EP0649471A1 - Recombinant cellulases - Google Patents

Recombinant cellulases

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Publication number
EP0649471A1
EP0649471A1 EP93912457A EP93912457A EP0649471A1 EP 0649471 A1 EP0649471 A1 EP 0649471A1 EP 93912457 A EP93912457 A EP 93912457A EP 93912457 A EP93912457 A EP 93912457A EP 0649471 A1 EP0649471 A1 EP 0649471A1
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Prior art keywords
cellulase
cdna
celd
enzyme
recombinant
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EP93912457A
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German (de)
French (fr)
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EP0649471A4 (en
Inventor
Gang Ping Xue
Colin George Orpin
James Harrison Aylward
Kari Steven Gobius
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Commonwealth Scientific and Industrial Research Organization CSIRO
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Commonwealth Scientific and Industrial Research Organization CSIRO
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Publication of EP0649471A4 publication Critical patent/EP0649471A4/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • 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)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • TITLE "RECOMBINANT CELLULASES" FIELD OF INVENTION
  • recombinant cellulases derived from anaerobic fungi and a method of production of recombinant cellulases and clones utilised in the method.
  • Cellulose is one of the most abundant polysaccharides in nature and consists of a polymer of glucose linked by S-1 , 4-glucosidic bonds. Conversion of cellulose to simple sugars (cellobiose and glucose) involves at least two types of hydrolases: endoglucanases which hydrolyse internal 3-1 , 4- glucosidic linkages in less ordered regions of cellulose and exoglucanases (mainly cellobiohydrolases) which cleave cellobiosyl units from non-reducing ends of cellulose chains.
  • Xylan similar to the structure of cellulose, consists of a backbone of /3-1 , 4-linked xylose units.
  • CM- cellulose carboxymethyl cellulose
  • MUC methylumbelliferyl cellobioside
  • many xylanases exclusively attack ⁇ - , 4-xylosidic linkages.
  • polysaccharide hydrolases have strict substrate specificity. Due to the similarity in the chemical nature of the substrates, cross specificity occurs not only between two types of cellulase, but also between cellulases and xylanases.
  • rumen anaerobic fungi such as Neocalli astix frontalis might produce multi-functional polysaccharide hydrolases (Gomez de Segura & Fevre, 1991; Li & Calza, 1991).
  • Multi-functional poly ⁇ saccharide hydrolases are of particular interest in genetic manipulation of rumen bacteria to enrich for the lignocellulose-degrading capacity.
  • Simultaneous enhancement of endoglucanase, cellobiohydrolase and xylanase activities would facilitate the disruption of the complex structure of lignocellulose, of which cellulose and xylan are the major components. It may also circumvent the rate-limiting problem which often occurs when only one of a complex of enzymatic reactions is enhanced.
  • Cellulose and hemicellulose are major components of ruminants' diets, consisting of 50- 80% by weight of plant tissue. Effective utilisation of plant feeds is therefore largely dependent on the production of cellulolytic and xylanolytic enzymes by microbial populations residing within the rumen. Compared with other components of the diet, degradation of cellulose and hemicellulose in the rumen is relatively slow and incomplete; digestion may be as low as 30% (Dehority, 1991). Thus, there is potential economic value in enhancing the plant fibre-degrading capacity by introducing plant polysaccharide hydrolase gene(s) into rumen micro-organisms using recombinant DNA techniques.
  • Neocallimastix patriciarum isolated from the sheep rumen, has a high capacity for cellulose degradation and can grow on cellulose as the sole carbohydrate source (Orpin & Munn, 1986; Williams & Orpin, 1987).
  • Cloning of cellulase genes from bacteria can be achieved by isolation of enzymatically active clones from genomic libraries established in E. coli .
  • this approach for isolation of cellulase genes from fungal genomic libraries with functional expression of cellulase is usually 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 RNAs 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 .
  • cellulases 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.
  • Bacteriophage vectors have an advantage in respect to the release of the cloned enzyme into cellulose-agar medium due to cell lysis.
  • commonly used bacteriophage expression vectors, ⁇ gtl 1 and its derivatives have polyclonal sites at the C-terminus of the LacZ peptide. The large part of LacZ peptide fused to the cloned enzyme often adversely affects the cloned enzyme activity.
  • a further object of the invention is to provide a method of cloning of cellulase cDNAs from an anaerobic rumen fungus which may encode the recombinant cellulase of the invention.
  • a further object of the invention is to provide cellulase clones which may be produced in the abovementioned method.
  • the method of cloning of the invention includes the following steps: (i) cultivation of an anaerobic rumen fungus; (ii) isolating total RNA from the culture in step (i);
  • RNA referred to in step (ii); (iv) constructing a cDNA expression library; (v) ligating cDNAs to a bacteriophage expression vector selected from ⁇ ZAP, ⁇ ZAP II or vectors of similar properties; (vi) screening of cellulase positive recombinant clones in a culture medium incorporating cellulose by detection of cellulose hydrolysis; and
  • step (vii) purifying cellulase positive recombinant clones.
  • anaerobic fungi particularly alimentary tract fungi, may be cultivated as described hereinbelow.
  • Neocallimastix patriciarum Neocallimastix frontalis
  • Neocallimastix h url eyensi s Neocallimastix h url eyensi s
  • Neocal l imas ti x s tan thorpensi s Sphaeromonas communis, Caecomyces equi , Piromyces communis, Piromyces equi , Piromyces dumbonica, Piromyces lethargicus, Piromyces mai , Ruminomyces elegans, Anaeromyces mucronatus, Orpinomyces bovis and Orpinomyces joyonii .
  • 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.
  • cellulose such as Avicel (ie. a form of microcrystalline cellulose)
  • After cultivation of the fungi total RNA may be obtained in any suitable manner.
  • 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 CsCl 2 cushion or alternative technique as described by Sambrook et. al.
  • 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. Alternatively a compound containing multiple uracil residues may be used such as poly (U)- Sephadex. 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 vi tro, 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: (i) having an E. coli promoter;
  • 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). It is also clear from the product summary which accompanies ' the ⁇ ZAPII vector as supplied by the manufacturer that in relation to fusion protein expression that such fusion proteins may only be screened with antibody probes. Clearly there was no contemplation that the ⁇ ZAPII vector could be utilised for screening of clones involving enzymic activity on a suitable substrate or any direct screening by biological activity. When it is realised that the present invention involves expression in a bacterial host cell such as E coli of a cDNA of eucaryotic origin (ie. fungal origin) then the novelty of the present invention is emphasised.
  • the screening of cellulase positive recombinant clones may be carried out by any suitable technique based on hydrolysis of cellulose.
  • the clones may be grown on culture media incorporating cellulose and hydrolysis may be detected by the presence of cellulase-positive plaques suitably assisted by a suitable colour indicator.
  • Cellulase positive recombinant clones may then be purified and the cDNA insert in the clones may then be excised into pBluescript (SK(-)).
  • E. coli promoter 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 recombinant cellulases may then be characterised and principal features that have been ascertained are as follows: (i) The cloned celA enzyme has high specific activity on crystalline and amorphous cellulase. The optimal pH and temperature for cellulose hydrolysis are pH5 and 40°C, respectively, (ii) The cloned celD enzyme is a multi-functional cellulase with a high activity of endoglucanase, cellobiohydrolase and xylanase.
  • celD cDNA can be truncated to code for three catalytically active domains. Each domain has endoglucanase, cellobiohydrolase and xylanase activity and cellulose-binding capacity.
  • the recombinant celA and celD enzymes also have very high activity on lichenan.
  • a combination of celA and celD enzymes can hydrolyse crystalline cellulose more efficiently.
  • Neocallimastix patriciarum (type species) was isolated from a sheep rumen by Orpin & Munn (1986) and cultivated in the laboratory for many years under selection by lignocellulose substrate.
  • the culture medium for N. patriciarum was described previously (Kemp et al. , 1984).
  • Microcrystalline cellulose (Avicel) was used as the sole carbohydrate source.
  • Host strains for cDNA cloning were E coli PLK-F and XL1-Blue obtained from Stratagene. E coli strains were grown in L-broth (Sambrook et al., 1989).
  • ⁇ ZAPII vector was obtained from Stratagene and the recombinant phage were grown in E coli strains according to the supplier's instructions. RNA isolation.
  • Frozen fungal mycelia were ground into fine powder with a mortar and pestle under liquid N 2 .
  • Powdered mycelia were homogenised in guanidinium thiocyanate solution (4M guanidinium thiocyanate, 0.5% (w/v) sodium lauryl sarcosine, 25 mM-sodiu citrate, pH7.0, 1mM-EDTA and 0.1 M-/3-mercaptoethanol) using a mortar and pestle for 5 min and then further homogenised with a Polytron at full speed for 2 min.
