EP0746607A1 - Xylanase recombinante - Google Patents

Xylanase recombinante

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Publication number
EP0746607A1
EP0746607A1 EP93912444A EP93912444A EP0746607A1 EP 0746607 A1 EP0746607 A1 EP 0746607A1 EP 93912444 A EP93912444 A EP 93912444A EP 93912444 A EP93912444 A EP 93912444A EP 0746607 A1 EP0746607 A1 EP 0746607A1
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EP
European Patent Office
Prior art keywords
xylanase
expression
polypeptide
cdna
clones
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93912444A
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German (de)
English (en)
Other versions
EP0746607A4 (fr
Inventor
Gang Ping Xue
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commonwealth Scientific and Industrial Research Organization CSIRO
Newcastle University of Upon Tyne
Babraham Institute
Original Assignee
Commonwealth Scientific and Industrial Research Organization CSIRO
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Application filed by Commonwealth Scientific and Industrial Research Organization CSIRO filed Critical Commonwealth Scientific and Industrial Research Organization CSIRO
Publication of EP0746607A1 publication Critical patent/EP0746607A1/fr
Publication of EP0746607A4 publication Critical patent/EP0746607A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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)
    • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
    • 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
    • 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

  • This invention relates to a recombinant xylanase derived from an anaerobic fungus and a method of production of the recombinant xylanase and clones utilised in the method.
  • Xylan is a major component of hemicellulose and the second major component of plant fibre.
  • Xylan consists of a backbone of ⁇ - ⁇ ,4-linked xylose units. The enzymic cleavage of ⁇ -1 ,4-xylosidic linkages is performed by endo- ⁇ -1 ,4-xylanases (xylanases).
  • xylanases endo- ⁇ -1 ,4-xylanases
  • Many microorganisms produce extracellular xylanases.
  • many xylanase genes were isolated from lignocellulolytic bacteria, but isolation of xylanase genes from fungi with functional expression in E. coli has not been documented prior to this invention.
  • Lignocellulolytic fungi usually produce more active xylanase than bacteria, in particular, the anaerobic fungus Neocallimastix patriciarum, isolated from the sheep rumen, has a high capacity for xylan degradation.
  • Cloning of xylanase genes from bacteria can be achieved by isolation of enzymatically active clones from genomic libraries established in E. coli.
  • this approach for isolation of xylanase genes from fungal genomic libraries with functional expression of xylanase is not possible. This is because fungi are eucaryotic microorganisms. Most eucaryotic genes contain introns and E. coli is unable to perform post-transcriptional modification of mRNAs in order to splice out introns. Therefore, enzymatically functional protein cannot normally be synthesised in clones obtained from a fungal genomic library.
  • the cDNA cloning approach can be used to overcome the post- transcriptional modification problem in E. coli.
  • xylanases in fungi are usually glycosylated and glycosylation is often required for biological activity of many glycosylated enzymes.
  • E. coli lacks a glycosylation mechanism. This problem can be solved if the cloned gene is transferred to an eucaryotic organism, such as yeast.
  • yeast eucaryotic organism
  • coli are (i) that many eucaryotic mRNAs contain translational stop codons upstream of the translational start codon of a gene which prevents the synthesis of the cloned protein from the translational start provided in the vector, and (ii) that synthesis of the cloned protein is based on fusion proteins and the biological function of the cloned protein is often adversely affected by the fused peptide derived from the cloning vector.
  • a further object of the invention is to provide a method of cloning of xylanase cDNAs from an anaerobic rumen fungus which may encode the recombinant xylanase of the invention.
  • a further object of the invention is to provide xylanase clones which may be produced in the abovementioned method.
  • the method of cloning of the invention includes the following steps:
  • step (ii) isolating total RNA from the culture in step (i);
  • step (iii) isolating poly A + mRNA from the total RNA referred to in step (ii);
  • step (i) above in relation to preparation of the recombinant xylanase from anaerobic fungi, particularly alimentary tract fungi, may be cultivated as described hereinbelow.
  • These fungi are strict anaerobes and may be exemplified by Neocallimastix patriciarum, Neocallimastix frontalis, Neocallimastix hurleyensis, Neocallimastix stanthorpensis, Sphaeromonas communis, Caecomyces equi, Piromyces communis, Piromyces equi, Piromycesdumbonica, Piromyces lethargicus, Piromyces mai, Ruminomyces elegans, Anaeromyces mucronatus, Orpinomyces bovis and Orpinomyces joyonii.
