Purification of oligouronides by immobilized L-histidine pseudoaffinity chromatography

Selective purification of alpha-(1,4)-oligogalacturonides was investigated using several pseudobioaffinity chromatography matrix with aminoacid L-histidine as pseudobiospecific ligand: (1) sepharose 4B-bisoxyran-histidine, (2) sepharose 4B-epoxy-histidine, (3) silica-oxyran-histidine and (4) CIM-disk-EDA-histidine. These anionic oligosaccharides prepared by enzymatic and chemical cleavage of polygalacturonic acid were used as models sugar in order to optimize the adsorption and elution parameters for a selective purification of bioactive oligouronides. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method using for separation and purification of biomolecules thanks to high mass transfer rate. In this way, this new monolithic CIM-disk system with L-histidine immobilized: immobilized histidine affinity chromatography (IHAC) constitutes a good tool allowing the fast and selective purification of bioactive oligouronides.     

Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation

The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.     

Application of calcium alginate immobilized and crude pectin lyase from Bacillus cereus in degumming of plant fibres

Abstract

In this study, the purified pectin lyase was immobilized in calcium alginate beads and compared with crude enzyme for application in degumming of buel and banana plant fibres. From the data of scanning electron microscopy (SEM), it was observed that untreated buel fibres were covered by non-cellulosic materials (pectin, hemicelluloses and waxes) and the surface of enzymatically treated buel fibres looked smoother. Also, the crude alkaline pectin lyase treated buel fibre exhibited a considerably cleaner surface, which suggested that the crude pectin lyase could provide better degumming effects in comparison to the immobilized pectin lyase. In case of banana fibre, the FTIR spectroscopy showed that both crude and immobilized alkaline pectin lyase treatments of banana plant fibres were equally efficient in degumming. The enzymatic degumming of buel and banana with crude pectin lyase resulted in maximum release of galacturonide after 24?h for buel and 15?h for banana fibre. The optimum temperature for degumming of buel and banana fibres with crude pectin lyase was found to be 50?°C and 45?°C, respectively. Also, the maximum galacturonide was released with 200 and 250?U of pectin lyase for buel and banana fibre, respectively.     

Isolation and characterization of extracellular pectin lyase from Penicillium canescens

Pectin lyase A (molecular weight 38 kD by SDS-PAGE, pI 6.7) was purified to homogeneity from culture broth of the mycelial fungus Penicillium canescens using chromatographic techniques. During genomic library screening, the gene encoding pectin lyase A from P. canescens (pelA) was isolated and sequenced, and the amino acid sequence was generated by applying the multiple alignment procedure (360 residues). A theoretical model for the three dimensional structure of the protein molecule was also proposed. Different properties of pectin lyase A were investigated: substrate specificity, pH- and temperature optimum of activity, stability under different pH and temperature conditions, and the effect of Ca2+ on enzyme activity. In the course of the laboratory trials, it was demonstrated that pectin lyase A from P. canescens could be successfully applied to production and clarification of juice.     

Binary immobilization of tyrosinase by using alginate gel beads and poly(acrylamide-co-acrylic acid) hydrogels

The use of the immobilized and the stable enzymes has immense potential in the enzymatic analysis of clinical, industrial and environmental samples. However, their widespread uses are limited due to the high cost of their production. In this study, binary immobilization of tyrosinase by using Ca-alginate and poly(acrylamide-co-acrylic acid) [P(AAm-co-AA)] was investigated. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the free and binary immobilized enzymes. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized tyrosinase were also examined. For the free and binary immobilized enzymes on Ca-alginate and P(AAm-co-AA), optimum pH was found to be 7 and 5, respectively. Optimum temperature of the free and immobilized enzymes was observed to be 30 and 35 degrees C, respectively. Reuse number, storage and thermal stability of the free tyrosinase were increased by a result of binary immobilization.     

An investigation into the pectolytic activity of the yeast Saccharomycopsis fibuliger

Saccharomycopsis fibuliger cells produce an inducible hydrolase, tentatively characterized as a polygalacturonase [poly(1,4-α-d-galacturonide) glycanohydrolase, EC 3.2.1.15], which is associated with the yeast cells and which causes the partial hydrolysis of pectin or poly-d-galacturonic acid. No evidence of pectinesterase (pectin pectyl hydrolyase, EC 3.1.1.11) or pectate lyase [poly(1,4-α-d-galacturonide) lyase, EC 4.1.1.1] activity has been found. Enzyme production took place at an optimum temperature of 28°C, whereas optimum activity was at ～45°C. The optimum pH for pectolytic activity was similar to the optimum pH for cell growth. A reduction in the concentration of dissolved oxygen in the culture medium and an increase in cell age caused an increase in the rate of pectin decomposition within the limits employed. Products of pectin decomposition consisted of a mixture of uronides including d-galacturonic acid.     

