WO2013038062A1 - Enzyme variants with improved properties - Google Patents
Enzyme variants with improved properties Download PDFInfo
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- WO2013038062A1 WO2013038062A1 PCT/FI2012/050884 FI2012050884W WO2013038062A1 WO 2013038062 A1 WO2013038062 A1 WO 2013038062A1 FI 2012050884 W FI2012050884 W FI 2012050884W WO 2013038062 A1 WO2013038062 A1 WO 2013038062A1
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- Prior art keywords
- laccase
- seq
- amino acid
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- variant
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
- D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
- D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
- D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2305/00—Use of specific compounds during water treatment
- C02F2305/02—Specific form of oxidant
- C02F2305/023—Reactive oxygen species, singlet oxygen, OH radical
Definitions
- the present invention relates to laccase variants and uses thereof as eco-friendly biocatalysts in various industrial processes. BACKGROUND OF THE INVENTION
- Laccases (EC 1 .10.3.2) are enzymes having a wide taxonomic distribution and belonging to the group of multicopper oxidases. Laccases are eco-friendly catalysts, which use molecular oxygen from air to oxidize various phenolic and non-phenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and produce water as the only side-product. These natural "green” catalysts are used for diverse industrial applications including the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. Another large proposed application area of laccases is biomass pretreatment in biofuel and pulp and paper industry.
- Laccases have a wide substrate specificity and they can oxidize many different substrate compounds. Owing to chemical properties of the substrates, they become more readily oxidized in different pH conditions, either alkaline or acidic. On the other hand, the advantageous pH range of action of different laccases may vary, which means that they have a preference to substrates within that range. For instance, relatives of CotA laccase are known to work best in acidic conditions.
- the present invention relates to laccase variants, which comprise a glutamine residue situated within 6 Angstrom (A) distance to the type 1 Copper ion in the 3-dimentional structure of the laccase variant.
- the laccase variant may comprise an amino acid sequence showing at least 50% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, comprising at least one amino acid variant selected from the group consisting of glutamine (Q) in a position which corresponds to the position 386 of the amino acid sequence depicted in SEQ ID NO:3 and a Proline- Tryptophan-Phenylalanine (PWF) sequence in a position which corresponds to the position 487-489 of the amino acid sequence depicted in SEQ ID NO:3.
- glutamine Q
- PWF Proline- Tryptophan-Phenylalanine
- the present laccase laccase variants have an increased enzymatic activity in alkaline conditions as compared to that of a corresponding control enzyme lacking said amino acid variants.
- the present invention also relates to nucleic acid molecules encoding the present laccase variants, vectors comprising said nucleic acid molecules, and recombinant host cells comprising said vector.
- the invention relates to a method of producing the present laccase variants.
- the method comprises the steps of i) culturing a recombinant host cell according to the present invention under conditions suitable for the production of the laccase variant, and ii) recovering the laccase variant obtained.
- the invention relates to various uses of the present laccase variants, especially in pulp delignification, textile dye bleaching, wastewater detoxifixation, and xenobiotic detoxification.
- Figure 1 is a schematic representation of T1 (Cu1 ) and T2/T3 (Cu4/Cu2-Cu3) copper sites of laccase CotA from Bacillus subtilis with indicated distances between the most important atoms (adopted from Enguita et al., "Crystal Structure of a Bacterial Endospore Coat Component", J. Biol. Chem., 278, 19416-19425, 2003). The area of MUT1 mutation is indicated by the dashed oval.
- Figure 2 shows the three-dimentional structure of Cu1 site ligand environment in radius of 6A elucidated from crystal structures of four evolutionary distant laccase (Bacillus Subtilis COTA protein, Streptomyces Coelicolor laccase, E.coli CuEO laccase, and Trametes Trogii laccase). Respective accession numbers in Structure Data Base 1 UVW, 3KW8, 2FQD and 1 KYA. Numeration of residues in B. subtilis laccase crystal structure is 9 residues less than that of the full size protein (a small N-terminal fragment was missing from the crystallized protein). A residue corresponding to the Glutamine 368 is depicted in black.
