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 .
Priority
This application is a National Stage Entry of PCT/IN2022/050439, filed 5/6/2022, which claims priority to foreign patent application IN202121020751, filed 5/7/2021. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55, filed 10/13/2023.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 10/13/2023 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Abstract
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In/ addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided.
The abstract of the disclosure is objected to because it recites the phrase “The presently disclosed invention relates to...”, which is unnecessary implied language, as described above. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b) for guidelines for the preparation of patent abstracts.
Specification
The disclosure is objected to because of the following informalities:
TRADE NAMES, TRADEMARKS, AND OTHER MARKS USED IN COMMERCE:
The use of the term Tween™ 80, see page 19 (line 9) and in Table 2 on pg. 20, which is a trade name or a mark used in commerce (Tween ™ 80 is a trademark for the chemical Polysorbate 80), has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks (see MPEP 608.01(v) and 608.01(u)).
Appropriate correction is required.
Claim Objections
Claims 16, 19 and 22 are objected to because of the following informalities:
Claim 16 recites the phrase “based on / with respect to cellulose content”. In this case, the use of “based on / with respect to” is improper. Does the slash mean that either term is to be used interchangeable? This should be amended to clarify the language. Only one of the terms “based on” or “with respect to” should be used.
In claim 19, line 3, the term “lytic polysaccharide monosaccharides (LPMOs)” appears to be a typo for the family of enzyme known as “lytic polysaccharide monooxygenases (LPMOs)” (see specification pg, 14).
In claim 22, the abbreviations of “HMF” and “2,3 BDO” are used without first introducing the full term. The terms should instead say: “hydroxymethylfurfural (HMF)”, which is also commonly known as 5-(hydroxymethyl)-furfural; and “2,3-Butanediol (2,3-BDO)”, respectively.
Appropriate correction is required.
Claim Interpretation
In claims 8 and 9, the further limitations recited have been interpreted as being optional. Claim 8 recites “when the submerged fermentation media optionally further comprises one or more inducers” and claim 9 further limits the inducers. The claims have been examined and the optional limitations compared to relevant prior art for the purposes of compact prosecution.
Further, in claim 8, the term “suitable times” included in the optional limitation is imprecise, but the claim is not considered indefinite as one of ordinary skill in the art understands to determine the suitable time for the inducers during fermentation.
Claim 22 recites that the free carbohydrates are “used for producing other biochemicals including furfural, HMF, lactic acid, or 2,3 BDO”. This does not practically limit the claimed method steps- instead this dependent claim is being interpreted as reciting a property of the carbohydrates obtained from the method. The phrase “used for” is being interpreted under the B.R.I. in light of the specification as a functional limitation of the product carbohydrates, and not as a requirement for additional method steps. Thus, this limitation is being interpreted as a functional property of the carbohydrates, i.e. an inherent feature of a composition or chemical, and has been afforded the proper patentable weight as described in MPEP §2112.01.
Claim Rejections - 35 USC § 112(b)
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-23 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, 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.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 1 recites “carbohydrates/sugars”, therefore the claim recites both the broad category of “carbohydrates” and the claim also recites “sugars”, which is the narrower statement of the limitation. The claims are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary, and therefore not required, or (b) a required feature of the claims. The scope of the claims is unclear because it cannot be determined if the claimed method is limited to only releasing sugars, or if all carbohydrates fall under the intended scope, including fiber, starches, and other molecules which are not sugars.
The term is also recited in claim 21. Meanwhile, claims 18 and 22 recite the term “carbohydrates/ free sugars”. These limitations are indefinite for essentially the same reasons discussed above. Further, the phrases “free carbohydrate...” and “...free sugar” are unclear and it is recommended to amend these to say, e.g., “the released carbohydrate”, to harmonize with the language of claim 1 (e.g. the language of the preamble).
Claim 1 also recites the limitation “at an increased efficiency” in lines 11-12. This is a relative term, which renders the claim indefinite. The term “increased efficiency” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. There is no measure for which the increase efficiency is to be compared against. The resulting claim scope is indefinite as one cannot determine how to assay the claimed efficiency.
