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 .
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-10, and the species diatomite (claim 6), polyethyleneimine (claim 7), and glutaraldehyde (claim 9) in the reply filed on 03/23/2026 is acknowledged.
Applicant’s election with traverse of the species α-glucan phosphorylase (claim 3) in the reply on 03/23/2026 is acknowledged. However, this election has been withdrawn necessitated by Applicant’s amendment changing “or” to “and” in claims 1-3; therefore, necessitating that all enzymes are expressed. Examination will proceed for all listed enzymes in claims 1-3.
Priority
The instant application filed on 01/03/2024 is a 371 of PCT/CN2021/123610 filed on 10/13/2021, which claims priority to CN202110757673.1 filed on 07/05/2021. The certified priority document for CN202110757673.1 is not in English; therefore, the effective filing date of the instantly claimed invention is 10/13/2021.
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 01/03/2024 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objections
Claim 8 is objected to because of the following informalities: claim 8 recites “PDADMAC”; however, abbreviations must be spelled out upon first use. This is an objection, not a rejection, because the instant specification defines PDADMAC as poly(diallyldimethylammonium chloride). Appropriate correction is required.
Claim Rejections - 35 USC § 112(b), Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 1-10 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 1 and 2 recite “fermenting to respectively obtain fermentation broths of Escherichia coli or Bacillus subtilis expressing a-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, tagatose 6-phosphate epimerase, and tagatose 6-phosphate phosphatase respectively, and mixing the above fermentation broths to obtain a fermentation mixture”; however, it is unclear if the fermentation broths containing Escherichia coli and Bacillus subtilis are mixed together to form a E. coli-B. subtilis mixture, or if the claimed invention pertains to five separate E. coli fermentation broths each individually expressing the different enzymes being mixed together, which is separate from five separate B. subtilis fermentation broths each individually expressing the different enzymes being mixed together. For the purpose of applying prior art, the Examiner has interpreted the claimed invention to be related to five separate E. coli or B. subtilis fermentation broths each individually expressing the five different enzymes which are mixed together. Therefore, the engineered bacterial strain expressing the enzymes is either E. coli or B. subtilis.
Claim 5 recites the limitation "the wet bacteria". There is insufficient antecedent basis for this limitation in the claim. No wet bacteria were previously recited.
Claim 5 recites the limitation “the bacteria suspension”. There is insufficient antecedent basis for this limitation in the claim. No bacteria suspension was previously recited.
Claim 8 recites “a molecular weight of 600-70,000”; however, it is unclear what the units are for the molecular weight. The instant specification does not state what the units are for polyethyleneimine; therefore, one of ordinary skill in the art would not readily understand what units apply to the molecular weight of polyethyleneimine. For the purposes of applying prior art, the Examiner has interpreted the units to be Daltons (Da).
Claim Rejections - 35 USC § 103, Obviousness
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-10 are rejected under 35 U.S.C. 103 as being unpatentable over Ma (CN 107988286; Date of Publication: May 4, 2018) in view of Yang (CN 104313009; Date of Publication: January 28, 2015 – cited in the IDS filed on 01/03/2024), Lantero (U.S. Patent No. 4,355,105; Date of Publication: October 19, 1982), and Gu (CN 104351483; Date of Publication: February 18, 2015).
Ma’s general disclosure relates to a whole-cell catalytic method for preparing tagatose, wherein the method includes transforming Escherichia coli with an α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene (see, e.g., Ma, English Translation, Abstract). Moreover, Ma discloses performing an enzymatic reaction of the E. coli cells to obtain tagatose, wherein the reaction has the advantage of low cost, low pollution, and high yield (see, e.g., Ma, English Translation, Abstract).
Regarding claims 1-2 pertaining to the fermentation broth of E. coli, Ma teaches transferring an α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene into E. coli engineered bacteria, respectively, to obtain five transformed strains (see, e.g., Ma, English Translation, “Contents of the invention”, pg. 3). Ma teaches that the transformed E. coli cells were fermented to obtain whole cells expressing the enzymes (see, e.g., Ma, English Translation, Embodiment 1, pg. 4). Ma teaches that the E. coli cells expressing the different enzymes were mixed to establish a whole-cell catalytic reaction system in order to obtain a tagatose product (see, e.g., Ma, English Translation, “Contents of the invention”, pgs. 3-4).
