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 .
DETAILED ACTION
Claims 9-10 and 12-18 are pending and under examination on their merits. Claims 1-8 and 11 are cancelled.
Response to Arguments
Applicant's arguments filed 2/10/2026 have been fully considered but they are not persuasive.
Applicant argues against the rejection of claims under 35 U.S.C. 103 over Denish in view of Hong and Kim and as evidenced by ZME Science on the grounds that Denish does not teach the following claim limitations: 1. performing a catalytic reaction by the enzyme on a substrate to enrich the cyanidin-3-diglucoside-5-qlucoside; 2. a concentration of the substrate in the catalytic reaction is 60 mg/mL, and a concentration of the enzyme is 0.5-2 mg/mL, and 3. a pH of the catalytic reaction is 6.0-6.5 (Arguments, middle of page 9).
In response, these arguments are not persuasive because Denish teaches the production of cyanidin-3-diglucoside-5-qlucoside by enzymatic reaction with red cabbage extract, which is enrichment. Denish teaches cyanidin-3-diglucoside-coumaroyl-5-glucoside (compound P3 of Fig. 1). Denish teaches that the enzyme PDB 1AUR (46 in Fig. 4C) hydrolyzes cyanidin-3-diglucoside-coumaroyl-5-glucoside (P3) into cyanidin-3-diglucoside-5-glucoside (P1): see Figure 4A, 4C, and Supplementary Table 11.1. With respect to the substrate concentration and the pH of the reaction, Denish teaches values that are similar to the claimed values. Denish teaches 100 mg/mL red cabbage extract (page 6, right column, paragraph 2, Protein purification and activity screening of esterases) and a pH of 7.5 (Supplementary Method 11.2, page 63, paragraph 1). It would have been obvious to the person of ordinary skill in the art before the effective filing date of the claimed invention to optimize both the pH and the substrate concentration by routine experimentation and the person of ordinary skill in the art would have had a reasonable expectation of success in doing so. Furthermore, per MPEP 2144.05(II)(A), generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. " Here, no such evidence is of record.
Applicant argues further against the rejection of claims under 35 U.S.C. 103 over Denish in view of Hong and Kim on the grounds that the claimed invention has distinctive technical features over the cited prior art references: the number of anthocyanin species in the crude extract is reduced (Arguments, point 2 on page 11) and the pH range of 6.0-6.5 maximizes enzyme activity (Arguments, point 3 on page 11). Applicant argues that the target of the enrichment of the present claims is a by-product of the enzymatic hydrolysis reaction of Denish (Arguments, paragraph 3 on page 12). Applicant argues that cyanidin-3-diglucoside-5-glucoside is prone to hydrolysis at Denish’s pH of 7.5, (Arguments, paragraph 4 on page 12).
These arguments are unpersuasive. Applicant’s claimed method does not require any specific amount of cyanidin-3-diglucoside-5-glucoside, nor does the claimed method exclude any additional reaction products. Furthermore, with respect to pH, Denish teaches that storage under acidic conditions enhances the stability of the colorant by reducing the fraction of hydrolyzed species (page 4, right column, Color expression and application of P2). Therefore, Applicant’s finding that reducing the pH reduces hydrolysis is taught by the prior art of Denish.
Applicant does not present new arguments with respect to the nonstatutory double patenting rejections of record that are not already addressed above.
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.
(The following rejections are necessitated by the amendment) Claims 9-10 and 12-18 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.
Claims 9-10 are indefinite for “wherein the target gene contains a nucleotide sequence as set forth in SEQ ID NO: 1 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 and the target gene encodes the enzyme having an activity of hydrolysis of acylated anthocyanin.” There are at least two different reasonable interpretations for this limitation in the claim, rendering the claim indefinite. In one interpretation, the requirement that the target gene encodes the enzyme having an activity of hydrolysis of acylated anthocyanin applies to the variant of SEQ ID NO: 1 (i.e. the target gene that has at least 90% identity to SEQ ID NO: 1). In a second interpretation, the requirement that the target gene encodes the enzyme having an activity of hydrolysis of acylated anthocyanin applies to both the target gene that contains a nucleotide sequence as set forth in SEQ ID NO: 1 as well as the target gene that has at least 90% identity to SEQ ID NO: 1. Note that the claim as drafted reads on any amino acid sequence of at least two amino acids in SEQ ID NO: 1 (“a nucleotide sequence”) rather than the nucleotide sequence as set forth in SEQ ID NO: 1.
