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 .
Claim Status
The amendment of 02/27/2026 has been entered. Claims 2-8, 10, 12-13, 15-16, 18-21 are pending (claim set as filed on 02/27/2026).
Claims 2-8, 10, 12-13, 15-16, 18-21 are currently under examination and were examined on their merits.
Information Disclosure Statement
The Information Disclosure Statement (IDS) filed on 02/27/2026 has been received and considered.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 2-3, 5-7, 12-13, 15-16, and 19-21 are newly rejected as necessitated by amendment under 35 U.S.C. 102(a) as being anticipated by Baum et al. (US2006/0191034 A1, published on 08/24/2006), hereinafter ‘Baum’, as evidenced by Schwardmann et al. (“Bacterial non-specific nucleases of the phospholipase D superfamily and their biotechnological potential”, published on 02/21/2020, Applied Microbiology and Biotechnology, Vol. 104, pages 3293-3304), hereinafter ‘ Schwardmann’, as evidenced by Hobel et al. (US 2017/0044209 A1, published on 02/16/2017), hereinafter ‘Hobel’, and as evidenced by Bravo-Anaya et al. (“Role of Electrostatic Interactions on Supramolecular Organization in Calf-Thymus DNA Solutions under Flow”, published on 10/28/2018, Polymers 2018, Vol. 10 (11), 1204, pages 1-19), hereinafter ‘Bravo-Anaya’.
Baum’s general disclosure relates “to the isolation and characterization of nucleotide sequences encoding novel insecticidal proteins secreted into the extracellular space from Bacillus thuringiensis and related strains” (see entire document, including abstract).
Regarding claims 2 and 5, pertaining to a method for reducing the viscosity and/or preventing an increase in viscosity of a fermentation broth, Baum teaches a method for reducing the viscosity of a fermentation broth comprising intact microorganisms after termination of the fermentation process (paragraph [0251]), comprising a step of introducing a protein having nuclease activity into the fermentation broth at the end of the fermentation process and before recovering an end product of the fermentation process (“Each sample was fermented in 1 milliliter of terrific broth (TB) per well … At the end of the fermentation time, twenty five units per milliliter of benzonase (…) were added to the culture broth to reduce viscosity and incubated at 30 C for an additional one hour. Solids, including cells and spores, were removed by centrifugation … and clarified supernatants were transferred in 200 microliter aliquots to shallow 96 well plates for storage at -80 C and further processing by ELISA analysis ”; paragraph [0251]). It is noted that benzonase is a nuclease as evidenced by Schwardmann (“NucA is commercially distributed and most popular under the trademark Benzonase® Endonuclease’’; page 3298, right column, paragraph 2)). Baum further discloses wherein the method is used to identify TIC901 related proteins in the supernatant (“An ELISA method was developed for identification of supernatant broths containing TIC901 related proteins”; paragraph [0252]), and therefore, TIC901 related proteins are considered the end product of the fermentation, which are recovered by separating the supernatant from solids including cells and spores (paragraph [0251]). It is noted that the instant specification teaches “process of recovering the end product (separation of the cells)” (specification, page 8, paragraph 2), and that “"start of recovering the microorganism or the target product" or "start of recovering the end product" means the separation of cells from the broth” (specification, page 15, paragraph 2). Baum further teaches wherein
the protein comprises a NucA nuclease (“benzonase”; paragraph [0251]). It is noted that benzonase is a NucA nuclease as evidenced by Schwardmann (“NucA is commercially distributed and most popular under the trademark Benzonase® Endonuclease’’; page 3298, right column, paragraph 2). Baum further teaches wherein
the intact microorganisms are not subjected to intended lysis or disruption after
termination of the fermentation process. The Examiner notes that Baum is silent on intended lysis or disruption after termination of the fermentation process before recovering the end product (paragraph [0251]), and further discloses wherein TIC901 related proteins are secreted into the growth medium (“TIC901, TIC1201, TIC407, TIC417, TIC431 and related proteins of the present invention are shown herein to be produced and secreted into the growth media by several stains of Bacillus thuringiensis”’; paragraph [0123]), and thus, recovery of the TIC901 related proteins does not require intended cell lysis or disruption. Baum further teaches wherein
the protein having nuclease activity is directly added to the fermentation broth (“At the end of the fermentation time, twenty five units per milliliter of benzonase (…) were added to the culture broth”; paragraph [0251]).
