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 .
Priority
This application is a National-Stage entry of PCT/CN2021/119423, filed 9/18/2021. Applicant’s claim for the benefit of a prior-filed application CN202110853890.0 filed 7/28/2021, under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 2/21/2024, 11/5/2024, 4/16/2025 are acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Election/Restrictions
Applicant's election with traverse of Group 1, claims 1-3, in the reply filed on 4/22/2026 is acknowledged. The traversal is on the grounds that there is no lack of unity because the cited reference Mattern-Dogru et al. (2002. European journal of biochemistry, 269:12, 2889-2896; "Mattern-Dogru") does not disclose the technical feature of the G19S mutation or a mutation combination comprising the G19S mutation based on SEQ ID NO: 1. Applicant argues that in claim 1, option (b) is also defined as a protein with the sequence having more than 80% identity with the protein sequence containing at least the G19S mutation as in option (a).
This is not found persuasive because under the B.R.I, claim 1 encompasses any esterase mutant comprising: (b) a protein with an amino acid sequence having 80% or higher identity to the protein of (a). There is no requirement in option (b), an alternative of (a) as indicated by the use of “or”, that the mutation G19S is required. In fact, because there can exist up to 20% sequence difference between (a) a protein having the amino acid sequence of SEQ ID NO: 1 and one or more of the recited mutations, the wild type esterase having 100% identity to SEQ ID NO: 1 is encompassed by alternative (b). The S19 of (a) could have been mutated to any other amino acid, including the original glycine residue. There is no requirement as currently presented in the claim, that option (b) has the G19S mutation as asserted by the Applicant. If Applicant intends for G19S to be required of all options in claim 1, then the claim must be amended to recite this.
The requirement is still deemed proper and is therefore made FINAL.
Claims 4-18 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. Applicant timely traversed the restriction (election) requirement in the reply filed on 4/22/2026.
Claims 1-3 were examined on the merits herein.
EXAMINER’S COMMENT
Although currently withdrawn from consideration, the Examiner notes that in claim 12, two periods are used, which does not conform with the requirements of 37 CFR 1.75. See MPEP § 608.01(m). To ensure rejoinder of this claim if any generic claims are found allowable, it is required to remove the unnecessary period, so that the claim meets all formal requirements.
Claim 12 also recites “is selected any one of the group consisting of: .... or... ”. The language used is indicative of a closed Markush group, so the claim should be amended to recite “is selected from the group consisting of... , and... .” See MPEP § 2173.05(h).
Withdrawn claim 6 recites “is selected from any one of the group consisting of: ... and ”. As discussed above, for the proper and traditional wording of a closed Markush group, this should be amended to recite: “is selected from the group consisting of... , and... .”
Specification
The disclosure is objected to because of the following informalities:
35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, requires the specification to be written in “full, clear, concise, and exact terms.” The specification is replete with terms which are not clear, concise and exact. The disclosure appears to be a literal translation into English from a foreign document and contains various idiomatic and grammatical errors. The specification should be revised carefully in order to comply with 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112. Examples of some unclear, inexact or verbose terms used in the specification are:
1) “Compared with a female parent”, which appears when discussing enzyme activity in [0061]; [0064]; [0068]; [0071]; [0075]; [0078]; and [0082] (the paragraph numbers for the specification refer to the numbering in the published application, US20250075195A1).
2) “reduces the difficulty of post-treatment”, which appears in the abstract, [0029], [0033], and [0087]. However there is no description of “post-treatment”. The meaning of this feature cannot be determined from the specification as filed because there is no practical explanation of “difficulty of post-treatment”, nor is this a recognized term in the art.
3) the phrase “ the recombinant plasmid is linked to the above DNA molecule” appears in the specification ([0011], [0047]) and claim 5 (withdrawn) recites “connected with the DNA molecule”. This is not a commonly used term in the art. A recombinant plasmid is DNA that comprises a nucleotide sequence that encodes a protein of interest, such as the esterase mutant of the instant invention. However, the generic language recited could be interpreted to mean that the plasmid is linked to the DNA molecule in any possible way, this could be through a covalent bond or crosslinking, which does not appear to be the intended disclosure.
