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1.
A simple one-step purification method, using expanded bed, ion-exchange chromatography, for the fractionation of nisin Z produced by Lactococcus lactis subsp. lactis A164 was developed. The highest dynamic binding capacity (0.92) of the adsorbent was obtained at a superficial velocity of 367 cm h(-1), resulting in approx. 2.7-fold bed expansion. The range of pH for the maximum adsorption was 3-4. The isocratic elution with 0.15 M NaCl led to approx. >90% recovery. Single-step purification of nisin Z from unclarified A164 culture broth resulted in 31-fold purification with a 90% yield.  相似文献   

2.
Two different recombinant human proteins were purified directly from Pichia pastoris whole cell fermentation broth, containing 30–44% biomass (wet weight percent), by strong cation exchange expanded bed adsorption chromatography. Expanded bed adsorption chromatography provided clarification, product purification and product concentration in a single unit operation at large scale (2000-l nominal fermentation volume). The efficiency of expanded bed adsorption chromatography resulted in a short process time, high process yield, and limited proteolytic degradation of the target proteins. The separations were operated using a 60-cm (d) column run at 14 l/min. For one protein, expanded bed adsorption chromatography resulted in an average product recovery of 113% (relative to fermentation supernatant) and a purity of 89% (n=10). For the other protein, the average product recovery was 99% (relative to fermentation supernatant) and the purity was 62.1 (n=10). Laboratory experiments showed that biomass reduced product dynamic binding capacity for protein 2.  相似文献   

3.
The recovery of cutinase of Fusarium solani pisi produced by the yeast Saccharomyces cerevisiae was studied in a fluidised bed adsorption system directly integrated with a productive fermenter (so-called direct product sequestration; DPS). The relative efficiency of this system was compared with the one of a conventional purification process by discrete sequences of fermentation, broth clarification, ultrafiltration and fixed bed anion exchange chromatography. By direct product sequestration of the extracellular heterologous cutinase it was possible, through only one unit operation: (i) to perform broth clarification, (ii) to obtain a high cutinase concentration factor, and (iii) to recover cutinase with a specific activity that equalled that obtained with the conventional purification process. It was also possible (iv) to substantially reduce the total process time, (v) to improve the overall yield, and (vi) to increase cutinase productivity. Furthermore, the procedure outlined is suitable for large scale bioprocess exploitation.  相似文献   

4.
Glycolipids produced byPseudomonas aeruginosa YPJ-80 were characterized by chromatographic and spectroscopic techniques as a mixture of two rhamnolipids. For recovery of glycolipids from the culture broth, various isolation methods including ultrafiltration, adsorption and solvent extraction were compared. Ultrafiltration showed the best results in terms of glycolipids recovery. Further purification for spectroscopic analysis was carried out by adsorption chromatography and preparation thin layer chromatography. From the spectroscopic analysis, such as IR spectroscopy, FAB-MS,1H-NMR and13C-NMR and hydrolysis analysis, the glycolipids were identified as L-α-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate and 2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate. Monorhamnolipid and dirhamnolipid lowered the surface tension of water to 28.1 mN/m and 29.3 mN/m, respectively.  相似文献   

5.
Immobilized metal ion affinity chromatography (IMAC) in expanded bed mode is used for purifying recombinant green fluorescent protein (GFP) overexpressed in Escherichia coli. The purification is carried out on two different matrices, i.e. Ni2+ Streamline™ and Ni2+ cross-linked alginate beads. The binding isotherms to both IMAC media followed the Langmuir model. The maximum binding capacity (qmax) of Ni2+ Streamline™ and Ni2+ cross-linked alginate for the GFP was 1,42,860 FU ml−1 and 18,000 FU ml−1, respectively. The expanded bed column chromatography using Ni2+ Streamline™ gave 2.7-fold purification with 89% of GFP recovery, while Ni2+ alginate gave 3.1-fold purification with 91% of GFP recovery. SDS-PAGE of purified GFP in both cases showed single band. The results obtained in the expanded bed chromatography are compared with those obtained in packed bed chromatography.  相似文献   

