Dual Enzyme System Research Articles

A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase.

In this study, a novel enzymatic approach to transform levulinic acid (LA), which can be obtained from biomass, into value-added (R)-4-aminopentanoic acid using an engineered glutamate dehydrogenase from Escherichia coli (EcGDH) was developed. Through crystal structure comparison, two residues (K116 and N348), especially residue 116, were identified to affect the substrate specificity of EcGDH. After targeted saturation mutagenesis, the mutant EcGDHK116C, which was active toward LA, was identified. Screening of the two-site combinatorial saturation mutagenesis library with EcGDHK116C as positive control, the k cat/K m of the obtained EcGDHK116Q/N348M for LA and NADPH were 42.0- and 7.9-fold higher, respectively, than that of EcGDHK116C. A molecular docking investigation was conducted to explain the catalytic activity of the mutants and stereoconfiguration of the product. Coupled with formate dehydrogenase, EcGDHK116Q/N348M was found to be able to convert 0.4 M LA by more than 97% in 11 h, generating (R)-4-aminopentanoic acid with >99% enantiomeric excess (ee). This dual-enzyme system used sustainable raw materials to synthesize (R)-4-aminopentanoic acid with high atom utilization as it utilizes cheap ammonia as the amino donor, and the inorganic carbonate is the sole by-product.

Cascade chiral amine synthesis catalyzed by site-specifically co-immobilized alcohol and amine dehydrogenases

Sustainable and efficient production of chiral amines was realized with an oriented co-immobilized dual-enzyme system via SiBP-tag.

Structure and Function of a Dual Reductase-Dehydratase Enzyme System Involved in p-Terphenyl Biosynthesis.

We report the identification of the ter gene cluster responsible for the formation of the p-terphenyl derivatives terfestatins B and C and echoside B from the Appalachian Streptomyces strain RM-5-8. We characterize the function of TerB/C, catalysts that work together as a dual enzyme system in the biosynthesis of natural terphenyls. TerB acts as a reductase and TerC as a dehydratase to enable the conversion of polyporic acid to a terphenyl triol intermediate. X-ray crystallography of the apo and substrate-bound forms for both enzymes provides additional mechanistic insights. Validation of the TerC structural model via mutagenesis highlights a critical role of arginine 143 and aspartate 173 in catalysis. Cumulatively, this work highlights a set of enzymes acting in harmony to control and direct reactive intermediates and advances fundamental understanding of the previously unresolved early steps in terphenyl biosynthesis.

Improved sensitive fluorescent/visible dual detection count plate for mold and yeast in food

An improved sensitive fluorescent/visible dual detection count plate for mold and yeast was designed to improve the mold and yeast detection efficiency and guarantee food security. The effect of substrate, co-solvent and nonionic surfactant on mold and yeast count plate detection performance was investigated. LAP/lipase as extracellular dual-enzyme system hydrolyzed 0.2 mg/mL of 5-bromo-4-chloro-3-indolyl phosphate disodium salt and 4-nitrophenyl phosphate disodium salt hexahydrate and released chromophores p-nitrophenol, and dehydroindigo dimer to chromogenic colony. Detection was achieved by recording the chromogenic mold and yeast colony-forming units (CFU). Moreover, 4 μL/mL of co-solvent water-ethanol was used as fluorescent sensitizer for colonies, while 0.1% Tween-20 (w/v), 0.1% Tween-80 (w/v) and 0.1% TritonX-100 (w/v) facilitated growth rate and obvious expression of extracellular dual enzyme system LAP/lipase. Counting results of the fluorescent/visible count plate (34 h) have not significantly different from the national standard GB 4789.15–2016 agar plate (5 days) (P > 0.05). Results of fluorescent/visible count plate for the detection of mold and yeast in actual food samples were 97.5% and 98.6% in accordance with the results of the national standard method. This work provided a convenient, rapid, and sensitive detection of mold and yeast.

Characterization and rational modification of aspartate 4-decarboxylase from Acinetobacter radioresistens for the production of l-alanine.

