DETAILED CORRESPONDENCE
Status of the Application
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-11 and 13 are pending in the application.
Applicant’s preliminary amendment to the claims, filed July 26, 2024, is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Priority
This application is filed under 35 U.S.C. 371 as a national stage of international application PCT/FR2023/050148, filed February 3, 2023, which claims foreign priority under 35 U.S.C. 119(a-d) to French application 22/00999, filed February 4, 2022. A certified copy of the foreign priority document has been filed in this application on July 26, 2024.
Should applicant desire to obtain the benefit of the filing date of the foreign priority application, a certified English translation of the application must be submitted. Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on July 26, 2024 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the IDS has been considered by the examiner.
Specification/Informalities
The specification is objected to because there is no heading “Brief Description of the Drawings” immediately preceding the description of the drawing figures beginning at p. 4, line 26. See MPEP 608.01(a).
Claim Objections
Claims 1-9 and 13 are objected to because of the following informalities:
Claim 1 is objected to in the recitation of “A thermostable protein expressing at least one xylanase activity, characterized in that it comprises or consists of a peptide sequence represented by SEQ ID NO: 1 comprising at least the mutations L34C and R38C” and in the interest of improving claim form, it is suggested that the claim be amended to recite (with markings to show changes made) “A thermostable protein havingand comprising or consisting of the amino acid sequence of SEQ ID NO: 1 modified to comprise, wherein amino acid numbering is relative to the amino acid sequence of SEQ ID NO: 1.”
Claim 2 is objected to in the recitation of “The protein according to claim 1, characterized in that the peptide sequence SEQ ID NO: 1 includes at least one mutation selected from T112E, Q145E and S187R” and in the interest of improving claim form, it is suggested that the claim be amended to recite (with markings to show changes made) “The protein according to claim 1, wherein the protein further comprisesthe group consisting of T112E, Q145E and S187R, wherein amino acid numbering is relative to the amino acid sequence of SEQ ID NO: 1.”
Claim 3 is objected to in the recitation of “The protein according to claim 2, characterized in that the peptide sequence SEQ ID NO: 1 includes at least the T112E mutation” and in the interest of improving claim form, it is suggested that the claim be amended to recite (with markings to show changes made) “The protein according to claim 2, wherein the protein further comprisesmutation T112E
Claim 4 is objected to in the recitation of “The protein according to claim 3, characterized in that the peptide sequence SEQ ID NO: 1 includes at least the S187R mutation” and in the interest of improving claim form, it is suggested that the claim be amended to recite (with markings to show changes made) “The protein according to claim , wherein the protein further comprisesmutation S187R
Claim 5 is objected to in the recitation of “encoding a protein” and in the interest of improving claim form, it is suggested that the claim be amended to recite (with markings to show changes made) “encoding the[[a]] protein.”
Claim 6 is objected to in the recitation of “The polynucleotide according to claim 5, characterized in that it comprises or consists of the nucleotide sequence SEQ ID NO: 2 presenting at each of positions 100-102 and 112-114, the codon TGT or TGC encoding for cysteine” and in the interest of improving claim form, it is suggested that the claim be amended to recite (with markings to show changes made) “The polynucleotide according to claim 5, wherein the polynucleotidemodified to comprise at each of positions 100-102 and 112-114, wherein nucleotide numbering is relative to the nucleotide sequence of SEQ ID NO: 2.”
Claim 7 is objected to in the recitation of “The polynucleotide according to claim 6, characterized in that the nucleotide sequence SEQ ID NO: 2 also has at least one of the following mutations, or any two of the mutations, or even the three following mutations: at the position 334-336, the GAA or GAG codon encoding for glutamic acid, at the position 433-435, the GAA or GAG codon encoding for glutamic acid, at the position 559-561, the codon CGT, CGC, CGA, CGG, AGA, AGG encoding for arginine” and in the interest of improving claim form, it is suggested that the claim be amended to recite (with markings to show changes made) “The polynucleotide according to claim 6, wherein the polynucleotide further comprises one, two, or three
codon GAA or GAG at the positions 334-336,
codon GAA or GAG at the positions 433-435,
or AGG encoding for arginine at the positions 559-561,
wherein nucleotide numbering is relative to the nucleotide sequence of SEQ ID NO: 2.”
