DETAILED CORRESPONDENCE
Status of the Application
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-40 are pending in the application.
Applicant’s claim listing filed April 9, 2026 is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Election/Restrictions
Applicant’s elections without traverse of:
Group I, claims 1-19, drawn to an engineered monoamine oxidase and a composition,
amino acid substitution at position 429 (species election A),
SEQ ID NO: 414 (species election B),
improved production of compound 2 (species election C), and
amino acid substitution at position 246 (species election E)
in the replies filed December 29, 2025 and April 9, 2026 are acknowledged.
Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 20-40 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim.
Claims 6, 8, 10, 15, and 16 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected species, there being no allowable generic or linking claim.
Claims 1-5, 7, 9, 11-14, and 17-19 are being examined on the merits with claims 2-5, 7, 9, 11, and 12 being examined only to the extent the claims read on the elected subject matter.
Priority
This application claims domestic priority under 35 U.S.C. 119(e) to US provisional application no. 63/319,135, filed March 11, 2022.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on August 15, 2024 and May 5, 2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the IDSs have been considered by the examiner.
Specification
The use of the term "NCBI," which is a trade name or a mark used in commerce, has been noted in this application (p. 18, paragraph [0045]). The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as TM, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claims 1-5, 7, 9, 11, 12, and 19 are objected to because of the following informalities:
Claim 1 is objected to in the recitation of “SEQ ID NOs: 6” in lines 3 and 6 and in the interest of improving claim form, it is suggested that “SEQ ID NOs: 6” be replaced with the singular “SEQ ID NO: 6.”
Claim 1 is objected to in the recitation of “amino acid positions” in line 5 and in the interest of improving claim form, it is suggested that “positions” in the noted phrase be replaced with the singular “position.”
Claims 1-5, 7, and 9 are objected to in the recitation of “80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%” and in the interest of improving claim form it is suggested that the noted phrase be amended to recite only “80%”.
Claim 9 is objected to in the recitation of “comprises at least one substitution” and in the interest of improving claim form and consistency, it is suggested that the noted phrase be amended to recite “further comprises at least one substitution.”
Claim 11 is objected to in the recitation of “to any even-numbered sequence set forth in SEQ ID NOs:6-1084” and in the interest of improving claim form and using proper Markush terminology, it is suggested that the noted phrase be amended to recite “to an even-numbered amino acid sequence selected from the group consisting of SEQ ID NO:6-1084.”
Claim 12 is objected to in the recitation of “a polypeptide sequence set forth in the even numbered sequences of SEQ ID NOs:6-1084” and in the interest of improving claim form and using proper Markush terminology, it is suggested that the noted phrase be amended to recite “a polypeptide sequence selected from the group consisting of SEQ ID NO:6-1084.”
Claim 19 is objected to in the recitation of “at least one engineered monoamine oxidase polypeptide provided in claim 1” and in the interest of improving claim form, it is suggested that the noted phrase be amended to recite “A composition comprising the engineered monoamine oxidase polypeptide of claim 1.”
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.
Claims 1-5, 7, 9, 11-14, and 17-19 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claim 1 (claims 2-5, 7, 9, 12-14, and 17-19 dependent therefrom) is indefinite in the recitation of “functional fragment thereof” because it is unclear from the claims and the specification as to what function(s) of the fragment is/are intended as being encompassed by “functional fragment thereof”. Also, it is unclear as to whether the recitation of “functional fragment thereof” is intended to refer to the recited polypeptide sequence of the engineered monoamine oxidase or is intended to refer to SEQ ID NO: 6. It is suggested that the applicant clarify the meaning of the phrase “functional fragment thereof”.
Claims 11 and 12 are confusing in the recitation of “wherein said engineered monoamine oxidase polypeptide comprises an amino acid sequence with at least 80% sequence identity to any even-numbered sequence set forth in SEQ ID NOs: 6-1084” and “wherein said engineered monoamine oxidase polypeptide comprises a polypeptide sequence set forth in the even numbered sequences of SEQ ID NOs: 6-1084,” respectively. Claims 11 and 12 encompass the embodiment of the engineered monoamine oxidase polypeptide comprising the amino acid sequence of SEQ ID NO: 6, yet given that the engineered monoamine oxidase of claims 11 and 12 has a substitution at amino acid 429 of SEQ ID NO: 6, the engineered monoamine oxidase polypeptide of claims 11 and 12 cannot comprise SEQ ID NO: 6. It is suggested that applicant clarify the meanings of claims 11 and 12.
