US20100184161A1 - Acidothermus celluloyticus xylanase - Google Patents

Acidothermus celluloyticus xylanase Download PDF

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US20100184161A1
US20100184161A1 US12/613,398 US61339809A US2010184161A1 US 20100184161 A1 US20100184161 A1 US 20100184161A1 US 61339809 A US61339809 A US 61339809A US 2010184161 A1 US2010184161 A1 US 2010184161A1
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xylanase
seq
amino acid
xyl
sequence
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Rebecca E. Parales
Alison M. Berry
Juanito V. Parales, JR.
Ravi D. Barabote
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University of California
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)

Definitions

  • the present disclosure relates to xylanases and methods for their expression and use. Specifically, the disclosure is related to a thermophilic xylanase (Xyl-1) and homologs thereof derived from Acidothermus cellulolyticus , and the use of these enzymes in hydrolyzing lignocellulose.
  • Xyl-1 thermophilic xylanase
  • homologs thereof derived from Acidothermus cellulolyticus
  • Lignocellulose is plant biomass composed of cellulose, hemicellulose, and lignin. Lignocellulose serves as an abundant and inexpensive source of fermentable biomass. However, one barrier to the utilization of lignocellulose is the tight crosslinking of the cellulose and hemicellulose to the lignin. Breaking down lignocellulose (i.e. separating cellulose and hemicellulose from lignin) is energy intensive, and thus inefficient. Efficient utilization of lignocellulosic biomass will enhance the economic competitiveness of bioconversion processes which must compete with petrochemical processes. It has been shown that xylanase enzymes can be used to efficiently break down lignocellulose.
  • Xylanase enzymes are important in a wide variety of biotechnological and industrial applications. These include prebleaching of kraft pulp in the pulp and paper industry, recovery of cellulose fiber in textiles, enhancing digestibility of animal feed and silage, clarification of juices and beer, separation of cereal gluten and starch, assorted applications in the bakery industry, as well as the production of xylo-oligosaccharides for pharmacological applications and food additives (1, 2, 9, 14, and 21). Furthermore, recent interest in biofuels production from lignocellulosic plant biomass has brought xylanases into renewed prominence (5). Collins et al. (4) recently reviewed the physicochemical and functional characteristics of xylanases from six different families, their mechanism of action, and industrial applications.
  • Xylanase production is commonly obtained from Trichoderma reesei and Trichoderma harzianum strain E58, both from the Forintek Canada Corp. culture collection. Although both fungi are prolific producers of extracellular xylanases, fungal growth and enzyme production can only be carried out at mesophilic temperatures (e.g., 28° C.). Consequently the fermentation requires considerable cooling water during fungal growth and is easily subjected to bacterial contamination. The xylanase enzymes produced are also thermally unstable, losing over 90% of their activities within a half hour of incubation at 50° C. As a result, enzymatic hydrolysis of lignocellulose using these enzymes has to be carried out at a lower temperature of about 37-45° C. This in turn lowers the hydrolysis efficiencies, necessitates asceptic conditions during hydrolysis, as well as preventing prolonged enzyme use without replacement. However, higher efficiency of hydrolysis can be obtained by using thermophilic xylanases.
  • thermophilic xylanases from fungal and bacterial microorganisms have been identified ( FIG. 1 ).
  • U.S. Pat. No. 5,935,836 discloses a thermophilic xylanase isolated from Actinomadura flexuosa that has an optimal pH of 6.0-7.0 and a temperature range of 70-80° C.
  • U.S. Pat. No. 5,395,765 discloses a xylanase derived from Rhodothermus , having activity over a pH range of 5-8 and thermostability at temperatures from 85-100° C.
  • a xylanase with a more acidic pH range is desired for the utilization of hemicellulose biomass in fermentation.
  • thermophilic cellulolytic bacterium Acidothermus cellulolyticus is described in Mohagheghi et al. (12), and the production of cellulase is described in Shiang et al. (19).
  • neither reference describes a purified xylanase that may be useful at a low pH and high temperatures.
  • U.S. Pat. No. 5,902,581 discloses a xylanase derived from Acidothermus cellulolyticus that is active at a pH range from 3.6-4.2 and that is thermostable at a range of 70-80° C.
  • this A. cellulolyticus xylanase does not have optimal activity at temperatures above 80° C. or at a pH range from 4.5-6.0.
  • a recombinant endo-beta-1,4-xylanase having an amino acid sequence with at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, or at least 93% sequence identity to SEQ ID NO: 1. More preferably the amino acid sequence of the recombinant endo-beta-1,4-xylanase has at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 1. In a particularly preferred embodiment, the amino acid sequence of the recombinant endo-beta-1,4-xylanase is SEQ ID NO: 1.
  • a region of the amino acid sequence of any of the recombinant endo-beta-1,4-xylanases described above conforms to consensus sequence: Pro-Xaa 1 -Pro-Xaa 2 -Pro (SEQ ID NO: 40).
  • Xaa i and Xaa 2 are each independently selected from no amino acid, any 1 amino acid, any 2 amino acids, or any 3 amino acids.
  • the amino acid sequence of any of the recombinant endo-beta-1,4-xylanases described above has a first Glu at a position corresponding to Glu-142 of SEQ ID NO: 1 and a second Glu at a position corresponding to Glu-259 of SEQ ID NO: 1.
  • the region of the amino acid sequence of any one of the recombinant endo-beta-1,4-xylanases described above is located between the first Glu and the second Glu.
  • the first Glu is located within an amino acid region having a sequence of Asp-Val-Ala-Asn-Glu (SEQ ID NO: 25); and the second Glu is located within an amino acid region having a sequence of Thr-Glu-Ala-Asp (SEQ ID NO: 26).
  • Xaa 1 is one amino acid selected from H is, Lys, or Arg; and Xaa 2 is one amino acid selected from Leu, Ala, Val, Ile, Pro, Phe, Met, or Trp.
