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Culture Broth (culture + broth)
Selected AbstractsProduction of Taxol fromPhyllosticta spinarum, an endophytic fungus ofCupressus sp.ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2008R. Senthil Kumaran Abstract Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235,,g/L) followed by PDB medium (125,,g/L). The results indicate that P.,spinarum is an excellent candidate for taxol production. The production rate was 4.7,×,103 -fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay. [source] Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growthEQUINE VETERINARY JOURNAL, Issue 7 2003J. S. Weese Summary Reasons for performing study: Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses. Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use. Objectives: To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C. perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro. Methods: Clostridium difficile toxins, C. perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests. Serial dilutions of smectite were tested for the ability to inhibit growth of C. perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C. perfringens in culture broth. Results: Clostridium difficile toxins A and B, and C. perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16. Partial binding of C. difficile toxins occurred at dilutions up to 1:256 while partial binding of C. perfringens enterotoxin occurred up to a dilution of 1:128. Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32. No inhibition of growth of C. difficile or C. perfringens was present at any dilution, and there was no effect on the action of metronidazole. Conclusions: Di-tri-octahedral smectite possesses the ability to bind C. difficile toxins A and B, C. perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole. Potential relevance: In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colifis and endotoxaemia in horses. [source] Isolation and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from KimchiJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2008M.S. Shin Abstract Aims:, Screening and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi, a traditional Korean fermented vegetable. Methods and Results:, A total of 1000 lactic acid bacteria were isolated from various Kimchi samples and screened for the production of bacteriocin. Pediocin K23-2, a bacteriocin produced by the Pediococcus pentosaceus K23-2 strain, showed strong inhibitory activity against Listeria monocytogenes. The bacteriocin activity remained unchanged after 15 min of heat treatment at 121°C or exposure to organic solvents; however, it diminished after treatment with proteolytic enzymes. The bacteriocin was maximally produced at 37°C, when the pH of the culture broth was maintained at 5·0 during the fermentation, although the optimum pH for growth was 7·0. The molecular weight of the bacteriocin was about 5 kDa according to a tricine SDS-PAGE analysis. Conclusions:,Pediococcus pentosaceus K23-2 isolated from Kimchi produces a bacteriocin, which shares similar characteristics to the Class IIa bacteriocins. The bacteriocin is heat stable and shows wide antimicrobial activity against Gram-positive bacteria, especially L. monocytogenes. Significance and Impact of the Study:, Pediocin K23-2 and pediocin K23-2-producing P. pentosaceus K23-2 could potentially be used in the food and feed industries as natural biopreservatives, and for probiotic application to humans or livestock. [source] A new spiroketal from Aspergillus terreus, an endophytic fungus in Opuntia ficusindica MillJOURNAL OF BASIC MICROBIOLOGY, Issue 2 2008Shao-Hua Wu Abstract A new spiroketal, named aspergiketal (1) was isolated from the culture broth of Aspergillus terreus, an endophytic fungus in the stems of the plant Opuntia ficusindica Mill., together with two known compounds, physcion (2) and asterric acid (3). Their structures were elucidated by spectroscopic methods including 2D-NMR techniques. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] New phenyl-ethanediols from the culture broth of Boletus edulisJOURNAL OF BASIC MICROBIOLOGY, Issue 2 2007Wan-Qiu Yang Abstract A new phenyl-ethanediol, (1S)-(4-acetylphenyl)-1, 2-ethanediol (1), and a new natural product, (1S)-(3-ethenylphenyl)-1, 2-ethanediol (2), were isolated from the culture broth of the basidiomycete Boletus edulis together with three related known compounds, 1-(4-ethylphenyl)-1, 2-ethanediol (3), 1-(3-ethylphenyl)-1, 2-ethanediol (4) and 1-(3-formylphenyl)-ethanone (5). Their structures were elucidated by spectroscopic methods including extensive 2D-NMR techniques. