantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These findings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering.

A Fungal Ascorbate Oxidase with Unexpected Laccase Activity.

Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. Af_AO1 had a 10-fold higher affinity to ascorbic acid (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.

Enzymes as Enhancers for the Biodegradation of Synthetic Polymers in Wastewater.

Synthetic polyesters are today the second-largest class of ingredients in household products and are entering wastewater treatment plants (WWTPs) after product utilization. One approach to improve polymer biodegradation in wastewater would be to complement current processes with polyester-hydrolyzing enzymes and their microbial producers. In this study, the hydrolysis of poly(oxyethylene terephthalate) polymer by hydrolases from wastewater microorganisms was investigated in vitro and under realistic WWTP conditions. An esterase and a cutinase from Pseudomonas pseudoalcaligenes and a lipase from Pseudomonas pelagia were heterologously expressed in Escherichia coli BL21-Gold(DE3) and were purified by a C-terminal His6 tag. The hydrolases were proven to hydrolyze the polymer effectively, which is a prerequisite for further biodegradation. The hydrolases maintained high activity up to 50 % upon lowering the temperature from 28 to 15 °C to mimic WWTP conditions. The hydrolases were also not inhibited by the wastewater matrix. Polyester-hydrolyzing enzymes active under WWTP conditions and their microbial producers thus have the potential to improve biological treatment of wastewater rich in synthetic polymers.

Polyol Structure Influences Enzymatic Hydrolysis of Bio-Based 2,5-Furandicarboxylic Acid (FDCA) Polyesters.

Polyesters of 2,5-furandicarboxylic acid (FDCA) have gained attention as they can be regarded as the bio-based alternatives to the petroleum-based polyesters of terephthalic acid. However, only little is known about the biodegradation and enzymatic hydrolysis of FDCA-based polyesters. This work aims to investigate the influence of different polyols on enzymatic hydrolysis of FDCA-based polyesters. A series of polyesters containing various polyols are synthesized and analyzed regarding susceptibility to enzymatic hydrolysis by cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1). FDCA-based polyesters' number average molecular weight (Mn ) ranged from 9360-35 800 g mol-1 according to gel permeation chromatography (GPC) analysis. Differential scanning calorimetry (DSC) analyses show decreasing glass transition temperature (Tg ) with increasing diol chain length. Crystallinity of all polyesters is below 1% except for polyesters containing 1,6-hexanediol, 1,8-octanediol, and 1,12-dodecanediol for which calculated crystallinities are 27, 37, and 30%, respectively. Thc_Cut1 hydrolyzes all tested polyesters with preference for polyesters containing 1,5-pentanediol and 1,9-nonanediol (57.7 ± 7.5 and 52.8 ± 4.0% released FDCA). Enzyme activity increases when the linear diol 1,3-propanediol is replaced by the branched analog 1,2-propanediol or ethoxy units are introduced into the polyester chain. The results will contribute to expand the knowledge of microbial biodegradation of FDCA-based polyesters.

Small cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilytica.

We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;114: 2481-2488. © 2017 Wiley Periodicals, Inc.

Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica.

To study hydrolysis of aromatic and aliphatic polyesters cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1) was expressed in P. pastoris. No significant differences between the expression of native Thc_Cut1 and of two glycosylation site knock out mutants (Thc_Cut1_koAsn and Thc_Cut1_koST) concerning the total extracellular protein concentration and volumetric activity were observed. Hydrolysis of poly(ethylene terephthalate) (PET) was shown for all three enzymes based on quantification of released products by HPLC and similar concentrations of released terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected for all enzymes. Both tested aliphatic polyesters poly(butylene succinate) (PBS) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were hydrolyzed by Thc_Cut1 and Thc_Cut1_koST, although PBS was hydrolyzed to significantly higher extent than PHBV. These findings were also confirmed via quartz crystal microbalance (QCM) analysis; for PHBV only a small mass change was observed while the mass of PBS thin films decreased by 93% upon enzymatic hydrolysis with Thc_Cut1. Although both enzymes led to similar concentrations of released products upon hydrolysis of PET and PHBV, Thc_Cut1_koST was found to be significantly more active on PBS than the native Thc_Cut1. Hydrolysis of PBS films by Thc_Cut1 and Thc_Cut1_koST was followed by weight loss and scanning electron microscopy (SEM). Within 96 h of hydrolysis up to 92 and 41% of weight loss were detected with Thc_Cut1_koST and Thc_Cut1, respectively. Furthermore, SEM characterization of PBS films clearly showed that enzyme tretment resulted in morphological changes of the film surface.

