DETAILED ACTION
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 .
Support for the amendments is within the instant application specification.
Applicants’ amendment to the claims filed on 4/17/2026 is acknowledged. This listing of claims replaces all prior listings of claims in the application.
Claims 1-24 are pending.
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-15, 17, 20-21, 22, 24) in the reply filed on 4/17/2026 is acknowledged.
Claims 1-15, 17, 20-21, 22, 24 are pending and examined on the merits.
Claims 16, 18-19, 23 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Priority
Acknowledgement is made of this national stage entry of PCT/NL2022/050425 of Non-provisional Application No. 18/580,343, filed on 7/20/2022, which claims foreign priority under 35 U.S.C. 119(a)-(d) to European Patent Application No. EP21186631.4, filing date 7/20/2021. The certified copy has been filed in the present application on 1/18/2024.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 1/18/2024, 2/7/2024 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Nucleotide and/or Amino Acid Sequences Disclosures
Examiner acknowledges Applicant’s submission of a substitute computer readable form (CRF) of the sequence listing (Sequence Listing XML). Examiner acknowledges Applicants submission of a Substitute Specification (including a mark-up version and a clean version) which directs incorporation by reference of the sequence listing. Additionally, Examiner acknowledges Applicant’s submission of a replacement sheet including amended Fig. 1 which clearly identifies the SEQ ID Nos for the various sequences in the figure.
Objection to the Claims
Claims 1 is objected to because it fails to appropriately notate the SEQ ID NO’s of recited ‘HXXFFD, H(X)XD, HXXD, HXSD. Applicant simply recites the sequences. It is suggested that Applicant include a SEQ ID NO to correspond with the recited sequences since the recited sequences are comprised of 4 or more amino acids.
Claim Rejections - 35 USC § 112
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 1-15, 17, 21-22, 24 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.
MPEP 2163.II.A.2.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states 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. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Claims 1-15, 17, 21-22, 24 are drawn to a method for oxyfunctionalization of a substrate of interest, comprising contacting the substrate with a source of hydrogen peroxide and a polypeptide having caleosin-like peroxygenase activity (EC 1.11.2.1), wherein the polypeptide is of bacterial origin and selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 60% pairwise sequence identity with any one of SEQ ID NOs: 2-10 of Figure 1, and comprising at least the following heme-coordinating motifs :i) HXXFFD; ii) H(X)XD, preferably H{XXD, more preferably HXSD, wherein X is any amino acid; and (b) a fragment of the polypeptide of (a) that has caleosin-like peroxygenase activity. The structure of the remaining amino acids of a polypeptide having caleosin-like peroxygenase activity (EC 1.11.2.1) having at least 60% sequence identity to SEQ ID NOs: 2-10 of figure 1 are a large number of sequences..
Claim 4 is drawn to the method according to claim 1 , wherein the polypeptide comprises a sequence that has at least 70% pairwise sequence identity with any one of SEQ ID NOs: 2-10 of Figure 1, or a fragment thereof that has caleosin-like peroxygenase activity. The structure of the remaining amino acids beyond a polypeptide having caleosin-like peroxygenase activity (EC 1.11.2.1) with at least 70% sequence identity to SEQ ID NOs: 2-10 of figure 1 are a large number of sequences.
In this case, the specification discloses the following representative polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1) as encompassed by the claims (i.e. SEQ ID NO: 2-10). Other than the above disclosed species, there is no prior-art or disclosed teaching as to the large number of amino acids that are the remaining amino acids of the claimed polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1). The breadth of the claims encompass any amino acids beyond polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1) with at least 60% and 70% sequence identity to amino acids of SEQ ID NOs: 2-10..
An adequate written description of a chemical invention also requires a precise definition, such as by structure, formula, chemical name, or physical properties, and not merely a wish or plan for obtaining the chemical invention claimed. See, e.g., Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 927, 69 USPQ2d 1886, 1894-95 (Fed. Cir. 2004). Here, the disclosure fails to teach which amino acids out of the numerous possibilities comprising the polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1).
