U.S. patent application number 10/401501 was filed with the patent office on 2003-10-30 for microbial production of epoxides.
Invention is credited to Clark, Thomas R., Roberto, Francisco F..
Application Number | 20030203456 10/401501 |
Document ID | / |
Family ID | 25192529 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203456 |
Kind Code |
A1 |
Clark, Thomas R. ; et
al. |
October 30, 2003 |
Microbial production of epoxides
Abstract
A method for microbial production of epoxides and other
oxygenated products is disclosed. The method uses a biocatalyst of
methanotrophic bacteria cultured in a biphasic medium containing a
major amount of a non-aqueous polar solvent. Regeneration of
reducing equivalents is carried out by using endogenous hydrogenase
activity together with supplied hydrogen gas. This method is
especially effective with gaseous substrates and cofactors that
result in liquid products.
Inventors: |
Clark, Thomas R.; (Lakewood,
CO) ; Roberto, Francisco F.; (Idaho Falls,
ID) |
Correspondence
Address: |
Stephen R. Christian
BBWI
PO BOX 1625
IDAHO FALLS
ID
83415-3899
US
|
Family ID: |
25192529 |
Appl. No.: |
10/401501 |
Filed: |
March 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10401501 |
Mar 28, 2003 |
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09805795 |
Mar 12, 2001 |
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6576449 |
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Current U.S.
Class: |
435/123 ;
435/252.1 |
Current CPC
Class: |
Y10S 435/822 20130101;
C12N 1/005 20130101; C12P 17/02 20130101 |
Class at
Publication: |
435/123 ;
435/252.1 |
International
Class: |
C12P 017/02; C12N
001/20 |
Goverment Interests
[0002] This invention was made with United States Government
support under Contract No. DE-AC07-94ID13223, now Contract No.
DE-AC07-99ID13727 awarded by the United States Department of
Energy. The United States Government has certain rights in the
invention.
Claims
We claim:
1. A method for regenerating reducing equivalents in a bioreactor
configured for utilizing reducing equivalents in a
methane-monooxygenase-catalyzed reaction for oxidizing a substrate
into an oxygenated product comprising: culturing a reconstituted
lyophilized whole cell enzyme preparation of methanotrophic
bacteria containing methane monooxygenase and hydrogenase in the
bioreactor, said bioreactor comprising a liquid phase comprising a
major amount of a non-polar non-aqueous solvent and a minor amount
of a polar aqueous medium; continuously dissolving an effective
amount of hydrogen gas in said non-polar non-aqueous solvent, such
that said hydrogen gas and NAD.sup.+are converted by the
hydrogenase into NADH, thereby regenerating reducing
equivalents.
2. The method of claim 1 wherein said oxygenated product is
propylene oxide and said substrate is propylene.
3. The method of claim 1 wherein said methanotrophic bacteria are
selected from the group consisting of Methylosinus, Methyloccus,
and Methylomonas, and mixtures thereof.
4. The method of claim 3 wherein said methanotrophic bacteria are
Methylosinus trichosporium OB3b.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional application of patent
application Ser. No. 09/805,795, filed Mar. 12, 2001.
BACKGROUND OF THE INVENTION
[0003] This invention relates to microbial production of chemicals.
More particularly, the invention relates to methods for microbial
production of epoxides and hydroxylated chemical feedstocks in a
multiphase reactor using gaseous inputs as precursors and for
regeneration of reducing equivalents.
[0004] Methane monooxygenase (EC 1.14.13.25) is a multicomponent
enzyme, produced by methanotrophic bacteria, which catalyzes the
incorporation of atmospheric oxygen into methane to form methanol.
This is the first in a series of reactions that ultimately provide
energy and carbon to the cell. Methanol dehydrogenase further
oxidizes methanol to form formaldehyde. Although reducing
equivalents are consumed initially by the monooxygenase, the
methanotrophic bacteria regenerate NADH by subsequent oxidations
mediated by formaldehyde and formate dehydrogenases, respectively,
the terminal product being CO.sub.2 (for an excellent review, see
J. D. Lipscomb, Biochemistry of the Soluble Methane Monooxygenase,
48 Annu. Rev. Microbiol. 371-399 (1994)).
[0005] Studies of methane monooxygenase refer to both a soluble
(sMMO) and membrane-bound or particulate (pMMO) enzyme. The
prevalent form in type II methanotrophs, such as Methylosinus
trichosporium OB3b, and type X methanotrophs, such as Methyloccus
capsulatus, is regulated in some fashion by the concentration of
copper in the growth medium. K. J. Burrows et al., Substrate
Specificities of the Soluble and Particulate Methane
Mono-oxygenases of Methylosinus trichosporium OB3b, 130 J. Gen
Microbiol. 3327-3333 (1984); S. H. Stanley et al., Copper Stress
Underlies the Fundamental Change in Intracellular Location of
Methane Monooxygenase in Methane-oxidizing Microorganisms: Studies
in Batch and Continuous Culture, 5 Biotechnol. Lett. 487-492
(1983). Type I methanotrophs such as Methylomonas methanica
typically express only pMMO, although one recent isolate has been
found to be an exception to that generalization. S.-C. Koh et al.,
Soluble Methane Monooxygenase Production and Trichloroethylene
Degradation by a Type I Methanotroph, Methylomonas methanica 68-1,
59 Appl. Environ. Microbiol. 960-967 (1993).
[0006] Components of sMMO from Methylomonas trichsporium OB3b that
retained a high specific activity were initially purified by B. G.
Fox et al., Methane Monooxygenase from Methylomonas trichsporium
OB3b. Purification and Properties of a Three-component System with
High Specific Activity from a Type II Methanotroph, 264 J. Biol.
Chem. 10023-10033 (1989). The enzyme comprises a 245 kDa
hydroxylase containing a hydroxo-bridged dinuclear iron cluster at
the active site of methane oxidation, a 15.8-kDa component B, and a
38.4-kDa iron-sulfur reductase with a flavin prosthetic group.
