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
All claims of the instant application are entitled to an effective filing date of 12/20/2019.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 11/17/2025 has been entered.
DETAILED ACTION
Claims 1-15 are canceled. Claims 16-33 are pending. Claims 23-30 are withdrawn from consideration as being drawn to a nonelected invention. Claims 16-22 and 31-33 are under consideration in this action.
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 18 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 18 broadens the scope of the phosphatase embodiment required in claim 16, from which claim 18 depends. Claim 16 requires the phosphatase to comprise an amino acid sequence that is at least 98% identical with the sequence according to SEQ ID NO: 41. Claim 18 recites wherein the phosphatase comprises an amino acid sequence that is at least 90% identical to the sequence according to SEQ ID NO: 13. SEQ ID NO: 13 is 96.6% identical to SEQ ID NO: 41. As such, claim 18 does not further limit the phosphatase embodiment of claim 16.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) 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.
Claims 16-22 and 31-33 are rejected under 35 U.S.C. 103 as being unpatentable over Moradian (Journal of the American Chemical Society, 1992 114(18), 6980-6987) in view of Sutiono, (Enzyme engineering of a haloacid dehalogenase-like phosphatase from Thermotoga neopolitana for optimization of substrate specificity, 2016), Wichelecki (WO 2018/169957) and Sieber (DE10 2017 002252), with evidence from New England Biolabs (2025). Moradian, Wichelecki and Sieber were relied upon in the action provided 02/18/2025, and Sieber and Wichelecki were provided in the IDS filed 06/17/2022.
Claim 16 requires two active method steps. In the first step, a composition comprising water, phosphate and at least one oligosaccharide or polysaccharide is added to at least six enzymes. The at least six enzymes are required to include at least one transferase, at least one phosphorylase, at least one mutase, at least one isomerase, at least one hydrolase and at least one phosphatase, wherein the phosphatase comprises an amino acid sequence that is at least 98% identical with the sequence according to SEQ ID NO: 41. The specification discloses that SEQ ID NO: 41 is a phosphatase variant with the following mutations: Y23H, V45R, P46T, E47Y, and G50L mutations. See the last full paragraph on page 16. Furthermore, claim 16 requires the oligosaccharide or polysaccharide to be glucose-based. According to the instant specification, starch is glucose-based. See the fourth paragraph from the bottom of page 3. Subsequently, the enzymes convert the oligosaccharide or polysaccharide to fructose. In the second step, at least one additional saccharide is added, and that at least one additional saccharide is selected from a list that includes disaccharide, trisaccharide, tetrasaccharide.
Claim 31 requires the at least one additional saccharide to be selected from maltose (i.e. disaccharide), maltotriose (i.e. trisaccharide) and/or maltotetraose (tetrasaccharide).
Regarding claims 16 and 31, Moradian teaches a multi-enzyme process for the conversion of starch to fructose. See the second paragraph in the left column on page 6982 and figure 2. In the batch process, all enzymes are combined in a Tris-HCl reaction buffer containing starch (a glucose-based polysaccharide), phosphate, and d-glyceraldehyde. Ten units each of phosphorylase-α, phosphoglucomutase (a mutase), phosphoglucose isomerase, and transaldolase (a transferase) are added. 3-Phosphoglycerate phosphatase, 1.3 U, is then added. See paragraphs 1-2 in “the process” section on page 6983. Moradian implies that water is present because Tris-HCl reaction buffer is prepared with water, as evidenced by New England Biolabs. See the first paragraph of New England Biolabs. In table VI, Moradian teaches that the 3-phosphoglycerate phosphatase uses fructose-6-phosphate as a substrate. Moradian suggests exploring alternative approaches to overcome an obstacle presented by nature: the unavailability of an enzyme catalyzing a key reaction, the direct hydrolysis of fructose-6-phosphate. See the first paragraph of the discussion. Furthermore, Moradian suggests that adding pullulanase (a hydrolase), or isoamylase (a hydrolase) would permit recovery of more of the hexose units in starch. See the last passage on page 6986. In the semi-continuous process, Moradian teaches repeatedly adding fresh reaction buffer containing starch. See the third paragraph in “the process” section on page 6983. In summation, Moradian teaches adding five enzymes one of which is 3-phosphoglycerate phosphatase to a composition comprising water, phosphate, and a glucose-based starch polysaccharide.