Properties of fibre-entrapped aspartase

Aspartase (l-aspartate ammonia lyase, EC 4.3.1.1) was extracted and purified from Escherichia intermedia cells. The enzyme was entrapped in cellulose triacetate porous fibres and the properties of the immobilized enzyme compared with those of the free enzyme. Similar behaviour was observed with regard to optimum pH, temperature, heat stability and kinetic constants. The stability of the entrapped enzyme was tested under operating conditions in a series of batch reactions. Good results were obtained for both the stability and the efficiency of the immobilized enzyme. The potential use of aspartase fibres for the production of l-aspartic acid is discussed.     

Production of lyases byPhoma exigua associated with seed-rot ofVigna radiata

Phoma exigua associated with seed-rot ofVigna radiata produced lyases which varied with the media tested. The production of lyases was higher in pectin-supplemented media.Vigna seed meal medium was not suitable for induction of lyase production. The pectin lyase and pectate lyase was maximum after 11 d of incubation by which time the pH was shifted to alkaline side. Temperature of 25 °C and pH 9 was found to be optimum for the activity of pectin lyase and pectate lyase. Fungicides (antracol and panoctine), phenols (pyrocatechol and gallic acid) and growth substances (gibberellic acid and yeast extract) adversely affected the enzyme secretion.     

Characteristics of the immobilized pectin lyase activity from a commercial pectolytic enzyme preparation

A commercial pectolytic enzyme preparation has been immobilized onto a nylon-polyethyleneimine copolymer, in order ot study the contribution of the pectin lyase activity inthe overall pectindegrading activitg. Optimal conditions for the immobilization process of the pectin lyase activity, such as chemical modification of the support, coupling pH and protein concentration, were determined. Kinetic parameters and temperature behaviour of both the soluble and immobilzied pectin lyase activitgy of the derivative were also determined. OPerational stability of the pectin lyase and overall viscosity reducring activities resulting in a halflife times of 3.8 and 8.5 days, respectively, for both pectolytic activities, when the immobilized derivatives were tested in a cross-flow reactor, using a highly esterified pedtin as substrate.     

Production of oligoglucuronans using a monolithic enzymatic microreactor

A glucuronan lyase (EC 4.2.2.14) was immobilized on a monolithic Convective Interaction Media (CIM((R))) disk. The immobilization yield was equal to 29% of the initial activity and 35% of the initial protein amount. Degradations of three glucuronans with various O-acetylation degrees were investigated and compared with degradations using free enzyme. The immobilized glucuronan lyase was inhibited by the O-acetylation degree like the free enzyme. (1)H NMR analyses were used to study the O-acetylation degree of oligoglucuronans and demonstrated that the average degrees of polymerization were inclusive between 4 and 13 after 24h of degradation. This first immobilization of a glucuronan lyase constitutes a new tool to produce oligoglucuronans.     

Methods for obtaining neutral and acid oligosaccharides from flax pectins

Esterified acid soluble pectins from flax (Linun usitatissimum L.) were degraded either with HCl or pectin lyase. Centrifugation and 2-propanol precipitation led to the isolation of two low molecular weight polygalacturonates after acid hydrolysis of pectins. However, after pectin lyase digestion and purification by size-exclusion HPLC, ¹H NMR analyses indicated that acetylated hairy regions, large methylated and acetylated oligogalacturonides together with small unsubstituted oligogalacturonides were produced. Thus, in a few steps, a panel of substituted neutral and acidic oligosaccharides was produced from a raw plant material. Such oligosaccharides could be useful for further fractionations such as chemical saponification and enzymatic removal of neutral sugar chains from the hairy regions. The procedures used for pectin extraction, for degradation, and for the purification of fragments seem appropriate for large-scale production of biologically active oligosaccharides from flax.Revisions requested 24 September 2004; Revisions received 4 November 2004     

Immobilization of recombinant pectate lyase from Clostridium thermocellum ATCC‐27405 on magnetic nanoparticles for bioscouring of cotton fabric