Claim 7 recites the phrase “with 5 to 10% active mycelia” in line 4. It is unclear what the percentage that is used is in reference to and therefore the metes and bounds of the limitation cannot be determined. Is this 5 to 10% of the total solution by weight (i.e. w/w)? Or is this in reference to percent enzyme activity? Or is this a percentage of “active” mycelia to “inactive mycelia”. This limitation should be amended to specify what the percent is in reference to and what units are used to perform the calculation.
Claim 10 recites the limitation "the mycelia" in line 1. There is insufficient antecedent basis for this limitation in the claim. There is no prior explicit recitation of mycelia or a mycelium in the claims on which claim 10 depends upon. The term has been interpreted for the purposes of compact prosecution to mean “the white rot fungi”, as recited in claim 3.
Claim 12 recites “involves one or more steps of treating with hot water or mild acid drying and mechanical shredding...”. The claim is indefinite because there is no manner in which to determine which steps are alternatives and which are required. In other words, the claim could be interpreted to mean that one or more steps of 1. treating with hot water ; 2. mild acid, or 3. drying is required and that then the mechanical shredding is absolutely required. The claim could also be interpreted to mean that only one of: 1. treating with hot water, 2. mild acid, 3. drying, or 4. mechanical shredding is necessary. The use of both “or” and “and” in the limitations following “one or more steps of” creates a problem of indefiniteness as one cannot determine if the mechanical shredding step is part of the “one or more steps”. Further, as another issue, there is no comma or similar separator between “mild acid” and “drying”, and the term could be interpreted as “mild acid drying” instead of two separate processes. Correction is recommended. Further, separating each steps into individual lines instead of in paragraph form is recommended (this can be applied to all of the claims).
In claim 14, line 2, the phrase “centrifugation, decantation methods” is missing a conjunction, either “and” or “or”. Therefore, one cannot determine if the claim requires all three of the recited steps (e.g. filtration, centrifugation, and decantation methods) or if the claim merely requires one of these methods (e.g. filtration, centrifugation, or decantation methods). The claim is indefinite because the claim does not properly set required steps of the method.
In claim 19, the phrase "such as", used in line 4, renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Removal of the exemplary phrase “such as AA9” is recommended.
Claim 20 recites that “the hydrolysis efficiency is in the range of about 40% to 80%”. This term and range is indefinite as there is no reasonable way for by which one is to determine by what calculation the claimed hydrolysis efficiency is found. As an enzymatic reaction’s efficiency is a product of reaction conditions, time, enzyme concentration, and starting product concentration, there are multiple dependent variables that must go into determining the “efficiency” of such a process. As an example, there is no recitation of the endpoint at which efficiency to is to be assessed. It appears that the 40% to 80% is in regards to how much of the starting material is converted into the related carbohydrate products. One of skill in the art would know that adding a saturating excess of enzymes will usually drive the process to yield more product. Because the efficiency of the hydrolysis is in comparison to variable starting concentrations and an unknown endpoint, the metes and bounds of the claim cannot be determined, as presented. The specification does not outline any specific assay, equation, or other knowledge through which one is to apply to determine the hydrolysis efficiency.
Claim 23 recites, in line 2, the limitation “comprises about 40 to 60% laccase”. There are no units or explanation provided for the percentages. There is no manner in which to determine the total against which the laccase is to be compared to. Is this by weight or by enzyme activity? Is the total amount in comparison to the full volume of the separated liquid, or is this in comparison to the total LME? Because the meaning of the amount of laccase cannot be determined, the resulting claim is indefinite. For the purposes of comparing this limitation to any relevant prior art, the B.R.I. of the term to mean laccase is 40 to 60% of the enzymes by activity or by weight will be used.
All other claims depend directly or indirectly from the rejected claims and are, therefore, also rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, for the reasons set forth above.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 15 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 15 recites “The process as claimed in claim 1, wherein treating the solids comprises one or more washes to provide the solids free from LME”. Claim 1 requires, in lines 8-9, “washing the solids with at least one of water, alkaline peroxide or ionic liquid to obtain washed solids free from LME”. A washing step is already recited in claim 1, and must be one of water, alkaline peroxide or ionic liquid. The washing step of claim 15 does not further limit this step, and there are no limitations for the component of the wash step in claim 15. Because the washes of claim 15 is essentially the same or broader than those of lines 8-9 in claim 1, the claim is improper.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
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.