Since Baum’s teaches intact microorganisms in the fermentation broth after the end of fermentation (see above), and further teaches the method step of introducing nuclease NucA into the fermentation broth at the end of the fermentation process and before recovering an end product of the fermentation process, wherein the protein having nuclease activity is directly added to the fermentation broth (see above), Baum teaches the method steps of both claims 2 and claim 5. Therefore, reducing viscosity and/or preventing an increase of viscosity of a fermentation broth (instant claim 2) or reducing thixotropy of a fermentation broth (instant claim 5) wherein the fermentation broth comprises intact microorganisms after termination of the fermentation process, would be inherent.
Regarding claim 3, pertaining to the end product, Baum teaches wherein the end product of the fermentation process is a target product produced by a microorganism (“identification of unique TIC901 related RFLP patterns and insecticidal protein coding sequences from Bacillus strains that produce in culture supernatant immunologically related TIC901 extracellular protein”, paragraphs [0249]-[0251]).
Regarding claim 5, pertaining to reducing thixotropy, Baum teaches the addition of a protein having nuclease activity to a fermentation broth at the end of fermentation (“At the end of the fermentation time, twenty five units per milliliter of benzonase (…) were added to the culture broth to reduce viscosity”; paragraph [0251]), wherein the protein having nuclease activity inherently reduces the thixotropy of the fermentation broth: Since Benzonase nuclease breaks down DNA, as evidenced by Schwardmann (“NucA is commercially distributed and most popular under the trademark Benzonase® Endonuclease’’, “NucA from Serratia marcescens ... It is active as a homodimer and cleaves all forms of nucleic acids into 1–8 bp fragments.; page 3298, right column, paragraph 2; page 3295, right column, paragraph 1), further since DNA is released from some cells during fermentation, as evidenced by Hobel et al. (“[d]uring fermentation some of the host cells producing the protein product of interest will break and the content of the cells, including DNA, will be released to the fermentation broth”; paragraph [0002]), and since DNA displays thixotropic behavior, as evidenced by Bravo-Anaya et al. (“thixotropic behavior of 10 mg/mL DNA solution”; page 3, paragraph 2), Baum inherently teaches wherein the protein having nuclease activity reduces the thixotropy of the fermentation broth after the end of the fermentation process.
Regarding claim 6, pertaining to the nuclease, Baum teaches wherein the NucA nuclease is NucA from Serratia (“benzonase”; paragraph [0251]), as evidenced by Schwardmann (“NucA is commercially distributed and most popular under the trademark Benzonase® Endonuclease”, “Benzonase® Nuclease … Serratia marcescens”; page 3298, right column, paragraph 2; see Table 3 on page 3299).
Regarding claim 7, pertaining to the microorganism, Baum teaches wherein the microorganism is Bacillus (“Bacillus strains that produce in culture supernatant immunologically related TIC901 extracellular protein”, “A Bt culture collection was arrayed into a 96-well format and stored as glycerol stocks at -80 C, and these were used as inoculum for micro-scale fermentations“; paragraphs [0249]-[0251]; note Bt, Bacillus thuringensis).
Regarding claim 12, pertaining to the method, Baum teaches wherein at least the following steps are encompassed:
(i) cultivating a microorganism capable of producing a target product (“Bacillus strains that produce in culture supernatant immunologically related TIC901 extracellular protein”, “A Bt culture collection was arrayed into a 96-well format and stored as glycerol stocks at -80 C, and these were used as inoculum for micro-scale fermentations … Each sample was fermented in 1 milliliter of terrific broth (TB) per well”; paragraphs [0249]-[0251]; note Bt, Bacillus thuringiensis),
(ii) adding the protein by direct addition to the fermentation broth (“twenty five units per milliliter of benzonase (…) were added to the culture broth”; paragraph [0251]),
(iii) obtaining a fermentation broth comprising a nuclease and a microorganism capable of producing target product and the target product (“twenty five units per milliliter of benzonase (…) were added to the culture broth to reduce viscosity and incubated at 30 C for an additional one hour. Solids, including cells and spores, were removed by centrifugation”, “supernatant broths containing TIC901 related proteins”; paragraphs [0251]-[0252] ). It is noted that Baum’s teachings on nuclease addition and subsequent separation of cells to obtain supernatants comprising TIC901 related proteins indicate that the fermentation broth after nuclease addition comprises a nuclease and a microorganism capable of producing target product and the target product.
