Preparation of and optimal module housings for hollow fibre membrane ion exchangers

Macroporous polyamide 6 hollow fibres can be polymer coated by a three-step procedure: first, reaction of the amino end groups with a bifunctional, double-bond-containing reagent; second, block polymerization with different monomers; and third, polymer analogue reactions with amines or sulphite salts to produce ion exchanger groups. The densities of double bonds are dependent on the amino densities and are in the range of 20-30 mumol/g polyamide 6. The ion exchanger fibres were packed in different types of module housings to get an optimal separation unit. The best housing seems to be a so-called single-dead-end arrangement of fibres. Three types of ion exchanger hollow fibres have been produced: a weak and a strong anion exchanger and a strong cation exchanger. The dynamic protein-binding capacities are in the range of 40 mg/ml membrane. Using these membrane modules, it is possible to separate proteins in the same way as with particle-based ion exchangers. Fast protein separations with low pressure drop are possible.     

Effect of the agitation on the adsorption and hydrolytic efficiency of cutinases on polyethylene terephthalate fibres

The effect of agitation on adsorption, desorption and hydrolytic efficiency of a native and a genetically modified cutinase (L182A) on polyethylene terephthalate fibres is reported in this paper. The effect of mechanical agitation was studied using a shaker bath with orbital agitation and a Rotawash machine with vertical agitation with and without extra steel discs inside the reaction pots. The results obtained indicate that mechanical agitation combined with enzymatic action enhances the adsorption and activity of cutinases towards PET (polyethylene terephthalate) fibres. L182A showed higher adsorption than the native enzyme for all the levels of mechanical agitation. Lower units of L182A lead to similar yields of terephthalic acid formed in all levels of mechanical agitation. The highest increase of hydroxyl surface groups was found for the genetically modified L182A at the lowest level of mechanical agitation with a shaker bath. These results indicate that enzymatic functionalization of PET is favoured with a process with lower levels of mechanical agitation.     

Factors affecting the activation of pulps with laccase

Activation of fibres by radical formation is the first step when aiming at oxidative coupling of new functional groups on the fibre bound lignin. In this work, factors affecting the amount of phenoxy radicals created to unbleached TMP, CTMP, softwood kraft and hardwood kraft pulp fibres in the laccase catalysed oxidation were determined by EPR. Laccase was able to catalyse the oxidation of all the pulps studied. The reactivity of the pulp was found to be affected by both the physical accessibility of lignin in the fibres and the chemistry of the surface lignin accessible to laccase. Laccase dosage, use of extra oxygen in the laccase catalysed radicalization reaction, treatment time and also the amount and type of low-molecular weight compounds (LMWC) present in the pulp were all found to contribute to the radical content of pulp fibres measured after the enzymatic reaction. It could not been excluded that two types of reactions take place during the radical formation in fibres. Within the fibre matrix there may be both fibre material bound and soluble lignin fragments differing with respect to accessibility, molecular weight or chemical structure which can be radicalized at various rates, and the formed radicals may also undergo cross-coupling reactions reducing the amount of the total radicals.     

Brain adenosine 5′-triphosphate–creatine phosphotransferase. Purification, thiol group reactivity and the amino acid sequence around the reactive thiol groups

1. The purification of creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) from ox brain by a method that is quicker, simpler and gives much higher yields than other published procedures is described. 2. Stoicheiometric inhibition studies with iodoacetate showed that the enzyme, like that from muscle, has two reactive thiol groups that are essential for enzyme activity. 3. The amino acid sequence around the essential thiol groups was determined and found to be virtually identical with that in creatine kinases from rabbit and ox muscle, and very similar to that found in arginine kinase; the evolutionary significance of this is discussed. 4. The identification of DNS-amino acids on thin layers of silica gel was found to have, in many cases, distinct advantages over that on polyamide layers.     