Hydrolysis of Ionic Phthalic Acid Based Polyesters by Wastewater Microorganisms and Their Enzymes.

Water-soluble polyesters are used in a range of applications today and enter wastewater treatment plants after product utilization. However, little is known about extracellular enzymes and aquatic microorganisms involved in polyester biodegradation and mineralization. In this study, structurally different ionic phthalic acid based polyesters (the number-average molecular weights (Mn) 1770 to 10 000 g/mol and semi crystalline with crystallinity below 1%) were synthesized in various combinations. Typical wastewater microorganisms like Pseudomonas sp. were chosen for in-silico screening toward polyester hydrolyzing enzymes. Based on the in-silico search, a cutinase from Pseudomonas pseudoalcaligenes (PpCutA) and a putative lipase from Pseudomonas pelagia (PpelaLip) were identified. The enzymes PpCutA and PpelaLip were demonstrated to hydrolyze all structurally different polyesters. Activities on all the polyesters were also confirmed with the strains P. pseudoalcaligenes and P. pelagia. Parameters identified to enhance hydrolysis included increased water solubility and polyester hydrophilicity as well as shorter diol chain lengths. For example, polyesters containing 1,2-ethanediol were hydrolyzed faster than polyesters containing 1,8-octanediol. Interestingly, the same trend was observed in biodegradation experiments. This information is important to gain a better mechanistic understanding of biodegradation processes of polyesters in WWTPs where the extracellular enzymatic hydrolysis seems to be the limiting step.

A new arylesterase from Pseudomonas pseudoalcaligenes can hydrolyze ionic phthalic polyesters.

Extracellular enzymes are assumed to be responsible for the initial and rate limiting step in biodegradation of polymers. Mainly enzymes with aliphatic esters as their natural substrates (e.g. lipase, cutinases) have until now been evaluated for polyester hydrolysis studies. However, the potential of enzymes with aromatic esters as their natural substrates (e.g. arylesterases) have been neglected although many types of polyester today contain aromatic moieties. Consequently, in order to elucidate biodegradation of phthalic polyesters in aquatic systems, a novel arylesterase (PpEst) was investigated related to hydrolysis of ionic phthalic polyesters. The hydrolysis of various ionic phthalic polyesters by PpEst was mechanistically studied. The polyester building blocks (terephthalic acid (TA), 5-sulfoisophthalic acid (NaSIP) and alkyl or ether diols) were systematically varied to investigate the impact on hydrolysis. PpEst effectively hydrolyzed all 14 synthetized ionic phthalic polyesters as indicated by released TA. However, no NaSIP was detected indicating that PpEst has a limited capacity to cleave bonds in close vicinity to the ionic monomer NaSIP. The systematic study indicated that increasing water solubility and hydrophilicity significantly enhanced hydrolysis. A higher release of TA was seen with increasing NaSIP ratio while up to 20 times more TA was released when alkyl diols were replaced by ether diol analogues. In contrast, cyclic and branched diols had a negative effect on hydrolysis when compared to linear diols. PpEst also revealed a linear release of TA over seven days for ether containing polyesters, indicating a very stable enzyme.