(iv) recovering the target product (“Solids, including cells and spores, were removed by centrifugation … and clarified supernatants were transferred”, “supernatant broths containing TIC901 related proteins”; paragraphs [0251]-[0252]).
Regarding claim 13, pertaining to the method, Baum teaches wherein at least the following steps are encompassed:
(i) cultivating a microorganism capable of producing a target product (“A Bt culture collection was arrayed into a 96-well format and stored as glycerol stocks at -80 C, and these were used as inoculum for micro-scale fermentations”; paragraph [0251]; note Bt, Bacillus thuringiensis),
(ii) obtaining a fermentation broth comprising the target product and the microorganism capable of producing the target product (“TIC901, TIC1201, TIC407, TIC417, TIC431 and related proteins of the present invention are shown herein to be produced and secreted into the growth media by several stains of Bacillus thuringiensis”; paragraphs [0123], [0251]-[0252]).
(iii) adding the protein to the fermentation broth (“twenty five units per milliliter of benzonase (…) were added to the culture broth”; paragraph [0251]),
(iv) recovering the target product (“Solids, including cells and spores, were removed by centrifugation … and clarified supernatants were transferred”, ““supernatant broths containing TIC901 related proteins”; paragraph [0251]-[0252]).
Regarding claims 15-16, pertaining to recovering the target product, Baum teaches wherein the step of recovering the target product is carried out within a time interval of one hour after the end of the fermentation process (“At the end of the fermentation time, twenty five units per milliliter of benzonase (..) were added to the culture broth to reduce viscosity and incubated at 30 C for an additional one hour”; paragraph [0251]).
Regarding claim 19, pertaining to recovering, Baum teaches wherein the recovering comprises separating the intact microorganisms from the fermentation broth comprising the protein having nuclease activity (“At the end of the fermentation time, twenty five units per milliliter of benzonase (SIGMA ALDRICH) were added to the culture broth to reduce viscosity and incubated at 30 C for an additional one hour. Solids, including cells and spores, were removed by centrifugation”; paragraph [0251]).
Regarding claim 20, pertaining to separating, Baum teaches wherein the separating comprises performing centrifugal separation (“Solids, including cells and spores, were removed by centrifugation”; paragraph [0251]).
Regarding claim 21, pertaining to a method for reducing the viscosity and/or preventing an increase in viscosity of a fermentation broth, Baum teaches a method for reducing the viscosity of a fermented broth (paragraph [0251]) comprising:
(i) performing a fermentation comprising a fermentation broth, wherein the
fermentation broth comprises intact microorganisms (“A Bt culture collection was arrayed into a 96-well format and stored as glycerol stocks at -80 C, and these were
used as inoculum for micro-scale fermentations … Each sample was fermented in 1 milliliter of terrific broth (TB) per well … At the end of the fermentation time, twenty five units per milliliter of benzonase (…) were added to the culture broth to reduce viscosity and incubated at 30 C for an additional one hour. Solids, including cells and spores, were removed by centrifugation … and clarified supernatants were transferred in 200 microliter aliquots to shallow 96 well plates for storage at -80 C and further processing by ELISA analysis ”; paragraph [0251]; note Bt, Bacillus thuringiensies). It is noted that Baum teaches wherein cells are removed from the broth, indicating that the fermentation broth comprises intact microorganisms.
