Characterization of a new alpha-L-fucosidase isolated from the marine mollusk Pecten maximus that catalyzes the hydrolysis of alpha-L-fucose from algal fucoidan (Ascophyllum nodosum)

Algal fucoidan is an alpha-L-fucose-based polysaccharide endowed with important biological properties for which the structure has not yet been fully elucidated. In an attempt to implement new enzymatic tools for structural study of this polysaccharide, we have found a fucosidase activity in the digestive glands of the common marine mollusk Pecten maximus, which is active on a fucoidan extracted from the brown algae Ascophyllum nodosum. We now report the purification and characterization of this alpha-L-fucosidase (EC 3.2.1.51). The enzyme was purified by three chromatographic steps, including an essential affinity chromatography based on the glycosidase inhibitor analog 6-amino-deoxymannojirimycin as the ligand. The purified alpha-L-fucosidase is a tetrameric glycoprotein of 200 kDa that hydrolyzes the synthetic substrate p-nitrophenyl alpha-L-fucopyranoside with a K(m) value of 650 microM. This enzyme has high catalytic activity (85 micromol x min(-1) x mg(-1)) compared with the other known fucosidases and also possesses an unusual thermal stability. The purified alpha-L-fucosidase is a retaining glycosidase. The activity of the purified fucosidase was determined on two structurally different fucoidans of the brown algae A. nodosum and Fucus vesiculosus to delineate glycosidic bond specificity. This report is to our knowledge the first demonstration of a fucosidase that can efficiently release alpha-L-fucose from fucoidan.     

Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography

C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A(620)/A(280) ratio) of C-phycocyanin isolated with STREAMLINE column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A(620)/A(650) index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin.     

扩张柱床吸附层析回收纯化灌流培养生产的单克隆抗体

用扩张柱床吸附层析技术,一步回收纯化连续灌流培养的单克隆抗体。用Streamline SP阳离子交换介质在固定床柱XK16/20上进行条件摸索,扩张床柱Streamline25和50分别用于小规模条件优化和中试规模放大。培养液中的低浓度单抗经此步处理,浓缩10倍以上,纯度提高5～7倍,回收率＞90%,制备周期比固定柱床层析缩短一半以上。 根据培养液中单抗浓度的不同,一次处理量为18～50L,纯化规模由实验室水平（400mg）扩大至中试水平（2g）,生产成本和工艺复杂性大为降低。应用扩张柱床吸附层析技术,建立单克隆抗体回收纯化工艺,具有经济、简便、高效实用和良好的可放大性。     

Novel method for purification of staphylococcal enterotoxin A

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Novel method for purification of staphylococcal enterotoxin A.

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Optimization of adsorption conditions of papain on dye affinity membrane using response surface methodology

The adsorption of papain on Reactive Blue 4 dye–ligand affinity membrane was investigated in a batch system. The combined effects of operating parameters such as initial pH, temperature, and initial papain concentration on the adsorption were analyzed using response surface methodology. The optimum adsorption conditions were determined as initial pH 7.05, temperature 39 °C, and initial papain concentration 11.0 mg/ml. At optimum conditions, the adsorption capacity of dye–ligand affinity membrane for papain was found to be 27.85 mg/g after 120 min adsorption. The papain was purified 34.6-fold in a single step determined by fast protein liquid chromatography. More than 85% of the adsorbed papain was desorbed using 1.0 M NaCl at pH 9.0 as the elution agent. The purification process showed that the dye–ligand immobilized composite membrane gave good separation of papain from aqueous solution.     

Lipase purification by affinity precipitation with a thermo-responsive polymer immobilized Cibacron Blue F3GA ligand

A thermo-responsive polymer (P_NNB) was synthesized with lower critical solution temperature 27.5°C and over 95% recovery. The adsorption of porcine pancreatic lipase on Cibacron Blue F3GA-conjugated P_NNB (P_NNB-CB) closely followed the bi-Langmuir adsorption isotherm. The maximum adsorption capacity was found at pH 5.0, with a ligand density of 18.4 μmol/g polymers. The optimized eluent was a 0.01 M phosphate buffer solution at pH 8.0 containing 20% ethylene glycol. Six adsorptiondesorption recycles indicated excellent reusability of the affinity adsorbent. P_NNB-CB was applied to separate porcine pancreatic lipase from its crude material giving a lipase activity recovery of 81.6% with a 16-fold purification factor. Lipase could be purified to single-band purity, according to gel electrophoresis. The purification strategy is therefore feasible and efficient for purifying proteins of interest.     

