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Biochemical Engineering Journal (v.47, #1-3)
Modelling and simulation of a transketolase mediated reaction: Sensitivity analysis of kinetic parameters by N.A. Sayar; B.H. Chen; G.J. Lye; J.M. Woodley (pp. 1-9).
In this paper we have used a proposed mathematical model, describing the carbon–carbon bond formation reaction between β-hydroxypyruvate and glycolaldehyde to synthesisel-erythrulose, catalysed by the enzyme transketolase, for the analysis of the sensitivity of the process to its kinetic parameters. The model was validated with experimental data. As a conclusion, kinetic parameters with a possible positive impact on reaction performance were identified and assessed in relation to operating conditions. This resulted in the identification of suitable catalyst and process development targets.
Keywords: Mathematical modelling; Biocatalysis; Kinetics; Sensitivity analysis; Enzyme; Transketolase
Process modelling and simulation of a transketolase mediated reaction: Analysis of alternative modes of operation by N.A. Sayar; B.H. Chen; G.J. Lye; J.M. Woodley (pp. 10-18).
A previously proposed model of a transketolase catalysed carbon–carbon bond formation reaction condensing β-hydroxypyruvate and glycolaldehyde to synthesisel-erythrulose has been extended to describe various modes of operation as an alternative to a batch process. The alternative continuous and fed-batch operations, with each substrate being fed separately and together have been analysed using the extended model. The analysis was carried out simulating the product concentration after a given time under defined process conditions. Comparison of product concentration and yield on catalyst as two process metrics were used to identify promising cases for further process development.
Keywords: Biocatalysis; Feeding strategy; Mathematical modelling; Simulation; Transketolase; Process analysis
Hepatocyte growth factor and epidermal growth factor promote spheroid formation in polyurethane foam/hepatocyte culture and improve expression and maintenance of albumin production by Hiroyuki Ijima; Hiroshi Mizumoto; Kohji Nakazawa; Toshihisa Kajiwara; Taku Matsushita; Kazumori Funatsu (pp. 19-26).
In our previous pre-clinical study with pig hepatic failure, an artificial liver with polyurethane foam (PUF)/primary porcine hepatocyte spheroids had superior curative effect. We examined the effect of hepatocyte growth factor (HGF), also known as scatter factor, on the quick formation of hepatocyte spheroids and albumin production. Spheroids were formed in the pores of PUF within 3 days regardless of addition of growth factors. In particular, spheroids were formed within 1 day in medium containing 100ng/ml HGF and 50ng/ml epidermal growth factor (EGF). 10,000ng/ml HGF was effective for albumin production, but the activity dramatically decreased after 6 days in EGF-free medium. On the other hand, 100ng/ml HGF was effective for albumin production in EGF-containing medium. Albumin production rate with ≥1000ng/ml HGF was about 1.5 times higher than that with 100ng/ml HGF. Furthermore, albumin production rate at 3 weeks was about 1.5 times higher than that at 2 days with 1000ng/ml HGF. The maintenance of albumin production rate depended on the activity of the individual cell and not cell growth. In other words, we were able to show the effectiveness of HGF for functional hepatocyte organoid formation in PUF pores.
Keywords: Tissue engineering; Culture medium; Bioartificial organs; Animal cell culture engineering; Cell engineering; Hepatocyte
Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids by Hidetaka Noritomi; Katsuyuki Suzuki; Manabu Kikuta; Satoru Kato (pp. 27-30).
The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25°C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60°C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF6] at 80°C.
Keywords: α-Chymotrypsin; Enzyme catalyzed; Ionic liquid; Peptide synthesis
Granular activated carbon single-chamber microbial fuel cells (GAC-SCMFCs): A design suitable for large-scale wastewater treatment processes by Daqian Jiang; Baikun Li (pp. 31-37).
