Enzyme Cutinase Research Articles

Enzymatic Degradation of Poly(Butylene Adipate‐co‐Terephthalate)/Poly(Lactic Acid) Blends

ABSTRACTPoly(lactic acid) (PLA) is regularly blended with poly(butylene adipate‐co‐terephthalate) (PBAT) to produce compostable plastic films used in packaging applications, for example. Since PLA and PBAT both contain ester bonds, these PBAT/PLA blends are interesting candidates for enzymatic recycling. This is a new, green recycling technique that allows the use of mild processing conditions to regenerate the pure building blocks of polymers, thereby enabling repolymerization to virgin‐quality plastic. In this work, various PBAT/PLA blends are prepared (20, 40, 60, and 80 wt% PBAT) and incubated with a cutinase enzyme from Humicola insolens (HiC). Enzymatic degradation is characterized by weight loss, scanning electron microscopy (SEM), and proton nuclear magnetic resonance (1H NMR) spectroscopy. Blends rich in PBAT are effectively degraded by HiC (up to 40% weight loss after 7 days of incubation at 70°C), while PLA‐rich blends are degraded to a lesser extent. HiC is capable of degrading both polymers, although for PLA, the extent of degradation is similar to control experiments without HiC. Furthermore, in the absence of an enzyme, a “shielding” effect is found for nonenzymatic degradation, where a hydrolytically stable PBAT matrix prevents the degradation of the PLA domains. This work provides insight into the enzymatic degradation of uncompatibilized polymer blends and opens up the possibility for the enzymatic recycling of commercial PBAT/PLA blends.

Hydrolysis rate and mechanism of water-dispersed polyesters with different sulfonate group contents in the cutinase-catalyzed system.

Water-dispersed polyester (WPET) has irreplaceable and wide application prospects in the textile field, but it is easy to form printing and dyeing wastewater during traditional fabric treatment. In this paper, cutinase-catalyzed green hydrolysis of WPET was achieved. The main purpose of this paper is to study the difference between two PEG-free WPETs with different -SO3- content in the cutinase-catalyzed hydrolysis process. Differences in solution properties, enzyme activity, SSIPA, and TPA release as well as functional groups and thermal properties of the hydrolysis products of the systems during cutinase hydrolysis were analyzed in detail for 14.1 % S-0 % PEG and 9.5 % S-0 % PEG. Results show that the structure of WPET has an influential effect on its hydrolysis rate in cutinase-catalyzed systems, which mainly originates from the -SO3- content. SSIPA released by WPET during hydrolysis has an inhibitory effect on cutinase enzyme activity. The hydrolysis process of WPET with higher -SO3- content releases more SSIPA, which in turn inhibits the enzyme activity of cutinase in the reaction system, ultimately leading to a slower hydrolysis rate of WPET. These findings provide new ideas for cutinase to target WPETs with different structures and are important for the green development of the textile industry.

Antifungal activity of clove (Eugenia caryophyllata) extract against Fusarium oxysporum cutinase enzyme in Tomato (Solanum lycopersicum) from in vitro study

Fusarium oxysporum f.sp. lycopersici presents a notable threat to tomato plants due to the hydrolytic activity of its cutinase enzyme, which facilitates the penetration of plant root tissues. Thus, reducing cutinase activity through the application of cloves as inhibitors offers an environmentally friendly alternative to hazardous chemical fungicides, showing great promise in controlling Fusarium wilt disease, particularly by targeting the cutinase enzyme of Fusarium oxysporum. This study involved GC-MS analysis of clove extract, fungal growth and cutinase activity assay using rhodamine-b olive oil media. Hence, this study aims to reveal the potential and mechanism of clove extracts as inhibitors of cutinase enzymes to reduce the penetration of the Fusarium oxysporum pathogen in tomato plants. The result shows that clove extract, containing approximately 20% antifungal compounds primarily eugenol (11% of total peak area), inhibits Fusarium oxysporum growth. It also reduces the hydrolytic activity of cutinase crucial for fungal penetration, evidenced by decreased fluorescence halos in tests with rhodamine-B and olive oil media under UV light after adding 1%, 2%, and 3% clove extract. Further in vivo studies are needed to explore its direct effects as a plant inhibitor.