Claim Rejections - 35 USC § 112(a)
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.
Claims 1, 12-14, and 17-19 are rejected under 35 U.S.C. 112(a) because the specification, while being enabling for an engineered monoamine oxidase comprising a polypeptide sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 6 and an A429G substitution at the position corresponding to amino acid 429 of SEQ ID NO: 6, does not reasonably provide enablement for all engineered monoamine oxidases as encompassed by the claims. 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.
The nature of the invention: According to the specification, it is necessary to provide improved engineered enzymes with reduced enzyme loading (wt/wt) requirements for the manufacturing of APIs and intermediates (paragraph [0019]).
The specification discloses that the monoamine oxidase biocatalysts of the present disclosure are engineered polypeptide variants of a previously engineered monoamine oxidase (SEQ ID NO: 6) derived from a fusion polypeptide of Aspergillus niger (SEQ ID NO: 2) and Aspergillus oryzae (SEQ ID NO: 4) monoamine oxidase homologs. These engineered monoamine oxidase polypeptides are capable of catalyzing the conversion of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane and related bicyclic proline analogs to (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-ene and related compounds (paragraph [0020]).
The breadth of the claims: The claims are drawn to an engineered monoamine oxidase comprising a polypeptide sequence comprising at least 80% sequence identity to SEQ ID NO: 6, or a functional fragment thereof, wherein said engineered monoamine oxidase polypeptide comprises a substitution at position 429 in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 6.
Regarding claim 1, the recitation of “engineered monoamine oxidase” is interpreted as requiring monoamine oxidase activity.
As stated above, it is unclear as to the function(s) that is/are exhibited by the recited “functional fragment thereof” and it is also unclear as to whether the phrase “functional fragment thereof” refers to the polypeptide sequence of the engineered monoamine oxidase or to SEQ ID NO: 6. Given a broadest reasonable interpretation, the recitation of “a functional fragment thereof” is interpreted as any two or more contiguous amino acids of the amino acid sequence of SEQ ID NO: 6.
Regarding claim 12, the recitation of “a polypeptide sequence” is interpreted as any two or more contiguous amino acids of the amino acid sequence of SEQ ID NO: 414.
Regarding claim 13, the “improved property” exhibited by the engineered monoamine oxidase polypeptide of claim 13 is unlimited.
The state of the prior art; The level of one of ordinary skill; and The level of predictability in the art: According to MPEP 2164.03, “…what is known in the art provides evidence as to the question of predictability” and “[I]f one skilled in the art cannot readily anticipate the effect of a change within the subject matter to which that claimed invention pertains, then there is lack of predictability in the art.”
Before the effective filing date, the reference of Ghislieri, D. (“Application of Engineered Amine Oxidases for the Synthesis of Chiral Amines,” Thesis submitted to University of Manchester, 2013; cited on the attached Form PTO-892) taught site-saturation mutagenesis at position 429 of a variant of Aspergillus niger monoamine oxidase (MAO-N) to replace A429 with all 19 other amino acids (pp. 44, bottom and p. 57, bottom to p. 58, middle).
However, as described above, the amino acid sequence of the engineered monoamine oxidase of claims 1, 12-14, and 17-19 encompasses essentially unlimited amino acid substitutions, deletions, insertions, and additions relative to SEQ ID NO: 6. Regarding the level of skill and knowledge in the art of amino acid modification, the reference of Singh et al. (Curr. Protein Pept. Sci. 18:1-11, 2017; cited on the attached Form PTO-892) 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). Also, the unpredictability associated with surface residue substitution is exemplified by the reference of Zhang et al. (Structure 26:1474-1485, 2018; cited on the attached Form PTO-892), which discloses that even a substitution of a surface residue that was predicted to be benign caused significant structural changes and unexpected effects on the function of a polypeptide (p. 1475, column 1).