Claims 8, 9, and 13 are objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim cannot depend from any other multiple dependent claim. See MPEP § 608.01(n).
Claim 8 is objected to in the recitation of “comprising a polynucleotide” and in the interest of improving claim form, it is suggested that the claim be amended to recite (with markings to show changes made) “comprising the[[a]] polynucleotide.”
Claim 9 is objected to in the recitation of “comprising an expression vector” and in the interest of improving claim form, it is suggested that the claim be amended to recite (with markings to show changes made) “comprising the[[an]] expression vector.”
Claim 13 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n).
Claim 13 is objected to in the recitation of “producing a protein” and “culturing a host cell” and in the interest of improving claim form, it is suggested that the claim be amended to recite (with markings to show changes made) “producing the[[a]] protein” and “culturing the[[a]] host cell, respectively.”
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.
Claims 1-11 and 13 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claim 1 (claims 2-11 and 13 dependent therefrom) is indefinite in the recitation of “expressing at least one xylanase activity.” Other than xylanase enzymatic activity, it is unclear from the claims and the specification as to any other xylanase activity or activities that is/are intended as being encompassed by the recitation of “expressing at least one xylanase activity.” Applicant may consider amending the phrase “expressing at least one xylanase activity” to recite “having xylanase activity.”
Claim 8 (claims 9 and 13 dependent therefrom) is indefinite in the recitation of “such as a plasmid.” The phrase "such as" 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). Applicant may consider deleting the phrase “such as a plasmid.”
Claim Interpretation
Claim 1 (claims 10, 11, and 13 dependent therefrom) is drawn to a thermostable protein expressing at least one xylanase activity, characterized in that it comprises or consists of a peptide sequence represented by SEQ ID NO: 1 comprising at least the mutations L34C and R38C. Outside of the L34C and R38C mutations, claim 1 does not limit the remaining amino acid sequence of the claimed thermostable protein. Given a broadest reasonable interpretation, other than the mutations L34C and R38C, the remaining amino acid sequence of the claimed thermostable protein encompasses any amino acid modifications (i.e., amino acid substitutions, deletions, insertions, and additions) to any amino acids relative to SEQ ID NO: 1 other than the L4C and R38C mutations.
Claims 2-4 (claims 10, 11, and 13 dependent therefrom) are drawn to the protein according to claim 1, characterized in that the peptide sequence SEQ ID NO: 1 includes at least one mutation selected from T112E, Q145E and S187R. Other than the mutations L34C and R38C and at least one additional mutation selected from T112E, Q145E and S187R, the remaining amino acid sequence of the claimed thermostable protein encompasses any amino acid modifications (i.e., amino acid substitutions, deletions, insertions, and additions) to any amino acids other than the L4C, R38C, and the at least one additional mutation selected from T112E, Q145E and S187R.
Claim 5 is drawn to a polynucleotide encoding a protein according to any one of claims 1 to 4.
Claim 6 is drawn to the polynucleotide according to claim 5, characterized in that it comprises or consists of the nucleotide sequence SEQ ID NO: 2 presenting at each of positions 100-102 and 112-114, the codon TGT or TGC encoding for cysteine. Other than the codon TGT or TGC at positions 100-102 and 112-114, the remaining nucleotide sequence of the claimed polynucleotide encompasses any nucleotide modifications (i.e., nucleotide substitutions, deletions, insertions, and additions) to any nucleotides other than the recited codons.