Production of thermostable recombinant cutinase using mixed food waste: A sustainable approach toward environmental remediation

Recently, there has been a significant surge in the utilization of single-use plastics. Cost-effective degradation of plastics is imperative to reduce the ecological burden. This study explores the overproduction and characterization of a PET (Polyethylene terephthalate) a degrading enzyme, cutinase, in the E. coli BL21 (DE3) expression system. Furthermore, this research delves into the utilization of mixed food waste (MFW) as a sustainable substrate for the production of Cutinase, replacing commercial glucose. The newly isolated strain, Aspergillus terreus, showed the highest glucoamylase activity of 76.10 ± 3.10 U/g. The enzymatic hydrolysis of mixed food waste resulted in 50 ± 0.4 g/L of glucose, used as feedstock for cutinase production. The crude enzyme activity was evaluated to be 11.64 U/mg, and a further 14.11-fold purification was achieved through IMAC. The purified recombinant enzyme showed its maximum activity at 70℃ and pH 7. The metal ion Na+ increased the enzyme activity by 26 % while Zn+2 significantly inhibited the same (47 % inhibition); other ions had little or no effect. Taguchi’s optimization of five variables showed that the concentration of food waste hydrolysate has the maximum impact on enzyme production. The treatment of commercially available PET bottles using the purified enzyme showed characterizable surface degradation effects. This is the first time food waste hydrolysate was used as a sole carbon source to produce recombinant cutinase. This approach can potentially pave the way for sustainable biorefinery and waste management.

Integration of pH Control into Chi.Bio Reactors and Demonstration with Small-Scale Enzymatic Poly(ethylene terephthalate) Hydrolysis.

Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1-2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.

Phylum-level studies of bacterial cutinases for unravelling enzymatic specificity toward PET degradation: an in silico approach.

The overwhelming use of PET plastic in various day-to-day activities led to the voluminous increase in PET waste and growing environmental hazards. A plethora of methods have been used that are associated with secondary pollutants. Therefore, microbial degradation of PET provides a sustainable approach due to its versatile metabolic diversity and capacity. The present work highlights the cutinase enzyme's role in PET degradation. This study focuses on the bacterial cutinases homologs screened from 43 reported phylum of bacteria. The reported bacterial cutinases for plastic degradation have been chosen as reference sequences, and 917 sequences have shown homology across the bacterial phyla. The dienelactone hydrolase (DLH) domain was identified for attaining specificity towards PET binding in 196 of 917 sequences. Various computational tools have been used for the physicochemical characterization of 196 sequences. The analysis revealed that most selected sequences are hydrophilic, extracellular, and thermally stable. Based on this analysis, 17 sequences have been further pursued for three-dimensional structure prediction and validation. The molecular docking studies of 17 selected sequences revealed efficient PET binding with the three sequences derived from the phylum Bacteroidota, the lowest binding energy of -5.9kcal/mol, Armatimonadota, and Nitrososphaerota with -5.8kcal/mol. The two enzyme sequences retrieved from the phylum Bacteroidota and Armatimonadota are metagenomically derived. Therefore, the present studies concluded that there is a high probability of finding cutinase homologs in various environmental resources that can be further explored for PET degradation.

Probing Enzymatic PET Degradation: Molecular Dynamics Analysis of Cutinase Adsorption and Stability.

Understanding the mechanisms influencing poly(ethylene terephthalate) (PET) biodegradation is crucial for developing innovative strategies to accelerate the breakdown of this persistent plastic. In this study, we employed all-atom molecular dynamics simulation to investigate the adsorption process of the LCC-ICCG cutinase enzyme onto the PET surface. Our results revealed that hydrophobic, π-π, and H bond interactions, specifically involving aliphatic, aromatic, and polar uncharged amino acids, were the primary driving forces for the adsorption of the cutinase enzyme onto PET. Additionally, we observed a negligible change in the enzyme's tertiary structure during the interaction with PET (RMSD = 1.35 Å), while its secondary structures remained remarkably stable. Quantitative analysis further demonstrated that there is about a 24% decrease in the number of enzyme-water hydrogen bonds upon adsorption onto the PET surface. The significance of this study lies in unraveling the molecular intricacies of the adsorption process, providing valuable insights into the initial steps of enzymatic PET degradation.