Enzymatic synthesis of l-alanine has the advantages of less byproducts, strong stereoselectivity, and high catalytic efficiency. Aspartate 4-decarboxylase (ASD) is used industrially in DL-aspartic acid resolution and l-alanine production because it catalyzes the decarboxylation of l-aspartic acid. In this study, the ASD gene from Acinetobacter radioresistens (ArASD) was cloned, and its enzymatic properties were analyzed. ArASD is a dodecamer and has the highest enzyme activity ever reported to date. The optimal conditions for ArASD catalysis are 50°C and pH 4.5. Site-directed mutagenesis was used to improve ArASD stability under acidic conditions to compensate for its weak acid resistance, and the variant N35D with higher catalytic ability was obtained. The conversion by N35 recombinant cells of l-aspartic acid to l-alanine was 92.5% at pH 4.5% and 99.9% at pH 6.0, whereas that of the wild-type recombinant cells was 29.7% and 31.4%, respectively. Aspartase from Escherichia coli (AspA) was employed with ArASD to construct a dual-enzyme system that catalyzes fumaric acid to l-alanine, and the conversion reached 97.1% using recombinant cells harboring the dual-enzyme system. This study explored the enzymatic properties of ArASD and an effective strategy for the acidic resistance modification of ASD. Moreover, the strain expressing the ArASD variant and AspA engineered in this study has great potential application for the l-alanine production industry, especially in the case of high optical purity requirements.

Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria

Marine Synechococcus cyanobacteria owe their ubiquity in part to the wide pigment diversity of their light-harvesting complexes. In open ocean waters, cells predominantly possess sophisticated antennae with rods composed of phycocyanin and two types of phycoerythrins (PEI and PEII). Some strains are specialized for harvesting either green or blue light, while others can dynamically modify their light absorption spectrum to match the dominant ambient color. This process, called type IV chromatic acclimation (CA4), has been linked to the presence of a small genomic island occurring in two configurations (CA4-A and CA4-B). While the CA4-A process has been partially characterized, the CA4-B process has remained an enigma. Here we characterize the function of two members of the phycobilin lyase E/F clan, MpeW and MpeQ, in Synechococcus sp. strain A15-62 and demonstrate their critical role in CA4-B. While MpeW, encoded in the CA4-B island and up-regulated in green light, attaches the green light-absorbing chromophore phycoerythrobilin to cysteine-83 of the PEII α-subunit in green light, MpeQ binds phycoerythrobilin and isomerizes it into the blue light-absorbing phycourobilin at the same site in blue light, reversing the relationship of MpeZ and MpeY in the CA4-A strain RS9916. Our data thus reveal key molecular differences between the two types of chromatic acclimaters, both highly abundant but occupying distinct complementary ecological niches in the ocean. They also support an evolutionary scenario whereby CA4-B island acquisition allowed former blue light specialists to become chromatic acclimaters, while former green light specialists would have acquired this capacity by gaining a CA4-A island.

Nanocomposite-based dual enzyme system for broad-spectrum scavenging of reactive oxygen species

A broad-spectrum reactive oxygen species (ROS)-scavenging hybrid material (CASCADE) was developed by sequential adsorption of heparin (HEP) and poly(L-lysine) (PLL) polyelectrolytes together with superoxide dismutase (SOD) and horseradish peroxidase (HRP) antioxidant enzymes on layered double hydroxide (LDH) nanoclay support. The synthetic conditions were optimized so that CASCADE possessed remarkable structural (no enzyme leakage) and colloidal (excellent resistance against salt-induced aggregation) stability. The obtained composite was active in decomposition of both superoxide radical anions and hydrogen peroxide in biochemical assays revealing that the strong electrostatic interaction with the functionalized support led to high enzyme loadings, nevertheless, it did not interfere with the native enzyme conformation. In vitro tests demonstrated that ROS generated in human cervical adenocarcinoma cells were successfully consumed by the hybrid material. The cellular uptake was not accompanied with any toxicity effects, which makes the developed CASCADE a promising candidate for treatment of oxidative stress-related diseases.

