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ABSTRACT: An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS-PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K(m) of 0.85 µM. The k(cat) and k(cat)/K(m) values were 13 s(-1) and 15 s(-1) µM(-1) respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K(i), of 25 pM.
Bioscience Biotechnology and Biochemistry 10/2010; 74(10):2154-7. · 1.28 Impact Factor
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ABSTRACT: There is a great demand for L-ornithine, which is used as a dietary supplement, and in the pharmaceutical industry. In the present study, when milk casein was hydrolyzed at 37 degrees C by using commercial digestive enzymes, namely, Pancreatin F and Protease A, a significant accumulation of L-ornithine in the hydrolysate and the simultaneous disappearance of L-arginine was noted. In a radiometric assay, transient but distinct arginase activity, which was sufficiently high for L-ornithine production, was detected in the hydrolysate for a certain period during casein hydrolysis. On the basis of the results of the enzymatic analyses, arginase was thought to be proteolytically generated from an inactive precursor, which may generally be contained in Pancreatin F, and ultimately degraded by further proteolysis. This conversion process using the above-mentioned digestive enzymes is useful for the production of L-ornithine directly from protein sources that are abundant in nature.
Bioprocess and Biosystems Engineering 08/2010; 33(6):773-7. · 1.81 Impact Factor
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ABSTRACT: In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610-2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells.
Bioscience Biotechnology and Biochemistry 02/2010; 74(2):370-4. · 1.28 Impact Factor
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ABSTRACT: Inactivation of baculoviruses was examined under various conditions by ELVA, which enzymatically labels the virus envelope with radioisotopes and detects the labeled viruses by host cell-specific binding. When baculoviruses were incubated in a 50-ml culture tube, they rapidly lost infective ability by more than 75% of the initial titer in 2 h even at an insect cell cultivation temperature of 27 degrees C. Altering pH from neutral significantly decreased the virus titer. Repetition of freezing and thawing of baculovirus solutions decreased the virus titer, but the addition of DMSO and glycerol prevented inactivation to some extent.
Bioscience Biotechnology and Biochemistry 08/2008; 72(7):1973-6. · 1.28 Impact Factor
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ABSTRACT: Recombinant human (rh) renin was expressed in Sf-9 insect cells. Baculovirus-infected Sf-9 cells produced active rh-renin in the late stage of cultivation. The rh-renin was purified after 5 d of culture by two steps of column chromatography. Approximately 0.61 mg of pure rh-renin was obtained from 200 ml of culture medium with a yield of 35.3%.
Bioscience Biotechnology and Biochemistry 11/2007; 71(10):2610-3. · 1.28 Impact Factor
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ABSTRACT: The present study developed a novel virus labeling and testing method, referred to as an envelope-labeled virus assay (ELVA), in which virus envelope is labeled in vitro by the action of phosphatidylethanolamine N-methyltransferase (PEMT) and tested through a host cell-specific binding. A recombinant strain (vGFPuv) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and Spodoptera frugiperda (Sf-9) insect cells were used as a model of viruses and host cells, respectively. The labeling mixture, which contained PEMT, [methyl-3H]S-adenosylmethionine (SAM), and a trace amount of detergent Triton X-100, brought about little change in virus titer of vGFPuv on a 1-h incubation, but was so toxic to Sf-9 cells as to immediately cause cell death. After being incubated with vGFPuv, therefore, the labeling mixture was neutralized by adsorptive removal of PEMT and Triton X-100 before Sf-9 cells were contacted with the mixture to extract the virus. The Sf-9 cells were then washed with a phosphate buffered saline (PBS), and lipid extracts with a 1% SDS solution were subjected to a liquid scintillation analysis for the determination of labeling efficiency. As a result, a significant amount of radioactivity was determined in the extracts, demonstrating the validity of ELVA for labeling and testing enveloped viruses. The conditions for the PEMT reaction and cell-virus binding were examined, and the lower detection limit of AcMNPV by ELVA was found to lie in the order of 10(3) plaque forming unit (pfu) per milliliter. Since the labeling reaction and detection of virus are based on neither immunological nor genetic characteristics of virus, ELVA is also expected to be a convenient and comprehensive test of other enveloped viruses.