Accordingly, one of skill in the art would not accept the disclosure of polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1) as any amino acids beyond polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1) with at least 60% and 70% sequence identity to amino acids of SEQ ID NOs: 2-10 as being representative of all polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1) as encompassed by the claims. As such, the specification, taken with the pre-existing knowledge in the art of polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1), fails to satisfy the written description requirement of 35 U.S.C. 112, first paragraph.
Scope of Enablement
Claims 1-15, 17, 21-22, 24 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1) such as SEQ ID NO: 2-10, it does not reasonably provide enablement for all polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1) with any amino acids amino acids beyond polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1) with at least 60% and 70% sequence identity to amino acids of SEQ ID NOs: 2-10 comprising of the amino acid sequence. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
The test of enablement is not whether any experimentation is necessary, but whether, if experimentation is necessary, it is undue.” In re Angstadt, 537 F.2d 498, 504, 190 USPQ 214, 219 (CCPA 1976). Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands (858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)) as follows: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. See MPEP § 2164.01(a). The Factors considered to be most relevant to the instant rejection are addressed in detail below.
(A)The breadth of the claims
Claims 1-15, 17, 21-22, 24 are drawn to a method for oxyfunctionalization of a substrate of interest, comprising contacting the substrate with a source of hydrogen peroxide and a polypeptide having caleosin-like peroxygenase activity (EC 1.11.2.1), wherein the polypeptide is of bacterial origin and selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 60% pairwise sequence identity with any one of SEQ ID NOs: 2-10 of Figure 1, and comprising at least the following heme-coordinating motifs :i) HXXFFD; ii) H(X)XD, preferably H{XXD, more preferably HXSD, wherein X is any amino acid; and (b) a fragment of the polypeptide of (a) that has caleosin-like peroxygenase activity. The structure of the remaining amino acids of a polypeptide having caleosin-like peroxygenase activity (EC 1.11.2.1) with at least 60% sequence identity to SEQ ID NOs: 2-10 of figure 1 are a large number of sequences.
Claim 4 is drawn to the method according to claim 1 , wherein the polypeptide comprises a sequence that has at least 70% pairwise sequence identity with any one of SEQ ID NOs: 2-10 of Figure 1, or a fragment thereof that has caleosin-like peroxygenase activity. The structure and function of the remaining amino acids beyond a polypeptide having caleosin-like peroxygenase activity (EC 1.11.2.1) with at least 70% sequence identity to SEQ ID NOs: 2-10 of figure 1 are a large number of sequences.
B) The nature of the invention; C)The state of the prior art; (D) The level of one of ordinary skill; and (E) The level of predictability in the art: As noted above, the scope of the claimed amino acids of the polypeptide with caleosin-like peroxygenase activity (EC 1.11.2.1) is a large number of sequences.
It is well-known in the prior art that the amino acid sequence of a polypeptide determines the polypeptide’s functional properties. The positions within a protein's sequence where modifications can be made with a reasonable expectation of success in obtaining a polypeptide having the desired activity/utility are limited in any protein and the result of such modifications is highly unpredictable. In addition, one skilled in the art would expect any tolerance to modification for a given protein to diminish with each further and additional modification, e.g., multiple substitutions. The reference of Singh et al. (Current Protein and Peptide Science, 2017; examiner cited) reviews various protein engineering methods and discloses that despite the availability of an ever-growing database of protein structures and highly sophisticated computational algorithms, protein engineering is still limited by the incomplete understanding of protein functions, folding, flexibility, and conformational changes [see p. 7, column 1, top]. The reference of Zhang et al. (Structure, 2018; examiner cited) discloses that a mutation of a residue that was predicted to be benign caused significant structural changes and unexpected effects on the function of a polypeptide [p. 1475, column 1].
