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.     

Lipase immobilisation: an equilibrium study of lipases immobilised on hydrophobic and hydrophilic/hydrophobic supports

This work investigates the enzyme-support equilibrium behaviour in immobilised lipase biocatalysts. Equilibrium data determines the maximum enzyme up-take by unit weight of support. Four lipases were immobilised on two polymeric supports, respectively. They were Lipase PS from Pseudomonas, Lipolase 100L from Humicola, SP871 from Rhizomucor miehel and QL from Alcaligenes. The supports were Accurel EP100 (a polypropylene material) and 45SAA (a polypropylene/silica composite). Experimentally, equilibrium was expressed in terms of lipase loading (LU/g support) versus residual lipase concentration (LU/dm³). Activity, efficiency and operational stability of the immobilised lipases were assayed by solvent-free esterification of oleic acid and octanol.Equilibrium data were modelled by the Langmuir, Freundlich and Redlich–Peterson formulae. It was found that Lipolase 100L/Accurel, PS/45SAA and SP871/45SAA systems conformed to the Langmuir behaviour, while Lipase PS/Accurel and SP871/Accurel systems followed the Freundlich behaviour and Lipolase 100L/45SAA, QL/45SAA and QL/Accurel EP100 resembled Redlich–Peterson behaviour. Whereas immobilisation on Accurel EP100 resulted in classical equilibrium isotherms with all four lipases, immobilisation on support 45SAA resulted in two-plateau equilibrium curves which included a step change in the isotherm for all lipases studied, except for SP871. Quantitatively, for 1 g lipase, Accurel and 45SAA had a maximum capacity of 140 and 260 kLU for PS, 112 and 550 kLU for Lipolase 100L, 320 and 800 kLU for SP871 and 18 and 29 kLU for QL, respectively.     

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.     

Lipase-catalyzed kinetic resolutions of racemic β- and γ-thiolactones

Several racemic β- and γ-thiolactones were synthesized and kinetic resolutions of them were executed using lipases. While a lipase from Pseudomonas cepacia (PCL) showed the highest enantioselectivity for (S)-form (>99% ee_S at 53% conversion, E > 100) in the kinetic resolution of racemic -methyl-β-propiothiolactone (rac-MPTL), it showed no hydrolysis activity in the kinetic resolution of -benzyl--methyl-β-propiothiolactone (rac-BMPTL), suggesting that the changes in the size of alkyl group from rac-MPTL to rac-BMPTL leads to lower hydrolysis activity and enantioselectivity. In contrast, racemic γ-butyrothiolactones were hydrolyzed by several lipases with low enantioselectivity, whereas a lipase from Candida antarctica (CAL) showed moderate enantioselectivity for (S)-form (>99% ee_S at 76% conversion, E = 11) in the kinetic resolution of racemic -methyl-γ-butyrothiolactone (rac-MBTL). Computer-aided molecular modeling was also performed to investigate the enantioselectivites and activities of PCL toward β-propiothiolactones. The computer modeling results suggest that the alkyl side chains of β-propiothiolactones and γ-butyrothiolactones interact with amino acid residues around hydrophobic crevice, which affects the activity of PCL.     

Covalent immobilization of pure isoenzymes from lipase of Candida rugosa

Covalent immobilization of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts ( ) with respect to the native pure lipases ( ). The percentage immobilization of lipases A and B is similar in both supports (33–40%). The remaining activity of the biocatalysts immobilized on agarose (70–75%) is greater than that of the enzymatic derivatives immobilized on SiO₂ (40–50%). The surface area and the hydrophobic/hydrophilic properties of the support control the lipase activity of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in SiO₂ (hydrophobic support) using the hydrolysis of the esters of (R) or (S) 2-chloropropanoic and of (R,S) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecificity of the native and the immobilized enzymes is the same.     

