Fusarium Solani Pisi Research Articles

Thermal Stabilization of Lipases Bound to Solid-Phase Triazine-Scaffolded Biomimetic Ligands: A Preliminary Assessment

Biomimetic ligands are synthetic compounds that mimic the structure and binding properties of natural biological ligands. The first uses of textile dyes as pseudo-affinity ligands paved the way for the rational design and de novo synthesis of low-cost, non-toxic and highly stable triazine-scaffolded affinity ligands. A novel method to assess and enhance protein stability, employing triazine-based biomimetic ligands and using cutinase from Fusarium solani pisi as a protein model, has been previously reported. This innovative approach combined the concepts of molecular modeling and solid-phase combinatorial chemistry to design, synthesize and screen biomimetic compounds able to bind cutinase through complementary affinity-like interactions while maintaining its biological functionality. The screening of a 36-member biased combinatorial library enabled the identification of promising lead ligands. The immobilization/adsorption of cutinase onto a particular lead (ligand 3′/11) led to a noteworthy enhancement in thermal stability within the temperature range of 60–80 °C. In the present study, similar triazine-based compounds, sourced from the same combinatorial library and mimicking dipeptides of diverse amino acids, were selected and studied to determine their effectiveness in binding and/or improving the thermal stability of several lipases, enzymes which are closely related in function to cutinases. Three ligands with different compositions were screened for their potential thermostabilizing effect on different lipolytic enzymes at 60 °C. An entirely distinct enzyme, invertase from Saccharomyces cerevisiae, was also assessed for binding to the same ligands and functioned as a ‘control’ for the experiments with lipases. The high binding yield of ligand 3′/11 [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid] to cutinase was confirmed, and the same ligand was tested for its ability to bind lipases from Aspergillus oryzae (AOL), Candida rugosa (CRL), Chromobacterium viscosum (CVL), Rhizomucor miehei (RML) and Rhizopus niveus (RNL). The enzymes CRL, CVL, RNL and invertase showed significant adsorption yields to ligand 3′/11—32, 29, 36 and 94%, respectively, and the thermal stability at 60 °C of free and adsorbed enzymes was studied. CVL and RNL were also stabilized by adsorption to ligand 3′/11. In the case of CRL and invertase, which bound but were not stabilized by ligand (3′/11), other ligands from the original combinatorial library were tested. Between the two alternative ligands, one was effective at stabilizing C. rugosa lipase, while none stabilized invertase.

One-Step Purification of Recombinant Cutinase from an E. coli Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand.

Cutinase from Fusarium solani pisi is an enzyme that bridges functional properties between lipases and esterases, with applications in detergents, food processing, and the synthesis of fine chemicals. The purification procedure of recombinant cutinase from E. coil extracts is a well-established but time-consuming process, which involves a sequence of two anionic exchange chromatography steps followed by dialysis. Affinity chromatography is the most efficient method for protein purification, the major limitation of its use being often the availability of a ligand selective for a given target protein. Synthetic affinity ligands that specifically recognize certain sites on the surface of proteins are highly desirable for affinity processes due to their cost-effectiveness, durability, and reusability across multiple cycles. Additionally, these ligands establish moderate affinity interactions with the target protein, making it possible to purify proteins under gentle conditions while maintaining high levels of activity recovery. This study aimed to develop a new method for purifying cutinase, utilizing triazine-scaffolded biomimetic affinity ligands. These ligands were previously screened from a biased-combinatorial library to ensure their binding ability to cutinase without compromising its biological function. A lead ligand, designated as 11/3', [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid], was chosen and directly synthesized onto agarose. Experiments conducted at different scales demonstrated that this ligand (with an affinity constant Ka ≈ 104 M-1) exhibited selectivity towards cutinase, enabling the purification of the enzyme from an E. coli crude production medium in a single step. Under optimized conditions, the protein and activity yields reached 25% and 90%, respectively, with a resulting cutinase purity of 85%.

Analysis of protein secretion in Bacillus subtilis by combining a secretion stress biosensor strain with an in vivo split GFP assay