Recombinant pectate lyase from family 1 polysaccharide lyase (PL1B) was immobilized on synthesized magnetic nanoparticles (MNPs) after 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride activation. At 70 mg/mL MNPs 100% binding of 1 mg/mL PL1B was achieved. The immobilized PL1B‐MNP displayed activity of 20.3 and 18.2 U/mg against polygalacturonic acid and citrus pectin, respectively, which was higher than the activity of free PL1B, on the same substrates of 17.8 and 16.2 U/mg. The immobilized PL1B‐MNP showed 32 fold and 14 fold enhanced thermal stability at 80°C and 90°C, respectively as compared with free PL1B at same temperatures. At high temperature the immobilized PL1B‐MNP retained its activity for a longer duration than free PL1B. The immobilized PL1B‐MNP could be reused till five cycles and after that it retained 70% of initial activity. It could be easily recovered from the reaction mixture with the help of a magnet. Bioscouring of cotton fabric was carried out with immobilized PL1B‐MNP which showed efficient removal of pectin from the fabric surface. The enhanced wettability of fabric resulted in the decrease of the water absorbing time period from 3 min taken by the free PL1B treated fabric to 15 s taken by the immobilized PL1B‐MNP treated fabric. As per our knowledge this is the first attempt of bioscouring of coarse cotton fabric by pectinase immobilized on magnetic nanoparticles. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:231–244, 2017     

The effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract

The effect of temperature and pH on the kinetics of ethanol production by free and calcium alginate immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract was investigated. With the free cells, the ethanol and biomass yields were relatively constant over the temperature range 25-35 degrees C, but dropped sharply beyond 35 degrees C. Other kinetic parameters, specific growth rate, specific ethanol production rate, and specific total sugar uptake rate were maximum at 35 degrees C. However, with the immobilized cells, ethanol yield remained almost constant in the temperature range 25-45 degrees C, and the specific ethanol production rate and specific total sugar uptake rate attained their maximum values at 40 degrees C. For the pH range between 3 and 7, the free-cell optimum for growth and product formation was found to be ca. pH 5. At this pH, the specific growth rate was 0.35 h(-1) and specific ethanol production rate was 2.83 g/g/h. At values higher or lower than pH 5, a sharp decrease in specific ethanol production rate as well as specific growth rate was observed. In comparison, the immobilized cells showed a broad optimum pH profile. The best ethanol production rates were observed between pH 4 and 6.     

Immobilization of polygalacturonase from Aspergillus niger onto activated polyethylene and its application in apple juice clarification

The present work is focused on efficient immobilization of polygalacturonase on polyethylene matrix, followed by its application in apple juice clarification. Immobilization of polygalacturonase on activated polyethylene and its use in apple juice clarification was not reported so far. Aspergillus niger Van Tieghem (MTCC 3323) produced polygalacturonase when grown in modified Riviere's medium containing pectin as single carbon source by fed-batch culture. The enzyme was precipitated with ethanol and purified by gel filtration chromatography (Sephacryl S-100) and immobilized onto glutaraldehyde-activated polyethylene. The method is very simple and time saving for enzyme immobilization. Various characteristics of immobilized enzyme such as optimum reaction temperature and pH, temperature and pH stability, binding kinetics, efficiency of binding, reusability and metal ion effect on immobilized enzymes were evaluated in comparison to the free enzyme. Both the free and immobilized enzyme showed maximum activity at a temperature of 45 degrees C and pH 4.8. Maximum binding efficiency was 38%. The immobilized enzyme was reusable for 3 cycles with 50% loss of activity after the third cycle. Twenty-four U of immobilized enzyme at 45 degrees C and 1 h incubation time increased the transmittance of the apple juice by about 55% at 650 nm. The immobilized enzyme can be of industrial advantage in terms of sturdiness, availability, inertness, low price, reusability and temperature stability.     

Effects of Temperature, pH, and NaCl on Growth and Pectinolytic Activity of Pseudomonas marginalis

The interaction of temperature (4, 10, 18, and 30°C), pH (6, 7, and 8), and NaCl (0, 2.5, and 5%) and their effects on specific growth rate, lag phase, and pectinolytic enzymes of Pseudomonas marginalis were evaluated. Response surface methodology was adapted to describe the response of growth parameters to environmental changes. To obtain good conditions of storage, the combined action of salt and temperature is necessary. At 4°C with an NaCl concentration of 5% and a pH of 7, the lag time was 8 days and no growth was observed at 4°C with 5% NaCl and a pH of 6. In the absence of salt, P. marginalis could grow regardless of temperature and pH. Pectate lyase and pectin lyase were produced by P. marginalis, while pectin methyl esterase activity was not observed in our culture conditions. The enzyme production depended on temperature, pH, and salt concentration but also on the age of the culture. Pectinolytic enzymes were abundantly excreted during the stationary phase, and even at 4°C, after 2 weeks of storage, enzyme activities in supernatant culture were sufficient to damage vegetables. Both bacterial growth and enzymatic production have to be taken into account in order to estimate correctly the shelf life of vegetables.     