[0007] Although not supporting growth, sMMO and pMMO will, in
addition to methane, adventitiously oxidize a variety of alkanes
and alkenes. C. T. Hou et al., Microbial Oxidation of Gaseous
Hydrocarbons: Epoxidation of C.sub.2 to C.sub.4 n-alkenes by
Methylotrophic Bacteria, 38 Appl. Environ. Microbiol. 127-134
(1979); D. I. Stirling et al., A Comparison of the Substrate and
Electron-donor Specificities of the Methane Monooxygenases from
Three Strains of Methane-oxidizing Bacteria, 177 J. Biochem.
361-364 (1979); H. Dalton, Oxidation of Hydrocarbons by Methane
Monooxygenases from a Variety of Microbes, 26 Adv. Appl. Microbiol.
71-87 (1980). The substrate range of sMMO, however, also includes
aromatic and alicyclic compounds, K. J. Burrows et al., supra; J.
Colby et al., The Soluble Methane Monooxygenase of Methylococcus
capsulatus (Bath). Its Ability to Oxygenate n-Alkanes, n-Alkenes,
Ethers, and Alicyclic, Aromatic, and Heterocyclic Compounds, 165 J.
Biochem. 395-402 (1977), ethers and heterocyclic compounds, J.
Colby et al., supra, and halogenated aromatics and alkenes, J.
Green & H. Dalton, Substrate Specificity of Soluble Methane
Monooxygenase. Mechanistic Implications. 264 J. Biol. Chem.
17698-17703 (1989).
[0008] Currently, there is considerable interest in enzyme function
in non-aqueous solvents (for reviews, see J. S. Dordick, Enzymatic
Catalysis in Monophasic Organic Solvents, 11 Enzyme Microb.
Technol. 194-211 (1989); P. Nickolova & O. P. Ward, Whole Cell
Biocatalysis in Nonconventional Media, 12 J. Ind. Microbiol. 76-86
(1993); G. J. Salter & D. B. Kell, Solvent Selection for Whole
Cell Biotransformations in Organic Media, 15 Crit. Rev. Biotechnol.
139-177 (1995)). Several beneficial and unexpected modifications of
typical enzyme behavior were noted in such systems. These include
enhanced enzyme activity, R. Batra & M. N. Gupta, Enhancement
of Enzyme Activity in Aqueous-organic Solvent Mixtures, 16
Biotechnol. Lett. 1059-1064 (1994), increased thermostability, and
alterations in substrate specificity, A. Zaks & A. M. Klibanov,
Enzymatic Catalysis in Organic Media at 100.degree. C., 224 Science
1249-1251 (1984).
[0009] Recent efforts have concentrated on applications that may be
less amenable to strictly aqueous approaches, since the substrates
are largely insoluble in water. Examples of such an approach
include degradation of sparingly soluble xenobiotics, M.
Ascon-Cabrera & J.-M. Lebeault, Selection of
Xenobiotic-degrading Microorganisms in a Biphasic Aqueous-organic
System, 59 Appl. Environ. Microbiol. 1717-1724 (1993), petroleum
fuel desulfurization, W. R. Finnerty, Organic Sulfur
Biodesulfurization in Non-aqueous Media, 72 Fuel 1631-1634 (1993),
and coal modification or solubilization, E. S. Olson et al.,
Non-aqueous Enzymatic Solubilization of Coal-derived Materials, 72
Fuel 1687-1693 (1993); C. D. Scott et al., The Chemical
Modification of Enzymes to Enhance Solubilization in Organic
Solvents for Interaction with Coal, 72 Fuel 1695-1700 (1993).
[0010] Production of propylene oxide from propylene for use as a
chemical feedstock has been investigated using immobilized whole
cells. L. E. S. Brink & J. Tramper, Production of Propene Oxide
in an Organic Liquid-phase Immobilized Cell Reactor, 9 Enzyme
Microb. Technol. 612-618 (1987); C. T. Hou, Propylene Oxide
Production from Propylene by Immobilized Whole Cells of
Methylosinus sp. CRL-31 in a Gas-solid Bioreactor, 19 Appl.
Microbiol. Biotechnol. 1-4 (1984).
[0011] T. R. Clark & F. F. Roberto, Methylomonas trichsporium
OB3b Whole-cell Methane Monooxygenase Activity in a Biphasic
Matrix, 45 Appl. Microbiol. Biotechnol. 658-663 (1996),
demonstrated soluble methane monooxygenase activity in a two-phase
(biphasic) matrix comprising a buffered aqueous phase and
2,2,4-trimethylpentane (isooctane) using reconstituted whole-cell
preparations of lyophilized Methylomonas trichsporium OB3b. The
rate of conversion of gaseous propylene to propylene oxide, a
non-metabolized liquid, was used as the primary measure of
enzymatic activity. Appreciable soluble methane monooxygenase
activity was detected when the volume of the aqueous phase
represented at least 1% of the total volume, although the initial
rate of product formation did increase as the volume of the aqueous
phase increased. In comparison to the aqueous system, the specific
rate and yields in the biphasic system were much less sensitive to
increases in the concentrations of formate and protein (i.e., the
methane monooxygenase). There was some evidence, however, that the
enzyme system was more stable in the biphasic matrix, since the
rate of propylene oxide formation remained linear for an extended
period of time. V.sub.(app.) in the biphasic system decreased by a
factor of 0.6 relative to the same parameter in the aqueous system.
Conversely, K.sub.m(app.) for propylene was 1.6 times greater in
the biphasic system. Hence, the apparent catalytic efficiency in
the aqueous system was four times that in the biphasic system, as
indicated by a decrease in the corresponding ratios of V.sub.(app.)
to K.sub.m(app.).
[0012] In view of the foregoing, it will be appreciated that
providing a method for microbial production of epoxides and
hydroxylated hydrocarbons would be a significant advancement in the
art.
BRIEF SUMMARY OF THE INVENTION
[0013] It is an object of the present invention to provide a method
for microbial production of epoxides and hydroxylated hydrocarbons
in a continuous production format.
[0014] It is also an object of the invention to provide a method
for microbial production of epoxides and hydroxylated hydrocarbons
that utilizes gaseous inputs and produces liquid products.
[0015] It is another object of the invention to provide a method
for microbial production of epoxides and hydroxylated hydrocarbons
that results in regeneration of reducing equivalents.