  • anaerobic alimentary tract fungi Caecomyces equi, Piromyces equi, Piromyces dumbonica and Piromyces mai are found in horses and thus are not located in the rumen of cattle like the other fungi described above.
  • the cultivation may proceed in appropriate culture media containing rumen fluid and also may contain cellulose such as Avicel (ie. a form of microcrystalline cellulose) as a carbon source under anaerobic conditions.
  • Avicel ie. a form of microcrystalline cellulose
  • the fungal cells may be harvested by filtration and subsequently lysed in appropriate cell lysis buffer by mechanical disruption.
  • a suitable RNA preserving compound may also be added to the fungal cells to maintain the RNA intact by denaturing RNAses which would otherwise attack the fungal RNA.
  • the total RNA may subsequently be isolated from the homogenate by any suitable technique such as by ultracentrifugation through a CsCI 2 cushion or alternative technique as described by Sambrook et. al. in Molecular Cloning; A Laboratory Manual 2nd Edition Cold Spring Harbor Laboratory Press in 1989.
  • An alternative method for preparation of total fungal RNA to that described above may be based on or adapted from the procedure described in Puissant and Houdebine in Bio-Techniques 148-149 in 1990.
  • Total fungal RNA in this alternative technique may also be isolated from the above homogenate by extraction with phenol chloroform at pH4 to remove DNA and associated protein. Total RNA obtained was further purified by washing with lithium chloride-urea solution.
  • Poly (A) + mRNA may then be isolated from the total RNA by affinity chromatography on a compound containing multiple thymine residues such as oligo (dT) cellulose.
  • a compound containing multiple uracil residues may be used such as poly (U)-
  • the poly (A) + mRNA may then be eluted from the affinity column by a suitable buffer.
  • a cDNA expression library may then be constructed using a standard technique based on conversion of the poly (A) + mRNA to cDNA by the enzyme reverse transcriptase.
  • the first strand of cDNA may be synthesised using reverse transcriptase and the second strand of the cDNA may be synthesised using E. coli DNA polymerase I.
  • the cDNA may subsequently be fractionated to a suitable size and may be ligated to the bacteriophage expression vector, preferably ⁇ ZAP or ⁇ ZAPII.
  • the cDNA library may then be amplified after packaging in vitro, using any suitable host bacterial cell such as a suitable strain of E. coli.
  • step (v) The choice of the bacteriophage expression vector in step (v) is important in that such expression vector should include the following features:
  • the fusion peptide derived from the vector should be as small as possible as the biological function of the cloned protein is usually adversely affected by the fused peptide derived from the vector. Therefore the polyclonal sites of the bacteriophage expression vector are suitably located at the N-terminus of lacZ peptides such as in ⁇ ZAPII.
  • vectors of similar properties to .ZAP or ⁇ ZAPII includes within its scope expression vectors having the abovementioned features (i), (ii), (iii) and (iv).
  • the screening of xylanase positive recombinant clones may be carried out by any suitable technique based on hydrolysis of xylan.
  • the clones may be grown on culture media incorporating xylan and hydrolysis may be detected by the presence of xylanase- positive plaques suitably assisted by a suitable colour indicator.
  • Xylanase positive recombinant clones may then be purified and the cDNA insert in the clones may then be excised into pBluescript (SK(-)) to provide an expression vector of simplified structure when compared to the ⁇ ZAP II construct which will enhance expression of the xylanase in E. coli.
  • Any suitable E. coli promoter may be used in the expression vector described above. Suitable promoters include lacZ, Tac, Bacteriophage T 7 and lambda-P L .
  • the cloned xylanases have high specific activity, (ii) the enzyme has no residual activity against cellulose, while many other xylanases possess some cellulase activity. This property of the xylanase is particularly useful in its application to pulp and paper industry to remove xylan and dissociate lignin from plant fibre without damaging cellulose fibre.
  • the high specific activity of the cloned xylanases is an excellent intrinsic property of this fungal xylanases.
  • the expression level of the present constructs of xylanase cDNAs can be further improved by manipulating the gene and promoters.