Single-step purification of lipase from Burkholderia multivorans using polypropylene matrix

Lipase from Burkholderia multivorans was purified with high yields directly from fermentation broth by a single-step purification protocol involving adsorption and desorption. The crude enzyme (lyophilized powder) from B. multivorans was loaded on Accurel (Membrana, Germany), a polypropylene matrix, using butanol as the solvent in a buffer at pH 9.0 and ambient temperature for a period of 12 h. The enzyme adsorbed onto the matrix with high specific activity (33 units mg^–1 protein). This was followed by desorption of the enzyme from the matrix using Triton X-100 as the eluent. The enzyme was finally recovered by precipitation with acetone (50%, v/v). Thus, an overall enzyme yield of 66% with a 3.0-fold purification was obtained. The purity of the enzyme was ascertained by SDS-PAGE. The phenomenon of adsorption and desorption on Accurel was studied for three more lipases, viz. Mucor meihei lipase (Sigma–Aldrich Co.), Lipolase (Novo Nordisk, Denmark) and Pseudomonas aeruginosa lipase (laboratory isolate).     

Purification and characteristics of fucoidanase obtained from <Emphasis Type="Italic">Dendryphiella arenaria</Emphasis> TM94

Dendryphiella arenaria TM94 is an obligate marine fungus. Fucoidanase expressed by TM94 by solid state fermentation was purified. The fermented solid medium was extracted with citric acid buffer, and the extracts were precipitated by acetone and separated on Sephadex G-100 chromatography. The specific fucoidanase activity of purified enzyme was 27-fold than that of the crude enzyme. The recovery of the enzyme was 17.69%. SDS-PAGE was used to identify the purity and the molecular weight of the fucoidanase. A single band appeared on SDS-PAGE gel which suggested that relatively pure fucoidanase has been obtained. The molecular weight of fucoidanase is 180 kDa and the isoelectric point was about pH 4.4. The purified fucoidanase appeared to have the maximum enzymatic activity at pH 6.0. K_M and the maximum velocity of the enzyme was 6.56 mg·mL⁻¹ and 6.55 mg·mL⁻¹·min⁻¹ by using fucoidan from Fucus vesiculosus as substrate. The enzyme may be a type of endo-fucoidanase which could hydrolyze high molecular weight fucoidan to low molecular weight fucoidan rather than to fucose.     

HZ-841吸附树脂精制银杏叶总黄酮

本文研究了用HZ-841吸附树脂精制银杏叶总黄酮的工艺。用10 BV 70％的乙醇分三次提取脱脂银杏叶粉中的银杏叶总黄酮,其收得率为4．8％,纯度为21．7％;用30BV纯净水、微波解冻提取30min,银杏叶总黄酮的收得率及纯度分别是2．63％和13．4％。HZ-841树脂对银杏叶总黄酮的动态吸附容量在pH=7．0时为0．265g／mL,树脂,动态吸附平衡时间为10min。酸度对HZ-841树脂吸附银杏叶总黄酮有显著影响,当pH=5．0时,其静态吸附量可达到0．322g／mL。吸附了银杏叶总黄酮的HZ-841树脂可用乙醇洗脱,当洗脱液pH=9．0、乙醇浓度为90％、洗脱流速3BV／h时,5BV洗脱液的收得率为1．8％。用无水乙醇洗脱的银杏叶总黄酮经过真空浓缩、干燥,获得的浅黄色粉末中银杏叶总黄酮含量为37．3％,产品收得率为2．41％。     

Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum)

The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20?mM tris-CaCl₂ buffer (pH 8.2) with a flow rate of 0.5?mL min^?1. The molecular weight and purity of ～23?kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697?U?mg^?1, fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.     

Simultaneous heat shock and in situ adsorption enhance plumbagin production in Plumbago indica root cultures

Plumbago indica L. is an important source of plumbagin, a commercially valuable bioactive compound. However, the uses of plumbagin are limited due to its low supply as well as low yields and slow growth of the plant sources. This study evaluated the use of a simple, easy, and low‐cost approach using heat shock (HS) and ultrasound (US), and an in situ adsorption using a nonpolar copolymer adsorbent styrene‐divynilbenzene resin (Diaion^® HP‐20) to enhance plumbagin production in Plumbago indica root cultures. Treatment with HS (60°C) for 10 min significantly increased the production of plumbagin (5.51 mg/g DW) by up to five‐fold, compared to the level in untreated root cultures (1.14 mg/g DW). In contrast, treatments with US alone or with HS treatment produced no satisfactory increase of plumbagin production. However, combined treatment of a 20‐day‐old root culture with HS (60°C, for 10 min) in the presence of Diaion^® HP‐20 (10 g/L) markedly increased the production up to 20.28 mg/g DW of plumbagin that was almost 14‐fold higher, compared to the level in an untreated root culture. Such an increase would be sufficient for commercial applications of this method to produce plumbagin.     