- Figure 3 shows an alignment of the two conserved regions containing T1 copper ligands derived from evolutionary distant laccases (Bacillus Subtilis COTA protein, Streptomyces Coelicolor laccase, E.coli CuEO laccase, and Trametes Trogii laccase). Corresponding crystal structures of the Cu1 surrounding are presented in Fig. 2. Empty arrows indicate the positions of Cu-1 ligands, black arrow indicates axial ligand. Panel C shows just a list of the sequences surrounding Q386 substitution (M1 ) identified from the 3-D structures. M1 position is framed.
- M1 Q386 substitution
- Figure 4 shows a multiple alignment of amino acid sequences that are related to COT1 (SEQ ID NO:1 ) and COT2 (SEQ ID NO:2) and were identified in a Blast search.
- Figure 5 shows a schematic representation of introducing MUT1 into
- Laccase type2 gene from Bacillus pseudomycoides are Laccase type2 gene from Bacillus pseudomycoides.
- Primers 1 and 2 represent terminal regions of the recombinant gene.
- Primers 3 and 4 represent fragments of the top and bottom strands of the mutated gene surrounding mutation site (X on the primers depicts the mutation).
- PCR reactions (1 ) and (2) produce two overlapping fragments of the gene (Fragment 1 and Fragment.2), both bearing the mutation.
- the third PCR reassembles the full length gene with mutation (black bar) at the desired position.
- Figure 6 illustrates measurements of relative activity of the present laccases at different pH.
- Panel A demonstrates the selection of the initial rate time range for the reactions. As this time depends on the amount of the enzyme in the reaction, a suitable dilution of the enzyme needs to be obtained for convenient measurement. In the present examples, 10 min time was within the linear range in all pH conditions.
- Panel B illustrates the photometric measurement of ABTS absorbance; maximal initial rates of the present laccases (with and without mutation - WT and Mutant, respectively).
- the present invention is based on a surprising finding that certain amino acid substitutions result in increased laccase activity especially in alkaline conditions.
- amino acid substitution is used herein the same way as it is commonly used, i.e. the term refers to a replacement of one or more amino acids in a protein with another. Artificial amino acid substitutions may also be referred to as mutations.
- alkaline is a synonym for the term “basic”.
- alkaline conditions refers to conditions having a pH value greater than 7.
- laccase activity is used herein to mean maximal initial rate of the oxidation reaction. Laccase activity may be determined by standard oxidation assays known in the art including, but not limited to measurement of oxidation of Syringaldazine by laccase according to Sigma online protocol, or according to Cantarella et al. ("Determination of laccase activity in mixed solvents: Comparison between two chromogens in a spectrophotometric assay", Biotechnology and Bioengineering V. 82 (4), pp 395-398, 2003). An example of determining relative laccase activity at different pH is presented in Example 2. Any substrate suitable for the enzyme in question may be used in the activity measurements. A non-limiting example of a substrate suitable for use in assessing the enzymatic activity of the present laccase variants is 2,6- Dimethoxyphenol (2,6-DMP).
- the term "increased (or improved) laccase activity” refers to a laccase activity higher than that of a corresponding non-mutated laccase enzyme under the same conditions. That is to say, for instance if enzymes A and B have equal activity at pH5, whereas at pH 9 the same preparation of enzyme A has a higher activity than that of enzyme B, then enzyme A is denoted as a laccase variant having "an increased laccase activity in alkaline conditions". Certain amino acid variants in certain positions of laccase protein disclosed herein result in increased laccase activity at alkaline pH at least by 50% as compared to the corresponding laccase enzymes omitting this amino acid variant. In some embodiments, an increased laccase activity in alkaline conditions means about 2-fold, and preferably 5- fold, higher laccase activity as compared to that of a corresponding non- mutated variant.
- Laccase molecules are usually monomers consisting of three consecutively connected cupredoxin-like domains twisted in a tight globule.