Regarding claim 3 pertaining to the enzymes, Ma teaches “α-glucan phosphorylase is derived from Thermotoga maritima, and the gene number on KEGG is TM1168; glucose phosphomutase is also derived from Thermotoga maritima, and the gene number on KEGG is TM0769; The isomerase is derived from Clostridium thermocellum, and the gene number on KEGG is Cthe0217; the 6-phosphate tagatose epimerase is from Thermoanaerobacter indiensis, and the enzyme coded by the gene is numbered WP_019907213.1 on NCBI; 6- Phosphotagatose phosphatase is derived from Archaeoglobus fulgidus, and the gene number on KEGG is AF_0444” (see, e.g., Ma, English Translation, “Embodiment 1”, pg. 4), all of which are thermostable.
Regarding claim 4 pertaining to enzymatic activity of the thermostable enzymes, Ma teaches that the whole cells expressing these thermostable enzymes were treated at 75oC for 5 minutes for cell permeabilization, followed by carrying out the whole-cell catalytic reaction at 70oC for 46 hours (see, e.g., Ma, Ma, English Translation, “Embodiment 1”, pg. 4). One of ordinary skill in the art would readily understand that since the whole-cell catalytic reaction is carried out for the cells expressing these thermostable enzymes at 70oC for 46 hours, that these enzymes have enzymatic activity at temperatures above 40oC.
Regarding claim 5 pertaining to the ratio of the ratio of the enzymes, Ma teaches “the amount of cells expressing α-glucan phosphorylase is 0.14 g DCW/mL, the expression The dosage of the cells of glucose phosphomutase is 0.08g DCW/mL, the dosage of the cells expressing glucose phosphoisomerase is 0.08g DCW/mL, and the dosage of the cells expressing 6-phosphate tagatose epimerase 0.5 g DCW/mL, the cells expressing 6-phosphate tagatose phosphatase were 0.5 g DCW/mL” (see, e.g., Ma, English Translation, Embodiment 1, pg. 4). Based on the teaching of Ma, the ratio of these mixed enzymes is calculated to be 1.75:1:1:6.25:6.25.
However, Ma does not teach: adding an inorganic soil to the fermentation mixture, and stirring homogeneously; further adding a flocculant to the fermentation mixture to flocculate bacteria, and then adding a cross-linking agent for cross-linking; filtering under vacuum to obtain a filter cake, extruding the filter cake to granulate into a strip with a rotary granulator, and then breaking into particles having a uniform length with a spherical shot blasting machine; and subjecting the resulting particles to boiling drying to obtain the immobilized cell for tagatose production (claim 1); or adding 1-10% w/v of inorganic soil to the fermentation mixture, and stirring homogeneously; further adding 0.1-2% w/v of flocculant to the fermentation mixture to flocculate bacteria, then adding 0.05-3% v/v of cross-linking agent, and cross-linking for 1-4 hours; filtering under vacuum to obtain a filter cake, extruding the filter cake to granulate into a strip with a rotary granulator, and then breaking the strip of immobilized cells into particles having a uniform length with a spherical shot blasting machine; and subjecting the resulting particles to boiling drying to obtain the immobilized cells for tagatose production, wherein the temperature at an air inlet for the boiling drying is controlled at 60-90°C (claim 2);or wherein the bacteria suspension obtained by mixing has an OD600 of 10-150 (claim 5); or wherein the inorganic soil is diatomite (claim 6); or wherein the flocculant is polyethyleneimine (claim 7); or wherein the flocculant is polyethyleneimine having a molecular weight of 600-70,000, or PDADMAC (claim 8); or wherein the cross-linking agent is glutaraldehyde (claim 9); or wherein the method further comprises a step of sieving the obtained immobilized cells to obtain morphologically uniform immobilized cells (claim 10).
Yang’s general disclosure relates to “a method for immobilizing whole cells of cellobiose epimerase, belonging to the technical field of food bioengineering. The method of the invention is to prepare the immobilized cells by flocculation embedding and cross-linking after the whole cells producing cellobiose epimerase are pretreated. The enzyme activity, enzyme activity recovery rate and pH stability of the enzyme prepared by the method of the present invention are all improved, and the production cost is reduced. In addition, the method has simple operation process and low energy consumption. The industrial production of lactulose provides a useful reference” (see, e.g., Yang, English Translation, Abstract).