Claim 7 (claims 8, 9, and 13 dependent therefrom) is drawn to the polynucleotide according to claim 6, characterized in that the nucleotide sequence SEQ ID NO: 2 also has at least one of the following mutations, or any two of the mutations, or even the three following mutations: at the position 334-336, the GAA or GAG codon encoding for glutamic acid, at the position 433-435, the GAA or GAG codon encoding for glutamic acid, at the position 559-561, the codon CGT, CGC, CGA, CGG, AGA, AGG encoding for arginine. Other than the codon TGT or TGC at positions 100-102 and 112-114 and at least one of the codons GAA or GAG encoding for glutamic acid at the position 334-336, GAA or GAG encoding for glutamic acid at the position 433-435, and the codon CGT, CGC, CGA, CGG, AGA, or AGG encoding for arginine at the position 559-561, the remaining nucleotide sequence of the claimed polynucleotide encompasses any nucleotide modifications (i.e., nucleotide substitutions, deletions, insertions, and additions) to any nucleotides other than the recited codons.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1-11 and 13 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor at the time the application was filed, had possession of the claimed invention.
MPEP 2163.II.A.2.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
MPEP § 2163 further states that “[s]atisfactory disclosure of a ‘representative number’ depends on whether one of skill in the art would recognize that the applicant was in possession of the necessary common attributes or features possessed by the members of the genus in view of the species disclosed. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus…Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are ‘representative of the full variety or scope of the genus,’ or by the establishment of ‘a reasonable structure-function correlation.’ Such correlations may be established ‘by the inventor as described in the specification,’ or they may be ‘known in the art at the time of the filing date.’"
The factors considered in the Written Description requirement are (1) level of skill and knowledge in the art, (2) partial structure, (3) physical and/or chemical properties, (4) functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the (5) method of making the claimed invention. Disclosure of any combination of such identifying characteristics that distinguish the claimed invention from other materials and would lead one of skill in the art to the conclusion that the applicant was in possession of the claimed species is sufficient." MPEP § 2163.
The claims have been interpreted as described in detail above. Given the substantial structural variation among the members of the respective genus of claimed proteins and encoding polynucleotides, the genus of claimed proteins and the genus of encoding polynucleotides are considered to encompass species with widely variant sequences.
The specification discloses the actual reduction to practice of the following representative species of the genus of thermostable proteins – a thermostable protein having xylanase activity and comprising the amino acid sequence of SEQ ID NO: 1 with the exception of L34C and R38C mutations relative to SEQ ID NO: 1, and optionally the mutation T112E, Q145E, S187R, or combinations thereof. Other than these disclosed representative species, the specification fails to disclose other thermostable proteins as encompassed by the claims.
Regarding the level of skill and knowledge in the art of amino acid modification, MPEP 2144.08.II.A.4.(c) states, "[i]n the area of biotechnology, an exemplified species may differ from a claimed species by a conservative substitution ("the replacement in a protein of one amino acid by another, chemically similar, amino acid... [which] is generally expected to lead to either no change or only a small change in the properties of the protein." Dictionary of Biochemistry and Molecular Biology 97 (John Wiley & Sons, 2d ed. 1989)). The effect of a conservative substitution on protein function depends on the nature of the substitution and its location in the chain. Although at some locations a conservative substitution may be benign, in some proteins only one amino acid is allowed at a given position. For example, the gain or loss of even one methyl group can destabilize the structure if close packing is required in the interior of domains. James Darnell et al., Molecular Cell Biology 51 (2d ed. 1990)."
The reference of Singh et al. (Curr. Protein Pept. Sci. 18:1-11, 2017; cited on the attached Form PTO-892) reviews various protein engineering methods and discloses that despite the availability of an ever-growing database of protein structures and highly sophisticated computational algorithms, protein engineering is still limited by the incomplete understanding of protein functions, folding, flexibility, and conformational changes (see p. 7, column 1, top).
The unpredictability associated with amino acid substitution is exemplified by the reference of Zhang et al. (Structure 26:1474-1485, 2018; cited on the attached Form PTO-892), which discloses that even a substitution of a surface residue that was predicted to be benign caused significant structural changes and unexpected effects on the function of a polypeptide (p. 1475, column 1).
More specific to the claimed invention, the specification discloses that “[m]utations are made to generate covalent and non-covalent interactions likely to give the enzyme greater thermostability. If a relationship does indeed exist between molecular structure and thermostability, improving the thermostability of an enzyme remains a difficulty despite the tools of protein engineering” (p. 2, lines 24-28).