  • Neocallimastix patriciarum type species was isolated from a sheep rumen by Orpin and Munn ( 1986) in Trans.
  • N. patriciarum was grown in a rumen fluid-containing medium as described in Kemp et. al. J. Gen. Microbiol. 130 27-37 ( 1984) in the present of 1 % Avicel at 39 °C and under anaerobic conditions for 48hr (Alternative culture media, such as described by Philips and Gordon in
  • Poly A + was purified from the total RNA by Oligo (dT) cellulose chromatography (Sambrook et. al., 1989). 6. Construction of a cDNA expression library of N. patriciarum.
  • the cDNA library was constructed, using Stratagene's ⁇ ZAP cDNA synthesis Kit, basically according to the manufacturer's instructions. The procedure is described briefly as follows: PolyA + , RNA was converted to the first strand cDNA by reverse transcriptase, using Xhol linker - oligo (dT) primer and 5-methyl dCTP. Double-stranded cDNA was synthesised from the first-strand cDNA by the action of RNase H and DNA polymerase I.
  • the cDNA was ligated with EcoR I adaptor, phosphorylated and digested with Xho1 to create cDNA with the EcoR I site at 5' region and the Xhol site at 3' region.
  • the cDNA was size-fractionated by 1 % low-melting point agarose gel electrophoresis and 1 .2-8Kb sizes of the cDNA were recovered by phenol extraction (Sambrook et. at., 1989).
  • the size-fractionated cDNA was then ligated to the EcoRI/XhoI digested ⁇ ZAPII vector.
  • the cDNA library was packaged in vitro and amplified using E. coli
  • Xylanase-producing phage plaques were surrounded by yellow haloes against a red background.
  • the xylanase-positive recombinant phage were purified to homogeneity by replating and rescreening the phage as above for 2-3 times.
  • the cDNA insert in xylanase-positive phage were excised into pBluescript SK (-) using R408 helper phage. 8. Xylanase and related-enzyme assays.
  • the cloned enzyme extracts from E. coli harbouring xylanase- positive recombinant plasmids were prepared by harvesting the cells by centrifugation. The cell pellet was suspended in 25mM Tris-CI/ 2mM EDTA containing lysozyme (0.25mg/ml) and incubated on ice for 60 mins. After freezing, thawing and homogenisation, the crude cell lysate was used for enzyme assays.
  • the enzymes were assayed for hydrolysis of xylan or other substrates at 40 °C in 50 mM Na-citrate, pH 6.5, except where otherwise indicated in the text.
  • the reducing sugars released from xylan or other plant polysaccharides (Avicel) were measured as described by
  • Xylanase activity on Kraft pulp was conducted as follows: Kraft pulp was suspended in tap water, and pH was adjusted to pH 7 with 1 M H 2 SO 4 . The xylanase extract was added to the Kraft pulp suspension and the reducing sugar released was measured as above.
  • Single-stranded plasmid DNA was prepared basically according to Stratagene's protocol. Sequencing of the resultant DNA was based on the protocol recommended by the manufacturer of the T7 DNA polymerase sequencing kit (Promega) .
  • a cDNA library consisting of 10 6 clones was constructed using mRNA isolated from N. patriciarum cells grown with Avicel as sole carbon source. Thirty-one recombinant bacteriophage, which hydrolysed xylan, were identified after an initial screening of 5 x 10 4 clones from the library and 1 6 strongly xylanase-positive phage and two weakly xylanase-positive phage were isolated and purified. Xylanase activity of these recombinant bacteriophage clones was initially analysed by scoring xylan-hydrolysis zones (Fig. 1 and Table 1 ).
  • pNPX30 has a similar length to pNPX21 but it has about 1 5-fold higher xylanase activity than pNPX21 . Because of the remarkable difference in enzyme activity between pNPX21 and pNPX30, the xylanase cDNA of pNPX30 clone was sequenced. The result shows that DNA sequence of pNPX30 shares the same sequence with pNPX21 in a large part of cDNA, but differ in both the 5' and 3' regions. (Fig. 3). pNPX30 cDNA is not full-length. Interestingly, the N-terminus of pNPX30 xylanase has six repeated arginine/glutamic acid residues (Fig. 4).