Biotechnology and Bioengineering 09/2006; 94(6):1017-24. · 3.95 Impact Factor
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ABSTRACT: Glutathione (L--glutamyl-L-cysteinylglycine) is physiologically synthesized through two ATP-dependent reactions catalyzed by -glutamylcysteine synthase and glutathione synthase. The present study was designed to produce glutathione without the aid of ATP by using glutathione-degrading enzymes, -glutamyl transpeptidase and aminopeptidase M, in reverse: intrinsically the former enzyme catalyzes the cleavage of glutathione to give L-cysteinylglycine and a -glutamyl moiety and the latter hydrolyzes the peptide linkage of L-cysteinylglycine. Both enzymes were simultaneously displayed on proteoliposomes, which were reconstituted from bovine kidney brush border membranes by a cholate dialysis method. The kinetic analysis using artificial substrates, L--glutamyl-p-nitroanilide for -glutamyl transpeptidase and L-leucine-p-nitroanilide for aminopeptidase M, revealed that the proteoliposome reconstitution significantly increased the enzyme activities: for both the enzymes the maximum reaction rates were increased and Michaelis constants with the respective substrates were decreased. When the proteoliposomes were incubated with the amino acids glycine, L-cysteine, and L-glutamate (or L-glutamine) at 37 °C, a new product was determined on HPLC analyses using ODS and cation-exchange columns, coinciding in retention time with authentic glutathione. This product was identified to be glutathione by LC–MS and 1H-NMR, after being purified by gel filtration using Sephadex G10 and HSKgel Toyopearl HW-40F in succession. When the incubation mixture contained acivicin and bestatin, specific inhibitors for -glutamyl transpeptidase and aminopeptidase M, respectively, glutathione was not produced at all. These results indicated that glutathione was produced by two-step reversible reactions of aminopeptidase M and -glutamyl transpeptidase from its constituent amino acids. The equilibrium glutathione concentration obtained with L-glutamine as a glutamyl donor substrate was about 3.5 times higher than that obtained with L-glutamate. The maximum pH for the glutathione production was 7.0–7.5, reflecting pH dependence of the activities of the enzymes. Copyright © 2004 Society of Chemical Industry
Journal of Chemical Technology & Biotechnology 09/2004; 79(11):1204 - 1211. · 2.17 Impact Factor
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ABSTRACT: The covalently cross-linked alginate gel beads were prepared by the reactions of Ca(2+)-doped alginate gel beads, which were formed by spraying a viscous alginate solution into a calcium chloride solution, with cyanogen bromide and following 1,6-diaminohexane. The cross-linking of alginate matrix decreased the mean bead diameter by about 30% and made the beads durable in some extent under alkaline conditions. The adsorption of metal ions on the covalently cross-linked alginate gel beads was rapid and reached at equilibrium within 30 min at 25 degrees C. Adsorption isotherms of Cu(II), Mn(II), and Ca2+ on the beads possessed a stepwise shape, which was firstly determined by Rorrer et al. [Ind. Eng. Chem. Res. 32 (1993) 2170] for cross-linked chitosan gel beads and explained by a pore-blockage mechanism. Higher selectivity was determined against Cu(II) over Mn(II) and Ca2+, especially at a low concentration region. These metal adsorption profiles for the covalently cross-linked alginate gel beads was almost the same as those for the un-cross-linked beads, indicating that the cross-linking reactions were performed without interfering the adsorption characteristics of alginate gel beads.