Claims 1-3, 11-18, and 20-23 are rejected under 35 U.S.C. 103 as being unpatentable over Inamdar et al. (US PGPub No. 20110129889, herein after “Inamdar”) in view of Tocco et al. ("Recent developments in the delignification and exploitation of grass lignocellulosic biomass." ACS Sustainable Chemistry & Engineering 9.6 (Feb 4, 2021): 2412-2432, herein after “Tocco”).
Inamdar teaches a process for production of ethanol from lignocellulosic material comprising cellulose, lignin and hemicellulose as main components, wherein the cellulosic fraction hydrolyzed into sugars and fermented to produce ethanol (Abstract, [0002]). The method of Inamdar comprises steps wherein: lignin-containing material is dissolved in alkali, preferably with NaOH; separating a cellulose fraction for further enzymatic treatment with one or more than one cellulolytic enzymes capable of hydrolyzing cellulose; and subjecting the enzyme hydrolysate to fermentation in the presence of ethanol-producing yeast, preferably S. cerevisiae (Abstract).
Inamdar teaches that a preferred substrate is used for ethanol production by fermentation is cellulosic materials which include lignocellulosic substances like straw, corncobs, wood wastes, bagasse and waste paper ([0004]), and specific examples include using sugar cane bagasse (Example I), grass waste (example II), byproducts of grains such as corn and/or wheat (examples III and VI), and corn wastes (examples IV and V), as in claim 11.
Inamdar teaches that the lignocellulosic material is pretreated or processed, including steps of size reduction, reducing it to a finely divided form ([0014], [0022]). Inamdar teaches that “The feedstock is subjected to mechanical action for size reduction to about 2 mm to about 20 mm and slurry is prepared with fresh water or the recycled condensate from the evaporation step, taking solids in the range of about 30% to about 95% depending upon the feedstock. In one embodiment of the invention the grass and DWG, the solids taken in the range of about 30% to about 50%”(see [0022]). Inamdar also teaches that the feedstock processing includes steps of hot water treatment and treatment with oxalic acid, as recited in claim 12 ([0023]).
Inamdar teaches that a slurry containing about 6% to about 25% by weight solids is subjected to a treatment with a mixture of water and about 0.5 to about 2% alkali, preferably sodium hydroxide (NaOH), at a temperature range of about 80°C to about 210°C for a period of about 5 to 360 minutes ([0025]). Inamdar teaches that such alkali treatment dissolves the lignin part and that cellulose and hemicelluloses part remain in cake (e.g. in the solid). Inamdar then teaches that the dissolved (soluble) lignin part is separated and washed with water and that the solid cake (i.e. solids free from lignin) is retained and treated/washed with water (“...filter cake consisting of rich cellulose is reslurried using water...”; [0025]).
Inamdar teaches that the filtered solids (cake) consisting of cellulose and hemicellulose (i.e. carbohydrates) is then subjected to enzymatic hydrolysis (saccharification) with cellulolytic enzymes capable of hydrolyzing the cellulose, including cellulase, β-glycosidase, endoglucanase, and cellobiohydrolase; for a period of about 4 hours to about 30 hours, preferably about 4 hours to 24 hours for complete hydrolysis of cellulose ([0026]). Inamdar further teaches that the hydrolysis releases simple carbohydrates including pentoses and hexoses and particularly glucose which is fermented to produce ethanol ([0016], [0026]-[0027]).
In regards to additional limitations in claim 16, Inamdar teaches using 0.5 to about 2% alkali, preferably NaOH, at a temperature range of about 80°C to about 210°C for a period of about 5 to 360 minutes ([0025]), which overlaps with the claimed treatment conditions of 60°C to 80°C for 60 to 90 minutes.