[0016] It is still another object of the invention to provide a
method for microbial production of epoxides and hydroxylated
hydrocarbons that uses a liquid phase comprising (i) a non-polar,
non-aqueous solvent or mixture of miscible non-polar, non-aqueous
solvents and (ii) an aqueous phase.
[0017] It is yet another object of the invention to provide a
method for microbial production of epoxides and hydroxylated
hydrocarbons wherein a hydrated biocatalyst can be immobilized on a
non-porous support material and submerged in the non-polar,
non-aqueous solvent phase.
[0018] It is a further object of the invention to provide a method
for microbial production of epoxides and hydroxylated hydrocarbons
wherein non-aqueous solvents have low vapor pressures, much higher
boiling points than reaction products, and are non-inhibitory to
methane oxidizing microorganisms.
[0019] It is a still further object of the invention to provide a
method for microbial production of epoxides and hydroxylated
hydrocarbons wherein water and other polar molecules, such as
reaction products, are insoluble in the non-aqueous solvents.
[0020] It is another object of the invention to provide a method
for microbial production of epoxides and hydroxylated hydrocarbons
wherein the gaseous substrates are readily soluble in the
non-aqueous solvents.
[0021] It is still another object of the invention to provide a
method for microbial production of epoxides and hydroxylated
hydrocarbons wherein the products are polar and readily soluble in
the aqueous phase.
[0022] It is yet another object of the invention to provide a
method for microbial production of epoxides and hydroxylated
hydrocarbons wherein the density of the non-aqueous phase is much
greater than the density of the aqueous phase such that a stable,
self-maintaining phase separation is obtained.
[0023] These and other objects can be addressed by providing a
method for microbial production of an oxygenated derivative of a
methane-monooxygenase substrate comprising:
[0024] (a) incubating a reconstituted lyophilized whole cell enzyme
preparation of methanotrophic bacteria containing methane
monooxygenase and hydrogenase in a bioreactor, said bioreactor
comprising a biphasic liquid phase comprising a major amount of a
non-polar non-aqueous solvent and a minor amount of a polar aqueous
medium, a non-aqueous solvent circulation circuit for circulating
said non-polar non-aqueous solvent through the bioreactor, and an
aqueous medium circulation circuit for circulating the polar
aqueous medium through the bioreactor in a counter-current manner
as compared to the non-polar non-aqueous solvent;
[0025] (b) continuously dissolving effective amounts of oxygen gas,
hydrogen gas, and a gaseous substrate in the non-polar non-aqueous
solvent, wherein the gaseous substrate is readily soluble in the
non-polar non-aqueous solvent and is susceptible to oxidation by
the methane monooxygenase to result in the oxygenated derivative
thereof, wherein the oxygenated derivative is a polar liquid;
[0026] (c) circulating the non-polar non-aqueous solvent in close
proximity to the reconstituted lyophilized whole cell enzyme
preparation of methanotrophic bacteria containing methane
monooxygenase and hydrogenase such that the methane monooxygenase
oxidizes the gaseous substrate into the oxygenated substrate;
[0027] (d) circulating the polar aqueous medium in a
counter-current manner through the circulating non-polar
non-aqueous solvent such that the oxygenated substrate is
partitioned into the polar aqueous medium; and
[0028] (e) removing and recovering the oxygenated substrate from
the polar aqueous medium and recycling the non-polar non-aqueous
solvent and the polar aqueous medium.
[0029] Many substrates and products can be used in this method, but
a preferred substrate is propylene, and the resulting preferred
oxygenated derivative is propylene oxide. Preferred methanotrophic
bacteria according to the invention are selected from the group
consisting of Methylosinus, Methyloccus, and Methylomonas, and
mixtures thereof. Especially preferred methanotrophic bacteria are
Methylomonas trichsporium OB3b. Preferred non-polar non-aqueous
solvents according to the invention are isooctane, hexane, silicone
oil, hexadecane, fluorocarbons, and mixtures thereof.
[0030] Another preferred embodiment of the invention comprises a
method for regenerating reducing equivalents in a bioreactor
configured for utilizing reducing equivalents in a
methane-monooxygenase-catalyzed reaction for oxidizing a substrate
into an oxygenated product comprising:
[0031] (a) incubating a reconstituted lyophilized whole cell enzyme
preparation of methanotrophic bacteria containing methane
monooxygenase and hydrogenase in the bioreactor, the bioreactor
comprising a liquid phase comprising a major amount of a non-polar
non-aqueous solvent and a minor amount of a polar aqueous medium;
and
[0032] (b) continuously dissolving an effective amount of hydrogen
gas in the non-polar non-aqueous solvent, such that the hydrogen
gas and NAD.sup.+are converted by the hydrogenase into NADH,
thereby regenerating reducing equivalents.
[0033] Still another preferred embodiment of the invention
comprises a method for preparing a lyophilized preparation of
methanotrophic bacteria for use as a biocatalyst comprising:
[0034] (a) culturing the methanotrophic bacteria, concentrating the
resulting cells, resuspending the concentrated cells, and then
chilling the resuspended cells at about 0.degree. C.;
[0035] (b) freezing the chilled cells at about -50.degree. C. using
a shell freezer;
[0036] (c) further cooling the frozen cells in a liquid nitrogen
bath and then freeze drying the resulting cells using a lyophilizer
for a period sufficient to obtain a powdered cell preparation;
and
[0037] (d) storing the powdered cell preparation under
refrigeration.
[0038] A still further preferred embodiment of the invention
comprises a method for propagating methanotrophic bacteria
comprising culturing the bacteria in a liquid medium comprising a
major amount of a non-aqueous polar solvent and a minor amount of a
known methanotrophic bacterial growth medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 shows a schematic diagram of a bioreactor for use in
microbial production of epoxides and hydroxylated chemicals
according to the present invention.
[0040] FIG. 2 shows the effect of the aqueous-phase concentration
on the specific rate of propylene epoxidation by lyophilized whole
cells of M. trichosporium OB3b in a biphasic matrix. Rates were
determined without the addition of exogenous formate (hatched bars)
and with 40.8 .mu.M formate (solid bars). The matrix (1000 .mu.l)
was composed of isooctane+indicated percentages of MOPS/Cys/Fe
buffer (pH 7.0)+190 .mu.g protein. Incubation was at 150 rpm at
21.degree. C.