- the active site of laccases contains four copper ions: a mononuclear "blue" copper ion (T1 site) and a three-nuclear copper cluster (T2/T3 site) consisting of one T2 copper ion and two T3 copper ions (Fig. 1 ).
- Laccases isolated from different sources are very diverse in primary sequences; however, they have some conserved regions in the sequences and certain common features in their three-dimensional structures.
- a comparison of sequences of more than 100 laccases has revealed four short conservative regions (no longer than 10 aa each) which are specific for all laccases (Kumar et al., "Combined sequence and structure analysis of the fungal laccase family", Biotechnol. Bioeng., 83, 386-394, 2003; Morozova et al., “Blue laccases", Biochemistry (Moscow), 72, 1 136-1 150, 2007).
- One cysteine and ten histidine residues form a ligand environment of copper ions of the laccase active site present in these four conservative amino acid sequences.
- the T1 site of the enzyme is the primary acceptor of electrons from reducing substrates.
- the potential of the enzyme T1 site also determines the efficiency of catalysis on oxidation of the majority of laccase substrates, and therefore T1 site is primary target for laccase protein engineering.
- the T1 site has as ligands two histidine imidazoles and the sulfhydryl group of cysteine, which form a trigonal structure (Fig. 1 ).
- the fourth residue in the immediate proximity of the copper 1 is so called axial ligand - methionine or phenylalanine (Met502 in Fig. 1 ).
- These ligands in the primary sequence are situated in the two conserved regions (third and fourth) at the distal end of the protein.
- Fig. 2 shows surrounding of copper 1 atom in 6 A radiuses of four evolutionary very distant laccases (sequence identity not more than 20%, length of the protein chain varies from 273 to 503 aa). All residues comprising copper 1 environment in these laccases are adjacent or proximal in the primary sequence to the copper ligands (two histidines and the cystein) and belong to the conserved regions, with one exception.
- a residue (marked dark in the Fig. 2, usually hydrophobic, in the depicted cases leucine or phenylalanine) is protruding into the environment of copper 1 atom from a distant part of the primary sequence.
- Fig. 3 shows fragments of aligned primary sequences of the laccases from Fig. 2. All residues depicted in the crystal structures in fair grey are situated in the regions depicted in panels A and B (conserved regions). Whereas the residue depicted in crystal structures black (Fig. 2, M1 position) is situated in the regions depicted on panel C. Panel C was not generated by alignment protocols owing to lack of sufficient homology in these part of the sequences), but the panel is only a list of sequences surrounding the MUT1 position elucidated from 3-D structure (marked black in Fig. 2).
- this region may be sequentially conserved, and thus MUT1 position may be elucidated from a sequence alignment. Whether sequentially conserved or not, this residue can be unambiguously identified in practically any laccase by being present in an about 5-6 A radius of copper 1 in proximity to the axial ligand of Copper 1 atom. To our best knowledge there is no glutamine in the copper-1 5-6 A environment in any of the laccases with a known three-dimensional structure.
- Amino acid variants presented by these mutations appear to be unique at corresponding positions among related polypeptide sequences since they were not identified in a protein search in BLAST, a public internet service which compares the query sequence to all sequences deposited in the public domain. The search revealed some closely related sequences only a few amino acids different from the queries and a whole range of homologous sequences with different degree of similarity (Table 1 ).
- sequences are listed in the order of decreasing similarity.
- subtilis BSn5 bj
- Mutations corresponding to the Q386 mutation and/or P487/W488/F489 triple mutation shown in SEQ ID NO:3 may be introduced to any of the amino acid sequences disclosed herein, or other homologous sequences, by standard methods known in the art, such as site-directed mutagenesis, in order to improve their laccase activity in alkaline conditions.
- Kits for performing site-directed mutagenesis are commercially available in the art (e.g. QuikChange® II XL Site-Directed Mutagenesis kit by Agilent Technologies). Further suitable methods for introducing the above mutations into a recombinant gene are disclosed e.g.