Regarding claims 17 and 18, Inamdar teaches that the pH for the saccharification enzyme hydrolysis is in the claimed range of “4.5 to 5.5” and that the temperature falls within the range of 40 to 80°C (see [0026]: “the temperature is kept at about 30°C to about 60°C, preferably at about 50°C to about 60°C,. more preferably about 55°C” and “the pH of the slurry is maintained in the range of about 4 to about 6, preferably in the range of about 4.8 to about 5.2”).
Regarding claim 20, Inamdar teaches that the hemicellulose pretreatment efficiency is may be from “about 50% to about 85%” and the fermentation efficiency is “in the range of about 70% to 85%”, in some examples ([0033]-[0038]). The other examples are within similar ranges, and thus, Inamdar teaches processes for pretreatment and fermentation of high efficiency.
Inamdar teaches that ethanol-producing fermentation is performed with yeast of the genera Pichia and/or Saccharomyces, preferably about 24 to about 48 hours, and preferably at 31°C to about 33°C, as recited in the instant claim 21.
However, Inamdar does not teach that the processed feedstock is treated with lignin modifying enzymes (LME), including enzymes from white rot fungi, and separating the soluble LMEs from the solid product material prior to washing the solids, as in the instant claims.
Tocco discusses the state of the art prior to the effective filing date in delignification of lignocellulosic biomass based on straw and related crops and wastes, and gives an overview on the means of current and predictable delignification protocols with a special emphasis on fungal ligninolytic enzymes (Abstract, Title). Tocco teaches that the delignification step is the most challenging and critical of the overall process, and teaches several delignification strategies used in the art, reproduced below (pg. 2417, right col):
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Tocco teaches that many of the listed pretreatments involve harsh conditions (high temperature and pressure, use of solvents, or extreme pH values) causing partial biomass degradation, therefore wastes as well as enzyme inhibitors are produced, making such processes unsuitable from economic and environmental points of view (pg. 2417, right col, lines 1-30).
Tocco teaches that excessive acidity in the pretreatment steps can promote unwanted side reactions and partial hydrolysis of both cellulose and lignin, and teaches that strong acids promote the formation of the undesired 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde, so milder conditions are generally adopted (pg. 2418, right col. “Chemical Pretreatments”). Tocco teaches that for nonenzymatic treatment a “mild acidic pretreatment to remove and partly hydrolyze hemicellulose, followed by an alkaline one to remove a substantial lignin fraction, could be the optimal solution”, which are conditions similar to those of Inamdar (pg. 2418, right col, “Chemical Pretreatments”).
Tocco teaches that basidiomycetous white rot fungi (WRF) are able to delignify lignocellulosic feedstock efficiently and teaches that biological pretreatment is considered a cleaner and greener approach compared to physical and chemical pretreatments (pg. 2419, right col). Tocco teaches that ligninolytic enzymes secreted by WRF are involved in natural delignification processes and could overcome drawbacks of other methods, and teaches that enzymatic delignification is attractive because it is faster than biological pretreatment and can operate in a wide range of pH (3-8) and temperature (25-80 °C), (pg. 2419, right col). Tocco also teaches that the main ligninolytic enzymes are laccases (LC, e.g. phenol oxidases) and heme-peroxidases (e.g. lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP)) (pg. 2419, right col, and Fig. 8). Figure 8 demonstrates “(a) laccase from Trametes versicolor, 1GYC; (b) lignin peroxidase from Phanerochaete chrysosporium, 1B85; (c) manganese peroxidase from P. chrysosporium, 1MNP, (d) Versatile peroxidase from Pleurotus eryngii, 3FJW”. Tocco elsewhere teaches the use of crude enzyme mixtures or enzymatic cocktails composed mainly of LCs, LiP, MnP, and VP- from organisms including Pleurotus ostreatus- is known to the art (pg. 2423, right col).
Tocco teaches that the efficiency of the enzymatic treatment process depends on several aspects, including biomass features and operating conditions including pH, temperature, mediator(s), oxygen, and use of surfactants (pg. 2423, right col).
In regards to washing or treating the solids with at least water, alkaline peroxide or ionic liquid, Tocco teaches that pretreatments with hydrogen peroxide (H2O2) under moderate alkaline conditions is well-known in the art (pg. 2419 left hand col). Tocco also teaches the use of ionic liquids (ILs) which have high solvating power and solubilizing power towards lignin is known in the art (pg. 2418, left col and Fig. 7).
