Method for producing reducing sugar from lignocellulosic substrates

Abstract

The invention relates to processes for the conversion of biomass into carbohydrates, notably reducing sugars. It provides means and methods for increasing the yield of enzymatic digestion of a biomass, in particular in methods wherein cellulose is converted into sugars using a cellulose-converting enzyme. More in particular, the invention provides a method for producing a reducing sugar from a lignocellulosic material, wherein the lignocellulosic material is contacted with a pectate lyase at a pH between 9 and 12, wherein the pectate lyase comprises an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence at least 70% identical with SEQ ID NO: 1.

Claims

1. A method for producing a reducing sugar from a lignocellulosic material, the method comprising: pretreating the lignocellulosic material with a pectate lyase (EC 4.2.2.2) at a pH between 9 and 12, treating the lignocellulosic material with at least one cellulase or hemicellulase; and obtaining at least 80% of the theoretical yield of reducing sugars from the lignocellulosic material; wherein the pectate lyase comprises the full length of the amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence at least 89% identical with the full length of SEQ ID NO: 1.

2. The method according to claim 1, wherein the reducing sugar is a fermentable sugar.

3. The method according to claim 1, wherein the lignocellulosic material is selected from the group consisting of corn stover, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, yard waste, wood and forestry waste, sugar cane, switchgrass, wheat straw, hay, barley, barley straw, rice straw, grasses, waste paper, sludge from paper manufacture, byproducts from paper manufacture, corn grain, corn cobs, corn husks, grasses, wheat, rice straw, sugar cane bagasse, sorghum, soy, trees, branches, wood chips, and sawdust.

4. The method according to claim 1, wherein the lignocellulosic material is contacted with a laccase (EC 1.10.3.2), simultaneously or sequentially with the pectate lyase, wherein the laccase comprises an amino acid sequence at least 90% identical with the full length of SEP ID NO: 4.

5. The method according to claim 4, wherein the laccase comprises the full length of the amino acid sequence according to SEQ ID NO: 4 or an amino acid sequence at least 95% identical with the full length of SEQ ID NO: 4.

6. The method according to claim 1, wherein the lignocellulosic material is also pretreated with a steam explosion step or ammonia fiber explosion.

7. The method according to claim 1, wherein the pectate lyase was produced by heterologous expression in Escherichia coli.

8. The method according to claim 1, wherein the pectate lyase has an amino acid sequence that is at least 93% identical to the full length of the sequence according to SEQ ID NO:1.

9. The method according to claim 1, wherein the pectate lyase has an amino acid sequence that is at least 96% identical to the full length of the sequence according to SEQ ID NO:1.

10. The method according to claim 1, wherein the pectate lyase has an amino acid sequence that is at least 98% identical to the full length of the sequence according to SEQ ID NO:1.

11. The method according to claim 1, wherein the pectate lyase has an amino acid sequence that is at least 99% identical to the full length of the sequence according to SEQ ID NO:1.

12. The method according to claim 4, wherein the laccase comprises an amino acid sequence at least 95% identical with the full length of SEQ ID NO: 4.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Methods for the enzymatic production of reducing sugars from lignocellulosic material are well known in the art. Enzymatic digestion of lignocellulosic material is most commonly performed using cellulases and/or hemicellulases.

(2) Cellulase is any of several enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis, the decomposition of cellulose and of some related polysaccharides. The name is also used for any mixture or complex of various such enzymes, that act serially or synergistically to decompose cellulosic material.

(3) Cellulases break down the cellulose molecule into monosaccharides (simple sugars) such as beta-glucose, or shorter polysaccharides and oligosaccharides. Cellulose breakdown is of considerable economic importance, because it makes a major constituent of plants available for consumption and use in chemical reactions. The specific reaction involved is the hydrolysis of the 1,4-beta-D-glycosidic linkages in cellulose, hemicellulose, lichenin, and cereal beta-D-glucans. Because cellulose molecules bind strongly to each other, cellulolysis is relatively difficult compared to the breakdown of other polysaccharides such as starch.

(4) Several different kinds of cellulases are known, which differ structurally and mechanistically. Synonyms, derivatives, and specific enzymes associated with the name cellulase include endo-1,4-beta-D-glucanase (beta-1,4-glucanase, beta-1,4-endoglucan hydrolase, endoglucanase D, 1,4-(1,3,1,4)-beta-D-glucan 4-glucanohydrolase), carboxymethyl cellulase (CMCase), avicelase, celludextrinase, cellulase A, cellulosin AP, alkali cellulase, cellulase, A 3, 9.5 cellulase, and pancellase SS. Cellulases and mixtures of different cellulases are commercially available.

(5) Five general types of cellulases may be distinguished, based on the type of reaction catalyzed:

(6) 1) Endocellulases (EC 3.2.1.4) randomly cleave internal bonds at amorphous sites that create new chain ends.

(7) 2) Exocellulases or cellobiohydrolases (EC 3.2.1.91) cleave two to four units from the ends of the exposed chains produced by endocellulase, resulting in tetrasaccharides or disaccharides, such as cellobiose. Exocellulases are further classified into type I, that work processively from the reducing end of the cellulose chain, and type H, that work processively from the nonreducing end.

(8) 3) Cellobiases (EC 3.2.1.21) or beta-glucosidases hydrolyse the exocellulase product into individual monosaccharides.

(9) 4) Oxidative cellulases depolymerize cellulose by radical reactions, as for instance cellobiose dehydrogenase (acceptor).

(10) 5) Cellulose phosphorylases depolymerize cellulose using phosphates instead of water.

(11) Apart from cellulases, hemicellulases may also be advantageously employed in the enzymatic production of reducing sugars from lignocellulosic material.

(12) Hemicellulose is a collective term for a group of enzymes that break down hemicellulose. Xylanase and galactanase are representative enzymes that belong to the hemicellulase group.

(13) Hemicellulose is a collective term for polysaccharides that can be obtained by alkaline extraction of the plant tissues and contains various lignocellulosic components of cell walls of plants (glucans, galactans, mannans, pentosans, xylans arabinoxylans, xyloglucans and glucomannans) with the exception of cellulose. They cannot be digested by human beings and are counted as roughage. In cereals, hemicelluloses are found in comparatively high proportions (2 to 12 percent) and also are referred to as fibrils.

