Affinity precipitation and macroaffinity ligand facilitated three-phase partitioning for refolding and simultaneous purification of urea-denatured pectinase

Protein refolding is an integral step in the recovery of protein activity from inclusion bodies. It is shown that affinity precipitation and macroaffinity ligand facilitated three-phase partitioning (MLFTPP) led to refolding of urea-denatured pectinase present in a commercial preparation, with simultaneous purification. Affinity precipitation consists of precipitation of the desired enzyme by complexing it with a suitable stimulus-sensitive macroaffinity ligand. This ligand in this case was alginate/esterified alginate. The complex of the polymer-pectinase could be precipitated by adding calcium ions. In MLFTPP (carried out by adding tertiary butanol and ammonium sulfate to the aqueous solution of crude enzyme and the polymer), the polymer or its complex with the enzyme form an interfacial precipitate between tert-butyl alcohol phase and aqueous phase. It is believed that in both processes, while molecular recognition of alginate/esterified alginate to pectinase facilitates their selective binding to the enzyme, the correct refolding is facilitated by preventing molecular aggregation of unfolded enzyme molecules. Three-phase partitioning with esterified alginate as the macroaffinity ligand gave 100% recovery with 4-fold purification. Affinity precipitation with 1% alginate gave 52% yield with 18-fold purification. On the other hand, use of 0.5% esterified alginate gave only 7-fold purification but with 75% recovery of activity.     

Development of a process for large-scale chromatographic purification of an alginate lyase from Klebsiella pneumoniae

Summary A process for purification of an alginate lyase, produced extracellularly by fermentation of Klebsiella pneumoniae, has been developed. The process includes two chromatographic steps and is well suited to large-scale operation. By hydrophobic interaction chromatography on Phenyl-Sepharose FF, followed by anion exchange chromatography on Q-Sepharose FF in a negative mode, the specific activity was increased from 0.09 units (U) mg ^–1 to more than 50 U mg^–1. Due to an extremely low product concentration in the fermentation broth, and large amounts of contaminating proteins, the chromatographic adsorbents had low capacities with respect to alginate lyase. By adsorption on the cation exchanger S-Sepharose FF, the capacity was so low that the enzyme could not be concentrated. The binding capacity of Phenyl-Sepharose FF was approximately 20-fold higher, and a three to tenfold concentration was obtained. The first stage of the process, hydrophobic interaction chromatography, has been applied to the isolation of alginate lyase from fermentation batches of 180 l. Several runs have resulted in a purified product with an average quantity of 30 000–35 000 U per fermentation, and an average specific activity of 4.5 U mg^–1. Although the raw material employed in this work has been particularly unfavourable, the process developed will also be applicable to raw materials with higher product concentrations. Offprint requests to: I. M. Aasen     

褐藻胶裂解酶基因的克隆表达与酶学性质

随着大型褐藻生产燃料乙醇以及褐藻寡糖重大药用价值的发现,褐藻胶裂解酶成为国内外多个领域的研究重点。文中对解藻酸弧菌上与褐藻胶降解相关的5个基因分别进行克隆表达,通过SDS-PAGE和酶活性定量测定,发现该基因簇中的4个基因有降解褐藻胶活性。对酶活最高的rAlgV3进行了诱导条件的优化、酶蛋白纯化及酶性质研究,发现优化诱导条件后重组酶rAlgV3的酶活由2.34×10~4 U/L上升为1.68×10~5 U/L,比优化前提高了7.3倍;对酶性质进行表征发现该酶在4–70℃均有活性,最适反应温度为40℃,在4–20℃酶相对稳定;该酶在pH 6.5-9.0环境下均有较高的酶活,最适pH为8.0;pH稳定性好,在pH 4.5–9.5环境下可以稳定存在;适量的NaCl浓度和Fe~(2+)、Fe~(3+)等离子具有促进酶活的作用,SDS和Cu~(2+)离子可明显抑制酶活力。对该酶的底物特性的研究发现,该酶不仅可以降解褐藻胶中的Poly-M片段,也能降解Poly-G片段,具有广泛底物特性;其降解海藻酸钠主要释放二糖和三糖,是一种内切酶。该酶对于第三代燃料乙醇的发展及褐藻寡糖的生产具有重要作用。     

