Ex Parte Usuda et al

Patent Trial and Appeal Board

Jul 12, 2013

12202484 (P.T.A.B. Jul. 12, 2013)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
__________

Ex parte YOSHIHIRO USUDA and KAZUHIKO MATSUI
1

__________

Appeal 2012-001609
Application 12/202,484
Technology Center 1600
__________

Before DEMETRA J. MILLS, MELANIE L. McCOLLUM, and
JEFFREY N. FREDMAN, Administrative Patent Judges.

McCOLLUM, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 involving claims to a method
for producing an L-amino acid. The Examiner has rejected the claims as
obvious. We have jurisdiction under 35 U.S.C. § 6(b). We affirm.
STATEMENT OF THE CASE
The Specification discloses “a method of culturing a bacterium
belonging to the family Enterobacteriaceae which is able to produce an

1
Appellants identify the Real Party in Interest as Ajinomoto Co., Inc. (App.
Br. 3).
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L-amino acid in a medium containing glycerol[, specifically crude glycerol,]
as the carbon source” (Spec. ¶ [0006]). The Specification states that
“[c]rude glycerol refers to industrially produced glycerol, which will contain
impurities” (id. at ¶ [0022]). The Specification also states that “[c]rude
glycerol is industrially produced by hydrolyzing fats or oils with water at a
high temperature and under high pressure, or during biodiesel fuel
production via the esterification reaction” (id.).
Claims 1, 4, 5, 9, 10, 14, and 15 are on appeal (App. Br. 3).
2

Claims 1, 10, and 15 are representative and read as follows:
1. A method for producing an L-amino acid comprising:
A) culturing a bacterium belonging to the Enterobacteriaceae family
which is able to produce an L-amino acid when cultured in a medium
containing glycerol as the carbon source, and
B) collecting the L-amino acid from the medium;
wherein the concentration of the glycerol in the medium at the start of the
culture is 1 to 30% w/v, and wherein crude glycerol is added to the medium.
10. The method according to claim 9, wherein the L-amino acid is
L-threonine, and the activity of an enzyme selected from the group
consisting of aspartokinase I, homoserine kinase, aspartate aminotransferase,
threonine synthase which are encoded by the thr operon, aspartate
semialdehyde dehydrogenase, and combinations thereof, is increased in the
bacterium.
15. The method according to claim 1, wherein said crude glycerol
contains ions in an amount of from 2 to 7% based on the weight of the crude
glycerol.

2
Claims 11-13 are also pending but have been withdrawn from
consideration (App. Br. 3).
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Claims 1, 4, 5, 9, 10, 14, and 15 stand rejected under 35 U.S.C.
§ 103(a) as obvious over Watanabe,
3
Gonzalez-Pajuelo,
4
Barbirato,
5
and
Colin
6
(Ans. 4-5).
The Examiner relies on Watanabe for teaching “that E. coli (ATCC-
21248) accumulates large quantities of L-threonine in the presence of about
20mg to about 100 mg of methionine and about 20 to about 800 mg of
L-valine or L-leucine per liter of nutrient medium,” that the “E. coli medium
comprises a carbon source, a source of nitrogen and a source of metallic
ion,” that, “[a]s a carbon source, it is possible to use monosaccharides, but
the bacteria can also use polyhydric alcohols, such as glycerine (i.e.,
glycerol),” and that “[s]uitable amounts of the carbon source range from
about 3-8% by weight of the medium” (id. at 5).
The Examiner relies on Gonzalez-Pajuelo for teaching “that
Clostridium butyricum VPI 3266 was grown and produced 1,3-propanediol
when cultured in the presence of raw glycerol obtained from biodiesel
production” (id.). The Examiner relies on Barbirato for teaching “that raw
glycerol is produced when agricultural crops are converted to produce ester
and bioethanol and that this raw glycerol was then converted into