BackgroundBacillus subtilis is one of the workhorses in industrial biotechnology and well known for its secretion potential. Efficient secretion of recombinant proteins still requires extensive optimization campaigns and screening with activity-based methods. However, not every protein can be detected by activity-based screening. We therefore developed a combined online monitoring system, consisting of an in vivo split GFP assay for activity-independent target detection and an mCherry-based secretion stress biosensor. The split GFP assay is based on the fusion of a target protein to the eleventh β-sheet of sfGFP, which can complement a truncated sfGFP that lacks this β-sheet named GFP1-10. The secretion stress biosensor makes use of the CssRS two component quality control system, which upregulates expression of mCherry in the htrA locus thereby allowing a fluorescence readout of secretion stress.ResultsThe biosensor strain B. subtilis PAL5 was successfully constructed by exchanging the protease encoding gene htrA with mCherry via CRISPR/Cas9. The Fusarium solani pisi cutinase Cut fused to the GFP11 tag (Cut11) was used as a model enzyme to determine the stress response upon secretion mediated by signal peptides SPPel, SPEpr and SPBsn obtained from naturally secreted proteins of B. subtilis. An in vivo split GFP assay was developed, where purified GFP1-10 is added to the culture broth. By combining both methods, an activity-independent high-throughput method was created, that allowed optimization of Cut11 secretion. Using the split GFP-based detection assay, we demonstrated a good correlation between the amount of secreted cutinase and the enzymatic activity. Additionally, we screened a signal peptide library and identified new signal peptide variants that led to improved secretion while maintaining low stress levels.ConclusionOur results demonstrate that the combination of a split GFP-based detection assay for secreted proteins with a secretion stress biosensor strain enables both, online detection of extracellular target proteins and identification of bottlenecks during protein secretion in B. subtilis. In general, the system described here will also enable to monitor the secretion stress response provoked by using inducible promoters governing the expression of different enzymes.

Biochemical Characterization and NMR Study of a PET-Hydrolyzing Cutinase from Fusarium solani pisi.

In recent years, the drawbacks of plastics have become evident, with plastic pollution becoming a major environmental issue. There is an urgent need to find solutions to efficiently manage plastic waste by using novel recycling methods. Biocatalytic recycling of plastics by using enzyme-catalyzed hydrolysis is one such solution that has gained interest, in particular for recycling poly(ethylene terephthalate) (PET). To provide insights into PET hydrolysis by cutinases, we have here characterized the kinetics of a PET-hydrolyzing cutinase from Fusarium solani pisi (FsC) at different pH values, mapped the interaction between FsC and the PET analogue BHET by using NMR spectroscopy, and monitored product release directly and in real time by using time-resolved NMR experiments. We found that primarily aliphatic side chains around the active site participate in the interaction with BHET and that pH conditions and a mutation around the active site (L182A) can be used to tune the relative amounts of degradation products. Moreover, we propose that the low catalytic performance of FsC on PET is caused by poor substrate binding combined with slow MHET hydrolysis. Overall, our results provide insights into obstacles that preclude efficient PET hydrolysis by FsC and suggest future approaches for overcoming these obstacles and generating efficient PET-hydrolyzing enzymes.

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.

Improved pEKEx2-derived expression vectors for tightly controlled production of recombinant proteins in Corynebacterium glutamicum

The Escherichia coli/Corynebacterium glutamicum shuttle vector pEKEx2 is an IPTG-inducible expression vector that has been used successfully for the synthesis of numerous proteins in C. glutamicum. We discovered that the leaky gene expression observed for pEKEx2-derived plasmids relates to reduced functionality of the plasmid-encoded repressor LacI carrying a modified C-terminus, while duplicate DNA sequences in the pEKEx2 backbone contribute to plasmid instability. We constructed the pEKEx2-derivatives pPBEx2 and pPREx2, which harbor a restored lacI gene and which lack the unnecessary duplicate DNA sequences. pPREx2 in addition enables fusion of target genes to a C-terminal Strep-tag II coding region for easy protein detection and purification. In the absence of inducer, the novel vectors exhibit tight gene repression in C. glutamicum, as shown for the secretory production of Fusarium solani pisi cutinase and the cytosolic production of green fluorescent protein and C. glutamicum myo-inositol dehydrogenase. Undesired heterogeneity amongst clones expressing cutinase from pEKEx2 was attributed to the loss of a vector fragment containing the cutinase gene, which likely occurred via homologous recombination of the identical flanking DNA sequences. Such loss was not observed for pPBEx2. Using pPREx2, IolG-Strep was successfully produced and purified to homogeneity by Strep-Tactin affinity chromatography, obtaining 1.5 mg IolG with a specific activity of 27 μmol·min−1·(mg protein)−1 from 100 mL culture. The tight gene repression in the absence of inducer and the improved plasmid stability make expression vectors pPBEx2/pPREx2 attractive alternatives to the available molecular tools for genetic manipulation and high-level production of recombinant proteins in C. glutamicum.

Screening of a genome-reduced Corynebacterium glutamicum strain library for improved heterologous cutinase secretion.