  • the region of the amino acid sequence of any of the recombinant endo-beta-1,4-xylanases described above is Pro-His-Pro-Leu-Pro (SEQ ID NO: 27).
  • any of the recombinant endo-beta-1,4-xylanases described above can be active at pH values of about 3 to 9. In preferred embodiments, any of the endo-beta-1,4-xylanases described above have a pH optimum of about 4.5-6.0. In yet other embodiments, any of the recombinant endo-beta-1,4-xylanases described above have a molecular weight of about 40-48 kD. In further embodiments, any of the recombinant endo-beta-1,4-xylanases described above have activity at a temperature of at least 80° C.
  • any of the recombinant endo-beta-1,4-xylanases described above are active at least from 80-120° C.
  • any of the recombinant endo-beta-1,4-xylanases described above have optimal activity at about 90° C.
  • any of the recombinant endo-beta-1,4-xylanases described above retain at least 50% of initial activity, at 90° C., after incubation for at least 20 min, at least 30 min, at least 45 min, at least 60 min, at least 90 min, at least 2 hr, at least 5 hr, at least 8 hr, at least 12 hr, at least 24 hr, or at least 48 hr, at 90° C.
  • any of the recombinant endo-beta-1,4-xylanases described above have a half-life of about 90 min at 90° C. in the presence of a xylan.
  • the xylan is birchwood xylan, beech wood xylan, or oat spelt xylan.
  • any of the recombinant endo-beta-1,4-xylanases described above further include a signal sequence peptide having amino acid sequence SEQ ID NO: 2.
  • any of the recombinant endo-beta-1,4-xylanases described above has a substrate selected from lignocellulosic biomass, xylan-containing material, or xyloglucan-containing material.
  • the present disclosure also pertains to a recombinant cell comprising a nucleic acid molecule encoding any of the recombinant endo-beta-1,4-xylanases described above.
  • the present disclosure further pertains to a recombinant cell that expresses a nucleic acid molecule that has a nucleotide sequence with at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, or at least 93% sequence identity to SEQ ID NO: 4, or its complementary sequence SEQ ID NO: 5.
  • the nucleic acid sequence of the nucleic acid molecule has at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 4, or its complementary sequence SEQ ID NO: 5.
  • the nucleic acid sequence of the nucleic acid molecule is SEQ ID NO: 4, or its complementary sequence SEQ ID NO: 5.
  • the present disclosure also pertains to a method of hydrolyzing lignocellulose by contacting the lignocellulose with a recombinant endo-beta-1,4-xylanase having an amino acid sequence with at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 1.
  • the lignocellulose is contacted with the recombinant endo-beta-1,4-xylanase at a temperature of at least 80° C., at least 85° C., at least 90° C., at least 95° C., or at least 100° C.; and a pH range from about 4.5-6.0.
  • the amino acid sequence of the recombinant endo-beta-1,4-xylanase is SEQ ID NO: 1.
  • the present disclosure further pertains to a method of hydrolyzing lignocellulose by contacting the lignocellulose with any of the recombinant endo-beta-1,4-xylanases described above.
  • the lignocellulose is contacted with the recombinant endo-beta-1,4-xylanase at a temperature of at least 80° C., at least 85° C., at least 90° C., at least 95° C., or at least 100° C.; and a pH range from about 4.5-6.0.
  • the lignocellulose of any of the methods described above is from a source selected from birchwood, oat spelt, switchgrass, corn stover, miscanthus, energy cane, sorghum, eucalyptus, willow, bagasse, hybrid poplar, short-rotation woody crop, conifer softwood, crop residue, yard waste, or a combination thereof.
  • the recombinant endo-beta-1,4-xylanase of any of the methods described above further includes a signal sequence peptide having amino acid sequence SEQ ID NO: 2.
  • FIG. 2 depicts a diagram of the A. cellulolyticus xyl-1 gene Acel — 0372.
  • FIG. 3 is an image of an agarose gel, depicting the results of PCR amplification of the xyl-1 gene Acel — 0372. Lane 1, molecular weight marker; lanes 2 and 3, PCR products from amplifications containing two different concentrations of A. cellulolyticus genomic DNA as the template.
  • FIG. 4 is an image of an agarose gel depicting the results of purified plasmid clones containing the amplified xyl-1 gene Acel — 0372.
  • Lane 2 contains a pK19 construct that does not contain the 1.4-kb PCR product.
  • FIGS. 5 (A) and 5 (B) depict the nucleotide coding sequence of the PCR product amplified from A. cellulolyticus (SEQ ID NO: 4), the nucleotide complementary sequence of the PCR product amplified from A. cellulolyticus (SEQ ID NO: 5), and the amino acid sequence encoded by the PCR product amplified from A. cellulolyticus (SEQ ID NO: 3).
  • FIGS. 6 (A), 6 (B), and 6 (C) depict the expression of Xyl-1 in A. cellulolyticus .
  • A RT-PCR analysis of the xyl-1 gene.
  • B RT-PCR analysis of an internal control housekeeping gene, gyrB. M: molecular-weight DNA ladder, lanes 1-4: exponential growth phase samples, 5-8: stationary growth phase samples, and 9: no-RT negative control to confirm the absence of contaminating genomic DNA.
  • Lanes 1 and 5 oat-spelt xylan-grown culture sample
  • 2 and 6 cellobiose-grown culture
  • 3 and 7 cellulose-grown culture
  • 4 and 8 glucose-grown culture.
  • (C) Representation of the peptide coverage of the Xyl-1 protein (389 aa) from tandem mass spectrometry of A. cellulolyticus culture supernatant. Hatched box indicates the N-terminal signal peptide, and black boxes indicate the positions of five non-overlapping peptides identified from the spectra.
  • the peptides are as follows, 1: HGNPPYHPPADSLR (SEQ ID NO: 6), 2: WQVVEPTQGTYDWSGGDR (SEQ ID NO: 7), 3: LVQFAQEHGQLVR (SEQ ID NO: 8), 4: HIVDEVTHFK (SEQ ID NO: 9), and 5: PAYTALQQTLALAAGAPHR (SEQ ID NO: 10).
