Influence of EDTA complexing agent on biopreparation of linen fabric

In this work the ‘EDTA–enzyme–substrate’ interaction was investigated in linen biopreparation. The effect of EDTA on the degradation of non-cellulosic constituents during bioscouring with pectinase enzyme was investigated in detail. Greige linen fabric was treated with pectinase, pectinase supplemented with ethylenediamine-tetra-acetic acid (EDTA), and EDTA pre-treatment followed by pectinase, in a tumble agitated bath. Adding EDTA to the enzyme solution accelerated the degree of hydrolysis and resulted in higher reducing sugar liberation and more efficient calcium ion extraction, indicating a synergistic effect of enzyme and EDTA. However, when EDTA was applied as a pre-treatment, a decrease in the efficiency of the subsequent enzymatic hydrolysis was observed.     

Hydrolysis of orange peel with pectinase and cellulase enzymes

Summary Hydrolysis of polysaccharides in comminuted orange peel by commercial cellulase and pectinase enzymes has been investigated. High levels of conversion to monomeric sugars were observed after treatment with pectinase enzyme, but cellulase enzyme achieved only limited solubilization. The combination of cellulase and pectinase enzymes appears to be a most efficient system for enzymatic hydrolysis of polysaccharides in orange peel.     

Enhancement of Catalytic Efficiencies of Xylanase,Pectinase and Cellulase by Microwave Pretreatment of their Substrates

Microwave pre-treatment of polygalacturonic acid, xylan and carboxymethylcellulose was found to improve the catalytic efficiencies of pectinase, xylanase and cellulase by 1.5, 2.3 and 1.6 fold, respectively. The microwave effect was distinguished from a pure thermal effect by irradiating at a constant temperature with the help of a non-contact infrared controller. The temperature and time of pre-treatment and substrate concentration during the pre-treatment were optimized by response surface methodology. Scanning electron microscopy revealed significant morphological changes in the substrate as a result of microwave pre-treatment. The time course of enzymatic hydrolysis in each case showed that the use of microwave pre-treated substrates gave higher catalytic rates. Also, a higher degree of bioconversion was observed in each case when microwave pre-treated substrates were used.     

Enhancement of Catalytic Efficiencies of Xylanase, Pectinase and Cellulase by Microwave Pretreatment of their Substrates

Microwave pre-treatment of polygalacturonic acid, xylan and carboxymethylcellulose was found to improve the catalytic efficiencies of pectinase, xylanase and cellulase by 1.5, 2.3 and 1.6 fold, respectively. The microwave effect was distinguished from a pure thermal effect by irradiating at a constant temperature with the help of a non-contact infrared controller. The temperature and time of pre-treatment and substrate concentration during the pre-treatment were optimized by response surface methodology. Scanning electron microscopy revealed significant morphological changes in the substrate as a result of microwave pre-treatment. The time course of enzymatic hydrolysis in each case showed that the use of microwave pre-treated substrates gave higher catalytic rates. Also, a higher degree of bioconversion was observed in each case when microwave pre-treated substrates were used.     

Enzymatic hydrolysis and extraction of arachidonic acid rich lipids from Mortierella alpina

A novel method for efficient arachidonic acid rich lipids extraction was investigated. Six different enzymes (papain, pectinase, snailase, neutrase, alcalase and cellulase) were used to extract lipids from Mortierella alpina. The effects of enzyme concentration, temperature and hydrolysis time on oil recovery were evaluated using factorial experimental design and polynomial regression for each enzyme. Hydrolysis time is found to be the most important parameter for all enzymes. The ratios of enzyme mixtures were also studied. It showed that the mixtures of pectinase and papain (5:3, v/v), pectinase and alcalase (5:1, v/v) were better combined effects on oil yields. The effects of hydrolysis time and temperature were then analyzed by response surface methodology, and oil recoveries were satisfactory (104.6% for pectinase and papain and 101.3% for pectinase and alcalase). In the whole process, the lipid composition was not affected by the enzyme treatments according to fatty acid profile.     