Evaluation of continuous ethyl ester synthesis from acid soybean oil using a dual immobilized enzyme system

The use of non-edible oils, waste oils, and animal fats have been investigated as a low-cost feedstock for biodiesel (fatty acid alkyl esters) synthesis. However, these alternative raw materials have some drawbacks due to the high content of free fatty acid (FFA). This work aimed to study the continuous enzymatic ethyl esters synthesis, in a solvent-free system, from the reaction of acid soybean oil (acid value, AV, of 0.5, 8.5, 50, and 90) and ethanol using a dual commercial immobilized lipase system (Novozym 435®, Lipozyme TL IM®, Lipozyme RM IM®). Initially, a dual lipase system was used in a single packed-bed reactor (PBR). The blend of Lipozyme TL IM and Novozym 435 had a positive synergistic effect on biodiesel synthesis: an increase of 76% in ethyl ester content was observed after 2 h using refined soybean oil. However, a reduction in the yield was observed in longer reaction times (25 h). Therefore, a continuous reaction system, operated with two different catalytic beds, was investigated. The use of two series reactors allowed high ethyl ester yield (>90%) and high acid conversion (>90%). The reaction using acid soybean oil (50 AV), and Lipozyme RM IM and Novozym 435 reached an ethyl ester content of 95% and 70% after 2 and 25 h, respectively. The results showed that using two different commercial immobilized lipases in intercalated packed-bed reactors enhanced biodiesel synthesis from acid soybean oil. The enzymatic system allows a high fatty acid conversion present in the acidic oil into ethyl ester.

Identification and characterization of two novel antioxidant peptides from silkworm pupae protein hydrolysates

Silkworm pupae are a kind of insect resource food that rich of good quality protein. To identify the peptide with high antioxidant activity from silkworm pupae protein hydrolysates, and provide the basis for the application of silkworm pupae protein hydrolysates and antioxidant peptides in functional foods, silkworm pupae proteins were hydrolysed by a dual-enzyme system consisting of acidic protease and neutral protease. The hydrolysates were purified sequentially by ultrafiltration, gel filtration chromatography and high-performance liquid chromatography (HPLC). The ABTS radical scavenging activity was used to evaluate antioxidant activity. Fractions with high activity were further analyzed by liquid chromatography- tandem mass spectrometry (LC–MS/MS). Two peptides, FKGPACA and SVLGTGC with molecular weights of 692.34 and 635.30 Da were obtained. To further determine the major active sites of FKGPACA and SVLGTGC, four peptides, FKGP, ACA, SVLG and TGC were artificially synthesized. ACA and TGC had higher ABTS radical scavenging activities than FKGP and SVLG. The main active sites of FKGPACA and SVLGTGC were possibly located in the ACA and TGC fragments, which are related to Cys, Ala or Thr residues. Both FKGPACA and SVLGTGC proved to good antioxidants even after high-temperature thermal processing for 1 h. After digestion with pepsin, the ABTS radical scavenging activity of FKGPACA was stable, while the ABTS radical scavenging activity of SVLGTGC decreased slightly. After further digestion with pancreatin, the ABTS radical scavenging activities of FKGPACA and SVLGTGC decreased by 10.59% and 43.56%, respectively. After digestion with chymotrypsin, the ABTS radical scavenging activities of FKGPACA and SVLGTGC were stable. The silkworm pupae protein hydrolysates and FKGPACA could be potentially used as natural antioxidants in functional foods.

A versatile biocatalytic nano-platform based on Fe3O4-filled and zirconia shrunk holey carbon nanotubes

The creation of biocatalytic nano-platform with high performance is the focus of attention in the field of enzyme catalysis. However, most enzyme nanoreactors suffer from poor versatility and recovery ability. Herein, we construct a versatile biocatalytic nano-platform with steady magnetic recyclability through hybrid magnetic carbon nanotubes (named by h-CNT/Fe3O4/ZrO2), realize the role of biocatalyst by the covalent attachment of lipase. On the other side, glucose oxidase (GOx) was immobilized on the h-CNT/Fe3O4/ZrO2 to prepare dual enzyme system (GOx@h-CNT/Fe3O4/ZrO2) for glucose detecting via colorimetric assay, this is its role as biosensor. In order to build an amphiphilic nano-bio interface, hydroxyl contained carbon nanotubes were selected as the substrate material of nano-platform. We punch lots of mesopores on the tube wall and inject Fe3O4 nanoparticles into inner nanotubes via these openings, then shrunk mesoporous by zirconia to maintain the magnetic recyclability. We believed such an architecture that can not only offer the comfortable bio-nano interfaces for enzyme but also effectively avoid the loss of magnetism thus brings constant recyclability.