As an emerging biotechnology capable of removing contaminants and producing electricity, microbial fuel cells (MFCs) hold a promising future in wastewater treatment. However, several main problems, including the high internal resistance ( Rin), low power output, expensive material, and complicated configuration have severely hindered the large-scale application of MFCs. The study targeted these challenges by developing a novel MFC system, granular activated carbon single-chamber MFC, termed as GAC-SCMFC. The batch tests showed that GAC was a good substitute for carbon cloth and GAC-SCMFCs generated high and stable power outputs compared with the traditional two-chamber MFCs (2CMFCs). Critical operational parameters (i.e. wastewater substrate concentrations, GAC amount, electrode distance) affecting the performance of GAC-SCMFCs were examined at different levels. The results showed that the Rin gradually decreased from 60Ω to 45Ω and the power output increased from 0.2W/m3 to 1.2W/m3 when the substrate concentrations increased from 100mg/L to 850mg/L. However, at high concentrations of 1000–1500mg/L, the power output leveled off. The Rin of MFCs decreased 50% when the electrode distance was reduced from 7.5cm to 1cm. The highest power was achieved at the electrode distance of 2cm. The power generation increased with more GAC being added in MFCs due to the higher amount of biomass attached. Finally, the multi-anode GAC-SCMFCs were developed to effectively collect the electrons generated in the GAC bed. The results showed that the current was split among the multiple anodes, and the cathode was the limiting factor in the power production of GAC-SCMFCs.
Keywords: Microbial fuel cells; Granular activated carbon; Wastewater treatment; Chemical oxygen demand (COD); Electrode distance; Multi-anode
Forward engineering of synthetic bio-logical AND gates by Kavita Iyer Ramalingam; Jonathan R. Tomshine; Jennifer A. Maynard; Yiannis N. Kaznessis (pp. 38-47).
The field of synthetic biology has produced genetic circuits capable of emulating functional paradigms seen in digital electronic circuits. Examples are bistable switches, oscillators, and logic gates. The present work combines detailed mechanistic-kinetic models and stochastic simulation techniques as well as the techniques of in vivo molecular biology to study the potential of a synthetic, single promoter AND gate. This device is composed of elements of the tet, lac, and λ-phage promoters and is responsive to the commonly used inducers IPTG and aTc, producing GFP as an output signal. The quantitative behavior of the AND gate phenotype is studied both in numero and in vivo as a function of promoter topology. The model is constructed from kinetic data obtained from the literature and yields clearly defined ON/OFF logical behavior at realistic inducer concentrations. These behaviors are matched with observed in vivo data obtained through fluorescence-activated cell sorting. The effect of incomplete repression by weaker LacI repressor is also investigated and quantified. The simulation results, coupled with in vivo data, not only identify important design degrees of freedom, but also provide parameters that can be used to guide future synthetic designs using these common regulatory elements.
Keywords: Logical AND gate; Multiscale models; Biocomputing; Stochastic simulations; Synthetic biology; Gene regulatory networks; Computer-aided design
Enhanced in situ dynamic method for measuring K L a in fermentation media by Nilesh Patel; Jules Thibault (pp. 48-54).
The overall oxygen mass transfer coefficient ( K L a) is often used as scale-up factor of fermentation systems. In fermenter scale-up, it is desired to achieve the same K L a values at the larger scale than the one that was obtained at a smaller scale during the development stage. It is therefore important to be able to measure K L a in situ during fermentation and to also determine the action to be taken to maintain its value at its design set point. These objectives can be obtained by measuring K L a using the dynamic method and enhancing the K L a information by immediately conducting a series of changes in agitation speed and/or aeration rate to determine the influence of these variables on K L a. This enhanced dynamic method is demonstrated with two filamentous microorganisms: Trichoderma reesei for the production of cellulase and Aspergillus niger for the production of citric acid. Two different types of bioreactor were used: a reciprocating plate bioreactor and a stirred (Rushton) bioreactor. It is shown that the proposed method can provide a simple way to measure the local variation of K L a and to adjust its value to its set point during the course of fermentation.
Keywords: Dissolved oxygen; Dynamic method; Filamentous fungi; Oxygen transfer; Scale-up; Submerged culture
Ovomucoid partitioning in aqueous two-phase systems by Fabíola Cristina de Oliveira; Jane Sélia dos Reis Coimbra; Luis Henrique Mendes da Silva; Edwin Elard Garcia Rojas; Maria do Carmo Hespanhol da Silva (pp. 55-60).