Moradian does not teach at least six enzymes including at least one hydrolase, and at least one phosphatase that comprises an amino acid sequence that is at least 98% identical with the sequence according to instant SEQ ID NO: 41; and Moradian does not teach adding at least one additional saccharide selected from a list that includes disaccharide (relevant to instant claim 16). However, Moradian suggests adding pullanase or isoamylase, which are hydrolases, and adding additional starch, but starch does not meet the instantly claimed at least one additional saccharide limitation.
Moradian does not teach adding at least one additional saccharide selected from maltose, maltotriose, and/or maltotetraose (relevant to instant claim 31).
Wichelecki teaches that enzymatic hydrolysis of starch can be catalyzed or enhanced by isoamylase and pullulanase, both of which hydrolyze α-1,6-glucosidic bonds. See [065]. Enzymes that can be used to hydrolyze starch to increase glucose-1-phosphate (G1P) include isoamylase and pullanase. See [078].
Moradian and Wichelecki do not teach at least six enzymes including at least one phosphatase that comprises an amino acid sequence that is at least 98% identical with the sequence according to instant SEQ ID NO: 41; and Moradian does not teach adding at least one additional saccharide selected from a list that includes disaccharide (relevant to instant claim 16).
Moradian and Wichelecki do not teach adding at least one additional saccharide selected from maltose, maltotriose, and/or maltotetraose (relevant to instant claim 31).
Sutiono teaches identifying amino acids that play a role in the substrate specificity of the haloacid dehalogenase from Thermatoga neapolitana (HADTn) and further increasing the specificity towards fructose-6-phosphate (F6P) by employing protein engineering. The HAD superfamily has phosphatase activity. See the first paragraph of section 1.4 on page 10. In appendix 1B on page 52, Sutiono teaches a wild type HADTn enzyme sequence that is a 96.3% query match to instant SEQ ID NO: 41. See the alignment in the office action appendix. Sutiono discloses that the only difference between the HADTn and 3KBB, a putative β-phosphoglucomutase from T. maritima, is that the HADTn has an arginine (R) at position 125, while 3KBB has a lysine (K) at that position; Sutiono suggests that position 125 is of negligible importance. See the first paragraph on page 40. Sutiono teaches employing site-directed mutagenesis to develop [HADTn] mutants, and investigate the role of different amino acids including P46 and E47. HADTnP46T shows almost doubled specificity towards F6P than wild type. See section 5.1 on page 46. Furthermore, Sutiono discloses that the glutamic acid at position 47 influences substrate recognition. See the abstract on page iv. In summation, Sutiono teaches P46T and R125K mutations, and Sutiono recognizes E47 as a variable position in the HADTn sequence.
Moradian, Wichelecki and Sutiono do not teach adding at least one additional saccharide selected from a list that includes disaccharide (relevant to instant claim 16).
Moradian, Wichelecki and Sutiono do not teach adding at least one additional saccharide selected from maltose, maltotriose, and/or maltotetraose (relevant to instant claim 31).
Sieber teaches a process which enables the conversion of di-, oligo- and polysaccharides to D-fructose through several catalytic steps. See paragraph [0010]. The process is generally suitable for the use of di-, oligo- and polysaccharides. For example, cellobiose, maltose or starch can be used to produce a hydrolysate rich in D-fructose. See [0014]. The saccharide compound is continuously fed. See claim 8. Sieber teaches enzymes with the following activities: phosphorylase, mutase, isomerase, and phosphatase. See claim 2. Thus, Sieber suggests that maltose can be continuously fed to either a batchwise or continuous process in which enzymes catalytically convert di-, oligo- and polysaccharides to D-fructose.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to: (1) add isoamylase and/or pullanase taught by Wichelecki to the batch process of Moradian; (2) modify the HADTn of Sutiono to include P46T, E47X and R125K substitutions, to further select any amino acid including tyrosine Y for position 47, and in process arrive at a modified HADTn sequence that is 98% identical to instant SEQ ID NO: 41; (3) further replace the 3-phosphoglycerate phosphatase of Moradian with the modified HADTn of Sutiono; and (4) combine the fresh reaction buffer containing starch of Moradian with the starch derivative maltose of Sieber.
