CHEM-E3140 Bioprocess technology II
Lecture 4
6.11.2015
Immobilized enzymes
Tero Eerikäinen
Different physical forms of enzymes
• Enzymes are sold in liquid, dried solids (e.g. freeze-dried, spray-dried,
fluidized-bed-dried, granulated), crystallized or immobilized form
• As foreign proteins for human beings dried, aerosol forming fine solids
may be harmful => allergy
• Dried solids are stable if kept dry; often some activity is lost during
drying; drying is costly; less storage costs than with liquid products
• Liquid products need stabilizing (biochemically and microbiologically)
agents (e.g. sorbitol or sugars and parabens, respectively)
• Enzyme crystallization lack theoretical basis => experimental technique
• Immobilized enzymes: less energy costs than drying, stable form, can be
used as such in reactors of any scale, some activity may be lost in
immobilization (sometimes activity is increased), process stability
increases
Think about which kind of phenomena could cause activity losses when an
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enzyme is immobilized.
Enzyme recycling
• Some bulk enzymes are so cheap or their share in product price so small
even in single use, that recycling is uneconomical (recycling cost vs.
enzyme cost) => batch process with homogeneous catalysis (e.g.
amylolytic enzymes acting on starch)
• In most cases enzyme immobilization (= it is turned into a solid, nonsoluble form) is the cheapest way to recycle; its costs come from:
• The carrier (solid) material
• The process of immobilization
• Possible loss of activity either true loss (inactivation) or apparent loss
because of mass-transfer limitations induced by immobilization
• Investment and running costs immo vs. free enzyme
• Other recycling options:
• Enzyme use in membrane reactors (soluble enzymes)
• Recovery of soluble enzymes from the reaction mixture by
membrane filtration (ultrafiltration, UF) or adsorption/desorption
• Costs: membrane filtration unit running costs (including membrane
renewals, energy costs, chemicals), enzyme inactivation; adsorbent
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costs, S/L unit investment and running costs, inactivation
Enzyme recycling: a special case
• Enzymatic hydrolysis of lignocellulosic (LC) materials:
• CBH I and CBH II have cellulose binding domain (CBD)
• These enzymes can be adsorbed on a new batch of LC after a total
hydrolysis of a previous batch
• Some hemicellulases are adsorbed as well
• β-glucosidase will not be adsorbed
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Protein surface can also be modified by mutations to direct the immobilization.
- Covalent attachment to carrier via Lys, Asp, Glu
- Attachment of protective groups (spacers) on the protein surface (increase stability)8
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Linking by glutaraldehyde
glutaraldehyde: OHC-(CH2)3-CHO
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From : Chem. Soc. Rev., 2013, 42, 6437
Industrial immobilized enzymes
• The cost of most industrial enzymes often a minor
component in overall process economics
• The additional costs can be substantial and hinder
the usage of immobilization, but
• Benefit realized from process advantages
• enabling continuous production
• improved stability
• the absence of the biocatalyst in the product stream.
• Applications for immobilized enzymes
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high-fructose corn syrup production
pectin hydrolysis
debittering of fruit juices
interesterification of food fats and oils
biodiesel production
carbon dioxide capture
• Many of the applications were developed rather
long time ago
Immobilization advantages and disadvantages
• Specialized
type of enzyme
formulation
• Is there a
differentiating
advantage?
+ Heterogeneous catalyst can be recovered and reused, retain activity for
long periods, suitable for a wide variety of processes, highly-selective
- Effectiveness of immobilized enzymes is often considerably less than that
of their soluble equivalents:
• due to diffusional constraints for macromolecules (ability of substrates to access
enzyme active sites and for products to exit to bulk solution)
Immobilized enzymes market share
• Biocatalysts only 3%
($160 million) from all
enzymes
• Still in 1990 20% of
industrial enzymes
were immobilized
• Benefit for customer
but not for enzymes
producer
• Benefit if part of the
sold process or product
development
Large scale industrial immobilized enzymes
• Customer can purchase a soluble form of the enzyme and prepare
an immobilized form of the enzyme themselves, or
• Outsource task to companies that specialize in immobilization, or
• Established companies can produce and immobilize their own
enzymes in parallel with process development
Enzyme cost contribution
• Incorrect assumption: industrial enzymes are very expensive and it is the reason
for immobilizations
• Most industrial enzymes $50 to $500 per kg enzyme protein
• E.g. cost of enzymes for starch-derived ethanol is around 1 cent per liter 
immobilization is not an economic solution
• Cost contribution of immobilized enzyme:
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number of times the enzyme is reused corresponds total productivity
few hundred $ per kg for specialty chemicals
few cents per kg for bulk chemicals
often in the range of $0.1 to $10 per kg product
• The utilization of immobilized enzymes requires a good understanding of
• the technical factors
• the economic factors
• the larger market forces
Productivities of enzymes and enzyme costs
• A penicillin G amidase (produces 6-APA* for derivatization): 1 kg of
immobilized enzyme produces in its operational life-time 1-2 tons of
6-APA
• A glucose isomerase (GI) produces 20 tons of HFCS** per kg
immobilized enzyme in its operational life-time
productivity (t HFCS/kg enz.)