4) the term “Firstly,” is used in [0037], which is an ordinal adverbs that is not typically used in this manner. Instead the term “First,” is recommended.
Other such phrasing may exist in the application, this is not meant to be an exhaustive list. Applicant’s assistance is requested in ensuring that all informalities are addressed.
Appropriate correction is required.
Claim Interpretation
Under the B.R.I. of the claim words, when viewed in light of the specification, claim 1 encompasses any esterase mutant comprising: (a) a protein having the amino acid sequence of SEQ ID NO: 1 with a mutation of one or more amino acids and having a esterase activity, having at least one of the mutations described therein, or (b) a protein with an amino acid sequence having 80% or higher identity to the protein of (a).
There is no requirement in option (b) that the mutation G19S, or any of the other mutations is required. Because there can exist up to 20% sequence difference between (a) a protein having the amino acid sequence of SEQ ID NO: 1 having one or more of the recited mutations, any and all esterases having up to 100% identity to SEQ ID NO: 1 are encompassed by alternative (b). There is no requirement in the claim, as presented, that option (b) has the G19S mutation. The S19 of (a) could have been mutated to any other amino acid, including the original glycine residue, as long as another mutation is present, under the B.R.I of the claim.
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.
Claims 1-3 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1, in option (a), recites a closed Markush group of alternatives for the groups of mutations. The language used is incorrect according to convention (see MPEP § 2173.05(h)). Claim 1 recites “the mutation comprises any one or more of the group consisting of: G19S, ...
G19S+H86A+M113I+S218F+F219Y+M137Y or G19S+H86A+M113I+S218F+F219Y+M137S;...”.
The phrase “comprises any one or more of the group” renders the claim term indefinite because the mutations recited include multiple combinations, many of which appear to be mutually exclusive, and it is unclear to what extent additional combinations are allowed. Because the language invokes a Markush group but also uses “any one or more of the group” there is no way to define the metes and bounds of the claim. For example, it would be impossible for “a protein having the amino acid sequence of SEQ ID NO: 1”, to have a combination of “G19S+H86S” and “G19S+H86A” because these combinations are exclusive.
Further as another example of indefiniteness, the claim recites: “G19S+H86S” and “G19S+F88R ”, but does not recite “G19S+H86S+F88R”. It is unclear if the combination of “G19S+H86S+ F88R” is excluded from the claim or included due to the recitation of “comprises any one or more of the group”.
Additionally, the conjunction “or” should instead say “and”, and for improved clarity, there should be a final comma between the penultimate and the last alternative.
For these reasons, one of ordinary skill in the art would not be reasonably apprised of the scope of claim 1.
It is suggested that, to overcome the rejection, this limitation should be amended to:
“wherein the mutation is selected from the group consisting of G19S, ...
G19S+H86A+M113I+S218F+F219Y+M137Y, and G19S+H86A+M113I+S218F+F219Y+M137S;”.
Claim 2 recites the phrase “wherein the esterase mutant has an amino acid sequence having 90% or higher, preferably 95% or higher, and more preferably 99% or higher identity to the protein of (a).” A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). Use of the term “preferably”, and the phrase “more preferably”, also renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. The resulting claim is considered indefinite because there is a question or doubt as to whether the features (e.g. 95% identity and 99% identity) introduced by the narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
All other claims depend directly or indirectly from the rejected claims and are, therefore, also rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, for the reasons set forth above.
Claim Rejections - 35 USC § 112(a)- Scope of Enablement
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 1-3 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 mutants of the esterase having SEQ ID NO: 1 and having the G19S mutation or any one of the specific combination recited in (a) of claim 1, does not reasonably provide enablement for any esterases having 80% sequence identity to that of SEQ ID NO:1, as recited in alternative (b). 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 use the invention commensurate in scope with the 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)). The factors include, but are not limited to: (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 invention are addressed in detail below.