Regarding claims 1-2 and 6 pertaining to the inorganic soil, Yang teaches adding diatomaceous earth (i.e., diatomite) as a filter aid, wherein the amount of diatomaceous earth is “0.1-1.0 g per 100 mL of bacterial suspension” (see, e.g., Yang, English Translation, “Contents of the invention”, pgs. 2-3). Therefore, the calculated amount of diatomaceous earth is 0.1-1% w/v (see, e.g., MPEP 2144.05(I)). Furthermore, Yang teaches that the diatomaceous earth is mixed evenly with the bacterial suspension to obtain a mixed solution (see, e.g., Yang, English Translation, Embodiment 3, pg. 4).
Regarding claims 1-2 pertaining to the addition of the flocculant, Yang teaches “the immobilized cells are prepared by flocculation embedding and glutaraldehyde crosslinking” (see, e.g., Yang, English Translation, “Contents of the invention”, pg. 3). Furthermore, Yang teaches the addition of “chitosan as a cationic flocculant to flocculate with cells, and then adds a certain concentration of sodium tripolyphosphate (TPP) as an anionic flocculant, and at a suitable concentration, chitosan and TPP will not form a homogeneous system, can well achieve the effect of flocculation and immobilization, and the immobilization efficiency can reach more than 90%” (see, e.g., Yang, English Translation, “Contents of the invention”, pg. 3). Yang teaches that the final concentration of chitosan and TPP, which are both used for flocculation, are 0.5% (w/v) and 0.012% (w/v) TPP, respectively (see, e.g., Yang, English Translation, Example 6).
Regarding claims 1-2 pertaining to addition of the cross-linking agent, Yang teaches “After the bacteria in the immobilization method are flocculated, 1-100 mL of a cross-linking agent solution with a volume fraction of 0-0.25% of the cross-linking agent can be added to each 100 mL of the floc of the bacteria after discarding the supernatant to perform cross-linking. The crosslinking reaction condition for adding the crosslinking agent is 0-5h at 4-30°C. The enzyme activity of the immobilized cells obtained after adding the cross-linking agent was higher, and the stability of the enzyme activity was enhanced” (see, e.g., Yang, English Translation, “Contents of the invention”, pg. 3). Furthermore, Yang teaches that the cross-linking agent is glutaraldehyde (see, e.g., Yang, English Translation, “Contents of the invention”, pg. 3).
Regarding claim 5 pertaining to the OD600, Yang teaches that the recombinant E. coli was fermented until the bacterial cell concentration in the fermentation broth reached an OD600 of 5-10 (see, e.g., Yang, English Translation, “Detailed ways”, pg. 4).
Lantero’s general disclosure relates to immobilizing cells containing enzymes “by forming a reaction product of the cell in an aqueous medium with glutaraldehyde, reacting the reaction product with polyethylenimine to flocculate the reaction product, and recovering the flocculated reaction product from the aqueous medium” (see, e.g., Lantero, Abstract).
Regarding claims 1-2 pertaining to filtering under a vacuum and extruding, Lantero teaches following the addition of glutaraldehyde and polyethylenimine to the cell mixture, the cell mixture “was collected by low speed centrifugation, and the resulting pellet was further dewatered by filtration on a Buchner funnel” (see, e.g., Lantero, Example 1, col 3, lines 3-6). Furthermore, Lantero teaches that the resulting cell cake was extruded through a 1.1-1.5 mm orifice by means of a syringe” (see, e.g., Lantero, Example 1, col 3, lines 6-8).
Regarding claims 1-2 pertaining to boiling drying, Lantero teaches drying the extruded cell cake for 15 hours at 60oC to obtain immobilized cells (see, e.g., Lantero, Example 2, col 5, lines 63-64).
Regarding claims 7-8 pertaining to the flocculant, Lantero teaches adding polyethylenimine at 5% (w/v) to a cell mixture until maximum flocculation appears (see, e.g., Lantero, Example 1). Furthermore, Lantero teaches “The selection of a particular polyethylenimine is not critical although those polymers having a molecular weight within the range from 1,800 to 60,000 are preferred (see, e.g., Lantero, col 2, lines 34-37).