HFCS cost ($/t)
1974
0.25-0.3
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1998
15-20
0.15
*: 6-aminopenicillanic acid
**: high fructose corn syrup (world production appr. 10 million tons/a)
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Glucose isomerase
• Glucose isomerase:
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= GI = xylose isomerase = D-xylose (ketol) isomerase = EC 5.3.1.5
one of the most important industrial enzymes
broad substrate specificity
efficiently converts D-glucose to D-fructose
HFCS sweetener for beverages and foodstuffs
Glucose isomerase
• Typically produced in aerobic fermentations
• batch times of 2–3 days.
• expressed intracellularly or associated with cellular mycelia
• Development targets:
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improved thermostability,
reduced dependence on metal ions for activity,
lower pH optima,
greater resistance to Ca2+ ions and other inhibitors
• Properties
• Km for D-glucose from 0.086 to 0.920 M (for D-xylose normally lower)
• Slightly endothermic isomerization ∆H about 5 kJ/mol
• Equilibrium constant (roughly) Keq = 1 (at 298 K)
Glucose isomerase
• Isomerization equilibrium is a function of temperature
• Higher temperatures favor fructose formation
• Commercial production at 60 °C, conversion around 50% fructose,
• At 90 °C the equilibrium mixture contains 56% fructose.
Glucose isomerase
• Almost all glucose isomerases exist as homotetramers.
• The Streptomyces rubinginosus enzyme (structure determined 1984)
is composed of four 43 kDa monomers
• Divalent metal ions (preferably Mg2+) needed for maximal catalytic
activity and stability.
• Two metal ions are bound near the active site, additional metal binding sites
provides conformational stability and contribute to maintenance of the
overall quaternary tetrameric structure of the active complex
• Inhibitors:
• Metal ions Ca2+, Zn2+, Ni2+ and Hg2+.
• Sugar alcohols xylitol, sorbitol, arabitol and mannitol
• Activity: glucose isomerase unit (GIU):
• the amount of enzyme required to isomerase 1 µmol of glucose to fructose
per minute under defined assay conditions
Immobilized glucose isomerase
• High Km for D-glucose (>0.1 M)  high enzyme concentrations
needed to push the reaction to near equilibrium within a practical
timeframe
• Many methods for the immobilization of GI have been developed and
commercialized over the years, beginning in 1967
• Immobilization of GI was considered a mature technology by the late
1970’s. Examples of the products here:
Whole cell preparations with cross-linking
• Takasaki’s method: GI-expressing cells (e.g. Streptomyces sp.) held at
elevated temperatures (60 to 80 °C) for short periods
• crosslinked matrix composed of denatured cellular protein and other
components is formed
• The advantage of the treatment:
• fixed the GI in a form that could be used in plug flow reactors,
• denaturing cellular proteases that might otherwise degrade the GI enzyme.
• Clinton Corn Processing Company, 1967, the process converted cornderived glucose syrup into a mixture containing 42% fructose.
• Chemical fixation techniques in the same era, cross-linking agents:
• inorganic salt solutions such as CoCl2, FeCl3, CaCl2
• organic acids including citrate
• glutaraldehyde (50 wt% solution) was determined to be a very effective fixing
reagent, first described in 1973
Process for large scale
cross-linking
immobilization of glucose
isomerase
• Crosslinked with glutaraldehyde
• Polyethylenimine and inorganic
carriers such as bentonite clay
and diatomaceous earth
• Dewatering
• Extrusion/marumerization
• Fluidized bed drying
• Product half-life of over 1 year
when used in a packed bed
reactor at 60 °C
Adsorption-based
methods
• Ionic adsorption of isolated GI to organic
and inorganic resins
• Adsorption of the GI to DEAE Sephadex
was first described by Tsmura and
coworkers in 1967
• Further improved method: GI was bound
to DEAE-cellulose either in the presence
or absence of associated cellular material
• Adsorption to polymeric anion-exchange
resins such as Amberlite IRA-938
• Granular DEAE-cellulose-polystyrene–
TiO2 resin to allow operation in deep-bed
reactors
• A key innovation was the ability to
regenerate the resin by addition of
soluble GI to compensate for the loss of
enzyme activity over time.