(14) We found that prior art methods employing one or more cellulase and/or hemicellulase for the production of reducing sugars from lignocellulosic material, such as the methods described in WO 2010/000858 may be greatly improved when the lignocellulosic material is treated or pretreated with a particular family of pectate lyases (E.C. 4.2.2.2) at a high pH. We provide herein a family of particularly suitable pectate lyases for that purpose.

(15) In a method according to the invention, wherein a lignocellulosic material is first pretreated with a member of this particular pectate lyase family at a pH between 9 and 12, the yields of reducing sugar obtained in its subsequent enzymatic digestion with at least one cellulase or hemicellulase were greatly improved.

(16) It was surprisingly found that polypeptides comprising an amino acid sequence according to SEQ ID NO: 1, or homologues thereof, improved the yield of reducing sugars with more than 20% as compared to the same process without the enzyme pretreatment or treatment with commercially available enzymes. Reducing sugars were obtained up to 80-85% of the theoretical yield.

(17) A method according to the invention may be employed using only a single cellulase or hemicellulase. In a preferred embodiment, however, a plurality of different cellulases or hemicelluloses is used. Commercially available mixtures of these enzymes are particularly preferred because of their availability and ease of use.

(18) The term homologues is to be interpreted as polypeptides comprising an amino acid sequence that is at least 70% identical to the amino acid sequence according to SEQ ID NO: 1. The skilled person is well aware of methods and means to make such homologues for instance by directed or random mutagenis, and screen them for activity in a standard assay, such as the one disclosed herein in example 5.

(19) The term high pH as used herein indicates a strong alkalic environment, such as a pH between 9 and 12, such as between 10 and 12 or between 11 and 12. It may also refer to a pH between 9 and 10, or 9 and 11, such as between 10 and 11.

(20) The term pectate lyase activity is used herein to indicate the ability of a polypeptide to cleave pectin using an eliminative cleavage of (1->4)-alpha-D-galacturonan yielding oligosaccharides with 4-deoxy-alpha-D-galact-4-enuronosyl groups at their nonreducing ends.

(21) Pectate lyases (E.C. 4.2.2.2) favor pectate, the anion, over pectin, the methylated ester, which is the preferred substrate of pectin lyase E.C. 4.2.2.10. Pectate lyases are also known under different names, such as alpha-1,4-D-endopolygalacturonic acid lyase, endo-alpha-1,4-polygalacturonic acid lyase, endogalacturonate transeliminase, endopectin methyltranseliminase, pectate transeliminase, pectic acid lyase, pectic acid transeliminase, pectic lyase, pectin trans-eliminase, PGA lyase, polygalacturonate lyase, polygalacturonic acid lyase, polygalacturonic acid trans-eliminase, polygalacturonic transeliminase and PPase-N.

(22) The term at least 70% is used herein to include at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 88%, 87%, 88%, 89%, 90% or more, such as 91%, 92%, 93%, 94%, 95%, 99%, 97%, 98%, 99%, or even 100%.

(23) As used herein, the degree of identity between two or more amino acid sequences is equivalent to a function of the number of identical positions shared by the sequences; i.e., % identity=number of identical positions divided by the total number of aligned positions100, excluding gaps, which need to be introduced for optimal alignment of the two sequences, and overhangs. The alignment of two sequences is to be performed over the full length of the polypeptides.

(24) The comparison (aligning) of sequences is a routine task for the skilled person and can be accomplished using standard methods known in the art. For example, a freeware conventionally used for this purpose is Align tool at NCBI recourse http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2s eq&LINK_LOC=align2seq, Other commercial and open software such as Vector NTI are also suitable for this purpose.

(25) The term lignocellulosic material refers to a material that comprises (1) cellulose, hemicellulose, or a combination thereof, and (2) lignin. The term also encompasses such material that has been pretreated in order to decrease the integrity of the material thereby making the lignocellulosic material more accessible to enzymatic digestion with cellulases and/or hemicellulases. Such pretreatment may include mechanical pretreatment, chemical pretreatment of enzymatic pretreatment.

(26) Examples of a lignocellulosic material that may advantageously be used in the methods of the invention include materials comprising corn stover, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, yard waste, wood and forestry waste, sugar cane, switchgrass, wheat straw, hay, barley, barley straw, rice straw, grasses, waste paper, sludge or byproducts from paper manufacture, corn grain, corn cobs, corn husks, grasses, wheat, wheat straw, hay, rice straw, sugar cane bagasse, sorghum, soy, trees, branches, wood chips, sawdust and any combination thereof.

(27) The term reducing sugar is used herein to refer to any sugar that is capable of acting as a reducing agent because it has a free aldehyde group or a free ketone group. All monosaccharides are reducing sugars, along with some disaccharides, oligosaccharides, and polysaccharides. The common dietary monosaccharides galactose, glucose and fructose are all reducing sugars. The amount of reducing sugars may conveniently be measured by a number of methods available in the art and known to the skilled person. Reducing sugar levels as referred herein were determined by the Dinitrosalicylic Acid Method (DNS method, Sadasivam S., Manickam A., Carbohydrates in Biochemical methods, New Age Internatioal Ltd Publishers, 2nd edition, 2005, p. 6).

(28) As detailed in the examples section, we incubated corn stover with enzyme solutions comprising a pectate lyase according to SEQ ID NO: 1 or its homologues according to SEQ ID NO: 2 or SEQ ID NO: 3 (93% and 89% identical to SEQ ID NO: 1 respectively). After an alkaline pretreatment at pH 10 for 120 minutes at 65 degrees Celsius, the corn stover was digested with a commercially available cellulase cocktail for biofuel applications for 72 hours at 60 degrees Celsius. We observed that the yield in reducing sugars improved from 430 mg reducing sugar per gram of corn stover to up to 550 mg, an improvement of 28%. Similar increases were observed when the corn stover was pretreated with the homologous enzymes according to SEQ ID NO: 2 or SEQ ID NO: 3 (table 1). Alkaline pretreatments were also performed at pH 9 and pH 12. Also in these cases, a remarkable improvement in the yield of reducing sugars was observed.

(29) Hence, in one embodiment, the invention relates to a method for producing a reducing sugar from a lignocellulosic material employing at least one cellulase or hemicellulase, wherein the lignocellulosic material is pretreated (i.e. contacted in a pretreatment step) with a pectate lyase at a pH between 9 and 12, wherein the pectate lyase comprises an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence at least 70% identical with SEQ ID NO: 1.