Fermentation of pectin by a newly isolated Clostridium thermosaccharolyticum strain

Abstract By enrichment on pectin a thermophilic anaerobic bacterium was isolated. This strain, identified as Clostridium thermosaccharolyticum , was capable of fast growth on pectin (μ_max 0.58 h⁻¹) forming acetate, butyrate, hydrogen, carbon dioxide, methanol and traces of ethanol. The optimum temperature for growth was 58°C and the optimal pH was 6. The initial breakdown of pectin was catalysed by methylesterase and polygalacturonate hydrolase activity; no polygalacturonate lyase activity was found.     

Purification and characterization of pectin lyase secreted by <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K _m and k _cat values were found to be 1 mg/ml and 76 sec⁻¹, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992.     

Pectin lyase from <Emphasis Type="Italic">Aspergillus giganteus</Emphasis>: Comparative study of productivity of submerged fermentation on citrus pectin and orange waste

The aim of this study was to investigate some of the factors affecting pectin lyase (PL) production by an Aspergillus giganteus strain, and to characterize this pectinolytic activity excreted into the medium. The highest activities were obtained with orange waste, citrus pectin and galacturonic acid as carbon sources. The highest activity, using citrus pectin as carbon source, was obtained in 11-day-old standing cultures, but the highest specific activity was obtained in 6.5-day-old shaken cultures, at pH 6.5 and 35°C. Using orange waste as carbon source, the highest activity was observed in 8-day-old standing cultures, at pH 7.0 and 30°C. Optimal assay conditions were pH 8.5–9.0 and 50°C. The PL activity showed thermal stability, with half-lives of 30 and 27 min when incubated at 45 and 50°C, respectively. High stability was observed at room temperature from pH 6.0 to 10.0; more than 85% of enzyme activity was preserved in this pH range. Under optimum conditions, the highest pectin lyase activity in the medium was 470 U/ml, with orange waste as carbon source.     

Cold-adapted yeasts as producers of cold-active polygalacturonases

Eight cold-adapted, polygalacturonase-producing yeasts belonging to four species were isolated from frozen environmental samples in Iceland. They were identified as Cystofilobasidium lari-marini, Cystofilobasidium capitatum, Cryptococcus macerans and Cryptococcus aquaticus species by sequence analysis of rDNA regions. Growth behavior of the isolates was investigated. All strains could grow at 2 degrees C. Addition of glucose to pectin-containing culture medium had a repressive effect on enzyme production except for C. aquaticus, which showed increased polygalacturonase activity. Optimal temperature for enzyme production for the Cystofilobasidium strains was 14 degrees C, while that for the Cryptococcus strains was lower. Among the isolates, C. lari-marini S3B produced highest levels of enzyme activity at pH 3.2. Preliminary characterization of the polygalacturonases in the culture supernatant showed the enzyme from Cystofilobasidium strains to be optimally active at 40 degrees C and pH 5, and that from the Cryptococcus strains at 50 degrees C and pH 4. The polygalacturonase from C. macerans started to lose activity after 1 h of incubation at 40 degrees C, while that from the other strains had already lost activity at 30 degrees C. All the strains except C. aquaticus produced isoenzymes of polyglacturonase. In addition to polygalacturonase, the Cystofilobasidium strains produced pectin lyase, C. aquaticus pectin esterase, and C. macerans pectin lyase, pectate lyase and pectin esterase.     

游离及固定化果糖基转移酶部分酶学性质的比较研究

 从诱变、筛选的米曲霉GX0 0 10菌株所产生的果糖基转移酶 ,经过纯化和固定化操作分别制备游离酶和固定化酶 ,对两者的酶学性质进行了比较研究 .结果表明 ,两者在蔗糖转化为蔗果低聚糖的酶促反应中 ,最适pH为 5 5,在pH5 0～ 7 5之间酶活性相对稳定 .游离酶和固定化酶的适宜温度范围分别是 4 5～ 52℃和 4 0～ 55℃ .在 55℃保温 60min ,酶活性保存率分别是 61 6%和 87 5% .固定化酶的热稳定性提高 .0 1mmol LHg2 +和 1mmol LAg+能完全抑制游离酶的活性 ,但只能部分抑制固定化酶的活性 ,1mmol L的Ti2 +能完全抑制两者的活性 .以蔗糖为底物时 ,游离酶的米氏常数Km=2 15mmol L ,而固定化酶Km =386mmol L .游离酶只能使用一次 ,固定化酶反复使用 54次后 ,剩余活力为 55 2 % .用 55% (W V)蔗糖溶液与固定化酶在pH5 0 ,4 6℃下作用 12h ,可获得61 5% (总低聚糖 总糖 )产物 ,其中蔗果五糖含量达到 7 2 % .