Claims 9-10 are further indefinite for “introducing a carrier of a target gene into an engineered bacterium.” This limitation has multiple reasonable interpretations. In one interpretation, the bacterium is engineered by introducing the target gene. In a second interpretation, the bacterium is further engineered by introducing the target gene. Under the second interpretation, the bacterium already has at least one genetic modification prior to the introduction of the target gene.
Claims 9-10 are further indefinite for “the substrate is selected from an anthocyanin, wherein the anthocyanin is selected from the group consisting of honeyberry extract, blueberry fruit extract, radish extract, purple cabbage extract, and purple sweet potato extract.” The claim combines broad (the substrate is selected from an anthocyanin) a narrow limitations (the anthocyanin is selected from the group consisting of honeyberry extract, blueberry fruit extract, radish extract, purple cabbage extract, and purple sweet potato extract) within the same claim, leading to ambiguity in the claim scope.
Claim 10 is also indefinite because it is unclear whether the preparation method of the biological enzyme is part of the claimed method. It is further unclear how the kit is part of the claimed method since the kit is not recited in the active method step.
Claim 12 is indefinite for “wherein the anthocyanin is at least one selected from a glycosylated anthocyanin, an acylated anthocyanin, an aggregate of small molecular weight anthocyanins, a mixture of the anthocyanins, or a crude extract of the anthocyanins.” The claim does not present a closed group of alternatives. Applicant may consider amending to “wherein the anthocyanin is at least one selected from a glycosylated anthocyanin, an acylated anthocyanin, an aggregate of small molecular weight anthocyanins, a mixture of the anthocyanins, and a crude extract of the anthocyanins” in order to obviate this rejection.
Claim 15 recites the limitation "the extract" in line 1. There is insufficient antecedent basis for this limitation in the claim because claim 15 depends from claim 9, which recites multiple extracts. Claim 15 is further indefinite because the claim recites “wherein the extract is extracted by a citric acid aqueous solution or acidified ethanol.” It is unclear whether the method further comprises extracting an additional material from the extract or whether this is a product-by-process limitation for the preparation of the extract, or whether the method further comprises preparing the extract by an unknown method step involving the citric acid aqueous solution or acidified ethanol (e.g. contacting radish with the citric acid aqueous solution or acidified ethanol to produce a radish extract).
Claim 16 recites “wherein a preparation method for the purple cabbage extract via the citric acid aqueous solution is as follows: mixing crushed purple cabbage or purple sweet potato with the citric acid aqueous solution at a ratio of material to liquid from 1 :2 to 1:10, and performing immersing and extracting at 50-60°C for 20-30 min.” it is unclear whether the preparation method for the purple cabbage extract is part of the claimed method or whether this is a product by process limitation. The claim does not say, for example, “The preparation method according to claim 15, wherein the method further comprises producing the purple cabbage by…” In addition, the claim recites “a ratio of material to liquid from 1:2 to 1:10.” It is unclear what material is required or whether the ratio is of material to liquid or liquid to material (both are ”a ratio”). Finally, the claim recites “a preparation method for the purple cabbage extract” but later recites “mixing crushed purple cabbage or purple sweet potato with the citric acid aqueous solution.” Therefore, it is unclear whether the extract is purple cabbage extract or purple sweet potato extract.
Claims 12-18 are rejected for depending from a rejected base claim and not rectifying the sources of indefiniteness discussed above.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
The following rejections are necessitated by the amendment.
Claims 9-10, 12-14, and 17-18 are rejected under 35 U.S.C. 103 as being unpatentable over Denish et al. (Science advances 7.15 (2021): eabe7871) in view of Hong et al. (Agric. Bioi. Chern., 55 (II), 2839-2845, 1991) and Kim et al. (Structure 5, 1571–1584 (1997)) as evidenced by ZME Science (2024, website).
“Enriching” is interpreted according to its plain and ordinary meaning: increasing the concentration of a specific component.