It is well-known in the art that even a single amino acid alteration can alter the folding of a polypeptide. See, e.g., MPEP 2144.08.II.A.4.(c), which states, “[i]n the area of biotechnology, an exemplified species may differ from a claimed species by a conservative substitution (“the replacement in a protein of one amino acid by another, chemically similar, amino acid... [which] is generally expected to lead to either no change or only a small change in the properties of the protein.” Dictionary of Biochemistry and Molecular Biology 97 (John Wiley & Sons, 2d ed. 1989)). The effect of a conservative substitution on protein function depends on the nature of the substitution and its location in the chain. Although at some locations a conservative substitution may be benign, in some proteins only one amino acid is allowed at a given position. For example, the gain or loss of even one methyl group can destabilize the structure if close packing is required in the interior of domains. James Darnell et al., Molecular Cell Biology 51 (2d ed. 1990).”
(F) The amount of direction provided by the inventor and (G) The existence of working examples: The specification discloses the following working examples of polypeptides having caleosin-like peroxygenase activity (EC 1.11.2.1) as encompassed by the claims (i.e. SEQ ID NO: 2-10 in fig. 1). Other than these working species, there is no prior-art or disclosed teaching as to the large number of sequences that encompass sequences with caleosin-like peroxygenase activity (EC 1.11.2.1) through genetic modification of genes encoding proteins or post-translational modifications, etc. via any modification technique such as deletion, mutation, etc. for the method for oxyfunctionalization of a substrate of interest. Other than these working examples, the specification fails to disclose any other working examples of a method for using polypeptides with caleosin-like peroxygenase activity (EC 1.11.2.1) for oxyfunctionalization of a substrate of interest.
In view of the overly broad scope of the claims, the lack of guidance and working examples provided in the specification, the high level of unpredictability, and the state of the prior art, undue experimentation would be necessary for a skilled artisan to make and use the entire scope of the claimed invention. Applicants have not provided sufficient guidance to enable one of ordinary skill in the art to make and use the claimed invention in a manner reasonably correlated with the scope of the claims. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of having the desired biological characteristics is unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988).
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 1-15, 17, 20-22, 24 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 (claims 2-15, 17, 21-22, 24 dependent thereof) attempts to incorporate by reference the polypeptide comprising an amino acid sequence having at least 60% pairwise sequence identity with any one of SEQ ID NOs: 2-10 recited in Figure 1 of the specification. However, as clearly noted in M.P.E.P. 2173.05(s), this kind of reference to table or figures is only permitted when there is no way to clearly describe the content of the table or figure in words within the claim. It is not, however, permitted merely for Applicant’s convenience. The 9 different polypeptides of figure 1 can be listed in the claim in order to overcome the instant rejection. This rejection can be overcome by deleting the reference to figure since there is a bona fide sequence listing in the case.
Regarding claims 1 (claims 2-8, 17, 21-22, 24 dependent thereof) 15, the phrases “preferably,” "more preferably" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. The metes and bounds of the claims are not clear. See MPEP § 2173.05(c)(I).
Claim 3 attempts to incorporate by reference substrates recited in Table 2 of the specification. However, as clearly noted in M.P.E.P. 2173.05(s), this kind of reference to table or figures is only permitted when there is no way to clearly describe the content of the table or figure in words within the claim. It is not, however, permitted merely for Applicant’s convenience. The 37 different substrates of table 2 can be listed in the claim in order to overcome the instant rejection. This rejection can be overcome by deleting the reference to Table 2 since there is a bona fide sequence listing in the case.
Regarding claim 6, the recitation of the phrase ‘enhanced expression’ in line 2 is indefinite because it is unclear what the scope of the phrase is intended to encompass structurally and functionally. It is unclear what the phrase ‘enhanced expression’ is intended to encompass. Said phrase is a relative phrase. Said claim does not establish a baseline or an exact quantitative value and fails to clearly define the scope of the invention. Without a specific quantitative definition as to what is encompassed in the phrase “enhanced expression” one of skilled cannot determine what is meant by “enhanced expression.” Accordingly, the metes and bounds upon which patent protection is sought cannot be ascertained from this phrase.
Regarding claim 15, the phrase "such as" in line 3 renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention or just mere examples, thus rendering the scope undiscernible. See MPEP § 2173.05(d).