[0041] FIG. 3 shows the effect of protein concentration on the rate
(nmol h.sup.-1) of propylene oxide formation in 1000 .mu.l aqueous
and biphasic matrices composed of respectively MOPS/Mg buffer
containing 1020 .mu.M formate (.box-solid.) or 98% v/v isooctane+2%
v/v MOPS/Mg containing 13.6 .mu.M formate (+). Protein amounts were
80-400 .mu.g, and incubation was at 200 rpm at 28.degree. C.
[0042] FIG. 4 shows yields of propylene oxide (nmol mg.sup.-1
protein) in 1000 .mu.l aqueous and biphasic matrices containing low
concentrations of protein. Matrices contained 80 .mu.g protein and
were composed of MOPS/Mg buffer or 98% v/v isooctane+2% v/v MOPS/Mg
buffer: Biphasic+13.6 .mu.M formate (*); aqueous+13.6 .mu.M formate
(+); aqueous+1020 .mu.M formate (.box-solid.). Incubation was at
200 rpm at 28.degree. C.
[0043] FIG. 5 shows relative hydrogenase activity in reconstituted
whole cell enzyme preparation from M. trichosporium OB3b cells in
98% (v/v) isooctane as a function of micromoles of headspace
hydrogen.
DETAILED DESCRIPTION
[0044] Before the present methods for microbial production of
epoxides and hydroxylated chemicals are disclosed and described, it
is to be understood that this invention is not limited to the
particular configurations, process steps, and materials disclosed
herein as such configurations, process steps, and materials may
vary somewhat. It is also to be understood that the terminology
employed herein is used for the purpose of describing particular
embodiments only and is not intended to be limiting since the scope
of the present invention will be limited only by the appended
claims and equivalents thereof.
[0045] The publications and other reference materials referred to
herein to describe the background of the invention and to provide
additional detail regarding its practice are hereby incorporated by
reference. The references discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior invention.
[0046] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
[0047] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0048] As used herein, "comprising," "including," "containing,"
"characterized by," and grammatical equivalents thereof are
inclusive or open-ended terms that do not exclude additional,
unrecited elements or method steps. "Comprising" is to be
interpreted as including the more restrictive terms "consisting of"
and "consisting essentially of."
[0049] As used herein, "consisting of" and grammatical equivalents
thereof exclude any element, step, or ingredient not specified in
the claim.
[0050] As used herein, "consisting essentially of" and grammatical
equivalents thereof limit the scope of a claim to the specified
materials or steps and those that do not materially affect the
basic and novel characteristic or characteristics of the claimed
invention.
[0051] As used herein, "oxygenated derivative of a
methane-monooxygenase substrate" means an oxidation product of a
methane-monooxygenase-catalyze- d reaction. For example, propylene
oxide is an oxygenated derivative of propylene.
[0052] As used herein, "methanotrophic bacteria" means bacteria
that produce methane monooxygenase, such as Methylomonas
trichsporium OB3b, Methyloccus capsulatus, and Methylomonas
methanica.
[0053] As used herein, "major amount" means greater than 50%, and
"minor amount" means less than 50%.
[0054] FIG. 1 shows an illustrative bioreactor suitable for use
according to the present invention. The bioreactor 10 comprises a
housing 12, preferably constructed of glass or another material
that is non-porous and unreactive to the contents of the
bioreactor. The housing 12 defines a chamber 14 for receiving the
contents of the bioreactor, which will be described below. At a
lower level in the chamber there is suspended a lower bead support
16, which is a porous layer that permits the passage of liquids and
gases, but retains glass beads and similar particles. Disposed on
the lower bead support is a layer of glass beads 18 or similar
material, which serves the functions of a diffuser layer and phase
separator. A layer comprising the immobilized biocatalyst 20 is
disposed on the layer of glass beads. The biocatalyst is a
reconstituted whole-cell preparation of lyophilized methanotrophic
bacteria, such as Methylomonas trichsporium OB3b. This biocatalyst
will be described in more detail below. Disposed above the
biocatalyst layer is an upper bead support 22, which can be the
same material as the lower bead support described above. Disposed
on the upper bead support is another layer of glass beads 24, which
functions as another diffuser layer. The chamber is partially
filled with a non-polar, non-aqueous solvent or mixture of miscible
non-polar, non-aqueous solvents that forms a non-aqueous solvent
layer 26, and disposed above the non-aqueous solvent layer is an
aqueous layer 28. There should be a headspace 30 above the aqueous
layer, which is not occupied with a liquid layer.
[0055] The bioreactor further comprises a non-aqueous solvent
circulation circuit 31 for circulating the non-aqueous solvent and
gases dissolved therein through the bioreactor and for injecting
gases into the bioreactor. This circuit comprises a non-aqueous
solvent exit port 32 disposed at a lower level of the housing and
preferably at a level below the lower bead support such that glass
beads, immobilized catalyst, and the like do not have access to the
non-aqueous solvent exit port. A non-aqueous solvent entrance port
34 is also disposed in the housing, but at a level above the
aqueous layer, i.e., in the portion of the housing adjacent to the
headspace. Coupled to the non-aqueous solvent exit port and the
non-aqueous solvent entrance port is a tube 36 having an in-line
pump 38 for pumping the non-aqueous solvent from the non-aqueous
solvent exit port to the non-aqueous solvent entrance port, thus
circulating the non-aqueous solvent through the bioreactor. A gas
injection port 40 is also disposed in the tube, through which
gases, such as oxygen, hydrogen, methane, propylene, and the like,
can be injected into the bioreactor. The gases are completely
soluble in the non-aqueous solvent, thus the gases are circulated
through the bioreactor by being dissolved in the non-aqueous
solvent.