Effects of N-acetylation degree on N-acetylated chitosan hydrolysis with commercially available and modified pectinases

Three types of N-acetylated chitosans (NACs) with different degrees of acetylation (DA) were prepared and used as a substrate for enzymatic hydrolysis with a commercially available pectinase and a modified one. Pectinase modification was conducted using polyalkyleneoxide-maleic anhydride copolymer (PEO-MA copolymer). The effects of DA on enzymatic reaction with native and modified pectinases were investigated experimentally. Initial hydrolysis rate and Michaelis-Menten kinetic parameters were measured by analysis of reducing sugars. DA of NAC strongly affected the hydrolytic characteristics of native and modified pectinases. N-acetylation of chitosan increased the initial hydrolysis rate and the enzyme-substrate affinity with respect to both pectinases: NACs with DA over 0.3 showed high initial hydrolysis rate and strong affinity between enzyme and substrate. Especially, when NAC with DA over 0.3 was treated with modified pectinase, the affinity became much stronger than the native pectinase.     

Enzymes and chelating agent in cotton pretreatment

Desized cotton fabric and cotton seed-coat fragments (impurities) have been treated with commercial cellulase (Celluclast 1.5 L), hemicellulase–pectinase (Viscozyme 120 L) and xylanase (Pulpzyme HC) enzymes. Seed-coat fragments hydrolyzed much faster than the cotton fabric itself. This relative difference in hydrolysis rates makes possible a direct enzymatic removal of seed-coat fragments from desized cotton fabric. Addition of chelating agents such as ethylenediamine-tetra-acetic acid (EDTA) markedly enhanced the directed enzyme action. Pretreatments carried out in acidic solution at pH 5 increased the lightness of seed-coat fragments, contrary to the samples treated in neutral medium at pH 7. Alkaline scouring resulted in darker seed-coat fragments except for the samples pretreated with Pulpzyme HC plus EDTA. This effect is similar to that observed in the biobleaching process in pulp and paper industry.     

Optimization of enzyme complexes for lignocellulose hydrolysis

The ability of a commercial Trichoderma reesei cellulase preparation (Celluclast 1.5L), to hydrolyze the cellulose and xylan components of pretreated corn stover (PCS) was significantly improved by supplementation with three types of crude commercial enzyme preparations nominally enriched in xylanase, pectinase, and beta-glucosidase activity. Although the well-documented relief of product inhibition by beta-glucosidase contributed to the observed improvement in cellulase performance, significant benefits could also be attributed to enzymes components that hydrolyze non-cellulosic polysaccharides. It is suggested that so-called "accessory" enzymes such as xylanase and pectinase stimulate cellulose hydrolysis by removing non-cellulosic polysaccharides that coat cellulose fibers. A high-throughput microassay, in combination with response surface methodology, enabled production of an optimally supplemented enzyme mixture. This mixture allowed for a approximately twofold reduction in the total protein required to reach glucan to glucose and xylan to xylose hydrolysis targets (99% and 88% conversion, respectively), thereby validating this approach towards enzyme improvement and process cost reduction for lignocellulose hydrolysis.     

Feulgen Cytophotometry of Pine Nuclei. II. Effect of Pectinase Used in Cell Sfparation

Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that hut denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.     

Feulgen cytophotometry of pine nuclei. II. Effect of pectinase used in cell separation.

Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that heat denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.     

Pectinase production by Bacillus subtilis and its potential application in biopreparation of cotton and micropoly fabric

An alkaline and thermostable pectinase production from Bacillus subtilis SS was optimized under submerged fermentation and its application was tested in textile industry for desizing and bioscouring of cotton and micropoly fabrics. Desizing of fabric was the best with 5 U/g pectinase treatment for 120 min at pH 9.5 and 65 °C. Under optimized conditions of bioscouring, desized cotton showed highest reducing sugar liberation and weight loss than desized micropoly. Along with enzyme, addition of chelating (EDTA) and wetting agent markedly enhanced the weight loss compared to single use of enzyme or EDTA alone. Agitation (50 ± 2) enhanced the weight loss values of cotton (1.9%) and micropoly fabric (1.7%) at pH 9.5 after treatment time of 2 h. Bioscouring of fabrics with pectinase resulted in enhancement of various physical properties of fabrics viz. whiteness (1.2%), tensile strength (1.6%) and tearness (3.0%) over conventionally alkaline scoured fabrics.     

Carboxypeptidase displaying differential velocity in hydrolysis of methotrexate, 5-methyltetrahydrofolic acid, and leucovorin.