6.
Efficient recombinant expression of N-acyl-l-aminoacylase 1 from pig kidney (pAcy1) was achieved in the prokaryotic host Escherichia coli. An optimized nucleotide sequence (codon adaptation index 0.95 for E. coli), was cloned into vector pET-52(b) yielding an E. coli-expressible pAcy1 gene. Formation of inclusion bodies was alleviated by co-expression of molecular chaperones resulting in 2.7- and 4.2-fold increased recovery of active pAcy1 using trigger factor or GroEL–GroES, respectively. Facile purification was achieved via StrepTag affinity chromatography. Overall, more than 80 mg highly active pAcy1 (94 U/mg) was obtained per liter of cultivation broth. The protein was analyzed for structural and functional identity, and the performances of further described expression and purification systems for pAcy1 were compared.  相似文献   

7.
Recombinant Bacillus brevis which carried an expression plasmid encoding the human epidermal growth factor (EGF) gene on a cryptic high-copy number plasmid, pHT926, extracellularly produced EGF in its biologically active form at a concentration of over 1.5 g L−1 in the culture broth in a 30-L jar fermenter. The culture broth also contained some other EGF compounds, which mainly consisted of oligomeric and polymeric forms with disulfide bonds. We developed a simple purification method for EGF, without prior cell removal from the culture broth, comprising cation exchange expanded bed adsorption followed by ultrafiltration with UF 10 000 and 3000 membranes. The EGF compounds were efficiently separated from the EGF in its native form in the expanded bed adsorption step. With this purification method, only EGF in its native form was recovered from the culture broth, with a yield of nearly 80%, and 90% purity. This efficient and economic system has made it possible to use EGF as a pharmaceutical in the livestock industry. Received 10 June 1998/ Accepted in revised form 10 September 1998  相似文献   

8.
Lactic acid fermentation process with L. casei CRL 686 was performed. The static adsorption isotherm over a strong anionic exchange resin, AmberliteTM IRA-400 was measured, and the static binding capacity parameters were quantified. Early recovery of lactic acid from this lactate producer from unclarified culture broth was performed in a liquid solid fluidized bed, with the resin as the solid adsorbent, and the dynamic adsorption capacity was calculated. Good agreement was found between static and dynamic binding capacity values. The fluidized bed height was twice the settled bed height and the overall process was controlled by the liquid solid mass transfer. This operation was also simulated by continuously well stirred tanks arranged in series and superficial solid deactivation as in a gas solid catalytic reactor. The deactivation process takes into account liquid channeling and agglomerations of solid induced by the viscosity of the broth and also by the cells during the adsorption. These patterns were also verified by experimental observations, and are in agreement with the results found in the literature. The breakthrough data together with others from previous works were satisfactorily fitted until the 90% dimensionless concentration was reached for both culture broths. The model could be used in future studies on predictions about the liquid solid fluidized bed behavior and other different operating conditions.  相似文献   

9.
Summary A strain of Saccharomyces uvarum produced extracellular invertase in a chemostat reactor using a medium containing corn steep liquor and sugars. The production of yeast invertase increased with increase in corn steep liquor concentration. The production rate of invertase was maximal at a dilution rate of 0.75 h–1. The production rate of invertase was found to be affected by the type of sugar substrate and fermentation temperature. The invertase in the crude broth could be purified by one-step DEAE chromatography. An 84% enzyme recovery with ninefold purification and 30-fold concentration could be achieved using this simple isolation procedure. Offsprint requests to: L. F. Chen  相似文献   

10.
A new bacteriocin, gassericin A, was purified from the culture fluid of Lactobacillus gasseri LA39 mainly by reverse-phase (RP) chromatography. The purification of gassericin A from a modified MRS broth, in which Tween 80 had been replaced by oleic acid, resulted in a 4500-fold increase in specific activity with a 6% recovery. Gassericin A was eluted as a single peak on the chromatogram from RP-HPLC and migrated by SDS-PAGE as a single band with a molecular weight of ca. 3.8 kDa. Gassericin A, a highly hydrophobic bacteriocin, was slightly soluble in water, but its solubility was increased by adding alcohol and acetonitrile. An amino acid analysis revealed that it was composed of 45.7% hydrophobic amino acids in the total residues of 35 amino acids. Gassericin A produced in the MRS broth associated strongly with Tween 80, although several further trials of dissociation were unsuccessful.  相似文献   