A method for efficient expression of Pseudomonas aeruginosa alginate lyase in Pichia pastoris

As an eco-friendly biocatalyst for alginate hydrolysis, bacteria-derived alginate lyase (AlgL) has been widely used in research and industries to produce oligosaccharides. However, the cost of AlgL enzyme production remains high due to the low expression and difficulty in purification from bacterial cells. In this study we report an effective method to overexpress the Pseudomonas aeruginosa AlgL (paAlgL) enzyme in Pichia pastoris. Fused with a secretory peptide, the recombinant paAlgL was expressed extracellularly and purified from the culture supernatant through a simple process. The purified recombinant enzyme is highly specific for alginate sodium with a maximal activity of 2,440 U/mg. The enzymatic activity remained stable below 45°C and at pH between 4 and 10. The recombinant paAlgL was inhibited by Zn²⁺, Cu²⁺, and Fe²⁺ and promoted by Co²⁺ and Ca²⁺. Interestingly, we also found that the recombinant paAlgL significantly enhanced the antimicrobial activity of antibiotics ampicillin and kanamycin against Pseudomonas aeruginosa. Our results introduce a method for efficient AlgL production, the characterization, and a new feature of the recombinant paAlgL as an enhancer of antibiotics against Pseudomonas aeruginosa.     

Three phase partitioning of carbohydrate polymers: separation and purification of alginates

Three phase partitioning, a technique described for protein purification, has been employed for precipitation and purification of three different commercial preparations of alginates. Three phase partitioning works by the addition of t-butanol to aqueous solution of the polymer containing 20–30% ammonium sulphate (w/v). Three phases formed are: upper t-butanol layer, interfacial polymer precipitate and lower aqueous phase. In all the three cases, the process optimization was carried out by varying ammonium sulphate concentration, volume of t-butanol, alginate concentration and temperature. Fluorescence spectroscopy was used to show that repeated cycles of TPP also resulted in considerable reduction in polyphenol content of a crude alginate preparation.     

De Novo Synthesis of Isocitritase in Peanut (Arachis hypogaea L.) Cotyledons

Germination of peanut seed is accompanied by a rapid increase in isocitritase (isocitrate lyase, EC 4.1.3.1) during the first 4 days. The presence of cycloheximide (50 μg/ml) during water imbibition inhibited the increase in isocitritase activity. Actinomycin D conversely did not inhibit isocitritase activity until the second day of imbibition while RNA synthesis was inhibited. Germination of peanut seed in ¹⁴C-reconstituted amino acids followed by fractionation of a 20 to 35% ammonium sulfate preparation on a Sephadex G-200 column (57-fold purification) showed that the active enzymic fraction coincided with a large peak of radioactivity. Germination of peanut seed in 45% D₂O followed by enzyme purification and CsCl equilibrium centrifugation revealed that all the enzyme from D₂O seed had a higher density than normal isocitritase. These data indicate that isocitritase in peanut seed is synthesized de novo.     

Purification of wheat germ amylase by precipitation

alpha-Amylase from various sources was found to bind alginate in free solution. The alginate-enzyme complex could be precipitated with Ca(2+). The enzyme activity could be recovered by dissolving the precipitate in 1 M maltose and precipitating alginate alone by addition of Ca(2+). Based upon these observations, alpha-amylase from wheat germ was purified with 68-fold purification and 72% recovery. The molecular weight estimated by SDS-PAGE was 18 kDa. The method also worked equally well with alpha-amylase for the whole wheat seed. The latter enzyme could be purified 54-fold with 70% activity recovery. The molecular weight of this second enzyme was estimated to be 45 kDa by SDS-PAGE.     

