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 .
DETAILED ACTION
This application is a 371 PCT/EP2022/066384.
The amendment filed don June 29, 2026 has been entered.
Election/Restrictions
Applicant elected without traverse of Group I with a species election of (1) SEQ ID NO:1 as the parent DNase and (2) T1l+S13Y+T22P+S27L+L33K+S39P+S42G+D56HS57W+S59V+T65V+V76L+Q109R+S116D+T127V+S144P+A147H+S167L+G175D as the amino acid modifications made in the parent DNase the reply filed on December 1, 2025 is acknowledged.
Claims 24-25 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on December 1, 2025.
Status of Claims
Claims 20-35 are pending.
Claims 24-25 are withdrawn.
Claims 20-23 and 26-35 are under examination.
Response to Amendments/Arguments
Claim Rejections - 35 USC § 112
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.
Maintained Rejection
Claim 20 and claims 21-23 and 26-35 depending therefrom 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.
The term “build-up” of slime in claim 20 is a relative term which renders the claim indefinite. The term “build-up”” 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. It is unclear as to how much slime is considered as “build-up”.
Applicant's arguments filed June 29, 2026 have been fully considered but they are not persuasive.
Applicant argues that the term “build-up” is not indefinite when the claim is read in light of the specification as according to MPEP 2173.05(b) (terms of degree are permissible the specification provides a standard form measuring the degree). Applicant argues that the specification consistently uses the term to describe the accumulation/formation of surface-attached microbial deposits and one or ordinary skill in the art would understand the metes and bounds with reasonable certainty: the term encompasses deposits, as objectively demonstrated by the microtiter assay using the real process water or by operationally improvements.
This is not found persuasive. MPEP 2173.05(b) states that the “claim is not indefinite if the specification provides examples or teachings that can be used to measure a degree even without a precise numerical measurement.”. In the instant case, while the specification uses the term “build-up” of slime to describe the accumulation/formation of surface-attached microbial deposits and reducing slime, the specification does not provide a standard form measuring the degree. Therefore, it is unclear as to how much slime is considered as “build-up”.
Hence the rejection has been maintained.
Withdrawn Rejection
Applicant’s arguments, see page 9 of the Remarks, filed June 29, 2026, with respect to claims 26-27 have been fully considered and are persuasive. Claims 26-27 has been amended to recite “mg/L” to clarify that the amount refers to the DNase. Therefore, the rejection of claims 26-27 under 35 U.S.C. 112(b) has been withdrawn.
New Rejection
Claim 35 is 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.
Claim 35 recites the limitation "according to claim 34, wherein the enzyme is a carbohydrate oxidase having at least 80% sequence identity to the mature polypeptide of SEQ ID NO:6”. There is insufficient antecedent basis for this limitation in the claim. Claim 34 recites “one or more enzyme selected…” but does not recite a carbohydrate oxidase” and claim 20 does not recite an “enzyme”. Therefore, it is unclear if the carbohydrate oxidase having at least 80% sequence identity to the mature polypeptide of SEQ ID NO:6 of claim 35 refers to the carbohydrate oxidase recited in claim 20 or to another carbohydrate oxidase. Clarification is requested.
Claim Rejections - 35 USC § 103
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.
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.
Withdrawn Rejection
Applicant’s arguments, see pages 10-11 of the Remarks, filed June 29, 2026, with respect to (A) claims 20-22, 28, and 31-33 and (B) claims 26-27 and 29-30 as applied to claims 20-22, 28, and 31-33 have been fully considered and are persuasive. Claim 20 has been amended to require a carbohydrate oxidase, which is not disclosed by the cited references. Therefore, (A) the rejection of claims 26-27and (B) the rejection of claims 26-27 and 29-30 as applied to claims 20-22, 28, and 31-33 under 35 U.S.C. 103 have been withdrawn.
Amended Rejection
Claim(s) 20-22, 28, and 31-35 is/are rejected under 35 U.S.C. 103 as being unpatentable over Johnsrud (Biotechnology for Solving Slim Problems in the Pulp and Pater Industry (Advances in Biochemical Engineering/Biotechnology, Vol. 57, pages 311-328, 1997 – cited previously on form PTO-892), Budny (US 2002/0037260 – cited previously on form PTO-892), Beier (US 2019/0211284 – cited previously on form PTO- 892), Xu (A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale. Eur J Biochem. 2001 Feb;268(4):1136-4 – cited previously on form PTO-892), and MNCO_MICNN (UniPortKB/Swiss-Prot Database. February 26, 2020 – cited previously on form PTO-892).
