Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
This application is a 371 of PCT/CN2021/076756 filed on 2/18/2021 which claims benefit of CHINA 202010683476.5 filed 7/16/2020. The effective filing date for prior art purposes is February 18, 2021.
DETAILED ACTION
Claims 1, 3, 6-10, 12-14 and 17-20 are pending. Claims 2, 4-5, 11 and 15-16 are cancelled. Claims 8-10, 12-13 and 18-20 are withdrawn.
Claims 1, 3, 6-7, 14 and 17 are under examination herein.
The rejection of claim 4 under 35 U.S.C. 112(b) as being indefinite is withdrawn in light of the cancellation of the claim.
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.
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.
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.
Maintained rejection: Claims 1, 3, 7 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Hong et al. (CN 110964710A, published on April 7, 2020; previously cited) in view of Uhlich et al. (“Immobilization of enzymes in photochemically cross-linked polyvinyl alcohol”, Enzyme and Microbial Technology, 1996, Vol. 19, pp.124-131; previously cited). As the original CN 110964710A document is in Chinese, an English translation is relied upon for support.
Regarding claim 1, Hong teaches an immobilized enzyme and an amino resin carrier for immobilizing the enzyme (abstract). Hong teaches the enzyme is selected from any one of transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase and nitrilase (abstract). Hong further teaches a method for preparing an immobilized enzyme comprising pretreating the cross-linking agent by using a high polymer to obtain a treatment cross-linking agent; modifying the amino resin carrier by using a treatment cross-linking agent to obtain a modified carrier, fixing the enzyme on a modified carrier to obtain an immobilized enzyme; wherein the enzyme is selected from any one of transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase and nitrilase (English translation p.3, 12th paragraph from top). Hong teaches that the PEG is selected from any one of PEG 400 – PEG 6000 and the PEI is selected with a molecular weight from 3 – 70kDa (English translation p.3, 12th paragraph from top).
Hong does not teach a PVA membrane comprising a PVA porous membrane.
However, Uhlich teaches immobilization of enzymes in photochemically cross-linked polyvinyl alcohol (title). Uhlich teaches polyvinyl alcohol (PVA) is frequently used as a matrix for the immobilization of various enzymes because of its easy availability, low price, and its chemical structure can cause a protein stabilization by attachment to the polymer chains p.124, 2nd column 1st paragraph). Uhlich teaches immobilizing two different enzymes, amyloglucosidase and invertase in the photocross-linked polymer films (p.125, 2nd column last paragraph).
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 system of Hong to replace the amino resin carrier taught by Hong with the cross-linked polyvinyl alcohol film taught by Uhlich to arrive at the claimed invention. Each of Hong and Uhlich teach enzyme immobilization in a matrix. One of ordinary skill in the art would have been motivated to use polyvinyl alcohol polymer as the matrix because Uhlich teaches that PVA is easily available, inexpensive and can cause protein stabilization by attachment to the polymer chains.
Regarding claim 3, Hong teaches a transaminase derived from Chromobacterium violacea DSM30191, or a transaminase derived from Arthrobacter citreus; a ketoreductase derived from Acetobacter sp. CCTCC M209061 or Candida medoniensis AKU4588; a monooxygenase is a cylclohexanone monooxygenase from Rhodococcus sp. phi1 or from Rhodococcus ruber-SD1; an ammonia lyase derived from Solenostemmon scutellarioides; an alkene reductase derived from Saccharomyces cerevisiae or Chryseobacterium sp. ca49; an imine reductase derived from Streptomyces sp. or Bacillus cereus; amino acid dehydrogenase is a leucine dehydrogenase derived from Bacillus cereus or a phenylalanine dehydrogenase derived from Bacillus sphaericus; and a nitrilase derived from Aspergillus niger CBS513.88 or Neurospora crassa OR74A (English translation p.2, claim 6).
Regarding claim 7, Hong teaches that for 1 gram of resin, 4mL of 20-25mg/mL enzyme solution was added (English translation p.10, Example 1).
Hong does not teach grams of free enzyme/ cm2 membrane.