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Purification of Aspergillus carbonarius polygalacturonase using polymeric membranesJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2008E. Nakkeeran Abstract BACKGROUND: Microfiltration (MF: 70,450 nm) and ultrafiltration (UF: 10,500 kDa) membranes were used to eliminate carbohydrates and other non-protein impurities from Aspergillus carbonarius culture broth containing polygalacturonase enzyme (EC 3.2.1.15) that would otherwise interfere with the purification processes and lead to enzyme loss. Further, diafiltration was attempted to improve the elimination of impurities as well as recovery of enzymes. RESULTS: MF resulted in removal of 2,25% carbohydrates with an enzyme recovery of 69,82% from the crude culture broth owing to the secondary layer formation. UF with 10 kDa membrane eliminated most of the carbohydrates (96%), phosphate salts and total acids with a recovery of 96% polygalacturonase and resulted in greater productivity. Using the above procedure, the enzyme was concentrated nearly 10-fold while the purity improved from 4.6 to 49.4 U mg,1 of dry matter. CONCLUSIONS: The results of this study focused on the elimination of carbohydrates and other non-protein impurities showed that UF could be used efficiently as a primary purification step during downstream processing of microbial culture broths containing enzymes. The present approach will ensure complete elimination of non-protein impurities thereby reducing the losses and difficulties in the subsequent purification steps. Copyright © 2008 Society of Chemical Industry [source] Production of delta-endotoxin by Bacillus thuringiensis subsp kurstaki and overcoming of catabolite repression by using highly concentrated gruel and fish meal media in 2- and 20-dm3 fermentersJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 8 2002Nabil Zouari Abstract Delta-endotoxin production by a strain of Bacillus thuringiensis subsp kurstaki exhibiting larvicidal toxicity towards lepidoptera was investigated in 2- and 20-dm3 fermenters, using gruel- and fish meal-based media. The results show clearly that in such complex media, aeration plays an important role in bioinsecticide production. Optimal aeration led to improvement of delta-endotoxin concentrations with decreases of final spore count and proteolytic activity. Moreover, in order to use high gruel concentrations, a fermenter configuration with an efficient aeration system should be used. In a 20-dm3 Biolafite fermenter, 59,g,dm,3 or 75,g,dm,3 gruel was used to produce bioinsecticides with a significant reduction of carbon catabolite repression of delta-endotoxin synthesis. This result is very interesting in order to produce high final delta-endotoxin concentrations in the culture broth. It was also concluded, by considering the key role of oxidative pathways in delta-endotoxin synthesis, that oxygen supply must be adequate for bioinsecticide production at high substrate concentrations. Moreover, the role of sodium chloride in improving delta-endotoxin production is dependent not only on protease synthesis and its effect on crystal stability, but also on the aeration level of the production medium. © 2002 Society of Chemical Industry [source] Enhanced ethanol production from enzymatically treated steam-exploded rice straw using extractive fermentationJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 8 2001Yoshitoshi Nakamura Abstract Alcohol fermentation of an enzymatic hydrolyzate of exploded rice straw was studied experimentally. Rice straw was treated under variable conditions, such as steam pressure and steaming time. The exploded rice straw was separated into water-soluble material, methanol-soluble lignin, Klason lignin, and a mixture of cellulose and a low molecular weight substance. The effects of steam explosion on the characteristics of the exploded rice straw were clarified from the point of view of the amounts of extractive components. Steam explosion was found to be effective for the delignification of rice straw and for increasing its susceptibility to enzyme hydrolysis and alcohol fermentation. The polysaccharides (cellulose and hemicellulose) in the rice straw treated at a steam pressure of 3.5,MPa with a steaming time of 2,min were hydrolyzed almost completely into monosaccharides, (ie glucose and xylose) by a mixture of Trichoderma viride cellulase (Meicelase) and Aspergillus aculeatus cellulase (Acucelase). The enzymatic hydrolyzate of exploded rice straw was converted into ethanol efficiently by Pichia stipitis and the ethanol yield from sugar was about 86%(w/w) of the theoretical value. The ethanol concentration in a membrane bioreactor coupled with a pervaporation system reached 50,gdm,3 and was about five times higher than that in the culture broth. The energy efficiency (ratio of combustion energy of ethanol produced to energy for steam explosion) reached a maximum value at a pressure of 3.5,MPa for 2,min. © 2001 Society of Chemical Industry [source] Verticase: a Fibrinolytic Enzyme