(30) Pre-treatment is carried out before or during hydrolysis with cellulases or hemicellualses. The goal of pre-treatment is to reduce the particle size, separate and/or release cellulose; hemicellulose and/or lignin and in this way increase the rate of hydrolysis.

(31) The lignocellulose-containing material may according to the invention be biologically pre-treated before or during hydrolysis in accordance with the methods as described herein.

(32) Preferably, the enzymatic pre-treatment is carried out prior to the hydrolysis. Alternatively, enzymatic pre-treatment may be carried out simultaneously with hydrolysis, such as simultaneously with addition of one or more cellulases or hemicellulases.

(33) The term enzymatic pre-treatment refers to any pre-treatment employing an enzyme which promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the lignocellulose-containing material. Known biological pre-treatment techniques involve applying lignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212; Ghosh, P., and Singh, A., 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv. Appl. Microbiol, 39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566, American Chemical Society, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson, L., and Hahn-Hagerdal, B., 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander, L., and Eriksson, K.-E. L., 1990, Production of ethanol from lignocellulosic materials: State of the art, Adv. Biochem. Eng./Biotechnol, 42: 63-95).

(34) The pre-treated lignocellulose-containing material is hydrolyzed enzymatically to break down especially hemicellulose and/or cellulose into fermentable sugars. According to the invention the enzymatic hydrolysis may also be performed in several steps.

(35) Some, if not most reducing sugars may be used as a substrate for fermenting micro-organisms. Such sugars are often referred to as fermentable sugars. The production of fermentable sugars is preferred because micro-organisms are preferred for the further synthesis of useful molecules from the sugars obtained in the methods as described herein, Hence, the invention also relates to a method as described above, wherein the reducing sugar is a fermentable sugar.

(36) In another preferred embodiment, the method may also be employed to increase the yield of reducing sugars obtained from a material comprising lignocellulose with a high content of lignin, such as wood, Hence, the methods according to the invention could be further improved if the pretreatment was conducted in the simultaneous presence of a lignin-degrading enzyme, such as a laccase, more preferably, an alkaline Cot A laccase. This was found to be particularly advantageous when employing high-lignin containing lignocellulosic material

(37) Furthermore, in spite of the fact that grassy biomass is considered to have a low lignin content, we surprisingly found that the method employing grassy materials as described above could also be further improved, if the pretreatment was conducted in the simultaneous presence of a lignin-degrading enzyme, such as a laccase, more preferably, an alkaline Cot A laccase. Hence, in one embodiment, the invention relates to a method as described above, wherein the lignocellulosic material is contacted with an enzyme capable of degrading lignin, such as a laccase, more in particular an alkaline laccase, even more in particular a CotA laccase, such as an alkaline Cot A laccase, simultaneously with the pectate lyase.

(38) The term alkaline laccase refers to an enzyme with laccase activity that is active at alkaline conditions.

(39) 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.

(40) Laccases were originally discovered in fungi, they are particularly well studied in White-rot fungi and Brown-rot fungi. Later on, laccases were also found in plants and bacteria. Laccases have broad substrate specificity; though different laccases can have somewhat different substrate preferences. Main characteristic of laccase enzyme is its redox potential, and according to this parameter all laccases can be divided in three groups (see, for example, Morozova, O. V., Shumakovich, G. P., Gorbacheva, M. a., Shleev, S. V., & Yaropolov, a. I. (2007). Blue laccases. Biochemistry (Moscow), 72(10), 1136-1150. doi:10.1134/50006297907100112): high redox potential laccases (0.7-0.8 V), medium redox potential laccases (0.4-0.7 V) and low redox potential laccases (<0.4V). It is believed that low redox potential limits the scope of substrates which the enzyme can possibly oxidize, and vice versa. All high redox potential laccases and the upper part of the medium redox potential laccases are fungal laccases. Industrial application of laccases is mostly if not entirely relying on fungal laccases.

(41) CotA is a bacterial laccase and is a component of the outer coat layers of bacillus endospore. It is a 65-kDa protein encoded by the cotA gene (Martins, O., Soares, M., Pereira, M. M., Teixeira, M., Costa, T., Jones, G. H., & Henriques, A. O. (2002). Molecular and Biochemical Characterization of a Highly Stable Bacterial Laccase That Occurs as a Structural Component of the Bacillus subtilis Endospore Coat. Biochemistry, 277(21), 18849-18859. doi:10.1074/jbc.M200827200). CotA belongs to a diverse group of multi-copper blue oxidases that includes the laccases. This protein demonstrates high thermostability, and resistance to various hazardous elements in accordance with the survival abilities of the endospore. The redox-potential of this protein has been reported to be around 0.5 mV, which places it in the range of medium-redox-potential laccases.

(42) As detailed in the examples section, we incubated corn stover with an enzyme solution comprising a pectate lyase according to SEQ ID NO: 1 together with a laccase according to SEQ ID NO: 4. After an alkaline pretreatment at pH 10 for 120 minutes at 65 degrees Celsius, the corn stover was digested with a commercially available cellulase cocktail for biofuel applications for 72 hours at 60 degrees Celsius. We observed that this simultaneous pretreatment resulted in a yield of 585 mg of reducing sugar per gram feedstock, an improvement of 36% when compared to the control preincubation without the enzymes, and an improvement of 6% over the pretreatment with the pectate lyase according to SEQ ID NO: 1 alone. Most remarkably, the yield of the method wherein the lignocellulosic material was pretreated with a pectate lyase and a laccase under alkaline conditions, closely approached the maximum theoretical yield of 650 mg reducing sugar per gram feedstock (table 1).

(43) In a preferred embodiment, the alkaline COT A laccase is a polypeptide comprising an amino acid sequence according to SEQ ID NO: 4 or an amino acid sequence at least 90% identical with SEQ ID NO: 4.

(44) The term at least 90% is used herein to include at least 91%, 92%, 93%, 94%, 95%, 99%, 97%, 98%, 99%, or even 100%.

(45) After preincubation, reducing sugars are preferably obtained from the pretreated material by enzymatic digestion of cellulose. Without wanting to be bound by theory, the inventors believe that the pretreatment steps as described herein make the cellulose more accessible to enzymatic digestion. Hence, the invention also relates to a method for making cellulose in a biomaterial more accessible to enzymatic digestion, such as cellulose-degrading enzymes.

