Note: Descriptions are shown in the official language in which they were submitted.
~L2~
Background of the Invention
This invention relates to a method of synthesizing the
amino acid L-serine. The amino acid L-serine is a valuable
commercial product that is employed in hyperalimentation
and nutritional compositions and is also used as an
intermediate or starting material in certain synthetic
processes. There thus is a demand for L-serine, and
various processes have been developed for producing it. A
number of these processes involve the production of
L-serine by fermentationO See, for example, Morinaga, Y.,
et al., Agric. Biol_. Chem. 45(6)1419-24 (1981)7 Morinaga,
Y., et al., Agric~ Biol. Chem. 45(6) 1425-1430 (1981).
Also, U.S.P. 3,616,224, issued to Shiio et al.~ (1971),
discloses a process for producing various amino acids,
including serine, by fermentation wherein strains of
certain bacteria are cultured on media containing methanol
as the source of assimilable carbon See also U.S.P.
3,943,038, issued to Mbrinaga et al. (1976), which
discloses a method for making serine and other amino acids
by culturing specific strains of various bacteria in an
aqueous culture medium in the presence of oxygen, hydrogen, -
and carbon dioxide.
A common shortcoming of the fermentation methods 1,
taught by the prior art is that the concentration of
L-serine produced in the broth and recovered is relatively
low, even after relatively long fermen~ations. ~i
Serine also can be produced by chemical synthesis
processes. See, for example, Kanedo (ed.), Synthetic
Production and Utilization of Amino Acids, Halsted Press
Books (1974). Often, these chemical procedures produce
serine as a racemic mixture of the D and L optical isomers
or as the less preferred D-isomer. DL- mixtures must be
resolved with methods that utilize D-serine catabolizing
bacteria, serine racemase, by L-amino acid acylase,
fractional crystallization of serine derivatives, or
i
.'
similar methods, thus adding to the cost of the product.
It is known that L-serine can be produced from
glycine. Bioloqical production of L-serine from glycine
has been accomplished with several microorganisms. See,
for example, Tanaka, Y., et al., J. Ferment. Technol.
59:447 (1981). As with the fermentation procedures, a
drawback to this method has been that the yield of L-serine
typically is not very high.
There thus is a need for a method of synthesizing
L-serine in high yields. It therefore is an object of this
invention to develop a method of synthesizing L-serine
wherein the serine is produced in high concentrations in a
solution from which it can be efficiently recovered.
It also is an object of this invention to provide an
enzymatic means of producing L-serine wherein the reaction
can be conducted in a batch or immobilized system.
:
Summary of the Invention
It has been discovered that L-serine can be
synthesized efficiently from glycine and formaldehyde in
the presence of biocatalytic amounts of the enzyme serine
hydroxymethyltransferase and the co-factor tetrahydrofolate - ¦
under serine-producing conditions. The enzyme can be
present in whole cellsr as a crude extractl or as a
purified enzyme and can be in immobilized or
non-immobilized form.
Detailed Description of the Invention
It is known that the enzyme serine hydroxymethyltrans-
ferase (hereinafter referred to as sHMr~ catalyses the
cleavage of serine to glycine in a reaction that is
dependent upon the co-factors pyridoxal-5'-phosphate and
tetrahydrofolate. The reaction yields glycine and
methylené tetrahydrofolate. See Schirck, L~, Advances in
,.', - . I
I
!
Enzymology 53:83 (1982). It now has been discovered that
the SHMT enzyme may be used efficiently as a biocatalyst to
produce L-serine from glycine and formaldehyde. The
reaction takes place in the presence of the co-factor
S tetrahydrofolate(THF)~ The source of the THF may be the
source of the SHMT, for THF is found in microorganism cells
which contain SHMT, or the THF may be added from an
exogenous source. ~ne advanta~e of this method is that it
produces only the L-serine optical isomer. Another
advantage is that after the reaction has been run the
output from the reactor contains only glycine and L-serine
rather than a complex mixture as one would expect to find
in a fermentation broth or as the result of chemical
synthesis.
It is surprising that L-serine can be synthesized from
glycine and formaldehyde, for formaldehyde is known to
chemically react with proteins and cause inactivation of
enzymes. French, D., et al., Advances in Protein Chemistry
2:277-335 (1945). In fact, SHMT is inactivated rapidly by
formaldehyde. It has been discovered, however, that the
enzyme can be protected by adding excess tetrahydrofolate
to the reaction mixture~ The THF reacts with the
formaldehyde, thus protecting the enxyme.
Chemical modification of SHMr also provides stability
and protection From formaldehyde inactivation. For
example, it has been found that reaction of imidoesters
with amino groups (see Means, G., et al., Chemical
Modification of Proteins, Holden-Day, Inc., 1971) on the
enzyme surface allows the enzyme to function in the
presence of higher concentrations of formaldehyde than the
non-modified enzyme.