  • Total cellular RNA was prepared from the homogenate either by. ultracentrifugation through a CsCl 2 cushion (Sambrook et al.
  • RNA pellet obtained after acid guanidinium thiocyanate/phenol/chloroform extraction and the first step of 2-propanol precipitation, was suspended in a LiCl/urea solution (6 M-urea, 3 M-LiCl, 1 mM-EDTA, pH 7.6). The suspension was shaken at 4°C for 1-2 h to remove contaminating protein and DNA. After centrifugation, the RNA pellet was briefly washed once with the LiCl/urea solution, twice with 75% (v/v) ethanol and then dissolved in 10 mM-Tris/HC1 /1 mM-EDTA, pH 8.0.
  • RNA was further purified by extraction with phenol/chloroform and ethanpol precipitation.
  • Poly(A) + RNA was selected by oligo(dT)-cellulose chromatography (Sambrook et al. 1989). General recombinant DNA techniques.
  • RNA isolation, restriction endonuclease digestion, ligation, transformation and preparation of RNA probes were performed basically according to procedures described by Sambrook et al. (1989).
  • Double-stranded cDNA was synthesised from mRNA isolated from N. patriciarum grown on the medium containing 1% (w/v) Avicel for 48 h and ligated with ⁇ ZAPII using a ZAP-cDNA synthesis kit, according to the manufacturer's instructions (Stratagene).
  • a cDNA library of 10 6 recombinants was obtained.
  • Recombinant phage were screened for cellulolytic activity by plating in 0.7% (w/v) soft agar overlays containing one of the following substrates 0.5% (w/v) carboxymethylcellulose (CM-cellulose) , 1 mm MUC or 0.1% xylan.
  • CM-cellulose hydrolysis was detected by the Congo red staining procedure (Teather & Wood, 1982). MUC hydrolysis was examined for fluorescence under UV light.
  • the cDNA inserts in CM- cellulose positive phage were recovered in the form of pBluescript (SK-) by in vivo excision, according to Stratagene's instructions. Construction of deletion mutants.
  • celD cDNA Deletion of celD cDNA was achieved by either removing a cDNA fragment with restriction enzymes or by exonuclease III digestion (Sambrook et al., 1989). The truncated celD cDNA was checked either by restriction mapping or by partial nucleotide sequencing at the insert terminals. DNA sequencing.
  • Single-stranded plasmid DNA was prepared basically according to Stratagene's protocol. Sequencing of the resultant DNA was performed using dideoxynucleotide method (Tabor and Richardson, 1987). Southern blot hybridisation. ⁇ DNA from the cellulase-positive clones was purified by a rapid mini-preparation method as follows. One millilitre of phage lysate from liquid culture was incubated with RNAase A (10 ⁇ g ml "1 ) and DNasel (1 ⁇ g ml" 1 ) at 37°C for 1 h and with proteinase K (1 mg ml" 1 ) at 37°C for 3 h and then extracted with phenol/chloroform.
  • the DNA was precipitated by ethanol, digested with EcoRI and Xho1 (the cDNA cloning sites), fractionated by electrophoresis on 1% (w/v) agarose gel and blotted onto Hybond N membrane (Amersham) . Procedures for hybridisation and signal detection were as described previously (Xue & Morris, 1992), using digoxigenin- labelled RNA probes prepared from the 3'-region-deleted cDNA. Hybridisation was carried out at 50°C in a hybridisation mixture of 50% (v/v) formamide, 0.8 M- NaCl, 50 mM-sodium phosphate (pH 7.2), 4mM-EDTA, 0.2% (w/v) SDS.
  • the reducing sugars released from cellulose, Lichenan or xylan were measured as described by Lever (1972).
  • the p-nitrophenyl groups released from p-nitrophenyl derivatives were measured as described by Deshpande et al. (1988).
  • the cell lysate prepared from E coli strain XL1-Blue harbouring non-recombinant pBluescript was used as control. Protein concentrations were determined by dye-binding assay using the Bio-Rad protein assay kit II according to the supplier's instructions.
  • cell lysates were incubated with 200 ⁇ l of pre-washed 5% (w/v) Avicel in 50 mM-sodium citrate (pH 5.7) at 0°C with continuous shaking for 1h. The unbound protein was removed after centrifugation and the Avicel pellet was washed three times with 50mM- sodium citrate (pH 5.7). The bound cellulase was assayed for enzyme activity as above.
  • a cDNA library was prepared from poly (A) + RNA isolated from N. patriciarum grown on Avicel as the sole carbohydrate source and was constructed in E coli using a ⁇ ZAPII vector. The library was initially screened for expression of endoglucanase activity on CM-cellulose plates. Two hundred CM-cellulose positive plaques were identified after screening 4 x 10 5 plaques from library.
  • CM-cellulose positive clones were screened for cellobiohydrolase activity first on MUC plates and were further tested for the ability to hydrolyse microcrystalline cellulose, by assaying the reducing sugar released after absorption of cellulase in the supernatant of the recombinant bacteriophage lysates to Avicel followed by incubation at 39°C for 3 hr (see cellulose-binding assay in Method). Eleven bacteriophage clones exhibited large hydrolysis zones on both CM-cellulose and MUC plates, as well as activity towards Avicel. These eleven clones were then tested for xylanolytic activity on xylan plates and all were positive.
  • Fig. 1 A restriction map of the longest cellulase cDNA sequence, designated celD (pCNP4.1) is shown in Fig. 1.
  • the remaining clone possessed an insert of 7.0 Kb designated as celE and also had a similar restriction pattern to celD, but contained two additional 1.15-Kb internal EcoRI-EcoRI fragments and a 1.7 Kbp cDNA (Fig 1).
  • Cross hybridisation analysis showed that CelD strongly hybridised to CelE using a nucleic acid probe prepared from CelD cDNA in which the 3' region was deleted.
  • celE clone is a related cDNA to celD.
  • Three other classes of cellulase cDNAs were isolated from the pool of CM-cellulose-positive clones by restriction mapping and cross-hybridisation. Restriction maps of three cellulase cDNAs (the longest cDNA insert for each type), designated celA (2.0 Kb), CelB (1.7 Kb) and CelC (1.6 Kb) respectively, are shown in Fig. 2. Southern hybridisation analysis showed these three cDNA inserts did not cross-hybridise to each other (Fig.
  • the substrate specificity of these recombinant cellulases was further characterised by quantitative measurement of the activity on various cellulosic substrates and xylan. As shown in Table 1, the celD enzyme was most active on CM-cellulose, but it also possessed cellobiohydrolase-like properties, as it was highly active on crystalline cellulose, MUC and p- nitrophenyl cellobioside (pNPC) as well as amorphous cellulose. The enzyme showed no activity on methylumbelliferyl glucoside (MUG) and p-nitrophenyl glucoside (pNPG), substrates for 3-glucosidase.
  • MUC methylumbelliferyl glucoside
  • pNPG p-nitrophenyl glucoside
  • lichenan a mixed glucan containing ⁇ - ⁇ , and ⁇ - ⁇ , 3 linkages
  • laminarin predominantly ⁇ - ⁇ , 3-glucan
  • the celD enzyme had very high activity towards lichenan (Table 1 ) and produced a large hydrolysis zone on lichenan- containing agarose gel plates, but did not produce a hydrolysis zone on laminarin plates (Fig. 4). This indicates that cleavage on lichenan is at the ⁇ - ⁇ , 4- linkages. Interestingly, a high xylanase activity was also present in the celD enzyme.
  • the celD enzyme was able to hydrolyse cellodextrins (containing 3-5 glucose units) to glucose and cellobiose. Its catalytic mode on these cellulosic substrates is of a typical endoglucanase (ie. it cleaved ⁇ - ⁇ , 4-glucosidic linkages at random positions, as shown in Fig. 5).
  • the hydrolysis products of microcrystalline cellulose were mainly cellobiose with a trace amount of glucose (Fig.5), indicative of cellobiohydrolase activity. It appears that it is a truly multi ⁇ functional plant polysaccharide-degrading enzyme.
  • celE enzyme Although a number of cellulases and xylanases have been shown to have multiple substrate specificity, most of them possess only residual activity (usually ⁇ 1%) towards the secondary substrate (Saarilahti et al. , 1990; Yague et al. , 1990; Hazlewood et al. , 1990; Flint et al., 1991; Taylor et al., 1987).
  • the substrate specificity of celE enzyme is similar to celD enzyme, but its activity was about 4-fold lower.