(A) The breadth of the claims:
The claims are drawn to esterases, encompassing any esterase mutant comprising:
(a) a protein having the amino acid sequence of SEQ ID NO: 1 with a mutation of one or more amino acids and having a esterase activity, having at least one of the mutations described therein, each of the combinations having the G19S mutation, or (b) a protein with an amino acid sequence having 80% or higher identity to the protein of (a).
Claim 2 further limits the sequence identity to 90% or higher, in the broadest recitation. 95% or higher and 99% or higher are also recited, as preferred embodiments but are found completely optional, as presented.
Claim 3 recites that the esterase is derived from Rauvolfia serpentina. The sequence of SEQ ID NO: 1 is a wild-type esterase from Rauvolfia serpentina.
Thus, the full breadth of the claims encompass (a) or (b), in the alternative, requiring an esterase activity and 80% or higher sequence identity to the protein of (a). 80% sequence identity allows for 20% variability, including mutations to any of the residues therein. This amounts to possible changes in about 52 of the 264 amino acids in SEQ ID NO:1, including any combination of any residue therein (264 amino acids * 0.2 = 52.8). Thus, the structure of the claimed proteins of the full breadth of option (b) amounts to more than 3.13 x 1066 possible sequences (19^52 = 3.13 x 1066).
Within this large group of sequences, not all mutations or combinations/permutations of the mutations will retain the critical esterase activity. The enablement of the claimed invention depends on the predictability of what sequences possess the esterase activity and which do not.
For claim 3, even if limited to 95%, there could be 4.21x1016 (19^13) different sequences within the breadth of option (b). This is greater than a trillion unique sequences. Regardless, the full breadth of claim 3 permits for 90% sequence identity to the wild-type parental sequence.
(B) The nature of the invention:
The invention, as best understood from the embodiments and the specification, comprises an improved esterase mutant, derived from the natural enzyme sequence from Rauvolfia serpentina (e.g. SEQ ID NO: 1), and having mutations that improve catalytic activity, stereoselectivity, or both (see e.g. Embodiments 1-7 and Tables 1-7, pages 10-21).
However, the broad nature of the claims is not commensurate with the instruction provided by the disclosure with regard to the immense amount of sequence variability and the number of possible mutations allowed by the presented claim language. The Embodiments and examples of the specification describe the activities of ~50 different combinations of mutations.
(C) The state of the prior art:
The amino acid sequence of an enzyme determines its structural and functional properties, which is well known to those skilled in the art. While the art discloses various esterases, including some similar to that of SEQ ID NO: 1, neither the specification as filed nor the art teaches a correlation between structure and function such that one of skill in the art can envision the structure of any and all esterases that fulfill the breadth of the claims. In addition, the art does not provide any teaching or guidance as to which changes can be made to the esterase of SEQ ID NO: 1 such that the resulting variants display the desired esterase activity, nor does the art teach the tolerance of esterases to structural modifications, such that one would be able to predict the combinations of mutations that are tolerated and which lose activity.
Mattern-Dogru et al. (“Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis.” European journal of biochemistry vol. 269,12 (2002): 2889-96, on IDS filed 11/5/2024), discloses a polyneuridine aldehyde esterase (PNAE) from the medicinal plant Rauvolfia serpentina, the same as that instantly claimed, and the characterization of this enzyme including performing site-directed mutagenesis (Abstract, Title, pg. 2889).
Mattern-Dogru discloses multiple esterase mutants in Table 2, including several that retain or have increased esterase activity (e.g. H17A, C257A, C213S/G152Q, C170A, C132A), but also some that have decreased or no esterase activity (e.g. C20A, H86A, S87A, D216A, H244A). Mattern-Dogru teaches there exists a catalytic triad of Ser87, Asp216 and His244 in this PNAE, and that the enzyme belongs to the α/β hydrolase superfamily (pg. 2895, left col).
Thus, just from this limited selection of mutations taught in Mattern-Dogru, it is evident that for every different mutation, even from among the residues associated with the active site (see Fig. 4), the effect on the esterase activity cannot be predicted.