Regarding claim 20, the phrase ‘…(a) cultivating the host cell of claim 19 under conditions conducive for production of the polypeptide…’ is indefinite for depending upon withdrawn claim 19. Based on claim language, Examiner is unable to ascertain which claim said depends thereof as claim 20 is drawn to cultivating a host cell, which is not recited in any of the elected claims. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements. Additionally, the recitation ‘under conditions conducive for production of the polypeptide’ is indefinite as Applicant has not clearly defined what is meant by ‘conditions conducive for production of the polypeptide.’ As recited, said limitation can include an indefinite number of conditions. It is suggested that Applicant clarify what is meant by said limitation.
Regarding claim 20 (claim 3 dependent thereof), the phrase "solubilized fraction (supernatant)" renders the claim indefinite because it is unclear whether the limitation(s) in brackets following the terms are part of the claimed invention. See MPEP § 2173.05(d). It is unclear whether the items inside of the brackets are examples or generic terms.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-2, 4-5, 7-13, 15, 17, 21-22, 24 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Vorgelegt et al (2013, Dissertation, Examiner cited) {herein Vorgelegt} as evidenced by Hong et al (2012, US2012/0301932Al, Examiner cited) {herein Hong}. See MPEP 2131.01 regarding multiple reference 102 rejections.
Claims 1-2, 4-5, 7-13, 15, 17, 21-22, 24 are drawn to a method for oxyfunctionalization of a substrate of interest, comprising contacting the substrate with a source of hydrogen peroxide and a polypeptide having caleosin-like peroxygenase activity (EC 1.11.2.1), wherein the polypeptide is of bacterial origin and selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 60% pairwise sequence identity with any one of SEQ ID NOs: 2-10 of Figure 1, and comprising at least the following heme-coordinating motifs :i) HXXFFD; ii) H(X)XD, preferably H{XXD, more preferably HXSD, wherein X is any amino acid; and (b) a fragment of the polypeptide of (a) that has caleosin-like peroxygenase activity.
With respect to claims 1-2, 4-5, 7-13, 15, 17, 21-22, 24, Vorgelegt teaches a method of the oxyfunctionalization of alkanes, alkenes and alkynes by unspecific peroxygenase (EC 1.11.2.1) (page 1). The method comprises catalyzing the selective mono-oxygenation of diverse organic compounds using only H2O2 as a co-substrate (page 7). Said peroxygenase is derived from (AaeUPO) Agrocybe aegerita (abstract) and inherently possess a highly conserved EF-hand loop motif based on the evidentiary reference of Hong et al (para 0087). It is the Examiner’s position that said enzyme inherently has caleosin-like peroxygenase activity since Applicant discloses that EC1.11.2.1 has said activity within the instant application claim 1. Furthermore, it is the Examiner’s position that the polypeptide taught by Vorgelegt consists of a fragment of the polypeptide of SEQ ID NO: 2 that has caleosin-like peroxygenase activity as the recitation ‘a fragment of’ only requires the sequence to have at least 2 contiguous bases. Vorgelegt further teaches the polypeptide is purified (page 11; see appendix A). Additionally, said polypeptide catalyzes the conversion of typical peroxidase substrates like ABTS [2,2`-azinobis-(3-ethylbenzothiazoline-6-sulfonate)] or DMP (2,6-dimethoxyphenol), oxidized aryl alcohols to their corresponding aldehydes and acids and was able to halogenate 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (MCD) (strong bromination and very weak chlorination) (page 12, para 1) and cyclohexene (page 85, para 1). Said cyclohexene is an aliphatic alkene. It is the Examiner’s position that said peroxygenase is a biocatalyst as it is derived from a microorganism. Multiple hydrocarbon oxidizing enzyme systems, as they have been reported for hydrocarbon degrading bacteria are also imaginable in fungi (page 41, para 2). A substrate of said polypeptide is 1-propene which is a non-cyclic aliphatic alkene (C3H6) (table 2). In addition, cyclobutene is a substrate of the polypeptide (table 2). Said substrate is a cyclic aliphatic alkene. Another substrate of the polypeptide is isoprene which is a terpene substrate (table 8).