(46) Cellulose-degrading enzymes are known in the art and commercially available. They are usually offered in combination preparations, for example, CELLIC CTEC3 or CTEC2 preparations (from Novozymes, Denmark) which are compositions of enzymes comprising cellulases, [beta]-glucosidases and hemi-cellulase; or CELLIC HTEC3 or HTEC2 (also from Novozymes, Denmark) which is a composition of enzymes comprising endoxylanase and cellulase.

(47) In summary, the invention relates to a method as described herein, wherein lignocellulosic material is contacted with an enzyme capable of degrading cellulose after being pretreated with an alkaline pectate lyase.

(48) Since most cellulose-degrading enzymes are active at neutral or acidic pH, the pH of the composition comprising the lignocellulosic material may be lowered to a suitable range after pretreatment. The skilled person is well aware of the means to accomplish that. The temperature may also be adjusted to an optimal temperature for enzymatic digestion using at least one cellulase and/or hemicellulase. This will entirely depend on the choice of the enzymes for this purpose, such is again well-known to the skilled person.

(49) Advantageously, the cellulose-degrading enzyme is selected from the group consisting of cellulase; hemi-cellulase; [beta] 1-4 endoglucanases (E.C. [beta] 1-4 exoglucanases (E.C. 3.2.1.9.1), [beta]-glucosidases (E.C. 3.2.1.2.1), and endoxylanases.

(50) In a further preferred method according to the invention, the lignocellulose material may advantageously be pretreated before the enzyme or enzymes are added.

(51) The term pretreated as used herein may therefore also refer to a treatment that occurs before the enzymatic treatment, either pectate lyases, laccases or cellulose-degrading enzymes or combinations thereof. Pretreatment may also consist of a steam treatment, such as a dilute acid steam treatment or a steam explosion treatment is applied to the biomass or lignocellulose material. One of the goals of the steam treatment is to depolymerize the lignin in the biomass to a sufficient extent to allow an enzyme or mixture of enzymes to convert the cellulose and hemi-cellulose in the biomass into less complex sugars in a subsequent step.

(52) The method as described herein may even be further improved when the lignocellulosic material is pretreated before the enzyme or enzymes are added. Preferably, such a pretreatement is a mechanical pretreatment. Hence, in one embodiment, the invention relates to a method as described herein wherein the lignocellulosic material is pretreated before the material is contacted with the pectate lyase, an enzyme capable of degrading cellulose (such as cellulase or hemicellulase, or an enzyme capable of degrading lignin, such as a laccase. Particularly preferred in this sense is a pretreatment by a steam explosion step or ammonia fiber explosion step.

(53) In a further preferred embodiment, the invention relates to a method as described herein wherein the pectate lyase has an amino acid sequence that is at least 89% identical to the sequence according to SEQ ID NO:1, preferably at least 93%, such as at least 96%, 97%, 98% or 99%.

(54) In another embodiment, the invention relates to a composition comprising a lignocellulosic material and a pectate lyase comprising an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence that is at least 70% identical with SEQ ID NO: 1, wherein the composition has a pH between 9 and 12.

(55) The lignocellulosic material of such a composition is then advantageously selected from the group consisting of corn stover, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, yard waste, wood and forestry waste, sugar cane, switchgrass, wheat straw, hay, barley, barley straw, rice straw, grasses, waste paper, sludge or byproducts from paper manufacture, corn grain, corn cobs, corn husks, grasses, wheat, wheat straw, hay, rice straw, sugar cane bagasse, sorghum, soy, trees, branches, wood chips and sawdust.

(56) Such compositions may additionally comprise an enzyme capable of degrading cellulose or an alkaline enzyme capable of degrading lignin. The enzyme capable of degrading cellulose is then preferably selected from the group consisting of cellulases and hemi-cellulases. This group encompasses [beta] 1-4 endoglucanases (E.G. 3.2.1.4), [beta] 1-4 exoglucanases (E.G. 3.2.1.9.1), [beta]-glucosidases (E.G. 3.2.1.2.1) and endoxylanases.

(57) The enzyme capable of degrading lignin may then advantageously comprise a laccase, such as an alkaline laccase or a CotA laccase or an alkaline CotA laccase comprising an amino acid sequence according to SEQ ID NO: 4 or an amino acid sequence at least 90% identical with SEQ ID NO: 4. The pectate lyase in such a composition may advantageously comprise an amino acid sequence that is at least 89% identical to the sequence according to SEQ ID NO: 1, preferably 93%, such as 96%, 97%, 98% or 99%.

(58) It is also particularly preferred when the method according to the invention is performed with a pectate lyase obtained by heterologous expression in Escherichia coli.

(59) In certain processes, the temperature of the biomass or lignocellulosic material to be treated may be in excess of the enzyme inactivation temperature. Since a high temperature may inactivate enzymes by denaturing its amino acid chain, the enzyme may advantageously be added to the biomass at a point below the enzyme inactivation temperature. The enzymes may be added within the functional temperature range(s) or at the optimal temperature(s) of the enzyme. To save energy, the enzymes may be added after the biomass has cooled below the inactivation temperature and that the enzymatic process is completed sufficiently before the temperature has dropped below the optimal functional temperature of the enzyme. Naturally, it is also an option to maintain a desired temperature by cooling or heating the biomass or lignocellulosic material. Adding a dilution liquid, such as water at a certain temperature, may be used to cool the biomass.

(60) In one embodiment, the enzyme pretreatment process may be performed at a specific temperature such as, for example at from 30 degrees C. to 70 degrees C.; 40 degrees C. to 65 degrees C.; or 45 degrees C. to 60 degrees C., or at room temperature or lower. More thermostable enzymes may be used when the temperature requirements are higher. Thermostable laccases, cellulases and pectate lyases are well known in the art.

(61) The contacting of the lignocellulosic material with an enzyme can be performed for a period of time up to one day. While longer enzymatic digestions are possible, such as two, three or four days, a shorter period of time such as 60 minutes, 2 hours, 10 hours, 20 hours, 30 hours, 40 hours, 60 hours or 72 hours or any time less than these values or any time between any of two of these values may be used for practical or economic reasons. In another preferred embodiment, the enzymatic digestions can take 50, 100, 150 or 200 hours or any time less than these values or any time between any of two of these values. In one embodiment, a preferred period of pretreatment is for about 2 hours.