The term “kit” in claim 10 is not interpreted as further limiting the claimed method because it is not recited in the active method step.
Regarding claims 9-10, Denish teaches cyanidin-3-diglucoside-coumaroyl-5-glucoside (compound P3 of Fig. 1). Denish teaches that the enzyme PDB 1AUR (46 in Fig. 4C) hydrolyzes cyanidin-3-diglucoside-coumaroyl-5-glucoside (P3) into cyanidin-3-diglucoside-5-glucoside (P1): see Figure 4A, 4C, and Supplementary Table 11.1. Denish teaches combining 10 µL of 100 mg/mL red cabbage extract (RCE) with the enzyme and reacting at room temperature for 24 hours (page 6, right column, paragraph 2, Protein purification and activity screening of esterases). Room temperature is approximately 20-22 °C as evidenced by ZME Science (paragraph 1 on page 2), which is within the claimed range. Likewise, 24 h is within the claimed range.
Denish teaches the enzyme PDB 1AUR (page 4, right column, paragraph 2), which is identical to the amino acid sequence encoded by the nucleotide SEQ ID NO: 1. See the amino acid sequence alignment in OA Appendix A (the amino acid sequence of 1AUR is on page 80 of the Supplementary Materials, Supplementary Table 11.3.).
Denish teaches transforming Escherichia coli (“engineered bacterium”) with a plasmid (carrier) encoding each protein (Supplementary Table 11.1 starting page 45), which includes the carboxylesterase 1AUR (ID #46 of Supplementary Table 11.1 on page 67). Denish teaches picking single colonies and inoculating terrific broth (Supplementary Method 11.3, bottom paragraph on page 63), then culturing the cells, inducing, and expressing the protein at 18 °C for 24 hours, after which cells are lysed and the protein is purified (page 6, right column, paragraph 2, Protein purification and activity screening of esterases). Since the cultures express the proteins, then the colonies picked were necessarily positive for the protein.
Denish’s protein 1AUR is encoded by Sequence ID No. 46 (Supplementary Materials page 80), which is codon-optimized for E. coli (page 6, right column, paragraph 2, Protein purification and activity screening of esterases).
Denish’s Sequence ID No. 46 is less than 90% identical to the instant SEQ ID NO: 1.
Kim teaches that the protein 1AUR is an esterase from Pseudomonas fluroescens (Title and page 1583, right column, bottom paragraph, Accession numbers).
Hong teaches the nucleotide sequence of the Pseudomonas fluroescens gene that encodes the esterase and clones the sequence into E. coli (Abstract). The instant SEQ ID NO: 1 is 100% identical to the nucleotide sequence of Fig. 2 of Hong (OA Appendix B).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace Denish’s Sequence ID No. 46 (PDB 1AUR) with Hong’s estB gene because they are art-recognized equivalents for the same purpose: they are both nucleotide sequences encoding the same amino acid sequence, the Pseudomonas fluorescens esterase (PDB 1AUR).
Denish teaches that both red cabbage and purple sweet potato extract contain a complex mixture of anthocyanins (page 1, right column, middle paragraph). Denish teaches that the enzyme is diluted to 1 mg/mL for enzyme activity assays and the concentration of the red cabbage extract is 100 mg/mL (Supplementary Method 11.2, page 63).
Denish teaches contacting red cabbage extract with the enzyme 1AUR but Denish does not teach contacting anthocyanins from purple sweet potato extract with the enzyme 1AUR. Denish does not teach the concentration of the enzyme in reaction with purple sweet potato extract or the concentration of the purple sweet potato extract.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the red cabbage extract with the purple sweet potato extract taught by Denish as another extract that contains anthocyanins. It would have been further obvious to contact the purple sweet potato extract with the enzyme 1AUR. The person of ordinary skill in the art would have been motivated to produce cyanidin-3-diglucoside-5-glucoside from substrates other than red cabbage in order to make the process more versatile. The person of ordinary skill in the art would have had a reasonable expectation of success given that Denish teaches that both red cabbage and purple sweet potato extract contain complex mixtures of anthocyanins.