This study evaluated the partitioning of ovomucoid from egg white, in aqueous two-phase systems (ATPS) composed of PEG 1500 and inorganic salt (lithium sulfate, sodium sulfate, magnesium sulfate, sodium carbonate or sodium citrate) at 25°C. The results showed a great effect of the electrolyte nature on the partition coefficient. The partition coefficient value ranges from 0.02 to 6.0. The highest partition coefficients were obtained from systems composed of sodium carbonate and the lowest in systems composed of magnesium sulfate. In the system containing magnesium sulfate, a recovery percentage greater than 90% was obtained.
Keywords: Aqueous two-phase; Extraction; Bioseparation; Partitioning; Egg white; Ovomucoid
Decolorization of practical textile industry effluents by white rot fungus Coriolus versicolor IBL-04 by Muhammad Asgher; Naseema Azim; Haq Nawaz Bhatti (pp. 61-65).
Textile industry discharges a vast amount of unused synthetic dyes in effluents. The discharge of these effluents into rivers and lakes leads to a reduction in sunlight penetration in natural water bodies, which, in turn, decreases both photosynthetic activity and dissolved oxygen concentration rendering it toxic to living beings. This paper describes the decolorization potential of a local white rot fungus, Coriolus versicolor IBL-04 for practical industrial effluents collected from five different textile industries of Faisalabad, Pakistan. Screening of C. versicolor IBL-04 on five effluents showed best decolorization results (36.3%) for Arzoo Textile Industry (ART) effluent in 6 days followed by Crescent Textile Industry (CRT), Itmad Textile Industry (ITT), Megna Textile Industry (MGT) and Ayesha Textile Industry (AST) effluents. Optimization of different process parameters for ART effluent decolorization by C. versicolor IBL-04 showed that manganese peroxidase (MnP) (486U/mL) was the lignolytic enzyme present in the culture filtrates with undetectable lignin peroxidase (LiP) and laccase. The MnP synthesis and effluent decolorization could be enhanced to 725U/mL and 84.4%, respectively, with a significant time reduction to 3 days by optimizing pH and temperature and using 1% starch as a supplementary carbon source.
Keywords: Textile industry effluents; Bioremediation; White rot fungi; Screening; Process optimization
Fermentative lactic acid production with a metabolically engineered yeast immobilized in photo-crosslinkable resins by Min-Tian Gao; Takashi Shimamura; Nobuhiro Ishida; Haruo Takahashi (pp. 66-70).
In this study, the immobilization technique involving photo-crosslinkable resin gels was used for lactic acid production. Saccharomyces cerevisiae OC-2T T165R , a metabolically engineered yeast that produces optically purel(+)-lactic acid, was immobilized in hydrophilic photo-crosslinked resin gels as a biocatalyst. Three resin gels, TEP 1, TEP 2 and TEP 3, were examined and all of them showed high performance as to lactic acid production. Resin gel TEP 1, which exhibited the highest productivity among the resin gels was used for 15 consecutive batch fermentations without decreases in productivity and mechanical deformation, indicating that it was a suitable carrier for long-term lactic acid fermentation. Moreover, the use of the immobilization technique can improve the productivity of the metabolically engineered yeast in the fermentation with or without extraction, showing promise for using the immobilized engineered yeast for lactic acid production.
Keywords: Immobilization; Lactic acid fermentation; Photo-crosslinkable resin gel; Yeast
Alkaline proteases and thermostable α-amylase co-produced by Bacillus licheniformis NH1: Characterization and potential application as detergent additive by Noomen Hmidet; Nedra El-Hadj Ali; Anissa Haddar; Safia Kanoun; Sellami-Kamoun Alya; Moncef Nasri (pp. 71-79).