Dual-Enzyme Crosslinking and Post-polymerization for Printing of Polysaccharide-Polymer Hydrogel.

Polymer hydrogels are ideal bioprinting scaffolds for cell-loading and tissue engineering due to their extracellular-matrix-like structure. However, polymer hydrogels that are easily printed tend to have poor strength and fragile properties. The gradually polymerized reinforcement after hydrogel printing is a good method to solve the contradiction between conveniently printed and high mechanical strength requirement. Here, a new succinct approach has been developed to fabricate the printable composite hydrogels with tunable strength. We employed the HRP@GOx dual enzyme system to initiate the immediate crosslinking of chondroitin sulfate grafted with tyrosine and the gradual polymerization of monomers to form the composite hydrogels. The detailed two-step gelation mechanism was confirmed by the Fluorescence spectroscopy, Electron paramagnetic resonance spectroscopy and Gel permeation chromatography, respectively. The final composite hydrogel combines the merits of enzymatic crosslinking hydrogels and polymerized hydrogels to achieve adjustable mechanical strength and facile printing performance. The dual-enzyme regulated polymer composite hydrogels are the promising bioscaffolds as organoid, implanted materials, and other biomedical applications.

Curing of Urushi under basic pH conditions by a dual enzyme system

Urushi (Oriental lacquer) is an environmentally friendly material obtained from the sap of lacquer trees. Under natural pH conditions (acidic–near neutral) Urushi curing is achieved by polymerization of the phenolic compound urushiol, a main component of Urushi. Curing is initiated by urushiol oxidation catalyzed by a specific laccase (UL) contained in the Urushi. However, Urushi cannot be cured by this enzyme under the strong basic pH conditions resulting from the addition of alkaline compounds. The aim of this study was to investigate why Urushi is not cured under strong basic pH conditions and find a way to solve this problem. H2O2 was generated in a mixture of urushiol and water, particularly in conditions of basic pH, that H2O2 inhibited UL activity, and that addition of H2O2 or NaOH substantially increased the curing time of Urushi. Addition of horseradish peroxidase (HRP), an enzyme catalyzing H2O2 decomposition, causes Urushi to be cured even in the strong basic conditions obtained following addition of NaOH. HRP’s main role in this system was inferred to be to prevent UL inhibition by H2O2. This achievement is expected to foster new uses of Urushi in the presence of basic compounds.

Layered Co-Immobilization of β-Glucosidase and Cellulase on Polymer Film by Visible-Light-Induced Graft Polymerization.

Exploring a suitable immobilization strategy to improve catalytic efficiency and reusability of cellulase is of great importance to lowering the cost and promoting the industrialization of cellulose-derived bioethanol. In this work, a layered structure with a thin PEG hydrogel as the inner layer and sodium polyacrylate (PAANa) brush as the outer layer was fabricated on low density polyethylene (LDPE) film by visible-light-induced graft polymerization. Two enzymes, β-glucosidase (BG) and cellulase, were separately coimmobilized onto this hierarchical film. As supplementary to cellulase for improving catalytic efficiency, BG was in situ entrapped into the inner PEG hydrogel layer during the graft polymerization from the LDPE surface. After graft polymerization of sodium acrylate on the PEG hydrogel layer was reinitiated, cellulase was covalently attached on the outer PAANa brush layer. Owing to the mild reaction condition (visible-light irradiation and room temperature), the immobilized BG could retain a high activity after the graft polymerization. The immobilization did not alter the optimal pH and temperature of BG or the optimal temperature of cellulase. However, the optimal pH of cellulase shifts to 5.0 after immobilization. Compared with the original activity of single cellulase system and isolated BG/cellulase immobilization system, the dual-enzyme system exhibited 82% and 20% increase in catalytic activity, respectively. The dual-enzyme system could maintain 93% of carboxymethylcellulose sodium salt (CMC) activity after repeating 10 cycles of hydrolysis and 89% of filter paper activity after 6 cycles relative to original activity, exhibiting excellent reusability. This layer coimmobilization system of BG and cellulase on the polymer film displays tremendous potential for practical application in a biorefinery.