[0056] The bioreactor further comprises an aqueous solvent
circulation circuit 41 for circulating the aqueous solvent through
the bioreactor and for recovering the product from the aqueous
solvent. An aqueous solvent exit port 42 is disposed in the housing
adjacent to the aqueous layer such that the aqueous solvent can
exit the chamber and be circulated through the aqueous solvent
circulation circuit. An aqueous solvent entrance port 44 is
disposed in the housing at a lower level thereof, preferably below
the level of the lower bead support. A tube 46 is disposed on both
the aqueous solvent exit port and the aqueous solvent entrance port
for connecting these two ports, and an in-line pump 48 is disposed
on the tube for pumping the aqueous solvent from the aqueous
solvent exit port to the aqueous solvent entrance port. Also
coupled to the tube is a product recovery member 50 for removing
the product from the aqueous solvent and recovering the product.
Still further, a reservoir 52 is also preferably present in the
aqueous solvent circulation circuit for holding an amount of
aqueous solvent, which can be directed back into the chamber by the
action of the pump.
[0057] In operation, the bioreactor functions as follows. The
non-aqueous solvent circulation circuit removes non-aqueous solvent
from the chamber, injects substrate gases in preselected amounts
into the non-aqueous solvent, and returns the non-aqueous solvent,
now containing the dissolved gases, to the chamber at the top of
the packed column. The flow rate can be much less than in a
corresponding aqueous system because the relative concentrations of
substrates are, in some cases, orders of magnitude higher than
could be achieved in water. Because of the immiscibility of water
in the non-aqueous solvent and the density differences between
these phases, a distinct aqueous layer 28 forms above the
non-aqueous layer 26. As the non-aqueous solvent passes through the
layer containing the catalyst, the substrate gases, i.e., oxygen,
hydrogen, and, for example, methane, come in close proximity with
the catalyst, which oxidizes the methane into methanol, a polar
product, by the action of methane monooxygenase. The methanol
product tends to partition out of the non-aqueous solvent and into
the much more polar aqueous phase. The hydrogen gas is used in
regenerating reducing equivalents by the hydrogenase-catalyzed
reaction that results in the conversion of NAD.sup.+ into NADH. As
described above, the oxygen and methane are used in oxidizing the
methane to methanol. Thus, if there were no replenishment of these
gases, the non-aqueous solvent would become depleted with respect
to the substrate gases. An aqueous phase introduced into the
chamber at the bottom of the packed column tends to collect or
"sweep up" any polar product as the aqueous solvent moves up
through the column because of the relative densities of the phases.
Therefore, product recovery can be achieved by collecting the
aqueous phase representing only a small percentage of the total
reactor volume. The aqueous phase is recycled following product
recovery. Since the mixed organic phase is also recycled, neither
liquid phase requires replacement, thus reducing both material and
waste disposal costs. It should be further noted that the
non-aqueous phase tends to circulate from top to bottom of the
bioreactor, as indicated by arrows 54. At the same time, the
aqueous phase tends to circulate from bottom to top of the
bioreactor, as indicated by arrows 56. Thus, the counter-current
circulation of the non-aqueous phase and the aqueous phase works to
increase the efficiency of partition of the polar reactions product
into the aqueous phase.
EXAMPLE 1
[0058] Materials and Methods
[0059] Culture maintenance. M. trichosporium OB3b (ATCC 35070) was
maintained at 30.degree. C. in serum vials containing Higgins
minimal nitrate salts medium (NSM) amended with 2 .mu.M Cu and, as
recommended by S. Park et al., Batch Cultivation of Methylomonas
trichsporium OB3b. 1. Production of Soluble Methane Monooxygenase,
38 Biotechnol. Bioeng. 423-433 (1991), 80 .mu.M Fe(II). The
headspace composition was adjusted to a 70:30 v/v mixture of air
and methane. The vials were shaken at a rate of 200-225 rpm.
Alternatively, the culture was maintained on Fe- and Cu-amended NSM
medium solidified with 1% w/v agarose (Sigma type 1-A). The plates
were incubated at 30.degree. C. in a vacuum desiccator containing
an atmosphere of 70% v/v air and 30% v/v methane.
[0060] Continuous-flow cultures. A 1-liter bioreactor (Bellco)
containing 550-600 ml NSM medium and 80 .mu.M Fe(II) was inoculated
with cells from the batch culture (8% v/v). The reactor contents
were stirred with an N.sub.2-driven magnetic impeller and sparged
with an air:methane mix (70:30 v/v), although initial growth lags
were reduced by sparging with 2% v/v CO.sub.2 in addition to air
and methane until log-phase growth commenced. S. Park et al.,
supra. The reactor was maintained in a continuous flow mode
(dilution rate, D=0.02 h.sup.-1) using a cartridge-type peristaltic
pump (Masterflex). Temperature was monitored using a type YM
thermistor and maintained at 30.degree. C. by a temperature
controller (Digi-Sense, Cole Parmer), input controller (Type 45500,
Thermolyne) and heat tape.
[0061] Lyophilization. The bioreactor was switched to batch mode
approximately 24 hours prior to cell harvest to allow the cells to
reach late log or stationary phase, as determined by comparison of
absorbance (600 nm) to a growth curve. The cells were harvested and
immediately centrifuged at 12,000 g (Sorvall RC5b, SS-34 rotor) for
10 minutes at 4.degree. C. The pellets were washed once at pH 7.0
with one of two MOPS [3-(N-morpholino)propanesulfonic acid]
buffers: MOPS/Mg was comprised of 25 mM MOPS and 5 mM MgSO.sub.4;
MOPS/Cys/Fe was comprised of 25 mM MOPS, 2 mM cysteine, and 0.2 mM
Fe(NH.sub.4).sub.2(SO.sub.4).sub.2.6H.sub.2O. B. G. Fox et al.,
supra; D. R. Jollie & J. D. Lipscomb, Formate Dehydrogenase
from Methylosinus trichosporium OB3b, 188 Methods Enzymol. 331-334
(1990). The presence of sMMO was verified by qualitative assays for
both propylene and naphthalene oxidation. G. A. Brusseau et al.,
Optimization of Trichloroethylene Oxidation by Methanotrophs and
the Use of Colorimetric Assay to Detect Methane Mono-oxygenase
Activity, 1 Biodegradation 19-29 (1990). The cells were resuspended
in a minimal volume of this same buffer containing 10 mM NADH
(Kodak), which was found to have cryoprotective properties, frozen
at -75.degree. C. for 1 hour or by immersion in liquid nitrogen,
and lyophilized.