Tocco teaches that even mild concentrations of H2O2 can interfere with peroxidase for use in enzyme treatments (Fig. 12 and pg. 2422, right col: “peroxidases are generally sensitive to H2O2 concentration”), thus teaching that steps of lignin modification with enzymes and alkaline peroxide treatment should be separate steps.
Therefore, before the effective filing date of the instant invention, to one of ordinary skill in the art, it would have been prima facie obvious to modify the process taught in Inamdar for production of ethanol from lignocellulosic material, comprising steps of pre-processing an agricultural feedstock, treating with alkaline, and hydrolyzing the materials with cellulolytic enzymes, by further performing an enzymatic delignification step, according to the teachings of Tocco, comprising treating the feedstock with lignin modifying enzymes (LME), and it would have been obvious to incorporate steps for separating the soluble LMEs from the solid product material and washing, for the expected benefits of an improved process for modification and utilization of lignocellulosic agricultural waste.
One of ordinary skill would have been motivated by the teachings of Tocco to provide lignin-modifying enzymes for lignocellulosic material treatment because the art recognizes the benefits of replacing other nonenzymatic pretreatments – typically involving steam, high pressure, or harsh acidic conditions - with natural processes. One would have reasonably predicted that the incorporation of an LME treatment would result in an efficient process for removing and/or modifying lignin, with economic and environmental benefits. The steps of separating, via filtration or other methods, and washing lignin-containing materials are known in the art, as evidenced in Inamdar. One of ordinary skill would be knowledgeable of the solubility of enzymes and the insolubility of the cellulose material. Removal of the enzymes and various washing steps amount to well-known steps to include in the process.
Regarding claims 2 and 3, these enzymes and sources of obtaining the enzymes are taught in Tocco. To one having ordinary skill in the art, it would have been obvious to obtain enzymes from fermentation of one or more white rot fungi species discussed in Tocco.
The limitations of claims 11 and 12 are explicitly discussed in Inamdar, which discloses many of the recited agricultural feedstocks, and discuses hot water, mild acid, and mechanical shredding steps (to about 2 mm - 20 mm) as set forth above.
Regarding claim 14, the combined teachings of Inamdar and Tocco make obvious the separation of the solid cellulosic fraction and a liquid fraction, because Inamdar teaches filtering the remove the liquids, which would include any soluble enzymes, and separating the solid material. One of ordinary skill would have been motivated by the teachings of Tocco because the enzymes should be removed before any steps using alkaline peroxide treatments, and one would predict that the enzyme mixture may be recycled.
Claim 15 is prima facie obvious, as the limitations therein do not further limit the invention compared to claim 1. Inamdar teaches steps of washing with water, and one of ordinary skill would arrive at the claimed method wherein the solids are separated from LMEs.
Regarding claim 16, Tocco teaches that pretreatments with H2O2 under moderate alkaline conditions is well-known in the art, and Inamdar teaches using 0.5 to about 2% NaOH at a temperature range of about 80°C to about 210°C for a period of about 5 to 360 minutes. When performing the method taught by the combination of Tocco and Inamdar, it would have been prima facie obvious to incorporate the alkaline hydrogen peroxide treatments of Tocco, and one having ordinary skill in the art would predictably arrive at the instantly claimed conditions.
Inamdar teaches conditions which overlap with the claimed treatment conditions of 60°C to 80°C for 60 to 90 minutes. MPEP § 2144.05.I. discusses that in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990), particularly when there is no showing of criticality of the claimed range.
Further, MPEP § 2144.05.II. describes that the determination of suitable or effective concentration of a known composition (or performing a known method) can be determined by one of ordinary skill in the art through the use of routine or manipulative experimentation to obtain optimal results, as these are variable parameters attainable within the art. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). In the instant case, the concentrations of H2O2, as well as the temperature and time of treatment, are known result-effective variables in the art, and the limitations of claim 16 would have been a matter of routine optimization of known parameters.
