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
The amendment filed 01/22/2026 has been entered. Claims 1-13, 15, 23, and 25 have been cancelled. Claims 14, 16-22, and 24 are pending and under examination.
Response to Arguments
With respect to the rejection of claims 16 and 17 under 35 USC 112(b), and the rejection of claims 14, 16-22, and 24 under 35 USC 103, Applicant's arguments filed 01/22/2026 have been fully considered but they are not persuasive.
Regarding amended claim 17, it is still unclear if the method requires adding Cu(II) ions during the fermentation step. The Examiner has suggested an amendment to overcome the 112(b) rejection.
Regarding the 103 rejections, amended claim 14 still recites “an” amino acid sequence. Therefore, any amino acid sequence that shares at least three contiguous amino acids with instant SEQ ID NOs: 2 or 3 meets the limitations of the instant claim. O’Connor teaches a method for the enzymatic conversion of tyrosol into hydroxytyrosol comprising incubating tyrosol with a Ralstonia solanacearum tyrosinase enzyme (Claim 1). O’Connor teaches that the Ralstonia solancearum tyrosinase amino acid sequence is SEQ ID NO:1 (Column 5, lines 20-36). SEQ ID NO:1 of O’Connor shares 91.4% sequence identity with instant SEQ ID NO:2. Therefore, the enzyme of O’Connor meets the limitations of instant claim 14.
Applicant argues that those in the art know that epimeric structures like erythorbic acid as a stereoisomer of ascorbic acid exhibit different effects. Applicant supports this argument by citing a reference that describes the enzymatic inhibitory function of completely different compound, N-acetygalactosamine and its epimer. Applicant does not provide any evidence that erythorbic acid and ascorbic acid have different reductive abilities. As stated, Fidler explicitly states that erythorbic acid is a stereoisomer of ascorbic acid with similar physiochemical properties and is used as an antioxidant, i.e., a reductant (Abstract)
Therefore, Applicant’s arguments are not considered persuasive.
Claim Rejections - 35 USC § 112(b)
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.
Claim 17 remains 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.
Regarding claim 17, it is unclear whether an additional method step of adding 0.02 mM Cu(II) ions after or during the fermentation is required, or whether the fermentation broth comprises at least 0.02 mM Cu(II) when the fermentation starts.
To obviate the rejection, Applicant may consider the following amendment which has been drafted by the Examiner:
Claim 17. The process of claim 16, wherein -Cu(II) ions are fed to a culture of the E. coli during at a concentration of at least 0.02 mM
Claim Rejections - 35 USC § 103
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.
Claims 14, 16, and 18-22 are rejected under 35 U.S.C. 103 as being unpatentable over O'Connor et al., (US Patent No. 10,822,594 B2) in view of Fidler et al., (Erythorbic acid as a potent enhancer of nonheme-iron absorption. Am J Clin Nutr. 2004 Jan;79(1):99-102).
Regarding claim 14, O’Connor teaches a method for the enzymatic conversion of tyrosol into hydroxytyrosol comprising incubating tyrosol with a Ralstonia solanacearum tyrosinase enzyme (Claim 1). O’Connor teaches that the Ralstonia solancearum tyrosinase amino acid sequence is SEQ ID NO:1 (Column 5, lines 20-36). SEQ ID NO:1 of O’Connor shares 91.4% sequence identity with instant SEQ ID NO:2 (see alignment below).
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Because the claim recites an amino acid sequence (not the amino acid sequence), any amino acid sequence that shares at least three contiguous amino acids with instant SEQ ID NOs: 2 or 3 meets the claim limitations.
O’Connor teaches the use of ascorbic acid (Claim 1) as a reductant (Column 8, lines 30-31), and teaches a tyrosol concentration of at least 75 mM and an ascorbic acid concentration of at least 150 mM (Claim 1), i.e., a substrate:reductant ratio of 1:2.
Although the ratio of O’Connor is different than that of the instant claim, MPEP§ 2144.05 II states: “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. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 ("The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.")” The selection of a specific substrate:reductant ratio would have been a routine matter of optimization on the part of the artisan of ordinary skill, said artisan recognizing that product amount and/or product purity may be affected by said ratio.
O’Connor does not teach the use of erythorbic acid or erythorbate as a protective reductant.
However, Fidler teaches that erythorbic acid is a stereoisomer of ascorbic acid with similar physiochemical properties and is used as an antioxidant, i.e., a reductant (Abstract).
Therefore, it would have been obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to substitute erythorbic acid as taught by Fidler for the ascorbic acid taught by O’Connor. One of ordinary skill would have had a reasonable expectation of success because substituting reducing agents for those of similar function is well within the knowledge of one of ordinary skill in the art, and the results are reasonably predictable.
Regarding claim 16, O’Connor teaches that recombinant E. coli was used to express the tyrosinase gene from R. solanacearum (Column 7, line 65) i.e., recombinant production. O’Connor teaches that strains were cultured in a rotary shaker in 5 mL broth overnight (Column 8, lines 4-7).
Page 12 of instant specification discloses that the terms “culture”, “growth”, and “fermentation” and also, “culture medium”, “growth medium”, and “fermentation medium” are used synonymously in the context of the present invention. Therefore, it is interpreted that the culture method taught by O’Connor is synonymous with “fermentation” as defined by the instant disclosure.
Regarding claim 18, it is interpreted that the claim means that the recombinant cells expressing the gene encoding the tyrosinase are directly reacted with tyrosol, i.e., the enzyme is not purified. O’Connor teaches that biotransformation of tyrosol to hydroxytyrosol was carried out using whole cells (Column 8, line 42).
