Evaluation of different parameters for the purification of inulinase using an ion exchange fixed bed

The main conditions affecting the downstream process for purification of inulinase from Kluyveromyces marxianus var. bulgaricus ATCC 16045 were investigated. Parameters including the elution mode, linear rate, ionic strength, and elution pH were evaluated in order to achieve the best purification factor and recovery. The best purification factor and recovery were achieved in run 6 using a gradient elution mode with concentrations of NaCl ranging from 0 to 1 M, a linear flow rate of 100 cm/h and a pH value of 4.1. Under these conditions, the recovery was ∼ 67.5% and the purification factor ∼ 6.6-fold.     

Evaluation of the effects of the parameters involved in the purification of clavulanic acid from fermentation broth by aqueous two-phase systems

The purification of clavulanic acid (CA), which is an important β-lactam antibiotic produced by submerged cultivation of Streptomyces clavuligerus, was studied through the use of phosphate and polyethylene glycol-based aqueous two-phase systems. The parameters’ effect on the yield and purification was evaluated through an experimental design and the preliminary results showed that the polyethylene molecular mass and tie-line length and phase volume ratio exerted the strongest effect on the yield and distribution coefficient in the range tested. In addition, the response surface methodology was used to optimize the distribution coefficient, yield, and purification factor. The optimal conditions of yield and purification factor are in the regions where polyethylene has a low molecular mass, pH close to the isoelectric point, and lower top phase volume. A 100% yield and a 1.5-fold purification factor are obtained when extracting CA by maximizing the conditions of an aqueous two-phase system.     

Recovery of endo-polygalacturonase using polyethylene glycol-salt aqueous two-phase extraction with polymer recycling

The partitioning behaviour of endo-polygalacturonase (endo-PG) and total protein from a clarified Kluyveromyces marxianus fermentation broth in polyethylene glycol (PEG)-ammonium sulfate and PEG-potassium phosphate (pH=7) aqueous two-phase systems was experimentally investigated. Both the enzyme and total protein partitioned in the bottom phase for these two kinds of systems. The enzyme partitioning coefficient can be lower than 0.01 in PEG8000-(NH₄)₂SO₄ ATPS with a large phase volume ratio and a moderate tie-line length, which implies the possibility of concentration operation using aqueous two phase partitioning. An ion-exchange separation of high purification efficiency was applied to analyze the clarified and dialyzed fermentation broth. A total purification factor of only 2.3 was obtained, which indicated the high enzyme protein content in the total protein of the fermentation broth. Consequently, the main purpose for separating endo-PG is concentration rather than purification. A separation scheme using an aqueous two-phase extraction process with polymer recycling and a dialysis was proposed to recover endo-PG from the fermentation supernatant of K. marxianus for commercial purpose. A high enzyme recovery up to 95% and a concentration factor of 5 to 8 with a purification factor of about 1.25 were obtained using the single aqueous two-phase extraction process. More than 95% polymer recycled will not affect the enzyme recovery and purification factor. Dialysis was used mainly to remove salts in the bottom phase. The dialysis step has no enzyme loss and can further remove small bulk proteins. The total purification factor for the scheme is about 1.7.     

The extractive purification of peroxidase from plant raw materials in aqueous two-phase systems

The extractive purification of peroxidase from Armoracia rusticana roots and Glycine max seed coats in temperature-induced and affinity microsphere-containing aqueous two-phase systems was stuied. The extractive purification of peroxidase from Glycine max seed coats was carried out in a temperature-induced aqueous two-phase system formed by Triton X-45, Triton X-100 and sodium acetate at pH 5.5 A 99% yield with a 6-fold purification factor was obtained. When the clear top phase was subjected to concanavalin-A affinity chromatography, the purification factor rose to 41 and the yield dropped to 28%. A two-step purification process for peroxidase from Armoracia rusticana roots was developed by adding concanavalin-A affinity microspheres to a PEG/phosphate aqueous two-phase system. The method allows a 60% recovery of high purity peroxidase (1,860 guaiacol units per mg). A lower recovery rate and degree of purification of this enzyme was achieved after temperature-induced aqueous two-phase partition or acetone precipitation and concanavalin-A affinity column chromatography.     

Purification and properties of the xylanases from the termite Macrotermes bellicosus and its symbiotic fungus Termitomyces sp.