Regarding the concentration of the substrate and the enzyme in the reaction, it would have been further obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to optimize by routine experimentation the concentration of the purple sweet potato extract and the enzyme in the reaction in order to maximize recovery of the cyanidin-3-diglucoside-5-glucside while minimizing the cost to perform the catalytic reaction. The person of ordinary skill in the art would have had a reasonable expectation of success in the optimization of both the 1AUR enzyme and the substrate (anthocyanins in the purple sweet potato extract).
Regarding the pH of the catalytic reaction, Denish also teaches that the pH of the catalytic reaction is buffered to 7.5 (Supplementary Method 11.2, page 63, paragraph 1), which is above the claimed range of pH 6.0 to 6.5. Denish teaches that storage under acidic conditions enhances the stability of the colorant by reducing the fraction of hydrolyzed species (page 4, right column, Color expression and application of P2).
It would have been obvious to the person of ordinary skill in the art before the effective filing date of the claimed invention to optimize by routine experimentation the pH of the catalytic reaction. The person of ordinary skill in the art would have included more acidic pH conditions in the range of optimization conditions based on the teaching of Denish that more acidic conditions reduce the hydrolysis of colorant products such as P2, which is structurally related to P1 or cyanidin-3-diglucoside-5-glucoside (see Fig. 4 of Denish). The person of ordinary skill in the art would have had a reasonable expectation of success in optimizing the pH of the catalytic reaction.
Regarding claim 12, Denish’s anthocyanins are from red cabbage extract and include glycosylated, acylated, and small molecular weight anthocyanins (see Figure 4A, and page 6, right column, paragraph 2). Denish teaches that both red cabbage and purple sweet potato extract contain a complex mixture of anthocyanins (page 1, right column, middle paragraph). Therefore, the person of ordinary skill in the art would have expected purple sweet potato extract to also contain glycosylated, acylated, and small molecular weight anthocyanins.
Regarding claims 13-14, the substrate for Denish’s 1AUR enzyme is red cabbage extract (page 6, right column, paragraph 2), which includes cyanidin-3-coumaroyl-diglucoside-5-O-glucoside (see compound P3 in Figure 1). The term O-glycoside refers to the sugar being linked to another group through an oxygen atom, as drawn in Denish’s P3 structure. Denish teaches that the enzyme is diluted to 1 mg/mL for enzyme activity assays and the concentration of the red cabbage extract is 100 mg/mL (Supplementary Method 11.2, page 63). The enzyme concentration of 1 mg/mL is within the claimed range of 0.5-2.0 mg/mL and the red cabbage extract concentration of 100 mg/mL is also within the claimed range (100-500 mg/mL). Since Denish teaches that both red cabbage and purple sweet potato extract both contain a complex mixture of anthocyanins (page 1, right column, middle paragraph), the person of ordinary skill in the art would have expected that cyanidin-3-coumaroyl-diglucoside-5-O-glucoside is also present in purple sweet potato extract and would have applied similar conditions to hydrolyze the substrate.
Regarding claim 17, Denish produces the enzyme in E. coli (page 6, right column, Protein purification and activity screening of esterases).
Regarding claim 18, Denish teaches that the concentration of the red cabbage extract (“substrate”) is 100 mg/mL, not 60 mg/mL (Supplementary Method 11.2, page 63). Denish teaches the substrate concentration 100 mg/mL for the hydrolysis of red cabbage extract, not purple sweet potato extract.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to optimize by routine experimentation the amount of substrate in the catalytic reaction based on the desired concentration of the product. The person of ordinary skill in the art would have had a reasonable expectation of success in optimizing the amount of purple sweet potato extract in the catalytic reaction.
Claims 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Denish et al. (Science advances 7.15 (2021): eabe7871) in view of Hong et al. (Agric. Bioi. Chern., 55 (II), 2839-2845, 1991) and Kim et al. (Structure 5, 1571–1584 (1997)) as evidenced by ZME Science (2024, website), as applied to claims 9-10, 12-14, and 17-18 above, further in view of Xu et al. (Bioresource Technology 101.18 (2010): 7151-7157).
Denish, Hong, and Kim do not teach that the purple sweet potato extract is extracted by citric acid aqueous solution at a ratio of solids to liquid of 1:10 or extracting at 60 °C for 20 to 30 min.