Regarding claim 20, Johnsrud discloses that a well-known and long-standing problem in paper manufacture is the proliferation of biological slimes on machinery, wherein slime seeds into process water (page 312, section 2). Johnsrud discloses that adequate control of microbial-induced slime deposits is an essential part of modern pulp and paper manufacture (page 316, section 3). Johnsrud discloses a method of preventing and disrupting biofilm build-up (which reads on preventing a build-up of slime or removing slime) caused by slime-forming species of microorganism and preventing a build-slime or removing slime by adding various enzymes to the process water (page 320, section 4.2.1). Johnsrud disclose that the striping of the extracellular polysaccharide layer prevents the microorganism from attaching to other surfaces and makes them more accessible to conventional biocide treatment (page 321, 1st full paragraph).
Regarding claim 28, the water of Johnsrud is process water (page 312, section 2).
Regarding claims 31-33, the surface of Johnsrud is from the paper/pulp making machinery (which reads on a manufacturing equipment) (page 312, section 2).
Johnsrud does not disclose a method of preventing a build-up of slime or removing slime from a surface by contacting water with a DNase and a carbohydrate oxidase.
Regarding claim 20, Budny discloses that biofilm is associated with paper pulping operations and discloses a composition for degrading biofilm comprising DNase (claims 32-34).
Regarding claim 20, Beier disclose that biofilm, slime of microorganism, is surround by a matrix of extracellular polymeric substances composed of extracellular DNA, proteins, and polysaccharides ([0003]). Beier discloses using DNase to degrade the biofilm ([0004]-[0005]). Beier discloses a method of treating a surface by contacting an aqueous wash liquor (which includes water) with DNase and treating the surface with the wash liquor ([0010]).
Regarding claim 21, Beier discloses a DNase having the amino acid sequence of SEQ ID NO:1, which has 100% sequence identity to the DNase of SEQ ID NO:1 of the instant application ([0008] and see the sequence alignment below).
Regarding claim 22, Beier discloses a DNase variant having at least 80% sequence identity to the DNase of SEQ ID NO:1 of the instant application, wherein the DNase variant has one or more amino acid substitutions selected from T1I, T1L, S13Y, T22P, S25P, S27l, S39P, S42G, S57W, S57Y, S59V, S59I, S59L, V76L, Q109R, S116D, S116E, T127V, S144P, A147H, S167L, G175D, and G175E ([0107]).
Regarding claims 20 and 35, Budny discloses use of two enzymes, a first enzyme to degrade the biofilm structure and a second enzyme to generate active oxygen to directly attack and kill bacteria that are exposed during the process of the degradation and removal of the biofilm ([0060]). Budny discloses that the first enzyme is a levanase ([0095]). Budny discloses that the second enzyme is a hexose oxidase of E.C. 1.1.3.5, which is synonymous with a carbohydrate oxidase, to degrade biofilm ([0061]).
Regarding claims 20 and 35, Xu discloses a carbohydrate oxidase having 100% sequence identity to the carbohydrate oxidase of SEQ ID NO:6 of the instant application (abstract and Fig. 2 at page 1138 and see the sequence alignment below). The carbohydrate oxidase of Xu belongs to E.C. 1.1.3.5 (see MNCO-MICNN).
Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to (A) enhance the method of Johnsrud by contacting the process water with the DNase of Beier to prevent or remove slime from the surface of the machinery, such as metals or plastics and (B) add an additional two sets of enzymes, a first enzyme, such as a levanase, and a second enzyme, such as a carbohydrate oxidase. One having ordinary skill in the art would have been motivated the use the DNase of Beier because said DNase of Beier breaks down biofilm/slime. One of ordinary skill in the art at the time the invention was made would have been motivated to add two additional sets of enzymes to (1) degrade the biofilm structure and (2) generate active oxygen to directly attack and kill bacteria that are exposed during the process of the degradation and removal of the biofilm. One of ordinary skill in the art would have had a reasonable expectation of success since Johnsrud discloses a method of preventing build-up of slime or removing slime from a surface during pulp or paper making process by degrading said slime, Budny discloses that biofilm is associated with paper pulping operations and discloses a composition for degrading biofilm comprising DNase, Beier discloses a method of degrading biofilm/slime using a DNase, Budny discloses use of two enzymes, first enzyme to degrade the biofilm structure and a second enzyme to generate active oxygen to directly attack and kill bacteria that are exposed during the process of the degradation and removal of the biofilm, and Xu discloses a carbohydrate oxidase having 100% sequence identity to the carbohydrate oxidase of SEQ ID NO:6 of the instant application.