[0062] Protein assay. Lyophilized cells were resuspended in 0.1 M
NaOH to concentrations between 0.05% and 0.1% w/v. The samples were
heated for 5 minutes in Eppendorf tubes submerged in a boiling
water bath. Samples comprising 50 .mu.l cell digest were added to
2.5 ml of BioRad protein reagent (diluted 1:4) and the absorbance
(595 nm) was read immediately using a Shimadzu UV-1201
spectrophotometer. Absorbance values were quantified by comparison
to a standard regression curve using bovine serum albumin as a
standard.
[0063] Naphthalene oxidation assay. The production of sMMO was
verified by demonstrating the capability of washed cells to
hydroxylate naphthalene, with a positive reaction for sMMO
determined by a color reaction upon the addition of tetrazotized
o-dianisidine dye. G. A. Brusseau et al., supra.
[0064] Propylene oxidation assay. Lyophilized cell preparations
were assayed for MMO activity, which is defined herein as the
quantity of propylene oxide formed per milligram of protein (total)
per hour. The reaction vessels consisted of vials with 10 ml
headspace sealed with aluminum caps and
polytetrafluoroethylene-lined butyl rubber septa. Lyophilized cells
were rehydrated to various degrees with buffer for aqueous and
biphasic activity assays. Sodium formate (Alfa) was then added to
the rehydrated cell suspensions for the purpose of regenerating
NADH. Isooctane (Fisher) was added to the appropriate reaction
vials. Assuming a total liquid-phase volume of 1000 .mu.l, the
final formate concentrations for the aqueous and biphasic systems
were, respectively, 1360 .mu.M and 13.6 .mu.M, unless otherwise
stated. The reaction mixtures were warmed to 28.degree. C., unless
otherwise stated, prior to the addition of substrate. Following
injection of propylene into the headspace (89 .mu.mol, aqueous;
44.6 .mu.mol, biphasic), the vials were shaken at 150 rpm or 200
rpm on a rotary shaker (New Brunswick G-24). The reaction was
stopped by the addition of methanol (74 mM or 124 mM), which was
found to inhibit the reaction completely in the biphasic system and
by approximately 99% in assays using an aqueous matrix. The entire
contents of the reaction vials were sacrificed for each
analysis.
[0065] Gas chromatography. Propylene oxide formation was quantified
with a Shimadzu model GC-14A gas chromatograph containing a glass
column (2.6 mm inner diameter, 3.1 m) packed with 80.times.100 mesh
Carbopack C+0.1% SP-1000 (Supelco). The gas chromatograph was
configured with an AOC-17 auto-injector and a Shimadzu CR-601
Chromatopac recorder. Helium was used as a carrier gas (20 ml/min).
The injector and detector (flame-ionization detector) temperatures
were respectively 200.degree. C. and 175.degree. C. Aqueous samples
were run isothermally at 80.degree. C. for 6 minutes. Isooctane
samples were determined as follows: 80.degree. C. for 2 minutes;
ramped at 40.degree. C./minute to a final temperature of
200.degree. C.; and held for 6 minutes at 200.degree. C. Product
formation was quantified in the single-time-point assay by
comparison of peak area to a standard regression curve.
[0066] Propylene available in solution was determined separately
under the same conditions listed above, but in the presence of the
lyophilized cell preparation. The extent of product partitioning
was examined by separating the isooctane phase from the hydrated
cells, normalizing the volumes, and determining the product
concentration.
[0067] Kinetic parameters. Since the range of liquid-phase
substrate concentrations was narrow in both matrices, despite the
headspace concentration, fitted plots of substrate versus velocity
were used in favor of approaches involving reciprocal plots.
V.sub.(app.) and K.sub.m(app.) were estimated by computer-fitting a
second-order polynomial expansion to plots of propylene
concentration against the rate of product formation (nmol propylene
oxide mg.sup.-1 protein h.sup.-1).
[0068] Results
[0069] Propylene and propylene oxide partitioning. Propylene
rapidly partitioned between the headspace and liquid phase in both
the aqueous and biphasic systems. The presence of lyophilized cells
and associated buffer salts did not affect the concentration of
propylene ([S]) in the liquid phase. The maximum solution
concentrations for the aqueous and biphasic matrices were
respectively 8.5 .mu.M and 26.7 .mu.M. The concentration of
propylene in solution remained constant during the course of
incubation because of a large excess present in the headspace.
[0070] Propylene oxide was not detected in normalized fractions
containing the hydrated cells. This indicated that measurement of
propylene oxide concentrations in the water-saturated isooctane
phase provided an accurate estimate of total product formation.
[0071] Aqueous phase concentration. Rehydration of the cellular
biocatalyst was a requirement for attaining appreciable rates of
formation of propylene oxide. As shown in FIG. 2, the
formate-stimulated rate increased ninefold when the aqueous-phase
concentration in the biphasic matrix was increased from 0.3% to
1.0% v/v. Propylene epoxidation in the absence of formate
represented a maximum of 11% of the total activity in the biphasic
system until the aqueous-phase content increased to 3% v/v, when a
marked increase in endogenous activity was noted. In this case,
however, the increase in endogenous activity did not result in a
proportional increase in the overall rate.
[0072] Protein concentration. The aqueous and biphasic matrices
differed with respect to an optimum enzyme concentration. FIG. 3
compares the effect of total protein, hence the concentration of
sMMO, on the rate of propylene oxide accumulation (nmol h.sup.-1)
in both matrices. Note that an increase in protein concentration in
the biphasic system resulted in a linear increase in the rate,
while a sigmoidal response was noted for the aqueous matrix with
respect to this same parameter. Clearly, the biphasic system was
much less responsive to increases in enzyme concentration.
Therefore, unlike the aqueous system, the specific activity (nmol
mg.sup.-1 protein h.sup.-1) in the biphasic system also declined
steadily with increasing concentrations of protein, with the
greatest rate attained at the lowest protein concentration
examined. There was also a considerable difference in the overall
rates (nmol h.sup.-1) for the two systems.