Immobilized cells biocatalyst for the production of S-acetylthio-2-methyl propionic acid

S-acetylthio-2-methylpropionic acid (S-AMPA) is an important chiral intermediary for numerous hypertension drugs such as captopril. S-AMPA can be produced by hydrolyzing the corresponding racemic methyl MAMP (S,R-methyl-β-acetylthioisobutyrate) by lipases or esterases that have the appropriate stereo specificity. Psudomonas fluorescens IFO 12055 possessing a highly specific lipase was used to process this reaction in the form of immobilized cells. Reaction kinetic and immobilization methods were also studied. Strong product inhibition was observed, that is, at 3% S-AMPA (namely 183 mM), activity was reduced by 50%. Spontaneous hydrolysis of the ester and thioester bonds was also observed, and was independent of the cells. Thus, reaction selectivity and yield must be optimized through adjusting the substrate concentration and total biocatalyst activity. Conventional calcium alginate (3% w/w) encapsulation was modified by adding 3% w/w polyethyleneimine (PEI) and cross-linked by a biologically derived agent, genipin (5.6 mM). This method was found to be satisfactory to produce stable and functioning biocatalyst and can maintain high reactivity for repeated 25 batches with e.e. values above 90%.     

Biocatalyst engineering exerts a dramatic effect on selectivity of hydrolysis catalyzed by immobilized lipases in aqueous medium

It has been found that enantioselectivity of lipases is strongly modified when their immobilization is performed by involving different areas of the enzyme surface, by promoting a different degree of multipoint covalent immobilization or by creating different environments surrounding different enzyme areas. Moreover, selectivity of some immobilized enzyme molecules was much more modulated by the experimental conditions than other derivatives. Thus, some immobilized derivatives of Candida rugosa (CRL) and C. antarctica-B (CABL) lipases are hardly enantioselective in the hydrolysis of chiral esters of (R,S)-mandelic acid under standard conditions (pH 7.0 and 25°C) (E<2). However, other derivatives of the same enzymes exhibited a very good enantioselectivity under nonstandard conditions. For example, CRL adsorbed on PEI-coated supports showed a very high enantio-preference towards S-isomer (E=200) at pH 5. On the other hand, CABL adsorbed on octyl-agarose showed an interesting enantio-preference towards the R-isomer (E=25) at pH 5 and 4°C. These biotransformations are catalyzed by isolated lipase molecules acting on fully soluble substrates and in the absence of interfacial activation against external hydrophobic interfaces. Under these conditions, lipase catalysis may be associated to important conformational changes that can be strongly modulated via biocatalyst and biotransformation engineering. In this way, selective biotransformations catalyzed by immobilized lipases in macro-aqueous systems can be easily modulated by designing different immobilized derivatives and reaction conditions.     

Enzymatic treatment of wool pre-treated with cetyltrimethylammonium bromide to achieve machine washability

Knitted wool fabric was pre-treated with cetyltrimethylammonium bromide in alkaline conditions in order to remove surface lipid to make the fibre more hydrophilic to enable more efficient subsequent treatment with the enzyme protease. As residual cetyltrimethylammonium bromide on the fibre surface causes an inhibitory effect towards protease, cetyltrimethylammonium bromide has to be removed from the wool. The efficacy of three different anionic surfactants in acid conditions was examined for removing residual cetyltrimethylammonium bromide from the treated wool. If the cetyltrimethylammonium bromide-treated knitted wool was washed with the anionic surfactant, Libraphos HC2A, in acidic conditions, followed by treatment with a chemically modified protease, machine-washable standards could be achieved. The possibility of whether native protease could be used instead of chemically modified protease in achieving shrink-resistance without excessive fibre damage was also studied, exploiting the tendency of residual cetyltrimethylammonium bromide to decrease the activity of the enzyme. It was found that if native protease or native protease in the presence of an enteric polymer was used to treat cetyltrimethylammonium bromide pre-treated wool, an improvement in shrink-resistance without excessive fibre degradation was observed. Machine washability could be achieved by optimizing the proportion of enteric polymer to native protease used to treat cetyltrimethylammonium bromide pre-treated wool. Up-scaling this process showed similar results. The dyeability of the wool with reactive dyes was unaffected by the treatment.     

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.     