  • FIG. 7 depicts the full 451-bp untranslated intergenic region between the Acel — 0373 and Acel — 0372 (xyl-1) open reading frames.
  • the sequence contains the xyl-1 promoter region.
  • the putative -10 and -35 sequences are shown in bold and the putative Shine-Dalgarno ribosomal binding site (RBS) is also shown in bold.
  • the Xyl-1 protein coding region is shown as a bold arrow at the end of the sequence.
  • Three inverted repeats (IR-1, IR-2, and IR-3) are shown with bold letters and boxes.
  • IR-1 GAAACTTTC (SEQ ID NO: 11)
  • IR-2 TTTCCGAAA
  • IR3 TCCGAAAATTTCGGA (SEQ ID NO: 13).
  • FIGS. 8 (A) and 8 (B) depict the purification of Xyl-1 by FPLC following heat treatment at 65° C. for 15 min.
  • A Protein elution from the ion exchange column during the FPLC run as KCl concentration was increased. The black line is UV absorbance at 280 nm, the thin grey line indicates conductivity, and the thick grey line indicates the KCl gradient.
  • B Activity profile (activity measured at 410 nm) at 52° C., pH 5.2 was used to identify the fractions containing Xyl-1 that were then combined and concentrated.
  • FIGS. 9 (A) and 9 (B) depict the purification of Xyl-1 from E. coli .
  • A Coomassie stained gel.
  • B In-gel assay (carried out at 52° C., pH 5.2). Leftmost lane, molecular weight markers (in kD).
  • CCE crude cell extract from E. coli clone
  • ⁇ H crude cell extract from E. coli clone after heat treatment (15 min at 65° C.
  • Main concentrated active fractions from the ion exchange column
  • Side concentrated side fractions.
  • Asterisks indicate Xyl-1.
  • FIG. 10 depicts the purification of Xyl-1 by hydroxyapatite chromatography.
  • SDS-PAGE (10% gel) analysis showing molecular weight markers (lane 1), crude cell extract from DH5 ⁇ (pK19-xyl-1) (lane 2), heat treated (65° C. for 20 min followed by centrifugation) crude cell extract from DH5 ⁇ (pK19-xyl-1) (lane 3), concentrated fractions from the hydroxyapatite column (lane 4).
  • FIG. 11 depicts the relative activity of the partially purified Xyl-1 at various pH values and temperatures. Activity is relative to the highest activity at each pH.
  • FIG. 12 depicts a multiple sequence alignment of Xyl-1 with other xylanases.
  • a 295 amino acid fragment of Xyl-1 (residues 67-361) was aligned with eleven other homologs using Clustal X version 2.0 (26).
  • Stars (*) indicate fully conserved sites, while colons (:) and periods (.) indicate sites with high and low degrees of conservation, respectively.
  • the bar above the A_ce sequence indicates the unique Xyl-1 region of five amino acids containing three prolines.
  • accession numbers of the proteins are as follows: A_ce ( A.
  • FIGS. 13 (A) and 13 (B) are images of an agarose gel that depicts an in-gel xylanase activity assay in E. coli crude cell extracts.
  • Lanes ST molecular weight standards (in kD);
  • A crude cell extracts from the vector control strain;
  • lane 2 crude cell extracts from the Xyl-1 clone;
  • B lane 3, crude cell extracts from the vector control after heat treatment (15 min at 65° C.);
  • lane 4 crude cell extracts from the Xyl-1 clone strain after heat treatment. Assayed at 52° C., pH 5.2.
  • FIG. 14 is an image of a polyacrylamide gel that depicts xylanase activity in concentrated culture supernatants from the E. coli clone and Acidothermus (after growth on cellobiose) in the in-gel assay.
  • Lanes ST molecular weight standards (in kD); lane 1, Acidothermus culture supernatant; lane 2, Xyl-1 clone culture supernatant; lane 3, E. coli vector control culture supernatant. Assayed at 52° C., pH 5.2.
  • FIG. 15 is an image of a polyacrylamide gel that depicts the activity of recombinant Xyl-1 in E. coli crude cell extracts at temperatures ranging from 0-100° C. in the in-gel assay at pH 5.2.
  • FIG. 16 is an image of a polyacrylamide gel that depicts activity of the recombinant Xyl-1 in E. coli crude cell extracts at pH values from 3-10 in the in-gel assay. Assays were carried out at 52° C.
  • FIG. 17 is an image of a polyacrylamide gel that depicts activity of the recombinant Xyl-1 in E. coli crude cell extracts following heat treatment at 80° C. for 20 min. The assay was carried out at 52° C. and at pH 5.2.
  • FIG. 18 depicts the activity of the recombinant Xyl-1 in E. coli crude cell extracts following heat treatment for 20 min. The assay was carried out at 52° C., pH 5.2.
  • FIG. 19 depicts the specific activity of the recombinant Xyl-1 in E. coli crude cell extracts at temperatures from 0-120° C. at pH 5.2.
  • FIGS. 20 (A) and 20 (B) depict the temperature and pH profile of purified Xyl-1.
  • Results are the averages of at least three independent experiments using the reducing sugars assay and oat spelt xylan as the substrate. Note that the activity ranges differ in A and B. Error bars indicate standard deviations.
  • FIGS. 21 (A) and 21 (B) depict the thermostability of recombinant Xyl-1.
  • A Stabilization of the cloned Xyl-1 by xylan substrates. Purified Xyl-1 was diluted 1:20 with oat spelt xylan (light grey), birchwood xylan (grey) or phosphate buffer (white), and incubated at 90° C. for the times indicated. Rates were determined by the reducing sugars assay and are relative to the unheated Xyl-1 (black). Results are the averages of three independent experiments and error bars indicate standard deviations.
  • B Activity retained by Xyl-1 in the presence of 3.8% oat spelt xylan. The half-life of Xyl-1 appears to be approximately 1.5 hr at 90° C.