Produced by Verticillium sp.JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 11 2007an Endophyte of Trachelospermum jasminoides Abstract Plant endophytes are among the most important resources of biologically active metabolites. Twenty-three endophyte strains residing in Trachelospermum jasminoides were cultivated in vitro with the cultures assayed for the fibrinolytic substance production. As a result, the culture of Verticillium sp. Tj33 was shown to be the most active. A fibrinolytic enzyme designated as verticase was subsequently purified from the supernatant of Verticillium sp. culture broth by a combination of DEAE-52, Sephadex G-75 and hydrophobic column chromatographies. Verticase, with its molecular mass of 31 kDa and pI of 8.5, was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. Verticase is an enzyme that hydrolyzes fibrin directly without activation of plaminogen. It was stable in a broad pH range from 4 through to 11 with the optimal reaction pH value and temperature shown to be around 9,10 and 50,60 °C, respectively. The fibrinolytic activity of verticase was severely inhibited by phenylmethylsulfony fluoride, indicating that verticase was a serine protease. [source] Sequence diversity of the peptaibol antibiotic suzukacillin-A from the mold Trichoderma virideJOURNAL OF PEPTIDE SCIENCE, Issue 5 2006Corina Krause Abstract From the culture broth of the mold Trichoderma viride, strain 63 C-I, the polypeptide antibiotic suzukacillin (SZ) was isolated. A peptide mixture named SZ-A was obtained by crystallization from crude SZ. Individual peptides from SZ-A were isolated by semipreparative HPLC and sequences were determined by HPLC-ESI-MS. The data confirm a general sequence of SZ-A published previously and in addition establish the individual sequences of 15 acetylated eicosa peptides with C -terminal alcohols. The major peptide SZ-A4 (21% of all peptides) shows the sequence: Ac-Aib-Ala-Aib-Ala-Aib-Ala6 -Gln-Aib-Lx9 -Aib-Gly-Aib12 -Aib-Pro-Vx15 -Aib-Vx17 -Gln-Gln-Fol. Amino acid exchanges of the peptaibol are located in position 6 (Ala/Aib), 9 (Vx/Lx), 12 (Aib/Lx), 17 (Aib/Vx) and possibly at position15 (Val/Iva) (uncommon abbreviations: Aib (,-aminoisobutyric acid); Iva (D -isovaline); Lx (L -leucine or L -isoleucine); Vx (L -valine or D -isovaline); Fol (L -phenylalaninol)). Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd. [source] Effect of Sublethal Hypoxia on the Immune Response and Susceptibility of Channel Catfish, Ictalurus punctatus, to Enteric SepticemiaJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 1 2007Thomas L. Welker The effect of sublethal hypoxia exposure on stress and immune responses and susceptibility to Edwardsiella ictaluri infection in juvenile channel catfish, Ictalurus punctatus, was investigated. Fish were monitored for temporal changes in glucose and cortisol concentrations before, during, and after 2 h exposure to sublethal hypoxia (<2 mg/L dissolved oxygen [DO]) and when maintained under normoxic conditions (6.0 ± 0.3 mg/L DO). Both blood glucose and plasma cortisol increased significantly in response to hypoxic conditions. Fish exposed to hypoxic or normoxic conditions were challenged with a high dose (1.3 × 107 colony-forming units [CFU]/mL) or a low dose (1.3 × 105 CFU/mL) of E. ictaluri or sterile culture broth by 30-min immersion bath. Approximately 1% of fish in both the normoxic and the hypoxic groups died when challenged with the low dose of E. ictaluri. However, when challenged with the high dose of E. ictaluri, catfish exposed to hypoxic conditions had significantly higher cumulative mortality (36 ± 12.1%) than those maintained under normoxic conditions (12 ± 1.1%). Total hemolytic complement and bactericidal activities and antibody response were lower in hypoxia-exposed channel catfish, indicating that increased susceptibility of channel catfish to E. ictaluri may be the result of the immunosuppressive effects of the stress response to hypoxia. [source] Structure elucidation of two new xanthone derivatives from the marine fungus Penicillium sp. (ZZF 32#) from the South China SeaMAGNETIC RESONANCE IN CHEMISTRY, Issue 11 2008Changlun Shao Abstract Two new xanthones, 8-(methoxycarbonyl)-1-hydroxy-9-oxo-9H-xanthene-3-carboxylic acid (1) and dimethyl 8-methoxy-9-oxo-9H-xanthene-1, 6-dicarboxylate (2) and one known xanthone methyl 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (3) were isolated from the culture broth of the mangrove fungus Penicillium sp. (ZZF 32#) collected from the South China Sea. Their structures were established by comprehensive analysis of one-dimensional (1D) and two-dimensional (2D) NMR data. The structure of compound 3 was confirmed by X-ray crystallography, which led to the suggestion that janthinone (4) might have the same structure as 3. Compounds 1,3 were inactive against KB or