One-Pot Biosynthesis of l -Aspartate from Maleate via an Engineered Strain Containing a Dual-Enzyme System

l-Aspartate has been widely used in medicine and the food and chemical industries. In this study, Serratia marcescens maleate cis-trans isomerase (MaiA) and Escherichia coli aspartase (AspA) were coupled and coexpressed in an engineered E. coli strain in which the byproduct metabolic pathway was inactivated. The engineered E. coli strain containing the dual-enzyme system (pMA) was employed to bioproduce l-aspartate from maleate with a conversion of 98%. We optimized the activity ratio of double enzymes through ribosome binding site (RBS) regulation and molecular modification of MaiA, resulting in an engineered strain: pMA-RBS4-G27A/G171A. The conversion of l-aspartate biotransformed from maleate using the pMA-RBS4-G27A/G171A strain was almost 100%. It required 40 min to complete the whole-cell catalysis, without the intermediate product and byproduct, compared to 120 min before optimization. The induction timing and the amount of inducer in a 5-liter fermentor were optimized for scale-up of the production of l-aspartate. The amount of produced l-aspartate using the cells obtained by fermentation reached 419.8 g/liter (3.15 M), and the conversion was 98.4%. Our study demonstrated an environmentally responsible and efficient method to bioproduce l-aspartate from maleate and provided an available pathway for the industrial production of l-aspartate. This work should greatly improve the economic benefits of l-aspartate, which can now be simply produced from maleate by the engineered strain constructed based on dual-enzyme coupling.IMPORTANCE l-Aspartate is currently produced from fumarate by biological methods, and fumarate is synthesized from maleate by chemical methods in industry. We established a biosynthesis method to produce l-aspartate from maleate that is environmentally responsible, convenient, and efficient. Compared to conventional l-aspartate production, no separation and purification of intermediate products is required, which could greatly improve production efficiency and reduce costs. As environmental issues are attracting increasing attention, conventional chemical methods gradually will be replaced by biological methods. Our results lay an important foundation for the industrialization of l-aspartate biosynthesis from maleate.

Co-immobilization of Short-Chain Dehydrogenase/Reductase and Glucose Dehydrogenase for the Efficient Production of (±)-Ethyl Mandelate

Derivatives of (±)-ethyl mandelate are important intermediates in the synthesis of numerous pharmaceuticals. Therefore, efficient routes for the production of these derivatives are highly desirable. The short-chain dehydrogenase/reductase (SDR) is a biocatalyst that could potentially be applied to the synthesis of (±)-ethyl mandelate; however, this enzyme requires the reduced form of the cofactor nicotine adenine dinucleotide (phosphate) (NAD(P)H), which is expensive. In this study, we developed a co-immobilization strategy to overcome the issue of NADPH demand in the SDR catalytic process. The SDR from Thermus thermophilus HB8 and the NAD(P)-dependent glucose dehydrogenase (GDH) from Thermoplasma acidophilum DSM 1728 were co-immobilized on silica gel. The properties and the catalytic abilities of this dual-enzyme system were evaluated. A final yield of 1.17 mM (±)-ethyl mandelate was obtained from the catalytic conversion of ethyl benzoylformate, with a conversion rate of ethyl benzoylformate to (S)-(+)-mandelate of 71.86% and in an enantiomeric excess of > 99% after 1.5 h. This system offers an efficient route for the biosynthesis of (±)-ethyl mandelate. In this study, we developed a co-immobilization strategy to overcome the issue of NADPH demand in the SDR catalytic process. The SDR from Thermus thermophilus HB8 and the NAD(P)-dependent glucose dehydrogenase (GDH) from Thermoplasma acidophilum DSM 1728 were co-immobilized on silica gel. Results showed that, this dual-system offers an efficient route for the biosynthesis of (±)-ethyl mandelate.