Xu teaches extracting anthocyanins from red cabbage using a conventional acidified water (CAW) approach by mixing the red cabbage with citric acid at a ratio of solids to liquids of 1:10 for 21 or 28 minutes at a temperature of 60 °C (Abstract and page 7153, left column, 2.3. Extraction experiments).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply the technique of Xu for the extraction of anthocyanins to the method of Denish modified by Hong and Kim. The person of ordinary skill in the art would have been motivated to improve Denish’s method by preparing the purple sweet potato extract using an extraction method that does not require organic solvents. The person of ordinary skill in the art would have had a reasonable expectation of success in applying Xu’s extraction technique to the method of Denish modified by Hong and Kim.
Xu’s temperature of 60 °C is within the claimed range of 50-60°C, both of the extraction times (21 or 28 minutes) are within the claimed range of 20-30 min, and the ratio of solids to liquids of 1:10 is also within the claimed range of from 1:2 to 1:10.
Although Xu does not explicitly recite “immersing” the purple sweet potato in the citric acid, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to immerse the purple sweet potato in the citric acid in order to maximize contact between the purple sweet potato and the citric acid and thus maximize extraction efficiency of the anthocyanins. The person of ordinary skill in the art would have had a reasonable expectation of success in the immersion of the purple sweet potato in the citric acid.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
The following rejections are necessitated by the amendment.
Claim 9-10, 12-14, and 17-18 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 3 of U.S. Patent No. 12,167,741 (‘741) in view of Denish et al. (Science advances 7.15 (2021): eabe7871), Hong et al. (Agric. Bioi. Chern., 55 (II), 2839-2845, 1991) and Kim et al. (Structure 5, 1571–1584 (1997)) and as evidenced by ZME Science (2024, website).
Claim 1 of ‘741 is drawn to a method of improving the stability of anthocyanin , Claim 3 of ‘741 requires that the anthocyanin comprises a glycosylated/acylated
anthocyanin.
Claim 3 of ‘741 does not recite introducing a carrier containing a target gene into an engineered bacterium, wherein the target gene contains a nucleotide sequence having at least 90% identity to SEQ ID NO: 1, selecting, cultivating, inducing, and lysing positive single colonies to obtain the enzyme, performing a catalytic reaction by the enzyme on a substrate to enrich cyanidin-3-diglucoside-5-glucoside, wherein a temperature of the reaction is 20-40 °C and a duration of the reaction is 10-30 h, the substrate is purple sweet potato extract, a concentration of the enzyme is 0.5-2 mg/mL, and a pH of the catalytic reaction is 6.0-6.5.
Regarding instant claims 9-10, Denish teaches transforming Escherichia coli (“engineered bacterium”) with a plasmid (carrier) encoding each protein (Supplementary Table 11.1 starting page 45), which includes the carboxylesterase 1AUR (ID #46 of Supplementary Table 11.1 on page 67). Denish teaches picking single colonies and inoculating terrific broth (Supplementary Method 11.3, bottom paragraph on page 63), then culturing the cells, inducing, and expressing the protein at 18 °C for 24 hours, after which cells are lysed and the protein is purified (page 6, right column, paragraph 2, Protein purification and activity screening of esterases). Since the cultures express the proteins, then the colonies picked were necessarily positive for the protein.
Denish teaches combining 10 µL of 100 mg/mL red cabbage extract (RCE) with the enzyme and reacting at room temperature for 24 hours (page 6, right column, paragraph 2, Protein purification and activity screening of esterases).
Room temperature is approximately 20-22 °C as evidenced by ZME Science (paragraph 1 on page 2), which is within the claimed range. Likewise, 24 h is within the claimed range. See MPEP 2131.03(I) for anticipation of claimed ranges.
Denish’s protein 1AUR is encoded by Sequence ID No. 46 (Supplementary Materials page 80), which is codon-optimized for E. coli (page 6, right column, paragraph 2, Protein purification and activity screening of esterases).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to prepare cyanidin-3-diglucoside-5-glucoside (a glycosylated and acylated anthocyanin) per the method of Denish and then to improve the stability of cyanidin-3-diglucoside-5-glucoside by the method of claim 3 of ‘741. The person of ordinary skill in the art would have had a reasonable expectation of success in the combination of the preparation method of cyanidin-3-diglucoside-5-glucoside with the method of claim 3 of ‘741 because cyanidin-3-diglucoside-5-glucoside is a glycosylated and acylated anthocyanin.