Alkalophilic Bacillus licheniformis NH1 strain produced at least five major extracellular proteases and a unique amylase as showed by zymography technique. The optimum pH and temperature for the proteolytic activity were 10.0 and 70°C, respectively, while those of amylolytic activity were 6.5 and 90°C, respectively. The alkaline proteases and thermostable α-amylase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1h at 40°C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various solid and liquid detergents. Wash performance analysis revealed that the NH1 crude enzyme could effectively remove a variety of stains, such as blood, chocolate and barbecue sauce. Considering its promising properties, B. licheniformis NH1 crude enzyme containing both α-amylase and proteases activities may be considered a potential candidate for future use in detergent processing industries.
Keywords: Bacillus licheniformis; NH1; Alkaline proteases; α-Amylase; Crude enzyme; Detergent
Modification of cellulase and its application to extraction of diosgenin from Dioscorea zingiberensis C.H. Wright by Yuqing Zhang; Linru Tang; Xuan An; Erhong Fu; Chaofan Ma (pp. 80-86).
In order to enhance the thermostability and efficiency of cellulase in the extraction of diosgenin from Dioscorea zingiberensis C.H. Wright, we applied polyethylene glycol (PEG) (400, 1000, 2000, and 4000) to modify cellulase. The modified cellulase, α-amylase and β-glycosidase were used to hydrolyze the material. The results show that the thermostability of modified cellulase is better than that of natural cellulase, the optimum pH value and temperature of modified cellulase are wider than that of natural cellulase, the activity of cellulase modified by activated PEG2000 is higher than that of cellulase modified by other modifiers, and its remaining activity is 58% of its initial value. With this technique, the purity of the product reaches 96%, the melting point is 201–204°C, the yield rate and the extraction rate of the diosgenin reaches 2.80% and 96.6%, respectively. IR spectra and1H NMR spectroscopy were used to confirm the structure of the product.
Keywords: Abbreviations; CC; cyanuric chloride; CC-PEG; the activated cyanuric chloride PEG; CMC; carboxymethylcellulose; FT-IR; Fourier transform infrared spectroscopy; HPLC; high-pressure liquid chromatography; MPEG; mono-methoxy polyethylene glycol; MWCO; molecular weight cut off; NMR; nuclear magnetic resonance; PEG; polyethylene glycol; TNBS; 2,4,6-trinitrobenzenesulfonic acidModification; Biocatalysis; Extraction; Bioseparation; Enzyme; Polyethylene glycol
Culture medium improvement for Isaria fumosorosea submerged conidia production by Ali Asaff; Francisco Escobar; Mayra de la Torre (pp. 87-92).
Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30mg/l of FeSO4·7H2O and 6–12mg/l of CuSO4·5H2O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2)×1012cell/l was achieved after 120h of liquid culture in a improved culture medium, containing 25ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).
Keywords: Optimization of culture medium; Submerged fermentation; Shake-flask culture; Biomass production; Submerged conidia; Isaria fumosorosea
Effect of various additives on enzymatic hydrolysis of castor oil by Virendra K. Rathod; Anirudha B. Pandit (pp. 93-99).
Hydrolysis of castor oil using lipase enzyme is carried out in a batch reactor at room temperature (35–40°C). In order to reduce the cost of enzyme catalyzed reaction, water in oil emulsion and a 3:1 ratio of oil to water is selected. The concentration of enzyme in the reaction mixture is optimized. The effect of various additives like solvent and salt which can enhance the rate of reaction is studied. It is found that the glycerol has no effect on the hydrolysis of oil. The reusability of the lipase enzyme has also been tested. The yield of enzymatic hydrolysis of castor oil is compared with those of coconut oil and olive oil.
Keywords: Enzyme; Castor oil hydrolysis; Stirred reactor; Optimization; Solvent; Glycerol
Genetic algorithm-based medium optimization for enhanced production of fluorescent pseudomonad R81 and siderophore by M.V.R.K. Sarma; Vikram Sahai; V.S. Bisaria (pp. 100-108).