  • FIGS. 22 (A) and 22 (B) depict the results of a TLC time course of Xyl-1 activity with oat spelt (A) and birchwood (B) xylans.
  • the TLC was developed as described in Materials and Methods. Lanes 1 and 8, standard compounds: X1, xylose; X3, xylotriose; X4, xylotetraose. Lane 2, unreacted xylan; lanes 3-7, increasing time of incubation in the presence of purified Xyl-1. Lane 3, 10 min; lane 4, 20 min; lane 5, 40 min; lane 6, 60 min; lane 7, 240 min.
  • SEQ ID NO: 1 shows the amino acid sequence of Xyl-1 without the N-terminal signal sequence.
  • SEQ ID NO: 2 shows the amino acid sequence of the N-terminal Xyl-1 signal sequence.
  • SEQ ID NO: 3 shows the amino acid sequence encoded by the PCR product amplified from A. cellulolyticus.
  • SEQ ID NO: 4 shows the nucleotide coding sequence of the PCR product amplified from A. cellulolyticus.
  • SEQ ID NO: 5 shows the nucleotide complementary sequence of the PCR product amplified from A. cellulolyticus.
  • SEQ ID NO: 6 shows the amino acid sequence of the first of five non-overlapping Xyl-1 peptides identified by tandem mass spectrometry of A. cellulolyticus culture supernatant.
  • SEQ ID NO: 7 shows the amino acid sequence of the second of five non-overlapping Xyl-1 peptides identified by tandem mass spectrometry of A. cellulolyticus culture supernatant.
  • SEQ ID NO: 8 shows the amino acid sequence of the third of five non-overlapping Xyl-1 peptides identified by tandem mass spectrometry of A. cellulolyticus culture supernatant.
  • SEQ ID NO: 9 shows the amino acid sequence of the fourth of five non-overlapping Xyl-1 peptides identified by tandem mass spectrometry of A. cellulolyticus culture supernatant.
  • SEQ ID NO: 10 shows the amino acid sequence of the fifth of five non-overlapping Xyl-1 peptides identified by tandem mass spectrometry of A. cellulolyticus culture supernatant.
  • SEQ ID NO: 11 shows the nucleotide sequence of the first of three inverted repeats (IR-1) located in the xyl-1 promoter region.
  • SEQ ID NO: 12 shows the nucleotide sequence of the second of three inverted repeats (IR-2) located in the xyl-1 promoter region.
  • SEQ ID NO: 13 shows the nucleotide sequence of the third of three inverted repeats (IR-3) located in the xyl-1 promoter region.
  • SEQ ID NO: 14 shows the nucleotide sequence of the forward primer used to PCR amplify Acel — 0372.
  • SEQ ID NO: 15 shows the nucleotide sequence of the reverse primer used to PCR amplify Acel — 0372.
  • SEQ ID NO: 16 shows the nucleotide sequence of a forward primer specific to the xyl-1 gene.
  • SEQ ID NO: 17 shows the nucleotide sequence of a reverse primer specific to the xyl-1 gene.
  • SEQ ID NO: 18 shows the nucleotide sequence of a forward gyrB gene-specific primer.
  • SEQ ID NO: 19 shows the nucleotide sequence of a reverse gyrB gene-specific primer.
  • SEQ ID NO: 20 shows the nucleotide sequence of the putative xyl-1 ribosomal binding site (RBS).
  • SEQ ID NO: 21 shows the nucleotide sequence of the conserved sequence found at the 3′ end of the A. cellulolyticus 16S ribosomal rRNA that is complimentary to the RBS sequence.
  • SEQ ID NO: 22 shows the amino acid sequence of the N-terminal sequence of the recombinant Xyl-1.
  • SEQ ID NO: 23 shows the conserved amino acid sequence of the first active site glutamate region of GH10 family xylanases.
  • SEQ ID NO: 24 shows the conserved amino acid sequence of the second active site glutamate region of GH10 family xylanases.
  • SEQ ID NO: 25 shows the amino acid sequence of the first active site glutamate region of Xyl-1.
  • SEQ ID NO: 26 shows the amino acid sequence of the second active site glutamate region of Xyl-1.
  • SEQ ID NO: 27 shows the amino acid sequence of the region containing three prolines close to the first active site glutamate of Xyl-1.
  • SEQ ID NO: 28 shows the amino acid sequence of a 295 amino acid fragment of Xyl-1 (i.e., residues 67-361)
  • SEQ ID NO: 29 shows the amino acid sequence of Catenulispora acidiphila DSM 44928 (GI:229247007) xylanase.
  • SEQ ID NO: 30 shows the amino acid sequence of Cellulosimicrobium sp. HY-12 (GI:162414427) xylanase.
  • SEQ ID NO: 31 shows the amino acid sequence of Cellulomonas fimi ATCC 484 (GI:73427793) xylanase.
  • SEQ ID NO: 32 shows the amino acid sequence of Streptomyces thermoviolaceus (GI:38524461) xylanase.
  • SEQ ID NO: 33 shows the amino acid sequence of Thermoascus aurantiacus (GI:13432255) xylanase.
  • SEQ ID NO: 34 shows the amino acid sequence of Phanerochaete chrysosporium (GI:167599628) xylanase.
  • SEQ ID NO: 35 shows the amino acid sequence of Thermobifida alba (GI:1621277) xylanase.
  • SEQ ID NO: 36 shows the amino acid sequence of Streptomyces avermitilis (GI:29828638) xylanase.
  • SEQ ID NO: 37 shows the amino acid sequence of Cryptococcus adeliensis (GI:2624008) xylanase.
  • SEQ ID NO: 38 shows the amino acid sequence of an uncultured bacterium (GI:18476191) xylanase.
  • SEQ ID NO: 39 shows the amino acid sequence of Thermotoga maritima (GI:71041762) xylanase.
  • SEQ ID NO: 40 shows the amino acid sequence of a Xyl-1 proline rich consensus sequence.
  • thermoostable refers to a threshold level of xylanase activity after an incubation period of 15 min at a temperature of 65° C.
