DETAILED ACTION
All objections and rejections raised in prior Office Actions are withdrawn unless restated below.
Claims 5, 7, 9, 11, 67 and 71-72 remain withdrawn.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Interpretation
Culturing is understood in the art as meaning propagation. From Taber’s Medical Dictionary, Culture, retrieved 08/03/2026, www.tabers.com/tabersonline/view/Tabers-Dictionary/737315/all/culture:
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All references to an amylase below are references to an alpha-amylase.
Claim Objections
Claim 32 is objected to because of the following informalities:
In claim 32, recitation of “the production” (repeated three times) should preferably be “production.” The use of the definite article “the” should be avoided unless grammatically needed.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-3, 6, 16-18, 31, 32 and 34 (all non-withdrawn claims) are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1, in part, recites:
wherein the liquefaction medium comprises:
. . .
(b) a second recombinant yeast host cell to obtain a second inactivated yeast product in the fermentation medium.
That is, claim 1 recites that the liquefaction medium comprises the second inactivated yeast product; however, the second inactivated yeast product is defined as “in the fermentation medium.” Further, the recited first inactive yeast product and the recited second inactivated yeast product appear to have the same structure except that the second yeast product is defined as “in the fermentation medium.”
As such, it is unclear if in embodiments wherein the second inactivated yeast product is implemented if one or both are required:
--second inactivated yeast product is in the liquefaction medium, and
--second inactivated yeast product is in the fermentation medium.
For this reason an ordinarily skilled artisan cannot determine how to avoid infringement of claim 1. All non-withdrawn claims depending from claim 1 are included in this rejection since claim 1 recites (a) first inactivated yeast product and/or (b) second inactivated yeast product such that all claims explicitly include a combination of the first inactivated yeast product and the second inactivated yeast as an expressly recited embodiment of all of claims 1-3, 6, 16-18, 31, 32 and 34. That is, none of the rejected claims are limited to the first inactivated yeast product excluding the second inactivated yeast product.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 17 and 18 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The purpose of the written description requirement is to ensure that the inventor had possession, at the time the invention was made, of the specific subject matter claimed. For a broad generic claim, the specification must provide adequate written description to identify the genus of the claim.
“A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) (“In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus.”). Regents of the University of California v. Eli Lilly & Co., 119, F.3d 1559, 1568, 43 USPQ2d 1398, 1405 (Fed. Cir. 1997).
“The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice . . ., reduction to drawings . . ., or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus.” MPEP 2163(II)(3)(a).
Furthermore, a “‘representative number of species’ means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. The disclosure of only one species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure ‘indicates that the patentee has invented species sufficient to constitute the gen[us].’ See Enzo Biochem, 323 F.3d at 966, 63 USPQ2d at 1615; Noelle v. Lederman, 355 F.3d 1343, 1350, 69 USPQ2d 1508, 1514 (Fed. Cir. 2004) (Fed. Cir. 2004) (‘[A] patentee of a biotechnological invention cannot necessarily claim a genus after only describing a limited number of species because there may be unpredictability in the results obtained from species other than those specifically enumerated.’). ‘A patentee will not be deemed to have invented species sufficient to constitute the genus by virtue of having disclosed a single species when … the evidence indicates ordinary artisans could not predict the operability in the invention of any species other than the one disclosed.’ In re Curtis, 354 F.3d 1347, 1358, 69 USPQ2d 1274, 1282 (Fed. Cir. 2004).” MPEP 2163(II)(3)(a).
Claims 17 and 18 recite a genus of amylolytic enzymes having trehalase activity or xylanase activity.
The specification (page 14) defines amylolytic enzyme/activity as follows: “As used herein, the expression "amylolytic enzyme" refers to a class of enzymes capable of hydrolyzing starch or hydrolyzed starch.” This is understood as a limiting definition that any embodiment amylolytic enzyme embodiment of the claims must have activity to hydrolyze starch.
In view of the above, claims 17 and 18 recite the following genera of enzymes:
--a genus of enzymes that are both an amylolytic enzyme and having trehalase activity including enzymes having SEQ ID NO: 70 or 71 (and 80% identity thereto); and
--a genus of enzymes that are both an amylolytic enzyme and having xylanase activity including enzymes having SEQ ID NO: 72 (and 80% identity thereto).
Trehalase activity is not an enzymatic activity understood in the art as related to ability to hydrolyze starch as required for an amylolytic enzyme. The specification does not demonstrate that any enzyme, including enzymes having SEQ ID NO: 71 and 71, has or is capable of having amylolytic activity and trehalase activity as recited. Gao et al. (Characterization, heterologous expression and engineering of trehalase for biotechnological applications, Systems Microbiol. Biomanuf. 2, 2022, 445-60) evidences that “Trehalase is a trehalose hydrolase that hydrolyzes one molecule of trehalose into two molecules of glucose (Fig. 1), wherein trehalose is a disaccharide of glucose linked by an alpha-1,1,-glycosidic bond. Gao, page 445, right col. Starch (a polysaccharide of glucose linked by alpha-1,4-glycosidic bonds) is a substantially different structure from trehalose such that an ordinarily skilled artisan at time of filing cannot recognize that activity as a trehalase and activity as an amylolytic enzyme would be present in the same enzyme in absence of evidence showing the same. That is, an enzyme having both trehalase activity and amylolytic enzyme is unpredictable in the art. While the specification asserts that SEQ ID NO: 70 or 71 has such dual activity, however, the operability of such an enzyme to have dual activity as recited cannot be predicted by ordinarily skilled artisans in view of the large structural difference between starch and trehalose.