KBv200 cells during cytotoxicity evaluations. Copyright © 2008 John Wiley & Sons, Ltd. [source] Production, purification and characterisation of a novel halostable xylanase from Bacillus sp.ANNALS OF APPLIED BIOLOGY, Issue 2 2010NTU-0 Bacillus sp. NTU-06 was used to produce xylanase, which is an important industrial enzyme used in the pulp and paper industry. The enzyme was purified by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The enzyme was active over a concentration range of 0,20% sodium chloride in culture broth, although its activity was optimal in 5% sodium chloride. A salinity stability test showed that 43% of the enzyme activity was retained after 4 h in 20% sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The enzyme was somewhat thermostable, retaining 20% of the original activity after incubation at 70°C for 4 h. The xylanase had Km and Vmax values of 3.45 mg mL,1 and 387.3 µmol min,1mg,1, respectively. The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of beta-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation are discussed. [source] Sensitivity to Hydrogen Peroxide of Growth and Hyaluronic Acid Production by Streptococcus zooepidemicus ATCC 39920ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING, Issue 5-6 2005M.D. Mashitah Abstract The sensitivity to hydrogen peroxide (H2O2) of growth and hyaluronic acid (HA) production by Streptococcus zooepidemicus ATCC 39920 was studied under various conditions. In sheep blood agar-plates, no detectable zone was observed even when the concentration of H2O2 was increased to 0.15 mM. With brain heart infusion-agar and chemically defined medium-agar plates, a profound zone was detected at 0.015 mM concentration of H2O2. To determine the minimal inhibitory concentration (MIC) of the strain in culture broth, various concentrations of H2O2 (0-200 mM) were maintained in the medium prior to fermentation. The result showed that for higher concentrations of H2O2 in the medium, the greater was the inhibition. Streptococcus is catalase-negative and known to produce H2O2 which may affect growth, HA production and glucose utilization. In order to determine at which growth phase H2O2 had the maximum inhibitory activity, a batch fermentation of S. zooepidemicus was conducted in shake flask culture. It was found that H2O2 production took place during the growth phase, and HA production started after the growth had reach late exponential phase when H2O2 in the culture media was depleted. This indicates that H2O2 produced by the cells did not affect cell growth but influenced HA production. [source] Farnesol production from Escherichia coli by harnessing the exogenous mevalonate pathwayBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Chonglong Wang Abstract Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two-phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5,mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway-only control. Biotechnol. Bioeng. 2010;107: 421,429. © 2010 Wiley Periodicals, Inc. [source] New milliliter-scale stirred tank bioreactors for the cultivation of mycelium forming microorganismsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Ralf Hortsch Abstract A novel milliliter-scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter-scale. A newly designed one-sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface-to-volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (kLa),>,0.15,s,1 were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter-scale stirred tank bioreactor (V,=,10,mL) and compared to a standard laboratory-scale stirred tank bioreactor with six-bladed Rushton turbines (V,=,2,000,mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter-scale stirred tank bioreactor was reduced compared to the laboratory-scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale-up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120,h. A high parallel reproducibility was observed on the milliliter-scale (standard deviation,<,8%) with up to 48 stirred tank bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear-thinning non-Newtonian behavior. The newly developed one-sided paddle impellers operated in unbaffled reactors on a 10 milliliter-scale with a magnetic inductive drive for up to 48 parallel bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100,h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically. Biotechnol. Bioeng. 