[0073] Formate concentration. The extent to which formate
stimulated activity in the aqueous system depended on the protein
concentration. At the high end of the concentration range, specific
rates of product formation increased as the formate concentration
was increased to 1320 .mu.M. At low concentrations of protein,
however, activity in the aqueous system never exceeded biphasic
activity. As shown in FIG. 4, the yield in the aqueous system was
less than that of the biphasic system whether low (13.6 .mu.M) or
high (1020 .mu.M) formate concentrations were used.
[0074] In contrast to the aqueous system, activity in the biphasic
system was generally unresponsive to additions of formate in excess
of 13.6 .mu.M. In fact, the maximum formate concentration in the
aqueous system (1360 .mu.M) was somewhat inhibitory in the biphasic
system and decreased the rate in one experiment by 21% (data not
shown).
[0075] Enzyme stability. There was evidence that sMMO and/or
formate dehydrogenase may have been stabilized (i.e., a greater
number of turnovers per unit time) in the biphasic matrix. FIG. 4
also summarizes yields (nmol mg-1 protein) in aqueous and biphasic
matrices containing a low concentration of protein. Negligible
aqueous activity resulted at low protein (80 .mu.g/ml) and formate
(13.6 .mu.M) concentrations. An increase in yield did result from
increasing formate availability (1020 .mu.M), indicating that the
conversion of substrate to product with only 13.6 .mu.M formate was
formate (NADH)-limited. However, product also ceased to accumulate
after 25 minutes at the much higher formate concentration although
yield was less than when using higher concentrations of protein. In
a separate experiment, the rate of product formation under optimum
conditions for the aqueous matrix (high concentrations of both
protein and formate) decreased by 46% over a 0.5-hour incubation
period. Therefore, substantial loss of sMMO activity was
experienced under aqueous conditions within 30 minutes regardless
of the enzyme concentration and amount of formate provided as
reductant.
[0076] Conversely, the yield in the biphasic system was
proportional to incubation time for a period of 60 minutes. Despite
greater yields than in the aqueous system, the specific activity
was also relatively constant during this period, demonstrating that
neither substrate depletion (propylene, O.sub.2, NADH) nor product
inhibition was responsible for the differences in yield.
[0077] Kinetic parameters. The data in Table 1 represent typical
values for the biphasic and aqueous whole-cell sMMO activity.
Greater bulk-phase substrate concentrations in the biphasic system
were somewhat offset by a higher K.sub.m(app.) than was determined
for the aqueous system. The apparent catalytic efficiency was four
times greater in the aqueous system than for the same preparation
in the biphasic system, as indicated by a decrease in the ratio of
corresponding values of V.sub.(app.) to K.sub.m(app.) for each
system.
1TABLE 1 Matrix V.sub.(app.) K.sub.m(app.) Aqueous 973 8.3 Biphasic
366 13.0
[0078] The correlation coefficients for second-order polynomial
curve fits: aqueous r2=0.984; biphasic, r2=0.822. The aqueous phase
consisted of MOPS/Mg buffer. The biphasic matric was comprised of
98% v/v isooctane+2% v/v MOPS/Mg buffer.
[0079] Discussion
[0080] The use of lyophilized cell preparations that catalyze
multi-enzyme reactions in biphasic systems has been reported for
yeast. F. Borzeix et al., Bi-enzymatic reaction for alcohol
oxidation in organic media: from purified enzymes to cellular
systems, 17 Enzyme Microb. Technol. 615-622 (1995). In the present
study, however, the primary advantages of using lyophilized cells
included ease of control of the aqueous content and generation of a
stable biocatalyst that contained both sMMO and formate
dehydrogenase activities, the latter being required for cofactor
regeneration.
[0081] A sufficient aqueous-phase content, however, was critical
for enzyme activity to occur. The system was not sufficiently
dissected to determine the extent of overlap of the individual
optimum aqueous-phase contents for formate dehydrogenase, sMMO, or
enzymes required for the utilization of endogenous sources of
reducing equivalents. A range in optima is indicated by the
increase in endogenous propylene oxidation activity upon increasing
the aqueous-phase content.
[0082] The rate of propylene oxide formation in the biphasic matrix
declined only slightly over a period exceeding 1 hour, indicating
that sMMO was not rapidly inactivated under these conditions. It
was also discovered that the rate of propylene oxide formation in
the biphasic system was largely unresponsive to increases in the
concentration of formate in excess of an optimum concentration,
which was, unexpectedly, much less than the optimum for the aqueous
system. Together, these findings are at least suggestive that
lability of formate dehydrogenase, D. C. Yoch et al., Formate
Dehydrogenase from the Methane Oxidizer Methylomonas trichosporium
OB3b, 172 J. Bacteriol. 4456-4463 (1990); D. R. Jollie & J. D.
Lipscomb, Formate Dehydrogenase from Methylomonas trichosporium
OB3b. Purification and spectroscopic characterization of the
cofactors, 266 J. Biol. Chem. 21853-21863 (1991), and not sMMO may
have been one factor that defined the overall rates of propylene
oxide formation in the biphasic system. Problems associated with
the use of dehydrogenases for regeneration of NADH by methanotrophs
have long been recognized. C. T. Hou et al., Epoxidation of Alkenes
by Methane Monooxygenase: Generation and Regeneration of Cofactor,
NADH.sub.2, by Dehydrogenase, 4 J. Appl. Biochem. 379-383
(1982).
[0083] It is significant that the K.sub.m(app.) for propylene
conversion by reconstituted lyophilized cell preparations in the
biphasic system, as described above, is within an order or
magnitude of previously reported values for the purified enzyme. J.
Green & H. Dalton, Steady-state Kinetic Analysis of Soluble
Methane Monooxygenase from Methylococcus capsulatus (Bath), 236 J.
Biochem. 155-162 (1986). This indicates that the reconstituted
cells may have damaged membranes and be somewhat leaky. An increase
in this kinetic parameter would not be surprising since the
presence of saturating concentrations of an organic solvent (in
this case, isooctane) might reduce the stability of the
enzyme-substrate complex, resulting in an apparent increase in the
K.sub.m(app.). J. S. Dordick, supra.