Further, the rationale supporting that the claims would have been obvious is that a method of enhancing a particular class of devices (preventing build-up of slime or removing slime from a surface using DNase) has been made part of the ordinary capabilities of one skilled in the art based upon the teaching of such improvement in other situations. One of ordinary skill in the art would have been capable of applying this known method of enhancement to a “base” device (method of preventing build-up of slime or removing slime from a surface of machinery during pulp or paper making process) in the prior art and the results would have been predictable to one of ordinary skill in the art because (1) the prior art contained a “base” device upon which the claimed invention can be seen as an “improvement;” (2) the prior art contained a “comparable” device that has been improved in the same way as the claimed invention; and (3) one of ordinary skill in the art could have applied the known “improvement” technique in the same way to the “base” device and the results would have been predictable to one of ordinary skill in the art.
Therefore, the above references render claims 20-22, 28, and 31-35 prima facie obvious.
Applicant's arguments filed June 29, 2026 have been fully considered but they are not persuasive. Applicant has addressed all rejections together.
Applicant argues that the cited references do not teach or suggest the claimed combination and argues the deficiencies of each reference.
This is not found persuasive.
In response to applicant's arguments against the 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).
Regarding Johnsrud, Budny discloses that biofilm is associated with paper pulping operations and discloses a composition for degrading biofilm comprising DNase (claims 32-34) and Beier disclose that biofilm, slime of microorganism, is surround by a matrix of extracellular polymeric substances composed of extracellular DNA, proteins, and polysaccharides ([0003]). Beier discloses using DNase to degrade the biofilm ([0004]-[0005]).
Regarding Budny, the claims of the instant application do not preclude anchored enzymes and MNCO-MICNN and Beir discloses the specific DNase recited in claims 21-22 and carbohydrate oxidase recited in claim 35. Applicant’s assertion of a synergist effect is addressed below.
Regarding Beier, the teachings of Beier can be applied to the instant invention because Beier and the instant claims are both directed to reducing biofilm. Budny discloses use of two enzymes, such as a levanase and a carbohydrate oxidase.
Regarding Hatcher, Budny discloses use of two enzymes, such as a levanase and a carbohydrate oxidase.
Regarding Xu, Budny discloses that biofilm is associated with paper pulping operations and discloses a composition for degrading biofilm comprising DNase and use of two enzymes, such as a levanase and a carbohydrate oxidase.
Applicant argues that (1) the Examiner’s rationale is hindsight-drive and (2) lacks the required motivation or suggestion in the art.
This is not found persuasive.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). In the instant case, a well-known and long-standing problem in paper manufacture is the proliferation of biological slimes on machinery, wherein slime seeds into process water was known in the art, see Johnsrud as discussed above. A method of preventing and disrupting biofilm build-up (which reads on preventing a build-up of slime or removing slime) caused by slime-forming species of microorganism and preventing a build-slime or removing slime by adding various enzymes to the process water was also known in the prior art, see Johnsrud as discussed above. Use of DNase, levanase, and carbohydrate oxidase for degrading biofilm was also known in the prior art, see Budny, Beir, and Xu as discussed above. Therefore, one having ordinary skill in the art would have been motivated to modify the method of Johnsrud by using the DNase of Beier because said DNase of Beier breaks down biofilm/slime. One of ordinary skill in the art at the time the invention was made would have been motivated to add two additional sets of enzymes to (1) degrade the biofilm structure (levanase) and (2) generate active oxygen to directly attack and kill bacteria that are exposed during the process of the degradation and removal of the biofilm (carbohydrate oxidase).
Applicant argues that Example 2 discloses unexpected results.
This is not found persuasive.
Applicant’s assertion of unexpected results is not commensurate in scope with the instant claims. The results of Examples 2-3 are limited to the combination of a specific DNase (DNase variants of SEQ ID NO:2, DNase-2, 3, 4, 5, 7, and 7) and a specific carbohydrate oxidase (mature carbohydrate oxidase of SEQ DI NO:6). However, the claims are directed to any DNase or DNase variants of SEQ ID NO:1, and any carbohydrate oxidase or any carbohydrate oxidase having at least 80% sequence identity to the mature polypeptide of SEQ ID NO:6.
