Cutinase Activity Research Articles

Production of cutinase from Fusarium falciforme and its application for hydrophilicity improvement of polyethylene terephthalate fabric.

Among 23 isolates of cutinase-producing fungi from Thailand, one strain of Fusarium falciforme PBURU-T5 exhibited the greatest cutinase activity (3.36 ± 0.12Uml-1) against p-nitrophenyl butyrate. This strain was found to produce an inducible cutinase when cultivated in the liquid mineral medium containing cutin from papaya peel as the sole carbon source. By optimizing the production condition based on the central composite experimental design, the maximal cutinase activity up to 4.82 ± 0.18Uml-1 was attained under the condition: 0.4% (w/v) papaya cutin as the carbon source, 0.3% (w/v) peptone as the nitrogen source, incubation temperature at 30°C for 4days, and initial pH 7.0. The crude enzyme was optimally active at 35°C and pH 9.0 which was suitable for textile industrial application. The treatment with the crude PBURU-T5 cutinase (100Ug-1 dry weight of fabric) could enhance the wetting time, water adsorption and moisture regain of polyethylene terephthalate fabric up to 1.9-, 1.2- and 1.3-fold, respectively, comparing with the conventional 1M NaOH treatment. The increment of these fabric properties by enzymatic treatment could facilitate the dyeing process and enhance the fabric softness. Thus, F. falciforme PBURU-T5 is the promising source of cutinase for the modification of the PET fabric surface.

High-level expression of Humicola insolens cutinase in Pichia pastoris without carbon starvation and its use in cotton fabric bioscouring

Huimcola insolens cutinase (HiC) was heterologously expressed in Pichia pastoris. To avoid a carbon starvation step, fermentation was conducted using combinations of sorbitol with glycerol and methanol in the cell growth and induction phases, respectively. The cutinase productivity (27.71 U mL−1 h−1) was 9.93 U mL−1 h−1 greater than that achieved using traditional two-phase methods, and a cutinase activity of 2660 U mL−1, using p-nitrophenyl butyrate as substrate, was achieved after only 96 h in a 3-L bioreactor. Subsequently, the combination of HiC with Thermobifida fusca cutinase (TfC) in cotton fabric bioscouring was evaluated by monitoring the wettability and dyeability of the fabric. Treatment with 20 U mL−1 of HiC at 80 °C for 5 min followed by 30 U mL−1 of TfC at 50 °C for 1 h gave the best results. The total treatment time was shorter and performance was better than those seen with the alkali method.

Stabilization of cutinase by covalent attachment on magnetic nanoparticles and improvement of its catalytic activity by ultrasonication

This paper reports on stabilization of serine cutinase activity by immobilizing it through cross linking with glutaraldehyde on magnetic nanoparticles (Fe-NPs) and intensification of catalytic activity by ultrasonic treatment. The optimum parameters were cross linking with 10.52 mM glutaraldehyde for 90 min using 1:2 (w/w) ratio of enzyme:Fe-NPs. The characterization of cutinase-Fe-NPs was done by different instrumental analysis. Ultrasonic power showed a beneficial effect on the activity of free and immobilized cutinase at 5.76 and 7.63 W, respectively, after 12 min. Immobilization and ultrasonic treatment led to increments in kinetic parameters (Km and Vmax) along with noticeable changes in the secondary structural fractions of cutinase. Cutinase-Fe-NPs showed augmented pH (4–8) and thermal stability (40–60 °C). Considerably higher thermal inactivation kinetic constants (kd, t1/2 and D-value) and thermodynamic constants (Ed, ΔH°, ΔG° and ΔS°) highlighted superior thermostability of cutinase-Fe-NPs. Cutinase-Fe-NPs and ultrasound treated cutinase-Fe-NPs retained 61.88% and 38.76% activity during 21-day storage, and 82.82 and 80.69% activity after fifth reusability cycle, respectively.

Genome-Wide Identification and Expression Analysis of Cutinase Gene Family in Rhizoctonia cerealis and Functional Study of an Active Cutinase RcCUT1 in the Fungal-Wheat Interaction.