Similarly, “The term "xylanase" is the name given to a class of enzymes which degrade the linear polysaccharide beta-1,4-xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell walls.” Specification, page 16. Again, starch does not contain xylose saccharide residues nor xylan as part of its structure. As such, it is unpredictable whether any individual enzyme can have the recited dual activity of an amylolytic enzyme and a xylanase, including the enzyme of SEQ ID NO: 72. An assertion that an enzyme may have such dual activity is not sufficient to overcome such unpredictability as to prove an adequate written description of the genus of dual activity enzymes recited.
“The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice . . ., reduction to drawings . . ., or by disclosure of relevant, identifying characteristics.” Here, there is no actual reduction to practice of any enzyme having dual activity as an amylolytic enzyme and a trehalase nor any enzyme having dual activity as an amylolytic enzyme and a xylanase as recited. While specific enzymes are named, any ability to have dual activity is not demonstrated. Further, there is no overt discussion of relevant, identifying characteristics for enzymes that would be predictably recognized to have dual activity as an amylolytic enzyme and a trehalase or having dual activity as an amylolytic enzyme and a xylanase. As such, the claims lack adequate written description for these reasons.
It is recommended the claims be amended to recite a trehalase and a xylanase without the requirement that the same further be an amylolytic enzyme.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1-3, 6, 16-18 and 34 is/are rejected under 35 U.S.C. 103 as being unpatentable over Aux (U.S. 2009/0221041 A1) further in view Callen et al. (U.S. 2003/0125534 A1), Wong et al. (Increased Expression and Secretion of Recombinant a-Amylase in Saccharomyces cerevisiae by Using Glycerol as the Carbon Source, J. Protein Chem. 21, 2002, 419-425) and Kovaleva et al. (Synthesis and secretion of bacterial a-amylase by the yeast Saccharomyces cerevisiae, FEBS Lett. 251, 1989, 183-86).
Aux, abstract, states:
The presently disclosed subject matter provides a process for starch liquefaction using at least two classes of α-amylase enzymes, wherein the starch hydrolysis pattern from at least two of these classes is different. At least one class of enzyme is provided to the liquefaction process in the form of transgenic plant material expressing at least one class of a-amylase enzyme or is provided in the form of a purified or partially-purified α-amylase enzyme preparation. The second or subsequent class(es) of α-amylase enzymes may be provided in the form of additional transgenic plant material expressing the second or subsequent class(es), or may be provided in the form of a second or subsequent purified or partially-purified α-amylase enzyme preparation.
Aux, in the claims, states:
21. A method for producing a biofuel comprising:
a) liquefying an aqueous slurry of starch-containing plant material [i.e. a liquefaction medium] in the presence of at least a first and a second class of α-amylase enzymes, wherein the first class of α-amylase enzymes exhibits a starch hydrolysis pattern that is different from the starch hydrolysis pattern of at least the second class of α-amylase enzymes to obtain a liquefact; and,
b) fermenting the liquefact to produce said biofuel [the same being a fermentation medium when subject to fermentation].
26. The method of claim 21, wherein the fermentation is a yeast fermentation.
“In various embodiments of the present invention, either the first or the second class of α-amylase enzymes, or both, is provided as a crude, purified or partially-purified preparation of the α-amylase enzyme. The exogenously-added α-amylase enzyme may be de novo synthesized, or may be isolated from an organism expressing the α-amylase enzyme prior to addition of the enzyme to the starch-containing plant material, or may be through the addition of a crude extract containing at least one enzyme useful in starch conversion.” Aux, para. [0028].
“Alpha-amylase enzymes can be expressed in and isolated from any number of eukaryotic and prokaryotic organisms. Appropriate expression cassettes, vectors, transformation, and transfection techniques for a particular organism of interest will be evident to one of skill in the art.” Aux, para. [0030].
“In another embodiment, fungal hosts, such as fungal host cells belonging to the genera Aspergillus, Rhizopus, Trichoderma, Nurerospora, Mucor, Penicillium, etc., such as yeast belonging to the genera Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Schwanniomyces, etc. may be used.” Aux, para. [0032].
“Additional methods for generating an enzyme extract are described in [several references cited].” Aux, para. [0034].
“[L]iquefying an aqueous slurry [i.e. a liquefaction medium] of starch-containing plant material in the presence of at least a first and a second class of α-amylase enzymes, wherein the first class of α-amylase enzymes exhibits a starch hydrolysis pattern that is different from the starch hydrolysis pattern of at least the second class of α-amylase enzymes to obtain a liquefact [i.e. a fermentation medium when subject to fermentation to produce ethanol],” is considered to be a disclosure of liquefying a liquefaction medium to obtain a fermentation medium as recited in claim 1. Aux, para. [0005], claim 21. “[F]ermenting the liquefact [i.e. fermentation medium] to produce said biofuel,” is considered to be a disclosure fermenting the fermentation medium with a fermenting to yeast (as recited in claim 26 of Aux) to obtain ethanol as a fermentation product.