11.
Gamma-aminobutyric acid (GABA) is a non-proteinaceous amino acid that is widely distributed in nature and acts as the major inhibitory neurotransmitter in the mammalian brain. This study aimed to find a separation method for getting high-purity GABA from a fermented broth. Firstly, a fermented broth with a high content of GABA (reaching 997 ± 51 mM) was prepared by fermentation with Lactobacillus brevis NCL912. GABA purification was conducted by successive centrifugation, filtration, decoloration, desalination, ion-exchange chromatography (IEC), and crystallization. Inorganic salt (Na2SO4) was removed from the both by desalination with 70% ethanol solution. A ninhydrin test strip was designed for the real-time detection of GABA during IEC. The recovery rate for the whole purification process was about 50%. The purified product was characterized by thin-layer chromatography and HPLC, and its purity reached 98.66 ± 2.36%.  相似文献   

12.
A new ribonuclease (RNase) with tobacco mosaic virus inhibition was isolated and purified from Bacillus cereus ZH14 through ammonium sulfate precipitation, ultrafiltration, ion-exchange chromatography of DEAE-Sephadex A-50 column, and gel chromatography of Sephacryl S-200HR column. The enzyme was purified approximately 134-fold with a recovery of 9.2%. The RNase had an MW of 75.6 kDa in SDS-PAGE, which differed from RNases reported previously. The inhibitory activity of the RNase in the purification process against tobacco mosaic virus was tested, and the percentage inhibition of the purified RNase (48 U/ml) reached 90%. The protein could tolerate 90°C and pH 4.0.  相似文献   

13.
Phenylboronates are competitive inhibitors of serine hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophenylbo ronate inhibited hepatic lipase activity with a K1 value of 55 μM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h−1 mg−1 protein with a total purification factor of 14 400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h−1 mg−1 The purification factor was 23 400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.  相似文献   

14.
Human epidermal growth factor is a polypeptide hormone having many diverse biological functions. This paper first presents the recovery results of human epidermal growth factor (hEGF) immediately from the fermentation broth of recombinant Escherichia coli by using an expanded bed system (a couple of STREAMLINE25 and ÄKTA explorer 100). The influences of operational conditions such as linear flow rate, gradient length of NaCl concentration, pH and sample concentration on the purification performances of hEGF in expanded and packed bed modes with STREAMLINE DEAE resin were systematically evaluated. After optimization, the practical recovery procedure in the expanded bed mode was carried out on a scaled-up system under the conditions of linear flow rates of 183 cm/h (upward) and 37 cm/h (downward), sample volume of 300 ml and column bed height of 13.8 cm which yielded a primary product of hEGF from the cell-free supernatant containing hEGF after centrifugation at 4000 rev/min for 15 min. As a result, the hEGF concentration in the product was higher than 20% (w/v), the concentration factor was greater than 4.3 and the total yield was higher than 80%, respectively. At the same time, the results of hEGF recovery by using expanded bed adsorption (EBA), packed bed chromatography (PBC) and salting out were compared. The results show that the procedure of hEGF recovery in expanded bed adsorption has some advantages over the other two procedures, because of its higher concentration factor, recovery yield, productivity, hEGF concentration in the primary product and shorter duration of purification run.  相似文献   

15.
-1,4-Xylanase, produced extracellularly by Bacillus amyloliquefaciens MIR 32, was isolated directly from the culture broth by adsorption on a cation exchanger, Amberlite IRC-50, in fluidized bed with a low degree of expansion. The enzyme was eluted from the adsorbent by increase in pH, with a recovery of 82.3% and purification of 5.3 fold. About 99.99% of the colony forming units, 82% of the contaminating neutral protease activity, and 100% of the reducing sugars present in the crude feedstock were removed at the end of the purification cycle.  相似文献   