An enzyme that catalyzes the hydrolysis of folic acid and the antifolate methotrexate nearly 20 times more rapidly than the hydrolysis of 5-methyltetrahydrofolate was extraced from a gram-negative bacterium tentatively identified as a Flavobacterium sp. The enzyme was purified 500-fold and found to have a molecular weight of about 53,000. Apparently a metallo-enzyme, it is inhibited by citrate and ethylenediaminetetraacetic acid (EDTA). Ca2+, Co2+, Mg2+, and Zn2+ reverse inhibition by EDTA, whereas Ca2+ and Zn2+ are weak activators in the absence of EDTA. The enzymatic reaction releases the carboxy-terminal glutamyl moiety of derivatives of pteroyl-mono-L-glutamic acid. Substituents on N5 of the pteridine ring decrease the velocity of hydrolysis. Some non-specificity for the terminal amino acid is expressed. The strikingly different rates of hydrolysis of methotrexate and 5-methyltetrahydrofolate have stimulated interest in this enzyme for its potential clinical value in improving the therapeutic index of methotrexate.     

Phosphatase systems in Paramphistomum explanatum Fischoeder, 1901.

Extracts of adult Paramphistomum explanatum have been shown to contain high concentration of acid phosphomonoesterase with maximum activity at pH 4.5. The enzyme has been characterized by an exhibition of an unexpected increase in the inhibitory action of a mercury at 1 mM concentration by EDTA. With a lower concentration of mercury (0.1 mM and below) EDTA gave partial protection against inhibition. Different concentrations of magnesium and cobalt activated the enzyme while fluoride, copper, arsenate, tartrate and p-mercuribenzoate brought about inhibition. EDTA, glycine, glutathione and sodium azide had no effect. There was an indication of the presence of alkaline phosphomonoesterase at pH 10.0. The Km for p-nitrophenyl phosphate hydrolysis was 0.45 mM at pH 4.5.     

Effect of various factors on the methylation of DNAse I

The effects of NaCl, EDTA and tRNA on methylation and enzymatic properties of deoxyribonuclease I (DNase I) were investigated. The methylation was carried out by S-methylmethionine (vitamin U) in the phosphate-citric buffer pH 4.0. It was found that 0.5 M NaCl decreases by about 30% the incorporation of CH3-groups into the DNase, whereas 1.5 M NaCl-by 47%. A similar, although a less pronounced effect, was exerted by tRNA within the concentration range of 1.36-34.7 microM. On the contrast, EDTA (0.01-0.05 M) stimulated the incorporation of CH3-groups by 15 and 30%, respectively. The functional properties of methylated DNase I in the presence of EDTA remained unaffected. The enzyme methylation in the presence of NaCl or tRNA caused deceleration of the 3H-DNA hydrolysis (by 15-30%) only within the first 20 min of the reaction.     

Electrostatic immobilization of pectinase on negatively charged AOT-Fe3O4 nanoparticles

Enzyme immobilization on magnetic nanoparticles (MNPs) has been a field of intense studies in biotechnology during the past decade. The present study suggests MNPs negatively charged by docusate sodium salt (AOT) as a support for pectinase immobilization. AOT is a biocompatible anionic surfactant which can stabilize MNPs. Electrostatic adsorption can occur between enzyme with positive charge and oppositely charged surface of MNPs (ca. 100 nm). The effect of three factors, i.e. initial enzyme concentration, aqueous pH and AOT concentration in different levels was investigated on pectinase immobilization. Maximum specific activity (1.98 U/mg enzyme) of immobilized pectinase and maximum enzyme loading of 610.5 mg enzyme/g support was attained through the experiments. Initial enzyme concentration is significantly important on both loading and activity of immobilized enzyme, while pH and AOT concentration only affect the amount of immobilized enzyme. Immobilized enzyme on MNPs was recovered easily through magnetic separation. At near pH of immobilization, protein leakage in reusability of immobilized enzyme was low and activity loss was only 10–20% after six cycles. Since pH is associated with immobilization by electrostatic adsorption, the medium pH was changed to improve the release of protein from the support, as well. MNPs properties were investigated using Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FT-IR) spectroscopy, and Dynamic Light Scattering (DLS) analysis.     