Regarding recitation that the liquefaction medium contains a first inactivated yeast product made from a first recombinant yeast host cell, wherein the first recombinant yeast host cell comprises a first heterologous nucleic acid molecule for expressing a first heterologous enzyme and the first inactivated yeast product comprises the first heterologous enzyme (and wherein the first heterologous nucleic acid molecule is operatively associated with a first promoter allowing expression of the first heterologous enzyme during propagation of the first recombinant yeast host cell), Aux as discussed above provides that “In various embodiments of the present invention, either the first or the second class of α-amylase enzymes, or both, is provided as a crude,” and that further such alpha-amylase can be expressed in and isolated from any number of eukaryotic and prokaryotic organisms including yeasts. Aux, paras. [0028] and [0032]. It is noted that para. [0028] of Aux is under a heading “Enzyme Extracts,” such that it is understood that cellular extracts containing the alpha-amylase are taught. When the host cell for expression is a yeast, the enzyme extract or crude is a yeast extract. “Furthermore, the partially-purified enzyme preparation may also be culture supernatant or crude extract collected from a cell population expressing and/or secreting the enzyme,” which is a yeast extract when collected from a yeast cell population. Aux, para. [0029].
The above is considered to be a disclosure of forming a crude enzyme extract from a recombinant yeast expressing a vector encoding an appropriate alpha-amylase and adding the same to the “aqueous slurry of starch-containing plant material” (i.e. liquefaction medium) as set forth in claim 21 of Aux wherein such a crude enzyme extract prepared from a yeast host cell is a first inactivated yeast product from a first recombinant yeast host cell, wherein the first recombinant yeast host cell comprises a first heterologous nucleic acid molecule for expressing a first heterologous enzyme and the first inactivated yeast product comprises the first heterologous enzyme being an alpha-amylase. More specifically, a crude enzyme extract from a recombinant yeast cell is within the broadest reasonable interpretation of a yeast extract. Corn is stated as a preferred starch-containing plant material. Aux, paras. [0012] and [0042].
Regarding recitation of the claims of “improve the yield of the fermentation product,” “In one embodiment, the liquefact is further processed to produce ethanol. In one embodiment, the use of at least two different classes of α-amylase enzymes in the liquefaction process results in a substrate that leads to higher ethanol yields compared to the ethanol yield from starch-containing plant material that is exposed to only one class of α-amylase enzymes.” Aux, para. [0043]. That is, Aux indicates that performing liquefaction increases product yield as compared to not performing any step of liquefaction.
Regarding specific identity of alpha-amylase (and how the same is expressed as recited in claim 1), Aux states: “Other amylases such as BD1 2870, Thermococcales derived amylases, unimodal or bimodal amylases may be combined essentially as described in Example 3 to show a synergistic dual mode of action benefit.” Aux, para. [0131]. “Alpha-amylases for example, such as those described in US Patent Publication US2003/0125534 [i.e. Callen] . . . may be further characterized in regards to their respective starch hydrolysis pattern and be further employed in starch hydrolysis as described herein.” That is, Aux directly cites Callen.
Callen, abstract, states:
The invention relates to alpha amylases and to polynucleotides encoding the alpha amylases. In addition methods of designing new alpha amylases and methods of use thereof are also provided. The alpha amylases have increased activity and stability at acidic, neutral and alkaline pH and increased temperature.
The alpha-amylases of Callen are expressly taught to be suitable for “particularly useful in corn-wet milling processes, detergents, baking processes, beverages and in oilfields (fuel ethanol), and also for liquefaction of starch. Callen, paras. [0003] and [0051].
“In yet another aspect, the invention provides an isolated nucleic acid encoding a polypeptide having a sequence as set forth in SEQ ID Nos.: 2, 4, 6, 10, 12, 14 .. . . 74. . . 80” Callen, para. [0018]. “Another aspect of the invention is an isolated nucleic acid encoding a polypeptide or a functional fragment thereof having a sequence as set forth in SEQ ID Nos.: 2, 4, 6, 10, 12, 14 . . . 74 . . . 80 (hereinafter referred to as “Group B amino acid sequences”), and sequences substantially identical thereto.” Callen, para. [0019].
“The polypeptides of Group B amino acid sequences, and sequences substantially identical thereto or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof, may also be used in the liquefaction and saccharification of starch. . . . In a preferred embodiment, the polypeptides or fragments thereof of this invention are thermostable at 90-95° C.” Callen, para. [0244].
SEQ ID NO: 74 of Callen has about 99% identity to recited SEQ ID NO: 63 as to be a variant of the same.
“Polynucleotides selected and isolated as hereinabove described are introduced into a suitable host cell. A suitable host cell is any cell which is capable of promoting recombination and/or reductive reassortment. The selected polynucleotides are preferably already in a vector which includes appropriate control sequences. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast.” Callen, para. [0127].
“Another aspect of the invention is a method of making a polypeptide having a sequence as set forth in Group B amino acid sequences, and sequences substantially identical thereto. The method includes introducing a nucleic acid encoding the polypeptide into a host cell, wherein the nucleic acid is operably linked to a promoter, and culturing the host cell under conditions that allow expression of the nucleic acid.” Callen, para. [0025]. The preceding from Callen, para. [0025], is a teaching of a first heterologous nucleic acid molecule encoding the amylase operatively associated with a control sequence (promoter) allowing for expression of the amylase during culturing (propagation).
Callen, in the claims, further provides:
106. A host cell as claimed in claim 47, 102, 103 or 105, wherein the host is selected from the group consisting of prokaryotes, eukaryotes, funguses, yeasts, plants and metabolically rich hosts.