Development of a full‐length human protein production pipeline

There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high‐throughput (HT) methods, we transferred the genes of 31 full‐length proteins into three expression vectors, and expressed the collection as N‐terminal HaloTag fusion proteins in Escherichia coli and two commercial cell‐free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip^® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full‐length human proteins in these three expression systems.     

Catalase purification from rat liver with iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) cryogel discs

In this study, iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) (PHEMAGA/Fe³⁺) cryogel discs were prepared. The PHEMAGA/Fe³⁺ cryogel discs were characterized by elemental analysis, scanning electron microscopy, Fourier transform infrared spectroscopy, swelling tests, and surface area measurements. The PHEMAGA/Fe³⁺ cryogel discs had large pores ranging from 10 to 100?µm with a swelling degree of 9.36?g H₂O/g cryogel. Effects of pH, temperature, initial catalase concentration, and flow rate on adsorption capacity of the PHEMAGA/Fe³⁺ cryogel discs were investigated. Maximum catalase adsorption capacity (62.6?mg/g) was obtained at pH 7.0, 25°C, and 3?mg/ml initial catalase concentration. The PHEMAGA/Fe³⁺ cryogel discs were also tested for the purification of catalase from rat liver. After tissue homogenization, purification of catalase was performed using the PHEMAGA/Fe³⁺ cryogel discs and catalase was obtained with a yield of 54.34 and 16.67 purification fold.     

Purification of Sulfated Fucoglucuronomannan Lyase from Bacterial Strain of <Emphasis Type="Italic">Fucobacter marina</Emphasis> and Study of Appropriate Conditions for Its Enzyme Digestion

A marine bacterial strain, Fucobacter marina, produced extracellular sulfated fucoglucuronomannan (SFGM) lyase when cultivated in the presence of crude SFGM obtained from fucoidan of Kjellmaniella crassifolia (brown algae) by cetyl pyridinium chloride fractionation. For the SFGM lyase assay, SFGM fraction separated from K. crassifolia fucoidan by anion exchange column chromatography was used as the substrate. The extracellular SFGM lyase was purified to homogeneity on an electrophoresis gel with 4240-fold purity at 13.8% yield. The enzyme proved to be a monomer, since gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis gave the same relative molecular mass of 67,000. The enzyme specifically digested SFGM but did not digest any other uronic-acid-containing polysaccharides tested. The optimum conditions for the enzyme reaction were around pH 7.5, 43°C, and 0.4 M NaCl concentration. The enzyme was strongly inhibited by CuCl₂ and ZnCl₂, and also by some sulfhydryl reagents.     

Reconstitution conditions for dried probiotic powders represent a critical step in determining cell viability

Aims: Resuscitation of dried cultures represents a critical control point in obtaining active and effective probiotic strains. This study examined the effects of various rehydration conditions on the viability of Bifidobacterium longum NCC3001 and Lactobacillus johnsonii La1. Methods and Results: Reconstitution conditions for these strains were optimized using a multivariate experimental design approach. Furthermore, using flow cytometry, the cell integrity was followed during reconstitution. By adjusting the pH, availability of a metabolizable sugar, reconstitution duration, powder matrix and ratio of powder to reconstitution solution, the recovery of Bif. longum NCC3001 and Lact. johnsonii La1 following reconstitution was increased eight‐ and two‐fold, respectively, over standard reconstitution in maximum recovery diluent. It was shown that pH had a significant effect on the recovery of Bif. longum NCC3001 and Lact. johnsonii La1. Conclusions: The recovery of dried probiotic cultures is greatly dependent on the reconstitution conditions. The maximum recovery of 11·7 ¹⁰log CFU g^?1Bif. longum NCC3001 was achieved at 30‐min reconstitution at pH 8, in the presence of 2%l ‐arabinose and a ratio of 1 : 100 of powder to diluent. Lact. johnsonii La1 showed highest recovery (9·3 ¹⁰log CFU g^?1) after reconstitution, when mixed with maltodextrin at pH 4. Significance and Impact of the Study: To achieve accurate viable probiotic numbers from dried probiotic cultures, the reconstitution conditions should be optimized for the strain used.     