Regarding claim 19, O’Connor teaches that in one embodiment, the substrate concentration is at least 175 mM (Column 3, lines 59-60). Therefore, it is interpreted than any concentration above 175 mM tyrosol may be used. The prior art concentration range overlaps with the claimed range, therefore, a prima facie case of obviousness exists.
Regarding claim 20, O’Connor teaches an ascorbic acid concentration of up to 400 mM (Column 3, line 42). It would have been obvious to one of ordinary skill in the art to substitute erythorbic acid for ascorbic acid based on the teachings of Fidler, and to have used erythorbic acid at the concentration taught by O’Connor as Fidler teaches that erythorbic acid is a stereoisomer of ascorbic acid with similar physiochemical properties and technologic applications (Fidler, Abstract; p. 99, column 2, para. 1).
Regarding claim 21, O’Connor teaches that biotransformation of tyrosol to hydroxytyrosol was carried out using whole cells, and teaches various concentrations of tyrosol (75, 100, 150, and 175 mM) were added to a working volume of 100 mL (Column 8, lines 35-45). Although O’Connor does not explicitly teach the percent volume of the fermentation broth, the selection of specific fermentation broth concentration is a routine matter of optimization on the part of the artisan of ordinary skill, said artisan recognizing that fermentation broth concentration would affect enzyme and subsequent product concentration. See MPEP § 2144.05 II.
Regarding claim 22, O’Connor teaches that the reaction is conducted for a period of time sufficient to allow the enzyme to convert at least 90% of tyrosol to hydroxytyrosol (Claim 1).
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over O'Connor et al., (US Patent No. 10,822,594 B2) and Fidler et al., (Erythorbic acid as a potent enhancer of nonheme-iron absorption. Am J Clin Nutr. 2004 Jan;79(1):99-102) as applied to claim 16 above, and further in view of Deri-Zenaty et al., 2019 (A coupled enzymatic reaction of tyrosinase and glucose dehydrogenase for the production of hydroxytyrosol. Applied Microbiology and Biotechnology (2020) 104:4945-4955).
The teachings of O’Connor and Fidler are incorporated into this rejection.
O’Connor does not teach at least 0.02 mM Cu(II) ions in the culture medium.
However, Deri-Zenaty teaches the conversion of tyrosol into hydroxytyrosol using a tyrosinase enzyme and a glucose dehydrogenase enzyme (Abstract). Deri-Zenaty teaches that the tyrosinase activity assay was performed in the presence of 0.01-0.04 mM of CuSO4 (p. 4948, Tyrosinase activity assay). Deri-Zenaty teaches adding Cu(II) ions after recombinant production of the enzyme and that tyrosinase is a copper containing enzyme (p. 4945, column 2, para. 2), i.e., an enzyme that utilizes copper in its catalytic role. Although Deri-Zenaty teaches the addition of Cu(II) ions during a different step, the method of Deri-Zenaty achieves the same result of increasing tyrosinase enzyme activity as the claimed invention. See MPEP 2144.04(IV)(C), which states: In reBurhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In reGibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.).
It would have been obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to add Cu(II) ions, as taught by Deri-Zenaty, to the reaction mixture containing tyrosinase and tyrosol as taught by O’Connor and erythorbic acid as taught by Fidler. Deri-Zenaty teaches that tyrosinase is a copper containing enzyme, therefore, one of ordinary skill in the art would have been motivated to add Cu(II) ions to a reaction mixture containing tyrosinase to optimize enzyme activity. One of ordinary skill in the art would have had a reasonable expectation of success because O’Connor and Deri-Zenaty are in the same field of endeavor of tyrosine-catalyzed enzymatic conversion of tyrosol into hydroxytyrosol.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over O'Connor et al., (US Patent No. 10,822,594 B2) and Fidler et al., (Erythorbic acid as a potent enhancer of nonheme-iron absorption. Am J Clin Nutr. 2004 Jan;79(1):99-102) as applied to claim 14 above, and further in view of Berninni et al., (WO 2008/110908 A1) as evidenced by Liebgott et al., (WO 2010/012871 A1).
The teachings of O’Connor and Fidler are incorporated into this rejection.
O’Connor and Fidler do not teach HTS extraction with ethyl acetate or removal of ethyl acetate via distillation.
However, regarding claim 24, Berninni teaches methods of producing hydroxytyrosol and hydroxytyrosol derivatives (Abstract) and teaches ethyl acetate extraction and removal of ethyl acetate by distillation (p. 8, Example 3).
It would have been obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to have extracted the hydroxytyrosol produced by reacting tyrosinase and tyrosol as taught by O’Connor, and erythorbic acid as taught by Fidler, using ethyl acetate, followed by removal of ethyl acetate by distillation, as taught by Berninni. One of ordinary skill in the art would have been motivated to do so because O’Connor teaches that separation of the product may comprise a solvent extraction step (Column 3, line 64), and extraction with ethyl acetate allows for a higher degree of purity, as evidenced by Liebgott. One of ordinary skill in the art would have had a reasonable expectation of success because O’Connor and Berninni are in the same field of endeavor of hydroxytyrosol production methods.
Conclusion
THIS ACTION IS MADE FINAL. 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 RACHEL EMILY MARTIN whose telephone number is (703)756-1416. The examiner can normally be reached M-Th 8:30-16:00, F 8:30-10:00 EST.
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/RACHEL EMILY MARTIN/Examiner, Art Unit 1657