Synthesis and characterization of a water-soluble affinity polymer for trypsin purification

A specific ligand bound polymer has been synthesized for the purpose of purification and stabilization of trypsin, an easily autodigestible enzyme. The affinity polymer was formed by copolymerizing N-acryloyl-m-aminobenzamidine, a strong trypsin inhibitor, and acrylamide in the absence of oxygen. Kinetic studies on the trypsin inhibition revealed that there was a strong binding between this enzyme and the polymer and the mechanism was of a competitive manner with an inhibition constant of 0.6 x 10(-3)M. Such an affinity polymer was also very effective in preventing trypsin from auto-digestion at 4 degrees C.Based on this finding and the principle of cross flow filtration, a new process has been developed for purification of trypsin from a solution containing chymotrypsin. The experimental data indicated that trypsin was bound to the polymer (MW > 10(5)) and remained in the retentate while unbound chymotrypsin was collected in the filtrate. This purification process has a capability of recovering 98% pure trypsin at 90% yield.     

Simultaneous refolding/purification of xylanase with a microwave treated smart polymer

Affinity precipitation with a smart polymer, Eudragit S-100 (a methyl methacrylate polymer), was exploited for simultaneous refolding and purification of xylanase. Affinity precipitation consisted of this reversibly soluble-insoluble polymer-binding xylanase selectively. The complex was precipitated by lowering the pH and xylanase was eluted off the polymer using 1 M NaCl. For refolding experiments, the commercial preparation of Aspergillus niger xylanase was denatured with 8 M urea. Addition of microwave irradiated Eudragit S-100 and affinity precipitation led to recovery of 96% enzyme activity by refolding. Simultaneously, the enzyme was purified 45 times. Thermally inactivated preparation, when subjected to similar steps, led to 95% recovery of enzyme activity with 42-fold purification. The strategy has the potential for recovering pure proteins in active forms from overexpressed proteins, which generally form inclusion bodies in E. coli.     

Development of a fermentation process for production of an alginate G-lyase from Klebsiella pneumoniae

A high-density-cell fermentation process for production of an exracellular alginat lyase from Klebseilla pneumoniae on a defined medium has been developed. The process employs a strategy using two carbon sources. One low-molecular-mass, low-viscosity carbon source (sucrose) with high water solubililty is used as the main carbons source for growth, while the high-molecular-mass and viscoous alginate in low concentration is used as an inducer for enzyme synthesis. The repression of algiante lyase production by sucrose and the growth inhibition that we observed at increased levels of ammonia were circumvented by a computer-assisted fed-batch addition of the carbon sources (succrose and alginate) and by supplying nitrogen source as ammonia in the pH control. No enzyme production was observed when dissolved oxygen limited growth at an oxygen uptake rate of 40%–50% of the maximum uptake rate. An optimal composition of the feeding solution (12.5 g alginate and 587.5 g sucrose 1^–1) was found both for the maximum final concentration of enzyme (1330 U 1^–1) and for the maximum volumetric rate of enzyme production (67 U 1^–1 h^–1). The enzyme production dependes of the growth rate in the linear growth phase, giving a maximum enzyme concentration at the highest growth rate tested. The final enzyme concentration shows a fiveflod increase compare with previously reproted daata where alginate was used as a carbon source. In addition, the ratio of alginate lyase by a factor of apporximately 15. A doubling in extracellular specific activity of the enzyme was observed, a property of significant interest, especially for purification of the enzyme. On the othr hand, the final dry cell weight concentration of the bacteria also increased by a factor of 15–20 thus giving a relatively lower specific productivity of 0.4 U (g cell dry weight)^–1 h^–1.     

Phosphoserine phosphatase of human brain: Partial purification,characterization, regional distribution,and effect of certain modulators including psychoactive drugs