Four xylanases were purified, two from the termite Macrotermes bellicosus workers (X1T and X2T) and two from its symbiotic fungus Termitomyces sp. (X1Mc and X2Mc). The analysis of the step required for the purification of X1T and X1Mc and the comparison of their different properties suggested that xylanases X1T and X1Mc were the same enzyme, X1. The determination of the reducing sugars by TLC revealed that X1 was an endoxylanase (EC 3.2.1.8) and X2T and X2Mc were exoxylanases (EC 3.2.1.37). The apparent molecular weights of the three xylanases, determined by SDS-polyacrylamide gel electrophoresis, were 36 kDa for X1, 56 kDa for X2T and 22.5 kDa for X2Mc. The optimal pH of the three xylanases was 5.5, and K_m values determined with birchwood xylan as substrate were 0.2% for X1, 0.1% for X2T and 0.3% for X2Mc, showing a high affinity for this substrate. The three enzymes differed also by their thermal stability.     

Large scale purification of factor X by hydrophobic chromatography

Factor X is a critical enzyme in the blood coagulation cascade, however, in recent years the coagulation zymogen factor X has received additional interest as a selective proteinase to allow production of functional eukaryotic proteins in a prokaryotic expression system. Traditional factor X purification schemes suffer from low yields, low capacity, lengthy dialysis steps, and contamination by the autoproteolytic activated enzyme factor Xa. By incorporating a reversible inhibitor of factor X activation, we were able to recover 67% of the factor X present without any detectable activated enzyme. Six liters of plasma could be processed onto a 50 mL phenylalanine-Sepharose hydrophobic chromatography column without saturating the matrix. The final product is devoid of detectable proteolytic activity. At time of use, the zymogen is specifically activated with a Sepharose-bound activating enzyme isolated from Russell's Viper Venom, resulting in factor Xa free of other detectable proteinases.     

Purification of a murine leukemia inhibitory factor from Krebs ascites cells

A factor capable of inducing terminal differentiation in the murine myeloid leukemia cell line M1 has been purified to apparent homogeneity from the medium conditioned by Krebs II ascites tumor cells. The factor, termed leukemia inhibitory factor (LIF) is a single chain glycoprotein of apparent Mr 58,000 which induces differentiation and inhibits proliferation of the M1 cell line but not the WEHI-3B D+ murine myeloid leukemic cell line and has no detectable proliferative activity on normal myeloid progenitor cells. It was purified using four successive high-efficiency purification steps--anion-exchange chromatography on DEAE-Sepharose; cation-exchange chromatography on CM-Sepharose; affinity chromatography on lentil lectin-Sepharose; and reverse-phase high-performance liquid chromatography on a phenyl-silica matrix--to a specific biological activity of approximately 1.25 X 10(8) units/mg with an overall purification of 12,000-fold and a yield of 73% for the activity failing to bind to DEAE-Sepharose. Sufficient quantities of the factor (12 micrograms, 200 pmol) have been purified to allow structural and functional analysis of the molecule and comparison with other know differentiation inducers.     

Overexpression of Arylsulfate Sulfotransferase as Fusion Protein with GlutathioneS-Transferase

A procedure has been developed for the overexpression and purification of milligram quantities of theKlebsiellaK-36 arylsulfate sulfotransferase (ASST). The structural gene was amplified by means of a polymerase chain reaction (PCR) technique and inserted into the plasmid vector pGEX-3X. The plasmid pGEX-100, carrying theKlebsiellaK-36astAstructural gene under the control of theEscherichia coli tacpromoter, was transformed into theE. colistrain BL21 (DE3). The ASST was produced inE. colias a fusion with glutathioneS-transferase. Conditions for protein production, isolation on glutathione Sepharose 4B, and Xa cleavage to generate active ASST were developed. The purification yielded approximately 0.7 mg of pure enzyme per liter of bacterial culture. Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties almost the same as those of the enzyme purified fromKlebsiellaK-36 cells. The purification procedure was very rapid and is suitable for obtaining considerable amounts of enzyme at a relatively high yield compared with its purifying method from the culture of theKlebsiellaK-36 strain.     

Rapid Affinity Purification Processes for Cyclodextrin Glycosyltransferase from <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">circulans</Emphasis>

Two rapid and easy-to-scale-up methods for the purification of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans were developed: affinity precipitation with starch and aqueous two-phase partition. The first method, optimised by a factorial design, gave an 80% CGTase adsorption at 11% starch and 1.6% ammonium sulphate, and a 65% recovery after elution with 10 mM α-cyclodextrin. The purification factor was 17. Aqueous two-phase partition yielded a 72% CGTase recovery in a two-step procedure; CGTase was obtained in the bottom phase with a purification factor of 37.     