(1) One would be motivated to add isoamylase and/or pullanase of Wichelecki to the batch process of Moradian, because Moradian suggests that isoamylase or pullanase can further improve hexose unit recovery from starch. There would be a reasonable expectation of success because Wichelecki discloses that isoamylase and pullulanase can increase glucose-1-phosphate yields from starch, and glucose-1-phosphate is the same intermediate taught by Moradian in figure 4.
(2) One would be motivated to apply the P46T, E47X and R125K substitutions to the HADTn of Sutiono, because Sutiono suggests that: a P46T substitution may double specificity towards F6P, the E47 position influences substrate recognition, and the R125K substitution associated with 3KBB is of negligible importance. There would be a reasonable expectation of success because Sutiono demonstrates applying site-directed mutations to HADTn. One would be further motivated to select tyrosine (Y) for the E47 position, because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because there are a finite number of amino acid residues to choose from. The resulting modified HADTn sequence of Sutiono with P46T, E47Y and R125K substitutions is 98% identical to instant SEQ ID NO: 41. See the office action appendix.
(3) One would be motivated to replace the 3-phosphoglycerate phosphatase of Moradian with the modified HADTn of Sutiono, because Moradian suggests exploring approaches to overcome an obstacle presented by nature: the unavailability of an enzyme that can catalyze the direct hydrolysis of fructose-6-phosphate; and Sutiono teaches employing protein engineering to increase specificity towards fructose-6-phosphate. There would be a reasonable expectation of success because both the 3-phosphoglycerate phosphatase of Moradian and the HADTn [as modified above] of Sutiono use fructose-6-phosphate as a substrate.
(4) One would be further prompted to add maltose, 4GT, of Wichelecki to the repeatedly added fresh reaction buffer of Moradian because Wichelecki suggests that 4GT can increase hexose yields by recycling maltose into longer maltooligosaccharides, which can be phosphorolytically cleaved by αGP to yield glucose-1-phosphate (G1P). Since Moradian teaches glucose-1-phosphate as an intermediate in the conversion of starch to fructose (e.g. see fig. 2), one would be motivated to increase the amount of glucose-1-phosphate as it affects fructose yield. There would be a reasonable expectation of success because Sieber suggests feeding maltose to batchwise processes in which phosphorylase, mutase, isomerase, and phosphatase enzymes catalytically convert saccharides to fructose.
Regarding claim 17, Moradian teaches starch. See “the process” section.
Wichelecki teaches converting a saccharide to glucose-1-phosphate. Wichelecki discloses that the saccharide is starch or a starch derivative selected from a group that includes maltose. See paragraph [033].
Regarding claim 18, Moradian teaches a batch process where all enzymes are combined (one-pot reaction). See the second paragraph in “the process” section.
Sutiono teaches, in appendix 1B on page 52, a wild type HADTn enzyme sequence that is a 99.7% query match to instant SEQ ID NO: 13. See the alignment in the office action appendix.
Sieber teaches Phosphatase from Thermotoga naphthophila DSM 13996 (SEQ ID NO: 1), which is a 100% identity match to instant SEQ ID NO: 13. See [0046], the original document and the alignment in the appendix of the office action mailed 08/15/2025.
Regarding claim 19, Moradian teaches phosphoglucomutase and phosphoglucose isomerase. See figures 2 and 4.
Wichelecki teaches converting starch to fructose in figure 4. Figure 4 includes PGM, i.e. phosphoglucomutase, and PGI, i.e. phosphoglucose isomerase. See paragraphs [032]-[033] for the abbreviations.
Sieber teaches phosphoglucomutase. See claim 3.
Regarding claim 20, Moradian teaches a semicontinuous process that is run at 37˚C for 170 hours, which is at least 24 hours as instantly claimed. See the third paragraph of “the process” section. However, Moradian discloses that no loss of enzyme activity is detected at both 25 and 37˚C. See the first paragraph in the “constructing the process” section.
Wichelecki teaches a reaction time that can be adjusted as necessary, and can range from about 8 hours to about 48 hours (e.g. an overlapping range with the instantly claimed at least 24-hour range). See paragraph [089].
Moradian, Wichelecki, Sutiono and Sieber do not teach enzymatically converting at least one oligosaccharide and/or polysaccharide to fructose at room temperature.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to optimize the semicontinuous process of Moradian by adjusting the 37˚C temperature to 25˚C (e.g. room temperature). One would be motivated to do so because Moradian suggests that no loss of activity is detected at 25˚C. There would be a reasonable expectation of success because Moradian demonstrates running the semicontinuous process for 170 hours at 37˚C and suggests that enzyme activity is not lost at both 25 and 37˚C. MPEP 2144.05(II) states that “[g]enerally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical” and that “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).