The enzymatic pathway believed to account for the
synthesis of L-serine from glycine is shown below:
formaldehyde ~ THF<-_> me~hylene-T~F
h ,~
S~MT
methylene THF ~ glycine <~~_> L-serine + TH~
It is preferred that the reaction be carried out under
anaerobic conditions, such as in a nitrogen atmosphere, to
prevent oxidation of the THF.
The substrates, the SHMT, and the cofactor, TH~, can
be reacted together in a variety of ways. Although the
order in which the reactants and catalysts are introduced
are not critical, it is preferred that the glycine and
form~ldehyde are added slowly to SHMT in the presence of
THF. The reaction is run under L-serine producing
conditions. When the E.coli S~MT gene (glyA) is used as
the source of the SHMT, these conditions generally includè
a reaction temperature of from about 4 to about 60C and a
pH in the range of about 4 to about ll. The preferred
reaction conditions include carrying out the reaction at a
temperature of from about 20 to about 45C and a pH of
about 6 to 8.5. If the temperature is below about 4C, the
reaction time is slowed considerably, and if the
temperature rises above about 60C the enzyme can be
denatured. Similarly, at a pH below about 4 or higher than
about ll, the enzyme can be inactivated~ 5HMT is an enzyme
central in the metabolism of microorganisms and higher
organisms; thus, there are many potential sources of the
enzyme. The ranges of conditions under which ~he reaction
to produce L-serine is run are related to the source of the
enzyme used. For example, enzymes obtained from the
thermophilic microorganisms could be used at a higher
temperature than is possible when the enzyme source is
E.coli.
The serine hydroxymethyltransferase may be in the form
of whole cells, a crude extract, or as a purified enzyme.
The enzyme may be used in immobilized or nonimmobilized
form. The enzyme is used in amounts sufficient to catalyze
the reaction.
. .
The enzyme may be obtained from microorganisms that have
been modified using conventional genetic engineering techniques
to produce it in high yields. See Stauffer, G., et al., Gene
14:63-72 (1981). The SHMT gene (glyA) may be isolated and
cloned into a plasmid which then can be used to transform suit-
able host cells resulting in hiah level SHMT expression. ~lu-
tant microorganisms which have been modified in their methio-
nine metabolism also will overproduce SHMT. See Stauffer, G.V.,
and Brenchley, J.E., Genetics 88, 221 (1978) and Stauffer, G.V.,
and Brenchley, J.E., J. Bacteriol. 129, 740 (1977). Using gene
cloning techniques the enzyme activity has been increased as
much as twenty fold and can represent more than ten percent of
the soluble protein of the cell. An E.coli strain (Gx1703)
transformed with such a plasmid, specifically, a derivative of
pBR322 into which the ~y~ gene has been cloned, pGx122, has
been deposited with the Northern Regional Research Laboratory
in Peoria, Illinois, as NRRL No. B-15215~ A Klebsiella aero-
genes strain (Gx1704) transformed with a similar but smaller
plasmid with an alteration causing high copy number, pGx139,
has been deposited with the American Type Culture Collection
in Rockville, Maryland as ATCC No. 39214, and a Salmonella ty-
phimurium strain (Gx1682) transformed with pGx139 has been de-
posited as ATCC No. 39215.
Further, when the gene is taken from a source such as E.
coli it can be modified by random mutagenesis or site directed
mutagenesis to produce an enzyme with improved slability. Al-
ternatively, the gene can be completely chemically synthesized
with multiple changes to improve the enzyme's stability during
the disclosed process.
The THF concentration typically ranges from about 0.15 to
about 50mM.
Using whole cells can provide a source of THF. If de-
sired, additional THF may be added to reach saturation levels,
which are dependent upon pH and temperature. For example,
at pH of about 7.5 and a reaction temperature of about 37C,
in an aqueous solution, THF may be added to
reach a concentration much greater than 50mM. The pH must
be adjusted while THF is dissolvin~. If the SH~ is added
as either a crude extract or a purified enzyme, an
independent source of THF is needed. The amount of THF
which can be added varies with the temperature, solvent
conditions and pH at which the reactior. is run.
It has been discovered that THF retains its activity
towards SH~r when immobilized. Immobilizing THF by
attachin~ it to a support which can be retained inside a
bioreactor used for carryiny out the reaction is
advantageous, for it enhances repeated use of the co-factor
after the synthesis of L-serine is complete. For example,
THF can be immobilized with soluble polymers, such as
dextranr polyethylene glycol, or polyethyleneimine.
Immobilization takes place by means of covalent attachment
in the first two instances and by ionic interaction in the
third. Covalent attachment generally occurs through the
carboxy groups of the THF with an amino group of the
support. Alternatively, using similar attachment methods,
the THF can also be attached to an insoluble support.