Fluorescent pseudomonad R81, a root-colonizing bacterium, is a potential bio-inoculant due to its plant growth promoting characteristics. It produces hydroxamate-type siderophore which is involved in disease suppression in plants. Genetic algorithm (GA) methodology was applied for the optimization of siderophore and cell mass production simultaneously in shake flask experiments. A total of 10 medium components were optimized within 80 experiments. A high siderophore concentration of 1.9g/L and cell mass concentration of 2.8g/L was achieved in the optimized medium. The application of GA was well suited for determination of optimum concentration levels of the medium constituents for a bi-objective function. GA was able to increase the siderophore concentration by 2.8-fold when compared to RSM-based optimization. Further, the batch fermentation of the GA-optimized medium in 14L bioreactor without pH control produced 2.2g/L siderophore in 36h, the highest reported so far. GA was also successfully used to estimate the kinetic parameters of the mathematical models of the batch fermentation.
Keywords: Bioreactor; Genetic algorithm; Kinetics; Optimization; Pseudomonad; Shake flask
Linoleic and α-linolenic fatty acids affect biomass and secondary metabolite production and nutritive properties of Panax ginseng adventitious roots cultured in bioreactors by Chun Hua Wu; Elena V. Popova; Eun Joo Hahn; Kee Yoeup Paek (pp. 109-115).
The effect of elicitation with linoleic (C18:2) and α-linolenic (C18:3) fatty acids (LLA and α-LNA) was investigated in Panax ginseng C.A. Meyer adventitious roots cultured in 5l balloon-type bioreactors. Fatty acids were added in culture medium at 0.0, 1.0, 2.5, 5.0, 10.0, and 20.0μmoll−1 at day 40, at the end of exponential growth phase. Roots were harvested and assayed at day 47. Elicitation with both LLA and α-LNA enhanced accumulation of total polyphenolics and flavonoids in roots compared with control without elicitation. The highest accumulation of flavonoids was observed at 5.0μmoll−1 for both elicitors. Total phenolics production reached its highest value of about 4.0mgg−1DW under the elicitation with 5.0μmoll−1 LLA and 5.0–20.0μmoll−1 α-LNA. Meanwhile, α-LNA was more effective than LLA for increasing biomass and ginsenoside production. The biomass of 11.1gDWl−1 and maximal total ginsenoside content of 7.9mgg−1DW were achieved at 5μmoll−1 α-linolenic acid. The essential polyunsaturated linoleic (C18:2) and α-linolenic (C18:3) fatty acids were accumulated in roots in response to elicitation while content of palmitic (C16:0) and oleic (C18:1) acids declined. The activities of SOD, G-POD and CAT were enhanced by two elicitors to similar extent while APX activity was preferably stimulated by α-LNA. Our results demonstrate that elicitation with α-linolenic acid stimulates production of biomass and secondary metabolites in bioreactor-cultured P. ginseng adventitious roots.
Keywords: Adventitious roots; Bioreactors; Biosynthesis; Elicitation; Optimization; Plant cell culture
Rheological characterization of culture broth containing the exopolysaccharide PS-EDIV from Sphingomonas pituitosa by Ellen Schultheis; Michael A. Dreger; José M. Muñoz-Villegas; José I. Escalante; Ezequiel Franco-Lara; Bernd Nörtemann (pp. 116-121).
Sphingomonas pituitosa excretes the capsular exopolysaccharide PS-EDIV into the culture broth augmenting considerably its fluid viscosity. Since this change particularly affects key processes like mixing and transport during the microbial production, this work was aimed at the rheological characterization of the polymer-containing culture broth of S. pituitosa. The study included investigations on basic properties of the culture broth, but also on the dependence of the biomass–polymer-solution properties on different physicochemical post-cultivation treatment steps like variations of temperature, pH-value or concentration of salts. The essential result is the characterization of the viscoelastic behavior of the culture broth, which was more gel-like than sol-like and exhibited slight elastic properties. This rheological behavior showed that the PS-EDIV culture broth formed non-Newtonian fluids, indicating that it is a pseudoplastic biopolymer, with yield stress appearance and exhibits thixotropic properties. Rheograms were fitted to the Herschel–Bulkley model. The amplitude sweep revealed a deformation of 21% as the limiting value of the linear viscoelastic interval. Furthermore, the PS-EDIV culture broth showed a high viscosity which was strongly influenced by salt type and concentration but weakly influenced by temperature and pH-value within the investigated experimental boundaries.