Crystal structure of the human acyl protein thioesterase I from a single X-ray data set to 1.5 A

BACKGROUND: Many proteins undergo posttranslational modifications involving covalent attachment of lipid groups. Among them is palmitoylation, a dynamic, reversible process that affects trimeric G proteins and Ras and constitutes a regulatory mechanism for signal transduction pathways. Recently, an acylhydrolase previously identified as lysophospholipase has been shown to function as an acyl protein thioesterase, which catalyzes depalmitoylation of Galpha proteins as well as Ras. Its amino acid sequence suggested that the protein is evolutionarily related to neutral lipases and other thioesterases, but direct structural information was not available. RESULTS: We have solved the crystal structure of the human putative Galpha-regulatory protein acyl thioesterase (hAPT1) with a single data set collected from a crystal containing the wild-type protein. The phases were calculated to 1.8 A resolution based on anomalous scattering from Br(-) ions introduced in the cryoprotectant solution in which the crystal was soaked for 20 s. The model was refined against data extending to a resolution of 1.5 A to an R factor of 18.6%. The enzyme is a member of the ubiquitous alpha/beta hydrolase family, which includes other acylhydrolases such as the palmitoyl protein thioesterase (PPT1). CONCLUSIONS: The human APT1 is closely related to a previously described carboxylesterase from Pseudomonas fluorescens. The active site contains a catalytic triad of Ser-114, His-203, and Asp-169. Like carboxylesterase, hAPT1 appears to be dimeric, although the mutual disposition of molecules in the two dimers differs. Unlike carboxylesterase, the substrate binding pocket and the active site of hAPT1 are occluded by the dimer interface, suggesting that the enzyme must dissociate upon interaction with substrate.     

Application of silica aerogel encapsulated lipases in the synthesis of biodiesel by transesterification reactions

Two types of commercial lipases preparations, one from Burkholderia cepacia, the other one from Candida antartica, were encapsulated in silica aerogels reinforced with silica quartz fibre felt and dried by the CO₂ supercritical technique. These immobilized biocatalysts were applied in biodiesel synthesis by transesterification of sunflower seed oil with methyl acetate. They were found to be efficient even with mixtures of both substrates without any solvent addition. The aerogel encapsulation technique made it possible to maintain the enzymes in a dispersion state similar to the dispersion prevailing in an aqueous solution, even for further use in organic hydrophobic media. In transesterification in excess iso-octane, the two lipases encapsulated in aerogels made from 40% MTMS, were found to have activities relatively close to each other and comparable with commercial Novozyme 435. On the other in transesterification with mixture of oil and methyl acetate without any solvent, the kinetics were severely limited by substrate diffusion inside the aerogels. This was particularly true with the C. antartica, so that the corresponding aerogel encapsulated enzyme was much less active than commercial Novozyme 435, although it improved after a few tests.     

Influence of Temperature on Stability of Multilamellar Liposomes in Wool Dyeing

Liposomes are lipid vesicles that are composed of amphiphile molecules and can carry hydrophobic and hydrophilic materials. In this research work liposomes used as carrier for transfer of dye molecules into wool fibers. The preparation and production of multilamellar liposomes (MLV) from Soya lecithin were carried out and the behavior of liposomes at different temperature was studied. The effect of different concentration of liposomes in the dye exhaustion profile of two dyes (Namely, Irgalan Blue FBL and Lanaset Blue 2R) at two different temperatures of 85°C and 95°C on the wool fabric was investigated. The results showed that presence of liposomes in the dye-bath helps to increase the dye absorption on the wool fabric before 80°C. Dyeing at higher temperature and longer time leads to a decrease in the final exhaustion along with increase in the liposomes concentration. Liposomes at high temperature converted to the disperse phospholipids unimers that may deposited on the fabric surface and may produce a hydrophobic barrier against absorption of dye. The presence of 1% o.w.f. (on weight of fabric) of liposomes at 85°C improved the dye exhaustion of Irgalan Blue FBL on the wool fabric. The wash fastness properties of samples which dyed in the dye-bath containing liposomes also improved.     

Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: I. Effect of pH and water content on reaction kinetics