- some embodiments of the present invention relate to laccase variants or mutants which comprise Glutamine (Q) in a position which corresponds to the position 386 of the amino acid sequence depicted in SEQ ID NO:3 (denoted as MUT1 ) and/or Proline-Tryptophan-Phenylalanine (PWF) triple mutation in a position which corresponds to the position 487-489 of the amino acid sequence depicted in SEQ ID NO:3 (MUT2), and have an increased laccase activity in alkaline conditions as compared to that of a corresponding non-mutated control variant (Table 2).
- Q Glutamine
- PWF Proline-Tryptophan-Phenylalanine
- Amino acid sequences revealed in the Blast search may be represented as a consensus sequence.
- SEQ ID NO:6 represent a consensus sequence of 33 amino acid sequences most closely related to the COT1 and COT2 query sequences.
- some embodiments of the present invention relate to laccase variants comprising an amino acid sequence depicted in SEQ ID NO:6 introduced with a MUT1 and/or MUT2 mutation.
- said amino acid sequence is selected from a group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:6, and any of the sequences shown Figure 4, further comprising a mutation corresponding to MUT1 and/or MUT2.
- the present invention relates to laccase variants which comprise an amino acid sequence having a degree of identity to any of the above-mentioned reference sequences of at least about 55%, preferably about 65%, more preferably about 75%, still more preferably about 85%, and even more preferably about 95%, 96%, 97%, 98%, or 99%, and which retain increased laccase activity in alkaline conditions.
- the degree of identity corresponds to any value between the above-mentioned integers.
- the comparison of sequences and determination of percent identity between two or more sequences can be accomplished using standard methods known in the art.
- the present laccase variants may comprise conservative amino acid substitutions as compared to any of the sequences depicted in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and Figure 4.
- conservative amino acid substitution refers to a replacement of an amino acid with a similar amino acid as known in the art. Conservative amino acid substitutions do not significantly affect the folding and/or activity of a protein sequence variant. Typical non-limiting examples of such conservative amino acid substitutions include substitution of glutamate for aspartate or vice versa.
- the present laccase variants may further comprise amino acid deletions and/or additions as long as they retain their increased laccase activity in alkaline conditions.
- the term "functional fragment” refers to a truncated laccase polypeptide retaining said increased enzyme activity in alkaline conditions.
- the term “conservative variant” refers to polypeptides comprising conservative amino acid substitutions, deletions and/or additions, and retaining their enzymatic properties, especially increased laccase activity in alkaline conditions.
- the present laccase polypeptides or proteins may be fused to additional sequences, by attaching or inserting, including , but not limited to, affinity tags, facilitating protein purification (S-tag, maltose binding domain, chtin binding domain), domains or sequences assisting folding (such as thioredoxin domain, SUMO protein), sequences affecting protein localization (periplasmic localization signals etc), proteins bearing additional function, such as green fluorescent protein (GFP), or sequences representing another enzymatic activity.
- affinity tags facilitating protein purification (S-tag, maltose binding domain, chtin binding domain
- domains or sequences assisting folding such as thioredoxin domain, SUMO protein
- sequences affecting protein localization periplasmic localization signals etc
- proteins bearing additional function such as green fluorescent protein (GFP)
- GFP green fluorescent protein
- Other suitable fusion partners for the present laccases are known to those skilled in the art.
- the present invention also relates to isolated polynucleotides encoding any of the laccase variants disclosed herein. Means and methods for cloning and isolating such polynucleotides are well known in the art.
- control sequences are readily available in the art and include, but are not limited to, promoter, leader, polyadenylation, and signal sequences.
- Laccase variants according to various embodiments of the present invention may be obtained by standard recombinant methods known in the art. Briefly, such a method may comprise the steps of i) culturing a desired recombinant host cell under conditions suitable for the production of a present laccase polypeptide variant, and ii) recovering the polypeptide variant obtained.
- a large number of vector-host systems known in the art may be used for recombinant production of laccase variants.
- Possible vectors include, but are not limited to, plasmids or modified viruses which are maintained in the host cell as autonomous DNA molecule or integrated in genomic DNA. The vector system must be compatible with the host cell used as well known in the art.