  • the enzyme encoded by celA possesses cellobiohydrolase properties. It has very high activity in hydrolysis of crystalline and amorphous cellulose, although it also has relatively weak activity on CM-cellulose (Table 1 ) .
  • the cellobiohydrolase-like properties of the celA enzyme was further confirmed by its hydrolysis pattern as cellobiose was the only product released from cellotetrose or Avicel by the celA enzyme (Fig. 6).
  • the celA enzyme also has very high activity on Lichenan and no activity on laminarin.
  • the enzyme properties of celB and celC resembled endo-glucanase (Table 1 and Fig. 6).
  • the pH and temperature profiles of celA and celD enzymes are shown in Fig. 7 and Fig. 8.
  • the celA and celD enzymes were active from pH4.5 to pH8.5 and preferably at pH5-7.
  • the thermostability of these enzymes was tested at temperature from 30°C-60°C.
  • the celA and celD enzymes are active preferably at 30°C- 50°C.
  • the recombinant enzymes remain active in hydrolysis of Avicel at 39°C for at least 21 hr (Fig. 9 and Fig. 10).
  • the hydrolysis rates of Avicel by celA or celD enzyme were not proportional to the enzyme levels tested (Fig. 9 and Fig. 10).
  • a combination of celA and celD enzymes performs much better in hydrolysis of Avicel than doubling the concentration of individual enzyme (Fig. 11), suggesting a complementary effect of the celA and ce-ZD enzymes.
  • pNX-Tac is a DNA construct as shown in Fig. 16 and has a DNA sequence as shown in FIG 17.
  • a combination of a recombinant xylanase such as pNX-tAC and celA and celD enzyme has demonstrated that co-operativity or synergy may occur in relation to biological activity on crude cellulosic substrates containing lignin and hemicellulose components. This activity is shown in Table 2.
  • Cellulose-binding capacity of celA and celD enzymes The cellulose-binding capacity of the celA and celD enzymes were assessed by a comparative assay of the enzyme activity with or without prior absorption to crystalline cellulose (Avicel).
  • the amount of reducing sugar released from Avicel after absorption of the enzyme to Avicel followed by extensive washing of the enzyme-substrate complex was 23.3 ⁇ g glucose equivalent min-• per mg protein (the crude cell lysate preparation), compared to 24.3 ⁇ g min "-1 per mg protein for the enzyme added without prior absorption.
  • This high recovery (95%) of the enzyme activity after absorption and washing suggests that the celD enzyme possesses a strong cellulose-binding capacity.
  • the recovery of celA enzyme after adsorption to Avicel and washing was 77%, slightly lower than celD enzyme. Presumably, the cellulose-binding capacity is important for efficient degradation of cellulose as a result of the close contact of the enzyme with this insoluble substrate.
  • celD cDNA can be truncated to code for three catalytically active domains, when each domain was fused in frame with the vector's lacZ translation initiation codon. These are designated domain I (pCNP4.2), domain II (pCNP4.4) and domain III (pCNP4.8), respectively.
  • domain II The subclone construction of domain I was obtained by deletion of a 2.75-Kb fragment at the 3'region of celD cDNA (the PvulII-Xhol fragment). Domain II contained sequence from the position 1.15 Kb to 2.3 Kb of celD cDNA and domain III from 2.3 Kb to 3.37 Kb.
  • the subclone construction of domain II (pCNP4.4) was achieved by deletion of a 1.15-Kbp EcoRI-PvuII fragment at the 5' region and exonuclease III digestion at the 3' region of celD cDNA and domain III by exonuclease III digestion from both the 5' region and 3' region of the celD.
  • the celD cDNA was sequenced (see Fig. 13) and graphical presentation of celD structure is shown in Fig. 14.
  • the amino acid sequences of three catalytic domains deduced from the nucleotide sequence are presented in Fig. 15.
  • the third catalytic domain is untranslated, because there is a translation stop codon at the end of the second domain.
  • Overall functional analysis has revealed the novel properties of celD enzyme. Although some cellulases and xylanases consist of two mono-functional catalytic domains (Saul et al., 1990; Gilbert et al.
  • celD cDNA encodes a highly active enzyme with endoglucanase, cellobiohydrolase and xylanase activities, capable of degrading a wide range of cellulosic materials and xylan.
  • the cloned celD enzyme can actively hydrolyse crystalline cellulose, presumably due to the presence of both endoglucanase and cellobiohydrolase activities which act synergistically in cellulolysis.
  • the celD cDNA contains sequences which can encode three functional domains; each domain possesses endoglucanase, cellobiohydrolase and xylanase activities in addition to strong cellulose-binding capacity.
  • the cellulose- binding capacity is important for efficient degradation of cellulose as a result of the close contact of the enzyme with this insoluble substrate.
  • celA and celD enzymes have very high activity in hydrolysis of lichenan.
  • a multi-functional enzyme could more efficiently degrade the polysaccharide complex existing in plant materials. Although a number of cloned cellulases showed multiple substrate specificity, most of them possess only residual activity (usually ⁇ 1%) towards the secondary substrate. There is no previous example of a cellulase or xylanase gene encoding three multi-functional catalytic domains with each possessing strong cellulose-binding capacity. The activity of the cloned celA and celD enzymes in E coli can be further increased by using stronger promoters. Potential applications of celA and celD cDNAs
  • Cellulose and hemicellulose represent the most abundant natural resource on earth.
  • Cellulose alone accounts for about 40% total biomass with an annual production of 4 x 10 10 tons (Coughlan, 1985), which was equivalent to 70 kg of cellulose synthesises per person each day, as calculated in 1983 by Lutzen et al. (1983).
  • Most plant materials consist of 40-60% cellulose and 15-30% hemicellulose (Dekker and Lindner 1979).
  • Efficient utilisation of plant materials by ruminant animals, such as sheep and cattle, are therefore largely dependent on production of cellulolytic and xylanolytic enzymes by microbial populations residing within the rumen (the enlarged forestomach of the ruminants).
  • cellulase cDNAs include transfer into some industrial strains of microorganisms for more efficient conversion of cheap plant material, even lignocellulosic wastes, to commercially valuable products, such as ethanol, butanol, acetic acid, citric acid and antibiotics.
  • the recombinant cellulases may also be used as a cellulase source for industrial applications.
  • celA and celD Cellulase are one of the sixteen important industrial enzymes.
  • the current world market for these enzymes is >750 million U.S. dollars with an annual growth rate of 5-10% in volume.
  • the potential use of the recombinant celD enzyme is listed below: 1.
  • To increase filtration rate of the beer in the brewing industry. Cellulase is added to wort to degrade 3-glucan which causes formation of gels and hazes in beer and hence decreases filtration rate of beer.
  • the enzyme may be added to waste water to remove cellulose residues in waste water recycling processes. It may also be used to facilitate drainage in paper making and the deinking of newsprint.
  • celA and celD as genetic material for modification of some economically important micro ⁇ organisms for improvement of cellulose utilisation 1. Modification of rumen bacteria for improvement of plant fibre digestion by sheep and cattle.
  • silage inoculant bacteria lactic acid bacteria
  • the invention also includes within its scope the following - (i) DNA sequences derived from celA, celB, celC, celD and celE cDNA clones; (ii) DNA sequences derived therefrom (i) including DNA sequences hybridisable therewith using a standard hybridisation technique as described in Sambrook et al. (1989);
  • the present invention could also cover the following compositions: celA, celB, celC, celD or celE enzymes in combination or mixtures of these enzymes as a pair, triplet or as a mixture of four enzymes, and these mixtures in combination with xylanase, eg. recombinant xylanase derived from Neocallimastix patriciarum.
  • Plasmid pCNP4.1 in E coli strain XL1-Blue has been deposited at the International Depository Australian Government Analytical Laboratories on June 22, 1992 under accession number N92/27543. Plasmid pCNPl has been deposited at the
  • hybridise refers to a standard nucleic acid hybridisation technique described by Sambrook et. al. (1989).
  • Neocallimasrix patriciarum contains two homologous catalytic domains.
  • CelB a gene coding for a bifunctional cellulase from the extreme thermophile 'Caldocellum saccharolyticum ' . Applied and Environmental Microbiology 56, 3117-3124. SCHWARZ, W.H., SCHIMMING, S. & STAUDENBAUER, W.L. (1987). High-level expression of Clostridium thermocellum cellulase genes in Esherichia coli .
  • Crude cell lysate preparations were used for the measurement of enzyme activities as described in Methods. The values given are representative of at least three assays and are expressed as nmol product (reducing sugar or ⁇ nitrophenol released) min - 1 (mg protein) -1 .