Exploring the pH dependence of an improved PETase

Enzymatic recycling of plastic and especially of polyethylene terephthalate (PET) has shown great potential to reduce its negative impact on our society. PET hydrolases (PETases) have been optimized using rational design and machine learning, but the mechanistic details of the PET depolymerization process remain unclear. Belonging to the carboxylic-ester hydrolase family with a canonical Ser-His-Asp catalytic triad, their observed alkaline pH optimum is generally thought to be related to the protonation state of the catalytic His. Here, we explore this aspect in the context of LCCICCG, an optimized PETase, derived from the leaf-branch compost cutinase enzyme. We use NMR to identify the dominant tautomeric structure of the six histidines. Five show surprisingly low pKa values below 4.0, whereas the catalytic H242 in the active enzyme displays a pKa value that varies from 4.9 to 4.7 when temperatures increase from 30°C to 50°C. Whereas the hydrolytic activity of the enzyme toward a soluble substrate can be modeled by the corresponding protonation/deprotonation curve, an important discrepancy is found when the substrate is the solid plastic. This opens the way to further mechanistic understanding of the PETase activity and underscores the importance of studying the enzyme at the liquid-solid interface.

Synthesis of Isoamyl Fatty Acid Ester, a Flavor Compound, by Immobilized Rhodococcus Cutinase.

Isoamyl fatty acid esters (IAFEs) are widely used as fruity flavor compounds in the food industry. In this study, various IAFEs were synthesized from isoamyl alcohol and various fatty acids using a cutinase enzyme (Rcut) derived from Rhodococcus bacteria. Rcut was immobilized on methacrylate divinylbenzene beads and used to synthesize isoamyl acetate, butyrate, hexanoate, octanoate, and decanoate. Among them, Rcut synthesized isoamyl butyrate (IAB) most efficiently. Docking model studies showed that butyric acid was the most suitable substrate in terms of binding energy and distance from the active site serine (Ser114) γ-oxygen. Up to 250 mM of IAB was synthesized by adjusting reaction conditions such as substrate concentration, reaction temperature, and reaction time. When the enzyme reaction was performed by reusing the immobilized enzyme, the enzyme activity was maintained at least six times. These results demonstrate that the immobilized Rcut enzyme can be used in the food industry to synthesize a variety of fruity flavor compounds, including IAB.

Efficient Mechano-Enzymatic Hydrolysis of Polylactic Acid under Moist-Solid Conditions

Quantitative mechano-enzymatic depolymerization of polylactic acid to lactic acid was achieved at 55 °C using the Humicola insolens cutinase enzyme in moist-solid reaction mixtures. The resulting lactic acid was easily recovered, and the crude product was pure enough to be used in further synthesis of a value-added compound, a known benzimidazole-based drug precursor. The presented mechano-enzymatic depolymerization strategy enables the closed-loop recycling of untreated polylactic acid under mild conditions, using a renewable, non-toxic catalyst and producing minimum solvent waste.

Mechanism and kinetics of enzymatic degradation of polyester microparticles using a shrinking particle-shrinking core model.

Generalized shrinking particle (SPM) and shrinking core (SCM) models were developed to the kinetics of heterogenous enzymatic degradation of polymer microparticles in a continuous microflow system. This enzymatic degradation was performed in a microfluidic device designed to both physically separate and immobilize the microparticles. Then time-resolved measurements were made using image processing of the physical changes of the particles during degradation. The kinetics of enzyme-polymer intermediate formation, enzymatic bond cleavage, and enzyme diffusion through the layer of degraded substrate (SCM only) were mathematically derived to predict the time-resolved degradation of the substrate. The proposed models were tested against the degradation of 15-25 μm particles of polycaprolactone (PCL) and poly (butylene adipate-co-terephthalate) (PBAT) by cutinase enzyme from Humicola insolens. Degradation of PCL microparticles followed the SPM model and its kinetics were found to be zero-order, while the SCM model applied to PBAT microparticles showed first-order kinetics. Further, the degradation of polybutylene succinate (PBS), and poly butylene-sebacate-co-terephthalate (PBSeT) microparticles demonstrated wide applicability of the method. The use of image processing simplifies the required analysis by eliminating the need to remove aliquots or concentrate effluent for additional analytical characterization.