Denish’s Sequence ID No. 46 is less than 90% identical to the instant SEQ ID NO: 1.
Kim teaches that the protein 1AUR is an esterase from Pseudomonas fluroescens (Title and page 1583, right column, bottom paragraph, Accession numbers).
Hong teaches the nucleotide sequence of the Pseudomonas fluroescens gene that encodes the esterase and clones the sequence into E. coli (Abstract).
The instant SEQ ID NO: 1 is 100% identical to the nucleotide sequence of Fig. 2 of Hong (OA Appendix B).
It would have been further obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace Denish’s Sequence ID No. 46 (PDB 1AUR) in the method of claim 3 of ‘741 modified by Denish with the estB gene of Hong because they are art-recognized equivalents for the same purpose: they both nucleotide sequences encoding the same amino acid sequence, which is amino acid sequence of the Pseudomonas fluorescens esterase (PDB 1AUR).
Regarding the substrate in instant claims 8-10, Denish teaches that both red cabbage and purple sweet potato extract contain a complex mixture of anthocyanins (page 1, right column, middle paragraph). Denish teaches that the enzyme is diluted to 1 mg/mL for enzyme activity assays and the concentration of the red cabbage extract is 100 mg/mL (Supplementary Method 11.2, page 63).
Denish teaches contacting red cabbage extract with the enzyme 1AUR but Danish does not teach contacting anthocyanins from purple sweet potato extract with the enzyme 1AUR. Denish does not teach the concentration of the enzyme in reaction with purple sweet potato extract or the concentration of the purple sweet potato extract.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the red cabbage extract with the purple sweet potato extract taught by Denish as another extract that contains anthocyanins. It would have been further obvious to contact the purple sweet potato extract with the enzyme 1AUR. The person of ordinary skill in the art would have been motivated to produce cyanidin-3-diglucoside-5-glucoside from substrates other than red cabbage in order to make the process more versatile. The person of ordinary skill in the art would have had a reasonable expectation of success given that Denish teaches that both red cabbage and purple sweet potato extract contain complex mixtures of anthocyanins.
Regarding the concentration of the substrate and the enzyme in the reaction, it would have been further obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to optimize by routine experimentation the concentration of the purple sweet potato extract and the enzyme in the reaction in order to maximize recovery of the cyanidin-3-diglucoside-5-glucside while minimizing the cost to perform the catalytic reaction. The person of ordinary skill in the art would have had a reasonable expectation of success in the optimization of both the 1AUR enzyme and the substrate (anthocyanins in the purple sweet potato extract).
Regarding the pH of the catalytic reaction, Denish also teaches that the pH of the catalytic reaction is buffered to 7.5 (Supplementary Method 11.2, page 63, paragraph 1), which is above the claimed range of pH 6.0 to 6.5. Denish teaches that storage under acidic conditions enhances the stability of the colorant by reducing the fraction of hydrolyzed species (page 4, right column, Color expression and application of P2).
It would have been obvious to the person of ordinary skill in the art before the effective filing date of the claimed invention to optimize by routine experimentation the pH of the catalytic reaction. The person of ordinary skill in the art would have included more acidic pH conditions in the range of optimization conditions based on the teaching of Denish that more acidic conditions reduce the hydrolysis of colorant products such as P2, which is structurally related to P1 or cyanidin-3-diglucoside-5-glucoside (see Fig. 4 of Denish). The person of ordinary skill in the art would have had a reasonable expectation of success in optimizing the pH of the catalytic reaction.
Regarding claim 12, Denish’s anthocyanins are from red cabbage extract and include glycosylated, acylated, and small molecular weight anthocyanins (see Figure 4A, and page 6, right column, paragraph 2). Denish teaches that both red cabbage and purple sweet potato extract contain a complex mixture of anthocyanins (page 1, right column, middle paragraph). Therefore, the person of ordinary skill in the art would have expected purple sweet potato extract to also contain glycosylated, acylated, and small molecular weight anthocyanins.