  • the rate of plant fibre hydrolysis was measured by assaying reducing sugar production by the method of Lever (1972).
  • the hydrolysis reaction was performed at 40 C for 2 days using Setaria stem with in vivo digestibility of 64.7%.
  • FIG. 3 Cross-hybridization of three cellulase cDNA inserts by Southern blot analysis. Plasmids containing celA (A), celB (B) and celC (C) were cut with .EcoRI and Xhol (the cDNA cloning sites) and fractionated on 1% (w/v) agarose gel. Digoxigenin-labelled RNA probes generated from 3'-region-deleted cDNA clones were used for hybridization : celA ' probe, left blot; celC probe, right blot. Large arrows indicate the cDNA inserts being hybridized.
  • the bands indicated by small arrows are the cloning vector being hybridized, as the RNA probes contain part of the sequence from the vector. Numbers on the margins indicate the sizes, in kb, of molecular markers (BstEII fragments of ⁇ DNA) .
  • Figure 4 Congo-red staining assay of the celD enzyme activity on lichenan and laminarin. Two microlitres of crude enzyme extract were placed onto wells cut in the agarose plates containing lichenan or laminarin as described in Methods. After incubation at 39°C for 2 hr and staining with Congo red, hydrolysis of substrates is indicated by presence of a yellow halo in the red background around the well.
  • FIG. 5 Analysis of products of cellulosic compounds hydrolysed by the celD enzyme.
  • Crude cell lysate was prepared from E. coli harbouring plasmid pCNP4.1 and low-molecular-mass compounds were removed by spin- dialysis using a Centricon-10 tube (Amicon). The enzyme preparation was incubated with cellodextrins: [cellotriose (G 3 ), cellotetraose (G 4 ) and cellopentaose
  • FIG. 6 Analysis of hydrolysis products of the celA, celB and CelC enzymes on cellulosic compounds.
  • Crude cell lysates were prepared from E. coli harbouring recombinant plasmids ( celA, celB and celC ) and the small molecules were removed by spin-dialysis using Centricon-10 tubes (Amicon). The enzyme preparations were incubated with cellodextrins ( 2 mg ml- 1 ); cellotriose (G 3 ), cellotetraose ( G 4 ) and cellopentaose (G 5 ) or with 1% (w/v) Avicel (C) as described in Methods. Products were identified by TLC.
  • Figure 7 Effect of pH on the activity of the recombinant cellulases.
  • Cellulase assays were performed at 40°C in 50 mM Na-citrate (pH4-7) or 25mM Tris-Cl/50mM NaCl (pH7.5-9.5) containing 1% Avicel for 22 hours.
  • Figure 8 Effect of incubation temperature on the activity of the recombinant cellulases.
  • Cellulase assays were performed in 50 mM Na-citrate ( pH 5 or 6 ) containing 1% Avicel for 22 hours. pH5. -*-.;pH6 -•-
  • Figure 9 Time course of cellulase hydrolysis by the cloned celA enzyme.
  • Cellulase hydrolysis was performed at 39°C in 50mM Na-citrate (pH6.0) containing 1% Avicel.
  • the celA enzyme (1-12 ⁇ L) was added to the reaction of a final volume of 500 ⁇ l.
  • Figure 10 Time course of cellulose hydrolysis by the cloned celD enzyme.
  • Cellulose hydrolysis was performed at 39°C in 50mM Na-citrate (pH6.0) containing 1% Avicel.
  • the celD enzyme (1-12 ⁇ L) was added to the reaction of a final volume of 500 ⁇ L.
  • FIG 11 Effect of celA and celD enzymes in combination on the rate of crystalline cellulose hydrolysis.
  • Cellulose hydrolysis was performed at 39°C in 50mM Na-citrate (pH6) containing 1% Avicel for 4 hr.
  • FIG. 12 Restriction map of celD cDNA and its deletion mutants.
  • the positions of the cleavage sites of EcoRI (E ) , Bg/II (B ) , Kpnl (K ) , PauII(P) and -Y ⁇ oI(X) are shown.
  • the positions of deletion mutants of celD are indicated by solid bars and numbers in kbp corresponding to the positions in pCNP4.1.
  • the enzyme activity of the clones was determined on substrate- containing agarose gel plates and cellulose-binding capacity was determined with Avicel: +, active, -, inactive; ND, not determined, CMC, CM-cellulose; Xyn, xylan; Av, Avicel: CB, cellulose-binding.
  • Catalytic domains present in triplicate.
  • Predicted amino acid identity of each catalytic domain is >95%.
  • A4 comprises only endoglucanases from anaerobic bacteria, including
  • Serine-, threonine- and proline-rich linker sequence separating each of the catalytic domains.
  • SEQ ID N0:1 refers to nucleotide sequence of
  • Neocallimastix patriciarum celD cDNA The sequence underlined is derived from pBluescript SK-vector and the EcoRI adaptor used for cDNA cloning.
  • SEQ ID NO:2 refers to translated sequence of domains I an d I I o f N eocallimastix patriciarum celD cD NA .
  • Translated polypeptide includes the N-terminus of the 3-galactosidase ⁇ -peptide (derived from nucleotides 1-111) and amino acids derived from the 5' oligonucleotide linker (nucleotides 112-124) used in cDNA library construction.
  • SEQ ID NO:3 refers to translated sequence of domain III of Neocallimastix patriciarum celD cDNA.
  • SEQ ID NO:4 refers to the sequence of the modified xylanase cDNA in pNX-Tac.
  • TTA GGT C ⁇ TT TA M C T N TA AA p G CC G A A w C C TT N A AA L T . CC G A >

Abstract

A method of cloning of cellulase cDNA clones with enzymatical activity from an anaerobic rumen fungus including the steps of: (i) cultivation of an anaerobic rumen fungus; (ii) isolating total RNA from the culture in step (i); (iii) isolating poly A+ mRNA from the total RNA referred to in step (ii); (iv) constructing a cDNA expression library; (v) ligating cDNA to a bacteriophage expression vector selected from μZAP, μZAPII or vectors of similar properties; (vi) screening of cellulase positive recombinant clones in a culture medium incorporating cellulose by detection of cellulose hydrolysis; and (vii) purifying cellulase positive recombinant clones. There is also provided recombinant cellulase fungal cDNA clones produced by the abovementioned method as well as the recombinant cellulase cDNA clones, derived from N. patriciarum, having the property of production of biologically functional cellulases in E. coli cells. There is also provided various cDNA molecules which may be utilised in the abovementioned method.

Description

TITLE "RECOMBINANT CELLULASES" FIELD OF INVENTION THIS INVENTION relates to recombinant cellulases derived from anaerobic fungi and a method of production of recombinant cellulases and clones utilised in the method.
BACKGROUND ART Cellulose is one of the most abundant polysaccharides in nature and consists of a polymer of glucose linked by S-1 , 4-glucosidic bonds. Conversion of cellulose to simple sugars (cellobiose and glucose) involves at least two types of hydrolases: endoglucanases which hydrolyse internal 3-1 , 4- glucosidic linkages in less ordered regions of cellulose and exoglucanases (mainly cellobiohydrolases) which cleave cellobiosyl units from non-reducing ends of cellulose chains. Xylan, similar to the structure of cellulose, consists of a backbone of /3-1 , 4-linked xylose units. The enzymatic cleavage of 3-1,4- xylosidic linkages is performed by endo-3-1 , 4-xylanases (xylanases). These three types of enzymes usually exist separately as individual proteins, each with unique substrate specificity. Many endoglucanases cleave only internal β-λ ,
4-glucosidic linkages, producing rapid depolymerisation of a model substrate, carboxymethyl cellulose (CM- cellulose); whereas cellobiohydrolases are able to hydrolyse crystalline cellulose and methylumbelliferyl cellobioside (MUC) and have no or little depolymerising activity against CM-cellulose. Similarly, many xylanases exclusively attack β- , 4-xylosidic linkages. However, not all polysaccharide hydrolases have strict substrate specificity. Due to the similarity in the chemical nature of the substrates, cross specificity occurs not only between two types of cellulase, but also between cellulases and xylanases. A large number of cloned cellulases from bacteria have been reported to possess some residual xylanolytic activity (usually < 1%) or vice versa (Saarilahti et al. , 1990; Yague et al. 1990; Hazelwood et al. , 1990; Flint et al., 1991; Taylor et al. , 1987) .