Characterization of AnCUT3, a plastic-degrading paucimannose cutinase from Aspergillus niger expressed in Pichia pastoris

Cutinases are hydrolytic enzymes secreted by phytopathogens to degrade cutin, the main polymeric component of plant cuticles. The multifaceted functionality of cutinases has allowed for their exploitation for catalytic reactions beyond their natural purpose. To diversify and expand the cutinase enzyme class, we identified five cutinase homologs from the saprotroph Aspergillus niger. One of these cutinases, AnCUT3, was over-expressed in Pichia pastoris and its biophysicochemical properties characterized. The purified recombinant AnCUT3 possessed an optimum temperature of 25 °C, an optimum pH of 5, and was stable at temperatures up to 50 °C (1 h incubation, melting point of 45.6 °C) and in a wide pH range. Kinetic studies of AnCUT3 using pNP ester substrates showed the highest catalytic efficiency, kcat/Km of 859 mM−1 s−1 toward p-nitrophenyl decanoate (C10). Although its calculated molecular mass is 27 kDa, AnCUT3 was expressed as two glycosylated proteins of molecular weights 24 and 50 kDa. Glycan profiling detected the presence of atypical paucimannose N-glycans (≤Man1-5GlcNAc) from recombinant AnCUT3, suggesting protein-dependent glycan processing of AnCUT3 in P. pastoris. AnCUT3 was also able to degrade and modify the surface of polycaprolactone and polyethylene terephthalate. Taken together, these features poise AnCUT3 as a potential biocatalyst for industrial applications.

Structural, functional, and molecular docking analyses of microbial cutinase enzymes against polyurethane monomers

Plastic waste is the biggest global problem in present times due to its constant bioaccumulation in the environment. During the last year, 367 Mt of plastics were produced in the world, of which 28.6 Mt correspond to polyurethane waste. Polyurethanes can be found in products such as adhesives, preservatives, and foams, and are often difficult to recycle. The fragmentation of plastic waste in the environment generates microplastics causing a long-term effect on our ecosystem. In search of its solution via bioremediation using enzymes, in our present study cutinase enzyme has been chosen, as it appears to be a novel candidate due to the wide variety of substrates it hydrolyzes and its presence in different microorganisms. According to physiochemical characteristics, it was found that microbial cutinase enzymes are majorly made up of aliphatic amino acids. A higher aliphatic index (more than 80) indicates the great thermostability of the enzyme. Moreover, the enzyme is found to be hydrophilic and structurally stable. The negative GRAVY value of the enzyme confirmed its stable interaction with water. It was also observed that all chosen protein models had an average of 91.10 % amino acids in the favorable regions of the Ramachandran plot. The studied microbial cutinase enzymes from both fungi Humicola insolens and actinobacterium Thermobifida fusca successfully coupled with the polyurethane resin monomers. The main interactions were found in the catalytic triad with bonds close to the urethane bonds of the ligand, in addition to having an average binding energy of − 6 kJ/mol. The interaction between the cutinases with the PUR resin as a ligand was found to be evident from our study with stable binding energies, which makes microbial cutinases potential enzymes for polyurethane waste bioremediation processes.

Understanding Consequences and Tradeoffs of Melt Processing as a Pretreatment for Enzymatic Depolymerization of Poly(ethylene terephthalate).

Melt extrusion pretreatment of poly(ethylene terephthalate) (PET) prior to enzymatic depolymerization with an unpurified leaf branch compost cutinase enzyme cocktail is explored to ascertain the efficiency gained by different processing methods on the enzymatic depolymerization of PET. Specific surface area (SSA) is investigated as a key factor in reducing depolymerization time. Higher SSA substrates (>5.6mm2 mg-1 ) show higher depolymerization rates (≈0.88gL-1 terephthalic acid [TPA] per day) and no induction phase, while lower SSA substrates (≈4.3, 4.4, and 5.6mm2 mg-1 ) show, after an initial induction phase, similar depolymerization rates (≈0.46, 0.45, and 0.44gL-1 TPA per day) despite increases in SSA of up to 30%. The mechanism of enzymatic depolymerization manifests in the appearance of anisotropic pitting. Longer incubation time used to overcome the induction phase in low SSA substrates allows for nearly full recovery of monomeric products, but manual pregrinding of extruded PET sharply increases SSA, depolymerization rate, and substrate crystallinity which may decrease the maximum recycled yield of the product materials. An estimate of the energy cost of increasing SSA is made and its effects on material properties are discussed. This work highlights key material structure and pretreatment aspects influencing the enzymatic recycling of PET.

