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 .
Status of Claims
Receipt of Arguments/Remarks filed on 12/10/2025 is acknowledged. Claims 1-23 are pending. Claims 1 and 4 were amended. New claims 20-23 were added. Claims 8-10 and 13-19 are withdrawn. Claims 1-7, 11-12, and 20-23 are under examination herein.
Withdrawn Objections and Rejections
The objection to claim 1 is withdrawn.
The rejection of claims 1-3 and 11-12 under 35 U.S.C. § 112(a) is withdrawn in view of applicant amendments and arguments.
New and modified rejections necessitated by amendment
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-5, 11-12, and 20-23 are rejected under 35 U.S.C. 103 as being unpatentable over Jiang et al., CN109266664A in view of Chen et al., Advanced drug delivery reviews; 65(10):1357-69 and Arima et al., The FEBS journal; 276(14):3894-903, as evidenced by pET-29a vector map and sequence.
Regarding claim 1, Jiang teaches a L-glutamate oxidase (LGOX) mutant, having an amino acid sequence according to SEQ ID NO: 2. SEQ ID NO: 2 taught by Jiang is 100% identical to instant SEQ ID NO: 3 (see Search Results 7/30/2025, file “us-17-947-596a-3.rag”, Result 2). Jiang teaches that the full LGOX from Streptomyces sp. X-119-6 is truncated to create the mutant enzyme (Jiang para. 45). Jiang teaches that the truncated LGOX has enhanced enzyme activity, catalyzing L-glutamate oxidase to produce α-ketoglutarate, ammonia, and hydrogen peroxide, i.e. oxidizing L-glutamate (Jiang para. 4, para. 7).
Regarding claims 2 and 3, Jiang teaches the LGOX mutant is derived from Streptomyces sp. X-119-6 (Jiang para. 45).
Regarding claim 11, Jiang teaches the polynucleotide encoding the mutant LGOX, with the sequence according to SEQ ID NO: 1 (Jiang para. 9).
Regarding claim 12, Jiang teaches an expression vector comprising the polynucleotide encoding the mutant LGOX (Jiang para. 67-69).
Regarding claim 20, the limitation, “which is obtained by a method of producing the L-glutamate oxidase mutant comprising…” is a product-by-process limitation. Patentability of product-by-process limitations is assessed based on the structure implied by the steps, not the manipulations of the recited steps. (see MPEP § 2113 subsection I). Therefore, any prior art that teaches the structure of the L-glutamate oxidase mutant of claim 1 reads on claim 20.
Additionally, Jiang teaches that the L-glutamate oxidase mutant is obtained using an expression unit (LGOX gene expression vector) containing a polynucleotide encoding the LGOX mutant (amplified gene fragment), and the LGOX protein is produced by expressing pET-29a-LGOX in bacteria (Jiang para. 68-74). The pET-29a plasmid from Novagen comprises a promoter for gene expression (see pET-29a ref).
Regarding claims 21-23, Jiang teaches a mutant of the protein having an amino acid sequence that is 100% identical to instant SEQ ID NO: 3 (Jiang SEQ ID NO: 2; para. 45).
Jiang does not teach a mutant with a peptide linker comprising one or two copies of GGGGS inserted into a site in the region between the α1 and α2 regions or between α2 and γ regions of L-glutamate oxidase, (claim 1), that the linker is inserted into both the region between the α1 and α2 regions and between α2 and γ regions (claim 4), or the sites recited in claim 5.
Regarding claim 4, Chen teaches properties of fusion protein linkers, including flexible linkers, which link protein domains together (Chen “Abstract”). Chen teaches that linkers are derived from naturally occurring multidomain proteins that are composed of two or more functional domains joined by a linker peptide, which function to maintain interdomain interactions and preserve biological activity (Chen pg. 1358 “2. General properties of linkers derived from naturally-occurring multi-domain proteins”). Chen teaches that flexible linkers are applied to join domains that require some movement or interaction, and are generally composed of small amino acids such as Gly, Ser, or Thr (Chen pg. 1359 “3.1. Flexible linkers”). The most commonly used flexible linkers are Gly and Ser residues, such as GGGGS (Chen pg. 1360 para. 1). This linker can be optimized by adjusting the number of residues to achieve appropriate separation of functional domains or maintain interactions (Chen pg. 1360 para. 1).
Regarding claims 4 and 5, Arima teaches the sequence and structure of LGOX from Streptomyces sp. X-119-6 (Arima “Abstract”). Arima teaches that LGOX has a hexameric structure, α2β2γ2, with a precursor having a homodimeric structure (Arima pg. 3895 para. 3). The precursor is cleaved by a protease to form the structure of the mature enzyme (Arima p. 3895 “Molecular mass of LGOX”).