Recent studies, based on partial enzyme purification, showed that rumen anaerobic fungi such as Neocalli astix frontalis might produce multi-functional polysaccharide hydrolases (Gomez de Segura & Fevre, 1991; Li & Calza, 1991). Multi-functional poly¬ saccharide hydrolases are of particular interest in genetic manipulation of rumen bacteria to enrich for the lignocellulose-degrading capacity. Simultaneous enhancement of endoglucanase, cellobiohydrolase and xylanase activities would facilitate the disruption of the complex structure of lignocellulose, of which cellulose and xylan are the major components. It may also circumvent the rate-limiting problem which often occurs when only one of a complex of enzymatic reactions is enhanced.
Cellulose and hemicellulose (mainly xylan) are major components of ruminants' diets, consisting of 50- 80% by weight of plant tissue. Effective utilisation of plant feeds is therefore largely dependent on the production of cellulolytic and xylanolytic enzymes by microbial populations residing within the rumen. Compared with other components of the diet, degradation of cellulose and hemicellulose in the rumen is relatively slow and incomplete; digestion may be as low as 30% (Dehority, 1991). Thus, there is potential economic value in enhancing the plant fibre-degrading capacity by introducing plant polysaccharide hydrolase gene(s) into rumen micro-organisms using recombinant DNA techniques. The isolation of a gene encoding a highly active enzyme able to degrade crystalline cellulose in a ruminal environment is considered to be one of the key steps in achieving this goal. In the past decade, isolation of cellulase genes from rumen micro-organisms was focused on bacteria. Most of the cloned cellulases from the rumen bacteria have little or no ability to degrade crystalline cellulose (Robinson and Chambliss, 1989; Hazlewood et al., 1990; Berger et al., 1989; Romaniec et al., 1989; Flint et al., 1989), though a few cloned cellulases exhibit some significant activity towards this substrate (Cavicchioli and Watson, 1991; Howard and White, 1988) .
The anaerobic fungus Neocallimastix patriciarum, isolated from the sheep rumen, has a high capacity for cellulose degradation and can grow on cellulose as the sole carbohydrate source (Orpin & Munn, 1986; Williams & Orpin, 1987).
Molecular biological aspects of fungal cellulases have been studied mainly in the aerobic fungi (Shoemaker et al. , 1983; Teeri et al., 1983; Chen et al. , 1987; Sims et al. , 1988; Azevedo et al., 1990). These studies have rapidly elucidated the complexity, structure and regulation of aerobic fungal cellulases. However, molecular characterisation of anaerobic fungal cellulases has been hampered by lack of information on the successful purification of individual cellulolytic enzymes from the fungal cellulase complexes. Thus, the preparation of antibodies or protein microsequencing for the design of oligonucleotide probes has not been possible.
Reference may also be made to other prior art which serves as background prior art prior to the advent of the present invention. Such prior art includes:
(i) Reymond et. al. FEMS Microbiology letters (1991) 107-112; (ii) Orpin et. al. Current Microbiology Vol 3 (1979) pp 121-124; (iii) Mountfort and Asher in "The Roles of Protozoa and Fungi in Ruminant Digestion" (1989) Pernambul Books (Australia); (iv) Joblin et. al. FEMS Microbiology Letters j55_ (1989) 119-122;
(v) Lowe et. al. Applied and Environmental
Microbiology June 1987 pp 1210-1215; and (vi) Lowe et. al. Applied and Environmental Microbiology June 1987 pp 1216-1223. Cloning of cellulase genes from bacteria can be achieved by isolation of enzymatically active clones from genomic libraries established in E. coli . However this approach for isolation of cellulase genes from fungal genomic libraries with functional expression of cellulase is usually 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 RNAs 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 . However, cellulases 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. Other problems which are often encountered in obtaining a biologically functional protein from a cDNA clone in E. 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.
Therefore, in the past, researchers in this field employed differential or cross hybridisation, antibody probes or oligonucleotide probes for the isolation of fungal polysaccharide hydrolase cDNA or genomic DNA clones. Relevant publications in this regard include Reymond et. al. ; Teeri et. al., referred to above; Shoemaker et. al. referred to above; Sims et. al. referred to above; Morosoli and Durand FEMS Microbiology Letters 5J_ 217-224 (1988); and Azevedo et. al. referred to above. However, these methods are very time-consuming, and quite often two stages of intensive cloning work are required for isolation of an enzymatically functional clone. For antibody or oligonucleotide probes, purification of the fungal cellulase is also required. It usually takes more than one year to obtain a functional enzyme clone using the above approaches. Isolation of fungal cellulase cDNAs by utilising an expression system in E. coli , has not been reported prior to the advent of this invention, probably at least partially due to failure in obtaining enzymatically functional cellulase clones resulting from the use of inappropriate expression vectors. Selection of expression vector systems is important. If plasmid expression vectors such as pUC vectors are used, and the cloned enzyme is trapped inside the cell, screening for cellulase clones by the convenient cellulose-agar plate technique becomes difficult. Bacteriophage vectors have an advantage in respect to the release of the cloned enzyme into cellulose-agar medium due to cell lysis. However, commonly used bacteriophage expression vectors, λgtl 1 and its derivatives, have polyclonal sites at the C-terminus of the LacZ peptide. The large part of LacZ peptide fused to the cloned enzyme often adversely affects the cloned enzyme activity.
In specific regard to the abovementioned Reymond et. al. (1991) reference there is described an attempt of molecular cloning of polysaccharide hydrolase (ie. cellulase) genes from an anaerobic fungus which is N. frontalis. In this reference a clone from a cDNA library derived from N. frontalis hybridized to a DNA probe encoding part of the exo- cellobiohydrolase (CBH 1) gene of Trichoderma reesei . However it was subsequently revealed by Reymond et. al. in a personal communication that the particular cDNA clone obtained from N. frontalis does not encode any polysaccharide hydrolase.
Moreover the Reymond et. al. reference did not describe the production of biologically functional enzymes from these clones.
BROAD STATEMENT OF INVENTION
It is an object of the invention to provide a recombinant cellulase from an anaerobic rumen fungus which may be of use commercially in relation to hydrolysis of cellulose or cellulose derivatives including plant cell walls.
A further object of the invention is to provide a method of cloning of cellulase cDNAs from an anaerobic rumen fungus which may encode the recombinant cellulase of the invention.
A further object of the invention is to provide cellulase clones which may be produced in the abovementioned method. The method of cloning of the invention includes the following steps: (i) cultivation of an anaerobic rumen fungus; (ii) isolating total RNA from the culture in step (i);
(iii) isolating poly A+ mRNA from the total
RNA referred to in step (ii); (iv) constructing a cDNA expression library; (v) ligating cDNAs to a bacteriophage expression vector selected from λZAP, λZAP II or vectors of similar properties; (vi) screening of cellulase positive recombinant clones in a culture medium incorporating cellulose by detection of cellulose hydrolysis; and
(vii) purifying cellulase positive recombinant clones. In step (i) above in relation to preparation of the recombinant cellulase, 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 h url eyensi s , Neocal l imas ti x s tan thorpensi s , Sphaeromonas communis, Caecomyces equi , Piromyces communis, Piromyces equi , Piromyces dumbonica, Piromyces lethargicus, Piromyces mai , Ruminomyces elegans, Anaeromyces mucronatus, Orpinomyces bovis and Orpinomyces joyonii . In regard to the above mentioned 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. After cultivation of the fungi total RNA may be obtained in any suitable manner. Thus initially 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 CsCl2 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 or by the method of Chomczynski and Sacchi in 1987. 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. Alternatively a compound containing multiple uracil residues may be used such as poly (U)- Sephadex. 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 λZAP or λZAPII. The cDNA library may then be amplified after packaging in vi tro, using any suitable host bacterial cell such as a suitable strain of E. coli .
The choice of the bacteriophage expression vector in step (v) is important in that such expression vector should include the following features: (i) having an E. coli promoter;
(ii) having a translation start codon;
(iii) having a ribosomal binding site;
(iv) 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 λZAPII.
It will be appreciated from the foregoing that if an expression vector is utilised as described above the chances of obtaining a biologically functional enzyme is greatly increased. Isolation of many enzymatically functional cellulase clones in the present invention as described hereinafter has proved the efficiency of this approach. To our knowledge this is the first record of isolation of cellulase cDNA clones with functional enzyme activity from anaerobic fungi based upon the expression of recombinant bacteriophage in E coli using an expression vector such as that described above. λZAP and λZAP II are examples of such expression vectors.
Therefore the term "vectors of similar properties" to λZAP or λZAPII includes within its scope expression vectors having the abovementioned features (i), (ii), (iii) and (iv). It is also clear from the product summary which accompanies' the λZAPII vector as supplied by the manufacturer that in relation to fusion protein expression that such fusion proteins may only be screened with antibody probes. Clearly there was no contemplation that the λZAPII vector could be utilised for screening of clones involving enzymic activity on a suitable substrate or any direct screening by biological activity. When it is realised that the present invention involves expression in a bacterial host cell such as E coli of a cDNA of eucaryotic origin (ie. fungal origin) then the novelty of the present invention is emphasised.