2010; 106: 443,451. © 2010 Wiley Periodicals, Inc. [source] Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Ting-Kuo Huang Abstract Plant cell culture production of recombinant products offers a number of advantages over traditional eukaryotic expression systems, particularly if the product can be targeted to and purified from the cell culture broth. However, one of the main obstacles is product degradation by proteases that are produced during cell culture, and/or the loss of biological activity of secreted (extracellular) products as a result of alteration in the protein conformation. Because proteolysis activity and target protein stability can be significantly influenced by culture conditions, it is important to evaluate bioprocess conditions that minimize these effects. In this study, a bioreactor strategy using a protocol involving pH adjustment and medium exchange during plant cell culture is proposed for improving the production of functional recombinant ,1 -antitrypsin (rAAT), a human blood protein, produced using several alternative expression systems, including a Cauliflower mosaic virus (CaMV) 35S constitutive promoter expression system, a chemically inducible, estrogen receptor-based promoter (XVE) expression system, and a novel Cucumber mosaic virus (CMV) inducible viral amplicon (CMViva) expression system developed by our group. We have demonstrated that higher medium pH help reduce protease activity derived from cell cultures and improve the inherent stability of human AAT protein as well. This strategy resulted in a fourfold increase in the productivity of extracellular functional rAAT (100 µg/L) and a twofold increase in the ratio of functional rAAT to total rAAT (48%) in transgenic N. benthamiana cell cultures using a chemically inducible viral amplicon expression system. Biotechnol. Bioeng. 2009;102: 508,520. © 2008 Wiley Periodicals, Inc. [source] Biomass recycling from a riboflavin cultivation with B. subtilis: Lysis, extract production and testing as substrate in riboflavin cultivationBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2006Karlheinz Bretz Abstract Autolysis of riboflavin-producing B. subtilis can be induced by pH, lack of carbon source, and the buffer system. Stress factors like temperature shift or oxygen dearth enhance the autolysis process. After cultivation of a riboflavin-producing strain, the pH of the whole culture broth was adjusted to 6.5,7.5. At a temperature of 40°C, autolysis started after 1 h. Adding a defined amount of commercially available endo- and exo-proteases enhanced both auto- and proteo-lysis. Optimization of endo- and exo-protease concentrations and of the time increased the degree of proteolysis. Additionally, the amount of DNA and Protein trapped in the riboflavin crystals could be significantly reduced by autolysis. After autolysis, the cultivation broth was centrifuged and the supernatant was cross-flow filtrated with a cut off of 10 kDa. Using this autolysate instead of yeast extract as a medium component for riboflavin production with B. subtilis, a riboflavin yield of 77% was obtained in comparison with the standard cultivation on yeast extract. © 2006 Wiley Periodicals, Inc. [source] Cellulases of Penicillium verruculosumBIOTECHNOLOGY JOURNAL, Issue 8 2010Valeria V. Morozova Abstract Nine major cellulolytic enzymes were isolated from a culture broth of a mutant strain of the fungus Penicillium verruculosum: five endo-1, 4-,-glucanases (EGs) having molecular masses 25, 33, 39, 52, and 70 kDa, and four cellobiohydrolases (CBHs: 50, 55, 60, and 66 kDa). Based on amino acid similarities of short sequenced fragments and peptide mass fingerprinting, the isolated enzymes were preliminary classified into different families of glycoside hydrolases: Cel5A (EG IIa, 39 kDa), Cel5B (EG IIb, 33 kDa), Cel6A (CBH II, two forms: 50 and 60 kDa), Cel7A (CBH I: 55 and 66 kDa), Cel7B (EG I: 52 and 70 kDa). The 25 kDa enzyme was identical to the previously isolated Cel12A (EG III). The family assignment was further confirmed by the studies of the substrate specificity of the purified enzymes. High-molecular-weight forms of the Cel6A, Cel7A, and Cel7B were found to possess a cellulose-binding module (CBM), while the catalytically active low-molecular-weight forms of the enzymes, as well as other cellulases, lacked the CBM. Properties of the isolated enzymes, such as substrate specificity toward different polysaccharides and synthetic glycosides, effect of pH and temperature on the enzyme activity and stability, adsorption on Avicel cellulose and kinetics of its hydrolysis, were investigated. [source] Selective Extraction of Free Astaxanthin from Haematococcus Culture Using a Tandem Organic Solvent SystemBIOTECHNOLOGY PROGRESS, Issue 4 2007Chang Duk Kang A novel tandem solvent process of dodecane and methanol was developed for the selective extraction of free astaxanthin from red encysted Haematococcus culture. The process consists of dodecane extraction for astaxanthin mixture from the culture (stage 1) and methanol extraction for free astaxanthin from the dodecane extract (stage 2). In the first stage, astaxanthin mixture was directly extracted to dodecane from the culture broth without cell harvest process, followed by a rapid separation of the dodecane extract