Keywords: Sphingan; Biorheology; Thixotropic behavior; Viscoelasticity
Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems by Daniela de Araújo Viana Marques; Beatriz Rivas Torres; Ana Lúcia Figueiredo Porto; Adalberto Pessoa-Júnior; Attilio Converti (pp. 122-126).
Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses; therefore, the knowledge of the volumetric mass transfer coefficient ( kL a) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the kL a values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium, in the absence of biomass. Aeration and agitation were selected as the independent variables using a 22 full factorial design. Both variables showed statistically significant effects on kL a, and the highest values of this parameter in both media for simple fermentation (241s−1) and extractive fermentation with ATPS (70.3s−1) were observed at the highest levels of aeration (5vvm) and agitation (1200rpm). The kL a values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number ( N3 D2) and superficial gas velocity ( Vs) determined in distilled water ( α=0.39 and β=0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium ( α=0.38 and β=0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation ( α=0.50 and β=1.0). A reasonable agreement was found between the experimental data of kL a for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions.
Keywords: Fermentation medium; Extractive fermentation medium; k; L; a; Oxygen transfer coefficient; Agitation; Aeration
Adsorptive bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003 by Guodong Wei; Xuepeng Yang; Wenyu Zhou; Jinping Lin; Dongzhi Wei (pp. 127-131).
An integrated bioprocess for the production of glycolic acid from ethylene glycol with Gluconobacter oxydans DSM 2003 and in situ product removal were investigated. A slight substrate inhibition was observed as substrate concentration was above 20g/l and the product inhibition was much stronger. Bioconversion of glycolic acid is an end-product-inhibited reaction. In order to increase the productivity of glycolic acid and reduce the end-product inhibition of bioconversion, an adsorptive bioconversion for glycolic acid production from ethylene glycol catalyzed by resting cells of G. oxydans DSM 2003, was developed by using anion exchange resin D315 as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2g/l, compared to 74.5g/l obtained from a conventional fed-batch mode.
Keywords: Gluconobacter oxydans; Glycolic acid; Ethylene glycol; Product inhibition; Integrated bioprocess; In situ product removal
A novel proteinaceous photolinker for simultaneous binding to an inert surface and a biomolecule by Saroj Kumar; Dileep Kumar Kannoujia; Azmi Naqvi; Pradip Nahar (pp. 132-135).
A simple and versatile method is developed for covalently binding a protein ligand onto a matrix irrespective of functional groups either on the ligand or the matrix. Prerequisite of the method is a novel proteinaceous photolinker having multiple light-activable functional groups. We have made photoreactive-BSA – a proteinaceous photolinker by the reaction of bovine serum albumin (BSA) with excess of 1-fluoro-2-nitro-4-azidobenzene (FNAB). When an enzyme is placed on an inert polystyrene matrix in presence of photoreactive-BSA and exposed to light the later forms highly reactive nitrenes some of which bind to the matrix and the rest to the ligand resulting simultaneous formation of covalent bonds with the matrix and the enzyme. The method is further exemplified by performing ELISA by covalent binding of antigen or antibody on a polystyrene microtiter plate in just 30min using photoreactive-BSA. ELISA carried out in less than 3h using photoreactive-BSA showed comparable results with that of conventional ELISA carried out in 18h. Thus the method is potentially useful for rapid ELISA or covalent immobilization of ligands onto an inert surface without prior activation.
Keywords: Abbreviations; FNAB; 1-Fluoro-2-nitro-4-azidobenzene; HRP; Horseradish peroxidase; BSA; Bovine serum albumin; HELISA; Heat-mediated enzyme-linked immunosorbent assayPhotoimmobilization; Enzyme; 1-Fluoro-2-nitro-4-azidobenzene; Photolinker; Covalent immobilization; Enzyme-linked immunosorbent assay