The removal of lipid from the surface of wool to promote the subsequent enzymatic process with modified protease for wool shrink resistance

Abstract

When treated with modified protease, wool shows shrink resistance without significant damage to the fiber. It was considered that if wool fiber was pre-treated to make it more hydrophilic, the subsequent treatment with modified protease would be more efficient. After wool was pre-treated with cetyltrimethylammonium bromide (CTAB) under alkaline conditions, the fiber became very hydrophilic due to the removal of surface lipid. After CTAB treatment, it was found that residual CTAB on the fiber significantly decreased enzyme activity. Therefore prior to enzyme treatment, CTAB was washed off the fiber with anionic surfactant under acidic conditions. It was found that the activity of modified protease towards wool improved if wool had been pre-treated with CTAB then washed with anionic surfactant. It was concluded that pre-treatment of wool with CTAB under alkaline conditions followed by washing with anionic surfactant improves the wettability of wool and therefore promotes more efficient treatment with modified protease, achieving improved levels of shrink resistance with no effect on strength of the fiber or coloration properties.     

Surface modification of wool with protease extracted polypeptides

Polypeptides were extracted from wool protein fibres using the serine type protease Esperase 8.0L (EC 3.4.21.62), a subtilisin from Bacillus sp., in a reducing solution. The extracted polypeptides, in aqueous liquor, were then applied to modify the fibre surface of wool fabric with or without additional protease. The treated wool fabric was subsequently treated with the cross-linking agent, glycerol diglycidyl ether, and then underwent a curing process to affix the polypeptide to the fibre. The resulting knitted fabric showed a very high level of shrink-resistance to machine washing, without excessive fibre damage. Shrinkage of 1-2% could be achieved after 5 times 5A washes with minimal (<1%) weight loss due to washing and a burst strength of 317 kPa.     

Effects of cutinase on the enzymatic shrink-resist finishing of wool fabrics

A novel microbial cutinase from Thermobifida fusca WSH04 was applied in the pretreatment of wool fabrics followed by protease treatment, aiming at improving the wettability of the samples by hydrolyzing the outmost bound lipids in the wool surface. Cutinase pretreatment could increase the efficacy of the subsequent protease treatment by improving the wettability, dyeability, and shrink-resistance of the wool fabrics. The data obtained by the XPS method showed the changes of elemental concentration in the wool surface after cutinase pretreatment. Compared with the fabrics treated with hydrogen peroxide and protease, the combination of cutinase and protease treatments produced better results in terms of wettability and shrink-resistance with less strength loss. The anti-felting property of the fabrics treated with the enzymatic resist-shrink technique is very promising to meet the commercial standard.     

Low cetyltrimethylammonium bromide concentrations induce reversible amorphous aggregation of tobacco mosaic virus and its coat protein at room temperature

Ordered and amorphous protein aggregation causes numerous diseases. Tobacco mosaic virus coat protein for many decades serves as the classical model of ordered protein aggregation ("polymerization"). It was also found to be highly prone to heat-induced amorphous aggregation and the rate of this aggregation could be easily manipulated by changes in solution ionic strength and temperature. Here, we report that rapid amorphous aggregation of this protein can be induced at 25 degrees C in phosphate buffer by low micromolar (start at about 15 microM) concentrations of cationic surfactant cetyltrimethylammonium bromide. At equilibrium four surfactant molecules bound to the protein subunit. As judged by circular dichroism and fluorescence spectroscopy data, the coat protein molecules retained their native structure upon the cetyltrimethylammonium bromide induced aggregation. No aggregation was observed at the higher surfactant concentrations (above 300 microM). Micromolar concentrations of anionic surfactant sodium dodecylsulfate rapidly reversed the cetyltrimethylammonium bromide induced aggregation of the coat protein due to formation of mixed surfactant-surfactant micelles. Cetyltrimethylammonium bromide (100-300 microM) also induced the reversible intact tobacco mosaic virus virion aggregation. The possible liability to the cetyltrimethylammonium bromide induced amorphous aggregation of other ordered aggregate-producing proteins has been discussed.     

Development and industrialisation of enzymatic shrink-resist process based on modified proteases for wool machine washability

There is currently considerable interest in the use of enzymes to achieve a variety of finishing effects on wool, but it is apparent that the extent of fibre degradation by enzymes is of major concern during their commercial application. Proteolytic enzymes are known to penetrate and degrade the internal wool structure during processing, causing fibre damage, rather than limiting the degradation to the cuticle cells. The ability to be able to control the exact location of proteolytic attack on wool protein structures will lead to the successful development of enzymatic treatments for achieving a variety of finishing effects for wool-containing products. This present work describes the modification of proteases so that enzymatic modification of wool fibres is restricted to the cuticle scales of the fibres.