Alternatively, if serine is synthesized in a batch
process, it is possible to recycle the THF. For example,
after the L-serine has been synthesized the reaction
solution can be passed through activated charcoal or an ion
exchan~e column, which will retain and separate the THF
from the L-serine product solution. The THF then can be
released and neutralized. Alternatively, the THF may be
~ modified by covalent attachment to a small molecule, such
; as a glucosamine to facilitate recovery of the cofactor.
After the L-serine has been synthesized, the reactor
solution is passed over a borate column. The borate binds
only to the THF-glucosamine, and the modified THF can be
released and recycled.
Glycine and formaldehyde preferably are added to the
tetrahydrofolate-SHMT mixture. The amount of glycine which
can be added varies with the pH, temperature, and solvent
~117~8
conditions of the reaction, but it generally can be added
until the saturation level is reached. ,
As stated previouslv, formaldehyde can be highly toxic
to SHMT. The formaldehyde, therefore, qenerally is added
slowly to the other cGmponents, and its addition is
regulated. The formaldehyde is added in an amount
sufficient to retain enzymatic activity, generally to
maintain a concentration of less than about 10mM higher
than the THF concentration used, although the formaldehyde ',
may be added to maintain a concentration as high as about
50mM greater than the THF concentration used. The greater
the concentration of THF in the system, the higher the
formaldehyde concentration can be maintained, for the THF
reacts favorably with the formaldehyde, thus protecting the
enzyme. The mechanism of the reaction of THF and
formaldehyde is described by Kallen, RoG ~ t et al., J. Biol.
Chem. 241(24) 5851-5863 (1966). The greater the enzyme
activity in the reactor the faster the formaldehyde is
added to maintain the desired concentration. I
It also has been discovered that it may be
advantageous to add a second SHMr co-factor, pyridoxa] 5 7-
phosphate~ to the reactants. Pyridoxal 5'-phosphate is
tightly bound to the enzyme. If L-serine is synthesîzed in
a reactor over an extended period of time, the pyridoxal
phosphate may be lost or inactivated, in which case
additional pyridoxal phosphate may be added. Loss of the
co-factor during the synthesis is indicated by loss of
yellow color of the reaction solution and loss of activity
by the serine hydroxymethyltransferase~ The concentration
of pyridoxal phosphate added to the reaction can vary from
0 to about 20 mM, as needed~ and preferably is from about ,
0.1mM to about 1mM.
The addition of excess pyridoxal phosphate can serve
an additional function. It has been discovered that in 1,
some microorganisms which have been transformed by plasmids
containing the ~ gene and which express high levels of
SHMT the addition of pyridoxal 5'-phosphate is necessary to
i
saturate the SHM~ present and enhance the enzyme activity
observed. One such microorgnaism is Klebsiella aerogenes
containing the plasmid pGx139 identified above.
- The synthesis reaction can be conducted in the
presence of any non-deleterious solvent. Examples of such
solvents include ethanol, methanol, isopropanol and
dioxane.
The following examples are intended to Eurther
illustrate this invention.
Example 1
Bacterial strains with and without plasmid pGx139 were
grown in LB medium (lOg/liter Bacto tryptone, 5g/liter
yeast extract 5g/liter NaCl) or minimal medium (10~5g/liter
K2HP04, 4~5g/liter KH2P04, l.Og/liter
(NH4)2S04 and 0.5g/liter sodium citrate . 2H20)
supplemented with 0.4% glucose or lactose. Amino acids
were added to 20ug/ml and vitamins to 1 ug/ml where
indicated. The specific activity of serine hydroxymethyl
transferase is expressed as nmoles ~-phenyl serine
~0 converted to benzaldehyde and glycine per minute per mg of
extractable protein after sonication of cells.
Assays were in 50mM phosphate buffer at pH 7~3 with
O.lmM pyridoxal phosphate and at 35mM -phenylserine at
20C. The appearance of benzaldehyde was monitored at
279nM in a recording spectrophotometer.
, . . . .
1~1~5~
SHMT
Specific Activity (nmoles/min/mg)
Minimal
Strain Plasmid LB medium Medium Supplement
E.coli
GX1698 - 39 N.D.*
GX1671pGx139 221 N.D.