3
Kiyoshi Watanabe et al., US 3,616,217, Oct. 26, 1971.
4
M. González-Pajuelo et al., Production of 1,3-propanediol by Clostridium
butyricum VPI 3266 using a synthetic medium and raw glycerol, 31 J. IND.
MICROBIOL. BIOTECHNOL. 442-446 (2004).
5
Fabien Barbirato et al., 1,3-propanediol production by fermentation: An
interesting way to valorize glycerin from the ester and ethanol industries, 7
INDUSTRIAL CROPS AND PRODUCTS 281-289 (1998).
6
Thierry Colin et al., Effects of Acetate and Butyrate During Glycerol
Fermentation by Clostridium butyricum, 43 CURRENT MICROBIOLOGY 238-
243 (2001).
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1,3-propanediol by a number of different bacteria” (id. at 5-6). The
Examiner concludes:
It would have been obvious for an artisan to take the E. coli
taught by Watanabe et al., culture it in the presence of crude
glycerol, and obtain[] threonine following culture . . . because
Gonzalez-Pajuelo et al. and Barbirato et al. teach that artisans
have been culturing bacteria in the presence of crude glycerol
and obtaining a product of interest following culturing.
(Id. at 6.)
The Examiner relies on Colin, as well as Gonzalez-Pajuelo, for
teaching “that the amount of sodium in crude glycerol affects the growth of
the microorganism and cell division” (id.). Thus, the Examiner concludes:
“[A]n artisan would understand that varying amounts of sodium in crude
glycerol would need to be tested in order to optimize the growth of the
microorganism and the amount of product (e.g. amino acid) made. As such,
the amount of sodium in crude glycerol is a matter of routine optimization.”
(Id.)
PRINCIPLES OF LAW
“The combination of familiar elements according to known methods
is likely to be obvious when it does no more than yield predictable results.”
KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007).
“[I]t is elementary that the mere recitation of a newly
discovered function or property, inherently possessed by things
in the prior art, does not cause a claim drawn to those things to
distinguish over the prior art. Additionally, where the Patent
Office has reason to believe that a functional limitation asserted
to be critical for establishing novelty in the claimed subject
matter may, in fact, be an inherent characteristic of the prior art,
it possesses the authority to require the applicant to prove that
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the subject matter shown to be in the prior art does not possess
the characteristic relied on.”
In re Best, 562 F.2d 1252, 1254-55 (CCPA 1977) (quoting In re Swinehart,
439 F.2d 210, 212-13 (CCPA 1971)). “Whether the rejection is based on
„inherency‟ under 35 U.S.C. § 102, on „prima facie obviousness‟ under 35
U.S.C. § 103, jointly or alternatively, the burden of proof is the same.” In re
Best, 562 F.2d at 1255.
Moreover, “it is well settled that unexpected results must be
established by factual evidence. „Mere argument or conclusory statements
in the specification does not suffice.‟” In re Geisler, 116 F.3d 1465, 1470
(Fed. Cir. 1997) (quoting In re De Blauwe, 736 F.2d 699, 705 (Fed. Cir.
1984)).
ANALYSIS
Watanabe discloses a process for producing L-threonine by cultivating
a mutant of E. coli, namely ATCC-21248, in a nutrient medium containing a
carbon source (Watanabe, col. 1, ll. 42-46 & col. 2, ll. 5-10). As the carbon
source, Watanabe discloses that “it is possible to use . . . polyhydric
alcohols, for example, glycerine,” and that “[s]uitable amounts of the carbon
source range from about 3 to 8 percent by weight of the medium” (id. at
col. 2, ll. 10-22).
Gonzalez-Pajuelo discloses the production of 1,3-propanediol by
Clostridium butyricum VPI 3266 using raw glycerol, obtained from the
biodiesel production process, as the carbon source (Gonzalez-Pajuelo,
Abstract). Barbirato discloses the production of 1,3-propanediol by
Clostridium butyricum using glycerol from ester production and from wine
stillage (Barbirato, Abstract). We agree with the Examiner that it would
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have been obvious to use crude glycerol, as described in Gonzalez-Pajuelo
and Barbirato, as a carbon source in the method described in Watanabe in
order to valorize crude glycerol (Ans. 6 & 17).
Appellants argue, however, that “crude glycerol contains a substantial
amount of salts, which is known by those in the art to increase the osmotic
pressure in the medium and cause an adverse effect on the bacterial growth”
(App. Br. 9). Therefore, Appellants argue that “simply combining the
teachings of Watanabe, Gonzalez, and Barbirato does not actually suggest
that simply substituting crude glycerol for glycerol will be successful for
producing any type of product” (id.). In support of this position, Appellants
rely on Underwood,
7
Record,
8
and Choi,
9
as well as Mu
10
(id. at 9-10). We
are not persuaded.
First, we agree with the Examiner that claim 1 does not require that all
of the glycerol in the medium be crude glycerol and therefore encompasses a
mixture of reagent glycerol and crude glycerol (Ans. 9). In addition, we are
not persuaded by Appellants‟ argument that one of ordinary skill in the art