Yang, Liuqing et al. (“Structural basis and enzymatic mechanism of the biosynthesis of C9- from C10-monoterpenoid indole alkaloids.” Angewandte Chemie (International ed. in English) vol. 48 (2009): 5211-5213, on IDS filed 11/5/2024) pertains to the cloning and expression of the PNAE from Rauvolfia serpentina (i.e. that of SEQ ID No:1) and studies in detail the structural features of this enzyme (see Abstract; pg. 5211, left col). Yang teaches that the structure of the binding pocket of PNAE determines its very high substrate specificity (Figure 1), and proposes that either systematic structure-guided or random mutagenesis could be utilized to find PNAE mutants with altered, including lowered, substate specificity (pg. 5213, left col). Yang teaches there is a characteristic catalytic triad in PNAE, which are in the order typical for the a/b fold: nucleophilic Ser87, acidic Asp216, and basic His244, suggesting a serine esterase mechanism (pg. 5212, right col, and Scheme 2). Yang et al. also discusses various residues related to the catalytic triad, which appear to be involved with substrate recognition (e.g. His86, Met245), and teaches residues that form a backbone oxyanion hole, including Gly19 and Phe88 (pg. 5212, right col: “After Ser87-assisted hydrolysis, 2a (Scheme 2) decarboxylates to the enolized enzyme product 3, in which the enolate anion is stabilized by hydrogen bonds of the backbone amides of Gly19 and Phe88 forming an oxyanion hole”).
However, it is not clear from the teachings of Yang whether the effects of any and all mutations can be predicted de novo. Particularly, Yang discusses the catalytic triad, necessary for the enzymatic activity, and thus one skilled in the art would recognize to avoid mutating these residues recklessly. However, due to allosteric interactions and other sources of unpredictability, there are no teachings that would lead one to predict the effect of every mutation found in all esterases with 80% similarity to SEQ ID NO: 1.
Further, in regards to the specific G19S mutation disclosed and studied in the instant specification, because Yang et al. teaches that Gly19 is helps form the backbone oxyanion hole, it does not appear to be reasonable that mutation of the relatively small and unreactive glycine to the bulkier polar serine side chain would result in a mutant having esterase activity with increased stereoselectivity, as discovered by the inventors. Thus, it is further evident from the knowledge in the art and the disclosure that the effects of such mutations are unpredictable.
Jones et al. (“Comparison of Five Protein Engineering Strategies for Stabilizing an α/β-Hydrolase.” Biochemistry vol. 56,50 (2017): 6521-6532. doi:10.1021/acs.biochem.7b00571) discusses that review of the previous stabilization of α/β hydrolase fold enzymes revealed many different strategies, but no comparison of strategies on the same enzyme and thus the authors compared five strategies to identify stabilizing mutations in a model α/β-hydrolase fold enzyme (Title, Abstract). Jones teaches that the five strategies used included one location agnostic approach (random mutagenesis using error-prone polymerase chain reaction), two structure-based approaches [computational design (Rosetta, FoldX) and mutation of flexible regions], and two sequence-based approaches (addition of proline at locations where a more stable homologue has proline and mutation to consensus), and while all strategies identified stabilizing mutations, the best balance of success rate, degree of stabilization, and ease of implementation was mutation to consensus (Abstract, Table 1, Discussion, pg. 6530). Jones concludes that the computational based methods performed poorly in predicting the effects of mutations (Discussion, pg. 6530).
Further, Jones states that “There was no clear pattern of which combinations were highly stabilizing and which were not. All single mutations were able to combine with others to form highly stable combination mutants, yet none were found in all of the highly stable mutants. The effect of any particular mutation seems to be highly dependent on what other mutations are present. Because combining stabilizing mutations identified in a wild-type background one at a time can result in unpredictable results, a stepwise combination of identified stabilizing mutations is inadvisable, as is making one combination mutant with all “good” mutations” (see pg. 6530, right col, last paragraph).
Thus, it is evident from Jones that even among very well studied enzyme structures (e.g. an α/β-Hydrolase fold), there is no reliable manner to de novo predict the effects of mutations, especially when considering all of the possible combinations of mutations.