For the reasons stated herein, the teachings of claims 1-2, 4-5, 7-13, 15, 17, 21-22, 24
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.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Vorgelegt et al (2013, Dissertation, Examiner cited) {herein Vorgelegt}, as applied to claims 1-2, 4-5, 7-13, 15, 17, 21-22, 24 as evidenced by Hong et al (2012, US2012/0301932Al, Examiner cited) {herein Hong}.
Claim 14 is drawn to the method of claim 8, wherein the vinyl arene substrate is styrene, 3- methylstyrene, indene or stilbene.
With respect to claim 14, Vorgelegt teaches a method wherein methyl styrene are substrates for the oxyfunctionalization of alkenes by unspecific peroxygenase (EC 1.11.2.1) (page 1 and para 89, para 2). Vorgelegt teaches the effect was even more pronounced in the case of methylstyrenes bearing larger isomeric groups (page 89, para 2). As such, it would be obvious to one of ordinary skill in that art to try using molecules such as styrene, 3- methylstyrene, indene or stilbene as substrates for the polypeptides since Vorgelegt teaches the effect was even more pronounced in the case of methylstyrenes bearing larger isomeric groups of which are the claimed styrene, 3- methylstyrene, indene and stilbene. Therefore there would be a reasonable expectation of success to arrive at the above invention. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 1-12, 17 are rejected under 35 U.S.C. 103 as being unpatentable over Hong et al (2012, US2012/0301932Al, Examiner cited) {herein Hong} in view of Aranda et al (Date of Publication: 3 February 2021, Examiner cited) {herein Aranda).
Claims 1-12, 17 are drawn to a method for oxyfunctionalization of a substrate of interest, comprising contacting the substrate with a source of hydrogen peroxide and a polypeptide having caleosin-like peroxygenase activity (EC 1.11.2.1), wherein the polypeptide is of bacterial origin and selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 60% pairwise sequence identity with any one of SEQ ID NOs: 2-10 of Figure 1, and comprising at least the following heme-coordinating motifs :i) HXXFFD; ii) H(X)XD, preferably H{XXD, more preferably HXSD, wherein X is any amino acid; and (b) a fragment of the polypeptide of (a) that has caleosin-like peroxygenase activity.
With respect to claims 1-12, 17, Hong teaches a method wherein a codon-optimized Aspergillus niger caleosin (Cal05s) polypeptide (appendix A) is engineered to over produce oil (abstract) of which oxyfunctionalization is a critical step in the production of oil. It is the Examiner’s position that said peroxygenase is a biocatalyst as it is derived from a microorganism. The caleosin polypeptide is linked to an enzyme that catalyzes acylation of diacylglycerol (para 0104). The recombinant oleaginous microorganism of the present invention comprises at least one genetic construct encoding a functional polyunsaturated fatty acid biosynthetic pathway (para 0110). Said engineered construct also comprises the limitation of the following heme-coordinating motif of HXXD, as recited in the instant application claim 1. It is the Examiner’s position that the polypeptide taught by Hong consists of a fragment of the polypeptide of SEQ ID NO: 3 that has caleosin-like peroxygenase activity as the recitation ‘a fragment of’ only requires the sequence to have at least 2 contiguous bases, of which is taught by Hong (see appendix B). Caleosins also possess a highly conserved EF-hand loop motif (para 0087). Said EF-hand loop motif has a glutamate residue corresponding to E129 (appendix C). Up to 12 cysteines were add to the N or C-terminus of the polypeptide (para 0107). Multiple cysteine residues are frequently used as an engineered protein tag as it allows for highly specific protein manipulation. Multiple cysteine residues facilitate disulfide bond formation and protein-membrane anchoring (para 0106). Said polypeptide is cloned into host microorganisms such as yeast, fungi and algae, of which are whole cells (para 0083). One of the substrates of said polypeptide is omega-3 polyunsaturated fatty acid (PUFA) which is an non -cyclic aliphatic alkene, contains a carboxyl and a carbon-carbon double bond at one end (para 0118).