Lipolase and Lipozyme are produced in large quantities (as a result of genetic engineering and overexpression) for the detergents market and provide a cheap source of highly active biocatalysts. Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RmL) have been isolated in partially purified form from commercial preparations of Lipolase and Lipozyme, respectively. These lipases were solubilized in Aerosol-OT (AOT)-stabilized water-in-oil (w/o) microemulsions in n-heptane. HIL and RmL activity in these microemulsions was assayed by spectrophotometric measurement of the initial rate of p-nitophenyl butyrate hydrolysis, and by chromatographic determination of the initial rate of octyl decanoate synthesis from 1-octanol and decanoic acid. The hydrolytic activity of HIL in microemulsions measured as a function of buffer pH prior to dispersal, followed a sigmoidal profile with the highest activities observed at alkaline pHs. This broadly matches the pH-activity profile for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. The hydrolytic activity of RmL in the same microemulsions, measured as a function of pH, gave a bell-shaped profile with a maximum activity at pH 7.5. Again, the observed pH-activity profile was similar to that reported for a purified RmL in a tributyrin-based aqueous emulsion assay. In contrast, the esterification activity exhibited by both HIL and RmL in AOT microemulsions over the available range pH 6.1 to 10.4, decreases as the pH increases, most likely reflecting the effect of substrate ionization. The dependence of the hydrolytic and condensation activity of HIL on R, the mole ratio of water to surfactant, were similar with both profiles exhibiting a maximum at R = 5. The hydrolytic and esterification activities of RmL followed similar R-dependent profiles, but the profiles in this case exhibited a maximum at R = 10. The water activities at these R values were directly measured as 0.78 and 0.9, respectively. Measured water activities were unperturbed by the presence of lipase at the concentrations used in these studies. (c) 1995 John Wiley & Sons, Inc.     

Hydrolases in supercritical CO<Subscript>2</Subscript> and their use in a high-pressure membrane reactor

The thermal stability and activity of enzymes in supercritical carbon dioxide (SC CO(2)) and near-critical propane were studied at a pressure of 300 bar in the temperature range 20-90 degrees C. Proteinase from Carica papaya was incubated in microaqueous SC CO(2) at atmospheric pressure in a nonaqueous system. Lipase stability in an aqueous medium at atmospheric pressure and in SC CO(2) as well as near-critical propane at 100 bar and 40 degrees C was studied. In order to investigate the impact of solvent on lipases, these were chosen from different sources: Pseudomonas fluorescences, Rhizpous javanicus, Rhizopus niveus and porcine pancreas. On the basis of our previous study on lipase activities in dense gases, a high-pressure continuous flat-shape membrane reactor was designed. The hydrolysis of sunflower oil in SC CO(2) was performed as a model reaction in this reactor. The reaction was catalyzed by the lipase preparation Lipolase 100T and was performed at 50 degrees C and 200 bar.     

The YbgC protein encoded by the ybgC gene of the tol-pal gene cluster of Haemophilus influenzae catalyzes acyl-coenzyme A thioester hydrolysis

This paper examines the catalytic function of the protein YbgC, encoded by the ybgC gene of the tol-pal gene cluster in Haemophilus influenzae. The YbgC protein, a homologue of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-coenzyme A thioesterase, conserves the active site Asp residue associated with thioesterase activity. The H. influenzae ybgC gene was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and tested for thioesterase activity towards acyl-CoA and acyl-N-acetylcysteamine thioesters. The YbgC protein catalyzes the hydrolysis of short chain aliphatic acyl-CoA thioesters, while the D18N YbgC mutant protein (prepared to serve as a control) does not.     

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.     

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(?) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).     

Enzymatic Hydrolysis of Esters Containing a Tetrazole Ring

The lipase‐catalyzed enantioselective hydrolysis of acetates containing tetrazole moiety was studied. Among all tested lipases, Novozyme SP 435 allowed to obtain optically active 4‐(5‐aryl‐2H‐tetrazol‐2yl)butan‐2‐ol and 1‐(5‐aryl‐2H‐tetrazol‐2yl)‐propan‐2‐ol and their acetates with the highest optical purities (ee = 95%‐99%) and excellent enantioselectivity (E>100). Some of the synthesized tetrazole derivatives were screened for their antifungal activity. Racemic mixtures of 4‐[5‐(4‐chlorophenyl)‐2H‐tetrazol‐2‐yl)butan‐2‐ol as well as pure enantiomers of this compound showed promising antifungal activity against F. sambucinum, F. oxysporum, C. coccodes, and A. niger. Chirality 26: 811–816, 2014. © 2014 Wiley Periodicals, Inc.     

Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein–associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

Unbiased proteomics with acyl resin-assisted capture reveals diverse novel substrates of the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) at the synapse, with potential implications for the pathogenesis of neuronal ceroid lipofuscinosis, disulfide bond formation, synaptic adhesion and additional critical synaptic functions.