Wheat (Triticum aestivum L.) is a staple food of more than 50% of global population. Rhizoctonia cerealis is the causal agent of sharp eyespot, a devastating disease of cereal crops including wheat. Cutinases produced by fungal pathogens play important roles in host-pathogen compatible interactions, but little is known about cutinases in R. cerealis. In this study, we identified a total of six cutinase encoding genes from R. cerealis genome, designated as RcCUT1–RcCUT6, analyzed their expression patterns during the infection, and determined virulence role for RcCUT1. All the proteins, RcCUT1–RcCUT6, contain a highly conserved GYSKG motif and another conserved C-x(3)-D-x(2)-C-x(2)-[GS]-[GSD]-x(4)-[AP]-H motif in the carbohydrate esterase 5 domain. The RcCUT1, RcCUT2, RcCUT4, and RcCUT5 are predicted to be secreted proteins containing four cysteine residues. These six cutinase genes had different expression patterns during the fungal infection process to wheat, among which RcCUT1 was highly expressed across all the infection time points but RcCUT6 was not expressed at all and the others were expressed only at certain time points. Further, RcCUT1 was heterologously expressed in Escherichia coli to obtain a purified protein. The purified RcCUT1 was shown to possess the cutinase activity and be able to induce necrosis, H2O2 accumulation, and expression of defense-related genes when infiltrated into wheat and Nicotiana benthamiana leaves. In contrast, RcCUT1 protein with serine mutation at the first motif had no cutinase activity, consequently lost the ability to induce necrosis. Noticeably, application of the purified RcCUT1 with R. cerealis led to significantly higher levels of the disease in wheat leaves than application of the fungus alone. These results strongly suggest that RcCUT1 serves as a virulence factor for the fungus. This is the first investigation of the cutinase genes in R. cerealis and the findings provide an important insight into pathogenesis mechanisms of R. cerealis on wheat.

Engineering a Chimeric Lipase-cutinase (Lip-Cut) for Efficient Enzymatic Deinking of Waste Paper

Lipase and cutinase belong to the esterase family and have biological applications in many fields. To develop more efficient biocatalysts that can be used for waste paper deinking, a chimeric lipase-cutinase (Lip-Cut) was constructed and successfully overexpressed in Pichia pastoris. The chimeric Lip-Cut exhibited lipase and cutinase activities that were 127% and 210% higher than their parent enzymes, respectively. Cut was superior to Lip in ink removal and improvement of paper brightness than Lip. The Lip-Cut displayed a better ink removal efficiency and paper brightness than that of the Lip, Cut, and Lip/Cut mixture. When the chimeric Lip-Cut was used, the ink removal efficiencies were 25.8% and 16.2% higher than that of the control-treated laser-printed paper and newspaper, which had sheet brightness values of 88% ISO and 59% ISO, respectively. The results demonstrated that the proper construction of bi-functional Lip-Cut could enhance the catalytic properties through the synergistic action of the two moieties because of the complementary advantages in the substrate specificities and catalysis patterns of both enzymes. This may provide an effective way to engineer more efficient bi-functional lipases and cutinases for deinking waste paper.

Reduction of Extracellular Proteases Increased Activity and Stability of Heterologous Protein in $${ Aspergillus}$$ A s p e r g i l l u s $${ niger}$$ n i g e r

The heterologous protein production in Aspergillus niger is often limited by the activity of the host extracellular proteases. To improve heterologous production, a transcription factor controlling expression of several extracellular protease-encoding genes, prtT, was deleted in A. niger PY11 and the mutant (An\(\Delta \) prtT) characterised. Extracellular proteolytic activity of An\(\Delta \) prtT was reduced as compared to the wild type, a result that was confirmed by RT-PCR analyses that showed reduced expression levels of several protease gene transcripts. To compare the efficiency of the mutant and parental (PY11) strains as hosts for heterologous protein production, the cutinase gene from Glomerella cingulate, under control of the glucoamylase A promoter, was integrated into each genome. The cutinase activity of PY11 and An\(\Delta \) prtT harbouring the G. cingulata cutinase gene was increased 20- and 36-fold higher, respectively, than the untransformed parental strains, suggesting that the ability of the mutant to produce heterologous protein is better than the wild type. Cutinase activity in culture filtrates prepared using both strains was stable at \(4\,^{\circ }\hbox {C}\) for extended periods; however, during incubation at \(25^{\circ }\)C the heterologous cutinase in culture filtrates prepared using An\(\Delta \) prtT retained 80% activity after a two-week incubation versus the less than 3% activity that was retained in culture filtrates prepared using PY11. This indicates that the reduction of extracellular proteases greatly improves the stability of heterologous proteins produced by A. niger.

Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes

ABSTRACTA total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200 rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent Km and Vmax values of the cutinase for p-nitrophenyl butyrate were 0.72 mM and 336.8 µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.