Arima teaches the amino acid sequence of LGOX (uniprot kb: Q8L3C7), which is 100% identical to instant SEQ ID NO: 1. Position 1 of instant SEQ ID NO: 1 aligns with position 15 as taught by Arima. Arima teaches that there are two α fragments, smaller and larger (i.e. α1 and α2), and that the C-terminus of the smaller α fragment is located at position 370, or position 356 of instant SEQ ID NO: 1 (Arima p. 3895 “Molecular mass of LGOX” para. 2; Fig. 1B). Arima teaches that the last amino acid of the α fragment is position 390, or 376 of instant SEQ ID NO: 1, and that the γ fragment starts at position 391, or 377 of instant SEQ ID NO: 1 (Arima Fig. 2). Thus, Arima teaches that the site between the α1 and α2 regions is between the 356th and 357th residues; and the site between the α2 and γ regions is between the 376th and 377th residues. These regions are identified as protease cleavage positions, where proteolysis occurs to form the mature enzyme structure (Arima Fig. 2).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of Jiang, Chen, and Arima, creating an LGOX mutant protein with a peptide linker inserted between the α1 and α2 and/or α2 and γ regions of LGOX. Jiang teaches modification of LGOX by deleting amino acid residues in order to enhance the stability and activity of the enzyme (Jiang para. 29). It would have been obvious to a skilled artisan that the truncated protein taught by Jiang could be further modified by inserting a peptide linker such as a flexible linker GGGGS, which is commonly used to connect regions of proteins as taught by Chen. It would have been obvious to insert a linker between two protein fragments, such as between residues 356 and 357 to connect the α1 and α2 fragments, or between residues 376 and 377 to connect the α2 and γ fragments, to promote flexibility and conformational changes.
A person of ordinary skill in the art would have been motivated to insert a peptide linker such as GGGGS between two regions of the LGOX protein because linkers such as flexible linkers are known in the art to enhance biological activity, increase expression yield, and allow for movement and conformational changes that can be beneficial to protein activity and interactions (Chen “Abstract"; pg. 1359-1360 “3.1. Flexible linkers”). A skilled artisan would have been motivated to insert a peptide linker between the α1-α2 and the α2-γ regions because these are locations of protease sites where cleavage occurs to form the mature protein structure (Arima Fig. 2) Thus, these sites are associated with changes to protein structure and a skilled artisan would have been motivated to insert a linker which promotes conformational changes and flexibility in these regions.
A skilled artisan would have a reasonable expectation of success in creating a LGOX mutant with a peptide linker inserted between the α2 and γ fragments or α1 and α2 regions because the insertion of peptide linkers is a common strategy for enhancing protein activity, stability, and function as taught by Chen, and protein linkers are naturally occurring in many multi-domain proteins in between protein regions (Chen “Abstract”; pg. 1358 “General properties of linkers derived from naturally-occurring multi-domain proteins). Given the teachings of Jiang that LGOX from Streptomyces can be successfully modified to enhance activity and stability, a skilled artisan could expect success in performing the common protein modification of adding a peptide linker.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Jiang, Chen, and Arima, as applied to claims 1-5, 11-12, and 20-23 above, and further in view of Utsumi et al., Biochemical and Biophysical Research Communications; 417(3):951-5.
Jiang, Chen, and Arima teach the L-glutamate oxidase mutant according to claim 1. These references do not teach that the protein has a mutation in the amino acid sequence of SEQ ID NO: 3 at any of the residues recited in claim 6.
Regarding claim 6, Utsumi teaches site-directed mutagenesis of LGOX from Streptomyces sp. X-119-6 to alter substrate specificity (Utsumi “Abstract”). Utsumi teaches mutations of R305A, H312A, and W564A (Utsumi pg. 953 first partial para.). As discussed above, position 15 of the WT LGOX sequence aligns with position 1 of instant SEQ ID NO: 1. Thus, these mutations correspond with R291A, H298A, and W550A in instant SEQ ID NO: 1.
Arima teaches the sequence and structure of LGOX as set forth above. Arima teaches that the structure of LGOX comprises two funnel-shaped entrances that extend from the surface of the protein to the interior near the active site (Arima pg. 3898 first full para.). Arima identifies amino acid residues involved in the formation of the funnel entrance, including Pro258 which corresponds to P244 in instant SEQ ID NO: 3; and Thr325, corresponding to T311 of SEQ ID NO: 3 (Arima Fig. 4). Arima teaches that the funnel of LGOX is an entry/exit point for substrate and product (Arima pg. 3901 first partial para.).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of Jiang, Chen, Arima, and, Utsumi, introducing a mutation to the protein at residues P244 or T311 of SEQ ID NO: 3. Both Jiang and Utsumi teach modification of the amino acid sequence of LGOX to alter activity. Utsumi teaches site-directed mutagenesis of amino acids in the active site of LGOX. It would have been obvious to a skilled artisan that site-directed mutagenesis, a common technique in protein engineering, could be performed in the LGOX sequence as taught by Jiang. It would have further been obvious to mutate residues P244 or T311 because, as taught by Arima, these residues are involved in the formation of the funnel entrance which is important for substrate binding.