The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genome-reduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. High-throughput phenotyping using robotics-integrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. The biomass-specific cutinase yield for strain GRS41_51_NprE was increased by~200%, although the growth rate was reduced by~60%. Importantly, the causative deletions of genomic clusters cg2801-cg2828 and rrnC-cg3298 could not have been inferred a priori. Strikingly, bioreactor fed-batch cultivations at controlled growth rates resulted in a complete reversal of the screening results, with the cutinase yield for strain GRS41_51_NprE dropping by~25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a 'high-performance' strain from batch screening into a 'low-performance' strain in fed-batch cultivation. In conclusion, future studies are needed in order to understand metabolic adaptations of C.glutamicum to both genomic deletions and different bioprocess conditions.

A secretion biosensor for monitoring Sec-dependent protein export in Corynebacterium glutamicum

BackgroundIn recent years, the industrial workhorse Corynebacterium glutamicum has gained increasing interest as a host organism for the secretory production of heterologous proteins. Generally, the yield of a target protein in the culture supernatant depends on a multitude of interdependent biological and bioprocess parameters which have to be optimized. So far, the monitoring of such optimization processes depends on the availability of a direct assay for the respective target protein that can be handled also in high throughput approaches. Since simple assays, such as standard enzymatic activity assays, are not always at hand, the availability of a general protein secretion biosensor is highly desirable.ResultsHigh level secretion of proteins via the Sec protein export pathway leads to secretion stress, a phenomenon that is thought to be caused by the accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. We have analyzed the transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins and found that, in both cases, the expression of the gene encoding a homologue of the extracytosolic HtrA protease was highly upregulated. Based on this finding, a C. glutamicum Sec secretion biosensor strain was constructed in which the htrA gene on the chromosome was replaced by the eyfp gene. The fluorescence of the resulting reporter strain responded to the secretion of different heterologous proteins (cutinase from Fusarium solani pisi and alkaline phosphatase PhoA from Escherichia coli) in a dose-dependent manner. In addition, three differently efficient signal peptides for the secretory production of the cutinase could be differentiated by the biosensor signal. Furthermore, we have shown that an efficient signal peptide can be separated from a poor signal peptide by using the biosensor signal of the respective cells in fluorescence activated cell sorting experiments.ConclusionsWe have succeeded in the construction of a C. glutamicum biosensor strain that allows for the monitoring of Sec-dependent secretion of heterologous proteins in a dose-dependent manner, independent of a direct assay for the desired target protein.

Catalytic Activation of Esterases by PEGylation for Polyester Synthesis

AbstractIn this work we explored PEGylation as an efficient strategy to improve esterase's catalytic performance. For this, we PEGylated three esterases, namely lipase from Candida antarctica B (CALB), lipase from Thermomyces lanuginosus (TL) and cutinase from Fusarium solani pisi (CUT) and evaluated their catalytic performance by using the biosynthesis of poly(ethylene glutarate) as model reaction. After PEGylation with a 5 kDa aldehyde‐PEG, CALB and cutinase revealed an increase of activity against p‐nitrophenyl butyrate hydrolysis (2‐fold of increase for CALB and 4‐fold of increase for cutinase). Unmodified and PEGylated lipase TL displayed however similar activity results. The polymerase activity of native and PEGylated esterases was also assessed. The data revealed a higher polymerase activity for the lipase TL and cutinase PEGylated forms (88 % conversion for PEG‐lipase TL and 34 % for PEG‐cutinase). Molecular dynamics were used to evaluate the effect of PEG on the geometry of the active site of enzymes with lid domain (TL and CALB). These studies corroborate the experimental data revealing a more open active site cavity for the PEGylated catalysts facilitating the catalysis.

Biocatalytic Degradation of Parabens Mediated by Cell Surface Displayed Cutinase.

Parabens are emerging environmental contaminants with known endocrine-disrupting effects. This study created a novel biocatalyst (named as SDFsC) by expressing the enzyme Fusarium solani pisi cutinase (FsC) on the cell surface of Baker's yeast Sacchromycese cerevisiae and demonstrated successful enzyme-mediated removal of parabens for the first time. Parabens with different side chain structures had different degradation rates by the SDFsC. The SDFsC preferentially degraded the parabens with relatively long alkyl or aromatic side chains. The structure-dependent degradability was in a good agreement with the binding energy between the active site of FsC and different parabens. In real wastewater effluent solution, the SDFsC effectively degraded 800 μg/L of propylparaben, butylparaben, and benzylparaben, either as a single compound or as a mixture, within 48 h. The estrogenic activity of parabens was considerably reduced as the parent parabens were degraded into 4-hydroxybenzoic acid via hydrolysis pathway by the SDFsC. The SDFsC showed superior reusability and maintained 93% of its initial catalytic activity after six rounds of paraben degradation reaction. Results from this study provide scientific basis for developing biocatalysis as a green chemistry alternative for advanced treatment of parabens in sustainable water reclamation.