(ii) terminating the fermentation, thereby obtaining a fermented broth (“At the end of the fermentation time, twenty five units per milliliter of benzonase (…) were added to the culture broth”; paragraph [0251]);
(iii) adding to the fermented broth an exogenous nuclease comprising a NucA nuclease, thereby obtaining a treated broth (“At the end of the fermentation time, twenty five units per milliliter of benzonase (…) were added to the culture broth to reduce viscosity”; paragraph [0251]). It is noted that Baum’s benzonase is a NucA nuclease as evidenced by Schwardmann (“NucA is commercially distributed and most popular under the trademark Benzonase® Endonuclease’’; page 3298, right column, paragraph 2); and
(iv) separating the intact microorganisms from the treated broth (“Solids, including cells and spores, were removed by centrifugation … and clarified supernatants were transferred”; paragraph [0251]);
wherein steps (ii)-(iv) lack intended lysis or disruption of the intact microorganisms. The Examiner notes that Baum is silent on intended lysis or disruption regarding the fermentation process and post-fermentation treatment (paragraph [0251]). Baum further teaches wherein TIC901 related proteins are secreted into the growth medium by the microorganisms (“TIC901, TIC1201, TIC407, TIC417, TIC431 and related proteins of the present invention are shown herein to be produced and secreted into the growth media by several stains of Bacillus thuringiensis”’; paragraph [0123]). As such, intended cell lysis or disruption for product recovery is not required.
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.
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 factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 2 and 7-8 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Baum et al. (US2006/0191034 A1, published on 08/24/2006), hereinafter ‘Baum’, as evidenced by Schwardmann et al. (“Bacterial non-specific nucleases of the phospholipase D superfamily and their biotechnological potential”, published on 02/21/2020, Applied Microbiology and Biotechnology, Vol. 104, pages 3293-3304), hereinafter ‘Schwardmann’, as evidenced by Hobel et al. (US 2017/0044209 A1, published on 02/16/2017), hereinafter ‘Hobel’, and as evidenced by Bravo-Anaya et al. (“Role of Electrostatic Interactions on Supramolecular Organization in Calf-Thymus DNA Solutions under Flow”, published on 10/28/2018, Polymers 2018, Vol. 10 (11), 1204, pages 1-19), hereinafter ‘Bravo-Anaya’.
Baum’s teachings have been set forth above.
Regarding claim 8, pertaining to the microorganism, Baum teaches wherein “[s]uitable hosts that allow for expression of the proteins of the present invention and related sequences include B. thuringiensis and other Bacillus species such as Bacillus subtilis, …, Bacillus megaterium” (paragraph [0151]). Additionally, Baum teaches that “[t]he present invention relates to a new family of nucleotide sequences encoding insecticidal proteins and insecticidal fragments thereof”, that “[i]in particular, the present invention is directed to exemplary proteins designated herein as TIC901, TIC1201, TIC407, TIC417, and, TIC431” (paragraph [0001]), wherein “[i]nsecticidal compositions can be produced from bacterial strains expressing the proteins of the present invention” (paragraph [0080]), and wherein “[f]ormulated bait granules containing … concentrated spent fermentation media or insecticidal proteins purified from the spores or spent fermentation media, …, can be applied to the environment of the pest (paragraph [0081]).
Baum does not expressly teach wherein the microorganism is selected from Hyprocrea jecorina, Trichoderma reesei, Trichoderma viride, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus
japonicus, Aspergillus nidulans, Humicola insolens, Humicola grisea, Streptomyces sp.,
Streptomyces violaceoruber, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus pumilus, Bacillus subtilis, Bacillus sp, Bacillus megaterium, or Geobacillus stearothermophilus (instant claim 8).
While Baum does not expressly teach wherein the microorganism is selected from Hyprocrea jecorina, Trichoderma reesei, Trichoderma viride, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Humicola insolens, Humicola grisea, Streptomyces sp., Streptomyces violaceoruber, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus pumilus, Bacillus subtilis, Bacillus sp, Bacillus megaterium, or Geobacillus stearothermophilus (instant claim 8), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined Baum’s teachings on a fermentation process for expressing and recovering proteins of interest (paragraph [0251]) with Baum’s teachings on host cells, to have created a method for reducing the viscosity of a fermentation broth comprising intact microorganisms after termination of the fermentation process, wherein the microorganism is Bacillus subtilis or Bacillus megaterium. One would have been motivated to do so, in order to develop a fermentation process that provides improved expression and thus increased yield of insecticidal proteins for preparing insecticides (paragraphs [0080]-[0081]).