7
S. A. Underwood et al., Lack of Protective Osmolytes Limits Final Cell
Density and Volumetric Productivity of Ethanologenic Escherichia coli
KO11 during Xylose Fermentation, 70 APPLIED & ENVIRONMENTAL
MICROBIOLOGY 2734-2740 (2004).
8
M. Thomas Record et al., Responses of E. coli to osmotic stress: large
changes in amounts of cytoplasmic solutes and water, 23 TIBS 143-148
(1998).
9
Won J. Choi, Glycerol-Based Biorefinery for Fuels and Chemicals, 2
RECENT PATENTS ON BIOTECHNOLOGY 173-180 (2008).
10
Ying Mu et al., Microbial production of 1,3-propanediol by Klebsiella
pneumoniae using crude glycerol from biodiesel preparations, 28
BIOTECHNOL. LETT. 1755-1759 (2006).
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would not have had reason to mix reagent glycerol with crude glycerol
(Reply Br. 4). As noted by Appellants, “the whole reason for using raw or
crude glycerol is to lower the cost of the chosen carbon source” (id.).
Although replacing all of the glycerol with crude glycerol may cause the
biggest reduction in cost, it is not clear to us why replacing a portion of the
glycerol with crude glycerol would not also lower the overall cost of the
carbon source. We also agree with the Examiner that providing this mixture
is one way to reduce the overall amount of impurities, such as salts (Ans. 9).
Moreover, we do not agree that the references relied upon by
Appellants teach away from the claimed method. As noted by Appellants,
Choi was “published after the filing date of the instant application” (App.
Br. 9). Thus, Appellants have not established that Choi is relevant to the
question of whether there would have been a reasonable expectation for
success at the time of the present invention.
Underwood states that there is “[l]imited cell growth and the resulting
low volumetric productivity of ethanologenic Escherichia coli KO11 in
mineral salts medium containing xylose” (Underwood, Abstract). However,
Underwood also discloses ways to deal with this problem, namely that
“[t]wo independent genetic modifications of E. coli KO11 . . . and the
addition of a metabolite, such as glutamate (11 mM) or acetate (24 mM), as
a supplement each increased the intracellular glutamate pool during
fermentation, doubled cell growth, and increased volumetric productivity”
and that this “apparent requirement for a larger glutamate pool for increased
growth and volumetric productivity was completely eliminated by the
addition of a protective osmolyte (2 mM betaine or 0.25 mM dimethyl-
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sulfoniopropionate)” (id.). Thus, we do not agree with Appellants that
Underwood teaches away from the claimed invention.
As noted by Appellants, “Record describes that the growth rate of the
bacteria decreases when external osmolarity increases as a result of the
addition of NaCl” (App. Br. 9 (citing Record, Fig. 1)). However, Record
also discloses that “Escherichia coli is capable of growing in environments
ranging from very dilute aqueous solutions of essential nutrients to media
containing molar concentrations of salts or nonelectrolyte solutes” (Record,
Abstract). Thus, rather than teaching away from the invention, we conclude
that Record suggests that E. coli is capable of growing in media containing
salts.
Furthermore, Mu discloses that “1,3-Propanediol (1,3-PD) was
produced by Klebsiella pneumoniae using crude glycerol obtained from
biodiesel production” (Mu, Abstract). As such, we agree with the Examiner
that Mu provides support for the position that “species of bacteria, other than
Clostridium, can be cultured in crude glycerol” (Ans. 10). We also agree
with the Examiner “that disclosed examples and preferred embodiments do
not constitute a teaching away from a broader disclosure or nonpreferred
embodiments” (id.).
We note Appellants‟ argument that the “results achieved by the
inventors by using crude glycerol in bacterial fermentation to obtain
increased production of a product were actually unexpected and surprising”
(App. Br. 9-10). However, Appellants have not pointed to sufficient
evidence that the claimed method provides an unexpectedly superior result.
We are therefore unpersuaded by this argument.
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With regard to claim 10, the Examiner finds that “this increase in
enzyme activity is inherent to the bacteria,” noting that Watanabe teaches
“that their bacteria produce about 10g/L of threonine, as compared to other
bacteria that produce 2-3g/ L” (Ans. 7). The Examiner concludes that “this
is indicative that an enzyme that synthesizes threonine (e.g. threonine
synthase) is increased” (id.).
Appellants argue that “it is unclear how [inherency] is possible since
this is an active step in the method” (App. Br. 11). We are not persuaded.
Although the Specification may describe ways to increase the activity of a
particular enzyme (Spec. ¶¶ [0042]-[0045]), we conclude that the Examiner
was reasonable in interpreting claim 10 to encompass a mutant bacterium
that has increased enzyme activity (Ans. 11-12). Moreover, Appellants
admit that “[w]hether this increase [in enzymatic activity] occurs
endogenously or exogenously is immaterial, as both are encompassed by the
claim” (Reply Br. 4). Thus, claim 1 encompasses an inherent increase in
endogenous enzymatic activity of the mutant bacterium of Watanabe, which
is associated with increased threonine production. Therefore, we conclude
that the Examiner has provided a sufficient basis to shift the burden to
Appellants to demonstrate that Watanabe‟s mutant bacterium does not have
increased activity of one or more of the enzymes recited in claim 10 (Ans. 7-
8). Appellants have not met this burden.
With regard to claim 15, Colin discloses:
During the preparation of culture media and fermentation, the
acids added and/or produced were neutralized by the addition of
sodium hydroxide. However, studies performed on Clostridium
pasteurianum . . . , C. botulinum . . . and C. butyricum . . . had
shown that sodium ion concentrations higher than 12 gL
-1