  • the pH and temperature optima of xylanases produced by pNPX21 and pNPX30 were investigated. These enzymes were active in a wide range of pH and preferably at pH 5 - 8. The thermostability of these enzymes was tested at temperatures from 30 °C - 60 °C. The enzymes are active at 30°C- 55 °C and preferably at 40°C - 50°C.
  • pNPX30 (and pNPX21 ) contains two large repeated domains. Three main constructs were produced from pNPX30.
  • pNXD-Tac pNPX30 plasmid (pNPX21 can also be used) was used as a template for in vitro DNA amplification by PCR for construction of pNXD- Tac using primer I and primer IV (Fig.5).
  • the amplified DNA was digested with EcoR 1 and Hind i 1 1 and ligated to EcoR1 and Hindi 1 1 digested pBTac2 (Boehringer) to produce pNXD-Tac.
  • pNXS-Tac pNXD-Tac plasmid was digested with Hind i 1 1 and blunted by filling-in with Klenow followed by partial digestion with Seal . After fractionation on LMT agarose gel, the 5.3Kb band was recovered from the gel and ligated to produce the pDGXS construct, which has xylanase activity.
  • pDGXS plasmid was used as a template for in vitro DNA amplification for construction of pNXS-Tac using primer I and primer II (Fig.5). The amplified DNA was digested with EcoR1 and Hindi 1 1 and ligated to EcoR1 and Hindi 1 1 digested pBTac2 vector to produce pNXS- Tac.
  • pNX-Tac pNPX30 plasmid (pNPX21 or other xylanase cDNAs listed in Fig.2 can be used) was digested with Rsal and a 709bp fragment as indicated in Fig.5 was isolated after fractionation on agarose gel electrophoresis. The 709 fragment was ligated to Sma1 and Pst1 digested pUC18 (Pst1 end was blunted with T4 DNA polymerase) .
  • This construct is designated pNXP2 and the xylanase activity of this construct with the right orientation of truncated xylanase cDNA from pNPX30 confirmed that this fragment of the cDNA encodes a caterlytically functional domain.
  • Two oligonucleotide primers, primer III and primer IV, (Fig.5) were then designed for PCR amplification of the pNXP2 xylanase cDNA insert. The PCR amplified fragment was digested with EcoR1 and Hind i 1 1 and ligated to EcoRI and Hind! 1 1 digested pBTac2 vector to produce pNX- Tac.
  • constructs are all modified at the N-terminal sequence of the truncated xylanase cDNA and a translational stop codon (TAA) was introduced into the end of the truncated xylanase coding region.
  • TAA translational stop codon
  • the expression of xylanase was controlled by the Tac promoter (Fig.6) and xylanases in these constructs are synthesised as nonfusion proteins.
  • the modified xylanase cDNA sequence in pNX-Tac is shown in Fig 7.
  • the specific activity of crude xylanase preparations of pNXD-Tac, pNXS-Tac and pNX-Tac clones were 228, 124 and 672 U/mg of total cellular protein of E.coli respectively, measured in 50mM Na-citrate buffer (pH6) and at 50°C (Fig.5).
  • the xylanase synthesised by the clone pNX-Tac was found mainly in the cell pellet, but a small amount of xylanase (about 5%) was released into the culture medium (Table 3).
  • the pNX-Tac xylanase has a temperature optimum at 50°C and retained > 80% of the maximum activity from 40°C to 55 °C, and 55% of the activity at 60°C (Fig.8).
  • pNX-Tac xylanase has a broad pH range (Fig 9) and is most active at pH5-7.5, 50% at pH8.5 and 20% at pH9.5.
  • the pNX-Tac xylanase has a high activity in the release of reducing sugar from Kraft pulp at 55 °C and in tap water (pH was adjusted to pH7 with H 2 SO 4 , see Fig.8) and remains active in the hydrolysis of xylan from the pulp at 55 °C and pH7 for at least 3hr (Fig.10)
  • the pNX-Tac xylanase is able to hydrolyse a significant amount of xylan from Eucalypt and Pine Kraft pulps (Table 4).
  • IPTG 2-fold higher xylanase yield than LBS.
  • Xylanase has many industrial applications, such as the pulp and paper industry, food processing, the feed industry and animal production industry.