EXAMPLES

Example 1: Preparation of a Polypeptide According to SEQ ID NO: 1

(62) The DNA construct disclosed in Takao et al., Biosci. Biotechnol. Biochem. (2001) 65: 322-329 encoding the polypeptide according to SEQ ID NO: 1 was optimized for expression in E. coli and commercially synthesized and cloned into a standard plasmid vector pET28a+ under the control of T7-RNA-polymerase promoter for expression in Escherichia coli BL21(DE3). The nucleotide sequence of the construct is provided herein as SEQ ID NO: 7

Example 2: Preparation of Variants of a Polypeptide According to SEQ ID NO: 1 with Pectate Lyase Activity

(63) Homologous protein sequences (according to SEQ ID NO: 2 and SEQ ID NO: 3) were generated by random mutagenesis of SEQ ID NO:s 7 and SEQ ID NO: 8 using error-prone PCR essentially as described (Curr Protoc Mol Biol. 2001 May; Chapter 8: Unit 8.3, doi: 10.1002/0471142727.mb0803s51, Random mutagenesis by PCR. Wilson DS1, Keefe A D) using a commercial random PCR mutagenesis kit (QuikChange II XL Site-Directed Mutagenesis kit by Agilent Technologies). More in particular, the DNA sequence of SEQ ID NO: 8 was obtained from SEQ ID NO: 7 encoding the polypeptide according to SEQ ID NO: 1. The DNA sequence of SEQ ID NO: 9 was obtained by random mutagenesis of SEQ ID NO: 8 encoding the polypeptide, according to SEQ ID NO: 2. SEQ ID NO: 9 is the DNA sequence encoding the polypeptide according to SEQ ID NO: 3.

(64) PCR fragments resulting from error-prone PCR were cloned to the plasmid vector pET28a+ under the control of T7-RNA-polymerase promoter for expression in Escherichia coli BL21(DE3), and screened for pectate lyase activity of the recombinant proteins.

(65) Active clones were subjected to further rounds of randomization using the same protocol. The polypeptide according to SEQ ID NO: 2 exhibited pectate lyase activity and was found to be 93% identical with SEQ ID NO: 1, The polypeptide according to SEQ ID NO: 3 also exhibited pectate lyase activity and was found to be 89% identical with SEQ ID NO: 1.

Example 3: Heterologous Expression of Polypeptides with Pectate Lyase Activity

(66) For recombinant expression in E. coli, recombinant genes were cloned into pET-28 commercial expression vector under the control of T7 bacteriophage promoter.

(67) Protein production was carried out in E. coli BL21(DE3) strain according to the plasmid manufacturer protocol available at http://richsingiser.com/4402/Novagen %20pET %20system%20 manual.pdf. The incubation temperature for protein production was 30 degrees C., which was found optimal for maximum yield of the active protein. Cells were lysed using lysis buffer (20 mM Sodium Citrate pH7.4, 1% Triton X100, 0.5 mM CaCl) and heated at 60 degrees C. for 20 minutes. Coagulated cell debris was removed by centrifugation. The thermostable recombinant pectate lyases were detected in the soluble fraction only, consistent with the notion that they were thermostable enzymes.

Example 4 Heterologous Expression of Polypeptides with Laccase Activity

(68) For recombinant expression in E. coli, recombinant genes were cloned into pET-28 commercial expression vector under the control of T7 bacteriophage promoter.

(69) Protein production was carried out in E. coli BL21(DE3) strain according to the plasmid manufacturer protocol available at http://richsingiser.com/4402/Novagen %20pET %20system%20manual.pdf. The incubation temperature for protein production was 30 degrees C., which was found optimal for maximum yield of the active protein. Cells were lysed using laccase lysis buffer (Sodium Citrate pH 7.4, 1% Triton X100, 1 mM CuCl2) and heated at 70 degrees C. for 20 min. Coagulated cell debris was removed by centrifugation. The thermostable recombinant laccases were detected in the soluble fraction only, consistent with the notion that they are thermostable enzymes.

Example 5: Pectate Lyase Activity Assay

(70) Pectate lyase activity assay was carried out essentially as described in Takao M, Nakaniwa T, Yoshikawa K, Terashita T, Sakai T., Purification and characterization of thermostable pectate lyase with protopectinase activity from thermophilic Bacillus sp. TS 47. Biosci Biotechnol Biochem. 2000 64:2360-7. In more detail, pectate lyase activity was assayed by measuring the increase in absorbance at 235 nm of the reaction mixture, Polygalacturonic acid (PGA) sodium salt from de-methylated citrus pectin (purchased from MegaZyme) was used as substrate. A reaction mixture containing 1 ml of 0.1% PGA in 10 mM Tris-HCl buffer, pH 8.0 and 0.5 mM CaCl2), and an appropriate amount of enzyme solution was incubated for 30 minutes at 60 degrees C.

(71) The reaction was stopped by placing the mixture in 100 degrees C. (boiling water bath) for 5 min. Pectate lyase activity was calculated from the difference in absorption of the reaction mixture at 235 nm at the start and at the end of the reaction.

(72) One unit of pectate lyase activity was defined as the enzyme amount oxidizing 1 micro mole of substrate per minute. Using absorption coefficient of the unsaturated bond at the 4-5 position of the uronic acid residue at 235 nm 4 600 mol-1cm-1.

Example 6: Laccase Activity Measurement

(73) The term laccase activity is used herein to mean the capability to act as a laccase enzyme, which may be expressed as the maximal initial rate of the specific oxidation reaction. Relative activity was measured by oxidation of syringaldazine. Reaction course was monitored by change in absorbance at 526 nM (extinction coefficient of syringaldazine at 526 nm is 65 000 M-1 cm-1). The appropriate reaction time was determined to provide initial rates of oxidation when color development is linear in time. Syringaldazine concentration in the reaction mixture was 1 mM to provide maximum initial rates (substrate saturation conditions).

(74) Typically, reactions were carried out in 1 ml volume of 50 mM Tris-HCl buffer pH 8, the substrate was preheated to the desired temperature (60 degrees Celsius) and reaction was initiated by the addition of the enzyme. After the reaction time has elapsed, absorbance at 526 nm of the reaction mixtures was determined by a spectrophotometer, and the absorbence of the blank sample (containing no enzyme) was subtracted.

(75) One unit of laccase activity was defined as the enzyme amount oxidising 1 micro mole of substrate per minute.