The screening of cellulase positive recombinant clones may be carried out by any suitable technique based on hydrolysis of cellulose. In this procedure the clones may be grown on culture media incorporating cellulose and hydrolysis may be detected by the presence of cellulase-positive plaques suitably assisted by a suitable colour indicator. Cellulase positive recombinant clones may then be purified and the cDNA insert in the clones may then be excised into pBluescript (SK(-)).
Any suitable E. coli promoter may be used in the expression vector described above. Suitable promoters include lacZ, Tac, Bacteriophage T7 and lambda-PL. The recombinant cellulases may then be characterised and principal features that have been ascertained are as follows: (i) The cloned celA enzyme has high specific activity on crystalline and amorphous cellulase. The optimal pH and temperature for cellulose hydrolysis are pH5 and 40°C, respectively, (ii) The cloned celD enzyme is a multi-functional cellulase with a high activity of endoglucanase, cellobiohydrolase and xylanase.
The optimal pH and temperature for cellulose hydrolysis are at pH5 and 40°C, respectively. (iii) celD cDNA can be truncated to code for three catalytically active domains. Each domain has endoglucanase, cellobiohydrolase and xylanase activity and cellulose-binding capacity. (iv) The recombinant celA and celD enzymes also have very high activity on lichenan. (v) A combination of celA and celD enzymes can hydrolyse crystalline cellulose more efficiently. DESCRIPTION OF THE PREFERRED EMBODIMENT
Method Microbial strains, vectors and culture media.
The anaerobic fungus Neocallimastix patriciarum (type species) was isolated from a sheep rumen by Orpin & Munn (1986) and cultivated in the laboratory for many years under selection by lignocellulose substrate. The culture medium for N. patriciarum was described previously (Kemp et al. , 1984). Microcrystalline cellulose (Avicel) was used as the sole carbohydrate source. Host strains for cDNA cloning were E coli PLK-F and XL1-Blue obtained from Stratagene. E coli strains were grown in L-broth (Sambrook et al., 1989). λZAPII vector was obtained from Stratagene and the recombinant phage were grown in E coli strains according to the supplier's instructions. RNA isolation.
Frozen fungal mycelia were ground into fine powder with a mortar and pestle under liquid N2. Powdered mycelia were homogenised in guanidinium thiocyanate solution (4M guanidinium thiocyanate, 0.5% (w/v) sodium lauryl sarcosine, 25 mM-sodiu citrate, pH7.0, 1mM-EDTA and 0.1 M-/3-mercaptoethanol) using a mortar and pestle for 5 min and then further homogenised with a Polytron at full speed for 2 min. Total cellular RNA was prepared from the homogenate either by. ultracentrifugation through a CsCl2 cushion (Sambrook et al. , 1989) or by the method of Chomczynski & Sacchi (1987) with the following modifications. The RNA pellet, obtained after acid guanidinium thiocyanate/phenol/chloroform extraction and the first step of 2-propanol precipitation, was suspended in a LiCl/urea solution (6 M-urea, 3 M-LiCl, 1 mM-EDTA, pH 7.6). The suspension was shaken at 4°C for 1-2 h to remove contaminating protein and DNA. After centrifugation, the RNA pellet was briefly washed once with the LiCl/urea solution, twice with 75% (v/v) ethanol and then dissolved in 10 mM-Tris/HC1 /1 mM-EDTA, pH 8.0. The RNA was further purified by extraction with phenol/chloroform and ethanpol precipitation. Poly(A)+ RNA was selected by oligo(dT)-cellulose chromatography (Sambrook et al. 1989). General recombinant DNA techniques.
DNA isolation, restriction endonuclease digestion, ligation, transformation and preparation of RNA probes were performed basically according to procedures described by Sambrook et al. (1989).
Construction and screening of the N. patriciarum cDNA library.
Double-stranded cDNA was synthesised from mRNA isolated from N. patriciarum grown on the medium containing 1% (w/v) Avicel for 48 h and ligated with λZAPII using a ZAP-cDNA synthesis kit, according to the manufacturer's instructions (Stratagene). A cDNA library of 106 recombinants was obtained. Recombinant phage were screened for cellulolytic activity by plating in 0.7% (w/v) soft agar overlays containing one of the following substrates 0.5% (w/v) carboxymethylcellulose (CM-cellulose) , 1 mm MUC or 0.1% xylan. 10 mM-isopropyl 3-D-thiogalactopyranoside (IPTG; an inducer for lacZp-controlled gene expression) was also included. CM-cellulose hydrolysis was detected by the Congo red staining procedure (Teather & Wood, 1982). MUC hydrolysis was examined for fluorescence under UV light. The cDNA inserts in CM- cellulose positive phage were recovered in the form of pBluescript (SK-) by in vivo excision, according to Stratagene's instructions. Construction of deletion mutants.
Deletion of celD cDNA was achieved by either removing a cDNA fragment with restriction enzymes or by exonuclease III digestion (Sambrook et al., 1989). The truncated celD cDNA was checked either by restriction mapping or by partial nucleotide sequencing at the insert terminals. DNA sequencing.
Single-stranded plasmid DNA was prepared basically according to Stratagene's protocol. Sequencing of the resultant DNA was performed using dideoxynucleotide method (Tabor and Richardson, 1987). Southern blot hybridisation. λDNA from the cellulase-positive clones was purified by a rapid mini-preparation method as follows. One millilitre of phage lysate from liquid culture was incubated with RNAase A (10μg ml"1) and DNasel (1μg ml"1) at 37°C for 1 h and with proteinase K (1 mg ml"1) at 37°C for 3 h and then extracted with phenol/chloroform. The DNA was precipitated by ethanol, digested with EcoRI and Xho1 (the cDNA cloning sites), fractionated by electrophoresis on 1% (w/v) agarose gel and blotted onto Hybond N membrane (Amersham) . Procedures for hybridisation and signal detection were as described previously (Xue & Morris, 1992), using digoxigenin- labelled RNA probes prepared from the 3'-region-deleted cDNA. Hybridisation was carried out at 50°C in a hybridisation mixture of 50% (v/v) formamide, 0.8 M- NaCl, 50 mM-sodium phosphate (pH 7.2), 4mM-EDTA, 0.2% (w/v) SDS. 5x Denhardt's solution, 0.2 mg yeast RNA ml"1 , 0.2 mg herring sperm DNA ml"1 (1 x Denhardt's solution is 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone). High-stringency washing was performed in 0.1 x SSC/0.1% (w/v) SDS at 68°C (1 X SSC is 0.15 M-NaCl, 15 mM-sodium citrate). Enzyme assays, cellulose-binding studies and product identification. E coli cells harbouring the recombinant plasmids were grown in LB medium to the end of the exponential phase in the presence of 1mM IPTG. Crude cell lysates prepared according to Schwarz et al. (1987) were used as enzyme sources. For standard quantitive assays, the enzyme preparations were incubated at 39°C for 30-60 min in 50 mM-sodium citrate (pH 5.7) with the following substrates: 0.5% (w/v) CM- cellulose (low viscosity, Sigma, 1% (w/v) amorphous cellulose (H3P04-swollen Avicel), 1% (w/v) Avicel (Merck), 0.05% (w/v) p-nitrophenyl cellobioside (pNPC, Sigma), p-nitrophenyl glucopyranoside (pNPG, Sigma), 0.25% (w/v) oat spelt xylan (Sigma) and 0.4% Lichenan. The reducing sugars released from cellulose, Lichenan or xylan were measured as described by Lever (1972). The p-nitrophenyl groups released from p-nitrophenyl derivatives were measured as described by Deshpande et al. (1988). The cell lysate prepared from E coli strain XL1-Blue harbouring non-recombinant pBluescript was used as control. Protein concentrations were determined by dye-binding assay using the Bio-Rad protein assay kit II according to the supplier's instructions. Qualitative assays were performed using 0.8% (w/v) agarose gel plates containing 0.2% (w/v) CM- cellulose, lichenan, laminarin or xylan or 1 mM MUC in 50 mM Na-citrate pH5.7. Hydrolysis zones were detected as described above.
For assays of cellulose-binding capacity of the cloned cellulase, cell lysates were incubated with 200μl of pre-washed 5% (w/v) Avicel in 50 mM-sodium citrate (pH 5.7) at 0°C with continuous shaking for 1h. The unbound protein was removed after centrifugation and the Avicel pellet was washed three times with 50mM- sodium citrate (pH 5.7). The bound cellulase was assayed for enzyme activity as above.