Chemosphere 05/2004; 55(1):57-64. · 3.21 Impact Factor
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ABSTRACT: Naturally occurring polysaccharides such as alginic acid and chitosan have been recognized as one of the most effective adsorbents to eliminating low levels of heavy metal ions from waste water stream. The present study intended to use alginic acid and chitosan simultaneously, which are expected to form a rigid matrix structure of beads due to electrostatic interaction between carboxyl groups on alginic acid and amino groups on chitosan, and to prepare alginate-chitosan hybrid gel beads. This could be achieved for the first time by using water-soluble chitosan, which was obtained by deacetylating chitin to 36-39% degree. The water-soluble chitosan dissolved in water could remain in solution in the presence of sodium alginate, and the homogeneous solution of chitosan and alginate was dispensed into a CuCl2 solution to give gel bead particles. The resultant beads were then reinforced by a cross-linking reaction of aldehyde groups on glutaraldehyde with amine groups on the chitosan. The cross-linking reaction made the beads durable under acidic conditions. The adsorption of Cu(II), Co(II), and Cd(II) on the beads was significantly rapid and reached at equilibrium within 10 min at 25 degrees C. Adsorption isotherms of the metal ions on the beads exhibited Freundlich and/or Langmuir behavior, contrary to gel beads either of alginate or chitosan showing a step-wise shape of adsorption isotherm.
Chemosphere 05/2004; 55(1):135-40. · 3.21 Impact Factor
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ABSTRACT: Spodoptera frugiperda Sf-9 insect cells were infected with recombinant Autographa californica nuclear polyhedrosis virus at a low multiplicity of infection (MOI) (0.1), and the effect of dissolved oxygen (DO) on the production of a polyhedrin promoter-driven recombinant protein (beta-galactosidase), intrinsic proteases (carboxyl and cysteine proteases), and the virus was determined. The DO concentrations used in the present study were 45%, 25%, 5%, and 1.3% of air saturation. At 5% DO the cell growth following viral infection was greatest and beta-galactosidase was about 5-fold increased in volumetric yield compared to that at 45% and 25% DO, whereas the growth at 1.3% DO was extremely poor. The virus titer in the medium at 4-8 d post-infection (dpi) was also highest at 5% DO, but the titer was significantly decreased by further increasing the culture time. This was in part attributed to the fact that baculovirus is susceptible to oxidative inactivation under aerobic conditions. The DO dependency of the specific oxygen consumption rate of virus-infected and uninfected Sf-9 cells was expressed by a Monod-type equation. A critical DO, above which the rate of oxygen utilization is not limited by DO, was estimated to be 3.5% of air saturation for virus-infected Sf-9 cells. These results indicated that for a baculovirus-infected Sf-9 insect cell culture of low MOI, the optimal DO was likely to be approximately 5% of air saturation, which is above the critical DO for the infected Sf-9 cells but sufficiently low to reduce the possibility of the oxidative inactivation of virus. For the production of carboxyl and cysteine proteases, the accumulation behavior and concentrations did not significantly vary with DO, except that a peak of cysteine protease activity was observed intracellularly only at 5% DO, coinciding with beta-galactosidase production.
Journal of Bioscience and Bioengineering 02/2002; 94(5):426-33. · 1.79 Impact Factor
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ABSTRACT: Spodoptera frugiperda Sf-9 insect cells that undergo apoptosis by the treatment of apoptosis-inducing reagents were individually determined as `comet cells' having a tail of fragmented DNA during single cell gel electrophoresis, the fragmented DNA being migrated from the cells under an electric field of the electrophoresis. However, the apoptosis induction of the cells infected with a recombinant strain of Autographa californica nuclear polyhedrosis virus (AcMNPV) was blocked, probably by an intrinsic anti-apoptotic p35 gene of the virus, because the virus-infected cells did not have a tail of fragmented DNA on a single cell gel electrophoresis. The virus-infected cells were individually discriminated from non-infected cells by determining the anti-apoptotic nature of the cells. At higher multiplicity of infection and under better aeration conditions of virus-infected cultures, the apoptosis-suppressive ratio, which represented a ratio of non-comet cells, was increased more rapidly. This apoptosis-suppressive behavior was a good benchmark for assessing successful infection of insect cells with AcMNPV during very early infectious period and forecasting the subsequent production of recombinant proteins.