Partial purification and characterization of bovine liver aspartyl beta-hydroxylase

In vitro hydroxylation of aspartic acid has recently been demonstrated in a synthetic peptide based on the structure of the first epidermal growth factor domain in human factor IX (Gronke, R. S., VanDusen, W. J., Garsky, V. M., Jacobs, J. W., Sardana, M. K., Stern, A. M., and Friedman, P. A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3609-3613). The putative enzyme responsible for the posttranslational modification, aspartyl beta-hydroxylase, has been shown to be a member of a class of 2-ketoglutarate-dependent dioxygenases, which include prolyl-4- and lysyl-hydroxylases. In the present study, we describe the solubilization with nonionic detergent of the enzyme from bovine liver microsomes and its purification using DEAE-cellulose followed by heparin-Sepharose. No additional detergent was required during purification. The partially purified enzyme preparation was found to contain no prolyl-4- or lysyl-hydroxylase activity. Using a synthetic peptide based on the structure of the epidermal growth factor-like region in human factor X as substrate, the apparent Km values for iron and alpha-ketoglutarate were 3 and 5 microM, respectively. The enzyme hydroxylated the factor X peptide with the same stereospecificity (erythro beta-hydroxyaspartic acid) and occurred only at the aspartate corresponding to the position seen in vivo. Furthermore, the extent to which either peptide (factor IX or X) was hydroxylated reflected the extent of hydroxylation observed for both human plasma factors IX and X.     

Purification, immobilization and stabilization of a highly enantioselective alcohol dehydrogenase from Thermus thermophilus HB27 cloned in E. coli

This paper shows the purification and immobilization of a very interesting thermophilic alcohol dehydrogenase from Thermus thermophilus HB27 cloned in Escherichia coli. The purification was based on a first thermal treatment of the crude extract, that leaves the target enzyme in the supernatant, followed by the adsorption of most contaminant proteins in a IMAC column (the target protein did not adsorb on these columns due to the poorness of His residues). Final purification factor was around a 9-fold factor (no other protein bands were detected in SDS-PAGE gels) with an overall yield around 80%. Covalent immobilization of the enzyme on very different supports only permitted to improve the enzyme stability by a 5–10-fold factor, very similarly to the results obtained by the adsorption of the enzyme on polyethyleneimine coated supports. This enzyme adsorbed by ionic exchange maintained the activity unaltered during immobilization which was a very rapid process, and was more stable than the covalent preparations in the presence of organic solvents, and the enzyme was quite strongly adsorbed on the support. Therefore, it was proposed as a good option to prepare industrial biocatalysts of the enzyme. This preparation was utilized in the asymmetric reduction of acetophenone to produce (S)-(−)-1-phenylethanol, with an enantiomeric excess of more than 99%.     

Mass spectrometry based analysis of human plasma‐derived factor X revealed novel post‐translational modifications

Human coagulation factor X is a central component of the blood coagulation cascade that converts, under its activated form, prothrombin into thrombin. Generation of thrombin is the final step of the clotting cascade that leads to the clot by polymerization of fibrinogen molecules into a fibrin network. Today, research of new by‐passing agents of the coagulation may contribute to an increased interest for human factor X, which may, in consequence, lead to the need of a more exhaustive picture of its structural features. Several post‐translational modifications of human factor X such as γ‐carboxylation/β‐hydroxylation of the N‐terminal light chain and N‐/O‐glycosylation of the activation peptide have been described. But, so far as we know, no comprehensive studies of its post‐translational modifications have been reported. In this article we report an exhaustive structural analysis of human factor X by mass spectrometry using successive protein and peptide mapping. Surprisingly, human factor X was found to be mostly O‐glucosylated on its light chain at Ser₁₀₆ position, Ser₉ of its activation peptide is phosphorylated at about 30% and its C‐terminal heavy chain is fully O‐glycosylated at Thr₂₄₉ by a mucin‐type O‐glycan (HexNAc‐Hex‐NeuAc). The knowledge of these post‐translational modifications is mandatory for the development of recombinant molecules.     

Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl₂, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over-expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.     

Purification of a new neurotrophic factor from mammalian brain.