Biocatalytic depolymerization of waste polyester mooring lines from oil and gas offshore platforms made of poly(ethylene terephthalate) (PET)

AbstractBACKGROUNDThe global concern of plastic pollution in the environment has consistently been emerging in the past years. As one of the main polymers constituting single‐use products, poly(ethylene terephthalate) (PET) is found in a plethora of packages for diverse sectors, and its recycling has been broadly addressed. However, other relevant industrial PET wastes have not been under investigation for recycling purposes, such as the polyester mooring lines (PMLs) used to anchor offshore oil and gas floating platforms. In this study, the fibers from either new or used PMLs were characterized according to different techniques (differential scanning calorimetry, thermogravimetric analysis, Fourier transform infrared spectroscopy, nuclear magnetic resonance and scanning electron microscopy–energy‐dispersive X‐ray spectroscopy), which allowed the interpretation of their chemical and thermal properties. The fibers were then used in depolymerization reactions under the catalysis of the cutinase enzyme from Humicola insolens and under different process conditions (fiber length, buffer composition, enzyme dosage, temperature, pH).RESULTSCharacterization of PMLs revealed inorganic incrustation in the external fibers, and very similar crystallinity degrees. The molecular weight, on the other hand, was found to be lower in new mooring lines (27 489 g mol−1) as compared to used mooring lines (35 813–36 785 g mol−1). Throughout depolymerization reactions, a total concentration increase of terephthalic acid of up to 7.4 times (2593 μmol L–1) was achieved.CONCLUSIONThe results proved the concept that the studied cutinase can act on PET chains. To the best of our knowledge, this is the first report to describe an enzyme‐catalyzed depolymerization of PET from mooring lines, and the results add relevant knowledge for a circular economy of plastic products.

Biosensor-Based Optimization of Cutinase Secretion by Corynebacterium glutamicum.

The industrial microbe Corynebacterium glutamicum is gaining substantial importance as a platform host for recombinant protein secretion. We recently developed a fluorescence-based (eYFP) C. glutamicum reporter strain for the quantification of Sec-dependent protein secretion by monitoring the secretion-related stress response and now demonstrate its applicability in optimizing the secretion of the heterologous enzyme cutinase from Fusarium solani pisi. To drive secretion, either the poor-performing PelSP or the potent NprESP Sec signal peptide from Bacillus subtilis was used. To enable easy detection and quantification of the secreted cutinase we implemented the split green fluorescent protein (GFP) assay, which relies on the GFP11-tag fused to the C-terminus of the cutinase, which can complement a truncated GFP thereby reconstituting its fluorescence. The reporter strain was transformed with different mutant libraries created by error-prone PCR, which covered the region of the signal peptide and the N-terminus of the cutinase. Fluorescence-activated cell sorting (FACS) was performed to isolate cells that show increased fluorescence in response to increased protein secretion stress. Five PelSP variants were identified that showed a 4- to 6-fold increase in the amount and activity of the secreted cutinase (up to 4,100 U/L), whereas two improved NprESP variants were identified that showed a ∼35% increase in secretion, achieving ∼5,500 U/L. Most of the isolated variants carried mutations in the h-region of the signal peptide that increased its overall hydrophobicity. Using site-directed mutagenesis it was shown that the combined mutations F11I and P16S within the hydrophobic core of the PelSP are sufficient to boost cutinase secretion in batch cultivations to the same level as achieved by the NprESP. Screening of a PelSP mutant library in addition resulted in the identification of a cutinase variant with an increased specific activity, which was attributed to the mutation A85V located within the substrate-binding region. Taken together the biosensor-based optimization approach resulted in a substantial improvement of cutinase secretion by C. glutamicum, and therefore represents a valuable tool that can be applied to any secretory protein of interest.

