Cutinase from Fusarium solani pisi was genetically modified near the active site, by site-directed mutagenesis, to enhance its activity towards polyethylene terephthalate (PET) and polyamide 6,6 (PA 6,6) fibers. The mutations L81A, N84A, L182A, V184A and L189A were done to enlarge the active site in order to better fit a larger polymer chain. Modeling studies have shown enhanced free energy stabilization of model substrate tetrahedral intermediate (TI) bound at the enzyme active site for all mutants, for both model polymers. L81A and L182A showed an activity increase of four- and five-fold, respectively, when compared with the wild type, for PET fibers. L182A showed the one- and two-fold higher ability to biodegrade aliphatic polyamide substrates. Further studies in aliphatic polyesters seem to indicate that cutinase has higher ability to recognize aliphatic substrates.
A bacterial cutinase from Thermobifida fusca, named Tfu_0883, was genetically modified by site-directed mutagenesis to enhance its activity on poly(ethylene terephthalate) (PET). The new mutations tailored the catalytic site for PET, increasing the affinity of cutinase to this hydrophobic substrate and the ability to hydrolyze it. The mutation I218A was designed to create space and the double mutation Q132A/T101A was designed both to create space and to increase hydrophobicity. The activity of the double mutant on the soluble substrate p-nitrophenyl butyrate increased two-fold compared to wild-type cutinase, while on PET both single and double mutants exhibited considerably higher hydrolysis efficiency. The replacement of specific amino acids at the active site was an effective approach for the improvement of the Tfu_0883 cutinase capacity to hydrolyze polyester surfaces. Thus, this study provides valuable insight on how the function and stability of enzymes can be improved by molecular engineering for their application in synthetic fiber biotransformation.
Enzymes are efficient catalysts designed by nature to work in physiological environments of living systems. The best operational conditions to access and convert substrates at the industrial level are different from nature and normally extreme. Strategies to isolate enzymes from extremophiles can redefine new operational conditions, however not always solving all industrial requirements. The stability of enzymes is therefore a key issue on the implementation of the catalysts in industrial processes which require the use of extreme environments that can undergo enzyme instability. Strategies for enzyme stabilization have been exhaustively reviewed, however they lack a practical approach. This review intends to compile and describe the most used approaches for enzyme stabilization highlighting case studies in a practical point of view.
We have shown that cutinase can be used to modify the surface of polyester, polyamide, and acrylic fibers. This enzyme can hydrolyze the esters groups of poly(ethyleneterephthalate) and the vinyl acetate, the comonomer of acrylic fibers. For the first time we have shown that cutinase is able to hydrolyze the amide bonds in polyamide 6.6. Hydroxyl and amine groups are formed at the fiber surface by enzyme action and detected by specific dyeing methods with reactive dyes.
Laccases (benzene diol: oxidoreductases, EC 1.10.3.2) are able to catalyze the oxidation of various compounds containing phenolic and aniline structures using dissolved oxygen in water. Laccase structural features and catalytic mechanisms focused on the polymerization of aromatic compounds are reported. A description about the most recent research on the biosynthesis of chemicals and polymers is made. Selected applications of this technology are considered as well as the advantages, shortcomings and future needs related with the use of laccases.
The ability of laccases from Trametes villosa (TvL), Myceliophthora thermophila (MtL), Trametes hirsuta (ThL) and Bacillus subtilis (BsL) to improve the dispersion properties of calcium lignosulfonates 398 in the presence of HBT as a mediator was investigated. Size exclusion chromatography showed an extensive increase in molecular weight of the samples incubated with TvL and ThL by 107% and 572% from 28400 Da after 17h of incubation, respectively. Interestingly, FTIR spectroscopy, (13)C NMR and Py-GC/MS analysis of the treated samples suggested no substantial changes in the aromatic signal of the lignosulfonates, a good indication of the ability of TvL/ThL-HBT systems to limit their effect on functional groups without degrading the lignin backbone. Further, the enzymatic treatments led to a general increase in the dispersion properties, indeed a welcome development for its application in polymer blends.
The enzymatic hydrolysis of polyamide fibres yields amino and carboxylic groups. These groups can be found in solution treatments as polyamide monomers and soluble oligomers. The amino groups can also be found at the surface of the fibres as end group chains. In this paper we report two methods to quantify the formation of these groups as a result of the enzymatic action. Soluble amino groups can be quantified with 2,4,6trinitrobenzenesulfonic acid (TNBS), which yields a coloured complex which can be determined spectrophotometrically. The amino groups on the fibre surface can be quantified by reaction with a wool reactive dye and determination of colour intensities after a dyeing procedure below the glass transition temperature of polyamide.
BackgroundMultidrug- (MDR) and extensively drug resistant (XDR) tuberculosis (TB) presents a challenge to disease control and elimination goals. In Lisbon, Portugal, specific and successful XDR-TB strains have been found in circulation for almost two decades.ResultsIn the present study we have genotyped and sequenced the genomes of 56 Mycobacterium tuberculosis isolates recovered mostly from Lisbon. The genotyping data revealed three major clusters associated with MDR-TB, two of which are associated with XDR-TB. Whilst the genomic data contributed to elucidate the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of Mycobacterium tuberculosis clinical isolates, revealed two major clades responsible for M/XDR-TB in the region: Lisboa3 and Q1 (LAM).The data presented by this study yielded insights on microevolution and identification of novel compensatory mutations associated with rifampicin resistance in rpoB and rpoC. The screening for other structural variations revealed putative clade-defining variants. One deletion in PPE41, found among Lisboa3 isolates, is proposed to contribute to immune evasion and as a selective advantage. Insertion sequence (IS) mapping has also demonstrated the role of IS6110 as a major driver in mycobacterial evolution by affecting gene integrity and regulation.ConclusionsGlobally, this study contributes with novel genome-wide phylogenetic data and has led to the identification of new genomic variants that support the notion of a growing genomic diversity facing both setting and host adaptation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-991) contains supplementary material, which is available to authorized users.
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