Applicant argues that Example 3 demonstrates a synergistic effect on the combined use of a DNase and a carbohydrate oxidase.
This is not found persuasive.
A greater than additive effect is not necessarily sufficient to overcome a prima facie case of obviousness because such an effect can either be expected or unexpected, see MPEP 716.02(a). In the instant case, the increased slime prevention comprising DNase-1 and carbohydrate is not unexpected because DNase-1 and carbohydrate oxidase have different enzymatic activities and affects and their combined used can contribute to an increased slime prevention. Biofilm, slime of microorganism, is surround by a matrix of extracellular polymeric substances composed of extracellular DNA, proteins, and polysaccharides. DNase degrades said DNA and degrades biofilm. Carbohydrate oxidase generates active oxygen to directly attack and kill bacteria that are exposed during the process of the degradation and removal of the biofilm. Therefore, Example 3 showing greater than additive slime prevention resulting from the claimed mixture a DNase and a carbohydrate oxidase is not sufficient to outweigh the evidence of obviousness because the teachings of the prior art would lead to a general expectation of greater than additive slime degradation when using mixtures of enzymes (DNase and a carbohydrate oxidase) degrading different components of biofilm.
Hence the rejections have been maintained.
Claim(s) 26-27 and 29-30 is/are rejected under 35 U.S.C. 103 as being unpatentable over Johnsrud (Biotechnology for Solving Slim Problems in the Pulp and Pater Industry (Advances in Biochemical Engineering/Biotechnology, Vol. 57, pages 311-328, 1997 – cited previously on form PTO-892), Budny (US 2002/0037260 – cited previously on form PTO-892), Beier (US 2019/0211284 – cited previously on form PTO- 892), Xu (A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale. Eur J Biochem. 2001 Feb;268(4):1136-4 – cited previously on form PTO-892), and MNCO_MICNN (UniPortKB/Swiss-Prot Database. February 26, 2020 – cited previously on form PTO-892) as applied to claims 20-22, 28, and 31-35 above, and further in view of Hatcher (US 3,773,623 – form PTO-892).
“[W]here 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.”, see MPEP 2144.
Regarding claims 26-27, Beier discloses that the amount of the DNase is 0.001-10 mg enzyme protein/L (ppm), which falls within the claimed range of 0.1 to 10 mg/L ([0257]-[0258]).
Regarding claims 26-27, Hatcher discloses a method of controlling slime for formation, such as retarding or removing, with an enzyme (abstract). Hatcher discloses using 1 ppm to 10 ppm of the enzyme, which falls within the claimed range of 0.1 to 10 mg/L (Column 3, lines 20-44).
Regarding claims 29-30, Hatcher discloses that the pH of the method is 6.7, which falls within the claimed range of 6.1 to 7.6 (Column 4, line 37).
Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was made to modify the method of Johnsurd by varying the amount of DNase and the pH of the process water. One of ordinary skill in the art at the time the invention was made would have been motivated to do so in order to optimize the method of preventing the build-up of slime or removing slime on the surface of the machinery for pulp or paper making. One of ordinary skill in the art would have had a reasonable expectation of success since Johnsrud discloses a method of preventing build-up of slime or removing slime from a surface during pulp or paper making process by degrading said slime, Budny discloses that biofilm is associated with paper pulping operations and discloses a composition for degrading biofilm comprising DNase, Beier discloses a method of degrading biofilm/slime using a DNase in an amount of 0.001-10 mg enzyme protein/L (ppm), and Hatcher discloses a method of controlling slime for formation, such as retarding or removing, with an enzyme in an amount of 1 ppm to 10 ppm and at a pH of 6.7.
Therefore, the above references render claims 20-22 and 26-35 prima facie obvious.
Applicant's arguments filed June 29, 2026 have been fully considered but they are not persuasive. Applicant has addressed all rejections together, see above.
Hence the rejections have been maintained.
Conclusion
Claims 20-35 are pending.
Claims 24-25 are withdrawn.