Highly pure 1,3‐propanediol: Separation and purification from crude glycerol‐based fermentation

A new separation and purification process was developed for recovering 1,3‐propanediol (1,3‐PD) from crude glycerol‐based fermentation broth with high purity. The downstream process integrated chitosan flocculation, activated carbon decolorization, fixed bed cation exchange resin adsorption, and vacuum distillation. Breakthrough curves were measured considering the effect of sample concentration, flow rate, temperature, and resin stack height. Yoon–Nelson model was proposed to fit the fixed bed adsorption. The characteristic column parameters were calculated. Optimal condition for adsorption was 1,3‐PD, 30.0 g/L; flow rate, 1.00 mL/min; stacking height, 30.0 cm; and temperature, 298 K. Ethanol‐water (75%, 1 mL/min) was used as eluent to separate 1,3‐PD and glycerol with 95.3% 1,3‐PD elution rate. After vacuum distillation, the overall purity and yield of 1,3‐PD were 99.2% and 80.8% in the purification process, respectively. This is a simple and efficient downstream strategy for 1,3‐PD purification.     

产胶原酶的蜡样芽胞杆菌发酵条件优化及酶的分离纯化

【目的】优化蜡样芽胞杆菌R75E菌株产胶原酶的条件,并通过蛋白分离纯化技术获得高纯度胶原酶。【方法】利用单因素及正交试验优化蜡样芽胞杆菌R75E产胶原酶的发酵条件及发酵培养基,将发酵液离心除菌后得到粗酶液,对其依次通过硫酸铵分级沉淀、Butyl FF疏水层析及SuperdexTM 200凝胶过滤层析等方法对目标胶原酶进行分离纯化,利用SDS-PAGE电泳检测其纯度。【结果】优化后发酵条件为培养温度41°C、接种量6%、培养时间36 h,优化后发酵培养基为葡萄糖10 g/L、蛋白胨5 g/L、起始p H 7.0,粗酶液酶活力较优化前提高了2.9倍;将该粗酶液经过一系列纯化后得到纯度超过90%的胶原酶产物,其纯化倍数和回收率分别为18.4和1.1%。【结论】获得蜡样芽胞杆菌R75E的最佳产酶条件,并对胶原酶分离纯化的方法进行了探索,为微生物胶原酶的开发应用奠定基础。     

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)1

Enolase from Synechococcus PCC 6301 was purified 1450‐fold to electrophoretic homogeneity and a final specific activity of 68 μmol of phosphoenolpyruvate produced·min^?1·mg protein^?1. Analytical gel filtration and nondenaturing and SDS‐gel electrophoresis demonstrated that this enolase exists as a 118‐kDa homodimer composed of 56‐kDa subunits. The purified enzyme displayed 1) a broad pH‐activity profile with maximal activity occurring at pH 8.0 and 7.5 for the forward and reverse reactions, respectively, 2) a forward‐to‐reverse maximal activity ratio of about 1.6, 3) a K_m (2‐phosphoglycerate) of 0.28 mM, and 4) an absolute requirement for a divalent metal cation cofactor that was best satisfied by Mg²⁺ (K_m=0.62 mM). Enolase activity increased by about 200% after the first purification step (60° C heat treatment), whereas addition of increasing amounts of a clarified extract led to a progressive 70% inhibition in the activity of the purified enzyme. This was reflected by a reduction in enolase's V_max from 73 to 22 U·mg^?1 and forward‐to‐reverse activity ratio from 1.6 to 1.3. This inhibition was negated when the clarified extract was either preincubated with trypsin or warmed to approximately 40° for 5 min. Results are indicative of a heat‐labile enolase inhibitor protein in Synechococcus PCC 6301. By contrast, the purified enolase lost no activity when incubated at 70° C for up to 5 min. This study represents the first purification of enolase from the Cyanophyceae. Characterization of the purified enzyme's physical and kinetic features has provided insights into the structural and functional properties of cyanobacterial enolase.     

Chitosan/cellulose-based beads for the affinity purification of histidine-tagged proteins

Chitosan/cellulose-based beads (CCBs) for the affinity purification of histidine-tagged proteins were prepared from chitosan/cellulose dissolved in ionic liquid as a solvent, and their structures were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. The affinity purification was used to separate hexahistidine-tagged (his-tagged) enhanced green fluorescent protein (EGFP) from Escherichia coli. The results showed that Zn²⁺–CCB exhibited more specific adsorption capacity toward the target protein compared with Ni²⁺–CCB and Cu²⁺–CCB. The maximum adsorption of EGFP was 1.84?mg/g of Zn²⁺–CCB, with 90% purity under the optimized conditions (ionic strength (1.0?M NaCl), pH (7.2) and imidazole concentration (500?mM)). In addition, a regeneration method for the sorbent was further developed by washing with ethylenediaminetetraacetic acid disodium and then reimmobilizing with metal ions. This technique is an alternative method for the purification of his-tagged proteins, making the process more economical, fast, stable, and large batch.