Ex Parte Usuda et alDownload PDFPatent Trial and Appeal BoardJul 12, 201312202484 (P.T.A.B. Jul. 12, 2013) Copy Citation 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). Appeal 2012-001609 Application 12/202,484 2 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). Appeal 2012-001609 Application 12/202,484 3 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). Appeal 2012-001609 Application 12/202,484 4 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 Appeal 2012-001609 Application 12/202,484 5 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 Appeal 2012-001609 Application 12/202,484 6 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). Appeal 2012-001609 Application 12/202,484 7 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- Appeal 2012-001609 Application 12/202,484 8 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. Appeal 2012-001609 Application 12/202,484 9 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 Appeal 2012-001609 Application 12/202,484 10 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 Appeal 2012-001609 Application 12/202,484 11 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 lp Copy with citationCopy as parenthetical citation