  • active and “activity” refer to an endo-beta-1,4-xylanase giving a positive result using the in-gel xylanase assay or the reducing sugar assay described below.
  • percent activity refers to the amount of endo-beta-1,4-xylanase activity measured at given experimental conditions compared to base-line xylanase activity. The measured xylanase activity at experimental conditions is divided by the base-line xylanase activity and multiplied by 100 to obtained percent activity.
  • base-line xylanase activity and “baseline control” refer to the amount of xylanase activity produced by an endo-beta-1,4-xylanase when assayed at 55° C. and pH 5.2 for 10 min.
  • half-life refers to the length of time necessary for an endo-beta-1,4-xylanase activity to drop by 50% (compared to base-line control) at a given temperature.
  • optimal activity refers to peak xylanase activity in a given temperature range or range of pH values.
  • percent “identical,” “percent identity,” and “percent sequence identity” are defined as amount of identity between a reference nucleic acid or amino acid sequence and at least one other nucleic acid or amino acid sequence. Percent sequence identity can be determined by comparing two optimally aligned sequences, wherein the portion of the sequence being compared may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., a nucleic acid or amino acid sequence of the disclosure), which does not comprise additions or deletions, for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions being compared and multiplying the result by 100 to yield the percentage of sequence identity.
  • Two sequences have percent identity if two sequences have a specified percentage of nucleic acids or amino acid residues that are the same (i.e., 75% identical over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • BLAST algorithm is described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • W wordlength
  • E expectation
  • BLASTP program is used with default settings of a wordlength of 3, and expectation (E) of 10
  • Acidothermus cellulolyticus 11B (ATCC 43068) is a thermophilic bacterium that was originally isolated from an acidic hot spring in Yellowstone National Park. A number of thermostable endoglucanases are produced by this organism, which are useful for degrading cellulose in the production of ethanol or other hydrocarbons for biofuel.
  • the genome of Acidothermus cellulolyticus 11B (henceforth referred to as A. cellulolyticus ) has been sequenced to completion (Refseq NC — 008578) and the sequence has been annotated.
  • the sequence and sequence annotation have provided information on the regulation and production of potentially useful enzymes (published as accession number NC — 008578). Among these potentially useful enzymes, the A.
  • cellulolyticus sequence annotation predicted a single putative endo-beta-1,4-xylanase, which is encoded by Acel — 0372 (from the A. cellulolyticus genome annotation). It is believed that there are no reports of this endo-beta-1,4-xylanase having been previously cloned, and the predicted molecular weight does not match the previously disclosed A. cellulolyticus xylanase (U.S. Pat. No. 5,902,581). Therefore, this predicted endo-beta-1,4-xylanase was cloned from A. cellulolyticus , and expressed as a recombinant protein.
  • the expressed protein encoded by Acel — 0372 was identified as a glycosyl hydrolase family 10 (GH10) endo-beta-1,4-xylanase enzyme.
  • This A. cellulolyticus GH10 family xylanase was named Xyl-1. Characterization of Xyl-1 revealed that it is a thermophilic xylanase that has a temperature optimum of 90° C. and a pH optimal range of about 4.5-6.0. Xyl-1 is also characterized by retaining xylanase activity at a temperature range of 25-120° C., and a pH range of 3-9. Xyl-1 also has a molecular weight of about 40-48 kD.
  • Xyl-1 is distinct from the A. cellulolyticus xylanase disclosed in U.S. Pat. No. 5,902,581 in that it has an optimal temperature range that is higher than the 70-80° C. range of the xylanase disclosed in U.S. Pat. No. 5,902,581, has a pH range that is less acidic than the pH range of 3.6-4.2 of the xylanase disclosed in U.S. Pat. No. 5,902,581, and is smaller in size than the 50-55 kD xylanase disclosed in U.S. Pat. No. 5,902,581.
  • A. cellulolyticus produces a second xylanase (i.e., Xyl-1), different in optimal temperature and pH range from the previously identified xylanase (U.S. Pat. No. 5,902,581).
  • Xyl-1 is useful in degrading (e.g., hydrolyzing) xylan-containing material, xyloglucan-containing material, and lignocellulosic biomass in the production of ethanol or other hydrocarbons for biofuel.
  • Xyl-1 can be used to hydrolyze lignocellulosic biomass from birchwood, oat spelt, switchgrass, corn stover, miscanthus, energy cane, sorghum, eucalyptus, willow, bagasse, hybrid poplar and other short-rotation woody crops, various species of conifer softwood, various crop residues, or yard waste in order to improve the availability of fermentable sugars in these substrates.
  • the hydrolysis step is preferably performed at temperatures from 50° C.-100° C. and at acidic pH values.
  • the stability of Xyl-1 over a wide range of temperatures and pH values provides versatility in industrial processes. Additionally, Xyl-1 provides long-lived and persistent xylanase activity under fluctuating conditions, which can occur in large-scale bioconversion systems. These conditions may further allow for the advantageous coupling of lignocellulose hydrolysis to lignocellulose fermentation.
  • Xyl-1 can also be used to bleach lignocellulosic pulp as a step in the process of making paper, to improve animal feed by making feed more digestible, or Xyl-1 can be used as a component in a detergent composition.
  • Acel — 0372 encodes a predicted secreted xylanase (Xyl-1).
  • A. cellulolyticus 11B (ATCC 43068) genomic DNA was purified as previously described (15), and oligonucleotide primers were designed in order to PCR amplify the complete Acel — 0372, the predicted 1.4-kb Xyl-1 gene from A. cellulolyticus ( FIG. 2 ).
  • the primer pair used was Xyl For (5′-GTG GTG GAG CTC GCA ATT CGT TCA CGT TGA GG-3′) (SEQ ID NO: 14) and Xyl Rev (5′-GTG GTG TCT AGA ACC ATC GAG TGG GAG TGA CG-3′) (SEQ ID NO: 15).
  • the underlined sequences indicate the added restriction sites for cloning, Sad and XbaI, respectively.