Given that the claimed genus of claimed thermostable proteins encompasses species having widely variant structures while the specification discloses only a relative few representative species of thermostable proteins, and given that there was a very high level of unpredictability in the art of amino acid modification at the time of the invention, the specification is considered to be insufficient to describe the claimed genus of thermostable proteins. In this case, the specification at best describes a research plan for making, testing, and identifying those species that are encompassed by the claimed genus of thermostable proteins, however, a plan for making the claimed invention is not sufficient to show possession at the time of filing. One of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the genus, and thus, that the applicant was not in possession of the recited genus.
For these reasons, it is the examiner’s position that the specification fails to adequately describe the claimed invention.
Claims 1-11 and 13 are rejected under 35 U.S.C. 112(a) because the specification, while being enabling for a thermostable protein having xylanase activity and comprising the amino acid sequence of SEQ ID NO: 1 with the exception of L34C and R38C mutations relative to SEQ ID NO: 1, and optionally the mutation T112E, Q145E, S187R, or combinations thereof, does not reasonably provide enablement for all thermostable proteins as broadly encompassed by the claims. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
“The test of enablement is not whether any experimentation is necessary, but whether, if experimentation is necessary, it is undue.” In re Angstadt, 537 F.2d 498, 504, 190 USPQ 214, 219 (CCPA 1976). Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands (858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)) as follows: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. See MPEP § 2164.01(a). The Factors considered to be most relevant to the instant rejection are addressed in detail below.
The nature of the invention: According to the instant specification, “[w]e are therefore looking for enzymes with xylanase activity, thermostable, capable of withstanding the heat treatments applied to it and which have a xylanase activity greater than that of the natural enzyme exposed to the same conditions. The development of a thermostable xylanase that can be used on an industrial scale as well as in the field of animal nutrition nevertheless faces another obstacle, that of profitable production… The invention provides a mutated xylanase which is a good compromise between thermostability and industrial production” (p. 2, lines 15-30).
The breadth of the claims: The claims have been interpreted as described in detail above.
The state of the prior art; The level of one of ordinary skill; and The level of predictability in the art: According to MPEP 2164.03, “…what is known in the art provides evidence as to the question of predictability.” “[I]f one skilled in the art cannot readily anticipate the effect of a change within the subject matter to which that claimed invention pertains, then there is lack of predictability in the art.” See MPEP § 2164.03.
Regarding the level of skill and knowledge in the art of amino acid modification, MPEP 2144.08.II.A.4.(c) states, "[i]n the area of biotechnology, an exemplified species may differ from a claimed species by a conservative substitution ("the replacement in a protein of one amino acid by another, chemically similar, amino acid... [which] is generally expected to lead to either no change or only a small change in the properties of the protein." Dictionary of Biochemistry and Molecular Biology 97 (John Wiley & Sons, 2d ed. 1989)). The effect of a conservative substitution on protein function depends on the nature of the substitution and its location in the chain. Although at some locations a conservative substitution may be benign, in some proteins only one amino acid is allowed at a given position. For example, the gain or loss of even one methyl group can destabilize the structure if close packing is required in the interior of domains. James Darnell et al., Molecular Cell Biology 51 (2d ed. 1990)."
The reference of Singh et al. (Curr. Protein Pept. Sci. 18:1-11, 2017; cited on the attached Form PTO-892) reviews various protein engineering methods and discloses that despite the availability of an ever-growing database of protein structures and highly sophisticated computational algorithms, protein engineering is still limited by the incomplete understanding of protein functions, folding, flexibility, and conformational changes (see p. 7, column 1, top).
The unpredictability associated with amino acid substitution is exemplified by the reference of Zhang et al. (Structure 26:1474-1485, 2018; cited on the attached Form PTO-892), which discloses that even a substitution of a surface residue that was predicted to be benign caused significant structural changes and unexpected effects on the function of a polypeptide (p. 1475, column 1).