• customers leasing the support and
purchasing soluble GI as needed to
maintain the desired level of enzyme
activity.
Comparison of two common carrier materials
• Alginate beads:
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Prepared by squeezing enzyme-alginate solution into Ca2+ -solution
Enzymes entrapped inside the beads
Soft, gel-like structure
Used in academic research
Compressible; leakage ?
• DEAE-GDC: DEAE –groups containing granulated derivatized cellulose
• Chemical/mechanical preparation
• Enzymes immobilized by adsorption and ionic forces (DEAE:
charge? In which unit operation is it used as attached to resins?)
• Hard, non-compressible material, microporous (Enzyme is in which
parts of the particles?)
Think about a column of 5 meters high and 1 m wide: which one is more
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suitable carrier material?
Immobilized GI process
• Largest commercial process involving an immobilized enzyme, yearly
amounts:
• IGI enzyme sold over 500 tons
• production of 10 million tons of HFCS
• Development of IGI made continuous processing possible and
improved economics
• Different reactor formats have been tested
• Today HFCS production reactors:
• Fixed bed reactors
• Arranged in parallel
• Continuously operated
Continuous vs. batch enzymatic processes
• Immobilization of an enzyme makes a continuous process possible and
so does also the use of enzymatic membrane reactors
• Batch vs. continuous:
• Batch: much more unproductive time, more labor intensive, larger
reactors, batch to batch variations in the process and product quality,
enzyme costs dictated mainly by the enzyme industry; on the other
hand it’s simple and basically homogenous type of catalysis
• Continuous immo: heterogeneous catalysis, possible mass-transfer
limitations, stability (process, biochemical, microbiological) problems
possible, error-prone (errors may destroy a big batch of an enzyme);
on the other hand its productivity (spatial and temporal) is usually
much higher and down-time dramatically shorter, in steady-state
reproducible (process & product) with constant quality
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Practical process arrangements
• Immobilized enzymes are used in a process in a tubular reactor (column)
and the substrate(s) are pumped through the reactor and during
residence time desired proportion of substrate(s) turned into product(s)
• For cheap, bulk products usually several reactors are in series and they
are at different stages of their life => constant production as the columns
are recharged periodically
• When the activity of the enzyme loading decreases it is also possible to
decrease accordingly the feed rate to keep product concentration and
yield and substrate conversion constant
• Enzymatic membrane reactors (EMR)
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Practical application of enzymes/continuous glucose
isomerization
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To achieve the equilibrium long reaction time and/or high enzyme dose is
needed (see separate figure) => in practice 42 % fru is enough
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A typical case in industry:
Enzyme dose per column: 1800 kg (e.g. 300 U/g enzyme product)
Product yield: 12 000 kg 42 % HFCS (DS) / kg enzyme
Practical life of an enzyme loading: 687 d (world record)
The feed of glucose syrup is lowered as the enzyme is loosing its
activity – enzyme loading changed when feed is 10 % of the initial
Production 1000 t HFCS (DS) / d in 20 columns
[HFCS = high-fructose corn syrup; DS: as dry solids]
How to increase fructose concentration beyond 42 %?
Would an increase of the enzyme dose increase the amount of fructose in
equilibrium state? What is the effect then? How about reaction
temperature? What other effects do the temperature have?
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Practical application of enzymes/ continuous glucose
isomerization
Feed rate of glucose syrup into an isomerization column during the
useful life of an enzyme loading
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Immobilized GI process
• Corn-derived starch is converted into D-glucose by liquefaction
(thermostable α-amylases) and saccharification (glucoamylase)
• The glucose liquor feed is fed in a down-flow manner into a series of
fixed bed IGI reactors arranged in parallel
• temperature 60 °C, pH 7,8…8.2, flowrate
• D-glucose syrup is converted into a HFCS-42 mixture containing
• 42% D-fructose, 50% D-glucose, 6% maltose, 2% maltotriose + traces of other
sugars
• Chromatographic enrichment to HFCS-90 which is blended with
HFCS-42 to produce HFCS-55 (used in soft drinks etc.)
• Optimizing targets:
• capital costs of enzyme reactors and chromatography columns
• operating lifetime of biocatalyst and ion exchange resins
• minimize side reactions and metal ion levels
• Corn-derived starch is
converted into D-glucose
• The glucose liquor feed is fed
in a down-flow manner into a
series of fixed bed IGI
reactors
• D-glucose syrup is converted
into a HFCS-42 mixture
containing
• Chromatographic enrichment
to HFCS-90
• HFCS-90 blended with HFCS42 to produce HFCS-55