- suitable host cells include bacteria (e.g. E.coli, bacilli), yeast (e.g. Pichia Pastoris, Saccharomyces Cerevisae), fungi (e.g. filamentous fungi) insect cells (e.g. Sf9).
- Recovery of a laccase variant produced by a host cell may be performed by any technique known to those skilled in the art. Possible techniques include, but are not limited to secretion of the protein into the expression medium, and purification of the protein from cellular biomass.
- the production method may further comprise a step of purifying the laccase variant obtained.
- thermostable laccases non-limiting examples of such methods include heating of the disintegrated cells and removing coagulated thermo labile proteins from the solution.
- non-limiting examples of such methods include ion exchange chromatography, and ultra-filtration of the expression medium. It is important that the purification method of choice is such that the purified protein retains its laccase activity.
- the present laccase variants may be used in a wide range of different industrial processes and applications, such as in pulp delignification, textile dye bleaching, wastewater detoxifixation, xenobiotic detoxification, and detergent manufacturing.
- the increased operable pH range of the disclosed laccase variants makes them particularly suitable for industrial waste water treatment processes.
- Mutations according to the present invention were introduced into various recombinant genes by standard site-directed mutagenesis.
- MUT1 L386Q substitution
- ZP_04150084 the gene of Multicopper oxidase, type 2 from Bacillus pseudomycoides
- ZP_04150084 which has approximately 50% sequence identity to the COT1 (SEQ ID NO:1 ) and COT2 (SEQ ID NO.2) laccases
- PCR amplifying the coding sequence of this gene accession number NZ_ACMX01000022
- 2,6-Dimethoxyphenol (2,6-DMP), which can be oxidized by wild type COT1 and COT2 laccases readily at pH 5 but much more slowly at pH 9, was chosen as the substrate.
- Initial rates of the reactions were measured in OD(500)/min.
- Initial rate (V) is velocity of the reaction in the time range when the colour develops linearly with time. Similar reactions were carried out at different substrate (2,6- DMP) concentrations (see protocol below). Then maximum initial rate (Vmax) was determined at each pH (this rate was observed at saturating substrate concentrations).
- the relative laccase activity at different pHs may be measured by any other substrate suitable for the laccase variant in question as long as the other substrate cab be oxidized at the same pH range (preferably pH 5 to pH 9). Also other parameters such as temperature may be adjusted to the particular laccase variant in question.
Abstract
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Priority Applications (9)
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EP12831162.8A EP2756076B1 (en) | 2011-09-15 | 2012-09-13 | Enzyme variants with improved properties |
SI201231019T SI2756076T1 (en) | 2011-09-15 | 2012-09-13 | Enzyme variants with improved properties |
PL12831162T PL2756076T3 (en) | 2011-09-15 | 2012-09-13 | Enzyme variants with improved properties |
DK12831162.8T DK2756076T3 (en) | 2011-09-15 | 2012-09-13 | ENZYM VARIETIES WITH IMPROVED PROPERTIES |
ES12831162.8T ES2634651T3 (en) | 2011-09-15 | 2012-09-13 | Enzyme variants with improved properties |
BR112014006009A BR112014006009A8 (en) | 2011-09-15 | 2012-09-13 | ENZYME VARIANTS WITH IMPROVED PROPERTIES |
US14/344,028 US20150159144A1 (en) | 2011-09-15 | 2012-09-13 | Enzyme variants with improved properties |
CA2848329A CA2848329C (en) | 2011-09-15 | 2012-09-13 | Enzyme variants with improved properties |
US15/366,962 US20170081643A1 (en) | 2011-09-15 | 2016-12-01 | Laccase variants having increased activity in alkaline conditions |