Ligninases remove phenolic inhibitors and facilitate yeast growth in lignocellulosic hydrolysate

Abstract Sugar-containing hydrolysates as byproducts of dissolving pulp production could be a source of alcohol production, but the presence of fermentation inhibitors hampers this utilization route. In the present study, nine types of phenolic inhibitors were found and their removal by ligninases gave satisfactory results. Laccase removed 73.1% of phenolic inhibitors and produced three new aromatic compounds, whereas the combination of laccase and lignin peroxidase (as a dual-enzyme treatment) resulted in a removal rate of 91.7% without new product formation. Xylose as the main sugar in the hydrolysate was retained with a 90% yield, regardless of the enzymatic treatment, which modified, however, the kinetics of yeast growth. Laccase improved cells’ maximum specific growth rate (v max) from 0.115 to 0.154 and reduced the saturation constant (K s ) from 53.4 to 42.5, whereas the dual-enzyme system improved v max to 0.206 and reduced K s to 42.44. The greater v max and the smaller K s imply a better growth rate, and accordingly, the dual-enzyme system is better suited for removing phenolic inhibitors.

Extraction, identification and structure-activity relationship of antioxidant peptides from sesame (Sesamum indicum L.) protein hydrolysate

To elucidate the sequence, origin and structure-activity relationship of antioxidant peptides from sesame protein, sesame protein was hydrolysed by a dual-enzyme system comprised alcalase and trypsin, then this hydrolysate was fractionated by ultrafiltration and preparative HPLC. Subsequently, peptides in the high antioxidant activity fraction were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (Nano-LC-ESI-MS/MS), finally the structure-activity relationship of antioxidant peptide with the strongest activity in the sesame peptides was illustrated by comparative molecular field analysis (CoMFA). The results showed that seven novel antioxidant peptides were discovered, their sequences were as follows, RDRHQKIG, TDRHQKLR, MNDRVNQGE, RENIDKPSRA, SYPTECRMR, GGVPRSGEQEQQ and AGEQGFEYVTFR. The SYPTECRMR was the hydrolysate of 2S albumin, the others derived from 11S globulin. The SYPTECRMR whose IC50 Values of DPPH and ABTS were 0.105 mg/mL and 0.004 mg/mL respectively exhibited the highest antioxidant activity among the seven sesame peptides. The active site of SYPTECRMR tended to locate on Cys6 and Met8. A positive correlation between Cys6, Met8, the bulky C-terminal amino acid residue (Arg9), the negative charged group around sulphur-containing amino acids and the antioxidant activity of SYPTECRMR was observed from the CoMFA model. The results presented herein suggested that sesame protein hydrolysates have potential applications in functional food due to their high antioxidant activity, the CoMFA model could provide insight into the structure-activity relationship of antioxidant peptide, which is useful to screen, identify and design the novel antioxidant peptide.

Production of d-psicose from d-glucose by co-expression of d-psicose 3-epimerase and xylose isomerase.

d-Psicose has been drawing increasing attention in recent years because of its medical and health applications. The production of d-psicose from d-glucose requires the co-expression and synergistic action of xylose isomerase and d-psicose 3-epimerase. To co-express these genes, vector pET-28a(+)-dual containing two T7 promoters and RBS sites and an Multiple Cloning Sites was constructed using the Escherichia coli expression plasmid pET-28a(+). The xylose isomerase gene from E. coli MG1665 and the d-psicose 3-epimerase gene from Agrobacterium tumefaciens CGMCC 1.1488 were cloned and co-expressed in E. coli BL21(DE3). After 24h incubation with the dual enzyme system at 40°C, the sugar conversion ratio from d-glucose to d-psicose reached 10%. The optimal conditions were 50°C, pH 7.5 with Co2+ and Mg2+. The d-psicose yields from sugarcane bagasse and microalgae hydrolysate were 1.42 and 1.69g/L, respectively.

Construction of a tunable multi-enzyme-coordinate expression system for biosynthesis of chiral drug intermediates.

Systems that can regulate and coordinate the expression of multiple enzymes for metabolic regulation and synthesis of important drug intermediates are poorly explored. In this work, a strategy for constructing a tunable multi-enzyme-coordinate expression system for biosynthesis of chiral drug intermediates was developed and evaluated by connecting protein-protein expressions, regulating the strength of ribosome binding sites (RBS) and detecting the system capacity for producing chiral amino acid. Results demonstrated that the dual-enzyme system had good enantioselectivity, low cost, high stability, high conversion rate and approximately 100% substrate conversion. This study has paved a new way of exploring metabolic mechanism of functional genes and engineering whole cell-catalysts for synthesis of chiral α-hydroxy acids or chiral amino acids.

A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films.

TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.