Improvement of enzyme stability for alkyl esters synthesis in miniemulsion systems by using media engineering

AbstractBACKGROUNDOil‐in‐water miniemulsions have been suggested to display potential for application as a ‘green’ system for enzymatic alkyl ester synthesis. Still, a realistic assessment of the feasibility of this approach for practical applications requires further insight, namely the relation between enzyme stability and reaction operational conditions. The objective of this work is within such scope. Accordingly, it extends previous research on esterification catalyzed by Fusarium solani pisi cutinase, by addressing medium‐chain length substrates, and aims to contribute to the optimization of reaction conditions and towards the design of an efficient set‐up.RESULTSThe esterification yield was significantly enhanced (e.g. 50% increase for octyl octanoate system) through the correction of the initial pH of the reaction medium to pH 6. Under such environment, conformational changes of the enzyme were negligible after 24 h, resulting in improved enzyme stability for all systems under study. Maximum esterification rate (3.59 mmol min‐1) and product yield (91%) were achieved for octyl decanoate synthesis. Fed‐batch mode of operation allowed for the production of 1 mol L‐1 hexyl octanoate, while retaining enzyme activity over 5 days. Consecutive batch runs allowed for the cumulative production of 1.1 mol L‐1 hexyl octanoate after four cycles.CONCLUSIONSMedium engineering enabled the use of miniemulsion systems for the synthesis of medium‐chain alkyl esters and increase in the final concentration of the intended product. Through a fed‐batch mode the enzyme stability and reutilization were tuned allowing long‐term utilization of the free enzyme which could not be achieved through a repeated batch system. © 2017 Society of Chemical Industry

Comparison of cutinases in enzymic deinking of old newsprint

In this study, the impact of cutinases from Thermobifida fusca and Fusarium solani pisi on the deinking of old newsprint were evaluated for the first time. When repulped old newsprint was treated with 8 U/g of T. fusca cutinase at pH 8 and 60 $$^{\circ }$$ C for 30 min, the brightness of the deinked papers reached 42.01%. With 8 U/g F. solani cutinase at pH 8.5 and 35 $$^{\circ }$$ C for 30 min, the brightness reached 41.62%. These brightness values are higher than that achieved using chemical deinking by 5.13 and 4.38%, respectively. These brightness values were also superior to that achieved using commercial lipase in this study and those previously reported using cellulases, hemicellulases, and laccase, which were higher than that achieved using chemical deinking by 2–4%. The mechanical properties of the deinked paper, including tensile index, tear index, and burst index, were also measured. The results showed that the two cutinases had different effects on the paper fibers. In summary, both T. fusca and F. solani pisi cutinases were able to remove ink from old newsprint more efficiently than alkaline chemistry or other enzymes. This study provides a potential strategy to deink efficiently old newsprint using cutinase.

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.

Potential of the solid-state fermentation of tomato by products by Fusarium solani pisi for enzymatic extraction of lycopene

The present study aims to evaluate the direct use of crude enzymes extract produced by solid state fermentation (SSF) of Fusarium solani pisi to improve lycopene extraction from tomato processing by products (TPB) and thus avoiding the downstream steps. Optimized production of the crude enzymes extract (23.90 μg/ml) was obtained after 7 days of incubation of TPB moistened with distilled water (1/3, w/v) at 30 °C. Afterwards, TPB was pretreated by the crude enzymes extract with pectinolytic, cellulolytic and cutinolytic activities and then subjected to ethanol extraction. The factors investigated included pH, temperature, particle size and solid-to-crude enzymes solution ratio and results provided the following optimal extraction conditions: pH = 8, T = 50 °C, solid-to-enzyme ratio = 1/30 (w/v) and TPB particle size between 0.8 and 1.25 mm. Lycopene recovery was moderately higher when extracted using F. solani crude enzymes extract compared to either pectinases and cellulases preparations. TPB enzymatic extract exhibited powerful antioxidant activity even better than the ethanol TPB extract. The obtained results strongly support the idea of using enzymes from SSF of F. solani as an effective means for recovering powerful antioxidant from tomato by products.