High-level expression and characterization of a novel cutinase from Malbranchea cinnamomea suitable for butyl butyrate production

BackgroundButyl butyrate has been considered as a promising fuel source because it is a kind of natural ester which can be converted from renewable and sustainable lignocellulosic biomass. Compared with the conventional chemical methods for butyl butyrate production, the enzymatic approach has been demonstrated to be more attractive, mainly owing to the mild reaction conditions, high specificity, low energy consumption, and environmental friendliness. Cutinases play an important role in the butyl butyrate production process. However, the production level of cutinases is still relatively low. Thus, to identify novel cutinases suitable for butyl butyrate synthesis and enhance their yields is of great value in biofuel industry.ResultsA novel cutinase gene (McCut) was cloned from a thermophilic fungus Malbranchea cinnamomea and expressed in Pichia pastoris. The highest cutinase activity of 12, 536 U/mL was achieved in 5-L fermentor, which is by far the highest production for a cutinase. McCut was optimally active at pH 8.0 and 45 °C. It exhibited excellent stability within the pH range of 3.0–10.5 and up to 75 °C. The cutinase displayed broad substrate specificity with the highest activity towards p-nitrophenyl butyrate and tributyrin. It was capable of hydrolyzing cutin, polycaprolactone, and poly(butylene succinate). Moreover, McCut efficiently synthesized butyl butyrate with a maximum esterification efficiency of 96.9% at 4 h. The overall structure of McCut was resolved as a typical α/β-hydrolase fold. The structural differences between McCut and Aspergillus oryzae cutinase in groove and loop provide valuable information for redesign of McCut. These excellent features make it useful in biosynthesis and biodegradation fields.ConclusionsA novel cutinase from M. cinnamomea was identified and characterized for the first time. High-level expression by P. pastoris is by far the highest for a cutinase. The enzyme exhibited excellent stability and high esterification efficiency for butyl butyrate production, which may make it a good candidate in biofuel and chemical industries.

Evaluation and application of constitutive promoters for cutinase production by Saccharomyces cerevisiae

Cutinase as a promising biocatalyst has been intensively studied and applied in processes targeted for industrial scale. In this work, the cutinase gene tfu from Thermobifida fusca was artificially synthesized according to codon usage bias of Saccharomyces cerevisiae and investigated in Saccharomyces cerevisiae. Using the α-factor signal peptide, the T. fusca cutinase was successfully overexpressed and secreted with the GAL1 expression system. To increase the cutinase level and overcome some of the drawbacks of induction, four different strong promoters (ADH1, HXT1, TEF1, and TDH3) were comparatively evaluated for cutinase production. By comparison, promoter TEF1 exhibited an outstanding property and significantly increased the expression level. By fed-batch fermentation with a constant feeding approach, the activity of cutinase was increased to 29.7 U/ml. The result will contribute to apply constitutive promoter TEF1 as a tool for targeted cutinase production in S. cerevisiae cell factory.

Cleavage Product Accumulation Decreases the Activity of Cutinase during PET Hydrolysis.

The Fusarium solani cutinase (FsC) is a promising candidate for the enzymatic degradation of the synthetic polyester polyethylene terephthalate (PET) but still suffers from a lack of activity. Using atomic MD simulations with different concentrations of cleavage product ethylene glycol (EG), we show influences of EG on the dynamic of FsC. We observed accumulation of EG in the active site region reducing the local flexibility of FsC. Furthermore, we used a coarse-grained mechanical model to investigate whether substrate binding in the active site causes an induced fit. We observed this supposed induced fit or "breath-like" movement during substrate binding indicating that the active site has to be flexible for substrate conversion. This guides rational design: mutants with an increased flexibility near the active site should be considered to compensate the solvent-mediated reduction in activity.

Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum.