Polyol Structure and Ionic Moieties Influence the Hydrolytic Stability and Enzymatic Hydrolysis of Bio-Based 2,5-Furandicarboxylic Acid (FDCA) Copolyesters.

A series of copolyesters based on furanic acid and sulfonated isophthalic acid with various polyols were synthetized and their susceptibility to enzymatic hydrolysis by cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1) investigated. All copolyesters consisted of 30 mol % 5-sulfoisophthalate units (NaSIP) and 70 mol % 2,5-furandicarboxylic acid (FDCA), while the polyol component was varied, including 1,2-ethanediol, 1,4-butanediol, 1,8-octanediol, diethylene glycol, triethylene glycol, or tetraethylene glycol. The composition of the copolyesters was confirmed by ¹H-NMR and the number average molecular weight (Mn) was determined by GPC to range from 2630 to 8030 g/mol. A DSC analysis revealed glass-transition temperatures (Tg) from 84 to 6 °C, which were decreasing with increasing diol chain length. The crystallinity was below 1% for all polyesters. The hydrolytic stability increased with the chain length of the alkyl diol unit, while it was generally higher for the ether diol units. Thc_Cut1 was able to hydrolyze all of the copolyesters containing alkyl diols ranging from two to eight carbon chain lengths, while the highest activities were detected for the shorter chain lengths with an amount of 13.6 ± 0.7 mM FDCA released after 72 h of incubation at 50 °C. Faster hydrolysis was observed when replacing an alkyl diol by ether diols, as indicated, e.g., by a fivefold higher release of FDCA for triethylene glycol when compared to 1,8-octanediol. A positive influence of introducing ionic phthalic acid was observed while the enzyme preferentially cleaved ester bonds associated to the non-charged building blocks.

PpEst is a novel PBAT degrading polyesterase identified by proteomic screening of Pseudomonas pseudoalcaligenes.

A novel esterase, PpEst, that hydrolyses the co-aromatic-aliphatic polyester poly(1,4-butylene adipate-co-terephthalate) (PBAT) was identified by proteomic screening of the Pseudomonas pseudoalcaligenes secretome. PpEst was induced by the presence of PBAT in the growth media and had predicted arylesterase (EC 3.1.1.2) activity. PpEst showed polyesterase activity on both whole and milled PBAT film releasing terephthalic acid and 4-(4-hydroxybutoxycarbonyl)benzoic acid while end product inhibition by 4-(4-hydroxybutoxycarbonyl)benzoic acid was observed. Modelling of an aromatic polyester mimicking oligomer into the PpEst active site indicated that the binding pocket could be big enough to accommodate large polymers. This is the first report of a PBAT degrading enzyme being identified by proteomic screening and shows that this approach can contribute to the discovery of new polymer hydrolysing enzymes. Moreover, these results indicate that arylesterases could be an interesting enzyme class for identifications of polyesterases.

Combining expression and process engineering for high-quality production of human sialyltransferase in Pichia pastoris.

The human ß-galactoside α2,6-sialyltransferase I, ST6Gal-I has drawn considerable interest for its use as biocatalyst for in-vitro glycoengineering of recombinantly produced therapeutic proteins. By attaching sialic acid onto the terminal galactoses of biantennary protein N-glycans, ST6Gal-I facilitates protein remodeling towards a humanized glycosylation and thus optimized efficacy in pharmacological use. Secreted expression of ST6Gal-I in Pichia pastoris is promising, but proteolysis restricts both the yield and the quality of the enzyme produced. Focusing on an N-terminally truncated (Δ108) variant of ST6Gal-I previously shown to represent a minimally sized, still active form of ST6Gal-I, we show here that protein expression engineering and optimization of bioreactor cultivation of P. pastoris KM71H (pPICZαB) synergized to enhance the maximum enzyme titer about 57-fold to 17units/L. N-Terminal fusion to the Flag-tag plus deletion of a potential proteolytic site (Lys(114)-Asn→Gln(114)-Asn) improved the intrinsic resistance of Δ108ST6Gal-I to degradation in P. pastoris culture. A mixed glycerol/methanol feeding protocol for P. pastoris growth and induction was key for enzyme production in high yield and quality. The sialyltransferase was recovered from the bioreactor culture in a yield of 70% using a single step of anion-exchange chromatography. Its specific activity was 0.05units/mg protein.