Voltametric monitoring of enzyme-mediated indigo reduction in the presence of various fibre materials

A comparative study of enzyme-mediated indigo reduction is presented as an environmentally-friendly alternative to alkaline sodium dithionite reduction. The effect of the mediator 1,8-dihydroxy-9,10-anthraquinone in enzymatic reduction was studied by means of voltammetry, both in the presence and absence of different textile materials (polyamide 6, polyamide 6,6 and cotton), and compared to chemically reduced indigo. It was observed that bio-catalytic formation of leuco indigo and its exhaustion on substrates is inversely proportional to the pH within the range of 7–11. Additionally, substrate coloration was strongly influenced by the mediator, resulting in in situ formation of leuco indigo. This effect was most pronounced for polyamide substrates. The reuse of an enzyme-mediated reduction bath for dyeing was assessed showing that the levelness of the obtained shade was either excellent or good at pH 9 and 11, respectively. The wash, perspiration, and light color fastness properties of all textile materials dyed with enzymatically-reduced indigo were comparable or even better than those obtained with chemically reduced indigo. The use of enzyme-mediated reduction of indigo combined with potential reuse of the reduction bath represents a cost effective and environmentally-friendly dyeing process that can be applied for the dyeing of natural cellulosic and synthetic polyamide fibres.     

Activation of polymeric materials towards enzymatic postgrafting and cross-linking

A methodology to activate inert polymeric materials to enzymatic functionalisation is described herein. Plasma irradiation can be used to graft compounds containing a moiety that is reactive towards an enzyme of interest. Subsequently, such enzyme can be used to either postgraft functional compounds or cross-link the polymeric materials. Argon plasma was utilised to graft 2-aminoethyl methacrylate onto cotton and wool fibres, introducing surface alkylamine groups to impart reactivity towards transglutaminase and tyrosinase. The efficiency of plasma grafting was verified by ATR-FTIR. Enzyme postgrafting of fluorescent peptides coupled with confocal microscopy was used to demonstrate transglutaminase activity towards cotton, a material typically inert to this enzyme. The grafting of alkylamines onto wool resulted in additional cross-linking by both enzymes, leading to significantly increased yarn breaking load and elongation at break. This technology permits the activation of inert materials towards enzymatic postgrafting, with applications in fields as diverse as textiles and biomaterials.     

Pepsin-catalyzed peptide synthesis

The use of pepsin as a catalyst for the synthesis of peptide bonds was investigated. It is shown that the enzyme enables the preparation of several protected dipeptides and tripeptides containing two adjacent aromatic residues of the type P-Al-Phe-Y, P-PHe-Ar-Y, or P-AR-Phe-Y where P and Y are amino and carboxyl protecting groups, AL is an aliphatic amino acid residue, and Ar is an aromatic, amino acid residue. They yields are in the rang 25–97%. The high yields, combined with the enzyme's stereospecificity, permit the isolation of optically pure enantiomers from racemic mixtures. For example, when Z-DL -Ph-OH is allowed to react with an excess of H-L -Phe-NH₂, the stereoisomer Z-L -Phe-L -Phe-NH₂ is obtained in practically quantitative yield. At the same time, the unreacted, optically pure Z-D -Phe-OH can be recovered (Z = carbobenzyloxy, Phe = phenylalanine). The advantages and disadvantages of the enzymatic coupling procedure as a possible routine method for peptide synthesis are discussed.     

Laccase-induced grafting on plasma-pretreated polypropylene

A new environmentally friendly strategy for the sustainable functionalization of inert man-made polymer surfaces is mapped out for the first time using a combination of plasma pretreatment and enzymatic postgrafting. The efficiency of enzymatic covalent binding is investigated by grafting methacrylate monomers possessing different amino groups, primary, tertiary, and quaternary, onto a polypropylene surface using plasma pretreatment. Subsequent enzymatic grafting, using laccase and guaiacol sulfonic acid (GSA), is determined by surface analytical techniques, such as attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The grafting of GSA in the presence of a laccase is proven by a 10-fold increase in sulfur compared to the control. The covalent coupling between GSA and primary amine groups is determined by HPLC-MS using hexylamine as a model substrate. The advantage of technology is in the strong covalent binding of functional groups onto the synthetic polymer's surface, which could then be suitably tailored by enzymes possessing substrate specificity and regional selectivity.     