[0084] The wide substrate range of sMMO, J. Colby et al., supra,
precluded the use of many solvents because of the potential for
competitive inhibition, a problem not entirely obviated by the use
of isooctane. This was indicated by the substantial decrease in the
ratio of V.sub.(app.) to K.sub.m(app.) for sMMO, J. Green & H.
Dalton, 236 J. Biochem. 155-162 (1986), in the biphasic system.
Maintenance of this system in a biphasic matrix, however, is
particularly interesting since this process involves not one, but
two separate multicomponent enzymes (methane monooxygenase and
formate dehydrogenase) and is most promising for processing a wide
variety of organic substrates.
EXAMPLE 2
[0085] In this example the procedure of Example 1 was followed
except that the bacteria used were mixed methanotrophs isolated
from raw water/sediment samples from Test Area North ("TAN")
injection well #1 at the Idaho National Engineering and
Environmental Laboratory, Idaho Falls, Id. These bacteria were
cocci or diplococci that appeared morphologically similar to M.
capsulatus. The cells were grown in a stirred bioreactor containing
NSM+80 mM Fe. After concentration, the cells were resuspended and
cooled in an ice/water bath. The cell suspension was added to
lyophilization jars at approximately 0.degree. C. and chilled to
approximately -50.degree. C. using a glycol shell freezer. The
frozen cells were cooled further in a liquid nitrogen bath and
placed on a lyophilizer for a period that usually extended
overnight (40 mT, -90.degree. C.). The resulting dry powder was
manually homogenized with a spatula, divided into aliquots, placed
in sealed headspace vials, and refrigerated prior to use in assays
for propylene oxide formation. To prevent condensation on the
lyophilized powder, the vials were allowed to warm to room
temperature prior to opening and use. The lyophilized preparations
were stored for six days at -73.degree. C. before use. Sodium
formate (Alfa) was added to the rehydrated cell suspensions for the
purpose of regenerating NADH in some samples ("(+) formate"), but
not in other samples ("(-) formate"). The results shown in Table 2
are based on assays performed in triplicate.
2TABLE 2 Preparation nmol*mg.sup.-1*h.sup.-1 (X .+-. s.sub.n-1) %
Washed Cell Activity Washed Whole Cells (-) formate 2106 .+-. 274
100 (+) formate 9201 .+-. 228 100 Reconstituted Lyophilized Cells
(-) formate 179 .+-. 212 8.5 (+) formate 7959 .+-. 1251 86.5
[0086] As was typical with many experiments, the endogenous methane
monooxygenase activity was reduced following lyophilization. The
level of activity obtained with lyophilized cell preparations with
formate added for regenerating reducing equivalents, was
unexpectedly high.
[0087] These results show that unexpectedly and surprisingly high
methane monooxygenase activity was obtained in cells Iyophilized
according to the present invention as compared to washed whole
cells.
EXAMPLE 3
[0088] In this example the procedure of Example 1 was followed
except that whole cells were used instead of lyophilized cells, the
cells were cultured in aqueous medium (NSM) without Mo and Ni
supplementation, the headspace gas compositions contained either
ambient or about 20% ambient (low) oxygen concentrations, and
sodium formate was not added to all cultures. Some cultures
contained added formate, whereas other cultures contained hydrogen
gas added to the headspace gas composition to assay endogenous
hydrogenase activity for regenerating reducing equivalents, and
other cultures contained no added formate or hydrogen, for assaying
endogenous sMMO activity. The data presented in Table 3 show the
rates of propylene oxide formation and represent the means values
for three vials per test group.
3 TABLE 3 Rate of Propylene Oxide Formation (nmol*mg-1
protein*h.sup.-1 Headspace Endogenous Formate Hydrogen Low Oxygen
6068 .+-. 102 5476 .+-. 552 9824 .+-. 410 Ambient Oxygen 6583 .+-.
705 11319 .+-. 524 11156 .+-. 358
[0089] These results show that hydrogen gas can be used to
regenerate reducing equivalents in whole cells cultured in aqueous
medium according to the reaction: 1
EXAMPLE 4
[0090] In this example, the procedure of Example 1 was followed
except that no formate was added to the reconstituted lyophilized
cells. Instead, various concentrations of hydrogen gas were added
to the headspace gas composition for assaying hydrogenase activity
for regenerating reducing equivalents. FIG. 5 shows the results
obtained in a biphasic system containing 98% isooctane. The assay
conditions were: t=90 minutes, 150 rpm, 25.5-27.0.degree. C.,
V.sub.T=1000 ml, 45 mmol propylene. These results show stimulation
of reconstituted hydrogenase activity over that of endogenous
activity. Formate dehydrogenase activity was negligible in this
experiment.
EXAMPLE 5
[0091] In this experiment M. trichosporium OB3b cells were cultured
according to the procedure of Example 1 except that a multiphasic
reactor system was used, as will be described below. Two problems
encountered with the multiphasic (minimal aqueous volume) systems
for culturing whole cells are that salt components may become
limiting after sufficient cell mass has been generated, and
sufficient iron must be supplied in a form that can be utilized by
the methanotrophic bacteria. To address these problems, cells were
cultured in a multiphasic system containing an aqueous phase, an
alkane, and a perfluorocarbon in varying proportions. NSM medium
was added at twice the concentration of Example 1. Chelated iron
(80 .mu.M) was used to maximize a stable concentration of available
iron for maximal sMMO activity.
[0092] Table 4 summarizes the compositions of various multiphase
matrices. The volumetric aqueous phase content was maintained at
40% (v/v), and hexadecane comprised the balance of the liquid
phase.
4 TABLE 4 Volume % Sample No. Hexadecane FC77 FC40 Aqueous 1 56 4 0
40 2 48 12 0 40 3 40 20 0 40 4 56 0 4 40 5 48 0 12 40 6 40 0 20
40
[0093] After more than two weeks of incubation under 30% (v/v)
methane atmosphere, growth was obtained. Subsamples were then
plated on NSM-agarose solid medium. Cell growth was rapid and
profuse, with no obvious morphological abnormalities evident by
phase constant microscopy. These results show that cells can be
maintained in media containing significant quantities of organic
solvents.
* * * * *