Claims 20-23 and 26-35 are rejected.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YONG D PAK whose telephone number is (571)272-0935. The examiner can normally be reached M-Th: 5:30 am - 3:30 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert Mondesi can be reached on 408-918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/YONG D PAK/Primary Examiner, Art Unit 1652
Sequence alignment between the DNase of SEQ ID NO:1 of the instant application (“Qy”) and the DNase of SEQ ID NO:1 of Beier (“Db”)
US-16-238-648A-1
Filing date in PALM: 2019-01-03
Sequence 1, US/16238648A
Publication No. US20190211284A1
GENERAL INFORMATION
APPLICANT: The Procter & Gamble Company
TITLE OF INVENTION: POLYPEPTIDE VARIANTS
FILE REFERENCE: CM04824C
CURRENT APPLICATION NUMBER: US/16/238,648A
CURRENT FILING DATE: 2019-01-03
PRIOR APPLICATION NUMBER: US 16/238,648
PRIOR FILING DATE: 2019-01-03
NUMBER OF SEQ ID NOS: 26
SEQ ID NO 1
LENGTH: 182
TYPE: PRT
ORGANISM: Bacillus cibi
Query Match 100.0%; Score 986; Length 182;
Best Local Similarity 100.0%;
Matches 182; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 TPPGTPSKSAAQSQLNALTVKTEGSMSGYSRDLFPHWISQGSGCDTRQVVLKRDADSYSG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 TPPGTPSKSAAQSQLNALTVKTEGSMSGYSRDLFPHWISQGSGCDTRQVVLKRDADSYSG 60
Qy 61 NCPVTSGSWYSYYDGVTFTNPSDLDIDHIVPLAEAWRSGASSWTTSKRQDFANDLSGPQL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 NCPVTSGSWYSYYDGVTFTNPSDLDIDHIVPLAEAWRSGASSWTTSKRQDFANDLSGPQL 120
Qy 121 IAVSASTNRSKGDQDPSTWQPPRSGAACGYSKWWISTKYKWGLSLQSSEKTALQGMLNSC 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 IAVSASTNRSKGDQDPSTWQPPRSGAACGYSKWWISTKYKWGLSLQSSEKTALQGMLNSC 180
Qy 181 SY 182
||
Db 181 SY 182
Sequence alignment between the carbohydrate oxidase of SEQ ID NO:6 of the instant application (“Qy”) and the carbohydrate oxidase of Xu/MNCO-MICNN (“Db”)
MNCO_MICNN
ID MNCO_MICNN Reviewed; 495 AA.
AC I1SB12;
DT 10-APR-2019, integrated into UniProtKB/Swiss-Prot.
DT 10-APR-2019, sequence version 2.
DT 09-APR-2025, entry version 69.
DE RecName: Full=Carbohydrate oxidase {ECO:0000303|PubMed:11179980};
DE EC=1.1.3.-;
DE EC=1.1.3.5 {ECO:0000269|PubMed:11179980};
DE AltName: Full=Lactose oxidase {ECO:0000303|PubMed:17154316};
DE Short=LaO;
DE Flags: Precursor;
GN Name=MnCO {ECO:0000303|PubMed:11179980};
OS Microdochium nivale (Pink snow mold) (Lanosa nivalis).
OC Eukaryota; Fungi; Dikarya; Ascomycota; Pezizomycotina; Sordariomycetes;
OC Xylariomycetidae; Xylariales; Microdochiaceae; Microdochium.
OX NCBI_TaxID=5520;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 23-46; 217-292;
RP 325-344; 358-369; 404-435 AND 442-462, FUNCTION, CATALYTIC ACTIVITY,
RP BIOPHYSICOCHEMICAL PROPERTIES, AND SUBCELLULAR LOCATION.
RC STRAIN=NN008551;
RX PubMed=11179980; DOI=10.1046/j.1432-1327.2001.01982.x;
RA Xu F., Golightly E.J., Fuglsang C.C., Schneider P., Duke K.R., Lam L.,
RA Christensen S., Brown K.M., Joergensen C.T., Brown S.H.;
RT "A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale.";
RL Eur. J. Biochem. 268:1136-1142(2001).
RN [2]
RP FUNCTION, AND CATALYTIC ACTIVITY.
RX DOI=10.1016/S1381-1177(00)00233-2;
RA Kulys J., Tetianec L., Schneider P.;
RT "Specificity and kinetic parameters of recombinant Microdochium nivale
RT carbohydrate oxidase.";
RL J. Mol. Catal., B Enzym. 13:95-101(2001).
RN [3]
RP FUNCTION, CATALYTIC ACTIVITY, AND BIOPHYSICOCHEMICAL PROPERTIES.