BackgroundTechnical bulk enzymes represent a huge market, and the extracellular production of such enzymes is favorable due to lowered cost for product recovery. Protein secretion can be achieved via general secretion (Sec) pathway. Specific sequences, signal peptides (SPs), are necessary to direct the target protein into the translocation machinery. For example, >150 Sec-specific SPs have been identified for Bacillus subtilis alone. As the best SP for a target protein of choice cannot be predicted a priori, screening of homologous SPs has been shown to be a powerful tool for different expression organisms. While SP libraries between closely related species were successfully applied to optimize recombinant protein secretion, this was not investigated for distantly related species. Therefore, in this study a Sec SP library from low-GC firmicutes B. subtilis is investigated to optimize protein secretion in high-GC actinobacterium Corynebacterium glutamicum using cutinase from Fusarium solani pisi as model protein.ResultsA homologous SP library (~150 SP) for recombinant cutinase secretion in B. subtilis was successfully transferred to C. glutamicum as alternative secretion host. Cutinase secretion in C. glutamicum was quantified using an automated micro scale cultivation system for online growth monitoring, cell separation and cutinase activity determination. Secretion phenotyping results were correlated to those from a previous study, in which the same SP library was used to optimize secretion of the same cutinase but using B. subtilis as host. Strikingly, behavior of specific SP-cutinase combinations was changed dramatically between B. subtilis and C. glutamicum. Some SPs showed comparable cutinase secretion performances in both hosts, whereas other SPs caused diametrical extracellular cutinase activities.ConclusionThe optimal production strain for a specific target protein of choice still cannot be designed in silico. Not only the best SP for a target protein has to be evaluated each time from scratch, the expression host also affects which SP is best. Thus, (heterologous) SP library screening using high-throughput methods is considered to be crucial to construct an optimal production strain for a target protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0604-6) contains supplementary material, which is available to authorized users.

Effect of Tris, MOPS, and phosphate buffers on the hydrolysis of polyethylene terephthalate films by polyester hydrolases.

The enzymatic degradation of polyethylene terephthalate (PET) occurs at mild reaction conditions and may find applications in environmentally friendly plastic waste recycling processes. The hydrolytic activity of the homologous polyester hydrolases LC cutinase (LCC) from a compost metagenome and TfCut2 from Thermobifida fusca KW3 against PET films was strongly influenced by the reaction medium buffers tris(hydroxymethyl)aminomethane (Tris), 3‐(N‐morpholino)propanesulfonic acid (MOPS), and sodium phosphate. LCC showed the highest initial hydrolysis rate of PET films in 0.2 m Tris, while the rate of TfCut2 was 2.1‐fold lower at this buffer concentration. At a Tris concentration of 1 m, the hydrolysis rate of LCC decreased by more than 90% and of TfCut2 by about 80%. In 0.2 m MOPS or sodium phosphate buffer, no significant differences in the maximum initial hydrolysis rates of PET films by both enzymes were detected. When the concentration of MOPS was increased to 1 m, the hydrolysis rate of LCC decreased by about 90%. The activity of TfCut2 remained low compared to the increasing hydrolysis rates observed at higher concentrations of sodium phosphate buffer. In contrast, the activity of LCC did not change at different concentrations of this buffer. An inhibition study suggested a competitive inhibition of TfCut2 and LCC by Tris and MOPS. Molecular docking showed that Tris and MOPS interfered with the binding of the polymeric substrate in a groove located at the protein surface. A comparison of the K i values and the average binding energies indicated MOPS as the stronger inhibitor of the both enzymes.

Optimization of medium parameters by response surface methodology (RSM) for enhanced production of cutinase from Aspergillus sp. RL2Ct.

Cutinases are hydrolytic enzymes which catalyzes esterification and trans-esterification reactions that make them highly potential industrial biocatalyst. In the present investigation microorganisms showing cutinase activity were isolated from plant samples. The strain showing maximum cutinase activity was identified by 18S rDNA sequencing as Aspergillus sp. RL2Ct and was selected for further studies. To achieve maximum enzyme production, the medium components affecting cutinase production were screened by Plackett–Burman followed by central composite design. The results obtained suggested that cutin, temperature and CaCl2 have influenced the cutinase production significantly with very high confidence levels. Cutinase production was maximum (663 U/mg protein) when using cutin prepared from orange peel as sole source of carbon. An overall 4.33-fold increase in the production of cutinase was observed after optimization of culture conditions (including 2.5-fold increase using RSM) during 24 h of incubation. The production time of Aspergillus sp. RL2Ct cutinase is significantly lower than the most of the earlier reported cutinase-producing fungus.

CglCUT1 gene required for cutinase activity and pathogenicity of Colletotrichum gloeosporioides causing anthracnose of Camellia oleifera