Gu’s general disclosure relates to “a preparation method of completely fermented oxytetracycline calcium granules. By taking oxytetracycline fermentation liquor as an initiating raw material, the preparation method is used for preparing granules of the oxytetracycline calcium by virtue of steps of calcifying an oxytetracycline fermentation liquor as a raw material, acidizing, filtering, crushing, pelletizing, straightening, drying and sieving. The preparation method disclosed by the invention can be used for overcoming the defects that a conventional spray drying process is high in energy consumption and not environmentally-friendly, and the completely fermented oxytetracycline calcium granules obtained by adopting a conventional method have the defect of inconvenience in use. The oxytetracycline calcium granules prepared by adopting the preparation method disclosed by the invention is uniform, compact, beautiful, good in mobility, unlikely to crush, convenient to use and capable of meeting the needs of a client very well” (see, e.g., Gu, English Translation, Abstract).
Regarding claims 1-2 pertaining to granulation and breaking the particles into a uniform length, Gu teaches obtaining an oxytetracycline calcium filter cake, grinding the filter cake to a particle size of 0.8 mm, passing the powder into a rotary squeezing granulator to obtain a particle diameter of 0.6 mm, followed by putting the granulated filter cake into a shot blasting machine (see, e.g., Gu, English Translation, Embodiments 1-5).
Regarding claim 10 pertaining to sieving, Gu teaches that after the particles are dried, the particles can be further sieved because the particles obtained after sieving are more even (see, e.g., Gu, English Translation, “Summary of the invention”, pg. 3 & Embodiments 1-5).
It would have been first obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a fermentation mixture comprising Escherichia coli expressing a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene, as taught by Ma, wherein inorganic soil, such as diatomite, is added to the fermentation mixture, as taught by Yang. One would have been motivated to do so because Yang teaches that the diatomaceous earth (i.e., diatomite) acts as a filter aid (see, e.g., Yang, English Translation, “Contents of the invention”, pg. 2). Furthermore, Yang teaches that the filter aid, such as diatomite, affects the enzyme recovery rate and the recovery rate of enzyme activity (see, e.g., Yang, English Translation, Embodiments 9-10). Additionally, Yang teaches use of a diatomite filter aid in obtaining immobilized E. coli cells for production of cellobiose epimerase (see, e.g., Yang, abstract), wherein a diatomite filter aid affects the amount immobilized cell granules, which affects the total enzyme amount and enzyme activity due to its filtering activity (see, e.g., Yang, English Translation, Examples 9-10). Moreover, Ma teaches expression of a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene in E. coli for production of tagatose (see, e.g., Ma, English Translation, Abstract). Therefore, based on the teachings of Ma and Yang, it would have been obvious to add a diatomite filter to the E. coli fermentation mixture in order to filter the immobilized cells for tagatose production.
It would have been secondly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a fermentation mixture comprising Escherichia coli expressing a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene, as taught by Ma, wherein the cells are flocculated and cross-linked, as taught by Yang. One would have been motivated to do so because Yang teaches that flocculation and cross-linking is used to obtain immobilized enzyme-producing cells (see, e.g., Yang, English Translation, “Contents of the invention”, pg. 2). Furthermore, Yang teaches that “The enzyme activity of the immobilized cells obtained after adding the cross-linking agent was higher, and the stability of the enzyme activity was enhanced” (see, e.g., Yang, English Translation, “Contents of the invention”, pg. 3). Moreover, Ma teaches expression of a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene in E. coli for production of tagatose (see, e.g., Ma, English Translation, Abstract). Based on the teachings of Ma and Yang, it would have been obvious to flocculate and cross-link the E. coli cells expressing a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene in order to increase the yield and stability of the enzymes. One would have expected success because Ma and Yang both teach expression of enzymes in E. coli host cells.