RA Tetianec L., Kulys J.;
RT "The specificity of recombinant Microdochium nivale carbohydrate oxidase.";
RL Biologija 2:38-41(2003).
RN [4]
RP CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, AND COFACTOR.
RX PubMed=17154316; DOI=10.1002/bit.21273;
RA Nordkvist M., Nielsen P.M., Villadsen J.;
RT "Oxidation of lactose to lactobionic acid by a Microdochium nivale
RT carbohydrate oxidase: kinetics and operational stability.";
RL Biotechnol. Bioeng. 97:694-707(2007).
RN [5]
RP CRYSTALLIZATION.
RX PubMed=19478452; DOI=10.1107/s1744309109017643;
RA Duskova J., Dohnalek J., Skalova T., Oestergaard L.H., Fuglsang C.C.,
RA Kolenko P., Stepankova A., Hasek J.;
RT "Crystallization of carbohydrate oxidase from Microdochium nivale.";
RL Acta Crystallogr. F 65:638-640(2009).
RN [6] {ECO:0007744|PDB:3RJ8, ECO:0007744|PDB:3RJA}
RP X-RAY CRYSTALLOGRAPHY (2.10 ANGSTROMS) OF 23-495 IN COMPLEX WITH FAD, AND
RP GLYCOSYLATION AT ASN-244.
RA Duskova J., Skalova T., Kolenko P., Stepankova A., Hasek J., Koval T.,
RA Ostergaard L.H., Fuglsang C.C., Dohnalek J.;
RT "Crystal structure and kinetic studies of carbohydrate oxidase from
RT Microdochium nivale.";
RL Submitted (APR-2011) to the PDB data bank.
CC -!- FUNCTION: Catalyzes the selective oxidation of C1 hydroxyl moieties on
CC mono-, oligo- and polysaccharides with concomitant reduction of
CC molecular oxygen to hydrogen peroxide. This results in the formation of
CC the corresponding lactones, which typically undergo spontaneous
CC hydrolysis. Carbohydrate oxidase is able to oxidize a variety of
CC substrates including D-glucose, D-galactose, D-xylose, D-maltose, D-
CC cellobiose, and lactose. In addition, among various oligosaccharides,
CC the enzyme preferred tetrameric dextrins, indicating a favorable
CC interaction of four linked glucose units with the substrate binding
CC pocket. {ECO:0000269|PubMed:11179980, ECO:0000269|Ref.2,
CC ECO:0000269|Ref.3}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=beta-D-glucose + O2 = D-glucono-1,5-lactone + H2O2;
CC Xref=Rhea:RHEA:11428, ChEBI:CHEBI:15379, ChEBI:CHEBI:15903,
CC ChEBI:CHEBI:16217, ChEBI:CHEBI:16240; EC=1.1.3.5;
CC Evidence={ECO:0000269|PubMed:11179980};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=D-galactose + O2 = D-galactono-1,5-lactone + H2O2;
CC Xref=Rhea:RHEA:59312, ChEBI:CHEBI:4139, ChEBI:CHEBI:15379,
CC ChEBI:CHEBI:15945, ChEBI:CHEBI:16240; EC=1.1.3.5;
CC Evidence={ECO:0000269|PubMed:11179980};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=D-cellobiose + O2 = D-cellobiono-1,5-lactone + H2O2;
CC Xref=Rhea:RHEA:59316, ChEBI:CHEBI:15379, ChEBI:CHEBI:16240,
CC ChEBI:CHEBI:17057, ChEBI:CHEBI:17863; EC=1.1.3.5;
CC Evidence={ECO:0000269|PubMed:11179980, ECO:0000269|Ref.3};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=beta-lactose + O2 = lactobiono-1,5-lactone + H2O2;
CC Xref=Rhea:RHEA:59352, ChEBI:CHEBI:15379, ChEBI:CHEBI:16240,
CC ChEBI:CHEBI:36218, ChEBI:CHEBI:143068; EC=1.1.3.5;
CC Evidence={ECO:0000269|PubMed:11179980};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=D-maltose + O2 = D-maltobiono-1,5-lactone + H2O2;
CC Xref=Rhea:RHEA:59344, ChEBI:CHEBI:15379, ChEBI:CHEBI:16240,
CC ChEBI:CHEBI:17306, ChEBI:CHEBI:143029; EC=1.1.3.5;
CC Evidence={ECO:0000269|PubMed:11179980, ECO:0000269|Ref.3};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=D-xylose + O2 = D-xylono-1,5-lactone + H2O2;
CC Xref=Rhea:RHEA:59324, ChEBI:CHEBI:15379, ChEBI:CHEBI:15867,
CC ChEBI:CHEBI:16240, ChEBI:CHEBI:53455;
CC Evidence={ECO:0000269|PubMed:11179980};
CC -!- COFACTOR:
CC Name=FAD; Xref=ChEBI:CHEBI:57692;
CC Evidence={ECO:0000269|PubMed:17154316};
CC Note=Binds 1 FAD per subunit in a bicovalent manner.