The amount of direction provided by the inventor and The existence of working examples: The specification discloses various working examples of the claimed engineered monoamine oxidase, e.g., an engineered monoamine oxidase comprising the amino acid sequence of SEQ ID NO: 414, which is the amino acid sequence of SEQ ID NO: 6 with a single A429G substitution, and which exhibits increased production of Compound 2 relative to SEQ ID NO: 6 (p. 88, Table 5.1).
The quantity of experimentation needed to make or use the invention based on the content of the disclosure: While methods of generating variants of a polypeptide were known in the art before the effective filing date, it was not routine in the art to screen by a trial and error process for all engineered monoamine oxidases as broadly encompassed by the claims and identify those having the desired activity and with any improved property as compared to SEQ ID NO: 6 and/or SEQ ID NO: 414.
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, and the amount of experimentation required, 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).
Claims 1, 12-14, and 17-19 are rejected under 35 U.S.C. 112(a) 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, 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.
MPEP § 2163 further states that “[s]atisfactory disclosure of a ‘representative number’ depends on whether one of skill in the art would recognize that the applicant was in possession of the necessary common attributes or features possessed by the members of the genus in view of the species disclosed. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus…Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are ‘representative of the full variety or scope of the genus,’ or by the establishment of ‘a reasonable structure-function correlation.’ Such correlations may be established ‘by the inventor as described in the specification,’ or they may be ‘known in the art at the time of the filing date.’"
The factors considered in the Written Description requirement are (1) level of skill and knowledge in the art, (2) partial structure, (3) physical and/or chemical properties, (4) functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the (5) method of making the claimed invention. Disclosure of any combination of such identifying characteristics that distinguish the claimed invention from other materials and would lead one of skill in the art to the conclusion that the applicant was in possession of the claimed species is sufficient." MPEP § 2163.
The claims are drawn to a genus of engineered monoamine oxidases comprising a polypeptide sequence comprising at least 80% sequence identity to SEQ ID NO: 6, or a functional fragment thereof, wherein said engineered monoamine oxidase polypeptide comprises a substitution at position 429 in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 6.
Regarding claim 1, the recitation of “engineered monoamine oxidase” is interpreted as requiring monoamine oxidase activity.
As stated above, it is unclear as to the function(s) that is/are exhibited by the recited “functional fragment thereof” and it is also unclear as to whether the phrase “functional fragment thereof” refers to the polypeptide sequence of the engineered monoamine oxidase or to SEQ ID NO: 6. Given a broadest reasonable interpretation, the recitation of “a functional fragment thereof” is interpreted as any two or more contiguous amino acids of the amino acid sequence of SEQ ID NO: 6.
Regarding claim 12, the recitation of “a polypeptide sequence” is interpreted as any two or more contiguous amino acids of the amino acid sequence of SEQ ID NO: 414.
Regarding claim 13, the “improved property” exhibited by the engineered monoamine oxidase polypeptide of claim 13 is unlimited.
The specification discloses representative species of the claimed engineered monoamine oxidase, e.g., an engineered monoamine oxidase comprising the amino acid sequence of SEQ ID NO: 414, which is the amino acid sequence of SEQ ID NO: 6 with a single A429G substitution and exhibits increased production of Compound 2 relative to SEQ ID NO: 6 (p. 88, Table 5.1). Other than the A429G substitution, the specification fails to disclose any other substitutions at the position corresponding to position 429 of SEQ ID NO: 6 that maintain monoamine oxidase activity and increase production of Compound 2 relative to SEQ ID NO: 6.
However, given a broadest reasonable interpretation, the amino acid sequences of the genus of engineered monoamine oxidase of claims 1, 12-14, and 17-19 are widely variant, encompassing essentially unlimited amino acid substitutions, deletions, insertions, and additions relative to SEQ ID NO: 6. Regarding the level of skill and knowledge in the art of amino acid substitution, the reference of Singh et al. (supra) 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). Also, the unpredictability associated with surface residue substitution is exemplified by the reference of Zhang et al. (supra), which discloses that even a substitution of a surface residue that was predicted to be benign caused significant structural changes and unexpected effects on the function of a polypeptide (p. 1475, column 1).