Regarding claims 17 and 18, Inamdar teaches that the pH for the saccharification enzyme hydrolysis is in the claimed range of pH 4.5 to pH 5.5 and that the temperature falls within the range of 40 to 80°C, as discussed above. See MPEP §§ 2144.05 and 2131.03.
In regards to claim 20, the examples of Inamdar demonstrate what appears to be hydrolysis processes at a 40% to 80% range of efficiency, as best understood under the B.R.I. in view of the indefiniteness rejection above. Regardless, the optimization of the pretreatments and the evaluation of the resulting efficiency would have been prima facie areas of routine optimization to one of ordinary skill, especially in view of all the teachings of the combined references. There are no novel or unpredictable steps in the method as claimed that would have resulted in an efficiency that is substantially different from that already achievable in the art.
The steps of claim 21 are taught in Inamdar, whereas for claim 22, the ability of the released carbohydrate products to make furfural, HMF, and lactic acid is discussed in Tocco. Thus these are well-known uses of the method’s product.
Claim 23 would have been prima facie obvious modifications of the method taught by the combination of Inamdar and Tocco and one would have been motivated to reuse the enzyme(s) as this would clearly result in an improved and more cost-efficient process, wherein the enzymes do not have to be produce freshly for each batch of feedstock. The selection of a concentration of laccase (in regards to the total amount of enzymes, presumably in view of the rejections under 112(b) discussed previously), would have been a matter of routine optimization to one having ordinary skill in the art. Tocco teaches that fungal laccase is the most used and critical enzyme of the enzyme mixture, and one would have recognized the concentration of the enzyme as a result-effective variable. Thus, the determining the optimal amount of laccase in the solution, to arrive at the claimed concentration of 40-60% (in comparison to total enzymes, presumably) would have been a predictable result.
As discussed above, MPEP § 2144.05 describes that the determination of a suitable or effective concentration of a known composition, when performing known methods, can be determined by one of ordinary skill in the art through the use of routine or manipulative experimentation to obtain optimal results, as these are variable parameters attainable within the art. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
From the teachings of the applied references, it is apparent that one of ordinary skill in the art would have had a reasonable expectation of success in producing the claimed invention, because each reference pertains to the processing and degradation of lignocellulosic material; the pre-processing steps, washing, filtering, and saccharification steps are taught in Inamdar, while Tocco teaches that such processing can be coupled with delignification with fungal enzymes.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the references, especially in the absence of persuasive evidence to the contrary.
Claims 1-3, 4-5, 7-10, 11-18, and 20-23 are rejected under 35 U.S.C. 103 as being unpatentable over Inamdar (US PGPub No. 20110129889) in view of Tocco (ACS Sustainable Chemistry & Engineering 9.6 (Feb 4, 2021): 2412-2432), as applied to claims 1-3, 11-18, and 20-23 above, and further in view of Elisashvili et al., (US Pat. No. US 8,753,844), to include the rejections of claims 4, 5, and 7-10.
The combined teachings of Inamdar and Tocco make obvious the claimed method for releasing carbohydrates from agricultural feedstocks, as discussed above, including providing lignin modifying enzymes from fermentation of white rot fungi, as in claims 2 and 3.
The relevant teachings of Inamdar and Tocco include all of those cited above.
However, the combination of Inamdar and Tocco does not teach that the lignin modifying enzymes are produced specifically with submerged fermentation, nor the particular additional limitations of claims 4-5, and 7-10.
Elisashvili (US 8,753,844) teaches methods, compositions, and systems for overproducing ligninolytic enzymes by growing fungi under conditions that promote expression of ligninolytic enzymes at high yields, using white-rot basidiomycetes that secrete one or more extracellular enzymes important for lignin degradation: lignin peroxidase, manganese-dependent peroxidase (MnP), and laccase (Abstract, Col 1, lines 45-48). Elisashvili states that “Non-limiting examples of fungi that can be used in the process disclosed herein include the white-rot fungi, such as Trametes versicolor, Phanerochaete chrysosporium, Phlebia radiata, Funalia trogii, and Cerrena unicolor (Col 3, lines 36-40). Elisashvili teaches that ligninolytic enzymes are produced by incubating the fungus in a submerged fermentation (Col 3, lines 45-65). Elisashvili teaches that "submerged fermentation" refers to fermentation in solution and allows for precise control of factors such as pH, temperature, oxygen and nitrogen diffusion, gas distribution, and nutrient distribution throughout the fluid fermentation mixture.