Example 7: Effect of Enzymatic Pretreatment on Reducing Sugar Yield

(76) Reducing sugars were produced from corn stover using a conventional process wherein the corn stover was mechanically pretreated by reducing the particle size, followed by an enzymatic pretreatment at alkaline conditions.

(77) Mechanical pretreatment involved reducing the particle size of corn stover in a Thomas knife mill (Arthur H. Thomas Company, Philadelphia, Pa., USA) followed by screening in a Tyler portable sieve shaker, model KX-24 (Combustion Engineering, Mentor, Ohio, USA). The particle size ranged from 8- to 200-mesh.

(78) Alkaline pretreatment was carried out in a 1 liter reactor wherein the temperature was controlled by water circulation through the jacket and thorough mixing was provided by propeller blades.

(79) The reactor was loaded with 55 gram (dry weight) of corn stover, and 450 ml of 0.5% NaOH was added. The pH of this suspension was found to be between 10 and 11.

(80) After the biomass was well mixed and equilibrated to 65 degrees Celsius, enzyme solution (10 ml of enzyme in lysis buffer) or the same volume of lysis buffer without the enzyme (control) was added and the reaction was allowed to continue for 120 minutes at 65 degrees Celsius.

(81) The pectate lyases disclosed herein were dosed at 10,000,000 units of pectate lyase (standard activity measured at pH 8.0, see example 5), per ton of dry corn stover material.

(82) Commercial preparation Pectinex was obtained from Novozymes, Denmark, and used at the same dosage according to specific activity as indicated by the manufacturer.

(83) The lignin degrading enzyme according to SEQ ID NO: 4 was used at 10 millikatal/ton of dry corn stover. One katal is defined as the amount of enzyme needed to convert 1 mole of substrate in 1 sec. A unit is defined as the amount of enzyme needed to convert 1 micromole of substrate in 1 minute, hence 10 millikatal equals 600,000 units.

(84) After pretreatment, the reactors were immediately cooled by passing ice-cold water through the jackets. Corn stover from two duplicate reactions treated under the same conditions was recovered, combined, and washed intensively with deionized water.

(85) The biomass was then suspended in 100 mM succinic acid at pH 5.0, to make up a suspension of 50 grams of dry weight of biomass per kilogram of suspension.

(86) Enzymatic hydrolysis of the pretreated biomass was carried out at 60 degrees Celsius for 72 hours using Alternafuel CMAX from Diadic International Inc (USA) a commercially available cellulase cocktail for biofuel applications. Dosing was performed according to the manufacturer's instructions.

(87) After the hydrolysis, reducing sugar levels were determined by the Dinitrosalicylic Acid Method (DNS method, Sadasivam S., Manickam A., Carbohydrates in Biochemical methods, New Age Internatioal Ltd Publishers, 2nd edition, 2005, p. 6). The results are shown in Table 1.

(88) We observed a large increase in yield when the lignocellulosic material was pretreated under alkaline conditions with a pectate lyase according to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, as compared to the same pretreatment without the enzymes.

(89) The yield in reducing sugars was even further improved when the biomass was simultaneously treated with a lignin-degrading enzyme according to SEQ ID No: 4. Notably, pretreatment with the commercial pectinase Pectinex from Novozymes had no effect on reducing sugar yield (table 1).

(90) Other lignocellulosic materials such as sugar cane, switchgrass, wheat straw and soft wood were also tested with the enzymes as disclosed herein, with equivalent results. The pH of the pretreatment compositions varied from pH 9 to pH 12.

(91) In all cases a clear advantage of the pretreatment with the pectate lyase enzymes was observed.

(92) TABLE-US-00001 TABLE 1 Effect of enzyme pretreatment on reducing sugar yield from lignocellulosic biomass. Theoretical Cellulase yield Percentage [mg sugar/ [mg sugar/ of Corn stover gram Improvement gram theoretical pretreatment feedstock] of yield [%] feedstock] yield [%] Reference 430 0% 650 66% SEQ ID NO: 1 550 28% 650 85% SEQ ID NO: 2 520 21% 650 80% SEQ ID NO: 3 530 23% 650 82% SEQ ID NO: 1 + 585 36% 650 90% SEQ ID NO: 4 Pectinex 420 2% 650 65%

Example 8: Sequences Provided Herein

(93) Amino acid sequence and nucleotide sequences are separately provided herewith according to the WIPO ST25 standard. For convenience, the sequences are also reproduced herein in table 2.

(94) SEQ ID NO: 1 is derived from the prior art and has been disclosed in Takao et al, Biosci. Biotechnol. Biochem. (2000) 64: 2360-2367 and in Takao et al., Biosci. Biotechnol. Biochem. (2001) 65: 322-329.

(95) SEQ ID NO: 2 was obtained by random mutagenesis of the DNA encoding SEQ ID NO: 1 (shown herein as SEQ ID NO: 5) as described in example 2.

(96) SEQ ID NO: 3 was obtained by random mutagenesis of the DNA encoding SEQ ID NO: 2 (shown herein as SEQ ID NO: 6). The DNA encoding the polypeptide according to SEQ ID NO: 3 is shown herein as SEQ ID NO: 7. The amino acids deviating from the wild type sequence of SEQ ID NO: 1 are shown in capital letters.

(97) The polypeptide with an amino acid sequence according to SEQ ID NO: 2 is a homologue of the polypeptide according to SEQ ID NO: 1, These two polypeptides have 385 of the 416 amino acids in common, in other words they are 93% identical.

(98) The polypeptide according to SEQ ID NO: 3 is also a homologue of the polypeptide according to SEQ ID NO: 1, These two polypeptides have 369 of the 416 amino acids in common, in other words they are 89% identical.

(99) SEQ ID NO: 4 and SEQ ID NO: 8 are the protein and DNA sequence respectively from a thermostable and alkaline Cot A laccase.