Regarding claim 21, Moradian teaches an initial starch concentration of 18.5 ± 1.1 mM, a starch converted concentration of 11.5± 1.5 mM and a fructose concentration of 11.4± 1.5, where the values represent the mean values analyzed at 3–8-hour intervals during the 170-hour run of the semicontinuous process. See table VIII and the second paragraph in the “(b) the complete process” section on page 6986. Therefore, Moradian indicates that on average 61% of the starch (e.g. saccharides present) in the composition is converted to fructose because (11.4mM ÷18.5mM)x100 is 61%; Moradian also indicates that at most 74.7% of the starch is converted because ((11.4+1.5)÷(18.5-1.1))x100 is 74%. Thus, Moradian indicates that at least 50% of the saccharides are converted over 170 hours (e.g. after 24 hours).
Regarding claim 22, Moradian teaches batch process and semicontinuous process at a temperature of 37˚C (e.g. a temperature between the instantly claimed 10-100 ˚C). See the first and second paragraph of “the process” section on page 6983.
Sieber teaches processes where the conversions are carried out between 10-100˚C and in an aqueous reaction medium having a pH between 3-12. See claim 9.
Regarding claim 32, Moradian teaches glucose-1-phosphate, glucose-6-phosphate and fructose-6-phosphate as intermediates in the multistep process for converting starch into fructose. See figure 2. Moradian teaches measuring concentrations of intermediates. See the paragraph spanning the left and right columns on page 6982.
Regarding claim 33, Moradian teaches batch process and semicontinuous process at a temperature of 37˚C (e.g. a temperature between the instantly claimed 20-90 ˚C). See the first and second paragraph of “the process” section on page 6983.
Sieber teaches processes where the conversions are carried out between 10-100˚C and in an aqueous reaction medium having a pH between 3-12. See claim 9.
Response to Arguments
Applicant's arguments filed 10/14/2025 have been fully considered to the extent that they apply to the new grounds of rejection set forth above, but they are not persuasive.
§ 103 rejection of claims 16-22 and 31-33
Applicant argues that the claimed subject matter exhibits unexpected results. Applicant references the specification, for example paragraphs [0220]-[00227], which Applicant asserts provides compelling, objective evidence that the phosphatase of SEQ ID NO: 41 exhibits surprisingly and unexpectedly superior properties for the claimed method. SEQ ID NO:41 is the “V45” variant (Y23HV45RP46TE47YG50L). The data show that after 18 minutes the V45R variant produces 1928.61 nmol of F6P. In contrast the wild-type phosphatase CE8 produces only 76.96 nmol in the same timeframe. This represents an approximately 25-fold increase, which is a dramatic and non-linear improvement that a person of ordinary skill in the art would not have predicted. The V45R variant demonstrates high F6P/G6P ratio indicating high selectivity for the desired substrate F6P. See the second paragraph on page 10 to the first passage on page 11 of the remarks.
This argument is not persuasive because the results relied upon are not commensurate in scope with the instant claims. The claims do not require the at least one phosphatase to comprise an amino acid sequence that is 100% identical to instant SEQ ID NO: 41, nor do the claims require the phosphatase to include the V45R mutation.
Applicant argues that Moradian teaches away from the idea that any phosphatase would work as Moradian teaches that “Much effort was devoted to identify enzymes to catalyze this transformation efficiently” (page 6983 paragraph 1 of the results section). See the first full paragraph on page 11 of the remarks.
This argument is not persuasive because this same teaching of Moradian provides motivation for using the HADTn phosphatase of Sutiono discussed above. MPEP 2123 states that “[a] reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill the art, including nonpreferred embodiments”. Merck & Co. v. Biocraft Laboratories, 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989). See also Upsher-Smith Labs. v. Pamlab, LLC, 412 F.3d 1319, 1323, 75 USPQ2d 1213, 1215 (Fed. Cir. 2005).
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KIMBERLY C BREEN whose telephone number is (571)272-0980. The examiner can normally be reached M-Th 7:30-4:30, F 8:30-1:30 (EDT/EST).
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657