polymer type aqueous two-phase systems (ATPSs) [78] and very few reports are
available on polymer/salt type ATPSs, [94] mainly due to the interference of
high salt concentrations with the biospecific interactions. More details such as
the theory available for AP, future trends, and its other applications are given
elsewhere [84, 95, 96].
Metal affinity partitioning exploits the affinity of transition metal ions for
electron-rich amino acid residues, such as histidine and cysteine, accessible on
the surfaces of proteins. When the metal ion is partially chelated and coupled to
a linear polymer, such as polyethylene glycol (PEG), the resulting polymerbound metal chelate can be used to enhance the partitioning of metal binding
proteins into the polymer-rich phase of a PEG-salt or PEG-dextran (DX) ATPS.
Since most proteins favor the salt-rich heavy phase of an aqueous two-phase
system, metal affinity partitioning can be a very efficient and selective means of
isolating and purifying a metal-binding protein from a crude mixture [97].
In most cases, the desired protein has been isolated in a single chromatographic step from clarified cell lysate, without further pretreatment [98]. Guinn
[95] and Sulkowski [99] successfully achieved the partitioning of recombinant
hemoglobin from crude cell lysate using Cu (II) IDA-PEG in a two-phase system
of PEG and magnesium sulfate. To our knowledge, this is the first recorded
successful attempt to apply metal affinity partitioning techniques to the
isolation of a recombinant protein from crude cell lysate. Successful demonstration of this technology sets the stage for its potential commercial use in the
isolation of native and non-native metal-binding proteins. More details in this
area can be found in a recent review article [96].
Extractive fermentation/bioconversion involves the integration of fermentation/bioconversion with one or more downstream processing step(s), such as extraction using ATPS, ultrafiltration, etc.An opportunity is thus created to explore
new types of industrially relevant bioreactor designs [100, 101]. Many examples
are given in the review articles by Diamond and Hsu [78] and Raghavarao et al.
[79]. Most of the bioconversions have been performed using polymer/polymer
type ATPSs. Lee and Chang [102] were among the first to employ the PEG/
potassium phosphate system successfully for the production of acrylamide from
acrylonitrile using Brevibactrium sp. The selective partitioning of the product
into the top phase was found to reduce the inhibition of the active bacterial
enzyme by both the substrate and the product. The same strategy is gaining
popularity under the name of aqueous two-phase fermentation (ATPF) [103, 104].
Over the past few years there has been considerable interest in the use of the
micro-gravity environment of earth orbit as a laboratory for understanding the
role of surface forces in liquid-liquid phase separation. The importance of
surface wetting forces on the phase separation of ATPSs in microgravity was
observed during Space Shuttle flight STS-26 in October 1988. Separation experiments were performed in a Plexiglas hand-held phase partition experiment
(PPE) module consisting of 18 chambers filled with PEG and DX in plastic extraction cavities [105, 106]. Evidence of surface tension-driven phase separation
in microgravity and its potential processing advantages led to the development
of a reusable platform, the ORSEP, for conducting multi-stage extraction using
ATPSs for both terrestrial and space-based processing applications [95, 96].
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K.S.M.S. Raghavarao et al.
available on polymer/salt type ATPSs, [94] mainly due to the interference of
high salt concentrations with the biospecific interactions. More details such as
the theory available for AP, future trends, and its other applications are given
elsewhere [84, 95, 96].
Metal affinity partitioning exploits the affinity of transition metal ions for
electron-rich amino acid residues, such as histidine and cysteine, accessible on
the surfaces of proteins. When the metal ion is partially chelated and coupled to
a linear polymer, such as polyethylene glycol (PEG), the resulting polymerbound metal chelate can be used to enhance the partitioning of metal binding
proteins into the polymer-rich phase of a PEG-salt or PEG-dextran (DX) ATPS.
Since most proteins favor the salt-rich heavy phase of an aqueous two-phase
system, metal affinity partitioning can be a very efficient and selective means of
isolating and purifying a metal-binding protein from a crude mixture [97].
In most cases, the desired protein has been isolated in a single chromatographic step from clarified cell lysate, without further pretreatment [98]. Guinn
[95] and Sulkowski [99] successfully achieved the partitioning of recombinant
hemoglobin from crude cell lysate using Cu (II) IDA-PEG in a two-phase system
of PEG and magnesium sulfate. To our knowledge, this is the first recorded
successful attempt to apply metal affinity partitioning techniques to the
isolation of a recombinant protein from crude cell lysate. Successful demonstration of this technology sets the stage for its potential commercial use in the
isolation of native and non-native metal-binding proteins. More details in this
area can be found in a recent review article [96].
Extractive fermentation/bioconversion involves the integration of fermentation/bioconversion with one or more downstream processing step(s), such as extraction using ATPS, ultrafiltration, etc.An opportunity is thus created to explore
new types of industrially relevant bioreactor designs [100, 101]. Many examples
are given in the review articles by Diamond and Hsu [78] and Raghavarao et al.
[79]. Most of the bioconversions have been performed using polymer/polymer
type ATPSs. Lee and Chang [102] were among the first to employ the PEG/
potassium phosphate system successfully for the production of acrylamide from
acrylonitrile using Brevibactrium sp. The selective partitioning of the product
into the top phase was found to reduce the inhibition of the active bacterial
enzyme by both the substrate and the product. The same strategy is gaining
popularity under the name of aqueous two-phase fermentation (ATPF) [103, 104].
Over the past few years there has been considerable interest in the use of the
micro-gravity environment of earth orbit as a laboratory for understanding the
role of surface forces in liquid-liquid phase separation. The importance of
surface wetting forces on the phase separation of ATPSs in microgravity was
observed during Space Shuttle flight STS-26 in October 1988. Separation experiments were performed in a Plexiglas hand-held phase partition experiment
(PPE) module consisting of 18 chambers filled with PEG and DX in plastic extraction cavities [105, 106]. Evidence of surface tension-driven phase separation
in microgravity and its potential processing advantages led to the development
of a reusable platform, the ORSEP, for conducting multi-stage extraction using
ATPSs for both terrestrial and space-based processing applications [95, 96].
172
K.S.M.S. Raghavarao et al.