(100) TABLE-US-00002 TABLE2 Aminoacidandnucleotidesequencesprovidedherein SEQ IDNO: Sequence 1 1 kelghevlkpydgwaaygegttggamaspqnvfvvtnrteliqalggnnhtnqynsvpki 61 iyvkgtidlnvddnnqpvgpdfykdphfdfeaylreydpatwgkkevegpleearvrsqk 121 kqkdrimvyvgsntsiigvgkdakikgggfliknvdnviirniefeapldyfpewdptdg 181 tlgewnseydsisiegsshiwidhntftdgdhpdrslgtyfgrpfqqhdgaldiknssdf 241 itisynvftnhdkvtligasdsrmadsghlrvtlhhnyyknvtqrlprvrfgqvhiynny 301 yefsnladydfqyawgvgvfsqiyaqnnyfsfdwdidpsliikvwskneesmyetgtivd 361 lpngrryidlvasynesntlqlkkevtwkpmfyhvihptpsvpalvkakagagnlh 2 1 kelghDvlkpydgwaSygegttggSmaspqnvYTvtnKtelVqalggnnhtnqynsvpki 61 iyvkgtiElnvddnnqpvgpEfykdphYdfeaylKeydpKKwgkkevSgpleearArsqk 121 kqkEriVvNvgsntsiigvgkdakiVgggfliknvdnviirniefeapVdyfpewdptdg 181 tlgewnseydsiTiegsHhiwidhntftdgdhpdKslgtyfgrpfqqhdgaldiknssdf 241 itisynvfKDhdkvtligasdsrmadEghlrvtlhhnyyknvtqrlprvrfgqvhiynny 301 yefsnladydfqyawgvgvEsKiyaqnnyfsfdwdidpsKiikvwskneesmyeSgtivd 361 lpngrryidlvasynesntlqlkkevGwkpmfyhvihptpsvpalvkakagagnlh 3 1 kelghDvlkpNdgwaSygegttggSEaspDnvYTvtnKSelVqalggnnhtnqynsTpki 61 iyvkgtiElnvddnnqpvgpEYyDdphYdfeaylKeydpKKwgkkevSgpleearArsqk 121 kqkEriVvNvgsntsiigvgkdakiVgggfliknvdnviirniefeapVdFfpewdptdg 181 EYgewnseydsiTieSsHhiwidhntftdgdhpdKslgtyfgrpfqqhdgaldiknssdf 241 itisynvfKDhdkvSligSsdsrKTdEghlKvtlhhnyyknvtqrlprvrfgqvhiynny 301 yefsnladydfqyawgvgvEsKiyaqnnyfsfdwdidpsKiikvwskneesmyeSgtivd 361 lpngrryidlvasynesntlqlkkevGwkpmfyhvihptpsvpalvkakagagnlh 4 1 mrrklekfvdslpimetlqpktkgknyyevkiqefkkklhrdlppttlwgynaqfpgpti 61 eansnepvevkwinelpnkhflpvdwsimnkdlpevrhvthlhggrtpsvsdgypeawyt 121 kdykevgsffkeevyrylneqrammlwyhdhtmgitrlnnyaglagayiirdkhekslnl 181 pegeyevpliiqdrtfnedgslfyptgpedggedlpnpsivpaflgdtvlvngkvwpyle 241 veprkyrfrilngsnarsyqlhldsnqevyqigsdggllekpvqmnkipiesseridvii 301 dfsqcdgdeivlkndlgpdadaedetneimkfkvskplkekdtsvipkrlstirslrnnk 361 isthrnlklvgstddfgrpllllnnkkwadpttekpkvgdtevwsfinttdfahpmhihl 421 ihfqvldrqpfdlerynhdgtiiytgpprapepnergwkdtvsapagqitrvigtfapyt 481 gnyvwhchilehedhdmmrpmkvidpkqrkdks 5 aaagaactgggtcatgaagttctgaaaccgtatgatggttgggcagcgtatggtgaaggt 60 acaaccggtggtgcaatggcaagtccgcagaatgtttttgttgttaccaatcgtaccgaa 120 ctgattcaggcactgggtggtaataatcataccaatcagtataattccgtgccgaaaatc 180 atctatgtgaaaggcaccattgatctgaacgtggatgataataatcagccggttggtccg 240 gatttctataaagatccgcattttgattttgaggcctatctgcgtgaatatgatccggca 300 acctggggtaaaaaagaagttgaaggtccgctggaagaagcacgcgttcgtagccagaaa 360 aaacagaaagatcgtatcatggtttatgtgggtagcaacaccagcattattggtgttggt 420 aaagacgcgaaaatcaaaggtggtggtttcctgattaaaaacgtggataatgtgatcatc 480 cgcaacatcgaatttgaagcaccgctggattattttccggaatgggatccgaccgatggc 540 accctgggtgaatggaatagcgaatatgatagcattagcattgaaggcagcagccatatt 600 tggattgatcacaatacctttaccgatggcgatcatccggatcgtagcctgggcacctat 660 tttggtcgtccgtttcagcagcatgatggcgcactggatatcaaaaatagcagcgatttt 720 atcaccatcagctacaacgtgtttaccaaccacgataaagttaccctgattggtgcaagc 780 gatagccgtatggcagatagcggtcatctgcgtgttaccctgcatcacaattattacaaa 840 aatgttacccagcgtctgcctcgtgttcgttttggtcaggttcatatctataacaactac 900 tatgagtttagcaacctggccgattatgattttcagtatgcatggggtgttggtgtgttt 960 agccagatttatgcacagaacaactatttcagcttcgattgggatattgatccgagcctg 1020 attatcaaagtttggagcaaaaatgaagaaagcatgtatgaaaccggcaccatcgttgat 1080 ctgccgaatggtcgtcgttatattgatctggttgcaagctataatgaaagcaataccctg 1140 cagctgaaaaaagaggttacctggaaaccgatgttctatcatgttattcatccgaccccg 1200 agcgttccggcactggttaaagcaaaagccggtgcaggtaatctgcat 1248 6 aaagaactgggtcatgatgtgctgaaaccgtatgatggttgggcaagctatggtgaaggt 60 acaaccggtggtagcatggcaagtccgcagaatgtttataccgttaccaataaaaccgaa 120 ctggttcaggcactgggtggtaataatcataccaatcagtataattccgtgccgaaaatc 180 atctatgtgaaaggcaccattgaactgaacgtggatgataataatcagccggttggtccg 240 gaattctataaagatccgcattatgattttgaagcctatctgaaagagtatgatccgaaa 300 aaatggggcaaaaaagaagttagcggtccgctggaagaagcacgcgcacgtagccagaaa 360 aaacagaaagaacgtattgttgtgaatgtgggtagcaacaccagcattattggtgttggt 420 aaagatgccaaaattgtgggtggtggtttcctgattaaaaacgtggataatgtgatcatc 