However, Hong does not teach the claimed polypeptide is of bacterial origin (claim 1)
With respect to claim 1, Aranda teaches Escherichia coli expression is in general preferred for structure-function studies and enzyme engineering (page 10, column 1, para 2). UPO expression
in E. coli as soluble and active enzymes has been reported (para 10, column 2, para 2).
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to apply the teachings of Hong et al. of a method for oxyfunctionalization of a substrate of interest or combine the teachings of Aranda because Aranda teaches Escherichia coli expression is in general preferred for structure-function studies and enzyme engineering (page 10, column 1, para 2).
One of ordinary skill in the art would be motivated to either use the teachings of Hong et al. by itself or combine the teachings Aranda because Aranda teaches UPO expression in E. coli as soluble and active enzymes (page 10, column 2, para 2). Hong would have a reasonable expectation of success that substituting a codon-optimized Aspergillus niger caleosin (Cal05s) polypeptide with the UPO from E.coli taught by Aranda because E. coli has a faster growth rate, superior metabolic versatility, and better suitability for expressing complex bacterial oxygenase enzymes (like Cytochrome P450s) without the need for eukaryotic post-translational modifications.
One of skill in the art would have a reasonable expectation of success to make and use the claimed method for oxyfunctionalization of a substrate of interest because Hong provides the basic polypeptide and its uses and methods of making it. Whereas, Aranda provides the teaching that Escherichia coli expression is in general preferred for structure-function studies and enzyme engineering (page 10, column 1, para 2). Therefore there would be a reasonable expectation of success to arrive at the above invention. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Conclusion
Status of the Claims
Claims 1-15, 17, 20-22, 24 are pending.
Claims 16, 18-19, 23 are withdrawn from further consideration pursuant to 37 CFR 1.142(b).
Claims 1-15, 17, 20-22, 24 are rejected.
No claims are in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ERICA NICOLE JONES-FOSTER whose telephone number is (571)270-0360. The examiner can normally be reached mf 7:30a - 4:30p.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath Rao can be reached at 571-272-0939. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/ERICA NICOLE JONES-FOSTER/Examiner, Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656
Appendix A
Instant Application SEQ ID NO: 2 vs Vorgelegt et al. EC 1.11.2.1
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390
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Appendix B
Instant Application SEQ ID NO: 3 vs Hong et al SEQ ID NO: 12 STIC search .rai result number 3
Query Match 20.0%; Score 173; Length 282;
Best Local Similarity 29.7%;
Matches 57; Conservative 31; Mismatches 72; Indels 32; Gaps 6;
Qy 4 DRADPSEA----------LETHITFFDVDRDGEITRDEIHQGLEELGFSPLVA--TVLAP 51
|| | |: |: |: |:| ||||:| : : | ||||: | : ||
Db 66 DRPDGDESYTKQFSDFTPLQQHVLFWDRDRDGQIYPWDTYIGFRELGFNMLFSFLAVLII 125
Qy 52 VLAIA LPRR--------------VEDLLEVRH-DDTGAFTSDGAFDEDAFEAWWRRTDRD 96
| : | | |: : : :| |: : :| | :|| : : |||
Db 126 NLNFSYPTRLAHSFLPDPWFRVYVDAVHKAKHGSDSNTYDPEGRFVPQSFENMFAKYDRD 185
Qy 97 GDGRLSRWEL--LCGSVALADDPISLGASVGELQLLHHLLAEDGGLSRASVLRFLDGSWF 154
||| |: || : | || ||:: | |: :|| : : | ||| |
Db 186 GDGALTLRELFDMMHGNRCAADPFGWGAAICEWGTTWLLIEKDGKVWKEDVRGVYDGSLF 245
Qy 155 AELIARRDAQSS 166
:: |:| |
Db 246 WKV---REANYS 254
Appendix C
Instant Application SEQ ID NO: 2 vs Hong et al SEQ ID NO: 12 (ABSS Alignment)
PNG
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381
806
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Greyscale
Prosecution Insights