We report the purification from pig brain of a factor supporting the survival of, and fibre outgrowth from, cultured embryonic chick sensory neurons. The purified factor migrates as one single band, mol. wt. 12 300, on gel electrophoresis in the presence of sodium dodecylsulphate (SDS) and is a basic molecule (pI greater than or equal to 10.1). Approximately 1 microgram factor was isolated from 1.5 kg brain. The final degree of purification was estimated to be 1.4 X 10(6)-fold, and the specific activity 0.4 ng/ml/unit, which is similar to that of nerve growth factor (NGF) using the same assay system. This factor is the first neurotrophic factor to be purified since NGF, from which it is clearly distinguished because it has different antigenic and functional properties.     

新一代糖化酶高产菌株的选育及其工业应用

【目的】建立对糖化酶生产菌种黑曲霉随机突变文库进行筛选的方法,以获得糖化酶酶活提高的突变菌株。【方法】以一株可产糖化酶的黑曲霉菌株Aspergillus niger X1为出发菌株,经硫酸二乙酯诱变获得突变文库,采用葡萄糖的结构类似物——2-脱氧葡萄糖进行筛选,并在筛选过程中逐渐提高2-脱氧葡萄糖浓度,定向选育具有2-脱氧葡萄糖抗性、高产糖化酶的突变株。【结果】获得的高产突变菌株DG36摇瓶发酵糖化酶产量比出发菌株A.niger X1提高22.2%–33.8%,经工业水平50 m~3罐发酵测试,突变株DG36发酵128 h糖化酶活可达49094 U/m L,在相同发酵时间内,其酶活较出发菌株A.niger X1提高32.8%,发酵时间缩短16.9%。【结论】本研究开发了一种以2-脱氧葡萄糖为抗性标记选育高产糖化酶突变株的方法,所得突变株DG36遗传性状稳定,与出发菌相比具有菌丝粗壮、产酶期提前、糖化酶活高、发酵时间短、有利于发酵后处理的优点。     

Purification and properties of the guanine nucleotide exchange factor (GEF) from HeLa cells and its role in the initiation of protein synthesis

A guanine nucleotide exchange factor (GEF), catalyzing the exchange of GDP bound to initiation factor eIF-2 for GTP, has been isolated from S3 HeLa cells as the eIF-2 X GEF complex and extensively purified by procedures originally developed for purification of GEF from rabbit reticulocytes. The HeLa cell factor resembles rabbit reticulocyte eIF-2 X GEF in polypeptide composition, catalytic activity, and inactivation by alpha-phosphorylated eIF-2.     

The human factor H-related gene 2 (FHR2): structure and linkage to the coagulation factor XIIIb gene

The human factor H-related gene 2 (FHR2) encodes a serum protein structurally and immunologically related to complement factor H. We describe the isolation and genomic organization of the human FHR2 gene from a yeast artificial chromosome library. The FHR2 gene is organized in five exosn and span about 7 kilobases (kb) of human genomic DNA. A comparison with the corresponding cDNA sequence (clone DDESK59) shows that the analyzed FHR2 gene has a deleted region within exon 4. A new splice acceptor site created in the truncated exon indicates that the analyzed gene could be translated to a truncated protein. Further, we demonstrate that the genes for FHR2 and subunit of coagulation factor XIII are located in the same 165 kb YAC DNA. Thus, the three structurally related genes FXIIIb, FHR2, and factor H are linked on human chromosome 1 in the regulators of complement activiation (RCA) gene cluster. The physical linkage of the FHR2 and the factor H genes provides additional evidence for a close relatedness of complement factor H and the factor H-related proteins. The linkage and the almost exclusive organization in short consensus repeat-containing domains indicates a close evolutionary relationship of the FXIIIb, FHR2, and factor H genes.The nucleotide sequence data reported in this paper have been submitted to the EMBL and GenBank nucleotide sequence databases and have been assigned the accession number X86564 (exon 1), X86565 (exon 2), X86566 (exon 3 and 4), and X86567 (exon 5)     

Purification of β-N-acetyl-d-glucosaminidase fromAspergillus niger using thermostabilization and heat as the initial step

A β-N-acetyl-d-glucosaminidase (EC 3.2.1.30) produced byAspergillus niger 419, was completely inactivated after heating 15 min at 65°C in 100 mM sodium phosphate buffer pH 7. The presence of 10% of polypropyleneglycol 1025 induced the thermal stability of the enzyme, the activity remaining unchanged after heating 60 min at 65°C. When this thermal treatment was used as the initial step of purification, the protein content of the crude extract was reduced by 98% without loss of the total initial enzymatic activity of the sample and a purification factor of 61. As the second and third step of purification DEAE-Sephacel, and Sephadex-G150 column chromatography were used, respectively. The final purification factor was 230 with a yield of 76%.