Regarding claims 13-14, the substrate for Denish’s 1AUR enzyme is red cabbage extract (page 6, right column, paragraph 2), which includes cyanidin-3-coumaroyl-diglucoside-5-O-glucoside (see compound P3 in Figure 1). The term O-glycoside refers to the sugar being linked to another group through an oxygen atom, as drawn in Denish’s P3 structure. Denish teaches that the enzyme is diluted to 1 mg/mL for enzyme activity assays and the concentration of the red cabbage extract is 100 mg/mL (Supplementary Method 11.2, page 63). The enzyme concentration of 1 mg/mL is within the claimed range of 0.5-2.0 mg/mL and the red cabbage extract concentration of 100 mg/mL is also within the claimed range (100-500 mg/mL). Since Denish teaches that both red cabbage and purple sweet potato extract both contain a complex mixture of anthocyanins (page 1, right column, middle paragraph), the person of ordinary skill in the art would have expected that cyanidin-3-coumaroyl-diglucoside-5-O-glucoside is also present in purple sweet potato extract and would have applied similar conditions to hydrolyze the substrate.
Regarding claim 17, Denish produces the enzyme in E. coli (page 6, right column, Protein purification and activity screening of esterases).
Regarding claim 18, Denish teaches that the concentration of the red cabbage extract (“substrate”) is 100 mg/mL, not 60 mg/mL (Supplementary Method 11.2, page 63). Denish teaches the substrate concentration 100 mg/mL for the hydrolysis of red cabbage extract, not purple sweet potato extract.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to optimize by routine experimentation the amount of substrate in the catalytic reaction based on the desired concentration of the product. The person of ordinary skill in the art would have had a reasonable expectation of success in optimizing the amount of red cabbage extract in the catalytic reaction.
Claims 15-16 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 3 of U.S. Patent No. 12,167,741 (‘741) in view of Denish et al. (Science advances 7.15 (2021): eabe7871), Hong, and Kim, and as evidenced by ZME Science (2024, website), as applied to claims 9-10, 12-14, and 17-18 above, further in view of Xu et al. (Bioresource Technology 101.18 (2010): 7151-7157).
See discussion of claim 3 of ‘741, Denish, Hong, Kim, and ZME Science above, which is incorporated into this rejection as well.
Regarding claims 15-16, claim 3 of ‘741 does not recite a that the purple sweet potato is extracted by citric acid aqueous solution at a ratio of solids to liquid of 1:10 or extracting at 60 °C for between 20 a 30 min. Neither does Denish, Hong or Kim teach this claim element.
Xu teaches extracting anthocyanins from red cabbage using a conventional acidified water (CAW) approach by mixing the red cabbage with citric acid at a ratio of solids to liquids of 1:10 for 21 or 28 minutes at a temperature of 60 °C (Abstract and page 7153, left column, 2.3. Extraction experiments).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply the technique of Xu for the extraction of anthocyanins in the method of claim 3 of ‘741 modified by Denish, Hong, and Kim. The person of ordinary skill in the art would have been motivated to improve the method of claim 3 of ‘741 modified by Denish, Hong, and Kim by preparing the purple sweet potato extract using an extraction method that does not require organic solvents. The person of ordinary skill in the art would have had a reasonable expectation of success in applying Xu’s extraction technique to the method of claim 3 of ‘741 modified by Denish, Hong, and Kim.
Xu’s temperature of 60 °C is within the claimed range of 50-60°C, both of the extraction times (21 or 28 minutes) are within the claimed range of 20-30 min, and the ratio of solids to liquids of 1:10 is also within the claimed range of from 1:2 to 1:10.
Although Xu does not explicitly recite “immersing” the purple sweet potato in the citric acid, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to immerse the purple sweet potato in the citric acid in order to maximize contact between the purple sweet potato and the citric acid and thus maximize extraction efficiency of the anthocyanins. The person of ordinary skill in the art would have had a reasonable expectation of success in the immersion of the purple sweet potato in the citric acid.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CANDICE LEE SWIFT whose telephone number is (571)272-0177. The examiner can normally be reached M-F 8:00 AM-4:30 PM (Eastern).
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Louise Humphrey can be reached at (571)272-5543. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/CANDICE LEE SWIFT/Examiner, Art Unit 1657