An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters.

Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. On the basis of reverse-phase high-performance liquid chromatography (RP-HPLC) analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter, a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the α/ß-hydrolases family. This study gives a clear hint that also micro-organisms in anaerobic habitats can degrade manmade PBAT.

Hydrolysis of synthetic polyesters by Clostridium botulinum esterases.

Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site.

Biomimetic Approach to Enhance Enzymatic Hydrolysis of the Synthetic Polyester Poly(1,4-butylene adipate): Fusing Binding Modules to Esterases.

Mimicking a concept of nature for the hydrolysis of biopolymers, the Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) was fused to a polymer binding module (PBM) to enhance the hydrolysis of the polyester poly(1,4-butylene adipate) (PBA). Namely, the binding module of a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (Thc_Cut1_PBM) was attached to the cutinase via two different linker sequences varying in length. In order to investigate the adsorption behavior, catalytically inactive mutants both of Thc_Cut1 and Thc_Cut1_PBM were successfully constructed by site-directed mutagenesis of serine 131 to alanine. Quartz crystal microbalance with dissipation monitoring (QCM-D) analysis revealed that the initial mass increase during enzyme adsorption was larger for the inactive enzymes linked with the PBM as compared to the enzyme without the PBM. The hydrolysis rates of PBA were significantly enhanced when incubated with the active, engineered Thc_Cut1_PBM as compared to the native Thc_Cut1. Thc_Cut1_PBM completely hydrolyzed PBA thin films on QCM-D sensors within approximately 40 min, whereas twice as much time was required for the complete hydrolysis by the native Thc_Cut1.

Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins.

Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased kcat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.

High-quality production of human α-2,6-sialyltransferase in Pichia pastoris requires control over N-terminal truncations by host-inherent protease activities.

BACKGROUND: α-2,6-sialyltransferase catalyzes the terminal step of complex N-glycan biosynthesis on human glycoproteins, attaching sialic acid to outermost galactosyl residues on otherwise fully assembled branched glycans. This "capping" of N-glycans is critical for therapeutic efficacy of pharmaceutical glycoproteins, making the degree of sialylation an important parameter of glycoprotein quality control. Expression of recombinant glycoproteins in mammalian cells usually delivers heterogeneous N-glycans, with a minor degree of sialylation. In-vitro chemo-enzymatic glycoengineering of the N-glycans provides an elegant solution to increase the degree of sialylation for analytical purposes but also possibly for modification of therapeutic proteins. RESULTS: Human α-2,6-sialyltransferase (ST6Gal-I) was secretory expressed in P.pastoris KM71H. ST6Gal-I featuring complete deletion of both the N-terminal cytoplasmic tail and the transmembrane domain, and also partial truncation of the stem region up to residue 108 were expressed N-terminally fused to a His or FLAG-Tag. FLAG-tagged proteins proved much more resistant to proteolysis during production than the corresponding His-tagged proteins. Because volumetric transferase activity measured on small-molecule and native glycoprotein acceptor substrates did not correlate to ST6Gal-I in the supernatant, enzymes were purified and characterized in their action on non-sialylated protein-linked and released N-glycans, and the respective N-terminal sequences were determined by automated Edman degradation. Irrespective of deletion construct used (Δ27, Δ48, Δ62, Δ89), isolated proteins showed N-terminal processing to a highly similar degree, with prominent truncations at residue 108 - 114, whereby only Δ108ST6Gal-I retained activity. FLAG-tagged Δ108ST6Gal-I was therefore produced and obtained with a yield of 4.5 mg protein/L medium. The protein was isolated and shown by MS to be intact. Purified enzyme exhibited useful activity (0.18 U/mg) for sialylation of different substrates. CONCLUSIONS: Functional expression of human ST6Gal-I as secretory protein in P.pastoris necessitates that N-terminal truncations promoted by host-inherent proteases be tightly controlled. N-terminal FLAG-Tag contributes extra stability to the N-terminal region as compared to N-terminal His-Tag. Proteolytic degradation proceeds up to residues 108 - 114 and of the resulting short-form variants, only Δ108ST6Gal-I seems to be active. FLAG-Δ108ST6Gal-I transfers sialic acids to monoclonal antibody substrate with sufficient yields, and because it is stably produced in P.pastoris, it is identified here as an interesting glycoengineering catalyst.

