DETAILED ACTION
Status of Application
The amendments and response filed 21 January 2026 are acknowledged and have been considered in their entireties. Claim 81 is cancelled; thus claims 77-78 and 82-86 remain pending; Claims 82-86 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claim. Thus, claims 77-78 are subject to examination on the merits.
Terminal Disclaimer
The terminal disclaimer filed on 22 October 2025 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of US Application No. 18175965 has been reviewed and is accepted. The terminal disclaimer has been recorded.
Withdrawn Rejection(s)
The rejection of claims 77-78 and 81 under 35 U.S.C. 103 as being unpatentable over Ohshiro et al. (Ag. And Biol. Chem., 1989 – cited on IDS) in view of Diderichsen et al. (JBC, 1990 – cited on IDS), Marlow et al. (ACS Chem. Biol., 2013 – cited on IDS) is withdrawn as none alone or in combination teach utilizing an expression comprising an AprE promoter and AprE signal peptide. However, it is noted, said rejection has been further modified below to reflect the new limitations.
Claim Rejections - 35 USC § 103
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 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(s) 77 and 78 are rejected under 35 U.S.C. 103 as being unpatentable over Ohshiro et al. (Ag. And Biol. Chem., 1989 – cited on IDS) in view of Diderichsen et al. (JBC, 1990 – cited on IDS), Marlow et al. (ACS Chem. Biol., 2013 – cited on IDS) and Chen et al. (IEEE 2009 3rd International Conference on Bioinformatics and Biomedical Engineering, pp. 1-4 – cited herein).
Ohshiro et al. teach that when various cations are added to the basal medium during ALDC cultivation/production, this increases enzymatic activity. And that specifically when zinc is added it increases the enzymatic activity five-fold, a rate much higher than the rest of the cations, from 3.34 munit/ml (when no cations are added) to 15.7 munit/ml in the presence of 0.005 to 0.3% zinc, or a relative activity percentage increase from 100% (when no cations are added) to 471% in the presence of 0.005 to 0.3% zinc (See Table II and Figure 1). The higher the zinc concentration, the more this inhibited cell growth (See p. 1916, 1st col., 1st paragraph), therefore concentrations of less than 0.3% were utilized and optimally, those of 0.005% (which translates to 174μM), which, absent any definition in the specification is about 150 μM. Said enzyme was then further purified in the presence of 0.005% zinc sulfate (See p. 1916, 1st col., 2nd paragraph). These concentrations clearly demonstrate zinc is a result effective variable not only for production purposes but also for activity purposes during fermentation and purification processes. Specifically, however, 0.005% zinc is the optimal concentration and this works out to be a concentration of about 174μM Zn2+, which as noted above, it deemed to be close enough to recited ranges upper end so as to be about 150 μM (See Applicant’s Remarks, 01/14/2025, p. 5, 2nd paragraph for specific calculations).
Oshiro et al., however, do not teach the ALDC enzyme is secreted in the host cell Bacillus subtilis (Difference One); they also do not teach an expression cassette system for expressing said ALDC in B. subtilis which utilizes aprE signal sequence and aprE promoter (Difference Two).
Marlow et al. teach the 3-D structure ALDC from B. brevis and confirm that ALDC’s are zinc dependent metalloenzymes requiring said zinc for enzymatic activity (See p. 2340, 1st col., 1st full paragraph; p. 2340, Overall Structure).
Diderichsen et al. teach expressing B. brevis ALDC in B. subtilis and that 74% of said ALDC is secreted and found in the supernatant; however, when utilizing a different host strain (E. coli) it is not secreted – See Abstract and p. 4318, last paragraph and p. 4320, 1st col.). They also teach ALDC and its flanking regions does not naturally possess a promoter but that it does seem to have a signal peptide active in B. subtilis (See Cloning of aldB in B. subtilis, p. 4318).
Chen et al. teach cloning the promoter fragment of Bacillus alcalophilus PB92 reveals a signal sequence and a promoter sequence. They created an expression cassette comprising the arpE promoter and the aprE signal sequence and tested its efficiency in expressing heterologous enzymes in B. subtilis (See abstract; Results A-D). They determined that this particular combination and expression cassette can be used to direct expression of heterologous genes/enzymes in B. subtilis. It is stated: “This promoter, together with the aprE signal peptide gene, represents a useful tool for the stable manipulation of a efficient secretion heterologous proteins from B. subtilis expression systems.” – See last line of Discussion.
Regarding difference one, therefore, it would have been obvious prior to the effective filing date of the claimed invention to substitute the ALDC expressing host cell of Oshiro et al. which is a Brevibacterium acetylicum that results in an internally expressed enzyme and to utilize instead B. subtilis of Diderichsen et al. as the host cell because this results in a secreted ALDC enzyme, thereby making purification and processing significantly more convenient because there is no need for additional steps of cell lysis. In addition, it would also be obvious to still utilize zinc in the cultivation medium and purification medium at a concentration of 174 μM which is about 150 μM. As taught by Diderichsen et al. simple centrifugation of the culture medium results in the ALDC enzyme preparation which can then be further purified (p. 4316, 2nd col., 2nd paragraph). This would be motivation in and of itself for one skilled in the art because it simplifies the ALDC production process. One skilled in the art would have a reasonable expectation of success in utilizing B. subtilis instead of B. acetylicum in the methods of Oshiro et al. which employ adding zinc to the cultivation medium and the purification process, because Diderichsen et al. teach expressing a heterologous ALDC results in said secretion in B. subtilis. In addition, there would be an expectation of success that when utilizing said B. subtilis that addition of the zinc to the cultivation medium and purification medium would also result in increased relative activity percentage increases from 100% (when no cations are added) to 471% when zinc is added (as taught in Oshiro et al. ) because Marlow et al. teach that ALDC is zinc dependent metalloenzyme and thus having sufficient zinc in the cultivation medium and purification medium would be expected to result in ALDC enzymes that are fully active compared to cultivation medium and purification medium where said zinc is scarce.