Biotechnology Letters 08/2001; 23(18):1473-1478. · 1.68 Impact Factor
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ABSTRACT: Perfluorocarbon (PFC) was used as an oxygen carrier in the cultures of insect cells and virus-infected insect cells. The cell suspensions were placed on a planar layer of PFC, which was re-oxygenated in an outer aeration unit and continuously recirculated, and were agitated by two sets of impeller blades, lower one of which was set in such a way that the ridge of the blade touched the PFC layer. The maximum cell density attained in the PFC-mediated aeration culture was higher than that in surface aeration culture. On viral infection, a recombinant protein yield was significantly high in the PFC-mediated aeration culture as compared with that in the surface aeration culture, though the production was largely decreased by setting apart the lower set of the blade from the PFC–medium interface. These results showed that the PFC-mediated aeration would be a useful technique for insect cell/baculovirus expression system. Overall mass-transfer coefficient KL for oxygen was examined in both the PFC-mediated aeration and surface aeration systems, by using a flask whose dimensions were identical to those of spinner flasks used for the cultures. The KL value in the PFC-mediated system was , 1.6 times higher than that in the surface aeration system, when impeller blades were positioned at PFC–medium and medium–air interfaces, respectively. However, the KL values in both the PFC-mediated and surface aeration systems were decreased and their differences were brought so close, as the blade was set apart from the interfaces. DO behavior in the cultures was well explained by the model calculation using the determined KL values and oxygen-consumption rates of viable cells. This calculation further suggested that crucial DO, under which recombinant protein productions were unsuccessful, was 0.24–0.5 ppm (3–7%) in the insect cell/baculovirus expression system.
Biochemical Engineering Journal 02/2001; · 2.64 Impact Factor
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ABSTRACT: The present study has demonstrated a novel immobilization support for membrane enzymes; the support is composed of agarose gel beads and polymerized phospholipid vesicles contained within the beads. A phosphatidylcholine analogue, bis-[12-(methacryloyloxy)dodecanoyl]-L--phosphatidylcholine (BMPC) [Regen, Singh, Oehme and Singh (1982) J. Am. Chem. Soc. 104, 791–795], was contained in Sepharose CL-6B beads through the formation and simultaneous entrapment of vesicles by a combination of phospholipid solubilization in organic solvent with the beads, complete removal of the solvent and sonication in a buffer. The vesicle membranes were then polymerized by UV irradiation, which stabilized the hybrid-type support. -Glutamyl transpeptidase, a membrane enzyme from bovine kidney, was immobilized in the beads by reconstitution in the polymerized BMPC vesicles contained within the beads. The enzyme catalytic activity, as indicated by apparent Michaelis-Menten kinetics, was almost identical with that of the enzyme reconstituted in unpolymerized BMPC vesicles. Polymerized BMPC vesicles significantly stabilized the enzyme to heat treatment when compared with unpolymerized BMPC vesicles and egg yolk phospholipid liposomes.
Biotechnology and Applied Biochemistry 05/1998; 27(3):197 - 204. · 1.53 Impact Factor
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ABSTRACT: In the baculovirus expression vector system (BEVS), intrinsic proteases concomitantly produced by infected insect cells have been generally regarded as a defect, because they sometimes degrade expressed recombinant proteins and decrease the productivity. The present study successfully used the proteolysis to generate active recombinant human- (rh) renin after the expression of inactive rh-prorenin. Sf-9 insect cells were infected with recombinant baculoviruses having human preprorenin cDNA in the site of polyhedron gene at several MOIs. At any MOIs, rh-prorenin was expressed in a late phase of infective cultures and processed to active rh-renin in a very late phase. The maximum volumetric yield of active rh-renin was obtained at MOIs of 1 and 10 pfu/cell. The protease activity was examined with an internally quenched fluorogenic substrate newly designed for the processing. The generation of rh-renin was coincided with a considerable increase in a protease activity that was classified into the cysteine protease family, and significantly suppressed by supplementing the culture medium with leupeptin, a cysteine protease inhibitor. This suggested that the cysteine protease was responsible to the processing of rh-prorenin to rh-renin.
Biochemical Engineering Journal.