More specific to the claimed invention, the specification discloses that “[m]utations are made to generate covalent and non-covalent interactions likely to give the enzyme greater thermostability. If a relationship does indeed exist between molecular structure and thermostability, improving the thermostability of an enzyme remains a difficulty despite the tools of protein engineering” (p. 2, lines 24-28).
Based on the evidence of record, one of skill in the art would recognize a high level of unpredictability in the art of amino acid modification.
The amount of direction provided by the inventor and The existence of working examples: The specification discloses the following working examples of the claimed thermostable protein – a thermostable protein having xylanase activity and comprising the amino acid sequence of SEQ ID NO: 1 with the exception of L34C and R38C mutations relative to SEQ ID NO: 1, and optionally the mutation T112E, Q145E, S187R, or combinations thereof. Other than these working examples, the specification fails to disclose other thermostable proteins as encompassed by the claims.
The quantity of experimentation needed to make or use the invention based on the content of the disclosure: In the Federal Circuit decision of Idenix Pharmaceuticals LLC v. Gilead Sciences Inc., 941 F.3d 1149, 1156 (Fed. Cir. 2019), the court stated that “the key enablement question is whether a person of ordinary skill in the art would know, without undue experimentation, which [species] would be effective….because of the many thousands of [species] which need to be screened for…efficacy, the quantity of experimentation needed is large and weighs in favor of non-enablement.” While methods for modifying the amino acid sequence of a polypeptide were known before the effective filing date, it was not routine in the art to screen by a trial and error process for a vast number of thermostable proteins as broadly encompassed by the claims.
In view of the broad scope of the claimed genus, the lack of guidance and working examples provided in the specification, and the high degree of unpredictability as evidenced by the prior art, undue experimentation would be necessary for a skilled artisan to make and use the entire scope of the claimed invention. Applicants have not provided sufficient guidance to enable one of ordinary skill in the art to make and use the claimed invention in a manner reasonably correlated with the scope of the claims. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of having the desired biological characteristics is unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988).
Allowable Subject Matter
The closest prior art is considered to be CN 105505806 A (cited on the IDS filed July 26, 2024) disclosing the hybrid xylanase polypeptide of SEQ ID NO: 2, which has a R38C mutation relative to instant SEQ ID NO: 1 (see attached Appendix for sequence alignment). However, the prior art of record does not teach or suggest a xylanase with a combination of L34C and R38C mutations relative to the amino acid sequence of SEQ ID NO: 1.
Conclusion
Status of the claims:
Claims 1-11 and 13 are pending.
Claims 1-11 and 13 are rejected.
No claim is in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DAVID J STEADMAN whose telephone number is (571)272-0942. The examiner can normally be reached Monday to Friday, 7:30 AM to 4:00 PM.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, MANJUNATH N RAO can be reached on 571-272-0939. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/David Steadman/Primary Examiner, Art Unit 1656
APPENDIX
BDD46211
ID BDD46211 standard; protein; 193 AA.
XX
AC BDD46211;
XX
DT 06-OCT-2016 (first entry)
XX
DE Hybrid xylanase (xynZL) polypeptide, SEQ ID 2.
XX
KW EC 3.2.1.8; Endo 1 4 beta xylanase; bleaching; brewing;
KW enzyme engineering; feed-additive; food;
KW genetically engineered microorganism; mutein; pulp; textile;
KW thermostable.
XX
OS Aspergillus niger.
OS Chimeric.
OS Synthetic.
OS Unidentified.
XX
FH Key Location/Qualifiers
FT Misc-difference 9
FT /note= "Wild-type Pro substituted by Tyr"
FT Misc-difference 14
FT /note= "Wild-type His substituted by Phe"
FT Misc-difference 17
FT /note= "Wild-type Phe substituted by Ser"
FT Disulfide-bond 38..191
FT Misc-difference 42
FT /note= "Wild-type Glu substituted by Asn"
FT Misc-difference 109
FT /note= "Wild-type Gly substituted by Ala"
FT Misc-difference 111
FT /note= "Wild-type Val substituted by Ala"
FT Misc-difference 160
FT /note= "Wild-type Asn substituted by Ile"
FT Misc-difference 164
FT /note= "Wild-type Lys substituted by Met"
FT Misc-difference 166
FT /note= "Wild-type Gly substituted by Ala"
XX
CC PN CN105505806-A.