16.
Large scale purification of a class IIa bacteriocin has been developed to recover pure carnocin KZ 213 produced by Carnobacterium piscicola 213. Most previous protocols reported in the literature for the purification of small peptides have used reversed phase chromatography but scale-up is difficult. The first step of this new protocol is hydrophobic interaction chromatography, the second and third steps are cation exchange chromatography. The protocol leads to a complete recovery of carnocin KZ 213 with 95% purity and to a concentration factor of 83. From 10 l culture supernatant, 5.8 mg carnocin KZ 213 have been produced with a specific activity of 8,500 UA g−1. The protocol is easy to implement for larger volumes.  相似文献   

17.
《Process Biochemistry》2004,39(5):565-570
Expanded bed purification of α-amylase and cellulase directly from unclarified fermentation broth was carried out on specially prepared composite affinity matrices. The concept used was incorporation of polymeric substrates/substrate analogue during cross-linking of cellulose to prepare rigid, porous, cross-linked composite affinity matrices for target enzymes. Of the several polymeric substrates/substrate-analogue used, alginic acid (AA) and microcrystalline cellulose (MCC) when used to prepare cross-linked composite matrices with cellulose, resulted in best affinity purification matrices for α-amylase and cellulase, respectively. These matrices were suitable for purification of the enzymes by batch, packed bed as well as expanded bed purification protocols. The optimized expanded bed protocol for α-amylase from Bacillus spp. B3 gave 51-fold purification on AA-CELBEADS with 69% recovery, whereas, cellulase from Bacillus spp. B21 was purified on MCC-CELBEADS to 18-fold purification with 97% recovery. The SDS-PAGE of both purified preparations showed single bands indicating significant purification on composite affinity adsorbents in a single step strategy.  相似文献   

18.
Abstract

The strain Paenibacillus curdlanolyticus MP-1 was used to obtain mutan-hydrolyzing enzymes. Different methods of precipitation and concentration of the post culture liquid were tested. All these methods produced satisfactory results in regard to the overall activity of mutanase and yielded active preparations of the enzyme. The best precipitation was obtained with propanol –98% of the initial enzyme activity was preserved with a purification of 2-fold. Salting out with ammonium sulfate at 50% saturation gave mutanase recovery of 77% and a purification of around 2-fold. Ultrafiltration yielded an about 10-fold concentrated preparation of the enzyme with a yield of 98%. Lyophilization and concentration of the culture broth (in the range from 5 to 20 times) in a vacuum evaporator yielded active crude preparations with mutanase recovery of 97%.  相似文献   

19.
Jia B  Jin ZH  Lei YL  Mei LH  Li NH 《Biotechnology letters》2006,28(22):1811-1815
Batch fermentation by Streptomyces pristinaespiralis with the addition of adsorbent resins was used to increase the production of pristinamycin. In consideration of the adsorption capacity and the desorption ability, a polymeric resin, JD-1, was finally selected. The maximum production of pristinamycin in Erlenmeyer flasks went up to 1.13 from 0.4 g l−1, by adding 12% (w/v) resin JD-1 into the culture broth at 20 h after inoculation. In a 3 l bioreactor, pristinamycin fermentation with the addition of 12% (w/v) resin JD-1 at 20 h after inoculation reached 0.8 g l−1, which was a 1.25-fold increase over fermentation without resin.  相似文献   

20.
Labrou NE 《Bioseparation》2000,9(2):99-104
Formate dehydrogenase (FDH, EC 1.2.1.2) from Candida boidinii was purified to homogeneity. The two step procedure comprised anion exchange chromatography (2.9-fold purification, 85% step yield, elution with 35 mM KCl), followed by dye-ligand affinity chromatography on immobilized Cibacron Blue 3GA (1.4-fold purification, 75% step yield, elution with 0.15 mM NAD+/2 mM Na2SO3). The procedure afforded FDH at 63.8% overall yield and a specific activity of 7.2 units/mg. The purity of the final FDH preparation was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), high performance gel filtration liquid chromatography (gfHPLC) and N-terminal amino acid sequencing. The analytical techniques showed the presence of a single polypeptide chain that corresponds to the molecular weight of 41 kDa (as determined by SDS-PAGE) and 81 kDa (as determined by gfHPLC).  相似文献   

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