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.     

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.     

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.     

羊毛防毡缩用蛋白酶的化学修饰

为减少防毡缩整理中蛋白酶对羊毛纤维主体结构的破坏作用,分别研究了戊二醛、微生物谷氨酰胺转氨酶(MTG)和水溶性碳二亚胺(EDC)对蛋白酶Savinase 16L的化学修饰,以期达到增大蛋白酶分子量,从而将水解作用限制在纤维表面的目的。主要通过体积排阻色谱、SDS-PAGE谱图以及荧光光谱研究修饰酶的分子量和结构变化。结果表明,戊二醛不能对蛋白酶分子进行有效修饰;MTG会被蛋白酶水解,无法催化酶分子间发生共价交联;而碳二亚胺既可以使蛋白酶分子间发生交联,又能将含有伯胺基的大分子修饰剂偶联到酶分子上。     

Combined use of mild oxidation and cutinase/lipase pretreatments for enzymatic processing of wool fabrics

A cutinase from Thermobifida fusca WSH04 and two lipases, L3126 and Lipex 100L, were applied to the enzymatic pretreatment of wool fabrics followed by protease treatment, aiming at hydrolyzing the outmost bound lipids on the wool surface. A mild oxidation with 2 g/L hydrogen peroxide (30%) was selectively carried out before the enzymatic treatments. The cooperative actions of mild oxidation, cutinase and lipase pretreatments during wool processing were investigated. The results showed that lipase pretreatment alone had less impact on the wettability and anti‐felting ability of wool fabrics than cutinase treatment. Combined use of cutinase and lipase pretreatments did not evidently improve the properties of the wool fabric compared with the individual cutinase pretreatment. By contrast, mild oxidation slightly enhanced the activity of cutinase toward the wool surface and promoted the subsequent proteolytic reactions. The wetting time and contact angle of the protease‐treated fabric deceased to 1.2 min and 55°, respectively; the area shrinkage decreased to 3.1%, with an acceptable strength loss from 489 to 418 N. The changes in the cuticle scales of the wool fibers, confirmed by scanning electron microscopy, further proved the cooperative actions of mild oxidation and cutinase pretreatment during enzymatic wool processing.     

Chemical modification of proteases for wool cuticle scale removal

Over the last few decades several enzymatic processes to improve properties of wool fabrics like felting tendency, shrink resistance, dyeing ability and handling characteristics have been described. Previous investigations into the use of proteases to hydrolyse the cuticles at the surface of wool fibres, resulted in high strength and weight losses. Therefore restriction of the enzyme activity to the wool surface or control of enzyme diffusion to the cortex cells is required.

To change the diffusion behaviour of proteases in wool fibres, the soluble polymer PEG was covalently attached to a protease from Bacillus lentus. Modified enzymes with different molecular weights were compared. These modified enzymes retained up to 80% of their activity in the standard assay while hydrolysis of wool fibres was successfully restricted to cuticles, resulting in a 90% decrease in weight losses compared to non-modified enzymes.     

Chemical modification of proteases for wool cuticle scale removal

Over the last few decades several enzymatic processes to improve properties of wool fabrics like felting tendency, shrink resistance, dyeing ability and handling characteristics have been described. Previous investigations into the use of proteases to hydrolyse the cuticles at the surface of wool fibres, resulted in high strength and weight losses. Therefore restriction of the enzyme activity to the wool surface or control of enzyme diffusion to the cortex cells is required.

To change the diffusion behaviour of proteases in wool fibres, the soluble polymer PEG was covalently attached to a protease from Bacillus lentus. Modified enzymes with different molecular weights were compared. These modified enzymes retained up to 80% of their activity in the standard assay while hydrolysis of wool fibres was successfully restricted to cuticles, resulting in a 90% decrease in weight losses compared to non-modified enzymes.     

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.     

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.     

Promotional effect of 1‐butyl‐3‐methylimidazolium chloride ionic liquid on the enzymatic finishing of wool

To improve the effects of protease finishing on wool, 1‐butyl‐3‐methylimidazolium chloride ionic liquid was employed as a pretreatment reagent. It was found that ionic liquid pretreatment significantly changed the wool surface characteristics. The Allwördern reaction showed that the epicuticle layer was damaged by the ionic liquid, and X‐ray photoelectron spectroscopy analysis further demonstrated that the surface elemental composition was significantly changed. Ionic liquid pretreatment remarkably improved the accessibility of protease to the wool and thus accelerated the hydrolysis rate of keratin. The properties of wool fabric after combined processing were also changed. Dyeability results showed that the color depth was increased but the wet rubbing and washing fastness of wool fabrics showed a decreased half grade. The wettability results demonstrate that the contact angle was further reduced after the comprehensive treatment because of the exposure of more proteins under the fatty‐acid layer. In addition, the shrink proofing of wool fabric was also enhanced after combined processing. In summary, ionic liquid modification presents a promising pretreatment method for protease processing of wool.     

Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases

We have identified and cloned 22 human cDNAs encoding novel members of the ubiquitin-specific protease (USP) family. Eighteen of the identified proteins contain all structural features characteristic of these cysteine proteinases, whereas four of them have been classified as non-peptidase homologues. Northern blot analysis demonstrated that the identified USPs are broadly and differentially distributed in human tissues, some of them being especially abundant in skeletal muscle or testis. Enzymatic studies performed with the identified USPs revealed that at least twelve of them are deubiquitylating enzymes based on their ability to cleave ubiquitin from a ubiquitin-beta-galactosidase fusion protein. These results provide additional evidence of the extreme complexity and diversity of the USP proteolytic system in human tissues and open the possibility to explore the relevance of their multiple components in the regulation of ubiquitin-mediated pathways in normal and pathological functions.     

An evaluation of the action of thioesterases on the surface of wool

The thioesterase activity of palmitoyl protein thioesterase (PPT1) and six commercial lipases was measured against the synthetic substrates, S-palmitoyl-N-acetylcysteamine (Ac-Cym-Pal) and S-(18-methyleicosanoyl)-N-acetylcysteamine (Ac-Cym-18-MEA). PPT1 showed good activity against Ac-Cym-Pal but relatively low activity against the longer chain substrate, Ac-Cym-18-MEA. The highest activity was given by Lipolase 100L type EX (Novozyme) and Lipoprotein Lipase (Sigma) with greater than 90% hydrolysis of Ac-Cym-18-MEA within 10 min at pH 7.4. Other lipases to show high levels of thioesterase activity include Lipex 100L (Novozyme), Lipomod 34P (Biocatalysts) and Lipozyme CALB L (Novozyme). Chemical analysis of wool fibre and fabric treated with the above enzymes under optimal conditions showed that there was no hydrolysis of 18-MEA or other covalently bound fatty acids from the fibre surface. No change in the wettability of the fabric surface was observed following enzyme treatments. Scanning electron micrographs of the fabric treated with the most active enzyme, Lipolase 100L type EX, revealed that the surface of the fibres appeared to have a coating that was not removed by extensive extraction. Reasons for the inability of PPT1 and the other esterases to hydrolyse 18-MEA from the wool fibre surface are discussed.     

A modified method for the detection of microbial proteases on agar plates using tannic acid

In routine assay for the screening of microbes producing proteases, 10% trichloroaceticacid (TCA) is flooded on the milk agar plates after inoculation and required incubation to precipitate the protein. However, the clarity of the hydrolyzed zone is not very sharp and distinct. We herein present an improved assay for detecting the presence of extracellular protease from microorganisms on agar plates. In this method 10% tannic acid is flooded on the milk agar plate (in place of, TCA) to observe the zone of hydrolysis. Tannic acid sharply increases the colour intensity of the plate, as it favours the precipitation of the unhydrolyzed protein in the plate, thereby improving the contrast between the intact zones and the enzymatic lyses zones of the substrate. Our results indicate that this method is useful to detect extracellular proteases produced by both fungi as well as bacteria. The method used in the present study is sensitive, and can be easily performed for screening of large number of microbial cultures. This is the first report on the use of tannic acid for the detection of microbial proteases.     

Localization and enzymatic activity profiles of the proteases responsible for tachykinin-directed oocyte growth in the protochordate, Ciona intestinalis

We previously substantiated that Ci-TK, a tachykinin of the protochordate, Ciona intestinalis (Ci), triggered oocyte growth from the vitellogenic stage (stage II) to the post-vitellogenic stage (stage III) via up-regulation of the gene expression and enzymatic activity of the proteases: cathepsin D, carboxypeptidase B1, and chymotrypsin. In the present study, we have elucidated the localization, gene expression and activation profile of these proteases. In situ hybridization showed that the Ci-cathepsin D mRNA was present exclusively in test cells of the stage II oocytes, whereas the Ci-carboxypeptidase B1 and Ci-chymotrypsin mRNAs were detected in follicular cells of the stage II and stage III oocytes. Double-immunostaining demonstrated that the immunoreactivity of Ci-cathepsin D was largely colocalized with that of the receptor of Ci-TK, Ci-TK-R, in test cells of the stage II oocytes. Ci-cathepsin D gene expression was detected at 2h after treatment with Ci-TK, and elevated for up to 5h, and then slightly decreased. Gene expression of Ci-carboxypeptidase B1 and Ci-chymotrypsin was observed at 5h after treatment with Ci-TK, and then decreased. The enzymatic activities of Ci-cathepsin D, Ci-carboxypeptidase B1, and Ci-chymotrypsin showed similar alterations with 1-h lags. These gene expression and protease activity profiles verified that Ci-cathepsin D is initially activated, which is followed by the activation of Ci-carboxypeptidase B1 and Ci-chymotrypsin. Collectively, the present data suggest that Ci-TK directly induces Ci-cahtepsin D in test cells expressing Ci-TK receptor, leading to the secondary activation of Ci-chymotrypsin and Ci-carboxypeptidase B1 in the follicle in the tachykininergic oocyte growth pathway.     