Because the claims encompass a widely variant genus of engineered monoamine oxidase polypeptides and in view of the unpredictable nature of amino acid substitutions, the disclosed representative species of engineered monoamine oxidase polypeptides do not reflect the wide variation among the members of the genus. As such, one of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the genus, and thus, that the applicant was not in possession of the claimed genus. The claimed subject matter is not supported by an adequate written description because a representative number of species has not been described.
Claim Rejections - 35 USC § 102/103
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.
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.
Claims 1-5, 7, 9, 11-14, 18, and 19 are rejected under 35 U.S.C. 102(a)(1) as anticipated by or, in the alternative, under 35 U.S.C. 103 as obvious over Ghislieri, D. (“Application of Engineered Amine Oxidases for the Synthesis of Chiral Amines,” Thesis submitted to University of Manchester, 2013; cited on the attached Form PTO-892; hereafter “Ghislieri”).
Applicant’s attention is directed to MPEP 2112.III regarding a rejection under 35 U.S.C. 102/103 when the prior art product seems to be identical except that the prior art is silent as to an inherent characteristic.
The claims are drawn to an engineered monoamine oxidase comprising a polypeptide sequence comprising at least 80% sequence identity to SEQ ID NO: 6, or a functional fragment thereof, wherein said engineered monoamine oxidase polypeptide comprises a substitution at position 429 in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 6. The claims have been interpreted as described in detail above.
Regarding instant claims 1-5 and 7, Ghislieri teaches wild-type Aspergillus niger monoamine oxidase (MAO-N) (p. 59, top) and a variant thereof referred to as D5 mutant, which has the mutations I246M, N336S, T384N, and D385S relative to the sequence of wild-type MAO-N (p. 59, Table S3). Ghislieri teaches site-saturation mutagenesis at position 429 of D5 mutant to replace A429 with all 19 other amino acids (pp. 44, bottom and p. 57, bottom to p. 58, middle), which includes glycine. A429 of MAO-N corresponds to position 429 of instant SEQ ID NO: 6 and I246 of MAO-N corresponds to position 246 of instant SEQ ID NO: 6 (see Appendix A for sequence alignment).
Ghislieri does not teach the amino acid sequences of the site-saturation mutants at position A429 have at least 80% sequence identity to SEQ ID NO: 6. However, as an example, the amino acid sequence of Ghislieri’s site-saturation mutant with glycine at position 429 has 86% sequence identity with instant SEQ ID NO: 6 (see Appendix B for sequence alignment). As such, the amino acid sequences of the site-saturation mutants of A429 of Ghislieri have at least 80% sequence identity to SEQ ID NO: 6.
Regarding instant claims 9 and 11, Ghislieri does not teach the amino acid sequences of the site-saturation mutants of A429 have at least 80% sequence identity to SEQ ID NO: 414. However, as an example, the amino acid sequence of Ghislieri’s site-saturation mutant with glycine at position 429 has 86% sequence identity with instant SEQ ID NO: 414 (see Appendix C for sequence alignment). As such, the amino acid sequences of the site-saturation mutants of A429 of Ghislieri have at least 80% sequence identity to SEQ ID NO: 414.
Regarding instant claim 12, the amino acid sequences of the site-saturation mutants of A429 of Ghislieri comprise a polypeptide sequence (i.e., any two or more contiguous amino acids) of SEQ ID NO: 414 (see Appendix C for sequence alignment).
Regarding instant claims 13 and 14, Ghislieri does not teach the site-saturation mutants of A429 exhibit at least one improved property compared to the engineered monoamine oxidase polypeptide of SEQ ID NOs: 6 and/or 414 including improved production of Compound 2 compared to SEQ ID NO: 6 and/or 414. However, according to MPEP 2112.01.I, when the structure recited in the reference is substantially identical to that that of the claims, claimed properties or functions are presumed to be inherent. Since the structures of the site-saturation mutants of A429 of Ghislieri are substantially identical to and are encompassed by claims 13 and 14, it is presumed that the site-saturation mutants of A429 of Ghislieri exhibit at least one improved property including improved production of Compound 2 compared to the engineered monoamine oxidase polypeptide of SEQ ID NOs: 6 and/or 414.