and the culture medium containing cell debris by simple settling. In the second stage, free astaxanthin was selectively collected to methanol from the dodecane extract, accompanied with saponification of astaxanthin-esters by the addition of NaOH to methanol. During saponification, use of the optimum NaOH concentration (0.02 M) and low temperature (4 °C) reaction minimized the degradation of free astaxanthin, resulting in a total recovery yield of free astaxanthin of over 85%. The free-astaxanthin-containing methanol extract was also simply separated from dodecane by gravity settling, after which the astaxanthin-free dodecane was effectively recycled to the first stage, yielding a stable extractability of astaxanthin mixture during repeated extraction. Our results indicate the potential of the proposed tandem solvent process as an alternative extraction technology for the high-value antioxidant Haematococcus astaxanthin. [source] Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG2a Monoclonal Antibody in a Wave Bioreactor-Perfusion Culture SystemBIOTECHNOLOGY PROGRESS, Issue 1 2007Ya-Jie Tang A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 ,m was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 × 105 and 200.5 × 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 × 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L·day) in perfusion culture were much higher than those (i.e., 22.3 × 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L·day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale. [source] Fluidized Bed Design Parameters Affecting Novel Lactic Acid Downstream ProcessingBIOTECHNOLOGY PROGRESS, Issue 6 2001Ana V. Sosa Lactic acid purification was directly done from fermentation utilizing a fluidized bed column refilled with a strong anionic exchange resin. The purpose of this work was to study the influence of two important design parameters, bed-diameter (D) and bed-height (H), in the lactic acid binding and elution capacity of the matrix. By changing the settled bed height from 2.5 to 5 cm for each diameter of column analyzed it was possible to obtain an 50% increase in the binding capacity of the resin in all experiments. This fact was attributed to a higher contact time between the culture broth and the anionic resin produced by the increase of back mixing and lactic acid residence time. [source] Dependence of Apparent Viscosity on Mycelial Morphology of Streptomyces fradiae Culture in Various Nitrogen SourcesBIOTECHNOLOGY PROGRESS, Issue 4 2000Du Bok Choi To examine what causes increased viscosity in culture broth in Streptomyces fradiae culture, various natural nitrogen sources were investigated. Extracellular protease activity increased with culture time and decomposed the natural nitrogen source into amino acids. In the case of gluten meal, after a culture time of 5 d, concentrations of glutamic acid and aspartic acid had increased to 600 and 200 mg/L, respectively, which were about 3- and 2-fold as high as levels in cultures under similar conditions using Pharmamedia. For various amino acids tested, the addition of glutamic acid or aspartic acid mixture to the culture medium raised the apparent viscosity to its highest demonstrated value, 260 mPa·s after 5 d of culture, which was 3-fold higher than without amino acids. Consumption of the decomposed glutamic acid and aspartic acid was dependent on the activities of glutamate dehydrogenase and aspartate aminotransferase, respectively. When ammonium ion was used as the nitrogen source, cell concentration reached 1.75 g/L measured as an intracellular nucleic acid concentration,which was about 2.3-fold higher than that with any other natural nitrogen source. However, apparent viscosity was only 75 mPa·s, a value one-third that of the amino acid mixture, and 70% of the pellets were bigger than 1.2 × 104 ,m2. In the case of gluten meal or the amino acid mixture, pellets bigger than 1.2 × 104 ,m2 comprised only 8%. This demonstrates that consumption of some amino acids affected the formation of filamentous morphology, which caused an increase in the apparent viscosity of the culture broth, and the apparent viscosity was not caused by the mycelial concentration but the mycelial morphology. [source] Genetically engineered Pseudomonas: a factory of new bioplastics with broad applicationsENVIRONMENTAL MICROBIOLOGY, Issue 10 2001Elías R. Olivera Summary New bioplastics containing aromatic or mixtures of aliphatic and aromatic monomers have been obtained using genetically engineered strains of Pseudomonas putida. The mutation (,) or deletion (,) of some of the genes involved in the ,-oxidation pathway (fadA,, fadB,,fadA or ,fad,BA mutants) elicits a strong intracellular accumulation of unusual homo- or co-polymers that dramatically alter the morphology of these bacteria, as more than 90% of the cytoplasm