CC {ECO:0000269|Ref.6};
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=0.97 mM for oxygen {ECO:0000269|PubMed:17154316};
CC KM=19 mM for cellobiose (at pH 6 and 30 degrees Celsius)
CC {ECO:0000269|PubMed:11179980};
CC KM=59 mM for cellobiose (at pH 6 and 40 degrees Celsius)
CC {ECO:0000269|PubMed:11179980};
CC KM=51 uM for cellobiose (at pH 7.2 and 25 degrees Celsius)
CC {ECO:0000269|Ref.3};
CC KM=59 uM for cellotriose (at pH 7.2 and 25 degrees Celsius)
CC {ECO:0000269|Ref.3};
CC KM=66 uM for cellotetraose (at pH 7.2 and 25 degrees Celsius)
CC {ECO:0000269|Ref.3};
CC KM=42 mM for glucose (at pH 6 and 40 degrees Celsius)
CC {ECO:0000269|PubMed:11179980};
CC KM=11 mM for maltose (at pH 6 and 40 degrees Celsius)
CC {ECO:0000269|PubMed:11179980};
CC KM=12 mM for maltose (at pH 7.2 and 25 degrees Celsius)
CC {ECO:0000269|Ref.3};
CC pH dependence:
CC Optimum pH is 5-7. {ECO:0000269|PubMed:11179980};
CC Temperature dependence:
CC Optimum temperature is 50 degrees Celsius.
CC {ECO:0000269|PubMed:11179980};
CC -!- SUBCELLULAR LOCATION: Secreted {ECO:0000269|PubMed:11179980}.
CC -!- PTM: The FAD cofactor is bound via a bicovalent 6-S-cysteinyl, 8alpha-
CC N1-histidyl FAD linkage. {ECO:0000269|Ref.6}.
CC -!- SIMILARITY: Belongs to the oxygen-dependent FAD-linked oxidoreductase
CC family. {ECO:0000305}.
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DR PDB; 3RJ8; X-ray; 2.40 A; A=23-495.
DR PDB; 3RJA; X-ray; 2.10 A; A=23-495.
DR PDBsum; 3RJ8; -.
DR PDBsum; 3RJA; -.
DR AlphaFoldDB; I1SB12; -.
DR SMR; I1SB12; -.
DR GlyCosmos; I1SB12; 2 sites, No reported glycans.
DR EvolutionaryTrace; I1SB12; -.
DR GO; GO:0005576; C:extracellular region; IEA:UniProtKB-SubCell.
DR GO; GO:0046562; F:beta-D-glucose oxidase activity; IEA:RHEA.
DR GO; GO:0071949; F:FAD binding; IEA:InterPro.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-KW.
DR Gene3D; 3.30.465.10; -; 1.
DR Gene3D; 3.40.462.20; -; 1.
DR InterPro; IPR012951; BBE.
DR InterPro; IPR016166; FAD-bd_PCMH.
DR InterPro; IPR036318; FAD-bd_PCMH-like_sf.
DR InterPro; IPR016169; FAD-bd_PCMH_sub2.
DR InterPro; IPR050416; FAD-linked_Oxidoreductase.
DR InterPro; IPR006094; Oxid_FAD_bind_N.
DR PANTHER; PTHR42973; BINDING OXIDOREDUCTASE, PUTATIVE (AFU_ORTHOLOGUE AFUA_1G17690)-RELATED; 1.
DR PANTHER; PTHR42973:SF39; FAD-BINDING PCMH-TYPE DOMAIN-CONTAINING PROTEIN; 1.
DR Pfam; PF08031; BBE; 1.
DR Pfam; PF01565; FAD_binding_4; 1.
DR SUPFAM; SSF56176; FAD-binding/transporter-associated domain-like; 1.