Regarding instant claim 18, Ghislieri teaches the site-saturation mutants of A429 were screened using a solid phase assay (paragraph bridging pp. 44-45; p. 58, middle) in which E. coli cells expressing the site-saturation mutants of A429 were transferred to a membrane and the cells on the membrane were partially lysed (p. 58, middle to bottom). As such, the site-saturation mutants of A429 of Ghislieri are considered to be immobilized onto a solid support.
Regarding instant claim 19, Ghislieri teaches cells and partially lysed cells comprising the site-saturation mutants of A429, which are encompassed by the composition of claim 19.
Therefore, Ghislieri anticipates claims 1-5, 7, 9, 11-14, 18, and 19 as written.
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 17 is rejected under 35 U.S.C. 103 as being unpatentable over Ghislieri in view of Rios-Solis et al. (Journal of Molecular Catalysis B: Enzymatic 120:100-110, 2015; cited on the attached Form PTO-892; hereafter “Rios-Solis”).
Claim 17 is drawn to the engineered monoamine oxidase polypeptide of claim 1, wherein said engineered monoamine oxidase polypeptide is purified.
The relevant teachings of Ghislieri as applied to claims 1-5, 7, 9, 11-14, 18, and 19 are set forth above.
Ghislieri does not teach purifying the site-saturation mutants of A429.
Rios-Solis teaches that to develop a biocatalyst for commercial application, it is necessary to screen several enzyme variants with a variety of substrates over a range of concentrations (p. 100, column 1). Rios-Solis teaches a high throughput substrate screening assay using clarified lysates of a MAO-N variant (p. 101, column 2, top).
In view of the combined teachings of Ghislieri and Rios-Solis, it would have been obvious to one of ordinary skill in the art before the effective filing date to use the high throughput substrate screening assay of Rios-Solis to screen the site-saturation mutants of A429 of Ghislieri, which would include preparing clarified lysates of the site-saturation mutants of A429 of Ghislieri. Clarified lysates of the site-saturation mutants of A429 of Ghislieri are considered to be at least partially purified. One would have been motivated and would have expected success because while Ghislieri taught making and screening a library of site-saturation mutants of A429, Ghislieri’s screening method used only a single substrate. However, Rios-Solis taught that to develop a biocatalyst for commercial application, it is necessary to screen several enzyme variants with a variety of substrates over a range of concentrations, and Rios-Solis taught a high throughput substrate screening assay using clarified lysates of a MAO-N variant.
Therefore, the engineered monoamine oxidase of claim 17 would have been obvious to one of ordinary skill in the art before the effective filing date.
Conclusion
Status of the claims:
Claims 1-40 are pending.
Claims 6, 8, 10, 15, 16, and 20-40 are withdrawn from consideration.
Claims 1-5, 7, 9, 11-14, and 17-19 are rejected.
No claim is in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DAVID J STEADMAN whose telephone number is (571)272-0942. The examiner can normally be reached Monday to Friday, 7:30 AM to 4:00 PM.
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, MANJUNATH N. RAO can be reached on 571-272-0939. 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.
/David Steadman/Primary Examiner, Art Unit 1656
APPENDIX A
AOFN_ASPNG
ID AOFN_ASPNG Reviewed; 495 AA.
AC P46882;
DT 01-NOV-1995, integrated into UniProtKB/Swiss-Prot.
DT 01-NOV-1995, sequence version 1.
DT 05-FEB-2025, entry version 102.
DE RecName: Full=Monoamine oxidase N;
DE Short=MAO-N;
DE EC=1.4.3.4;
GN Name=maoN;
OS Aspergillus niger.
OC Eukaryota; Fungi; Dikarya; Ascomycota; Pezizomycotina; Eurotiomycetes;
OC Eurotiomycetidae; Eurotiales; Aspergillaceae; Aspergillus;
OC Aspergillus subgen. Circumdati.
OX NCBI_TaxID=5061;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], AND PROTEIN SEQUENCE OF 165-175.
RX PubMed=7770050; DOI=10.1007/bf00293144;
RA Schilling B., Lerch K.;
RT "Cloning, sequencing and heterologous expression of the monoamine oxidase
RT gene from Aspergillus niger.";
RL Mol. Gen. Genet. 247:430-438(1995).