480 cgcaacatcgaatttgaagcaccggtggattattttccggaatgggatccgaccgatggc 540 accctgggtgaatggaatagcgaatatgatagcattaccattgaaggcagccatcatatt 600 tggatcgatcacaatacctttaccgatggcgatcatccggataaaagcctgggcacctat 660 tttggtcgtccgtttcagcagcatgatggcgcactggatatcaaaaatagcagcgatttt 720 atcaccatcagctacaacgtgtttaaagaccatgataaagtgaccctgattggtgcaagc 780 gatagccgtatggcagatgaaggtcatctgcgtgttaccctgcatcacaattattacaaa 840 aatgttacccagcgtctgcctcgtgttcgttttggtcaggttcatatctataacaactac 900 tatgagtttagcaacctggccgattatgactttcagtatgcatggggtgttggtgttgaa 960 agcaaaatctatgcccagaacaactatttcagcttcgattgggatattgacccgagcaaa 1020 attatcaaagtttggagcaaaaacgaagaaagcatgtatgaaagcggtacgattgttgat 1080 ctgccgaatggtcgtcgttatattgatctggttgcaagctataatgaaagcaataccctg 1140 cagctgaaaaaagaggttggttggaaaccgatgttctatcatgttattcatccgaccccg 1200 agcgttccggcactggttaaagcaaaagccggtgcaggtaatctgcat 1248 7 aaagaactgggtcatgatgtgctgaaaccgaatgatggttgggcaagctatggtgaaggt 60 acaaccggtggtagcgaagcaagtccggataatgtttataccgttaccaataaaagcgaa 120 ctggttcaggcactgggtggtaataatcataccaatcagtataattccaccccgaaaatc 180 atctatgtgaaaggcaccattgaactgaacgtggatgataataatcagccggttggtccg 240 gaatattatgatgatccgcattatgattttgaagcctatctgaaagagtatgatccgaaa 300 aaatggggcaaaaaagaagttagcggtccgctggaagaagcacgcgcacgtagccagaaa 360 aaacagaaagaacgtattgttgtgaatgtgggtagcaacaccagcattattggtgttggt 420 aaagatgccaaaattgtgggtggtggtttcctgattaaaaacgtggataatgtgatcatc 480 cgcaacatcgaatttgaagcaccggttgatttttttccggaatgggatccgaccgatggt 540 gaatatggcgaatggaatagcgaatatgatagcattaccatcgaaagcagccatcatatt 600 tggatcgatcacaatacctttaccgatggcgatcatccggataaaagcctgggcacctat 660 tttggtcgtccgtttcagcagcatgatggcgcactggatatcaaaaatagcagcgatttt 720 atcaccatcagctacaacgtgtttaaagaccatgataaagtgagcctgattggttcaagc 780 gatagccgtaaaaccgatgaaggtcatctgaaagttaccctgcatcacaactattacaaa 840 aatgttacccagcgtctgcctcgtgttcgttttggtcaggttcatatctataacaactac 900 tatgagtttagcaacctggccgattatgactttcagtatgcatggggtgttggtgttgaa 960 agcaaaatctatgcccagaacaactatttcagcttcgattgggatattgacccgagcaaa 1020 attatcaaagtttggagcaaaaacgaagaaagcatgtatgaaagcggtacgattgttgat 1080 ctgccgaatggtcgtcgttatattgatctggttgcaagctataatgaaagcaataccctg 1140 cagctgaaaaaagaggttggttggaaaccgatgttctatcatgttattcatccgaccccg 1200 agcgttccggcactggttaaagcaaaagccggtgcaggtaatctgcat 1248 8 atgcgtcgcaaactggaaaaatttgttgatagcctgccgattatggaaaccctgcagccg 60 aaaaccaaaggcaaaaactattatgaggtgaaaatccaagagtttaaaaaaaaactgcac 120 cgtgatctgcctccgaccaccctgtggggttataatgcacagtttccgggtccgaccatt 180 gaagcaaatagcaatgaaccggttgaagtgaaatggattaatgagctgccgaacaaacat 240 tttctgccggttgattggagcatcatgaataaagatctgccggaagttcgtcatgttacc 300 catctgcatggtggtcgtaccccgagtgttagtgatggttatccggaagcatggtatacg 360 aaagattataaagaagtgggcagcttcttcaaagaagaggtttatcgttatctgaatgaa 420 cagcgtgcaatgatgctgtggtatcatgatcataccatgggtattacccgtctgaataac 480 tatgcaggtctggcaggcgcatatatcattcgtgataaacatgaaaaaagcctgaatctg 540 cctgaaggcgaatatgaagttccgctgattattcaggatcgcacctttaatgaagatggc 600 agcctgttttatccgaccggtccggaagatggcggtgaggatctgccgaatccgagcatt 660 gttccggcatttctgggtgataccgttctggttaatggtaaagtttggccgtatctggaa 720 gttgaaccgcgtaaatatcgttttcgtattctgaatggtagcaacgcccgtagctatcag 780 ctgcatctggatagcaatcaagaagtgtatcagattggttcagatggtggtctgctggaa 840 aaaccggtgcagatgaacaaaattccgattgaaagcagcgaacgcattgatgtgattatc 900 gattttagccagtgtgatggtgatgagattgtgctgaaaaatgatctgggtccggatgca 960 gatgccgaagatgaaaccaatgaaatcatgaaattcaaagtgagcaaaccgctgaaagag 1020 aaagataccagcgttattccgaaacgtctgagcaccattcgtagcctgcgtaataacaaa 1080 attagcacccatcgtaatctgaaactggttggtagcaccgatgattttggtcgtcctctg 1140 ctgctgctgaacaacaaaaaatgggcagatccgaccacagaaaaaccgaaagttggcgat 1200 accgaagtttggagctttattaacaccaccgattttgcacatccgatgcatattcatctg 1260 atccattttcaggttctggatcgtcagccgtttgatctggaacgttataatcatgatggc 1320 accattatctataccggtccgcctcgtgcaccggaaccgaatgaacgtggttggaaagat 1380 acagttagcgcaccggcaggtcagattacccgtgttattggcacctttgcaccgtatacc 1440 ggtaattatgtttggcattgtcatatcctggaacacgaagatcacgatatgatgcgtccg 1500 atgaaagttattgatccgaaacagcgtaaagataaa 1536