Bulk trials have demonstrated that novel modifications of the enzyme enable the reaction of the enzyme with wool to be controlled, so that less degradation of the wool occurs than in similar treatments with the native protease. An anti-felting effect has been achieved without any significant weight loss being caused by the modified protease during the treatment. This novel enzymatic process leads to environmentally friendly production of machine washable wool.     

Restricting detergent protease action to surface of protein fibres by chemical modification

Due to their excellent properties, such as thermostability, activity over a broad range of pH and efficient stain removal, proteases from Bacillus sp. are commonly used in the textile industry including industrial processes and laundry and represent one of the most important groups of enzymes. However, due to the action of proteases, severe damage on natural protein fibres such as silk and wool result after washing with detergents containing proteases. To include the benefits of proteases in a wool fibre friendly detergent formulation, the soluble polymer polyethylene glycol (PEG) was covalently attached to a protease from Bacillus licheniformis. In contrast to activation of PEG with cyanuric chloride (50%) activation with 1,1′-carbonyldiimidazole (CDI) lead to activity recovery above 90%. With these modified enzymes, hydrolytic attack on wool fibres could be successfully prevented up to 95% compared to the native enzymes. Colour difference (ΔE) measured in the three dimensional colour space showed good stain removal properties for the modified enzymes. Furthermore, half-life of the modified enzymes in buffers and commercial detergents solutions was nearly twice as high as those of the non-modified enzymes with values of up to 63 min. Out of the different modified proteases especially the B. licheniformis protease with the 2.0-kDa polymer attached both retained stain removal properties and did not hydrolyse/damage wool fibres.     

The influence of enzymatic treatment on wool fibre properties using PEG-modified proteases

The main contribution of the presented work was to introduce the use of proteases modified with the soluble polymer polyethylene glycol (PEG) in the bio-finishing process of wool fibres, to target enzyme action to the outer parts of wool fibres, i.e. to avoid the diffusion and consequent destroying of the inner parts of the wool fibre structure, in the case of native proteases using.

Different proteolytic enzymes from Bacillus lentus and Bacillus subtilis in native and PEG-modified forms were investigated and their influence on the modification of wool fibres morphology surface, chemical structure, as well as the hydrolysis of wool proteins, the physico-mechanical properties, and the sorption properties of 1:2 metal complex dye during dyeing were studied. SEM images of wool fibres confirmed smoother and cleaner fibre surfaces without fibre damages using PEG-modified proteases. Modified enzyme products have a benefit effect on the wool fibres felting behaviours (14%) in the case when PEG-modified B. lentus is used, without markedly fibre damage expressed by tensile strength and weight loss of the fibre. Meanwhile the dye exhaustion showed slower but comparable level of dye uptake at the end of the dyeing.     

Structural analysis of alpha-helical proteins from wool using cysteine labelling and mass spectrometry

A simple reduction/labelling/extraction protocol has been developed to fractionate cortical matrix proteins from filament proteins in wool. Through differential labelling of cysteine residues their relative accessibility in the wool fibre has been investigated. This has allowed the preliminary development of a map of the chemical functionality that is accessible within wool fibres under native conditions. Protein analyses of wool subjected to mechanical action, wet chemical permonosulphate/sulphite treatment and dry argon plasma treatment revealed that none of these detectably improved the accessibility of functional groups at the wool cortex. It is anticipated that this analytical method can be extended to improve the sensitivity and scope with which chemical functionality within native fibres can be mapped and lead to a better understanding of the potential limits/opportunities for fibre modification.     

Extremozymes for improving wool properties

The project ‘EXTRETEX’ funded by the German Federal Foundation Environment (DBU, Osnabrück, Germany) aims at the improvement of wool properties dyeability, handle, felting behaviour and degree of whiteness by means of enzymes derived from extremophilic micro-organisms. In this paper the effects of a commercial thermo- and alkalistable protease on wool with regard to the degree of whiteness, the dyeability and the felting behaviour are presented. A method to treat wool top and wool fabric was developed on a laboratory scale in which the protease was integrated into the pre-washing step of a dyeing process. This treatment method was than scaled up and tested on an industrial winch beck for fabric. With this method—the addition of enzyme in the pre-washing step—the degree of whiteness is generally enhanced. Dyeing untreated and the enzyme-treated wool with Lanasol Blue 8G leads to an improved dyestuff uptake and a distinctive difference in the colour shade for the latter. Microscopy pictures of fibre cross-sections of these samples display a more even distribution of the dyestuff and a better penetration in the enzyme-treated wool fibres but the colour fastness of the enzyme-treated wool is decreased. Though the felting behaviour of the protease treated wool is significantly improved the felting tendency is still too high for an antifelting finish. An increased damage of the enzyme-treated wool in comparison with the untreated one was not observed.     