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a secondary aliphatic amine + O2 + H2O = a primary amine + an
CC aldehyde + H2O2; Xref=Rhea:RHEA:26414, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:15379, ChEBI:CHEBI:16240, ChEBI:CHEBI:17478,
CC ChEBI:CHEBI:58855, ChEBI:CHEBI:65296; EC=1.4.3.4;
CC -!- COFACTOR:
CC Name=FAD; Xref=ChEBI:CHEBI:57692;
CC -!- SUBCELLULAR LOCATION: Peroxisome {ECO:0000305}.
CC -!- SIMILARITY: Belongs to the flavin monoamine oxidase family.
CC {ECO:0000305}.
CC ---------------------------------------------------------------------------
CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms
CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR EMBL; L38858; AAA98490.1; -; Genomic_DNA.
DR PIR; S55273; S55273.
DR PDB; 2VVL; X-ray; 2.45 A; A/B/C/D/E/F/G/H=1-495.
DR PDB; 2VVM; X-ray; 1.85 A; A/B=1-495.
DR PDB; 3ZDN; X-ray; 2.55 A; A/B/C/D=1-495.
DR PDBsum; 2VVL; -.
DR PDBsum; 2VVM; -.
DR PDBsum; 3ZDN; -.
DR AlphaFoldDB; P46882; -.
DR SMR; P46882; -.
DR PaxDb; 5061-CADANGAP00009588; -.
DR VEuPathDB; FungiDB:An12g03290; -.
DR VEuPathDB; FungiDB:ASPNIDRAFT2_1084380; -.
DR VEuPathDB; FungiDB:ATCC64974_41670; -.
DR VEuPathDB; FungiDB:M747DRAFT_358493; -.
DR eggNOG; KOG0029; Eukaryota.
DR BRENDA; 1.4.3.4; 518.
DR EvolutionaryTrace; P46882; -.
DR GO; GO:0005777; C:peroxisome; IEA:UniProtKB-SubCell.
DR GO; GO:0097621; F:monoamine oxidase activity; IEA:UniProtKB-EC.
DR Gene3D; 3.90.660.10; -; 2.
DR Gene3D; 3.50.50.60; FAD/NAD(P)-binding domain; 2.
DR InterPro; IPR002937; Amino_oxidase.
DR InterPro; IPR036188; FAD/NAD-bd_sf.
DR InterPro; IPR001613; Flavin_amine_oxidase.
DR InterPro; IPR050703; Flavin_MAO.
DR PANTHER; PTHR43563; AMINE OXIDASE; 1.
DR PANTHER; PTHR43563:SF1; AMINE OXIDASE [FLAVIN-CONTAINING] B; 1.
DR Pfam; PF01593; Amino_oxidase; 1.
DR PRINTS; PR00757; AMINEOXDASEF.
DR SUPFAM; SSF51905; FAD/NAD(P)-binding domain; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Direct protein sequencing; FAD; Flavoprotein; Oxidoreductase;
KW Peroxisome.