  • the enzymes produced by these recombinant xylanase clones have no cellulase activity and have the pH and temperature profile (especially the genetically modified xylanase clone, pNX-Tac) fitted to conditions used for the enzymatic pre-treatment of pulp. Therefore it is believed that the xylanases of the present invention are applicable to the paper and pulp industry.
  • the xylanase of the invention could find a valuable application in the sugar industry and in relation to the treatment of bagasse or other products containing xylan for more efficient disposal as well as for the treatment of feedstock to improve nutritional value.
  • the genetically modified xylanase gene can also be used for modification of rumen bacteria to improve plant fibre utilization by ruminants.
  • the invention includes within its scope not only the recombinant xylanase described above but also xylanases derived from other anaerobic fungi as described above which may be prepared by the methods described herein.
  • the invention also includes within its scope: (i) DNA sequences derived from these xylanase cDNAs (particularly the sequences in pNPX30, pNXD-Tac, pNXS-Tac and pNX-Tac) and DNA sequences capable of hybridising thereto using a standard nucleic acid hybridisation technique as described in
  • Plasmid pNX-Tac in E. coli strain JM83 has been deposited at the International Depository ie. Australian Government Analytical Laboratories 17 March 1993 under accession number N93/1221 1 .
  • the cloning method of the invention is based upon obtaining a large number of recombinant xylanase clones with strong xylanase activity from an anaerobic rumen fungus such as N. patriciarum which were functionally expressed in E. coli.
  • This approach for isolation of fungal xylanase or other plant polysaccharide hydrolases such as cellulases has not been documented prior to this invention.
  • the approach used in this invention is very efficient and requires only a single cloning step to obtain biologically functional recombinant xylanases from an anaerobic fungus. Therefore it takes much less time to obtain biologically functional xylanase clones from a fungal source compared to previous approaches for isolation of plant polysaccharide hydrolases from fungi which are described in the prior art discussed above.
  • ⁇ NPX11-28 were isolated from initial screening.
  • ⁇ NPX29 and ⁇ NPX30 were isolated after further screening of N.patricia m r cnDMNAft l llihbrraonry/ 19
  • Crude enzyme extracts were used for enzyme assay. The reactions were carried out at 40°C in 50 mM Na- ⁇ trate, pH6.5, containing 0.25% xylan or 1% Avicel.
  • E.co/7 strain JM83 harbouring pNX-Tac plasmid was grown in L-broth at 30°C for 17hrs.
  • Xylanase activity was measured in 50mM Na-citrate pH6 containing 0.25% Xylan at 50°C and the reducing sugar released was measured as described in the method.
  • the crude xylanase extract from pNX-Tac clone was incubated with 6%(W/V) pulp suspension in tap water at pH 7.0. The hydrolysis was carried out at 52°C for 3 hours.
  • E.co!i strain JM83 harbouring pNX-Tac plasmid was grown in the specified media containing 50 ⁇ g/ml Amp at 30°C and IPTG was added at the beginning of the cultivation.
  • E. ⁇ li strain JM83 harbouring pNX-Tac plasmid was grown in LBMG containing 50 ⁇ g/ml Amp and 0.5 mM IPTG at 30°C for 24 hours.
  • V.NPX21 and ⁇ NPX26 respectively.
  • Neocallimastix patriciarum cDNA library Neocallimastix patriciarum cDNA library.
  • restriction enzymes B, BstXI; E, EcoRI; H, Hpal; K, Kpnl; P, Pvull; S, Sad; Sc, Seal; X, Xhol.
  • Figure 4 The amino acid sequence of pNPX30 xylanase.