In review of the above:
Aux teaches a liquefaction method for production of ethanol form corn wherein “In various embodiments of the present invention, either the first or the second class of α-amylase enzymes, or both, is provided as a crude, purified or partially-purified preparation of the α-amylase enzyme. Aux, para. [0028].
Aux, para. [0032], and Callen, para. [0127], states that yeast including Cerevisiae are a suitable host cell for production of amylase used for liquefaction.
“Another aspect of the invention is a method of making a polypeptide having a sequence as set forth in Group B amino acid sequences, and sequences substantially identical thereto. The method includes introducing a nucleic acid encoding the polypeptide into a host cell, wherein the nucleic acid is operably linked to a promoter, and culturing the host cell under conditions that allow expression of the nucleic acid.” Callen, para. [0025].
As such, Callen teaches that in producing an appropriate amylase in any host cell including yeasts, a first heterologous nucleic acid sequence is operatively associated with a first promoter allowing expression of the first heterologous enzyme during culturing, that is propagation, of a first recombinant yeast host cell.
Neither Aux nor Callen exemplify the actual production of amylase in a yeast or Saccharomyces cerevisiae host cell. “Alpha-amylase enzymes can be expressed in and isolated from any number of eukaryotic and prokaryotic organisms. Appropriate expression cassettes, vectors, transformation, and transfection techniques for a particular organism of interest will be evident to one of skill in the art.” Aux, para. [0030].
That is, techniques for expression of heterologous proteins in common host cells including yeasts are well understood and established as to be “evident to one of skill in the art.” Regardless, Wong et al. exemplifies expression of amylase in S. cerevisiae.
From Wong, abstract:
Saccharomyces cerevisiae transformed with plasmids containing the barley α-amylase gene was cultured, and enzyme activity and cell density were monitored at various time intervals. Proteins in yeast extract and culture medium were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Western blots of intra- and extracellular proteins were sequentially probed with anti-amylase antibody and anti-rabbit horseradish peroxidase conjugate, followed by chemiluminescent detection. The enzyme activity of recombinant barley α-amylase secreted by the yeast clone DY150[pYEX-Amy1] showed a significant increase when the culture medium included glycerol as the carbon source. The enhancement reached a 4.5-fold increase at 120 hr, and the effect was strain-nonspecific. Intra- and extracellular proteins increased significantly with time in both the yeast clone and the control grown in YEPG (2% yeast extract, 1% bacto-peptone, 2% glycerol). Proteins in YEPD (2% yeast extract, 1% bacto-peptone, 2% glucose) and YEPG cultures showed very different band patterns, indicating that the metabolic pathway was altered. Western blot analysis indicated that the recombinant amylase accumulated inside yeast cells, at a relatively low level, compared with that in the culture medium.
From Wong, Sec. 2.2:
The cDNA coding for the barley a-amylase 1 isozyme was isolated from a barley cDNA library. The truncated cDNA was 59 end-filled, and cloned downstream of the phosphoglycerate kinase promoter in the pYEX vector [i.e. nucleic acid encoding alpha-amylase is operatively linked to a promoter for expression].
The above is a description of a first recombinant yeast host cell having a first heterologous nucleic acid molecule operatively associated with a first phosphoglycerate kinase promoter allowing for expression of the first heterologous enzyme being alpha-amylase.
Wong, Sec. 2.4, describes:
“About 0.1 g of 0.2-mm glass beads was added to the sample mixture, followed by vigorous vortexing for 2 min.” This is understood as bead beating.
“The increase in enzyme activity in YEPG per unit cell mass was even more striking (Table 1). The yeast cell density in YEPG was about one fourth of that in YEPD, with the exception of the first 24 hr of incubation, during which cell growth in YEPG was only one tenth of that in YEPD. This initial phase of extremely slow growth in YEPG corresponds to the induction period for the enzyme activity curve in Fig. 1. The cell growth had to reach a critical density before the enzyme was synthesized
secreted into the culture medium.” Wong, page 422, right col.
Fig. 1 of Wang shows amylase activity during culturing with hours of the x-axis and Table 1 shows the cell density over time:
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As can be seen above, in both media amylase activity and cell density increases significantly between 24 and 120 hours, which shows that heterologous expression of the amylase encoded by the first heterologous nucleic acid sequence is occurring during propagation of a recombinant host cell. As indicated above in Claim Interpretation section, culturing is understood in the art as cell propagation, which is further supported by the teachings of Wong. That is, the promoter employed by Wong allows for expression of a nucleic acid encoding an alpha-amylase during propagation of a recombinant yeast host cell.
Wong, sec. 2.4 and abstract, teach forming a yeast extract from the described recombinant yeast by vortexing with glass beads (bead beating) wherein both secreted and intracellular protein are present. Wong analyzes the yeast extract by SDS-PAGE and does not actively employ the amylase in a process. However, as discussed above, Aux teaches the use of a cell extract containing amylase proteins to be added to a liquefaction reaction wherein such amylase needs to be active.
As such, in producing an amylase to be used in the methods of Aux by expression in any host cell including yeast host cells, an ordinarily skilled artisan at time of filing would understand that the cell extract would not be treated in a manner that would cause amylase deactivation. However, Kovaleva has further teachings regarding formation of S. cerevisiae cell extracts containing active amylase.