Claims 2-4 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Baum et al. (US2006/0191034 A1, published on 08/24/2006), hereinafter ‘Baum’, as evidenced by Schwardmann et al. (“Bacterial non-specific nucleases of the phospholipase D superfamily and their biotechnological potential”, published on 02/21/2020, Applied Microbiology and Biotechnology, Vol. 104, pages 3293-3304), hereinafter ‘Schwardmann’, as evidenced by Hobel et al. (US 2017/0044209 A1, published on 02/16/2017), hereinafter ‘Hobel’, and as evidenced by Bravo-Anaya et al. (“Role of Electrostatic Interactions on Supramolecular Organization in Calf-Thymus DNA Solutions under Flow”, published on 10/28/2018, Polymers 2018, Vol. 10 (11), 1204, pages 1-19), hereinafter ‘Bravo-Anaya’, in view of Hobel et al. (US 2017/0044209 A1, published on 02/16/2017), hereinafter ‘Hobel’.
Baum’s teachings have been set forth above.
As discussed above, Baum teaches the addition of a protein having nuclease activity to a fermentation broth at the end of fermentation to reduce viscosity (“At the end of the fermentation time, twenty five units per milliliter of benzonase (…) were added to the culture broth to reduce viscosity”; paragraph [0251]), wherein the protein having nuclease activity breaks down DNA, as evidenced by Schwardmann (“NucA is commercially distributed and most popular under the trademark Benzonase® Endonuclease’’, “NucA from Serratia marcescens ... It is active as a homodimer and cleaves all forms of nucleic acids into 1–8 bp fragments.; page 3298, right column, paragraph 2; page 3295, right column, paragraph).
Baum does not teach wherein the target product produced is selected from an enzyme, a hormone, an immunoglobulin, a vaccine, an antibiotic, an amino acid, a sugar or a vitamin (instant claim 4).
For clarity, Hobel is cited in this rejection as a secondary teaching for claim 4. Hobel’s general disclosure is related to a “simple and very effective method for removing residual host cell DNA from a fermentation broth (see entire document, including paragraph [0014]).
Regarding claim 4, pertaining to the target product, Hobel teaches “[p]roduction of protein products by fermentation is a well know process and it is used for production in industrial scale of many different proteins of interest” (paragraph [0002]), and wherein “[i]n a preferred embodiment, the protein of interest is an enzyme” (paragraph [0036]).
Additionally, Hobel teaches wherein “[d]uring fermentation some of the host cells producing the protein product of interest will break and the content of the cells, including
DNA, will be released to the fermentation broth”, that “DNA in the fermentation broth may increase the viscosity leading to a higher energy demand for stirring and or handling the fermentation broth (paragraph [0002]), and wherein a method for reducing the level of DNA in a fermentation includes a heating step where the broth is heated to a temperature of at least 70° C (see abstract). Hobel further discloses that “in order not to denature the protein of interest such as an enzyme, it is important to keep the temperature of the fermentation broth below the denaturing temperature of the
protein.” (paragraph [0083]).
While Baum does not teach wherein the target product produced is selected from an enzyme, a hormone, an immunoglobulin, a vaccine, an antibiotic, an amino acid, a sugar or a vitamin (instant claim 4), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have combined Baum’s method for reducing viscosity of a fermentation broth with Hobel’s teachings on a fermentation process for producing an enzyme, in order to develop a method for reducing viscosity in a fermentation broth comprising intact microorganisms after termination of the fermentation process, wherein the target product is an enzyme. One would have been motivated to do so to create an improved fermentation process for producing an enzyme of interest wherein the viscosity of a fermentation broth is significantly decreased so that handling of downstream processes for recovering the enzyme of interest is reduced. One would further be motivated to use Baum’s nuclease treatment instead of Hobel’s heating step to remove DNA from a fermentation broth to avoid the risk of heat induced denaturing of the enzyme. A skilled artisan would have reasonably expected success in combining Baum’s teachings with Hobel’s teachings, since both references are directed to fermentation processes for producing a target product.