Uhlich teaches using 50 U of enzyme per membrane spread to an area of 10 cm2 (p.125, 1st column last paragraph – 2nd column top paragraph). Uhlich teaches that the enzyme solution was 75 U/mg for amyloglucosidase (p.125, 1st column – Materials). Thus, 50 U of amyloglucosidase is equivalent to 0.67 mg of enzyme per 10cm2 membrane, which is about 0.067 mg/cm2, which overlaps with the claimed range of free enzyme/cm2 membrane.
Regarding claim 14, Hong teaches the transaminase is derived from Chromobacterium violacea DSM30191 having the amino acid sequence shown in SEQ ID NO:2, which has 99.13% identity with instant SEQ ID NO:1, and comprises a mutation at the 7th site, where the threonine at the 7th site is mutated to a cysteine. The sequence alignment is shown below, with the instant SEQ ID NO:1 on top and Hong’s SEQ ID NO:2 listed on the bottom.
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Maintained rejection: Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Hong et al. (CN 110964710A, published on April 7, 2020; previously cited) in view of Uhlich et al. (“Immobilization of enzymes in photochemically cross-linked polyvinyl alcohol”, Enzyme and Microbial Technology, 1996, Vol. 19, pp.124-131; previously cited) as applied to claim 1 above, and further in view of Wang et al. (“Immobilization of lipase enzyme in polyvinyl alcohol (PVA) nanofibrous membranes”, Journal of Membrane Science, 2008, Vol. 309, pp.73-81; previously cited) and Ji et al. (“Direct immobilization of laccase on titania nanoparticles from crude enzyme extracts of P. ostreatus culture for micro-pollutant degradation”, Separation and Purification Technology, 2017, Vol. 178, pp.215-223; previously cited). As the original CN 110964710A document is in Chinese, an English translation is relied upon for support.
The teachings of Hong et al. and Uhlich et al. are discussed above.
Regarding claim 6, Hong and Uhlich do not teach wherein the enzyme is a crude enzyme.
However, Wang teaches lipase enzyme from Candida rugosa has been successfully immobilized in 100-500 nm diameter fibers via electrospinning of aqueous mixtures of lipase and polyvinyl alcohol (PVA) (abstract). Wang teaches that the catalytic activity of the fiber bound lipase was the same as the crude enzyme, showing no adverse effects from either electric charges or PVA on the structure or functions of the enzyme proteins (abstract).
Ji teaches direct immobilization of laccase on titania nanoparticles from crude enzyme extracts (title). Ji teaches that industrial application is constrained by the high cost of purified enzyme preparations (abstract). Ji teaches directly immobilizing in-house crude enzyme extracts onto functionalized TiO2 nanoparticles with the resultant biocatalytic nanoparticles having comparable performance to the immobilized purified commercial enzymes (abstract).
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 the immobilized enzyme system of Hong and Uhlich to replace the purified transaminase enzyme with crude enzyme as taught by Wang and Ji. One of ordinary skill in the art would have been motivated to use crude enzymes, because both Wang and Ji teach that crude enzyme had comparable performance to purified enzyme. One of ordinary skill in the art would have found it beneficial use crude enzyme because Ji teaches that industrial application is constrained by the high cost of purified enzyme preparations, and crude in-house enzyme preparations were much less expensive without any loss in enzyme performance.
Maintained rejection: Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Hong et al. (CN 110964710A, published on April 7, 2020; previously cited) in view of Uhlich et al. (“Immobilization of enzymes in photochemically cross-linked polyvinyl alcohol”, Enzyme and Microbial Technology, 1996, Vol. 19, pp.124-131; previously cited) as applied to claim 1 above, and further in view of Falqi et al. (“Preparation of Miscible PVA/PEG Blends and Effect of Graphene Concentration on Thermal, Crystallization, Morphological, and Mechanical Properties of PVA/PEG (10wt%) Blend”, International Journal of Polymer Science, 2018, Volume 2018, Article ID 8527693, 10 pages; previously cited).
The teachings of Hong et al. and Uhlich et al. are discussed above.
Regarding claim 17, Hong teaches a mass ratio of PEG to glutaraldehyde is 1:1 to 10:1 and a mass ratio of the PEI to glutaraldehyde is 3:1 to 1:5 (English translation, p.2, claim 4).