In brief, Kovaleva, abstract, teaches: a-Amylase from Bacillus amyloliquefaciens, synthesized in yeast Saccharomyces cerevisiae without substitution of the signal sequence, is efficiently secreted from yeast cells: 6&70% of the overall amount of the enzyme is found in the culture fluid. In contrast to many yeast secretory proteins, which accumulate in the periplasmic space and in the cell wall, intracellular a-amylase is localized mainly in the cytoplasm.
“Cell extracts were prepared by vortexing yeast cells with glass beads,” which is within the broadest reasonable meaning of “bead beating.” Kovaleva, page 183, left col.
Table 1 of Kovaleva shows the activity of amylase in cell extracts is measured indicating that a yeast cell extract was formed with alpha-amylase activity formed by vortexing with glass beads, i.e. bead beating.
Aux, in the claims, states:
21. A method for producing a biofuel comprising:
a) liquefying an aqueous slurry of starch-containing plant material [including corn] in the presence of at least a first and a second class of α-amylase enzymes, wherein the first class of α-amylase enzymes exhibits a starch hydrolysis pattern that is different from the starch hydrolysis pattern of at least the second class of α-amylase enzymes to obtain a liquefact; and,
b) fermenting [with a yeast cell] the liquefact to produce said biofuel [including ethanol].
The above is a process for improving the yield of a fermentation product made from a fermenting yeast cell in a fermenting medium, the process comprising: (i) liquefying a liquefaction medium to obtain a fermentation medium; and (ii) fermenting the fermentation medium with the fermenting yeast cell to obtain a fermentation product being ethanol, so as to improve the yield of the fermentation product. As discussed, Aux states that performance of the liquefying step has an effect of improving ethanol yield “so as to improve the yield of the fermentation product.” In one embodiment, the liquefact is further processed to produce ethanol by fermenting with a fermenting yeast cell. “In one embodiment, the use of at least two different classes of α-amylase enzymes in the liquefaction process results in a substrate that leads to higher ethanol yields compared to the ethanol yield from starch-containing plant material that is exposed to only one class of α-amylase enzymes,” or by logical extension to no enzyme/amylase treatment at all. Aux, para. [0043].
Regarding recitation in claim 1 of the liquefaction medium comprises a first inactivated yeast product made from a first recombinant yeast host cell, wherein the liquefaction medium comprises:(a) a first inactivated yeast product made from a first recombinant yeast host cell, wherein the first recombinant yeast host cell comprises a first heterologous nucleic acid molecule for expressing a first heterologous enzyme and the first inactivated yeast product comprises the first heterologous enzyme, wherein the first heterologous nucleic acid molecule is operatively associated with a first promoter allowing expression of the first heterologous enzyme during propagation of the first recombinant yeast host cell, as discussed, Aux teach addition of at least one amylase enzyme to the liquefaction medium.
Paras. [0028]-[0034] of Aux contains an entire section discussing use of enzyme extracts including from yeast. “Furthermore, the partially-purified enzyme preparation may also be culture supernatant or crude extract collected from a cell population expressing and/or secreting the enzyme.” Aux, para. [0029]. As such, Aux teaches that it is appropriate to add amylase enzyme to a liquefaction medium in the form of a crude extract from a cell expressing the amylase such that an ordinarily skilled artisan at time of filing would have been motivated to do the same in view of the same express teachings of Aux.
Aux directly cites Callen. Callen, para. [0025], states that in is generally understood that amylase is produced by culturing the host cell under conditions that allow expression of the nucleic acid operatively associated with a promoter, wherein culturing is understood as meaning propagation in the art. In particular, Wong and Kovaleva teach specific methods for production of amylase in S. cerevisiae (i.e. yeast cells) that meets the claim limitation of a first inactivated yeast product made from a first recombinant yeast host cell, wherein the first recombinant yeast host cell comprises a first heterologous nucleic acid molecule for expressing a first heterologous enzyme and the first inactivated yeast product comprises the first heterologous enzyme, wherein the first heterologous nucleic acid molecule is operatively associated with a first promoter allowing expression of the first heterologous enzyme during propagation of the first recombinant yeast host cell, as discussed above.
At the time of filing an ordinarily skilled artisan would have been motivated to add an appropriate amylase to the liquefaction medium and methods of Aux in the form of a cell/yeast extract (a first inactivated yeast product), since Aux in paras. [0028]-[0034] extensively discusses the appropriateness of the use of crude cell extracts such that Aux has an express teaching, suggestion and motivation to do the same. Regarding the use of a yeast host cell that results in a first inactivated yeast product containing an alpha-amylase, Aux, para. [0032], directly suggests that yeasts including Saccharomyces be used as a host cell for producing amylase. Further, “Unlike Escherichia coli, S. cerevisiae is a generally recognized as safe (GRAS) organism.” Wong, page 420, left col. As such, production amylase in S. cerevisiae offers further safety advantages when the ethanol product may be used for food or feed purposes. “The products of this liquefaction can be concentrated and purified for food and other applications such as cleaning agents, textile agents, and animal feed.” Aux, para. [0042]. As such, an ordinarily skilled artisan at time of filing would have been motivated to produce an appropriate alpha-amylase in a yeast host cell and to express an amylase as taught by Wong and/or Kovaleva for these reasons, the method of at least Wong, as discussed, teaching: a first inactivated yeast product made from a first recombinant yeast host cell, wherein the first recombinant yeast host cell comprises a first heterologous nucleic acid molecule for expressing a first heterologous enzyme and the first inactivated yeast product comprises the first heterologous enzyme, wherein the first heterologous nucleic acid molecule is operatively associated with a first promoter allowing expression of the first heterologous enzyme during propagation of the first recombinant yeast host cell.