Claims 2 and 10 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Baum et al. (US2006/0191034 A1, published on 08/24/2006), hereinafter ‘Baum’, as evidenced by Schwardmann et al. (“Bacterial non-specific nucleases of the phospholipase D superfamily and their biotechnological potential”, published on 02/21/2020, Applied Microbiology and Biotechnology, Vol. 104, pages 3293-3304), hereinafter ‘Schwardmann’, as evidenced by Hobel et al. (US 2017/0044209 A1, published on 02/16/2017), hereinafter ‘Hobel’, and as evidenced by Bravo-Anaya et al. (“Role of Electrostatic Interactions on Supramolecular Organization in Calf-Thymus DNA Solutions under Flow”, published on 10/28/2018, Polymers 2018, Vol. 10 (11), 1204, pages 1-19), hereinafter ‘Bravo-Anaya’, in view of EMD (“Benzonase® Nuclease”, published 2008, downloaded from https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/342/154/benzonase-nuclease-ms.pdf; pages 1-16), hereinafter ‘EMD’.
Baum’s teachings have been set forth above.
Baum does not teach wherein the nuclease is added to the fermentation process at a final concentration of at least 10 mg, at least 1 mg, at least 0.1 mg, at least 0.01 mg, at least 0.001 mg, at least 0.0001 mg nuclease per 100 ml fermentation broth (instant claim 10).
EMD’s general disclosure relates to the nuclease Benzonase (“Benzonase® Nuclease is a genetically engineered endonuclease from Serratia marcescens”; page 2, paragraph 1).
Regarding claim 10, pertaining to nuclease concentrations, EMD teaches nuclease concentrations of 2.5 U/mL and 25 U/ml for reducing viscosity (see photograph with labels and legend on page 5 below paragraph 4) which correspond to 0.00025 mg/100mL and 0.0025 mg/100 mL, respectively, based on the specific activity of about 1 × 106 U/mg protein as taught by EMD (see specific activity in upper Table on page 8).
While Baum does not teach wherein the nuclease is added to the fermentation process at a final concentration of at least 10 mg, at least 1 mg, at least 0.1 mg, at least 0.01 mg, at least 0.001 mg, at least 0.0001 mg nuclease per 100 ml fermentation broth (instant claim 10), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have combined Baum’s method with EMD’s teachings on nuclease concentrations, in order to have created a method wherein the nuclease is added to the fermentation process at a final concentration of 0.00025 mg/100mL or 0.0025 mg/100 mL. One would have been motivated to do so, in order to optimize viscosity reduction of the fermentation broth. A skilled artisan would have reasonably expected success in combining Baum’s and EMD’s teachings since both references are directed to the use of the nuclease benzonase for reducing viscosity.
Claims 2, 6, and 18 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Baum et al. (US2006/0191034 A1, published on 08/24/2006), hereinafter ‘Baum’, as evidenced by Schwardmann et al. (“Bacterial non-specific nucleases of the phospholipase D superfamily and their biotechnological potential”, published on 02/21/2020, Applied Microbiology and Biotechnology, Vol. 104, pages 3293-3304), hereinafter ‘ Schwardmann’, as evidenced by Hobel et al. (US 2017/0044209 A1, published on 02/16/2017), hereinafter ‘Hobel’, and as evidenced by Bravo-Anaya et al. (“Role of Electrostatic Interactions on Supramolecular Organization in Calf-Thymus DNA Solutions under Flow”, published on 10/28/2018, Polymers 2018, Vol. 10 (11), 1204, pages 1-19), hereinafter ‘Bravo-Anaya’, in view of Rajarajan (“A Study on the Diversity and Production of Microbial Extracellular Nucleases: Potential Anti-Biofilm Enzymes”, published 2013, Newcastle University, PhD thesis, pages 1-163, and Appendix A and B, pages 1-11), hereinafter ‘Rajarajan’.
Baum’s teachings have been set forth above.