Quantitative succinate-dehydrogenase histochemistry. II. A comparison between visual and quantitative msucle fibre typing.

Most muscles exhibit a mosaic pattern of staining intensities of their muscle fibres after the histochemical reaction for succinate dehydrogenase (SDH). Visually these muscle fibres are usually classified into three groups: with low (A-fibres), intermediate (B-fibres), and high (C-fibres) enzymatic activity (staining intensity). Cytospectrophotometric methods were employed to investigate whether discrete groups of muscle fibres could be discerned, comparable to those found after the visual classification. The classifications were based on quantitative parameters of the total absorbance per cell and the distribution of the coloured endproduct over the fibre cross area.     

Conjugation of enzymes on polymer nanoparticles covered with phosphorylcholine groups

We investigated the bioconjugation of enzymes on polymer nanoparticles covered with bioinert phosphorylcholine groups. A water-soluble amphiphilic phospholipid polymer (PMBN) was specially designed for preparation of nanoparticles and conjugation with enzymes on them. The PMBN was prepared by random copolymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and p-nitrophenylester bearing methacrylate. The PMBN was used as an emulsifier and a surface modifier to prepare the poly(l-lactic acid) nanoparticles by a solvent evaporation technique in aqueous medium. The nanoparticles covered with phosphorylcholine groups were stably dispersed in an aqueous solution and a phosphate buffered saline. The diameter and surface zeta-potential of the nanoparticles were ca. 200 nm and -6 mV, respectively. The p-nitrophenyl ester groups, which are active ester units for the amino groups of the protein, were located at the surface of the nanoparticles. Both acetylcholine esterase and choline oxidase were co-immobilized (dual-mode conjugation) by the reaction between the p-nitrophenyl ester group and the amino group of these enzymes. The enzymatic reactions on the nanoparticles were followed using a microdialysis biosensor system with a microtype hydrogen peroxide electrode in the probe. The nanoparticles conjugated with these enzymes could detect the acetylcholine chloride as hydrogen peroxide, which is a product of the enzymatic reactions on the surface of the nanoparticles in the probe. Namely, continuous enzyme reactions could be occurring on the surface of the nanoparticles. It is concluded that the nanoparticles are a promising tool for a highly sensitive and microdiagnostic system.     

Biotransformations in synthetic fibres

Recent studies clearly indicate that the modification of synthetic polymers with enzymes is an environmentally friendly alternative to traditional chemical methods requiring harsh conditions. Some work already performed on polyamide 6.6 (nylon 6.6), polyethyleneterephthalate (PET) and polyacrylonitrile (PAN) revealed that surface functionalization of these materials is a key requirement for an extensive range of applications, such as textiles, electronics, biomedical field and others. Research performed on PET with lipases, cutinases and other esterases has previously been reported, whilst enzymatic treatment of PAN with nitrilases and cutinase has also been the subject of study. However, at present, few studies have been done on nylon fabrics, mainly with esterases and proteases. This work is intended as a brief review of research in the area of biocatalytic functionalization of synthetic fibres, with a special focus on work recently performed by our research group with cutinase from Fusarium solani pisi.     