More specifically, Wong and Kovaleva teach that significant amounts of expressed amylase can be retained intracellularly in S. cerevisiae and is accessible by forming a cell/yeast extract. Kovaleva (Table 1), as discussed, teaches formation of a yeast extract by vortexing yeast cells with glass beads (bead beating) to produce a yeast extract (i.e. first inactivated yeast product) with active amylase.
Upon practice of the above, the features of claims 1-3 are reached.
Regarding claim 6, Wong, abstract, describes: “The enzyme activity of recombinant barley a-amylase secreted by the yeast clone DY150[pYEX-Amy1],” which is a description that the first heterologous nucleic acid molecule allows for expression in a secreted form. As discussed, Wong and Kovaleva teach that a significant amount of amylase can fail to secrete and be retained intracellularly.
Regarding claims 16 and 17, as discussed above, Aux teaches that the enzyme to be added to a liquefaction medium is specifically an alpha-amylase.
Regarding claim 18, “Alpha-amylases for example, such as those described in US Patent Publication US2003/0125534 [i.e. Callen] . . . may be further characterized in regards to their respective starch hydrolysis pattern and be further employed in starch hydrolysis as described herein.”
Callen, abstract, states:
The invention relates to alpha amylases and to polynucleotides encoding the alpha amylases. In addition methods of designing new alpha amylases and methods of use thereof are also provided. The alpha amylases have increased activity and stability at acidic, neutral and alkaline pH and increased temperature.
The alpha-amylases of Callen are expressly taught to be suitable for “particularly useful in corn-wet milling processes, detergents, baking processes, beverages and in oilfields (fuel ethanol), and also for liquefaction of starch. Callen, paras. [0003] and [0051].
“In yet another aspect, the invention provides an isolated nucleic acid encoding a polypeptide having a sequence as set forth in SEQ ID Nos.: 2, 4, 6, 10, 12, 14 .. . . 74. . . 80” Callen, para. [0018]. “Another aspect of the invention is an isolated nucleic acid encoding a polypeptide or a functional fragment thereof having a sequence as set forth in SEQ ID Nos.: 2, 4, 6, 10, 12, 14 . . . 74 . . . 80 (hereinafter referred to as “Group B amino acid sequences”), and sequences substantially identical thereto.” Callen, para. [0019].
“The polypeptides of Group B amino acid sequences, and sequences substantially identical thereto or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof, may also be used in the liquefaction and saccharification of starch. . . . In a preferred embodiment, the polypeptides or fragments thereof of this invention are thermostable at 90-95° C.” Callen, para. [0244].
SEQ ID NO: 74 of Callen has about 99% identity to recited SEQ ID NO: 63 as to be a variant of the same.
As such, Aux contains a direct teaching, suggestion and motivation to employ the alpha-amylases of Callen. As such, in addition to any of the specific amylases discussed in the art above, an ordinarily skilled artisan at time of filing would have been motivated to employ the amylase having SEQ ID NO: 74 of Callen (having about 99% identity to recited SEQ ID NO: 63) including expressing the same in a S. cerevisiae host cell and forming an inactivated yeast product (yeast extract) of the same as discussed above in view of the teachings of Wong and Kovaleva. Callen, para. [0127], has further teachings that a host cell for expression of the alpha-amylases taught therein can be yeast.
Regarding claim 34, as discussed, Aux teaches that a fermentation product is ethanol.
Claim(s) 1-3, 6, 16-18, 31, 32 and 34 (all non-withdrawn claims) is/are rejected under 35 U.S.C. 103 as being unpatentable over Aux (U.S. 2009/0221041 A1) further in view Callen et al. (U.S. 2003/0125534 A1), Wong et al. (Increased Expression and Secretion of Recombinant a-Amylase in Saccharomyces cerevisiae by Using Glycerol as the Carbon Source, J. Protein Chem. 21, 2002, 419-425) and Kovaleva et al. (Synthesis and secretion of bacterial a-amylase by the yeast Saccharomyces cerevisiae, FEBS Lett. 251, 1989, 183-86) as applied to claims 1-3, 6, 16-18 and 34 above, and further in view of McBride et al. (WO 2014/035458 A1). This rejection expressly applies to all pending claims subject to examination as not being withdrawn.
Regarding claims 31 and 32, McBride, abstract, further relates to ethanol fermentation using S. cerevisiae. McBride, para. [0032] states: “In some embodiments of the invention, the recombinant yeast host cell comprising at least one saccharolytic enzyme further comprises a deletion or alteration of one or more glycerol producing enzymes. In some embodiments, the recombinant yeast host cell further comprises a deletion or alteration of GPD1.” “Because glycerol is a major by-product of anaerobic production of ethanol, many efforts have been made to delete cellular production of glycerol.” McBride, para. [0351].