Hong and Uhlich do not teach a mass ratio of polyethylene glycol to PVA is 5:4 to 75:4.
However, Falqi teaches preparation of miscible PVA/PEG blends for films (abstract). Falqi teaches biodegradable polymeric materials have been found useful for drug delivery, pharmaceuticals Falqi teaches varying ratios of PVA:PEG with PEG content ranging from 5 wt% - 20 wt% and PVA content ranging from 80 wt% to 100 wt%, as shown in Table 1. Falqi further teaches preparing other blends with PEG loadings including 30, 40, 50, 60 and 70 wt%, which overlaps with the applicant’s claimed range of PEG:PVA of 5:4 (55.5%) to 7:4 (95%).
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 the PVA porous membrane of Hong and Uhlich to vary the polyethylene glycol (PEG) to PVA mass ratio from 5:4 to 7:4 to arrive at the claimed invention. One of ordinary skill in the art would reasonably expect that varying the mass ratio of PEG to PVA would predictably result in a PVA membrane useful for immobilizing enzymes, because these mass ratios are taught by Falqi, and it was known in the art at the time of invention that PVA-PEG could be used to create membranes for immobilizing enzymes.
Response to Arguments
Applicant argues that the technical features "the three-dimensional structured PVA porous membrane has a three-dimensional structure that are formed by protrusions or grooves; the PVA porous membrane further has polyethylene glycol and/or polyethyleneimine; a molecular weight of the polyethylene glycol is PEG 400-PEG 6000, and a molecular weight of the polyethyleneimine is 3KDa-70KDa" of present claim 1 have not been disclosed in Hong (See Remarks dated 11/5/25, p.10, last 2 paragraphs).
Applicant argues that the specification records the technical effects brought by the three-dimensional structure formed by protrusions and grooves, which allows increased loading of enzymes and guarantees high activity and stability of the enzymes; and reproduces Table 2 from embodiment 1, providing conversion rates of a planar PVA membrane immobilized enzyme and the three-dimensional structured PVA membrane immobilized enzyme during repeated use (See Remarks dated 11/5/25, p.11 – top of p.12).
Applicant argues that that the conversion rate of the three-dimensional structured PVA membrane immobilized enzyme was higher than that of the planar PVA membrane immobilized enzyme, and this conversion rate is completely beyond the technical effect that could be predicted by simply increasing the specific surface area of the PVA membrane (See Remarks dated 11/5/25, p.12, 1st paragraph).
Applicant argues that polyethylene glycol and polyethyleneimine of the present application enrich the pore structure within the PVA porous membrane, thereby further increasing the enzyme load and the firmness of entrapment, arguing the data in Table 9 indicates that the molecular weight of the polyethyleneimine brings about an unexpected technical effect, which is not disclosed or provided with relevant technical teachings or technical suggestion in Uhlich (See Remarks dated 11/5/25, p.12, last 2 paragraphs). Applicant argues that dependent claims 3, 6-7, 14 and 17 depend from claim 1 and should also be allowable for the reasons discussed previously for independent claim 1 (See Remarks dated 11/5/25, p.13, top 2 paragraphs).
Applicant's arguments filed November 5, 2025 have been fully considered but they are not persuasive. In response to applicant's arguments against the Hong and Uhlich references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., conversion rate of the enzyme; enzyme activity after reuse; enzyme load; firmness of the entrapment) are not recited in the rejected claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
The claims are directed towards a structure requiring a PVA porous membrane and an enzyme entrapped on the PVA porous membrane. As discussed in the rejection above, Hong teaches an immobilized enzyme on a carrier, including PEG and PEI of the required molecular weights required in claim 1. Uhlich teaches immobilized enzymes in a PVA matrix, which is a three-dimensional structure. The combined teachings of Hong and Uhlich teach a cross-linked PVA film with immobilized enzymes having PEG and PEI, thereby teaching the required structural limitations of claim 1.
Conclusion
THIS ACTION IS MADE FINAL. 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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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/DEEPA MISHRA/Examiner, Art Unit 1657
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