The Chemical Synthesis of Biochemically Active Oligoribonucleotides Using Dimethylaminomethylene Protected Purine H-Phosphonates

Abstract

Dimethylaminomethylene was applied as the protecting group for the exocyclic amino groups of adenosine and guanosine in the automated chemical synthesis of oligoribonucleotides on a polymer bound support. The dimethyl-aminomethylene protecting group can be removed at room temperature under conditions where the concomitant loss of the 2′-protection group can be excluded. The transformation of 2′-O-(t-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl) protected nucleosides to 3′-H-phosphonates yields synthons, well suited for the automated chemical synthesis of oligoribonucleotides. Using these H-phosphonate monomers, a coupling time of two minute: is sufficient to obtain average coupling yields of more than 98 %. Synthesized RNA is recognized as a substrate in an enzymatic reaction, forms the expected secondary structures and is suitable for NMR structural investigations.     

Enzymatic surface modification of cellulose acetate fibre by cutinase-CBM (carbohydrate-binding module) fusion proteins

Abstract

In this paper, two types of bacterial fusion protein, cutinase-CBM_Cel6A and cutinase-CBM_CenA, were used to modify the surface of cellulose acetate fibre. The enzyme binding on cellulose acetate fibres and the hydrolysis of acetyl groups were monitored. Water absorbency and dye uptake were measured to assess the extent of enzymatic modification. The results demonstrated that cutinase-carbohydrate-binding module (CBM) has a greater effect on cellulose acetate fibres than that of cutinase. The use of non-ionic surfactant Triton X-100 could further improve enzymatic modification of cellulose acetate fibres in terms of wettability and dyeability. Scanning electron microscopy confirmed that both cutinase-CBMs could lead to the formation of carving characters on the surface of treated cellulose acetate fibres. Our studies provide a foundation for the potential application of cutinase-CBM in the surface modification of cellulose acetate fibre.     

Immobilization of thermolysin to polyamide nonwoven materials

In the last few years, an increasing number of biotechnological techniques have been applied to the restoration and conservation of works of art, paintings, old maps, and papers or books. Enzymes can solve problems that give restorers difficulties, although for many applications it is not possible to use soluble enzymes; therefore, it is necessary to look for suitable carriers for immobilization. Different methods for covalent immobilization of enzymes to polyamide nonwovens were tested, using thermolysin as an example. Two distinct strategies were pursued: (1). controlled, partial hydrolysis of the polymer and subsequent binding of the enzyme to the released amino and carboxy groups; and (2). attachment of reactive groups directly to the polyamide without disintegrating the polymeric structure (O-alkylation). Different spacers were used for covalent fixation of the enzyme in both cases. The enzyme was fixed to the released amino groups by glutaraldehyde, either with or without a spacer. Either way, active enzyme could be immobilized to the matrix. However, intense treatment caused severe damage to the stability of the nonwoven fabric, and reduced the mechanical strength. Conditions were investigated to conserve the nonwoven fabric structure while obtaining near-maximum immobilized enzyme activity. Immobilization of the enzyme to the released carboxy group after acid hydrolysis was performed using dicyclohexylcarbodiimide. In comparison to the enzyme bound via the amino group, the yield of immobilized enzyme activity was slightly lower when benzidine was taken as spacer and still lower with a 1,6-hexanediamine spacer. O-alkylation performed with dimethylsulfate caused severe damage to the nonwoven fabric structure. Considerably better results were obtained with triethyloxonium tetrafluoroborate. As the spacers 1,6-hexanediamine and adipic acid dihydrazide were used, activation for immobilizing thermolysin was performed with glutaraldehyde, adipimidate, and azide. With the exception of azide, all combinations of spacers and activation reagents gave high yields of immobilized enzyme activity. Thermolysin immobilized by this technique showed a remarkably improved stability with respect to elevated temperature, extreme pH values, and reduced polarity. The nonwoven fabric can be stored for weeks without loss of enzyme activity by washing with distilled water and drying.     

Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media

The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR(1) (R(1): Et, Al, Cam) and H-Asp-(OR(2))-Phe-NH(2) (R(2): H, Bu(t)) catalyzed by alpha-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at a(w) = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at a(w) = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH(2) with beta-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.     