DR PROSITE; PS51387; FAD_PCMH; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Direct protein sequencing; FAD; Flavoprotein; Glycoprotein;
KW Metal-binding; Nucleotide-binding; Oxidoreductase; Secreted; Signal; Zinc.
FT SIGNAL 1..22
FT /evidence="ECO:0000269|PubMed:11179980"
FT CHAIN 23..495
FT /note="Carbohydrate oxidase"
FT /id="PRO_0000446664"
FT DOMAIN 55..229
FT /note="FAD-binding PCMH-type"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00718"
FT CARBOHYD 244
FT /note="N-linked (GlcNAc...) asparagine"
FT /evidence="ECO:0007744|PDB:3RJ8, ECO:0007744|PDB:3RJA"
FT CARBOHYD 417
FT /note="N-linked (GlcNAc...) asparagine"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00498"
FT CROSSLNK 92..154
FT /note="6-(S-cysteinyl)-8alpha-(pros-histidyl)-FAD (His-
FT Cys)"
FT /evidence="ECO:0000269|Ref.6"
FT HELIX 24..31
FT /evidence="ECO:0007829|PDB:3RJA"
FT HELIX 42..47
FT /evidence="ECO:0007829|PDB:3RJA"
FT STRAND 60..64
FT /evidence="ECO:0007829|PDB:3RJA"
FT HELIX 68..80
FT /evidence="ECO:0007829|PDB:3RJA"
FT STRAND 85..90
FT /evidence="ECO:0007829|PDB:3RJA"
FT HELIX 97..99
FT /evidence="ECO:0007829|PDB:3RJA"
FT STRAND 101..104
FT /evidence="ECO:0007829|PDB:3RJA"
Query Match 100.0%; Score 2630; Length 495;
Best Local Similarity 100.0%;
Matches 495; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MRSAFILALGLITASADALVTRGAIEACLSAAGVPIDIPGTADYERDVEPFNIRLPYIPT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MRSAFILALGLITASADALVTRGAIEACLSAAGVPIDIPGTADYERDVEPFNIRLPYIPT 60
Qy 61 AIA QTQTTAHIQSAVQCAKKLNLKVSAKSGGHSYASFGFGGENGHLMVQLDRMIDVISYN 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AIA QTQTTAHIQSAVQCAKKLNLKVSAKSGGHSYASFGFGGENGHLMVQLDRMIDVISYN 120
Qy 121 DKTGIAHVEPGARLGHLATVLNDKYGRAISHGTCPGVGISGHFAHGGFGFSSHMHGLAVD 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 DKTGIAHVEPGARLGHLATVLNDKYGRAISHGTCPGVGISGHFAHGGFGFSSHMHGLAVD 180
Qy 181 SVVGVTVVLADGRIVEASATENADLFWGIKGAGSNFGIVAVWKLATFPAPKVLTRFGVTL 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SVVGVTVVLADGRIVEASATENADLFWGIKGAGSNFGIVAVWKLATFPAPKVLTRFGVTL 240
Qy 241 NWKNKTSALKGIEAVEDYARWVAPREVNFRIGDYGAGNPGIEGLYYGTPEQWRAAFQPLL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 NWKNKTSALKGIEAVEDYARWVAPREVNFRIGDYGAGNPGIEGLYYGTPEQWRAAFQPLL 300
Qy 301 DTLPAGYVVNPTTSLNWIESVLSYSNFDHVDFITPQPVENFYAKSLTLKSIKGDAVKNFV 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 DTLPAGYVVNPTTSLNWIESVLSYSNFDHVDFITPQPVENFYAKSLTLKSIKGDAVKNFV 360
Qy 361 DYYFDVSNKVKDRFWFYQLDVHGGKNSQVTKVTNAETAYPHRDKLWLIQFYDRYDNNQTY 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 DYYFDVSNKVKDRFWFYQLDVHGGKNSQVTKVTNAETAYPHRDKLWLIQFYDRYDNNQTY 420
Qy 421 PETSFKFLDGWVNSVTKALPKSDWGMYINYADPRMDRDYATKVYYGENLARLQKLKAKFD 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 PETSFKFLDGWVNSVTKALPKSDWGMYINYADPRMDRDYATKVYYGENLARLQKLKAKFD 480
Qy 481 PTDRFYYPQAVRPVK 495
|||||||||||||||
Db 481 PTDRFYYPQAVRPVK 495
Prosecution Insights