XX
CC PD 20-APR-2016.
XX
CC PF 16-JAN-2016; 2016CN-10046663.
XX
PR 16-JAN-2016; 2016CN-10046663.
XX
CC PA (UYXI-) UNIV XINXIANG MEDICAL.
XX
CC PI Liu Z, Wang Y, Zhou C, Zhu X, Li T;
XX
DR WPI; 2016-27446B/51.
DR N-PSDB; BDD46210.
XX
CC PT Constructing xylanase hybrid enzyme engineering strain, comprises taking
CC PT xynZL gene as template, PCR amplifying using YS and YX primers,
CC PT recovering the PCR products, double digesting expression vector,
CC PT recycling, ligating, and transforming.
XX
CC PS Disclosure; SEQ ID NO 2; 11pp; Chinese.
XX
CC The present invention relates to a novel method for constructing a
CC genetically engineered bacterial strain. The method comprises: (a) taking
CC a xynZL gene as a template; (b) performing PCR amplification using PCR
CC primers; (c) recovering the PCR products; (d) double digesting the
CC corresponding expression vector; (e) recycling and ligating the product;
CC (f) transforming it into Escherichia coli DH5 alpha competent cells; (g)
CC applying the transformed solution on lysogeny broth plates containing
CC ampicillin resistance; and (h) screening the positive clones. The method
CC of the present invention is useful for preparing a hybrid xylanase enzyme
CC engineered strain, where the hybrid xylanase has improved heat stability
CC and can be used in food processing, brewing, pulp, bleaching,
CC pharmaceuticals, feed additives, pharmaceuticals and textiles. The
CC present sequence represents a hybrid xylanase (comprising an Aspergillus
CC niger XynZF-2 N-terminal fragment replaced with a N-terminal fragment (34
CC amino acids) of a xylanase EvXyn11 and comprising the following mutations
CC P9Y/H14F/F17S/E42N/K164M/G166A/N160I/V111A/G109A and a disulfide bond
CC Cys38-Cys191) which can be used in food processing, brewing, pulp,
CC bleaching, pharmaceuticals, feed additives, pharmaceuticals and textiles.
XX
SQ Sequence 193 AA;
Query Match 91.3%; Score 968; Length 193;
Best Local Similarity 93.2%;
Matches 179; Conservative 1; Mismatches 12; Indels 0; Gaps 0;
Qy 2 AQTCLTSPQTGFHNGFFYSFWKDSPGTVNFCLLEGGRYTVEWSNVGNFVGGKGWNPGSAQ 61
||||||| |||| || |||||||||||||||||: | ||| |||||||||||||||||||
Db 2 AQTCLTSYQTGFFNGSFYSFWKDSPGTVNFCLLDAGCYTVNWSNVGNFVGGKGWNPGSAQ 61
Qy 62 DITYSGTFTPSGNGYLSVYGWTTDPLIEYYIVESYGDYNPGSGGTYKGTVTSDGSVYDIY 121
||||||||||||||||||||||||||||||||||||||||||||||| | ||||||||||
Db 62 DITYSGTFTPSGNGYLSVYGWTTDPLIEYYIVESYGDYNPGSGGTYKATATSDGSVYDIY 121
Qy 122 TATRTNAASIQGTATFTQYWSVRQNKRVGGTVTTSNHFNAWAKLGMNLGTHNYQIVATEG 181
|||||||||||||||||||||||||||||||||||||| ||| | |||||||||||||||
Db 122 TATRTNAASIQGTATFTQYWSVRQNKRVGGTVTTSNHFIAWAMLAMNLGTHNYQIVATEG 181
Qy 182 YQSSGSSSITVQ 193
||||||||| ||
Db 182 YQSSGSSSICVQ 193
Prosecution Insights