Singh et al. (“Protein Engineering Approaches in the Post-Genomic Era.” Current protein & peptide science vol. 19,1 (2018): 5-15. doi:10.2174/1389203718666161117114243) reviews several protein engineering approaches (Abstract) and states that despite the availability of ever-growing databases 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 (pg. 11, left col, last paragraph). Singh discusses the use of irritative directed evolution, rational and semi-rational design strategies, utilized to engineer proteins to having improved or novel functions (Fig. 1). However, Singh also teaches that for such design strategies, robust assays and large scale screening is required to test the effects of a great number of proteins with random or semi-directed mutagenesis (pages 7-8).
From the teachings of Singh, it is clear that even with modern advances, proteins having mutations must be produced and tested in order to understand the effects of mutation.
Since the ability to predict the mutational effect of any position to any residue remains largely unsolved, means for predicting changes in esterase activity of all of the esterases having 80% (or 90%) to that of SEQ ID NO: 1 is highly unpredictable.
(D) The relative skill of those in the art:
The relative skill of those in the art is high, however one skilled would know from the knowledge in the art and the high amount of experience that the effects of different mutations, even among relatively similar residues, at all of the different positions of the enzymes can not be predicted without the actual production and assaying of the different mutations. Further, one would recognize that as more mutations are made (i.e. double and triple mutants), the complexity involves gets exponentially larger. Thus, one of skill in the art would be appraised of the difficult in predicting the effects of protein mutations de novo.
(E) The predictability or unpredictability of the art:
The instant invention encompasses a very large amount of possible enzyme mutants, within the full scope of 80% identity to SEQ ID NO:1, as discussed above. To determine whether any and all of the mutations have required esterase activity, one would have to predict by either sequence similarity or knowledge in the art, the effects of every mutation on the enzyme activity.
It is well established that “the scope of enablement varies inversely with the degree of unpredictability of the factors involved”. MPEP§ 2164.03 states that “[i]n cases involving unpredictable factors, such as most chemical reactions and physiological activity, more may be required. In re Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970) (contrasting mechanical and electrical elements with chemical reactions and physiological activity). See also In re Wright, 999 F.2d 1557, 1562, 27 USPQ2d 1510, 1513 (Fed. Cir. 1993); In re Vaeck, 947 F.2d 488, 496, 20 USPQ2d 1438, 1445 (Fed. Cir. 1991)... “in art areas having a high degree of uncertainty (i.e. the unpredictable arts) it is not reasonably predictable from the disclosure of one species, what other species will work”.
The biological arts are often unpredictable, often requiring each embodiment or species of an enzyme or cellular composition to be individually assessed for the desired activity. There would have been very little predictability in the production of any and every possible mutant encompassed by the claims, and each would have to be tested for esterase activity.
For esterases of the α/β hydrolase superfamily, the unpredictability in producing mutations is attested to in the findings of Mattern-Dogru et al. and Jones et al., as discussed above. Therefore, one would turn to the instant specification for further instruction.
(F) The amount of direction or guidance presented and (G) the existence of working examples:
The specification provides several specific examples of mutants within the 80% sequence similarity of SEQ ID NO: 1 having esterase activity (see Embodiments 1-7). There are ~50 such combinations in Tables 1-7, and these include the specific combinations named in (a) of claim 1. However, the number of examples reduced to practice is many magnitudes smaller than the trillions or more possible combinations of mutations encompassed by option (b) of the claims.
The specification fails to provide any direction to the structural elements required in of the esterase, including particular residues or structural features within SEQ ID NO: 1 that can be modified and/or those that must be present for a variant having the recited % sequence identity to possess the claimed esterase activity. Further, no recognized correlation between structure and function has been presented.
The specification thus does not provide additional guidance for predicting the effectiveness of all of the possible mutations that are encompassed by the instant claim, in such a way that the effect of the mutation on the enzyme’s function can be predicted from a sequence.