FT CHAIN 1..495
FT /note="Monoamine oxidase N"
FT /id="PRO_0000099865"
FT REGION 1..23
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT MOTIF 493..495
FT /note="Microbody targeting signal"
FT /evidence="ECO:0000255"
FT COMPBIAS 1..19
FT /note="Polar residues"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT STRAND 7..10
FT /evidence="ECO:0007829|PDB:2VVM"
FT TURN 11..13
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 14..17
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 26..29
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 40..45
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 49..60
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 65..68
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 70..75
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 80..83
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 86..89
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 100..108
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 115..118
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 122..124
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 127..133
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 138..140
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 142..157
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 159..162
FT /evidence="ECO:0007829|PDB:2VVM"
FT TURN 163..167
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 180..184
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 188..195
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 196..198
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 201..215
FT /evidence="ECO:0007829|PDB:2VVM"
FT TURN 219..221
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 224..233
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 238..246
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 247..250
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 254..266
FT /evidence="ECO:0007829|PDB:2VVM"
FT TURN 267..269
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 271..276
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 279..284
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 286..293
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 298..306
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 310..315
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 316..320
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 324..332
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 339..346
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 348..352
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 353..357
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 359..362
FT /evidence="ECO:0007829|PDB:2VVL"
FT STRAND 365..371
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 377..383
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 385..387
FT /evidence="ECO:0007829|PDB:2VVL"
FT TURN 391..393
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 395..403
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 412..417
FT /evidence="ECO:0007829|PDB:2VVM"
FT TURN 420..422
FT /evidence="ECO:0007829|PDB:2VVM"
FT TURN 424..426
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 427..430
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 437..446
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 452..454
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 457..459
FT /evidence="ECO:0007829|PDB:2VVM"
FT STRAND 461..463
FT /evidence="ECO:0007829|PDB:2VVM"
FT HELIX 467..485
FT /evidence="ECO:0007829|PDB:2VVM"
SQ SEQUENCE 495 AA; 55617 MW; 0E614FF09D3C5B3D CRC64;
Query Match 86.9%; Score 2337; Length 495;
Best Local Similarity 86.9%;
Matches 430; Conservative 21; Mismatches 44; Indels 0; Gaps 0;
Qy 1 MTSRDGYQWTPETGLTQGVPSLGVISPPTNIEDTDKDGPWDVIVIGGGYCGLTATRDLTV 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTSRDGYQWTPETGLTQGVPSLGVISPPTNIEDTDKDGPWDVIVIGGGYCGLTATRDLTV 60
Qy 61 AGFKTLLLEARDRIGGRSWSSNIDGYPYEMGGTWVHWHQSHVWREITRYKMHNALSPSFN 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AGFKTLLLEARDRIGGRSWSSNIDGYPYEMGGTWVHWHQSHVWREITRYKMHNALSPSFN 120
Qy 121 FSRGVNHFQLRTNPTTSTYMTHEAEDELLRSALHKFTNVDGTNGRTVLPFPHDMFYVPEF 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 FSRGVNHFQLRTNPTTSTYMTHEAEDELLRSALHKFTNVDGTNGRTVLPFPHDMFYVPEF 180
Qy 181 RKYDEMSYSERIDQIRDELSLNERSSLEAFILLCSGGTLENSSFGEFLHWWAMSGYTYQG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 RKYDEMSYSERIDQIRDELSLNERSSLEAFILLCSGGTLENSSFGEFLHWWAMSGYTYQG 240
Qy 241 CMDCLISYKFKDGQSAFARRFWEEAAGTGRLGYVFGCPVRSVVNERDAVRVTARDGREFA 300
|||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||
Db 241 CMDCLISYKFKDGQSAFARRFWEEAAGTGRLGYVFGCPVRSVVNERDAARVTARDGREFA 300
Qy 301 AKRLVCTIPLNVLSSVHFSPPLSPQRMAAANIGHVNQCVKVHAEVSCPDMRSWSGISYPF 360
||||||||||||||:: ||| || :|::| |||| | |||||| |||||:||:|||
Db 301 AKRLVCTIPLNVLSTIQFSPALSTERISAMQAGHVNMCTKVHAEVDNKDMRSWTGIAYPF 360
Qy 361 NKLAYAIGDGTTPAGNTHIVCLGGAHNHIQPEEDVEATKMAVENMSPGNMDIKRLVFHNW 420
||| ||||||||||||||:|| | |||||:||| | || ::|| :||||||||
Db 361 NKLCYAIGDGTTPAGNTHLVCFGTDANHIQPDEDVRETLKAVGQLAPGTFGVKRLVFHNW 420
Qy 421 CKDEFAKGAWFFAPPQLLSKSLDELRCRHGNVLFANSDWALGWRGFIDGAIEEGTRAAVT 480
|||||||||||: | ::|: | || :| |:||||||||||| |||||||||||||
Db 421 VKDEFAKGAWFFSRPGMVSECLQGLREKHRGVVFANSDWALGWRSFIDGAIEEGTRAARV 480
Qy 481 VIEELRPAPAVRSHL 495
|:||| |:: |
Db 481 VLEELGTKREVKARL 495
APPENDIX B
PNG
media_image1.png
600
686
media_image1.png
Greyscale
APPENDIX C
PNG
media_image2.png
600
690
media_image2.png
Greyscale
Prosecution Insights