Phosphoserine phosphatase (PSPase), a cytosolic enzyme has been purified 106 fold from human brain, by employing conventional protein purification techniques. The use of MgCl₂ (10 mM) and chloroform treatment, during purification enabled the removal of non-specific proteins. The final enzyme preparation exhibited a broad pH optimum of 5.6–6.6 and could dephosphorylate bothl andd enantiomers of the phosphoserine, but with different Km values for O-P-L serine (3.6×10^–5M) and O-P-D serine (1×10^–4M). Enzyme activity was found to be specific for phosphoserine, whereas other phosphoesters including phosphothreonine and phosphoproteins such as casein and phosvitin were found to be poor substrates. The enzyme activity was uncompetitively inhibited byl-serine. Further the PSPase activity was inhibited by vanadate, (41%), trifluoperazine (23%), chlorpromazine (34%) at an equimolar concentration of 1 mM, whereas lithium and ethanol did not influence the enzyme activity. Minor tranquilizers such as diazepam and chlordiazepoxide activated the enzyme activity to an extent of 13% and 59% respectively. In addition, species and regionwise heterogeneity was observed with respect to distribution of enzyme activity in six major areas of human, rabbit and rat brains.     

Purification and characterization of a Na+/K+ dependent alginate lyase from turban shell gut Vibrio sp. YKW-34

An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The k_cat/K_m value for alginate (heterotype) was 1.7 × 10⁶ s⁻¹ M⁻¹. The enzyme activity was completely lost by dialysis and restored by addition of Na⁺ or K⁺. The optimal activity exhibited in 0.1 M of Na⁺ or K⁺. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA).     

Enhancing purification of α-amylase by superparamagnetic complex with alginate/chitosan/β-cyclodextrin/TPP

The main aim of this study was to synthesize the superparamagnetic nanoparticles coated by alginate/chitosan/β-cyclodextrin to purify α-amylase. Isolated bacteria were identified by morphological, biochemical and taxonomic molecular studies. FTIR- spectrometer, VSM, X-ray instruments and Malvern Zetasizer were used to characterize nanoparticles characteristics. The morphological structures and the elemental composition of the nanoparticles were studied by using FESEM and EDS, respectively. The molecular weight of enzyme was determined using SDS-PAGE, and the enzyme activity detected by zymographic analysis. FTIR studies showed the presence of Fe–O–Fe in the Fe₃O₄ and verified the interaction between chitosan, β-cyclodextrin and alginate. The saturation magnetization for superparamagnetic and coated superparamagnetic nanoparticles was indicated 39 and 1.9?emu?g^?1, respectively. The maximum intensity of the XRD peak indicated the presence of the Fe₃O₄. FESEM and EDS analysis showed that the nanoparticles were regular and spherical in shape and corresponded to the Fe and O elements. Enzyme purification by synthesized nanoparticles was achieved 13.84?U?mg^?1; purification fold of 3.50. The molecular weight of α-amylase was about 22?kDa. The highest activity of α-amylase was observed at 70?°C, pH 9.3 and Ca²⁺-independent. As a conclusion, the coated superparamagnetic nanoparticles showed more applications in enzyme purification comparing to the conventional methods.     

Free polymeric bioligands in aqueous two-phase affinity extractions of microbial xylanases and pullulanase

Two reversibly soluble-insoluble polymers (viz. Eudragit S-100 and alginate) were used as free macroaffinity bioligands in polyethylene glycol (PEG)/salt two-phase systems for separation of enzymes. Incorporation of Eudragit S-100 and alginate in the PEG phase led to considerable selectivity in separation of microbial xylanases and pullulanase, respectively. Xylanase from Aspergillus niger was recovered 93% with 56-fold purification, whereas the enzyme from Trichoderma reesei and Bacillus amyloliquefaciens was obtained with 93% activity recovery (31-fold purification) and 90% activity recovery (32-fold purification), respectively. From Bacillus acidopullulyticus pullulanase, 85% enzyme activity recovery with 44-fold purification was obtained. The approach described here shows the potential of developing into a general approach for use of reversibly soluble-insoluble macroaffinity ligand in two-phase affinity extraction.     

Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain

Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)–bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.     