Repeated Enzymatic Hydrolysis of Polygalacturonic Acid, Chitosan and Chitin Using a Novel Reversibly-soluble Pectinase with the Aid of κ-carrageenan

Aspergillus niger pectinase, together with κ-carrageenan, could be precipitated in the presence of 0.2% KCl and re-dissolved by ten-fold dilution of the salt. The free as well as this reversibly-soluble (rs) enzyme were evaluated for hydrolysis of polygalacturonic acid, chitosan and chitin. The rs-enzyme showed 92%, 80% and 74% activity (as compared to the corresponding amount of enzyme when present as a free enzyme) towards the three substrates, respectively. There was no significant change in the pH and temperature optima of the rs-enzyme. This preparation could be reused six times without loss of any detectable polygalacturonase activity. This biocatalyst design was found to be efficient for the hydrolysis of polygalacturonic acid, chitosan and chitin.     

A smart bioconjugate of alginate and pectinase with unusual biological activity toward chitosan

The commercial preparation of pectinase (Pectinex Ultra SP-L) was conjugated to alginate by noncovalent interactions by employing 1% alginate during the conjugation protocol. The optimum "immobilization efficiency" was 0.76. The pH optimum and the thermal stability of the enzyme remained unchanged upon conjugation with alginate. The soluble bioconjugate showed a 3-fold increase in V(max)/K(m) as compared to the free enzyme when the smart biocatalyst was used for chitosan hydrolysis. Time course hydrolysis of chitosan thus showed higher conversion of chitosan into reducing oligosaccharides/sugars. The smart bioconjugate could be reused five times without any detectable loss of chitosanase activity.     

Repeated Enzymatic Hydrolysis of Polygalacturonic Acid,Chitosan and Chitin Using a Novel Reversibly-soluble Pectinase with the Aid of κ-carrageenan

Aspergillus niger pectinase, together with κ-carrageenan, could be precipitated in the presence of 0.2% KCl and re-dissolved by ten-fold dilution of the salt. The free as well as this reversibly-soluble (rs) enzyme were evaluated for hydrolysis of polygalacturonic acid, chitosan and chitin. The rs-enzyme showed 92%, 80% and 74% activity (as compared to the corresponding amount of enzyme when present as a free enzyme) towards the three substrates, respectively. There was no significant change in the pH and temperature optima of the rs-enzyme. This preparation could be reused six times without loss of any detectable polygalacturonase activity. This biocatalyst design was found to be efficient for the hydrolysis of polygalacturonic acid, chitosan and chitin.     

A comparison of the non-specific acid phosphomonoesterase activity in the larva of Phocanema decipiens (Nematoda) with that of the muscle of its host the codfish (Gadus morhua).

The non-specific phosphomonoesterase (enzyme I) extracted from the larva of the codworm (Phocanema decipiens) is different from the enzyme (enzyme II) from the muscle of its host, the codfish (Gadus morhua). The pH optima were 4.0 and 4.5, and the KM values for p-nitrophenyl phosphate hydrolysis were 1.8 mM and 6.5 mM for enzymes I and II respectively. The specific specific activity in units (0.01 mumol/min) per mg protein was 4.80 +/- 0.85 and 0.54 +/- 0.07 for enzymes I and II respectively. The specific activity from uninfected muscles was only 0.39 (SD +/- 0.017) units per mg of protein. Both enzymes were inhibited by NaF, HgCl2, and cysteine but were stimulated by 2-mercaptoethanol. EDTA and iodoacetamide had no effect on enzyme I but enzyme II was activated by EDTA and inhibited by iodoacetamide. Cadmium ions inhibited both the enzymes but a conspicuous feature with enzyme II was in the increase in percentage inhibition by lowering the concentration of CD2+.     

Augmentation of the biodisposition of simple sugars from sorghum biomass mediated with biotechnologic processes

The results of the enzymatic hydrolysis of pectin, hemicellulose and cellulose in the biomass of sweet sorghum (Sorghum vulgare var. saccharatum, L.) are reported. Some commercial enzymatic preparations were used: Maxazym CL 2000 (with prevailing cellulase activity), Rapidase C 80 (with prevailing pectinase activity) Rohament PC (mainly with pectinase and cellulase activities) and Rohament O (mainly with pectinase and hemicellulase activities). The treatment with Rohament PC, Rohament O and Rapidase C 80 gives an increase of the glucose content higher than the effect induced by Maxazym CL 2000. On the other hand, the cellulase and pectinase combined treatment (Maxazym CL 2000 + Rapidase C 80 or Maxazym CL 2000 + Rohament O) shows a good synergistic effect in the degradation of the plant cell wall cellulosic material.