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US14/344,028 A-371-Of-International US20150159144A1 (en) | 2011-09-15 | 2012-09-13 | Enzyme variants with improved properties |
US15/366,962 Continuation US20170081643A1 (en) | 2011-09-15 | 2016-12-01 | Laccase variants having increased activity in alkaline conditions |
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EP (1) | EP2756076B1 (en) |
BR (1) | BR112014006009A8 (en) |
CA (1) | CA2848329C (en) |
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ES (1) | ES2634651T3 (en) |
HU (1) | HUE033314T2 (en) |
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PT (1) | PT2756076T (en) |
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Cited By (11)
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WO2014146712A1 (en) * | 2013-03-20 | 2014-09-25 | Metgen Oy | Method for saving energy in paper production |
WO2014146713A1 (en) * | 2013-03-20 | 2014-09-25 | Metgen Oy | Method for improving the fermentable sugar yield from lignocellulosic substrates |
WO2015144679A1 (en) * | 2014-03-24 | 2015-10-01 | Metgen Oy | Laccase variants with improved properties |
WO2015155363A1 (en) * | 2014-04-11 | 2015-10-15 | Metgen Oy | Laccase variants with improved properties |
WO2015158803A1 (en) * | 2014-04-16 | 2015-10-22 | Metgen Oy | Laccase variants with improved properties |
WO2015185393A1 (en) * | 2014-06-05 | 2015-12-10 | Henkel Ag & Co. Kgaa | Detergent containing at least one laccase as a dye-transfer inhibitor |
WO2016090059A1 (en) | 2014-12-02 | 2016-06-09 | Novozymes A/S | Laccase variants and polynucleotides encoding same |
WO2017102542A1 (en) | 2015-12-15 | 2017-06-22 | Metgen Oy | Method for producing mechanical pulp from a biomass comprising lignocellulosic material |
WO2018019707A1 (en) * | 2016-07-25 | 2018-02-01 | Metgen Oy | Method for lignin depolymerisation |
WO2019145288A1 (en) | 2018-01-23 | 2019-08-01 | Metgen Oy | Alkaline laccase variants with improved properties |
WO2020193452A1 (en) | 2019-03-26 | 2020-10-01 | Metgen Oy | Endoglucanase variants with improved properties |
Families Citing this family (3)
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CA3130763A1 (en) | 2019-02-25 | 2020-09-03 | Ginkgo Bioworks, Inc. | Biosynthesis of cannabinoids and cannabinoid precursors |
CN110106153B (en) * | 2019-05-24 | 2020-12-29 | 江南大学 | Multi-copper oxidase mutant with improved salt tolerance |
CN114703212B (en) * | 2022-03-01 | 2024-03-29 | 东华大学 | Method for modifying laccase by using specific segment random mutation method and laccase strain LAC123 |
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WO1997009431A1 (en) * | 1995-09-01 | 1997-03-13 | Novo Nordisk Biotech, Inc. | Blue copper oxidase mutants with enhanced activity |
EP1826266A1 (en) * | 2006-02-23 | 2007-08-29 | Helmholtz-Zentrum für Infektionsforschung GmbH | Polypeptides with laccase activity |
CN102115722A (en) * | 2010-12-02 | 2011-07-06 | 东北林业大学 | Bacillus subtilis ls02 laccase and application thereof |
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US20030199068A1 (en) * | 2002-03-05 | 2003-10-23 | Bott Richard R. | High throughput mutagenesis screening method |
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Also Published As
Publication number | Publication date |
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EP2756076A4 (en) | 2015-04-22 |
CA2848329A1 (en) | 2013-03-21 |
BR112014006009A8 (en) | 2017-09-12 |
US20170081643A1 (en) | 2017-03-23 |
SI2756076T1 (en) | 2017-12-29 |
US20150159144A1 (en) | 2015-06-11 |
CA2848329C (en) | 2020-11-03 |
PT2756076T (en) | 2017-08-01 |
HUE033314T2 (en) | 2017-11-28 |
BR112014006009A2 (en) | 2017-06-13 |
EP2756076B1 (en) | 2017-04-26 |
PL2756076T3 (en) | 2017-10-31 |
ES2634651T3 (en) | 2017-09-28 |
EP2756076A1 (en) | 2014-07-23 |
DK2756076T3 (en) | 2017-08-21 |
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