  • the amino acid residues underlined come from the N-terminus of LacZ peptide and encoded by
  • Xylanase assays were performed in 50 mM Na-citrate (pH7) and 0.25%

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Abstract

Un procédé de clonage des clones de la xylanase à partir d'un champignon de rumen anaérobie comprend les étapes suivantes: (i) culture d'un champignon de rumen anaérobie; (ii) isolement de tout l'ARN de la culture de l'étape (i); (iii) isolement de tout l'ARNm poly A+ de tout l'ARN de l'étape (ii); (iv) établissement d'une bibliothèque d'expressions de l'ADNc; (v) ligature de l'ADNc à un vecteur d'expression bactériologique sélectionné parmi μZAP, μZAPII ou des vecteurs à propriétés similaires; (vi) tamisage des clones positifs recombinants de la xylanase dans un milieu de culture incorporant le xylane par détection de l'hydrolyse du xylane; et (vii) purification des clones positifs recombinants de la xylanase. L'invention décrit également les clones positifs recombinants de la xylanase produits par ce procédé ainsi que les clones positifs recombinants de la xylanase ayant les propriétés suivantes: (i) production de zones d'élimination du xylane dans une culture contenant l'ADNc de la xylanase dérivé du N. patriciarum; (ii) activité dans l'hydrolyse du xylane mais pas d'activité dans l'hydrolyse de la CMC ou de la cellulose cristalline. Diverses molécules de l'ADNc pouvant être utilisées dans le procédé ci-dessus sont également décrites.
EP93912444A 1992-06-17 1993-06-17 Xylanase recombinante Withdrawn EP0746607A4 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
AUPL298592 1992-06-17
AUPL2985/92 1992-06-17
AUPL323892 1992-06-29
AUPL3238/92 1992-06-29
AUPL810093 1993-04-01
AUPL8100/93 1993-04-01
PCT/AU1993/000294 WO1993025671A1 (fr) 1992-06-17 1993-06-17 Xylanase recombinante

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EP0746607A1 true EP0746607A1 (fr) 1996-12-11
EP0746607A4 EP0746607A4 (fr) 1997-03-12

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EP (1) EP0746607A4 (fr)
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BR (1) BR9306576A (fr)
CA (1) CA2138399A1 (fr)
FI (1) FI945889A (fr)
NZ (1) NZ252937A (fr)
WO (1) WO1993025671A1 (fr)

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US5935836A (en) * 1994-07-29 1999-08-10 Rohm Enzyme Finland Oy Actinomadura xylanase sequences and methods of use
US5871730A (en) * 1994-07-29 1999-02-16 Universite De Sherbrooke Thermostable xylanase DNA, protein and methods of use
US7816129B2 (en) 1994-07-29 2010-10-19 Ab Enzymes Gmbh Production and secretion of proteins of bacterial origin in filamentous fungi
US5892010A (en) * 1996-07-15 1999-04-06 The Regents Of The University Of California Genes from the 20Q13 amplicon and their uses
US7455964B1 (en) 1996-07-15 2008-11-25 The Hospital For Sick Children Genes from the 20q13 amplicon and their uses
US5801021A (en) 1995-10-20 1998-09-01 The Regents Of The University Of California Amplifications of chromosomal region 20q13 as a prognostic indicator in breast cancer
US7049424B2 (en) 1996-07-15 2006-05-23 The Regents Of The University Of California Genes from the 20Q13 amplicon and their uses
AU3602097A (en) 1996-07-15 1998-02-09 Regents Of The University Of California, The Genes from 20q13 amplicon and their uses
CN103131684B (zh) * 2013-01-09 2015-09-30 中国农业科学院饲料研究所 一种具有C端多余序列的木聚糖酶XynA及其基因和用途、提高木聚糖酶催化率的方法
WO2018124988A1 (fr) 2016-12-28 2018-07-05 Istanbul Teknik Universitesi Procédé d'amélioration de la production de méthane à partir de microalgues
WO2018124984A1 (fr) 2016-12-28 2018-07-05 Istanbul Teknik Universitesi Méthode d'amélioration du potentiel du biogaz de digestions anaérobies avec des champignons du rumen
CN113913410B (zh) * 2021-10-29 2022-09-23 甘肃省科学院生物研究所 一种牦牛瘤胃厌氧真菌木聚糖酶基因工程菌的构建及其应用

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EP0463706A1 (fr) * 1990-07-24 1992-01-02 Gist-Brocades N.V. Clonage et expression de gènes de xylanases d'origine fongique
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EP0746607A4 (fr) 1997-03-12
FI945889A (fi) 1995-02-08
FI945889A0 (fi) 1994-12-14
CA2138399A1 (fr) 1993-12-23
NZ252937A (en) 1996-10-28
JPH07508647A (ja) 1995-09-28
BR9306576A (pt) 1999-01-12
WO1993025671A1 (fr) 1993-12-23

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