is occupied by these macromolecules. The introduction of a blockade in the ,-oxidation pathway, or in other related catabolic routes, has allowed the synthesis of polymers other than those accumulated in the wild type (with regard to both monomer size and relative percentage), the accumulation of certain intermediates that are rapidly catabolized in the wild type and the accumulation in the culture broths of end catabolites that, as in the case of phenylacetic acid, phenylbutyric acid, trans -cinnamic acid or their derivatives, have important medical or pharmaceutical applications (antitumoral, analgesic, radiopotentiators, chemopreventive or antihelmintic). Furthermore, using one of these polyesters (poly 3-hydroxy-6-phenylhexanoate), we obtained polymeric microspheres that could be used as drug vehicles. [source] Purification of Aspergillus carbonarius polygalacturonase using polymeric membranesJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2008E. Nakkeeran Abstract BACKGROUND: Microfiltration (MF: 70,450 nm) and ultrafiltration (UF: 10,500 kDa) membranes were used to eliminate carbohydrates and other non-protein impurities from Aspergillus carbonarius culture broth containing polygalacturonase enzyme (EC 3.2.1.15) that would otherwise interfere with the purification processes and lead to enzyme loss. Further, diafiltration was attempted to improve the elimination of impurities as well as recovery of enzymes. RESULTS: MF resulted in removal of 2,25% carbohydrates with an enzyme recovery of 69,82% from the crude culture broth owing to the secondary layer formation. UF with 10 kDa membrane eliminated most of the carbohydrates (96%), phosphate salts and total acids with a recovery of 96% polygalacturonase and resulted in greater productivity. Using the above procedure, the enzyme was concentrated nearly 10-fold while the purity improved from 4.6 to 49.4 U mg,1 of dry matter. CONCLUSIONS: The results of this study focused on the elimination of carbohydrates and other non-protein impurities showed that UF could be used efficiently as a primary purification step during downstream processing of microbial culture broths containing enzymes. The present approach will ensure complete elimination of non-protein impurities thereby reducing the losses and difficulties in the subsequent purification steps. Copyright © 2008 Society of Chemical Industry [source] Ultra scale-down approaches for clarification of mammalian cell culture broths in disc-stack centrifugesBIOTECHNOLOGY PROGRESS, Issue 6 2009Ferhana Zaman Abstract Ultra-scale down (USD) methodology developed by University College London for cell broth clarification with industrial centrifuges was applied to two common cell lines (NS0 and GS-CHO) expressing various therapeutic monoclonal antibodies. A number of centrifuges at various scales were used with shear devices operating either by high speed rotation or flow-through narrow channels. The USD methodology was found effective in accounting for both gravitational and shear effects on clarification performance with three continuous centrifuges at pilot and manufacturing scales. Different shear responses were observed with the two different cell lines and even with the same cell line expressing different products. Separate particle size analysis of the treated broths seems consistent with the shear results. Filterability of the centrifuged solutions was also evaluated to assess the utility of the USD approach for this part of the clarification operation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Recovery of Poly(3-hydroxybutyrate) from Coagulated Ralstoniaeutropha Using a Chemical Digestion MethodBIOTECHNOLOGY PROGRESS, Issue 4 2000Hee Wook Ryu For economic recovery of poly(3-hydroxybutyrate) (PHB) from culture broths of Ralstonia eutropha containing PHB, Al-based and Fe-based coagulants were used in the pretreatment step. The coagulated cells were then separated by centrifugation, and PHB was extracted by chemical digestion with a sodium hypochlorite/chloroform dispersion solution. The practical upper limits of dosage were found to be 1,500 mg-Al/L and 1,000 mg-Fe/L, respectively, for Al- and Fe-based coagulants. When the harvested cells were treated with a 50% sodium hypochlorite/chloroform dispersion solution, PHB recovery and purity were 90,94% and 98,99%, respectively. The influence of the use of coagulants on the PHB recovery process was found to be insignificant. Despite the residual Al and Fe in the recovered PHB (less than 450 mg-Al/kg-PHB and 750 mg-Fe/kg-PHB, respectively), no detectable amounts of Al and Fe were leached from films made of the recovered PHB under acidic conditions. The use of Fe-based coagulants is less recommended because the Fe impurity can cause an unwanted colorization problem in the final product. [source] | ||||||||||||||||||||||||||||