Detergent Formulations for Wool Domestic Washings Containing Immobilized Enzymes

The stability of immobilized and native Esperase, a commercial serine protease, was studied by incubating the enzymes in four formulations containing the same amount of anionic and non-ionic surfactants. The results show that the activity of the immobilized enzyme is not affected by the presence of detergents while the native enzyme lost 50% of activity after 20 min of incubation in these four formulations. The washing performance of the detergents prepared with the immobilized Esperase was studied on cotton and wool fabric samples stained with human blood and egg yolk, using as control the detergent containing native Esperase. The best stain removal for cotton samples stained with human blood was achieved using the detergent with immobilized Esperase. Several physical tests confirmed that wool keratin was not degraded by the immobilized Esperase, validating the ability to use formulated detergents containing this immobilized enzyme for safe wool domestic washing.     

An ethoxylated alkyl phosphate (anionic surfactant) for the promotion of activities of proteases and its potential use in the enzymatic processing of wool

Pretreatments of wool fabrics with cationic, anionic or non-ionic surfactants were investigated to reduce surface tension and improve the wettability of the fibres in order to promote protease activity on the fibres in subsequent processes. Results showed that an ethoxylated alkyl phosphate (specific anionic surfactant) as well as the widely used non-ionic surfactant was compatible with proteases in the enzymatic treatment of wool. There is therefore a potential for using specific anionic surfactants to achieve efficient enzymatic scouring processes.     

Simultaneous protease and transglutaminase treatment for shrink resistance of wool

A bioprocess for machine washable wool, combining the advantages of both protease and transglutaminase in a simultaneous enzymatic treatment has been developed. This process reduced the felting tendency of woven wool fabrics by 9% at the expense of only 2% weight and tensile strength loss. In contrast to previously described protease-based processes for shrink resistant wool, the anti-felting properties achieved in the simultaneous enzymatic treatment produced insignificant fibre damage, confirmed also by scanning electron images of the fabrics.     

Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases.

Native poly(hydroxybutyrate) (PHB) granules, purified PHB and artificial amorphous PHB granules were examined as putative substrates for hydrolysis by the intracellular depolymerase system of Rhodospirillum rubrum and the extracellular depolymerase of Pseudomonas lemoignei. The R. rubrum depolymerizing system requires pretreatment of granules with a heat stable 'activator' fraction; the activator can be replaced by mild trypsin treatment. Artificial granules were prepared with a cationic detergent, cetyltrimethylammonium bromide (CTAB) and an anionic detergent, (sodium cholate). Cholate and CTAB PHB granules were hydrolyzed by both enzyme systems; however, some differences were noted. Cholate granules were hydrolyzed in the absence of the R. rubrum activator fraction. Activator was required for the hydrolysis of CTAB granules but could be replaced by heparin in the extracellular depolymerase system but not in the intracellular depolymerase system. A Triton X-114 extract of native PHB granules inhibited the hydrolysis of trypsin-activated granules by the intracellular depolymerase. The inhibition was reversed by the activator fraction. Detergent extracts of granules activated with the R. rubrum activator were unable to inhibit the hydrolysis of trypsin-activated granules. These data suggest that the activator acts to modify an inhibitor present on native granules.     

The determination of molecular weights of biologically active proteins by cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis

A novel cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis system which is useful for the separation of native forms of proteins consistent with their molecular weights is reported here. Many proteins examined in this system demonstrated the same association patterns which have been shown by other techniques to exist under nondenaturing conditions. In addition, biological activity could be assayed directly in the gel after electrophoresis. Based on the peculiar characteristics of cetyltrimethylammonium bromide, a possible explanation which may account for the behavior of proteins in this system is presented.     

Chitosan contribution on wool treatments with enzyme

In a previous research work, it was observed that the application of biopolymer chitosan (CHT) on wool fabrics before the enzymatic treatment promotes an increase of the weight loss. In order to deep on the role played by CHT, several experimental conditions have been selected according to a hybrid experimental design and different parameters, such as weight loss and shrink-resist properties, have been controlled. To enhance the CHT sorption on the wool fibre surface, wool was submitted previously to a water-vapour low-temperature plasma treatment. The weight loss results reveal that the enzyme effect increases by increasing the CHT concentration applied to untreated wool. However, CHT concentration does not have any influence when wool has been previously treated with plasma. It is deduced that the surface free energy of wool fibres plays an important role on the enzyme activity. Therefore, the results obtained reveal that the main contribution of CHT on hydrophobic surface of untreated wool fibres is to confer hydrophilicity to wool. Furthermore, CHT tends to coat the wool fibres by film formation reducing apparently the fibre damage promoted by enzyme treatment and also reducing the wool shrinkage.     