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ABSTRACT: Nanofiltration experiments of glutathione and its related amino acids (l-glutamate, l-cysteine, glycine, and l-glutamine) were carried out using NTR-7450 nanofiltration membrane in order to determine the potential use of nanofiltration in the enzymatic glutathione synthesis, in which the reaction equilibria are need to be driven to production by separating glutathione from starting substrates. In the experiments with a single component system, the rejection of NTR-7450 membrane for the electrolytes corresponded well to the ratio of their anionic species varying with pH. Difference in the rejection between glutathione and some amino acids was maximal in the neutral pH region: at pH 7.4, the rejection for glutathione and l-glutamate was almost 100%, whereas that for l-cysteine, glycine, and l-glutamine was below 30%. In the presence of divalent metal ions, the rejection for glutathione and l-glutamate decreased with increasing the metal concentration, probably due to partial neutralization of negative charges of the electrolytes forming complexes with the metal ions. In the experiments with a multi-component system, glutathione was completely blocked again by the membrane in a range of glutathione concentration from 0.1 to 5.0 mM in the presence of 1.0, 2.0, or 5.0 mM each of l-glutamine, glycine, and l-cysteine. The amino acid rejection decreased with increasing the amino acid concentrations. These results suggest that nanofiltration would be applicable to the separation of glutathione from substrates, l-cysteine, glycine, and l-glutamine, in the enzymatic glutathione synthesis.
Biochemical Engineering Journal.
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ABSTRACT: In insect cell-baculovirus expression systems for recombinant protein production, it is sometimes necessary to supplement cultures with protease inhibitors to protect recombinant proteins against proteolysis. To date, however, there is no information available concerning protease activities in inhibitor-supplemented cultures. The aim of the present study was to investigate intracellular and extracellular protease activities in cultures of virus-infected Sf-9 insect cells which were supplemented with inhibitors against carboxyl and cysteine proteases produced during culture. Prior to the supplementation culture, the cell toxicity of several protease inhibitors was determined. As a result, pepstatin A (carboxyl protease inhibitor) and E64, cystatin, leupeptin, and antipain (cysteine protease inhibitors) tested in this study showed no apparent negative effects on the growth and viability of noninfected Sf-9 insect cells at low concentrations. In addition, E64 and pepstatin A could rapidly permeate virus-infected Sf-9 cells and inhibit the respective intracellular protease activities. A virus-infected culture with a multiplicity of infection of 1 was carried out with E64 and pepstatin A which were added to the culture medium at 2 d post-infection. As a result of inhibitor supplementation, the cellular activity for recombinant protein biosynthesis was reduced by 5–30%. However, a significant reduction in carboxyl and cysteine protease activities was observed not only in the medium but also intracellularly. This is the first study that directly demonstrates a reduction in extracellular and intracellular protease activities in protease inhibitor-supplemented cultures of virus-infected insect cells.
Journal of Bioscience and Bioengineering.
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ABSTRACT: The oxygen consumption profile of Sf-9 insect cells was determined under various conditions using a 250 ml spinner flask. The specific oxygen consumption rate, q, of Sf-9 was scarcely changed with cell density and growth phases except for stationary phase. However, the q was varied with dissolved oxygen (DO) concentration and described by a Monod-form equation in the range of 20–34 °C. For uninfected Sf-9, the maximum specific oxygen consumption rate, qmax, in the equation decreased with decreasing temperature, whereas the saturation constant, KS, remained constant independent of temperature up to 28 °C, at which Sf-9 cultures were usually performed. This indicated that the cellular physiology of Sf-9 was not altered in the temperature region. On a viral infection, the qmax increased by 30–40% irrespective of temperature, and the KS increased more largely at higher temperature.The virus-infected Sf-9 culture was performed at 20 and 28 °C using a 2 l spinner flask with a working volume of 1 l, and DO behavior and recombinant protein production were determined. The oxygenation was done by supplying only air to mimic a large-scale culture of oxygenation-limited conditions. As a result, only the 20 °C culture was successful to keep DO concentration above the critical DO concentration (3KS) and produce a large amount of green fluorescent protein, a recombinant model protein, but not the 28 °C culture. The DO behavior in both the 20 and 28 °C cultures was well simulated by model calculation using the oxygen consumption parameters obtained above. These results indicated that a low temperature culture was useful to circumvent oxygen starvation of virus-infected Sf-9 cells and to successfully produce recombinant proteins.
Biochemical Engineering Journal.