McBride is directed towards fermentations of lignocellulosic hydrolysates. Regardless, the discussion in McBride of “glycerol is a major by-product of anaerobic production of ethanol” is the case regardless of the specific source of fermentable sugars fermented to ethanol. Since in methods of Aux, as in McBride, ethanol and not glycerol is the desired product, an ordinarily skilled artisan at the time of filing would have been motivate to utilize a S. cerevisiae having genetic modification for reducing production of GDP1 (a native enzyme that functions to produce glycerol, a genetic modification for reducing production of a native enzyme that produces glycerol) as taught by McBride in order to achieve the benefit of reduced glycerol production that can be coproduced in ethanol fermentation with S. cerevisiae.
Response to arguments
Applicant argues:
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In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Balasundaram et al. discussed production of amylases in yeast cells in depth and was cited for teaching the same.
Applicant argues:
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In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., propagation-specific promoter) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Claim 1 recites “a first promoter allowing expression of the first heterologous enzyme during propagation,” which is a generic promoter and not a “propagation specific promoter.”
That is, the broadest reasonable interpretation of “wherein the first heterologous nucleic acid molecule is operatively associated with a first promoter allowing expression of the first heterologous enzyme during propagation of the first recombinant yeast host cell” is 1) propagation is any minimum or minor amount of cell division, and 2) any generic promoter allowing for expression of a heterologous nucleic acid.
“Another aspect of the invention is a method of making a polypeptide having a sequence as set forth in Group B amino acid sequences, and sequences substantially identical thereto. The method includes introducing a nucleic acid encoding the polypeptide into a host cell, wherein the nucleic acid is operably linked to a promoter, and culturing the host cell under conditions that allow expression of the nucleic acid.” Callen, para. [0025].
“The specification need not disclose what is well-known to those skilled in the art and preferably omits that which is well-known to those skilled and already available to the public.” MPEP 2164.05(a).
MPEP 2164.05(a) discusses the scope of disclosure in a specification for enablement purposes. However, by extension, the same rule applies to the scope of disclosure required by the prior art in order to enable claims under examination. “Alpha-amylase enzymes can be expressed in and isolated from any number of eukaryotic and prokaryotic organisms. Appropriate expression cassettes, vectors, transformation, and transfection techniques for a particular organism of interest will be evident to one of skill in the art.” Aux, para. [0030]. This teaching of Aux is understood to be an accurate statement regarding the state of the prior art, which is that methods for recombinant expression of enzymes in eukaryotic (i.e. yeasts) and prokaryotic organism “will be evident to one of skill in the art” such that the same is well-known to those skilled and already available to the public and is preferably not discussed with particular detail in the prior art and by extension in the body of the rejection.
Further, Another aspect of the invention is a method of making a polypeptide having a sequence as set forth in Group B amino acid sequences, and sequences substantially identical thereto. The method includes introducing a nucleic acid encoding the polypeptide into a host cell, wherein the nucleic acid is operably linked to a promoter, and culturing the host cell under conditions that allow expression of the nucleic acid.” Callen, para. [0025]. The prior art is interpreted in view of a person having ordinarily skill in the art at time of filing, which in this instance is an individual with at least an undergraduate degree in a biological science or similar technical field. Such a person would understand that “culturing” is a synonym for “propagation” and does not require the same to be explained. Disclosures “need not disclose what is well-known to those skilled in the art and preferably omits that which is well-known to those skilled and already available to the public.” MPEP 2164.05(a). From Taber’s Medical Dictionary, Culture, retrieved 08/03/2026, www.tabers.com/tabersonline/view/Tabers-Dictionary/737315/all/culture:
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As such, Callen, para. [0025], teaches expression of a heterologous nucleic acid molecule encoding amylase operative associated with a control sequence (which is a promoter) allowing for expression of the heterologous nucleic acid molecule encoding amylase during culturing/propagation of a recombinant cell.
Such an ordinarily skilled artisan does not require specific instruction regarding how to express an amylase in an appropriate host cell including yeast cells and how to form a cell extract of the same. Regardless, it is noted Balasundaram discusses expression of amylase in S. cerevisiae host in depth with many art citations including how to form a cell extract by bead milling.
Regarding the recombinant cell being yeast or S. cerevisiae, “A reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill in the art, including nonpreferred embodiments.” MPEP 2121(I). It is recognized that neither Aux nor Callen is particularly focused on yeast host cells. Nevertheless, Aux explicitly teaches “In another embodiment, fungal hosts, such as fungal host cells belonging to the genera Aspergillus, Rhizopus, Trichoderma, Nurerospora, Mucor, Penicillium, etc., such as yeast belonging to the genera Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Schwanniomyces, etc. may be used.” Aux, para. [0032]. As such, Aux contains an explicit teaching, suggestion and motivation to utilize any suitable host cell for amylase expression including Saccharomyces (yeast) which teaching is not obviated by being part of the broad disclosure of Aux. Further, Balasundaram discusses expression of amylase in S. cerevisiae host in depth and the advantages of expression of amylases in the same.
Applicant argues:
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In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., propagation-specific promoter) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Aux is cited for teaching these features.
Balasundaram and Schuster were cited for general knowledge in the prior art for how to express amylases in S. cerevisiae including alpha-amylases as cell-wall bound product or as a cytoplasmic product as recited in dependent claims and not to be duplicative of the teachings of Aux and Callen.