Performance linked to residence time distribution by a novel wool-based bioreactor for tertiary sewage treatment

Laboratory-scale experiments were carried out using up-flow 7 L Submerged Aerated Filter reactors packed with wool fibre or commercial plastic pall rings, Kaldnes, (70% by volume) support media for the tertiary treatment of sewage. The performance of the wool bioreactor was more consistent than that with Kaldnes medium, for both TOC removal (93%) and SS removal (90%). Both plastic and wool-packed bioreactors achieved complete nitrification at the load of about 0.4 kgCOD/m³/day. The sludge yield of the wool bioreactor was almost half that of the bioreactor with Kaldnes suggesting that wool could retain residual organics and particulates. The wool however was degraded and it was concluded that wool would have to be considered as additional sacrificial adsorption capacity rather than an alternative medium. The performance was linked to the residence time distribution studies and these changes in the wool structure. Biomass growth increased the retention of the tracer in the wool reactor by, it was suggested, exposing a greater surface area. Results from the plastic media on the other hand showed increased mixing possibly by increasing the mobility of the plastic. Aeration increased the mixing in both reactors, and patterns were in all cases predominantly well-mixed.     

Atomic force microscopy of supported lipid membranes and their complexes with polycations

The method of atomic force microscopy has been used to investigate the morphology of mica-supported bilayer lipid membranes and stability of their complexes with a cationic polymer, poly-(N-ethyl-4-vinylpyridinium bromide). Lipid bilayers with a minimum of defects were obtained by the fusion of monolamellar neutral or mixed anionic bilayer vesicles (liposomes) on the mica surface, followed by excessive solvent removal by means of rapid rotation of a plate in horizontal plane (spin-coating). It has been shown that the cationic polymer does not interact with the bilayers, where the outer leaflet (i.e., the monolayer exposed to the surrounding aqueous solution) is made of an electroneutral phosphatidylcholine (PC). At the same time, the polymer irreversibly binds to the bilayer containing an anionic lipid.     

A novel “trifunctional protease” with reducibility,hydrolysis, and localization used for wool anti-felting treatment

Proteases can cause unacceptable fiber damage when they are singly applied to wool anti-felting treatment which can make wool textiles machine-washable. Even if protease is attached by synthetic polymers, the modified protease plays a limited role in the degradation of keratin with dense structure consisting of disulfide bonds in the scales. Here, to obtain “machine-washable” wool textiles, a novel “trifunctional protease” with reducibility, hydrolysis, and localization is developed by means of covalent bonding of protease molecules with poly (ethylene glycol) bis (carboxymethyl) ether (HOOC-PEG-COOH) and l-cysteine using carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling, aiming at selectively degrading the scales on the surface of wool. The formation of polymer is confirmed with size exclusion chromatography (SEC) and Fourier transform infrared spectroscopy (FT-IR). Ellman’s test and fluorescence microscopy reveal that the modified protease can reduce disulfide bonds and restrict hydrolysis of peptide bonds on the wool scales. Furthermore, when applied to wool fabrics, the modified protease reach better treatment effects considering dimensional stability to felting (6.12%), strength loss (11.7%) and scale dislodgement proved by scanning electron microscopy (SEM), alkali solubility, wettability, and dyeability. This multifunctional enzyme is well-designed according to the requirement of the modification of wool surface, showing great potential for eco-friendly functionalization of keratin fibers rich in disulfide linkage.

     

New method for detecting rhamnolipids excreted byPseudomonas species during growth on mineral agar

Summary A new semi-quantitative agar plate test for the detection of extracellular rhamnolipids has been developed. These biological anionic tensides (biosurfactants) form an insoluble ion pair with the cationic tenside cetyltrimethylammonium bromide and the basic dye methylene blue which was included in mineral agar plates. On the light blue agar, productive colonies ofPseudomonas spec. were surrounded by dark blue halos. The test is specific for anionic biosurfactants and can be applied to other glycolipid producing microorganims.     

Bio‐antifelting of wool based on mild methanolic potassium hydroxide pretreatment

Covalently bound lipids cover the wool surface and make enzymatic degradation of wool scales very difficult. In this paper, methanolic potassium hydroxide (MPH) pretreatment was used prior to enzymatic treatment of wool with protease, aiming at hydrolyzing the outmost lipids on the wool surface and promoting the subsequent proteolytic reaction. The efficacy of lipid removal from the fiber surface and the properties of the protease‐treated wool were evaluated. The results indicated that mild MPH pretreatment with 0.10 mol/L MPH for 10 min improved the wettability of the wool without adverse impacts on its mechanical properties. The wetting time and area shrinkage of the wool fabric reached 0.5 s and 5.6%, respectively, and the strength loss was within the acceptable range. Pretreatment with high concentrations of MPH for longer times led to significant damage to the wool fibers and caused heavy strength loss, without improving the antifelting properties after protease treatment. Thus, the combination of mild MPH and protease treatments endowed the wool with desirable properties in contrast to the treatment with protease alone.