QTL Mapping of Enzymatic Saccharification in Short Rotation Coppice Willow and Its Independence from Biomass Yield

Short rotation coppice (SRC) willows (Salix spp.) are fast-growing woody plants which can achieve high biomass yields over short growth cycles with low agrochemical inputs. Biomass from SRC willow is already used for heat and power, but its potential as a source of lignocellulose for liquid transport biofuels has still to be assessed. In bioethanol production from lignocellulose, enzymatic saccharification is used as an approach to release glucose from cellulose in the plant cell walls. In this study, 138 genotypes of a willow mapping population were used to examine variation in enzymatic glucose release from stem biomass to study relationships between this trait and biomass yield traits and to identify quantitative trait loci (QTL) associated with enzymatic saccharification yield. Significant natural variation was found in glucose yields from willow stem biomass. This trait was independent of biomass yield traits. Four enzyme-derived glucose QTL were mapped onto chromosomes V, X, XI, and XVI, indicating that enzymatic saccharification yields are under significant genetic influence. Our results show that SRC willow has strong potential as a source of bioethanol and that there may be opportunities to improve the breeding programs for willows for increasing enzymatic saccharification yields and biofuel production.     

Peptide enzymatic microsynthesis, using carboxypeptidase Y as the catalyst: application to stepwise synthesis of Leuenkephalin

In order to develop the use of carboxypeptidase Y (CPD-Y, EC 3.4.12.1) as a catalyst for radioactive hormonal synthesis, the stepwise synthesis of a pentapeptide, Leu-enkephalin, was studied on a microscale. Each peptide bond was formed by enzymatic catalysis, using microquantities of the precursors (amino acid or peptide esters as acyl-components and amino acid ester or amide as nucleophiles). The high condensation yields obtained suggests that CPD-Y can be a useful tool for preparation of radioactive hormonal peptides.     

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.     

Enzymatic synthesis of arginine-based cationic surfactants

A novel enzymatic approach for the synthesis of arginine N-alkyl amide and ester derivatives is reported. Papain deposited onto solid support materials was used as catalyst for the amide and ester bond formation between Z-Arg-OMe and various long-chain alkyl amines and alcohols (H2N-Cn2, HO-Cn; n = 8-16) in organic media. Changes in enzymatic activity and product yield were studied for the following variables: organic solvent, aqueous buffer content, support for the enzyme deposition, presence of additives, enzyme loading, substrate concentration, and reaction temperature. The best yields (81-89%) of arginine N-alkyl amide derivatives were obtained at 25 degrees C in acetonitrile with an aqueous buffer content ranging from 0 to 1% (v/v) depending on the substrate concentration. The synthesis of arginine alkyl ester derivatives was carried out in solvent-free systems at 50 or 65 degrees C depending on the fatty alcohol chain length. In this case, product yields ranging from 86 to 89% were obtained with a molar ratio Z-Arg-OMe/fatty alcohol of 0.01. Papain deposited onto polyamide gave, in all cases, both the highest enzymatic activities and yields. Under the best reaction conditions the syntheses were scaled up to the production of 2 g of final product. The overall yields, which include reaction, Nalpha-benzyloxycarbonyl group (Z) deprotection and purification, varied from 53 to 77% of pure (99.9% by HPLC) product.     

Enzymes as reagents in the synthesis of peptides

The use of enzymes to catalyse peptide bond formation and for manipulating blocking groups during peptide synthesis is discussed. The history of solubility-controlled peptide condensations in the presence of proteolytic enzymes is traced. General techniques for obtaining improved yields of soluble condensation products are outlined along with special conditions which sometimes favour the enzymatic condensations of peptide fragments. Progress in the use of enzymes to manipulate blocking groups on α-amino groups, α-carboxyl groups, and on the sidechain functional groups of amino acid residues are examined. Some anticipated developments in the use of enzymes as reagents in peptide synthesis and semisynthesis are discussed.