For analysis of hydrolysis products of cellulosic substrates, crude E coli lysates containing the cloned cellulases were spin-dialysed to remove small molecules using Centricon concentrators (Amicon) . The dialysed enzyme preparations were incubated at 39°C in 50 mM-sodium citrate (pH 5.7) with 1% (w/v) Avicel or cellodextrins (2 mg ml"1) containing 3-6 glucose units. In order to examine the intermediate and end hydrolysis products of cellodextrins, samples were taken at five incubation times (30 min, 1 h, 2h, 4h and 30h), using appropriate amounts of enzymes to ensure partial as well as complete digestion. Hydrolysis products of cellulosic substrates were identified by thin-layer chromatography (TLC) using silica gel plates and a solvent system of ethyl acetate/water/methanol (3:3:4, by vol.). The positions of sugars on the plate were visualised by spraying with the diphenylamine reagent as described by Lake & Goodwin (1976) and authentic cellodextrins (Merck) were used for identification. Results and Discussion Isolation of cellulase cDNAs from N. patriciarum cDNA expression library.
A cDNA library was prepared from poly (A)+ RNA isolated from N. patriciarum grown on Avicel as the sole carbohydrate source and was constructed in E coli using a λZAPII vector. The library was initially screened for expression of endoglucanase activity on CM-cellulose plates. Two hundred CM-cellulose positive plaques were identified after screening 4 x 105 plaques from library. These CM-cellulose positive clones were screened for cellobiohydrolase activity first on MUC plates and were further tested for the ability to hydrolyse microcrystalline cellulose, by assaying the reducing sugar released after absorption of cellulase in the supernatant of the recombinant bacteriophage lysates to Avicel followed by incubation at 39°C for 3 hr (see cellulose-binding assay in Method). Eleven bacteriophage clones exhibited large hydrolysis zones on both CM-cellulose and MUC plates, as well as activity towards Avicel. These eleven clones were then tested for xylanolytic activity on xylan plates and all were positive.
Analysis of the selected clones by restriction mapping revealed that ten of the eleven clones (the size of cDNA inserts ranging from 1.6 Kb to 3.9 Kb) shared the same restriction pattern. A restriction map of the longest cellulase cDNA sequence, designated celD (pCNP4.1) is shown in Fig. 1. The remaining clone possessed an insert of 7.0 Kb designated as celE and also had a similar restriction pattern to celD, but contained two additional 1.15-Kb internal EcoRI-EcoRI fragments and a 1.7 Kbp cDNA (Fig 1). Cross hybridisation analysis showed that CelD strongly hybridised to CelE using a nucleic acid probe prepared from CelD cDNA in which the 3' region was deleted. Thus it is most likely that the ten clones originate from the same gene and the celE clone is a related cDNA to celD. Three other classes of cellulase cDNAs were isolated from the pool of CM-cellulose-positive clones by restriction mapping and cross-hybridisation. Restriction maps of three cellulase cDNAs (the longest cDNA insert for each type), designated celA (2.0 Kb), CelB (1.7 Kb) and CelC (1.6 Kb) respectively, are shown in Fig. 2. Southern hybridisation analysis showed these three cDNA inserts did not cross-hybridise to each other (Fig. 3), using nucleic acid probes prepared from CelA and CelC clones with the 3'regions of the cDNA insert removed by digestion with Xhol and the enzyme at the upstream restriction site (see Fig 2). Similarly, CelD did not hybridise to celA, celB and CelC using high stringency conditions.
Enzymatic properties of the Recombinant cellulases
The substrate specificity of these recombinant cellulases was further characterised by quantitative measurement of the activity on various cellulosic substrates and xylan. As shown in Table 1, the celD enzyme was most active on CM-cellulose, but it also possessed cellobiohydrolase-like properties, as it was highly active on crystalline cellulose, MUC and p- nitrophenyl cellobioside (pNPC) as well as amorphous cellulose. The enzyme showed no activity on methylumbelliferyl glucoside (MUG) and p-nitrophenyl glucoside (pNPG), substrates for 3-glucosidase. Other cellulosic substrates tested were lichenan (a mixed glucan containing β-Λ , and β-Λ , 3 linkages) and laminarin (predominantly β-Λ , 3-glucan) . The celD enzyme had very high activity towards lichenan (Table 1 ) and produced a large hydrolysis zone on lichenan- containing agarose gel plates, but did not produce a hydrolysis zone on laminarin plates (Fig. 4). This indicates that cleavage on lichenan is at the β-Λ , 4- linkages. Interestingly, a high xylanase activity was also present in the celD enzyme. Analysis of hydrolysis products by TLC showed that the celD enzyme was able to hydrolyse cellodextrins (containing 3-5 glucose units) to glucose and cellobiose. Its catalytic mode on these cellulosic substrates is of a typical endoglucanase (ie. it cleaved β-Λ , 4-glucosidic linkages at random positions, as shown in Fig. 5). However, the hydrolysis products of microcrystalline cellulose were mainly cellobiose with a trace amount of glucose (Fig.5), indicative of cellobiohydrolase activity. It appears that it is a truly multi¬ functional plant polysaccharide-degrading enzyme. Although a number of cellulases and xylanases have been shown to have multiple substrate specificity, most of them possess only residual activity (usually < 1%) towards the secondary substrate (Saarilahti et al. , 1990; Yague et al. , 1990; Hazlewood et al. , 1990; Flint et al., 1991; Taylor et al., 1987). The substrate specificity of celE enzyme is similar to celD enzyme, but its activity was about 4-fold lower. The enzyme encoded by celA possesses cellobiohydrolase properties. It has very high activity in hydrolysis of crystalline and amorphous cellulose, although it also has relatively weak activity on CM-cellulose (Table 1 ) . The cellobiohydrolase-like properties of the celA enzyme was further confirmed by its hydrolysis pattern as cellobiose was the only product released from cellotetrose or Avicel by the celA enzyme (Fig. 6). The celA enzyme also has very high activity on Lichenan and no activity on laminarin. The enzyme properties of celB and celC resembled endo-glucanase (Table 1 and Fig. 6).
The pH and temperature profiles of celA and celD enzymes are shown in Fig. 7 and Fig. 8. The celA and celD enzymes were active from pH4.5 to pH8.5 and preferably at pH5-7. The thermostability of these enzymes was tested at temperature from 30°C-60°C. The celA and celD enzymes are active preferably at 30°C- 50°C. The recombinant enzymes remain active in hydrolysis of Avicel at 39°C for at least 21 hr (Fig. 9 and Fig. 10). The hydrolysis rates of Avicel by celA or celD enzyme were not proportional to the enzyme levels tested (Fig. 9 and Fig. 10). However, a combination of celA and celD enzymes performs much better in hydrolysis of Avicel than doubling the concentration of individual enzyme (Fig. 11), suggesting a complementary effect of the celA and ce-ZD enzymes.
It has also been ascertained that recombinant xylanases may be produced by the method of the invention using substantially the same experimental protocols described above. One such xylanase termed pNX-Tac is a DNA construct as shown in Fig. 16 and has a DNA sequence as shown in FIG 17.
A combination of a recombinant xylanase such as pNX-tAC and celA and celD enzyme has demonstrated that co-operativity or synergy may occur in relation to biological activity on crude cellulosic substrates containing lignin and hemicellulose components. This activity is shown in Table 2. Cellulose-binding capacity of celA and celD enzymes The cellulose-binding capacity of the celA and celD enzymes were assessed by a comparative assay of the enzyme activity with or without prior absorption to crystalline cellulose (Avicel). The amount of reducing sugar released from Avicel after absorption of the enzyme to Avicel followed by extensive washing of the enzyme-substrate complex was 23.3 μg glucose equivalent min-• per mg protein (the crude cell lysate preparation), compared to 24.3 μg min"-1 per mg protein for the enzyme added without prior absorption. This high recovery (95%) of the enzyme activity after absorption and washing suggests that the celD enzyme possesses a strong cellulose-binding capacity. The recovery of celA enzyme after adsorption to Avicel and washing was 77%, slightly lower than celD enzyme. Presumably, the cellulose-binding capacity is important for efficient degradation of cellulose as a result of the close contact of the enzyme with this insoluble substrate. Functional domains of celD enzyme To investigate the locations of catalytic and cellulose-binding domains of the celD enzyme and to elucidate whether the multiple substrate specificity of the enzyme is due to the presence of different catalytic domains, a series of deletion analyses of celD cDNA was conducted. As shown in Fig. 12, celD cDNA can be truncated to code for three catalytically active domains, when each domain was fused in frame with the vector's lacZ translation initiation codon. These are designated domain I (pCNP4.2), domain II (pCNP4.4) and domain III (pCNP4.8), respectively. The subclone construction of domain I was obtained by deletion of a 2.75-Kb fragment at the 3'region of celD cDNA (the PvulII-Xhol fragment). Domain II contained sequence from the position 1.15 Kb to 2.3 Kb of celD cDNA and domain III from 2.3 Kb to 3.37 Kb. The subclone construction of domain II (pCNP4.4) was achieved by deletion of a 1.15-Kbp EcoRI-PvuII fragment at the 5' region and exonuclease III digestion at the 3' region of celD cDNA and domain III by exonuclease III digestion from both the 5' region and 3' region of the celD. Interestingly, all three domains possessed the same pattern of substrate specificities as the enzyme produced by the untruncated celD cDNA. Moreover, all three domains had cellulose-binding capacity. Recovery of the enzyme activity after absorption to Avicel and subsequent washing ranged from 70% to 80%. This is slightly lower than the enzyme from the untruncated celD cDNA.