Colletotrichum gloeosporioides is the causal agent of Camellia oleifera anthracnose, mainly infecting fruits and leaves. The fungus secretes degrading enzymes to destroy the cuticle of aerial plant parts and help infect the host successfully. To validate whether a cutinase gene (CglCUT1) was required for cutinase activity and pathogenicity of C. gloeosporioides, the CglCUT1 gene was cloned and analyzed. The characterization of CglCUT1 predicted protein suggests that the cloned DNA encoded a cutinase in C. gloeosporioides affecting C. oleifera. The CglCUT1 showed a high homology to those from C. gloeosporioides causing papaya anthracnose and C. capsici causing pepper anthracnose, as well as those of other ascomycetes. The whole CglCUT1 gene was knocked-out and the knockout mutant (∆CglCUT39) was subsequently complemented using Agrobacterium tumefaciens mediated transformation. The knockout transformants exhibited significant decreases in cutinase activity and virulence compared with the wild-type strain. The complemented transformants of the disrupted transformant ∆CglCUT39 showed a significant increase in cutinase activity and virulence compared with the disrupted transformant ∆CglCUT39. This study suggests that the CglCUT1 gene has a positive effect on fungal virulence of the hemibiotrophic C. gloeosporioides on C. oleifera.

Production of secretory cutinase by recombinant Saccharomyces cerevisiae protoplasts.

Background During heterologous protein production using recombinant microbes, the protein tends to accumulate in the cell and may not be secreted. Here, we studied the production of secretory cutinase (heterologous protein) by recombinant Saccharomyces cerevisiae protoplasts.Findings Recombinant S. cerevisiae cells (i.e., cells into which the cutinase gene was transferred) secreted trace amounts of cutinase into the broth. Approximately 28 % of the cutinase produced in the cells localized to the cell walls and/or between cell wall and cell membrane (CW). In comparison with cell culture, protoplasts in a static culture secreted measurable amounts of cutinase into the broth. Protoplasts were protected from physical and osmotic stresses by entrapping them in a membrane capsule with a low-viscous liquid-core of 1.92 % w/v calcium-alginate. To increase secretory cutinase production, the entrapped protoplasts were cultivated in a shake flask at low osmotic pressure without disruption. During 60 h of cultivation, the extracellular cutinase activity of the free protoplasts at 29.3 atm and protoplasts entrapped in the capsule at 17.2 atm were 0.13 and 0.39 U/mL, respectively.ConclusionsThis is the first report which demonstrates that the efficient production of a secretable enzyme by using protoplasts isolated from recombinant microbes. This system described here is useful to produce products that accumulate in the CW.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-1806-4) contains supplementary material, which is available to authorized users.

Enhanced extracellular expression of gene-optimized Thermobifida fusca cutinase in Escherichia coli by optimization of induction strategy

Our previous study demonstrated that when Thermobifida fusca cutinase was expressed in Escherichia coli without mediation of a signal peptide, it could release to the culture medium via the enhanced membrane permeability, which was based on its limited phospholipid hydrolysis activity. The goal of the present work was to achieve highly efficient extracellular production of the recombinant signal peptide-free cutinase in E. coli. The codons of the T. fusca cutinase gene were optimized for expression in E. coli, and a recombinant expression system was constructed using this optimized gene. After that, the induction strategy was optimized using high-cell-density cultivation in a 3-L fermentor. Results showed that the optimal induction condition was at a dry cell weight (DCW) of 13gL−1, and an IPTG/lactose combination induction strategy is proposed, in which IPTG is added once in a final concentration of 25.0μM, and lactose is fed at a rate of 0.5gL−1h−1. In this condition, an extracellular cutinase activity of 2258.5UmL−1 (5.1gL−1) was achieved, which represented the highest cutinase production ever reported, and demonstrated the potential of this system for the industrial production of cutinase.

The Role of Cutinase and its Impact on Pathogenicity of Colletotrichum truncatum

The phytopathogenic fungus, Colletotrichum truncatum infects and colonises chili fruit through direct penetration of the cuticle. The cutinase gene of C. truncatum (CtCut1), a cutin degrading enzyme was identified, cloned and shown to be essential in breaching the cuticle of chili fruit. The expression of CtCut1 gene was studied through RNA-mediated gene silencing and its impact on fungal pathogenicity was demonstrated. The vector, pAA1 encoding a hairpin RNA of GFP and CtCut1 was constructed and transformed into C. truncatum pathotype F8-3B (virulent strain). F8-3B-pAA1 transformants exhibited reduced patterns of infection with one isolate having a 45.8% reduction in cutinase activity (reduction in CtCut1 transcript) in comparison to the wild type. Importantly, CtCut1-deficient strains were unable to infect detached chili and soybean hosts as efficiently as the wild type. There was a delay in the infection period by the transformants. Nevertheless, artificial wounding of the cuticle enabled these F8-3B-pAA1 transformants to infect and colonise host tissues, resulting in typical anthracnose disease lesions. Coupled with microscopy, these data suggested that the defect in pathogenicity was likely due to a failure in penetration of the host cutin. Knowledge of the plant-fungal interactions arose from the development of a fungal transformation system for C. truncatum and implementation of RNAi technology. This technology thus provides an alternative genetic tool for studies of gene function, particularly of essential pathogenicity genes.