GX1703pGx139 141 533 glucose,
phenylalanine,
thiamine
GX1703pGx122 218 682 glucose,
phenylalanine,
thiamine
Salmonella typhimurium LT2
GX1682 - 28 10 glucose
tryptophan
17 lactose,
tryptophan
GX1682pGx139 152 ~ tryptophan
:~ ~ 306 lactose,
tryptophan
Klebsiella aerogenes
GX1704 - 36 NoD~
GX1704pGx139 590 114 glucose
223 108 lactose
Strain Genotype
E.coli
GX1698 trpEA , tna2, serB , lac~q ts 402
0 GX1671 thi, ara, strR, glyA, ~serB trpR] , lacIql,
tn5
GX1703 ,~lyA, pheA, thi, lac , ara, strR
' I
I
7~
1 0
Salmonella typhimurium LT2
GX1682 trpBEDC43 F' lacIq ts420 pro
Klebsiella aeroqenes
GX1704 lsd (L-serine deaminase mucant)
* not determined
E.coli strain GX1671 which contains plasmid pGx139, is a
representative additional strain that has been used as the
source of SHMT in the method of this invention
Example 2
One colony of Escherichia coli strain GX 1703
(containing pGx122), which has been deposited as NRRL
No. B-15215 was inoculated into 100ml of culture medium I
described below, and was shaken at 37C overnight.
Culture Medium I:
K2HPo4 10.59/1
KH2PO4 4.5g/1
(NH4)2SO4 1g/1
Sodium citrate - 2H20 0.5g/1
phenylalanine 20ug/ml
20 Vitamin B1 lug/ml
Ampicillin 100ug/ml
MgSO4 2mM
FeSO4 5mg/ml
Cells were cGllected by centrifugation of cell culture
medium and used as enzyme source. Glycine [12mM, final
concentration~ was mixed with tetrahydrofolate (5mM, final
concentration) pyridoxal-phosphate (1mM), and formaldehyde
(lOmM, final concentration) under a nitrogen blanket~ The
pH was maintained at 7.6 with 0.1M potassium phos~hate
buffer and the final volume of this reaction mixture was
100 ml.- Reaction was started by mixing the above mentioned
reaction solution and cells at 37C with shaking. After 8
, :,
1 1
hours, 5mM serine was produced (the yield was 45% based on
glycine). All of the enzyme activity was retained. Glycine
and serine concentrations were determined using high perfor-
mance liquid chromatography.
Example 3
One colony of Klebsiella aerogenes strain Gx1704 (con-
taining pGx139), which has been deposited as ATCC No. 39214,
was inoculated into 100ml of culture medium II described be-
low and was shaken at 30C overnight.
Culture Medium II.
Tryptone 10g/1
NaCl 10g/1
Yeast Extract 5g/1
Glucose 10g/1
Cells were collected by centrifugation of cell culture
medium and used as the enzyme source.
The procedure of Example 2 for serine production was
followed with the exception that Klebsiella aerogenes was
used in place of E.coli. After 8 hours, 7~ serine was pro-
duced (the yield was 64% based on glycine). All the enzyme
activity was retained.
Example 4
The procedure of Example 2 was followed wi-th the excep-
tion that partially purified serine hydroxymethyltransferase
from E.coli (strain Gx1703 [containing pGx122]) was used.
Cells were broken by sonification at 4C. The supernatant
collected after centrifugation was mixed with ammonium sul-
fate (50% saturation) at 4C and pH 7.5. After removing the
solid by the centrifugation, the enzyme was forced out of
the solution by increasing the ammonium sulfate content to
100% sa~uration. The enzyme was collected by centrifugation
and dialyzed. 10mM serine was
12
produced after 4 hours of reaction. The yield was 90
based on glycine. All enzymatic activity was
retained.
_ample 5
The procedure of Example 4 was followed with the
exception that initial glycine concentration was 340mM
and forrnaldehyde (original concentration of 2M) was
introduced at a rate of 1ml per hour. After 12 hours,
119mM serine was produced.
Example 6
The procedure of Example 4 was followed with the
exception that glycine (original concentration of 2M) and
formaldehyde (original concentration of 2M) were introauced
at the same rate of`1ml per hour. After 5 hours, 57mM
serine was produced~
Example 7
The procedure of Example 4 was followed with the
exception that THF was modified. Dextran 15 g, Pharmacia
T40) was dissolved in water to a final concentration of 5~.
Dextran solution was oxidized by 0.l M NaIO~ for l hour
at room temperature. Oxidized dextran was precipitated out
by adding ethanol to 60% (v/v)O This step was repeated
twice~ Oxidized dextran was dissolved in l00 ml of 0.2 M
1.6-hexanediamine (HMD) at pH 9Ø Sodium borohydride (0.2
9) was added after 30 and 60 minutes respectively.
HMD-dextran was dialyzed against water overnight and
lypholized. THF (5 mM) was mixed with l0 mM
l-ethyl-3-(3-dimethylaminopropyi~ carbodiimide and 0.5%
HMD-dextran at pH 7.0 under nitrogen atmosphere. The
precipit-ate fo-rmed was collected by centrifugation and was
washed twice with 0.5 M NaCl solution. The solid THF was
~ " ~
,, ;~ ',
''' ~.Z~,7~58' 1
13
mixed with reaction mixture as described in Example 3,
After 3 hours of rec~ion, 7 mM serine was prodoced.
.
~ . .
:
: ~ , : :
' :
: .