Partial purification and characterization of a mannuronan-specific alginate lyase fromPseudomonas aeruginosa

Production of a thick exopolysaccharide coat (alginate) by mucoid strains ofPseudomonas aeruginosa has been shown to contribute to the pathogenicity and persistence of these bacteria in the lungs of patients with cystic fibrosis. Previous studies have shown that some mucoidP. aeruginosa strains produce an enzyme(s) capable of degrading this alginate coat. In this study, an alginate lyase from mucoidP. aeruginosa strain WcM#2 was isolated and characterized. Lyase production was enhanced by the addition of 0.2–0.3m NaCl to the growth media. The lyase was eluted from an alginate-Sepharose affinity column with 0.5m NaCl, which can serve as a simple one-step purification protocol for obtaining semi-pure functional alginate lyase. Fractionation of the enzyme preparation on a Sephadex G-75 sizing column showed that the enzyme has an apparent molecular weight of 40,000, whereas sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested a molecular weight of approximately 43,000. The affinity-purified enzyme had a pH optimum of 9.0, its activity was enhanced in the presence of 0.3m NaCl, and it showed substrate specificity for polymannuronic acid blocks. These results demonstrate the presence of a mannuronan-specific alginate lyase inP. aeruginosa that differs in several respects from previous reports ofP. aeruginosa alginate lyases.     

IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO2 SEQUESTERER

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co²⁺, Cu²⁺, and Fe³⁺, increased the enzyme activity, whereas CA activity was inhibited by Pb²⁺, Hg²⁺, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The maximum CO₂ sequestration potential was achieved with immobilized CA (480 mg CaCO₃/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO₂ sequestration.     

The isolation and preliminary characterization of apotransketolase from human erythrocytes

Human erythrocyte apotransketolase (EC 2.2.1.1) has been isolated with greater than 400 fold purification, and free of glyceraldehyde-3-phosphate dehydrogenase. The preparation has an absolute requirement for thiamin pyrophosphate in order to exhibit enzyme activity. Neither thiamin nor thiamin monophosphate could substitute for this requirement, nor were they inhibitory separately or together at concentrations of 1 mM. The K_m for thiamin pyrophosphate was 0.4 μM. The K_m for ribose-5-phosphate was 3 × 10^?4M and for xylulose-5-phosphate 1.8 × 10^?4M.     

温和气单孢菌YH311硫酸软骨素裂解酶的分离纯化与固定化

通过硫酸铵沉淀、QAESephadex-A50柱层析及Sephadex-G150凝胶过滤等纯化步骤,对源自温和气单孢菌YH311的ChSase进行了分离纯化。结果表明,ChSase经上述纯化步骤后被纯化了55倍,其最终纯度可达95%以上,比活为31.86u/mg。经SDSPAGE及IFE测定可知该酶的分子量约为80kD,等电点为4.3～4.8。将纯化后的ChSase用海藻酸钠或纤维素固定化后,ChSase的热稳定性及贮存稳定性均可得到大幅度的提高：固定化酶用80℃水浴处理120min或于4℃冰箱放置30d后仍可保留50%以上的相对活力;但固定化酶的收率较低,仅为18.56%和18.86%。     

Purification and partial characterization of an extracellular alginate lyase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> isolated from brown seaweed

The extracellular enzyme alginate lyase produced from marine fungus Aspergillus oryzae isolated from brown alga Dictyota dichotoma was purified, partially characterized, and evaluated for its sodium alginate depolymerization abilities. The enzyme characterization studies have revealed that alginate lyase consisted of two polypeptides with about 45 and 50 kDa each on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed 140-fold higher activity than crude enzyme under optimized pH (6.5) and temperature (35°C) conditions. Zn²⁺, Mn²⁺, Cu²⁺, Mg²⁺, Co²⁺ and NaCl were found to enhance the enzyme activity while (Ca²⁺, Cd²⁺, Fe²⁺, Hg²⁺, Sr²⁺, Ni²⁺), glutathione, and metal chelators (ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid) suppressed the activity. Fourier transform infrared and thin-layer chromatography analysis of depolymerized sodium alginate indicated the enzyme specificity for cleaving at the β-1,4 glycosidic bond between polyM and polyG blocks of sodium alginate and therefore resulted in estimation of relatively higher polyM content than polyG. Comparison of chemical shifts in ¹³C nuclear magnetic resonance spectra of both polyM and polyG from that of sodium alginate also showed further evidence for enzymatic depolymerization of sodium alginate.