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inhibited cell growth. At concentrations lower than 12 gL
-1
, the
inhibitory effect of sodium ions was not pronounced. During
our study, the quantities of sodium ions added during acid
neutralization never exceeded 8 gL
-1
.
(Colin 242.) Given this teaching, as well as the disclosure in Gonzalez-
Pajuelo that “raw glycerol contains other substances such as sodium salts
and heavy metals in concentrations that might interfere with cell division”
(Gonzalez-Pajuelo 445), we agree with the Examiner that “an artisan would
understand that varying amounts of sodium in crude glycerol would need to
be tested in order to optimize the growth of the microorganism and the
amount of product (e.g. amino acid) made” (Ans. 6).
Appellants argue that “there would have been no motivation to
practice the invention as claimed with any expectation of success based on
the knowledge in the art and the issues in production of L-amino acids via
fermentation” (App. Br. 12). While we agree with Appellants that neither
Colin nor Gonzalez-Pajuelo specifically relate to bacterium belonging to the
Enterobacteriaceae family, Appellants admit that “crude glycerol contains a
substantial amount of salts, which is known by those in the art to increase
the osmotic pressure in the medium and cause an adverse effect on the
bacterial growth” (id. at 9). In addition, as noted in the Specification:
In the biodiesel fuel production process, the alkaline catalyst
method is typically used for the transesterification, and acids
are added for neutralization. As a result, crude glycerol
containing water and impurities is produced, and typically is
about 70 to 95% pure by weight. Crude glycerol produced in
the biodiesel fuel production contains, in addition to water,
residual methanol, alkali salts such as NaOH which acts as a
catalyst, and an acid, such as K2SO4, which acts to neutralize
the alkali. Although it depends on the manufacturer and the
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production method, the content of such salts and methanol can
be several percent.
(Spec. ¶ [0022] (emphasis added).) Thus, we agree with the Examiner that it
would have been obvious to optimize the amount of salts in the crude
glycerol, such as by selecting crude glycerol having the claimed amount of
ions.
CONCLUSION
The evidence supports the Examiner‟s conclusion that Watanabe,
Gonzalez-Pajuelo, Barbirato, and Colin suggest the methods of claims 1, 10,
and 15. Claims 4, 5, and 9 have not been argued separately and therefore
fall with claim 1. Claim 14 has been argued with claim 15 and therefore
falls with claim 15. 37 C.F.R. § 41.37(c)(1)(vii).
TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).

AFFIRMED

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