  • PCR was performed using the following program using Pfu: an initial denaturation at 95° C. for 3 min, followed by 25 cycles of 95° C. for 1 min, 55° C. for 1 min, 70° C. for 1 min, and then a final extension at 70° C. for 5 min.
  • the PCR yielded a 1.4-kb product, which is the xyl-1 gene ( FIG. 3 ).
  • the resulting 1.4-kb PCR product was purified from an agarose gel with a QIAquick Gel Extraction kit (Qiagen, Valencia, Calif.), digested with Sad and XbaI and ligated to SacI-XbaI-digested pK19 (16).
  • E. coli DH5 ⁇ cells were transformed with plasmid DNA by standard procedures (18). Clones were confirmed by restriction digestion with Sad and XbaI ( FIG. 4 ).
  • DNA sequence analysis was performed on the positive pK19 clones containing the xyl-1 gene ( FIG. 5 ). Fluorescent automated DNA sequencing was carried out at the University of California, Davis sequencing facility with an Applied Biosystems 3730 automated sequencer. Nucleotide and amino acid sequence analyses were performed using the Vector NTI software suite (Invitrogen, Carlsbad, Calif.). The complete sequence of the xyl-1 gene was identical to the sequence reported for Acel — 0372.
  • A. cellulolyticus cultures were grown in supplemented mineral medium as described previously (12). Oat spelt xylan, cellulose, cellobiose, or glucose were provided individually as carbon sources at 0.5%. Cells were grown to mid exponential phase or stationary phase and cells were harvested by centrifugation at 10,000 ⁇ g for 15 min at 4° C. The cell pellet was frozen in liquid nitrogen and cells were disrupted by grinding with a mortar and pestle prechilled in liquid nitrogen, in the presence of sterilized sand. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen), with several rounds of DNase digestion with RNase-free DNase (Qiagen), and cleaned with the RNeasy Plant Mini Kit.
  • Primers specific to the xyl-1 gene (primers: 5′-CAAAGGAAAGATCTGGCAATG-3′ (SEQ ID NO: 16) and 5′-TGAGCATCCCGTCGTAGTAGT-3′ (SEQ ID NO: 17)) were used to amplify a 485-bp product from 100 ng total RNA template using reverse transcriptase polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the Qiagen OneStep RT-PCR kit was used. The amplified products were analyzed on agarose gels and photographed using the Red gel imaging system (Alpha Innotech).
  • the gyrB gene-specific primers 5′-GACCCGACCGAGGTTTATTAC-3′ (SEQ ID NO: 18) and 5′-GCCGAACTTGTTCACCAAATA-3′ (SEQ ID NO: 19) were used to amplify a 270-bp product that was used to normalize for the RNA loaded as well as for semi-quantification of the xyl-1 expression.
  • RT-PCR was repeated using a second batch of RNA that was extracted independently under all growth conditions. Identical results were obtained in duplicate experiments, and data from one of the replicates are presented.
  • A. cellulolyticus was grown on oat spelt xylan to stationary phase and culture supernatant was harvested by centrifugation.
  • the culture supernatant was concentrated by a factor of 50 using an Amicon stirred ultrafiltration cell carrying a polyethersulfone membrane with 5-kDa molecular weight cutoff (Millipore).
  • Amicon stirred ultrafiltration cell carrying a polyethersulfone membrane with 5-kDa molecular weight cutoff (Millipore).
  • the sample was subjected to liquid chromatography followed by tandem mass spectrometry (LC/MS/MS) at the University of California Davis Proteomics Core facility.
  • the transcriptional expression of the A. cellulolyticus xyl-1 gene was studied during exponential as well as stationary phases of growth on oat spelt xylan, cellulose, cellobiose, and glucose.
  • the RT-PCR analysis revealed that xyl-1 was expressed in xylan- and cellulose-grown cultures ( FIG. 6A ).
  • the gene was also expressed at low levels in cellobiose-grown cultures during stationary phase. No expression was detectable in glucose-grown cultures at any time or in cellobiose-grown cultures during exponential phase. In xylan- and cellulose-grown cultures, xyl-1 expression appeared to be essentially the same during the exponential and stationary phases of growth.
  • the gyrB gene was used as an internal control to normalize the expression levels of the xyl-1 gene ( FIG. 6B ).
  • Tandem mass spectrometry verified the presence of Xyl-1 in the culture supernatant ( FIG. 6C ) and indicated that the N-terminal 25 aa signal peptide, which targets proteins for secretion, was cleaved from the mature protein.
  • the xyl-1 gene was determined to be on the negative strand and is separated by 147 by from the divergently oriented upstream gene, and 451 by from the downstream gene on the negative strand. The functions of both of these flanking genes appear to be unrelated to xylanases. Thus, xyl-1 does not appear to be part of a gene cluster or operon. Analysis of the 451-bp upstream promoter region revealed a putative Shine-Dalgarno ribosomal binding site (RBS) six nucleotides upstream of the translational start.
  • RBS Shine-Dalgarno ribosomal binding site
  • the RBS sequence (5′ TGGAGG 3′) (SEQ ID NO: 20) is complementary to the conserved CCTCCT (SEQ ID NO: 21) sequence found at the 3′ end of the A. cellulolyticus 16S ribosomal rRNA, with only a one-base mismatch ( FIG. 7 ). Putative-35 and -10 sequences were also identified ( FIG. 7 ) that match well with the consensus promoter motifs proposed in the closely-related actinomycete, Streptomyces (20), and are likely to be the ⁇ 70 promoter sequences of the A. cellulolyticus xyl-1 gene.
  • the distal IR-1 sequence forms a short palindrome (5′ GAAA-C-TTTC 3′) (SEQ ID NO: 11) and the downstream IR-3 palindrome extends three nucleotides longer (5′ TCCGAAA-A-TTTCGGA 3′) (SEQ ID NO: 13), whereas the Box 3 palindrome is one-nucleotide longer than the Box 1 in Streptomyces .