Applicant argues:
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The burden is on applicant to establish that results are unexpected and significant. MPEP 716.02(b). "[A]ppellants have the burden of explaining the data in any declaration they proffer as evidence of non-obviousness." Ex parte Ishizaka, 24 USPQ2d 1621, 1624 (Bd. Pat. App. & Inter. 1992); MPEP 716.02(b). “An affidavit or declaration under 37 CFR 1.132 [or data from the specification] must compare the claimed subject matter with the closest prior art to be effective to rebut a prima facie case of obviousness.” MPEP 716.02(e). "Expected beneficial results are evidence of obviousness of a claimed invention, just as unexpected results are evidence of unobviousness thereof." In re Gershon, 372 F.2d 535, 538, 152 USPQ 602, 604 (CCPA 1967); MPEP 716.02(c)(II). “Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the ‘objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support.’" MPEP 716.02(d).
Example III, and Figures 8 through 11 describes all examples including a commercial enzyme, which is not recited in any of the claims. For this reason, the examples are not commensurate in scope with the claims for any unexpected results. Further, none of the rejected claims require an amylase. Further, the addition of higher amounts of enzymes in the form of 0.2% identified commercial enzyme and enzymes contained in yeast extract (inactivated yeast product) would be actively expected to support increased breakdown of starch that can support increased ethanol production including combination of different amylase enzymes that have different activities in view of the teachings of Aux. "Expected beneficial results are evidence of obviousness of a claimed invention, just as unexpected results are evidence of unobviousness thereof." In re Gershon, 372 F.2d 535, 538, 152 USPQ 602, 604 (CCPA 1967); MPEP 716.02(c)(II).
That is, Fig. 13 provides the following examples wherein all examples include commercial enzyme:
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Table 2 (page 59) of the specification describes the M19211 strain as expressing two heterologous enzymes: P. furiosus alpha amylase (SEQ ID NO: 64) and T. hydrothemalis alpha-amylase (SEQ ID NO: 63).
As such, the liquefactions argued to have unexpected results from Fig. 13 have three enzymes present:
Enzyme 1: commercial enzyme
Enzyme 2: P. furiosus alpha amylase (SEQ ID NO: 64); and
Enzyme 3: T. hydrothemalis alpha-amylase (SEQ ID NO: 63).
Aux discusses that two different amylase enzymes can have complementary affects that are synergistic. “[W]hile not bound by any particular theory or mechanism, it is believed that using the two different classes of α-amylase enzymes together in a liquefaction reaction will produce a substrate that will give higher concentrations of ethanol and less residual sugars/starch in a fermentation process than the use of either class alone.” Aux, para. [0013].
The data form the specification, appears to describe a similar scenario wherein utilization of multiple enzyme (i.e. at least three), the commercial enzyme and enzyme in inactivated yeast product expressing two different alpha-amylases have a synergistic effect that may be superior to the use of the commercial enzyme alone. There is no way to determine what the effect would be for using the inactivated yeast product alone. Further, as far as the claims do not recite three different enzymes as shown in Working Examples of the specification, the claims are not commensurate in scope with the data from the specification argued to support unexpected results.
Example IV and Figures 14 and 15 of the specification are not understood to describe inactivated yeast product is added to a “nutrient rich commercial mash” (i.e. a fermentation medium) and not to a liquefaction medium as recited in claim 1.
There is no example of the effect of adding the same enzyme as a purified enzyme as compared to as part of an inactivated yeast product at an equivalent amount. The burden is on applicant to establish that results are unexpected and significant. MPEP 716.02(b). An affidavit or declaration under 37 CFR 1.132 [or data from the specification] must compare the claimed subject matter with the closest prior art to be effective to rebut a prima facie case of obviousness.” MPEP 716.02(e).
Aux for example demonstrates that it is known in the prior art to apply one or two amylase enzyme to a liquefaction for ethanol production. For example, Aux, Table 3, show addition of Bac (Bacillus amylase) alone and CA (corn amylase) alone and not in combination with other enzymes, along with these enzymes in combination. The purpose of addition of amylase enzyme is “During the gelatinization process, there is a dramatic increase in viscosity. To enable handling during the remaining process steps, the starch must be thinned or “liquefied”. This reduction in viscosity can be accomplished by enzymatic degradation in a process referred to as liquefaction. During liquefaction, the long-chained starch molecules are degraded into smaller branched and linear chains of glucose units (dextrins) by an enzyme, such as α-amylase (i.e., α-amylase).” Aux, para. [0002]. This understood as a direct teaching that not performing such liquefaction will be detrimental to downstream ethanol production (i.e. decreasing viscosity benefits ethanol production).
That is, the specification provides for: An unidentified commercial enzyme plus two different enzymes expressed by a yeast cell present in an inactivated yeast product may increase ethanol yield compared to the unidentified commercial enzyme alone. However, none of the above is a comparison to the closes prior art (Aux) of addition of one or two amylase enzymes to a liquefaction being the actual Bac (Bacillus amylase) alone and/or CA (corn amylase) employed by Aux.
Stated in other words, the evidence of record only shows that supplementation of a uncharacterized commercial enzyme with two additional enzymes of SEQ ID NOS: 63 and 64 has a benefit over using the commercial enzyme alone to which the claims are not commensurate in scope.
It is further noted that claim 1 may not require any amylase added to a liquefaction. That is, embodiments of claim 1 include adding a protease to a liquefaction medium or to a fermentation medium. As such, even if unexpected results were shown for some addition of amylase to a liquefaction medium, the same would not be commensurate in scope to the claims that do not require addition of amylase to a liquefaction medium.
Conclusion
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652
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