Characterisation of new aqueous two-phase systems comprising of Dehypon®LS54 and K4484®Dextrin for potential cutinase recovery

A new aqueous two-phase system composed of a non-ionic surfactant, Dehypon®LS54 (abbreviated as Ls54) and a starch derivative, K4484®Dextrin (abbreviated as Dx), was prepared in different compositions and the formed phases were characterized for physical properties such as density and viscosity. The system was later investigated to be applied for the extraction cutinase enzyme. The phase formation behaviour subjected to temperature and pH were studied. The system equilibrium represented by a binodal diagram, was constructed by using cloud point method. The binodal curve was suitably fitted with Merchuk equation. The correlated tie lines associated with the equation were determined by the use of lever arm rule of the mass phase ratio. The results showed that the tie line length (TLL) had a strong influence on system’s properties such as phase volume ratio and viscosity difference, however, the density of systems were not strongly affected. Unlike pH, the temperature changes clearly affected the phase formation ratio. Furthermore, cutinase incubation in the Ls54 and Dx solutions had resulted small variation in cutinase activity, suggesting that the presence of the phase-forming polymer did not reduce the stability of the targeted enzyme. The effects of pH, tie line length and phase volume ratio on partition coefficient, recovery and purification of the enzyme were further investigated. The optimal pH of system containing 22%w/w Ls54 and 12.5%w/w Dx was found at pH 8.0 with the partition coefficient, kcut 1.32, yield 65% and purification factor of 6.92.

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Cutinase is a multifunctional esterase with potential industrial applications. In the present study, a truncated version of the extracellular Thermobifida fusca cutinase without a signal peptide (referred to as cutinase(NS)) was heterologously expressed in Escherichia coli BL21(DE3). The results showed that the majority of the cutinase activity was located in the culture medium. In a 3-liter fermentor, the cutinase activity in the culture medium reached 1,063.5 U/ml (2,380.8 mg/liter), and the productivity was 40.9 U/ml/h. Biochemical characterization of the purified cutinase(NS) showed that it has enzymatic properties similar to those of the wild-type enzyme. In addition, E. coli cells producing inactive cutinase(NS)S130A were constructed, and it was found that the majority of the inactive enzyme was located in the cytoplasm. Furthermore, T. fusca cutinase was confirmed to have hydrolytic activity toward phospholipids, an important component of the cell membrane. Compared to the cells expressing the inactive cutinase(NS)S130A, the cells expressing cutinase(NS) showed increased membrane permeability and irregular morphology. Based on these results, a hypothesis of "cell leakage induced by the limited phospholipid hydrolysis of cutinase(NS)" was proposed to explain the underlying mechanism for the extracellular release of cutinase(NS).

The galactolipase activity of some microbial lipases and pancreatic enzymes

AbstractSeveral well known microbial lipases were screened for their ability to hydrolyze synthetic medium chain monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). Fusarium solani cutinase and Thermomyces lanuginosus lipase (TLL) were found to hydrolyze MGDG at high rates (984 ± 62 and 450 ± 41 U/mg, respectively). These activities remained however lower than those measured with pancreatic lipase‐related protein 2 (PLRP2) on the same substrate. As previously observed with PLRP2, galactolipid–bile salt mixed micelles were found to be the best substrate form for microbial enzymes. The galactolipid to bile salt molar ratios for measuring maximum galactolipase activities were found to be similar to those previously established with PLRP2, suggesting that bile salts have mainly an effect on the substrate and not on the enzyme itself. The galactolipase activity of cutinase and TLL, as well as human and guinea pig PLRP2s were also measured using galactolipid monomolecular films. Enzymes having a lid (TLL and human PLRP2) were found to act at higher surface pressures than those with no lid (cutinase and guinea pig PLRP2). In silico docking of medium chain MGDG and DGDG in the active site of guinea pig PLRP2 and TLL reveals some structural analogies between these enzymes.Practical applications: Galactolipase activity already finds applications in baking process with the partial lipolysis of galactolipids present in wheat flour and the in situ production of emulsifier molecules. More generally, these enzymes could be used for the enzymatic modification of galactolipids, the main plant lipids. These lipids are considered to be the most abundant at the surface of the earth and thus constitute the largest resource of fatty acids.