Kinetic model for the esterification of ethyl caproate for reaction optimization

Abstract The present work aims to achieve additional insight on a mechanism describing the fundamental steps involved in the esterification reactions catalyzed by cutinase. The synthesis of ethyl caproate has been used as a model system to obtain a suitable kinetic model to estimate the activation energies involved in the various steps of the reaction pathway. Kinetic measurements have been made for the enzymatic esterification of caproic acid with ethyl alcohol catalyzed by recombinant Fusarium solani pisi cutinase expressed in Saccharomyces cerevisiae SU50. Different temperature conditions, from 25 to 50 °C, were tested for two different alcohol/acid molar ratios ( R = 1 and R = 2). The third ordered Ping Pong Bi Bi mechanism with alcohol inhibition was shown to be able to describe the experimental results. The model shows that the productivity decreases as the reaction temperature increases.

Production of heterologous cutinases by E. coli and improved enzyme formulation for application on plastic degradation

Background: The hydrolytic action of cutinases has been applied to the degradation of plastics. Polyethylene terephthalate (PET) have long half-life which constitutes a major problem for their treatment as urban solid residues. The aim of this work was to characterize and to improve stable the enzyme to optimize the process of degradation using enzymatic hydrolysis of PET by recombinant cutinases. Results: The wild type form of cutinase from Fusarium solani pisi and its C-terminal fusion to cellulose binding domain N1 from Cellulomonas fimi were produced by genetically modified Escherichia coli . The maximum activity of cutinases produced in Lactose Broth in the presence of ampicillin and isopropyl β-D-1-thiogalactopyranoside (IPTG) was 1.4 IU/mL. Both cutinases had an optimum pH around 7.0 and they were stable between 30 and 50ºC during 90 min. The addition of glycerol, PEG-200 and (NH 4 ) 2 SO 4 to the metabolic liquid, concentrated by ultra filtration, stabilized the activity during 60 days at 28ºC. The treatment of PET with cutinases during 48 hrs led to maxima weight loss of 0.90%. Conclusions: Recombinant microbial cutinases may present advantages in the treatment of poly(ethylene terephthalate) PET through enzymatic treatments.

Lipases efficiently stearate and cutinases acetylate the surface of arabinoxylan films

This is the first report on successful enzyme catalyzed surface esterification of hemicellulose films. Enzyme catalyzed surface acetylation with vinyl acetate and stearation with vinyl stearate were studied on rye arabinoxylan (AX) films. Different surface analytical techniques (FT-IR, TOF-SIMS, ESCA, CA) show that lipases from Mucor javanicus, Rhizopus oryzae and Candida rugosa successfully surface stearate AX films and that a cutinase from Fusarium solani pisi surface acetylates these films. The specificities of cutinase and lipases were also compared, and higher activity was observed for lipases utilizing long alkyl chain substrates while higher activity was observed for cutinase utilizing shorter alkyl chain substrates. The contact angle analysis showed films with increased initial hydrophobicity on the surfaces.

Automation Highlights from the Literature

Automation Highlights from the Literature

Protein stabilization with a dipeptide‐mimic triazine‐scaffolded synthetic affinity ligand

Protein stabilization was achieved by a novel approach based on the adsorption and establishment of affinity-like interactions with a biomimetic triazine-scaffolded ligand. A synthetic lead compound (ligand 3'/11, K(a) ≈ 10(4) M(-1)) was selected from a previously screened solid-phase library of affinity ligands for studies of adsorption and stabilization of cutinase from Fusarium solani pisi used as a model system. This ligand, directly synthesized in agarose by a well-established solid-phase synthesis method, was able to strongly bind cutinase and led to impressive half-lives of more than 8 h at 70 °C, and of approximately 34 h at 60 °C for bound protein (a 25- and 57-fold increase as compared with the free enzyme, respectively). The ligand density in the solid matrix was found to be a determinant parameter for cutinase stabilization. It is conceivable that the highly stabilizing effect observed results from the binding of more than one ligand residue to the enzyme, creating specific macromolecular configurations that lock structural mobility thus improving molecular stability.

Fusarium solani pisi cutinase-catalyzed synthesis of polyamides

Polyamides, or nylon, are widely used in fiber and engineering plastic materials, due to their good mechanical and thermal properties. Synthesis of oligomers from nylon-4,10, nylon-6,10, and nylon-8,10 were performed via polycondensation of diamines (1,4-butanediamine, 1,6-hexanediamine, and 1,8-diaminooctane) and diester (diethyl sebacate). These reactions were catalyzed by immobilized cutinase from Fusarium solani pisi on Lewatit beads, cutinase in the form of cross-linked enzyme aggregates (CLEA), or by immobilized Lipase B from Candida antarctica (N435). The highest maximal degree of polymerization (DPmax), up to 16, can be achieved in the synthesis of nylon-8,10 catalyzed by CLEA cutinase in diphenyl ether at 70°C. By performing a reaction at cutinase optimal temperature (70°C), CLEA cutinase in the synthesis of nylons shows good catalytic activity, like N435.