Baum does not teach wherein the protein having nuclease activity comprises a NucB nuclease (instant claim 2), wherein the NucB nuclease is from Bacillus, Brevibacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Lysinibacillus, Oceanobacillus, Paenibacillus, Staphylococcus, Streptococcus, Streptomyces, Thermoactinomyces, Acinetobacter, Agro bacterium, Burkholderia, Enterobacter, Erwinia, Escherichia, Lysobacter, Methylomonas, Mesorhizobium, Photobacterium, Pseudomonas, Rhizobium, Serratia, Xenorhabdus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus jirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis (instant claim 6), wherein the nuclease is a NucB nuclease from B. amyloliquefaciens (instant claim 18).
Rajarajan’s general disclosure relates to “microbial nucleolytic enzymes for applications for the control of harmful biofilms” (see entire document, including abstract).
Regarding claims 2, 6, and 18, Rajarajan teaches a NucB nuclease from Bacillus amyloliquefaciens, and further teaches wherein said nuclease is secreted (“List of nucleases surveyed across prokaryotic and eukaryotic organisms based on the location defined as secreted (+), intracellular (-) or not defined (ND)”, “Bacillus amyloliquefaciens FZB42 … NucB…nucB…+”; see Table A.1 on page 4 of the Appendix A). Additionally, Rajaran teaches wherein “biofilms are associated with disease persistence and biofouling and are comprised of adhered microbes within a hydrated matrix rich in polysaccharides, proteins and extracellular DNA (eDNA)”, and that “eDNA is an important structural component and its degradation by deoxyribonucleases may be a novel approach to eradicate biofilm related problems.” (see abstract).
While Baum does not teach wherein the protein having nuclease activity comprises a NucB nuclease (instant claim 2), wherein the NucB nuclease is from Bacillus, Brevibacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Lysinibacillus, Oceanobacillus, Paenibacillus, Staphylococcus, Streptococcus, Streptomyces, Thermoactinomyces, Acinetobacter, Agro bacterium, Burkholderia, Enterobacter, Erwinia, Escherichia, Lysobacter, Methylomonas, Mesorhizobium, Photobacterium, Pseudomonas, Rhizobium, Serratia, Xenorhabdus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus jirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis (instant claim 6), wherein the nuclease is a NucB nuclease from B. amyloliquefaciens (instant claim 18), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Baum’s method with Rajarajan’s NucB nuclease from Bacillus amyloliquefaciens, in order to create a superior method for reducing viscosity in the fermentation broth wherein the nuclease is a NucB nuclease from B. amyloliquefaciens. One would have been motivated to do so, in order to increase degradation of nucleic acid for maximal reduction of viscosity in a fermentation broth after fermentation. A skilled artisan would have reasonably expected success in the combination of Baum’s and Rajarajan’s teachings since Rajaran teaches a NucB nuclease from Bacillus amyloliquefaciens (Table A.1 on page 4 of the Appendix A), and Baum teaches the addition of a nuclease to a fermentation broth (paragraph [0251]).
Response to Arguments
Applicant has traversed the previous rejections under 35 U.S.C. 102(a) and under 35 U.S.C. 103 in the reply filed on 02/27/2026 (remarks, pages 6-10). Applicant's arguments have been fully considered but they are not persuasive.
In Applicant’s reply, Applicant states that “Baum teaches a method in which certain B. thuringiensis strains are intentionally fermented to late log or early stationary phases of growth in which sporulation is induced which results in cell autolysis” (remarks, page 6), and that “[c]laims 2 and 21 recited a method which is not taught by Baum, who intends for sporulation and the resulting autolysis to occur” (remarks, page 7).
The Examiner responds that no lysis or disruption step after fermentation is taught by Baum. Baum teaches: “[A]t the end of the fermentation time, twenty five units per milliliter of benzonase (SIGMA ALDRICH) were added to the culture broth to reduce viscosity and incubated at 30 C for an additional one hour. Solids, including cells and spores, were removed by centrifugation” (paragraph [0251]). Baum’s teachings on removal of cells by centrifugation further indicate that the fermentation broth comprised intact microorganisms after termination of the fermentation process.
Conclusion
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.
Correspondence Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SANDRA ZINGARELLI whose telephone number is (703)756-1799. The examiner can normally be reached M-F 9-5.
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, Sharmila Landau can be reached at (571) 272-0614. 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.
/SANDRA ZINGARELLI/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
Prosecution Insights