Lecture 11 Test Questions – Flashcards
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| Why are immobilized biocatlysts used? |
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| method for reuse and stabilization of biocatalysts |
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| when should immobilization of enzymes occur in cells or as isolated enzymes? |
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| immobilization in cells: for high enzyme density in a cell or when cofactor regeneration is required otherwise immobilization of isolated enzymes |
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| what are important properties for immobilization and carriers for immobilization properties? |
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| 1. protein surface propreties, functional groups, ionic charge, and hydrophobic groups carriers for immobilization properties: porous systems, high internal surface area for adsorption or covalent binding; functionalization of carrier surface |
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| growth of natural biofilms leads to increased ____,____,___and ____ |
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| volume, thickness, channels, sloughing |
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| What is biofilm thickness a function of? |
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| age (time), nutrients and shear stress |
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| grwoth in an artifical matrix leads to increased___ but usually only ___ activity |
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| cell density pellicular activity |
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| provide 7 reasons and corresponding limitations for enzyme immobilizations? |
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| Reasons Reuse of enzyme, reducing cost -- limitations cost of carriers and immobilization continuous processing- mass transfer limitations facilitated process control- problems with cofactors and regeneration low residence time (high volumetric activity) problems with multienzyme systems optimization of product yield easy product separation and recovery (changes in properties- selectivity) stabilization by immobilization (activity loss during immobilization) |
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| What are two critical properties of traditional immobilized enzyme supports? |
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| surface area and pore size |
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| what are 8 advantages of immobilized cells? |
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| high cell concentrations cell reuse, less $$ in cell recovery and recycle little or no cell washout at high dilution rates high volumetric productivities (due to high cell concentration and flow rate) favorable microenvironmental conditions, hence higher product yields and rates improved genetic stability protection against shear damage multi step cofactor requiring biosynthetic reactions are feasible |
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| what are 6 disadvantages of immobilized cells? |
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| products need to be excreted important diffusional limitations high heterogeneity mechanical disruption of matrix due to gas evolution and cell growth complex modeling poor control of microenvironmental conditions |
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| what are two methods for immobilization on different carriers? |
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| 1. adsorption; equilibrium (T,pH, I) prevention of desorption by cross-linking 2. covalent binding; activation of the carrier reaction with functional enzyme groups |
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| What are the advantages of immobilization by adsorption (ionic or non-ionic)? |
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| simple very mild (no chemicals) |
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| What are the disadvantages of immobilization by adsorption (ionic or non-ionic)? |
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| low adhesion capacity pore size (>350 A) pore surface area (loading capacity) low adhesion strength, reversible unless cross linked low fluid velocity in fixed bed reactors no control of catalyst density over time |
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| What are two factors that affect the space-time yield of immobilized biocatalysts? |
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| catalyst density mass transfer rate particle size (smaller particle--> closer you can get to the maximal mass transfer rate) |
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| chemical cross-linking of cells with bi-functional reagents results in ___ and ____. however cross linking is effective for ____ and ___ without purification |
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| membrane damage and cell death cell permeabilization and enzyme stabilization |
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| what are 6 characteristics of cross-linked enzyme crystals (CLECs) for biocatalysis and drug delivery? |
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| 1. micro crystals not x ray quality crystals 2. retain activity in harsh environments 3. can be lyophilized and reconstituted 4. can be stored at room temperature 5. resist proteolysis 6. resist denaturation by solvents |
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| enzyme immobilization in nanomaterials are known for ___ while enzyme immobilization by entrapment is characterized by ___ and ____ |
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| high surface to volume ratio very large pores, low mechanical strength |
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| The following are illustrations (A,B, C,D,E and F) of immobilization methods for microorganisms- provide the name of each of the 6 methods |
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| as the hardening of gel beads increases using divalent cations, the enzyme/cell stability ____ |
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| decreases |
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| prepare two examples of commercial immobilized glucose isomerase (GI) that are prepared as cross-linked cells, in addition to the enzyme source, immobilization method and typical initial space velocity for each example |
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| a) ferm culture--> centrifuge--> homogenization--> treatment with glutaraldehyde--> granulation in humid condition00> drying--> waxhing and drying freeze--> melting--> water and washing--> drying crude enzyme--> column chromatography--> UF--> gel filtration--> UF --> ammonium sulfate crystallization--> recrystallization |
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| enzyme encapsulation and artificial cells can be immobilized within ___- that may contain ___ bound to nanoparticles., |
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| To avoid sterically hindered active sites multi point enzyme attachment with spacers are utilized as depicted in the following diagram. Describe how enzymes are immobilized on glyoxyl agarose |
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| enzyme will be immobilized via an area rich in lys resides |
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| describe how enzymes are immobilized on mana agarose |
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| the enzyme will be immobilized via an area rich in asp and glu residues |
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| describe how enzymes are immobilized on glutaraldehyde agarose |
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| enzyme will be immobilized by the n-terminal amine |
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| What are 8 advantages of immobilized whole cells compared to immobilized enzymes |