(H) The quantity of experimentation necessary:
It evident from the art and the instant disclose that small sequence changes, even with relatively conservative mutations, can have unpredictable effects on enzymatic activity. Considering the state of the art as discussed by the cited art above and the high unpredictability and the lack of guidance provided in the specification, beyond the G19S containing mutations named specifically in Tables 1-7, one of ordinary skill in the art would be burdened with undue experimentation to make and use all of the claimed esterase mutants having 80% sequence identity to SEQ ID NO: 1, because each of the trillions of claimed sequences, including many permutations and combinations of mutations, would have to be produced, isolated, and tested.
It is thus the Examiner’s position that one skilled in the art could not practice the invention commensurate in the scope of the claims without undue experimentation.
Claims 1-3 are thus rejected under 112(a) for lack of enablement commensurate with the full scope of the claims.
Claim Rejections - 35 USC § 102
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 1-3 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Mattern-Dogru et al. (“Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis.” European journal of biochemistry vol. 269,12 (2002): 2889-96, on IDS filed 11/5/2024), with supporting evidence from GenBank Accession No. Q9SE93 (PNAE_RAUSE, polyneuridine aldehyde esterase from Rauvolfia serpentina, cited on IDS filed 11/5/2024).
As discussed above, claim 1 recites (a) and (b) in the alternative, and there are no particular mutant site requirements for option (b), merely that there is 80% or higher sequence identity to the protein of (a). Because 80% sequence identity allows for 20% variability, including mutations to any of the residues therein, there is no limitation that requires G19S, or any of the combinations of mutations recited in (a), to be present.
Mattern-Dogru et al. discloses a polyneuridine aldehyde esterase (PNAE) from the medicinal plant Rauvolfia serpentina (Benth. ex. Kurz.) and the characterization of this enzyme including performing site-directed mutagenesis (Abstract, Title, pg. 2889).
Mattern-Dogru discloses multiple mutations, including, inter alia, the mutation H86A (Table 2), which is also disclosed in some of the combinations of (a) herein. Further, multiple of the esterase mutants disclosed in Table 2 retain or have increased esterase activity (e.g. H17A, C257A, C213S/G152Q, C170A, C132A).
When regarding the full breadth of the claimed enzymes, including the sequence identity requirements in option (b), the sequence of the wild-type PNAE enzyme taught in Mattern-Dogru is 100% identical to that of SEQ ID NO:1, as evidenced by the sequence alignment included below to the sequence of PNAE from Rauvolfia serpentina (UniProt entry Q9SE93, PNAE_RAUSE), which is the same as that of Mattern-Dogru (see Fig. 3).
Thus, Mattern-Dogru discloses esterase mutants comprising a mutated protein with an amino acid sequence having 80% or higher identity to an esterase comprising the sequence of SEQ ID NO:1, as encompassed by (b) of claim 1.
Regarding claim 2, the sequence of Mattern-Dogru, having at least one of the mutations in Table 2, would amount to an esterase mutant, having less than 100% but greater than 99% identity to the sequence of SEQ ID NO: 1 (wherein % identity = (264-1)/264 * 100 = 99.6%).
Regarding claim 3, Mattern-Dogru discloses that the sequence originated from the plant Rauvolfia serpentina.
For these reasons, claims 1-3 are rejected under 35 U.S.C. § 102 as being anticipated by the disclosure of Mattern-Dogru.
Citation of Pertinent Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Sosa et al., U.S. Patent No. 7,576,260 to Ceres Inc., and other related patent documents (e.g. U.S. 8,022,273, U.S. 11,162,108, among others) disclose a sequence that is 100% identical to that of SEQ ID NO: 1 for use in the production of transgenic plants.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ANDREW TERRY MOEHLMAN whose telephone number is (571)270-0990. The examiner can normally be reached M-F 9am-5pm EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Anand Desai can be reached at 571-272-0947. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/A.T.M./Examiner, Art Unit 1655
/ANAND U DESAI/Supervisory Patent Examiner, Art Unit 1655
APPENDIX: Sequence alignment of SEQ ID NO:1 and Q9SE93-PNAE_RAUSE
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