Regarding the particular conditions of claim 7, Elisashvili teaches that the fermentation is performed for between 7–14 days in one embodiment, or up to about 21 days (Col 5, lines 44-50); at a pH of 5.5 (see “fermentation medium” in Col 7, lines 14-22) and at a temperature of at least 15°C to up to 30°C, preferably at 27°C (Col 5, lines 35-42). Elisashvili also states that “the mycelium homogenate can be transferred into sterile medium in the proportion 1:10-1:20.” (Col 5, lines 20-23), thus teaching that the active growing mycelia is inoculated in a proportion of 5% to 10%, as in claim 7.
Elisashvili teaches that the submerged fermentation includes one or more additional stimulators of ligninase production (i.e. inducers), including copper sulphate (see claim 14) or xylidine (Col 4, line 66- Col 5, line 5), as recited in claims 8 and 9.
Regarding claim 10, Elisashvili teaches that the ligninases may be recovered from the spent medium by separating the cells from the medium by centrifugation or filtration and obtaining the proteins (Col 6, lines 25-34).
Therefore, before the effective filing date of the instant invention, to one of ordinary skill in the art, it would have been prima facie obvious to modify the process made taught by the combination of Inamdar and Tocco for the delignification and saccharification of lignocellulosic material, to further include steps of preparing the lignin modifying enzymes by performing submerged fermentation of white rot fungi, with species known to the art, using the methods taught in Elisashvili for improved production of extracellular enzymes, including lignin peroxidase, manganese-dependent peroxidase, and laccase.
One would have been motivated by the teachings of Elisashvili to perform submerged fermentation for the predictable improved production of these enzymes from white rot fungi because Elisashvili teaches that such liquid fermentation allows for precise control of factors such as pH, temperature, and nutrient distribution. These techniques would have been known to one of ordinary skill in the art at the time. Further, the particular conditions described in the instant claim 7 are all disclosed in the description and preferred embodiments of Elisashvili. Thus, the selection of these parameters would have been a matter of judicious selection to one having ordinary skill in the art. Regardless, the parameters of pH, temperature, and time for fermentation are well known and established in the art, that these would all be considered routine optimization in order to improve the process and yield greater amounts of enzyme. See MPEP § 2144.05, and In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
In regards to claims 8 and 9, the fully optional limitations therein would have been obvious over the teachings of Elisashvili, as the reference explicitly teaches the use of additional inducers, including copper sulphate or xylidine to improve the enzyme production.
Regarding claim 10, one having ordinary skill in the art would have been motivated by the teachings of Elisashvili, which explicitly describes removing the enzymes from the biomass (i.e. the solids) for further use. Further, Tocco teaches the use of ligninolytic enzymes secreted by WRF and isolated, as described previously. One of ordinary skill in the art would recognize to collect the spent media (i.e. the “clear broth”), which contains the secreted enzymes.
From the teachings of the cited references, it is apparent that one of ordinary skill in the art would have had a reasonable expectation of success in producing the claimed invention, because each reference pertains to the processing and use of lignocellulosic material, while Tocco and Elisashvili both teach the production and optimization of WRF enzymes for modifying and degrading lignin.
Thus, the limitations of claims 4, 5, and 7-10 would have been obvious to one of ordinary skill in the art, especially in the absence of persuasive evidence to the contrary.
Claims 1-3 and 11-23 are rejected under 35 U.S.C. 103 as being unpatentable over Inamdar (US PGPub No. 20110129889) in view of Tocco (ACS Sustainable Chemistry & Engineering 9.6 (Feb 4, 2021): 2412-2432), as applied to claims 1-3, 11-18, and 20-23 above, and further in view of Eibinger (“Cellulose surface degradation by a lytic polysaccharide monooxygenase and its effect on cellulase hydrolytic efficiency.” The Journal of biological chemistry vol. 289,52 (2014): 35929-38. doi:10.1074/jbc.M114.602227), to include the rejection of claim 19.