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| 1. multi-step and synthetic reactions possible 2. intracellular enzymes are often more stable 3. eliminates enzyme purification costs 4. cofactor regeneration possible as long as cell membrane interact and cells are viable 5. interfering enzymes can be inactivated or deleted genetically 6. can alter cell specific activity by altering gene expression in vivo protein stability 7. useful when cells are permeable to substrates and products (low molecular weight) 8. useful when processing very large volumes of liquid |
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| What are 4 facts to keep in mind regarding microbial cell immobilization |
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| 1. immobilization means restriction of cell mobility within a defined space 2. immobilization has nothing to do with cell growth 3. if the kinetics of bioconversion are described by a michaelis menten type of kinetic expression and the cells are not growing 4. models for interaction of diffusion and reaction for surface immobilized or entrapped biocatalysts apply directly to immobilized cells |
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| What are 6 disadvantages of immobilized cells? |
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| 1. lower specific activity 2. presence of interfering enzyme activities 3. reduced substrate permeability 4. little control of loss of in vivo enzyme activity during immobilization, following immobilization 5. high biomass loading required to maintain high productivity 6. mass transfer limitations can be severe depending on geometry, thickness 7. viable cells can grow in the presence of nutrients/ cell lysis, leave adsorbent, plug reactor |
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| enzyme pH profile may ____ or _____ upon immobilization of carrier bound cross linked enzymes. Enzyme active site can be _____ blocked. Pore microenvironment may differ from the ___ environment |
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| broaden or shift sterically bulk liquid |
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| What is synthesized by covalent bonds formed between enzymes or between enzyme support surface? |
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| crystal lattice CLECs |
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| Provide 5 examples of bio-printed biomolecules |
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| 1. cell adhesion molecules which promote cell attachment: poly-L lysine, laminin, fibronectin, adhesion peptides Antibodies, AB fragments, AB fusion proteins antibodies for AB cell interactions to make patters of cells enzymes for surface catalysis (glucose test strip) residual "free" surface sites often blocked w BSA prior to recognition binding step |
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| List 4 characteristics of biomolecular activity after printing? |
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| 1. mechanical stress can lead to irreversible conformational change 2. proteins more susceptible to denaturation during printing than during adsorption from solution 3. 10%-40% loss of activity following printing compared to adsorbed activity 4. loss of activity differs for different types of biomolecules |
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| protein microarrays are used for _______ and ________ |
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| screening binding assays |
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| differences in surface wettability determines ____ when printing biomolecules. Surfaces may be nano-structured with ____, ___ and ___ |
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| transfer efficiency patches, lines, posts |
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| What substrates are used for printing biomolecules? |
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| What are three examples of microcontact printing methods? |
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| 1. printing cell adhesion proteins |
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| What are 6 characteristics of affinity contact printing? |
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| * covalently immobilized biomolecules on PDMS stamp, cross- linked * adsorb receptor molecules onto bound proteins- affinity capture step * release stamp bound protein to substrate surface during printing * reusable stamp * can have multiple site of different affinity * can have high resolution |
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| what are 8 characteristics of microcontact printing? |
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| protein inks applied by adsorption to a stamp surface poly dimethylsiloxane (PDMS) or PS stamps formed on mold * surface modified PDMS hydrophobic, promotes deposition of proteins from solution in monolayer (rinsed) * hydrophilic stamp surfaces also possible * can be high resolution * protein size not a factor * stacking of proteins in multiple layers possible * subtractive printing possible |
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| describe or illustrating the difference between bio-printing methods such as contact processing, microcontact printing, and affinity contact printing |
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| what are 8 limitations of existing immobilized biocatalyst technologies |
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| what are 10 characteristics of deposition of biocatalysts onto surfaces to produce biocatalytic plastics, biocatalytic coatings, and microbial inks? |
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| biocatalytic plastics are coatings of ___ embedded enzymes which are active and stable materials for ____ |
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| Rank the productivity of cumulative hydrogen by viable cell containing fiber based materials for bioprocess intensification, such as gentle blending of cells, moderate blending of cells or vigorous blending of cells- highest to lowerst |
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| how do viable cells colonize the pores of ceramic particles? |
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| cells adsorbed into particle pores followed by a growth phase to fill in the pores with biomass |
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| silica or ceramic particles can be ___ with ____ surface areas and ___ pore size |
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| uniform with high surface area but with small pore size |
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| what 6 characteristics of hydrogel cell immobilization methods for synthesizing gel, slabs, fibers, and beads? |
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| 1. natural polyelectrolyte hydrogens use thermo-gelation 2. stabilized by divalent cations 3. highly porous, high diffusion coefficients 4. matrix soluble (chelator), biodegradable 5. relatively weak can be strengthed by glutaraldehyde cross linking 6. low or no solubility synthetic gels using polymerization by cross-linking |
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| alginate gels are formed by ____ cross linking of ___ |
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| divalent L-gluronic acid |
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| what is a type of divalent cation that regulates gel strength? |
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| pB 2+, Ba+2 |
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| gel bead density can be altered by ____ while microbial growth in gel beads can alter ____ and ____. beads can also be surface cross linked to increase ____ |
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| inorganic particles cell distribution and reactivity mechanical strength |
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| the radial distribution of microcolonies can be _____. microcolonies eventually grow to the ____and ____ |
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| quantified gel slab or slab surface and erupt |