It would have been thirdly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a fermentation mixture comprising Escherichia coli expressing a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene, as taught by Ma, wherein the cells are filtered under vacuum and boil dried, as taught by Lantero. One would have been motivated to do so because Lantero teaches that filtering under vacuum results in dewatering of the cell pellet (see, e.g., Lantero, Example 1, col 3, lines 3-6). Furthermore, Lantero teaches immobilization of whole microbial cells and recovery of these immobilized cells from an aqueous medium following flocculation and cross-linking (see, e.g., Lantero, col 2, lines 16-23). Additionally, Lantero teaches further drying of the cell cake obtained from vacuum filtration by means of boil drying for 15 hours at 60oC to obtain immobilized cells (see, e.g., Lantero, Example 2, col 5, lines 63-64). Moreover, Ma teaches expression of a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene in E. coli for production of tagatose (see, e.g., Ma, English Translation, Abstract). Additionally, Ma teaches an aqueous catalytic reaction system for production of tagatose from E. coli expressing a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene (see, e.g., Ma, English Translation, Embodiment 1). Ma also teaches that boil drying the cells can result in permeabilization of the cells (see, e.g., Ma, English Translation, Embodiment 1). Therefore, based on the teachings of Ma and Lantero, it would be obvious to immobilize E. coli cells expressing a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene, followed by vacuum filtering the cells, because this would result in recovery of a cell cake comprising immobilized cells expressing a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene, wherein the immobilized cells are permeabilized. One would have expected success because Ma and Lantero both teach expression of enzyme in E. coli host cells.
It would have been fourthly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a fermentation mixture comprising Escherichia coli expressing a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene, as taught by Ma, wherein the fermentation mixture is vacuum filtered and dried, as taught by Lantero, followed by granulating the particles with a rotary granulator and shot blasting machine, as taught by Gu. One would have been motivated to do so because Gu teaches grinding the filter cake to a particle size of 0.8 mm, passing the powder into a rotary squeezing granulator to obtain a particle diameter of 0.6 mm, followed by putting the granulated filter cake into a shot blasting machine (see, e.g., Gu, English Translation, Embodiments 1-5). Furthermore, Gu teaches that after the particles are dried, the particles can be further sieved because the particles obtained after sieving are more even (see, e.g., Gu, English Translation, “Summary of the invention”, pg. 3 & Embodiments 1-5). The combined teachings of Ma and Lantero teach production of a dried cell cake containing immobilized E. coli cells expressing a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene for tagatose production (see, e.g., Ma, English Translation, Embodiment 1 & Lantero, Example 2, col 5, lines 63-64). Therefore, based on the teachings of Ma, Lantero, and Gu, it would be obvious to granulate the cell cake comprising the immobilized E. coli cells expressing a α-glucan phosphorylase gene, a glucose phosphomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene because this would result in production of uniform particles comprising immobilized cells for tagatose production. One would have expected success because fermentation for production of products. Additionally, Ma and Lantero both teach production of E. coli cells expressing enzymes, and Lantero and Gu both teach vacuum filtering to obtain filter cakes.
Regarding claim 2’s percentage limitation for the flocculant, those working in the biological and/or pharmaceutical arts would understand that adjustments of particular convention working conditions (e.g., concentrations, amounts, percentages, etc.) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan. For example, Yang teaches that the addition of chitosan for flocculation by itself results in low efficiency of the final enzyme immobilization; however, addition of TPP to the chitosan results in immobilization efficiency of more than 90% (see, e.g., Yang, English Translation, “Contents of the invention”, pg. 3). Additionally, Yang teaches that manipulating the amount of chitosan and/or TPP results in alterations in the recovery rate of the enzyme (see, e.g., Yang, English Translation, Example 7). Therefore, one of ordinary skill in the art would reasonably understand that the percentage of the flocculant will influence the amount of flocs produced. This is motivation for someone of ordinary skill in the art to practice or test the parameter widely to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning concentration, it would be prima facie obvious that one of ordinary skill in the art would recognize these limitations are result effective variables which can be met as a matter of routine optimization.
Regarding claim 5’s OD600 limitations for the E. coli fermentation culture, those working in the biological and/or pharmaceutical arts would understand that adjustments of particular convention working conditions (e.g., concentrations, amounts, percentages, etc.) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan. For example, Yang teaches that an OD600 of 0.6 is a logarithmic phrase for E. coli (see, e.g., Yang, English Translation, “Detailed ways”, pg. 4), wherein E. coli is growing rapidly. Therefore, one of ordinary skill in the art would reasonably understand that the higher the OD600 value, the more E. coli bacteria there are within the fermentation culture. This is motivation for someone of ordinary skill in the art to practice or test the parameter widely to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning concentration, it would be prima facie obvious that one of ordinary skill in the art would recognize these limitations are result effective variables which can be met as a matter of routine optimization.
Conclusion
Claims 1-10 are rejected.
No claims are allowed.
Correspondence Information
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/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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