Characterization of a multifunctional α2,3-sialyltransferase from Pasteurella dagmatis.

A new multifunctional α2,3-sialyltransferase has been discovered in Pasteurella dagmatis. The enzyme, in short PdST, was identified from the P. dagmatis genome by sequence similarity with sialyltransferases of glycosyltransferase family GT-80. In addition to its regioselective sialyltransferase activity (5.9 U/mg; pH 8.0), purified PdST is alternatively active at low pH as α2,3-sialidase (0.5 U/mg; pH 4.5) and α2,3-trans-sialidase (1.0 U/mg; pH 4.5). It also shows cytidine-5'-monophosphate N-acetyl-neuraminic (CMP-Neu5Ac) hydrolase activity (3.7 U/mg; pH 8.0) when no sialyl acceptor substrate is present in the reaction. After sialyltransferase PmST1 from P. multocida, PdST is the second member of family GT-80 to display this remarkable catalytic promiscuity. A unique feature of PdST, however, is a naturally occurring Ser-to-Thr substitution within a highly conserved Y(112)DDGS(116) sequence motif. In PmST1, the equivalent Ser(143) is involved in binding of the CMP-Neu5Ac donor substrate. Reversion of the natural mutation in a T116S-PdST variant resulted in a marked increase in α2,3-trans-sialidase side activity (4.0 U/mg; pH 4.5), whereas the major sialyltransferase activity was lowered (3.8 U/mg; pH 8.0). The Michaelis-Menten constant for CMP-Neu5Ac was decreased 4-fold in T116S mutant when compared with wild-type PdST (KM=1.1 mM), indicating that residue 116 of PdST contributes to a delicate balance between substrate binding and catalytic activity. D-Galactose and various ß-D-galactosides function as sialyl acceptors from CMP-Neu5Ac, whereas other hexoses (e.g. D-glucose) are inactive. Structure comparison was used to rationalize the particular acceptor substrate specificity of PdST in relation to other GT-80 sialyltransferases that show strict α2,3-regioselectivity, but are flexible in using α/ß-galactosides for sialylation.

Fusion of binding domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis.

A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli . Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s(-1) on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s(-1)/ µM (native enzyme) to 0.21 and 0.33 s(-1)/µM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.

Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis.

Modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 revealed that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site could be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate). To investigate this hypothesis in more detail, selected amino acids of surface regions outside the active site of Thc_Cut2, which hydrolyzes PET much less efficiently than Thc_Cut1 were exchanged by site-directed mutagenesis. The mutants were expressed in E. coli BL21-Gold(DE3), purified and characterized regarding their specific activities and kinetic parameters on soluble substrates and their ability to hydrolyze PET and the PET model substrate bis(benzoyloxyethyl) terephthalate (3PET). Compared to Thc_Cut2, mutants carrying Arg29Asn and/or Ala30Val exchanges showed considerable higher specific activity and higher kcat /KM values on soluble substrates. Exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for 3PET and PET. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on PET films. These findings clearly demonstrate that surface properties (i.e., amino acids located outside the active site on the protein surface) play an important role in PET hydrolysis.