Regarding Difference Two, while neither Oshiro, Marlow and Diderichsen teach the use of aprE promoters and signal sequences, Diderichsen do teach that ALDC seems to naturally lack a promoter and does comprise some sort of natural signal sequence such that 74% of the enzyme is found in the supernatant of B. substilis expressing cells. It would thus be obvious to optimize the expression of ALDC in said B. subtilis, further incorporating the zinc concentrations of Oshiro because said ALDC is a zinc-dependent enzyme as taught by Marlow, and to utilize the expression cassette of Chen et al. in order to direct ALDC expression and secretion in B. subtilis as in the methods of Diderichsen because Chen et al. have established that the aprE promoter and aprE signal sequence expression cassette are highly effective for expression and secretion of proteins of interest in B. subtilis. This would be motivation in and of itself for one skilled in the art seeking to make substantial amounts of the highly desirable and essential ALDC utilized in the beer fermentation industry (See introduction of Diderichsen). One skilled in the are would have a reasonable expectation of success in utilizing the expression cassette of Chen et al. comprising the aprE promoter and aprE signal sequence for expressing ALDC in B. subtilis in the methods of expressing ALDC taught by Diderichsen because Chen et al. establish this combination provides for efficient expression and secretion of heterologous proteins and enzymes in B. subtilis.
As such, the claims are deemed prima facie obvious in view of the combined references.
Applicant’s Response and Examiner’s Rebuttal:
Applicant’s traverse the previous rejection of record. Said rejection was withdrawn in favor of the modified one above, however, Applicant’s remarks are still pertinent to the current rejection and will be addressed.
First Applicant’s assert that none of the references, alone or combined, teach expressing ALDC within an expression cassette comprising an aprE signal sequence and an aprE promoter. The Examiner acknowledges this and has addressed the new limitation with Chen et al. which provide guidance, motivation and a reasonable expectation of success for utilizing an expression cassette comprising an aprE signal sequence and an aprE promoter for expressing heterologous enzymes in B. subtilis.
Second, Applicant’s assert that the claims as amended are not obvious because the optimal concentration for zinc addition as taught by Oshiro et al. is 174 μM which is outside the claimed range of 60-150 μM. It is asserted the prior arts range is now excluded and one skilled in the art would not modify it below the optimal concentration (See Remarks, pp. 6-7) and there is nothing to suggest lower concentrations would provide comparable or improved secretion yields in B. subtilis.
The Examiner has considered there arguments but do not find them convincing. First, paragraph 0140 (PG-Pub) states that the zinc is added to the cultivation to “increase activity and/or stability of the ALDC” but does not mention or suggest that the zinc has anything to do with secretion efficiency. Secretion efficiency in the instant case would likely be a direct effect of the signal peptide, here the aprE secretion sequence as taught by Chen et al. In addition, Applicant’s have not met the burden of establishing the claimed range is critical to the success of producing and secreting ALDC in B. subtilis host cell as compared to the close concentration of Oshiro et al. – See MPEP 2144.05(III)(A). In addition, as noted, absent any definition, the ranges do appear to overlap absent any definitive meaning of the term “about”.
Third Applicant’s stipulate that the prior art does not address the interaction between zinc concentration and the secretion apparatus. None of the references, alone or combined, address this. And a skilled artisan would have no basis to belive the combination of the aprE secretion system and 160-150 μM zinc supplantation would yield and improved or even functional ALDC (Remarks, p. 7).
The Examiner has considered these arguments but asserts there is nothing in the instant claims, nor nothing in the specification at all that suggests zinc has anything to do with the secretion apparatus. Example is drawn to zinc’s influence on activity of ALDC when it is supplied in the cultivation medium; Examples 5 and 6 are drawn to modulation of specific activity of aldB samples by adding zinc to a fermentation sample; and Example 7 is drawn to purification of aldB in the presence of zinc. With regard to the asserted combination of Bacillus specific secretion architecture based on aprE promoter and signal peptide and zinc concentration range explicitly designed for cultivation resulting in a synergistic and non-obvious production strategy, it is noted any assertion of synergy must actually be backed up by data/evidence. Here there is none, rather, just an assertion of it, which is insufficient to establish that any kind of synergy exists – See MPEP 2145(I).
Applicant’s final argument (Remarks, p. 7), again goes back to the fact that none of the references alone or combined teach an aprE secretion system (promoter and secretion signal) for ALDC to be produced in B. subtilis. However, as noted above, this has been addressed by the new reference of Chen et al. who establish guidance, motivation and a reasonable expectation of success for utilizing an expression cassette comprising an aprE signal sequence and an aprE promoter for expressing heterologous enzymes in B. subtilis. This, coupled with Diderichsen who state ALDC lacks a clear promoter, anyone skilled in the art wishing to overexpress said ALDC enzyme in B. subtilis would want to add well known and established promoter, as well as secretion signal, such as that established by Chen et al.
Thus, for these reasons, the rejection, as modified above, is made/maintained.
Conclusion
No claim is 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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/SUZANNE M NOAKES/Primary Examiner, Art Unit 1656 05 March 2026