  • the A. cellulolyticus IR-2 sequence (5′ TTTC-C-GAAA 3′) (SEQ ID NO: 12) is almost identical to the Streptomyces Box 2 sequence.
  • the xyl-1 IR-2 element overlaps with IR-1 ( FIG. 7 ). Furthermore, in A. cellulolyticus , the IR-1 and IR-2 regions are located downstream of the putative-35 and -10 elements and closer to the translational start site, while both of the corresponding boxes are upstream of the promoter, and farther away from the first codon in Streptomyces.
  • E. coli DH5 ⁇ cells containing the cloned xyl-1 gene were grown in a minimal salts medium containing 10 mM glucose, 1 mM thiamine and 100 ⁇ g/ml kanamycin at 30° C. for 48 hr. Cells were harvested by centrifugation (14,000 ⁇ g at 4° C. for 15 min), washed once with 10 mM phosphate buffer (pH 5.2), resuspended in the same buffer and stored frozen at ⁇ 20° C. Frozen cell suspensions were removed from the freezer and allowed to thaw on ice. The thawed cell suspension was then passed once through a French pressure cell maintaining the internal cell pressure a constant 18,000 psi.
  • Unbound proteins were eluted from the column with 100 ml of the same buffer at a rate of 1.0 ml/min. Bound proteins were eluted with a linear gradient from 0 to 1.0 M KCl in phosphate buffer at the same flow rate and 5 ml fractions were collected.
  • Extracts of the E. coli clone were subjected to heat treatment at 65° C. for 15 min prior to loading the extract onto an ion exchange column.
  • Xyl-1 protein eluted as one major peak with activity ( FIG. 8 ).
  • This heat treatment eliminated many of the E. coli proteins and resulted in a significant purification of Xyl-1 ( FIG. 9 ).
  • the concentrated protein was >90% pure, as determined by SDS-PAGE analysis ( FIGS. 9 and 10 ).
  • Approximately 2 mg of purified Xyl-1 was obtained from 4 L of E. coli culture.
  • the recombinant Xyl-1 had a molecular weight of approximately 40-48 kD on SDS gels ( FIGS. 9 and 10 ), which is consistent with the predicted molecular weight based on the deduced amino acid sequence of the processed protein (40.3 kD).
  • a temperature vs. pH profile with the partially purified Xyl-1 demonstrated that the enzyme has a broad temperature range with optimal activity at approximately 90° C. and a broad pH range with an optimum between pH 4.5 and 6.0 ( FIG. 11 ).
  • the N-terminal sequence of the recombinant Xyl-1 expressed in E. coli was found to be HGNPPYHPPAD (SEQ ID NO: 22).
  • the first amino acid of this sequence mapped to position 26 in the full-length protein sequence, indicating that the predicted signal sequence of Xyl-1 was properly cleaved in the heterologous host, E. coli ( FIG. 5 ).
  • Xyl-1 expressed in E. coli lacks the secretion signal sequence.
  • a BLAST search was performed with the A. cellulolyticus GH10 family xylanase Xyl-1 amino acid sequence.
  • the top hit was from a xylanase of another actinomycete, Catenulispora acidiphila DSM 44928, which has 75% sequence identity with Xyl-1.
  • a recent biochemically-characterized xylanase from the actinomycete Cellulosimicrobium sp. HY-12 was the second best hit with 55% sequence identity to Xyl-1 (13).
  • Xyl-1 has valine-to-alanine and leucine-to-alanine substitutions in the two regions, resulting in DVANE (SEQ ID NO: 25) and TEAD (SEQ ID NO: 26) sequences, respectively ( FIG. 12 ).
  • DVANE SEQ ID NO: 25
  • TEAD SEQ ID NO: 26
  • homologous endo-beta-1,4-xylanases of the present disclosure may have a first glutamate at a position corresponding to Glu-142 of SEQ ID NO: 1.
  • the first glutamate is located within an amino acid region having the sequence of Asp-Val-Ala-Asn-Glu (SEQ ID NO: 25).
  • Homologous endo-beta-1,4-xylanases of the present disclosure may have a second glutamate at a position corresponding to Glu-259 of SEQ ID NO: 1.
  • the second glutamate is located within an amino acid region having the sequence of Thr-Glu-Ala-Asp (SEQ ID NO: 26).
  • T_ma A similar under-representation of these two amino acids was previously noted in the T_ma (7) protein and is thought to contribute to its high thermostability. However, most other features noted to provide thermostability to T_ma could not be identified in Xyl-1.
  • Xyl-1 contains a region of 5 amino acids, PHPLP (SEQ ID NO: 27), immediately downstream of the first active site glutamate that is different from its two closest homologs (C_ac and C_sp), as well as from other GH10 family xylanases ( FIG. 12 ).
  • the amino acid region includes three prolines in alternating positions.
  • Homologous endo-beta-1,4-xylanases of the present disclosure may have an amino acid region that includes three prolines that has an amino acid sequence that has at least 80% sequence identity with SEQ ID NO: 27.
  • the amino acid region has amino acid sequence SEQ ID NO: 27.
  • homologous endo-beta-1,4-xylanases may have an amino acid region that includes three prolines that has an amino acid sequence homologous to SEQ ID NO: 27, where the amino acid sequence has a conservative amino acid substitution in place of any one of the three prolines.
  • Homologous endo-beta-1,4-xylanases may also have an amino acid region that includes three prolines that has an amino acid sequence homologous to SEQ ID NO: 27, where the two non-proline amino acids of the region may be any amino acids.
  • Xyl-1 activity was monitored using a modified version of the previously described zymogram assay (8). Briefly, proteins were separated using SDS polyacrylamide gel electrophoresis (10%). After the gel was run, it was placed in 10 mM phosphate buffer (pH 5.2) and incubated with gentle shaking at 52° C. for 15 min. The buffer was removed and replaced with 1% xylan (1% xylan from oat spelts (Sigma) in 10 mM phosphate buffer [pH 5.2]) and incubated with gentle shaking at 52° C. for 15 min. The xylan solution was removed and the gel briefly rinsed with distilled water.