The combined teachings of Inamdar and Tocco make obvious the claimed method for releasing carbohydrates from agricultural feedstocks, as discussed above, including the use of one or more cellulolytic enzymes, including cellulase, for the hydrolysis of the treated feedstock, as recited in claim 17.
The relevant teachings of Inamdar and Tocco include all of those cited above.
However, the combination of Inamdar and Tocco does not teach that the hydrolysis also comprises the addition of one or more accessory proteins, as required in claim 19.
Eibinger teaches that the enzyme lytic polysaccharide monooxygenase (LPMO) depolymerizes cellulose and that LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation (see Abstract, ”Background”, and “Conclusion” in the summary on pg. 35929). Eibinger teaches that LPMO and cellulases are used together on solid cellulose surface, resulting in a synergistic effect on the degradation of different cellulosic substrates (Figure 4). Eibinger concludes that “Dynamic interplay between LPMO and CBH I [cellulase] activity appears to be beneficial for degradation of large, hence highly resistant crystalline substrate structures” (see pg. 35967, and Abstract).
Therefore, before the effective filing date of the instant invention, to one of ordinary skill in the art, it would have been prima facie obvious to modify the process made obvious by the combination of Inamdar and Tocco for the processing, delignification, and saccharification of lignocellulosic material, to further modify the method to include one or more lytic polysaccharide monooxygenase (LPMO) for the predictable benefit of improved hydrolysis towards cellulosic substrates, as taught in Eibinger.
One would have been motivated to include polysaccharide monooxygenase (LPMO), (i.e. an accessory protein”) because Eibinger teaches the beneficial synergistic effects of mixing LPMO with cellulase enzymes when performing hydrolysis of large, resistant, crystalline cellulose substrates. See MPEP § 2143.I.(A) regarding the combination of prior art elements according to known methods to yield predictable results.
From the teachings of the applied references, it is apparent that one of ordinary skill in the art would have had a reasonable expectation of success in producing the claimed invention, because each reference pertains to the processing and degradation of lignocellulosic material, Inamdar teaches performing hydrolysis with cellulolytic cellulase, and Eibinger demonstrates that LPMO successfully improves the hydrolysis activity of cellulases, with synergy.
Thus, the additional limitations of claim 19 would have been obvious to one of ordinary skill in the art, especially in the absence of persuasive evidence to the contrary.
Citation of Pertinent Art
The prior art made of record below and not relied upon is considered pertinent to applicant's disclosure.
Zhang et al. (US Pat No. 9,447,539) pertains to methods for the pretreatment of lignocellulosic material, and enzymatic hydrolysis carried out with an enzyme selected from the group consisting of cellulases, ligninases, hemicellulases, xylanases, lipases, pectinases, amylases, and/or proteinases (Abstract and claims 11 and 14 therein).
Forootanfar et al. (“Insights into laccase producing organisms, fermentation states, purification strategies, and biotechnological applications.” Biotechnology progress vol. 31,6 (2015): 1443-63. doi:10.1002/btpr.2173) is a review article that provides an overview of laccase-producing organisms, their physiological roles, the currently available screening methods, laccase activity assays, laccase mediators, inducers, and inhibitors, as well as a description of the fermentation states and purification strategies (Abstract).
The following NPL references are authored by the inventors of the instant application, and disclose many, if not all of the features of the instant invention. These two references are not valid as prior art, however, they are cited and provided herein for reference.
Ambatkar et al. "Functional screening and adaptation of fungal cultures to industrial media for improved delignification of rice straw." Biomass and Bioenergy 155 (2021): 106271, available online 6 November 2021.
Ambatkar et al. "Optimized process for the production of fungal peroxidases and efficient saccharification of pre-treated rice straw." Bioresource Technology Reports 17 (2022): 100913, available online 21 December 2021.
Conclusion
No claims are allowable.
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/A.T.M./Examiner, Art Unit 1655
/ANAND U DESAI/Supervisory Patent Examiner, Art Unit 1655