Modification of Esperase® by covalent bonding to Eudragit® polymers L 100 and S 100 for wool fibre surface treatment

The protease Esperase® was modified by covalent bonding with two grades of a reversible soluble-insoluble co-polymer of methacrylic acid and methyl-methacrylate, namely Eudragit® L 100 and Eudragit® S 100. The optimum reaction conditions and washing protocol were investigated and it was found that Esperase® modified with Eudragit® L 100 showed greater activity than if modified with Eudragit® S 100. This should be expected as there is a greater quantity of active sites, namely carboxyl groups, per mass of Eudragit® L 100 in comparison with Eudragit® S 100 to interact with the enzyme. Gel filtration confirmed that Eudragit® L 100 covalently bonded to Esperase®. Treatment of the modified Esperase® on wool showed that the enzyme modified with Eudragit® L 100 had greater activity towards the wool and appeared more effective in shrink resistant finishing.     

Stabilization mechanism of MPEG modified trypsin based on thermal inactivation kinetic analysis and molecular modeling computation

Thermal inactivation kinetic analysis and molecular modeling computation were jointly utilized to illuminate the detailed stabilization mechanism of trypsin caused by methoxypolyethylene glycol (MPEG) modification. First, trypsin was modified by MPEG (molecular mass 350 Da) to enhance its thermal stability. As expected, the modified trypsin was more stable against temperature than the native form. Second, a new kinetic model, which has the ability of taking the thermal denaturation and autolysis effects of proteases into account, was established and used to analyze the thermal inactivation process of the native and modified trypsin. The kinetic analysis showed that the increased thermal stability of MPEG modified trypsin is the joint result of a reduction in autolysis and a decrease in thermal denaturation. Finally, the molecular modeling technique was also employed to calculate some structural information change, i.e. solvent accessible surface, intramolecular hydrogen bond and root mean square fluctuation, between the native and modified trypsin. The results of molecular modeling computation demonstrated that (i) the steric hindrance caused by MPEG chain would result in the decreased rate of autolysis, (ii) the decreased rate of thermal denaturation should be ascribed to the increased number of hydrogen bond, not the result of the increased molecular rigidity.     

Kinetic study of thermal inactivation for native and methoxypolyethylene glycol modified trypsin

Bovine pancreatic trypsin (Ti) has been modified with four kinds of methoxypolyethylene glycol (MPEG, molecular masses 350, 750, 2000 and 5000 Da) to enhance thermostability. The MPEG-modified Ti was more stable against temperature than the native form, the larger molecular mass moiety of MPEG showing higher thermostabillty. To investigate the mechanism of thermal inactivation, a new kinetic model, which has the ability of taking the thermal denaturation and autolysis effects of the proteases into account, has been used to analyze the thermal inactivation process of the native and modified Ti in detail. The kinetic analysis showed that the stabilization effect caused by MPEG modification was the result of a decrease in autolysis rate and a decrease in the rate of thermal denaturation. In addition, the possible mechanism of reduced autolysis and lower thermal denaturation rate were also discussed.     

A study of the influence of structure on the effectiveness of chitosan as an anti-felting treatment for wool

The effect of chitosan on the resistance of wool fabric to felting on washing has been studied using nine structurally different samples of chitosan. Structural differences examined include molecular weight, level of N-acetylation, and the nature and concentration of homologous N-acyl groups. No strong dependency of shrinkage on molecular weight or level of N-acetylation has been found, but increasing the hydrophobic character of chitosan through the incorporation of a number of long-chain N-acyl groups gives improved antifelting behaviour, compared to chitosan itself, at the same level of add-on.     

Treatment of wool fibres with subtilisin and subtilisin-PEG

In this work the diffusion of serine proteases into wool fabrics and yarns was studied. The proteases used were free subtilisin and subtilisin-PEG (the same enzyme that was covalently cross linked to polyethylene glycol). It is shown that the adsorption and diffusion is facilitated by the pre-treatment performed, being the alkaline surfactant washing and bleaching the most effective in what concerns enzyme adsorption. Furthermore, this study suggests that the diffusion of proteases into wool is dependent on the size of the protease. The free enzyme penetrates into wool fibre cortex while the modified bigger enzyme is retained only at the surface, in the cuticle layer. Also, proteins without proteolytic activity do not adsorb considerably on wool due to its hydrophobic nature, therefore the diffusion is facilitated by hydrolytic action.

These results have important practical implications for the establishment of enzymatic wool finishing processes, since they allow for control of the enzyme hydrolysis, which was the major drawback of this environmental friendly option to the conventional chlorine treatments.