  • the gel was then placed in a solution of Congo red (0.2% in water) and incubated at room temp with gentle shaking for 20 min. The gel was destained with 1 M NaCl until the “cleared” band(s) could be visualized.
  • the in-gel assays were carried out from 4° C.-100° C. in heated or cooled water baths at pH 5.2. Activity at 0° C. was monitored in an ice bath. All solutions were preincubated to the assay temperature.
  • the assay was also modified to test the pH range of the enzyme by using phosphate buffer from pH 2.0 to pH 10.
  • E. coli cells expressing the recombinant Xyl-1 were tested for xylanase activity, using oat spelt xylan as the substrate. Extracts of E. coli carrying the cloned gene had xylanase activity (tested at 52° C., pH 5.2), and activity was retained after a heat treatment of 65° C. for 15 min ( FIG. 13 ). No activity was present in extracts of the strain carrying pK19 only (vector control). Activity was also observed with birchwood xylan (data not shown). Some of the protein was present in the E. coli culture supernatant and it exhibited electrophoretic mobility identical to that of the exported xylanase present in concentrated A. cellulolyticus culture supernatants from cells grown with cellobiose as the carbon source ( FIG. 14 ).
  • In-gel xylanase activity was measured using the zymogram assay described in Example 7.
  • Xylan substrates (oat spelt xylan or birchwood xylan, Sigma) were made by adding 1% xylan to unbuffered 10 mM phosphate solution. The slurry was incubated at 55° C. for 20 min with gentle shaking. Insoluble material was allowed to settle at room temperature. The supernatant was removed and the pH was adjusted with HCl or NaOH. Two ml of xylan suspension was added to a screw cap tube and incubated in a water bath at the desired temperature for 10 min.
  • Xylanase was added and 200 ⁇ l samples were taken at various time points and added to 800 ⁇ l PABAH (0.5% p-hydroxybenzoic acid hydrazide in 0.5 M NaOH). Samples were boiled for exactly 5 min, allowed to cool at room temp for 10 min and the absorbance at 410 nm was determined. Results were compared to a standard curve for xylose. This assay was carried out at various temperatures and pH values to determine the temperatures and pH values at which the xylanase is active. Activity was also tested in an autoclave at 121° C. To carry out this assay, the components were mixed on ice and placed in the autoclave set to 121° C. for 10 min. The amount of reducing sugars released was determined in the presence of PABAH as described above. Protein concentrations were determined by the Bradford method (3) using bovine serum albumin as the standard.
  • Xyl-1 has an optimal activity at approximately 90° C., which is approximately 225% higher relative to the activity determined at 55° C., when stabilized in the presence of xylan ( FIG. 19 ).
  • the specific activity of Xyl-1 from crude cell extracts was high, at temperatures between 55 and 100° C., with incubation with substrate, and the optimal activity was at approximately 90° C. ( FIG. 19 ).
  • Specific activity of Xyl-1 in crude cell extracts was measured as mg of xylose per min per mg of unpurified protein. Activity was tested in an autoclave in order to test activity at a temperature higher than 100° C. (the autoclave was set at 121° C.), and some activity was retained even in the autoclave (900 ⁇ g xylose/min/mg protein).
  • the activity of purified Xyl-1 was also characterized over a range of temperatures and pH values using the reducing sugars assay.
  • Purified Xyl-1 was active from 30 to 100° C., with an optimum temperature for activity of approximately 90° C. ( FIG. 20 ).
  • Specific activity of purified Xyl-1 was measured as mg of xylose per min per mg of purified Xyl-1.
  • Optimal activity was observed between pH 4.5 and pH 6.0 ( FIG. 20A ), but significant activity was present at pH values between pH 3 and pH 9 ( FIG. 20B ).
  • the optimum pH for the enzyme varied depending on the assay temperature.
  • Xyl-1 was active between pH 4 to pH 6 with an optimum pH of 5.0.
  • Purified Xyl-1 (1 mg/ml) was diluted 1:20 with 10 mM phosphate buffer (pH 6), or 4% oat spelt or birchwood xylan in 10 mM phosphate buffer (pH 6), mixed, and incubated on ice for a minimum of 15 min to ensure sufficient time for Xyl-1 to interact with the xylan before heat treatment. Aliquots of the control and xylan-pretreated Xyl-1 were then incubated at 90° C. for 10, 20, 40 or 60 min and immediately returned to ice. Activity was then determined using the reducing sugars assay (described in Example 5 above) at 90° C. and pH 6.
  • FIG. 21A The contribution of xylan substrates to the thermal stability of Xyl-1 was investigated.
  • Xyl-1 retained approximately 74 ⁇ 18%, 63 ⁇ 12%, 24 ⁇ 3%, and 5 ⁇ 1% of its activity after 10, 20, 40 and 60 min, respectively, at 90° C. ( FIG. 21A ).
  • no significant loss of activity was detected even after 1 hr at 90° C. in the presence of either oat spelt or birchwood xylan.
  • the major products from oat spelt xylan had R f values between those of xylose and xylotriose. These may also represent acylated oligosaccharide products of Xyl-1 action or backbone xylosyl residues that carried sugar side groups (e.g., arabinosyl residues) in the intact polysaccharide.
  • Lignocellulosic material such as switchgrass and corn stover
  • a fermentation organism engineered to express the recombinant Xyl-1, extracts containing the enzyme, or purified enzyme may be treated with a fermentation organism engineered to express the recombinant Xyl-1, extracts containing the enzyme, or purified enzyme. Treatment may be performed by combining the switchgrass or corn stover with organisms engineered to express the enzyme. Alternatively, the recombinant Xyl-1 may be added during the lignocellulose fermentation process.

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CN102245763A (zh) 2011-11-16
WO2010074821A1 (en) 2010-07-01
EP2367935A4 (en) 2012-07-04
MX2011006723A (es) 2011-08-03
EP2367935A1 (en) 2011-09-28

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