A person of ordinary skill in the art would have been motivated to make this mutation because modifications to amino acid residues can reveal specific residues that are critical for enzyme function, which is important for engineering enzymes with enhanced or altered activity or substrate binding (Utsumi pg. 954 para. 2-3). As LGOX is commercially useful as a biosensor, engineering this enzyme for better function or substrate specificity is highly relevant and desirable (Utsumi pg. 951 “Introduction”; Arima pg. 3895 “Introduction”). A skilled artisan would have been motivated to modify residues involved in formation of the funnel entrance such as P244 or T311 because the funnel is important for substrate recognition and enzyme activity, and changes to residues in this region would be expected to alter enzyme activity or structure.
A skilled artisan would have a reasonable expectation of success in mutating these residues in LGOX as site-directed mutagenesis is an established technique in protein engineering and has been successfully used to alter LGOX activity as taught by Utsumi. As residues 244 and 311 are known to be important, a skilled artisan could reasonably expect to successfully alter enzyme activity by mutating these residues.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Jiang, Chen, Arima, and Utsumi as applied to claim 6 above, and further in view of Livingstone et al., Bioinformatics; 9(6):745-56.
Jiang, Chen, Arima, and Utsumi, teach the LGOX mutant according to claim 6, as set forth above. These references do not teach the specific amino acid mutations according to claim 7.
Regarding claim 7, Livingstone teaches physiochemical properties of amino acids and the identification of conservative amino acid substitutions (Livingstone “Abstract”; pg. 745 para. 1). Livingstone teaches that T and S have similar properties, as both are polar, uncharged amino acids (Livingstone Fig. 1).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of Jiang, Chen, Arima, and Utsumi with the teachings of Livingstone, creating an L-glutamate oxidase mutant with a mutation T311S. As discussed above, it would have been obvious to mutate this residue given its role in the LGOX funnel entrance. It would further be obvious to make the mutation of T to S, as these amino acids have similar properties. Given the similarity, it would be expected that a T to S substitution would not have drastic changes to enzyme activity, such as completely eliminating catalytic activity, but could still impact protein structure and substrate interaction given the position of this residue in the funnel entrance.
A person of ordinary skill in the art would have been motivated to make this mutation because substitutions of amino acids in an enzyme are an established technique for revealing important residues in protein structure and function, as discussed above. Given the utility of LGOX as a biosensor, it would be of interest to make mutations in this enzyme to understand the role of particular residues in activity and substrate specificity.
A skilled artisan would have a reasonable expectation of success in making a mutation of T to S at position 311, as it is known that amino acids having similar properties are less likely to have a drastic impact on protein function but can still provide insight into the role of particular residues or modulate enzyme substrate specificity. As site-directed mutagenesis for LGOX and other enzymes is established in the art, a skilled artisan could reasonably expect to be successful in making a T311S modification.
Response to Arguments
In light of amendments to the claims, the rejection of claims 1-3 and 11-12 under 35 U.S.C. § 102 has been withdrawn. However, upon further consideration, new grounds of rejection of claims 1-5, 11-12, and 20-23 are made under 35 U.S.C. § 103 as set forth above. Given these new grounds of rejection, the arguments presented regarding claims rejected under 35 U.S.C. § 102 are moot. Responses to pertinent arguments are set forth below.
35 U.S.C. § 103
Regarding applicant arguments directed to the peptide linker now required by claim 1, a new rejection is made under 35 U.S.C. § 103 to address this limitation as discussed above.
Regarding applicant arguments that the LGOX mutant has advantageous effects, specifically improved activity with the introduction of flexible linkers, these arguments are not persuasive.
It is noted that the instant claims are directed to a product, an LGOX enzyme with peptide linkers and sequence identity as recited in claim 1. Such an enzyme is obvious in view of the teachings of Jiang, Chen, and Arima as discussed above. As the prior art teaches the same structure as instantly claimed, it is considered to have the same functional capabilities as the claimed product. Regarding the insertion of a flexible linker into the LGOX protein between α1 and α2 and/or α2 and γ regions, using linkers such as GGGGS to connect protein domains is established in the prior art as taught by Chen. As the regions of the LGOX protein are known, as taught by Arima, a skilled artisan would have found it obvious to insert linkers between regions of the LGOX protein associated with protein structural changes, and would have been motivated to do so as the use of flexible linkers is associated with various benefits including increased biological activity, yield, and stability of proteins (Chen “Abstract"; pg. 1359-1360 “3.1. Flexible linkers”). Thus, the structure of the LGOX mutant as recited in claim 1 is rendered obvious in view of the prior art.
Conclusion
Claims 1-7, 11-12, and 20-23 are rejected. No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/EMILY F EIX/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653