Hydrolytic Degradation of Polyethylene Terephthalate by Cutinase Enzyme Derived from Fungal Biomass–Molecular Characterization

Polyethylene Terephthalate (PET) is utilized worldwide in plastic items, and its aggregation in the earth has turned into a worldwide concern. The hydrolysis of PET was done by cutinase and lipase enzyme. Lipase and cutinase enzymes were extracted from the fungal species isolated from soil. The isolated fungal species were identified as Aspergillus tamarii and Penicillium crustosum by 18S rRNA sequencing. Biodegradation test, cutinase enzyme-catalyzed PET hydrolysis, and different assays were conducted to elucidate PET hydrolysis by the extracted enzymes. These components incorporate cell surface connection inside biofilms, enzyme catalysts engaged with oxidation or hydrolysis of the plastic polymer, metabolic pathways in charge of take-up and osmosis of plastic parts, and synthetic factors favorable or inhibitory to the biodegradation procedure. The degradation of plastic was identified with terephthalic acid release (TPA) release as they are made of two monomeric units. Characterization studies such as FTIR and SEM analysis have exhibited loss of weight or change in physical properties, such as elasticity and the examination of Spectroscopic.

Functionalization Strategies and Fabrication of Solvent-Cast PLLA for Bioresorbable Stents

Actual polymer bioresorbable stents (BRS) generate a risk of device thrombosis as a consequence of the incomplete endothelialization after stent implantation. The material-tissue interactions are not fully controlled and stent fabrication techniques do not allow personalized medical solutions. This work investigates the effect of different functionalization strategies onto solvent-cast poly(l-lactic acid) (PLLA) surfaces with the capacity to enhance surface endothelial adhesion and the fabrication of 3D printed BRS. PLLA films were obtained by solvent casting and treated thermally to increase mechanical properties. Surface functionalization was performed by oxygen plasma (OP), sodium hydroxide (SH) etching, or cutinase enzyme (ET) hydrolysis, generating hydroxyl and carboxyl groups. A higher amount of carboxyl and hydroxyl groups was determined on OP and ET compared to the SH surfaces, as determined by contact angle and X-ray photoelectron spectroscopy (XPS). Endothelial cells (ECs) adhesion and spreading was higher on OP and ET functionalized surfaces correlated with the increase of functional groups without affecting the degradation. To verify the feasibility of the approach proposed, 3D printed PLLA BRS stents were produced by the solvent-cast direct writing technique.

Complete genome sequencing and comparative CAZyme analysis of Rhodococcus sp. PAMC28705 and PAMC28707 provide insight into their biotechnological and phytopathogenic potential.

Study of carbohydrate-active enzymes (CAZymes) can reveal information about the lifestyle and behavior of an organism. Rhodococcus species is well known for xenobiotic metabolism; however, their carbohydrate utilization ability has been less discussed till date. This study aimed to present the CAZyme analysis of two Rhodococcus strains, PAMC28705 and PAMC28707, isolated from lichens in Antarctica, and compare them with other Rhodococcus, Mycobacterium, and Corynebacterium strains. Genome-wide computational analysis was performed using various tools. Results showed similarities in CAZymes across all the studied genera. All three genera showed potential for significant polysaccharide utilization, including starch, cellulose, and pectin referring their biotechnological potential. Keeping in mind the pathogenic strains listed across all three genera, CAZymes associated to pathogenicity were analyzed too. Cutinase enzyme, which has been associated with phytopathogenicity, was abundant in all the studied organisms. CAZyme gene cluster of Rhodococcus sp. PAMC28705 and Rhodococcus sp. PAMC28707 showed the insertion of cutinase in the cluster, further supporting their possible phytopathogenic properties.

Isolation and identification of keratinophilic fungi in soil samples from excavation area of ancient city of Stratonikeia, Turkey and determination of its enzyme potentials

Aim: To isolate and identyfy keratinophilic fungi from soil samples excavated excavation area within the ancient city of Stratonikeia, Turkey and determination of their enzyme potentials. Stratonikeia, a city in the interior of Caria, located at Eskihisar Village, in the Yatagan district of Mugla province of Turkey. Methodology: Keratin bating technique was applied for isolating of dermatophytes and keratinophilic fungi. Fungal isolate were identified by phenotyping and genotyping methods. Screening of protease, keratinase, cellulose, lipase and cutinase enzyme was carried at solid medium. Results: Non-dermatophyte species, viz., Aspergillus fumigatus, Engyodontium album, Chrysosporium keratinophilum, Lecanicillium lecani and Purpureocillium lilacinum were identified. Protease, keratinase and cellulase were determined at moderate and high levels, while lipase and cutinase were not recorded. Interpretation: Non-dermatophyte strains having high keratinase, cellulase and protease activities are not only involved in pathogenesis, but also have a great ecological significance due to keratin degrading potential.