The celD cDNA was sequenced (see Fig. 13) and graphical presentation of celD structure is shown in Fig. 14. The amino acid sequences of three catalytic domains deduced from the nucleotide sequence are presented in Fig. 15. In the untruncated celD, the third catalytic domain is untranslated, because there is a translation stop codon at the end of the second domain. Overall functional analysis has revealed the novel properties of celD enzyme. Although some cellulases and xylanases consist of two mono-functional catalytic domains (Saul et al., 1990; Gilbert et al. 1992) or possess a single multi-functional domain (Foong et al., 1991), there is no previous example of a polysaccharide hydrolase cDNA encoding three multifunctional catalytic domains, with each catalytic domain possessing cellulose-binding capacity. A multi¬ functional enzyme would be beneficial for the rumen fungus in its natural environment where these polysaccharide substrates exist in a complex structure. Usually, several types of polysaccharide hydrolases are required to form a multi-enzyme complex acting co¬ operatively on these natural substrates. Main features of celD cDNAs from the rumen anaerobic fungus, Neocallimastix patriciarum 1. celA cDNA encodes a highly active cellobiohydrolase which efficiently hydrolyses both crystalline and amorphous cellulose.
2. celD cDNA encodes a highly active enzyme with endoglucanase, cellobiohydrolase and xylanase activities, capable of degrading a wide range of cellulosic materials and xylan.
3. The cloned celD enzyme can actively hydrolyse crystalline cellulose, presumably due to the presence of both endoglucanase and cellobiohydrolase activities which act synergistically in cellulolysis.
4. The celD cDNA contains sequences which can encode three functional domains; each domain possesses endoglucanase, cellobiohydrolase and xylanase activities in addition to strong cellulose-binding capacity. The cellulose- binding capacity is important for efficient degradation of cellulose as a result of the close contact of the enzyme with this insoluble substrate.
5. celA and celD enzymes have very high activity in hydrolysis of lichenan.
A multi-functional enzyme could more efficiently degrade the polysaccharide complex existing in plant materials. Although a number of cloned cellulases showed multiple substrate specificity, most of them possess only residual activity (usually < 1%) towards the secondary substrate. There is no previous example of a cellulase or xylanase gene encoding three multi-functional catalytic domains with each possessing strong cellulose-binding capacity. The activity of the cloned celA and celD enzymes in E coli can be further increased by using stronger promoters. Potential applications of celA and celD cDNAs
Cellulose and hemicellulose (consisting mainly of xylan) represent the most abundant natural resource on earth. Cellulose alone accounts for about 40% total biomass with an annual production of 4 x 1010 tons (Coughlan, 1985), which was equivalent to 70 kg of cellulose synthesises per person each day, as calculated in 1983 by Lutzen et al. (1983). Most plant materials consist of 40-60% cellulose and 15-30% hemicellulose (Dekker and Lindner 1979). Efficient utilisation of plant materials by ruminant animals, such as sheep and cattle, are therefore largely dependent on production of cellulolytic and xylanolytic enzymes by microbial populations residing within the rumen (the enlarged forestomach of the ruminants). Compared with other components of the diet, degradation of cellulose and hemicellulose in the rumen is relatively slow and it can be as low as 30% (Dehority, 1991). Thus, there is potential economic value in enhancing the plant fibre-degrading capacity of rumen micro-organisms by introducing plant polysaccharide hydrolase gene(s) using recombinant DNA techniques. Isolation of a gene encoding a highly active enzyme which is able to degrade crystalline cellulose and xylan in a ruminal environment is considered to be one of the key steps in achieving this goal. celA and celD cDNAs, isolated from a rumen anaerobic fungus, may possess advantages over other cellulase genes from non- ruminal origin, for use as genetic material for transfer into rumen bacteria.
Other potential applications of these cellulase cDNAs include transfer into some industrial strains of microorganisms for more efficient conversion of cheap plant material, even lignocellulosic wastes, to commercially valuable products, such as ethanol, butanol, acetic acid, citric acid and antibiotics. The recombinant cellulases may also be used as a cellulase source for industrial applications.
Industrial use of the recombinant cellulases celA and celD Cellulase is one of the sixteen important industrial enzymes. The current world market for these enzymes is >750 million U.S. dollars with an annual growth rate of 5-10% in volume. The potential use of the recombinant celD enzyme is listed below: 1. To increase filtration rate of the beer in the brewing industry. Cellulase is added to wort to degrade 3-glucan which causes formation of gels and hazes in beer and hence decreases filtration rate of beer. 2. For waste water treatment in the pulp and paper industry and starch industry. The enzyme may be added to waste water to remove cellulose residues in waste water recycling processes. It may also be used to facilitate drainage in paper making and the deinking of newsprint.
3. For use in the dietary food, medicine and cosmetic industries. Recent study has shown that modified cellulose by partial enzymatic depolymerisation was found to be a useful product in these industries.
4. Other uses include clarification of fruit juices, vegetable processing, bread making, animal feed preparation and research purposes. The use of celA and celD as genetic material for modification of some economically important micro¬ organisms for improvement of cellulose utilisation 1. Modification of rumen bacteria for improvement of plant fibre digestion by sheep and cattle.
2. Modification of silage inoculant bacteria (lactic acid bacteria) to stimulate conversion of cellulosic material to microbial protein and increase nutritive value of silage as animal feeds.
3. Modification of nitrogen-fixing microbes in compost preparations or plant residues to improve degradation of cellulosic material which is used as energy to support the growth of nitrogen-fixing bacteria.
4. Modification of ethanol-producing microbes such as Saccharomyces cerevisiae or Zymomonas mobilis for conversion of cellulosic material such as agricultural wastes to ethanol for industrial use.
The invention also includes within its scope the following - (i) DNA sequences derived from celA, celB, celC, celD and celE cDNA clones; (ii) DNA sequences derived therefrom (i) including DNA sequences hybridisable therewith using a standard hybridisation technique as described in Sambrook et al. (1989);
(iii) celA, celB, celC, celD and celE enzymes having the features described herein; (iv) polypeptides having amino acid sequences derived from celA, celB, celC, celD and celE cDNA.
It will also be appreciated that the present invention could also cover the following compositions: celA, celB, celC, celD or celE enzymes in combination or mixtures of these enzymes as a pair, triplet or as a mixture of four enzymes, and these mixtures in combination with xylanase, eg. recombinant xylanase derived from Neocallimastix patriciarum. Plasmid pCNP4.1 in E coli strain XL1-Blue has been deposited at the International Depository Australian Government Analytical Laboratories on June 22, 1992 under accession number N92/27543. Plasmid pCNPl has been deposited at the
International Depository Australian Government Analytical Laboratories on June 22, 1993 under accession number N93/28000.
The term "essentially" as used herein refers to 70-100% identity with the sequences shown in Fig. 13 and Fig. 15. The term "hybridise" as used herein refers to a standard nucleic acid hybridisation technique described by Sambrook et. al. (1989).
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Table 1 Activity of the cloned cellulases on various substrates.
ND - Not Determined
Crude cell lysate preparations were used for the measurement of enzyme activities as described in Methods. The values given are representative of at least three assays and are expressed as nmol product (reducing sugar or ^nitrophenol released) min- 1 (mg protein)-1.
Table 2
Effect of celA and celD enzymes and pNX-Tac xylanase in combination on hydrolysis rate of natural plant material
Relative rate of hydrolysis
control 0.056 pNX-tac Xylanase 1 .07
CelA and CelD enzymes plus pNX-Tac xylanase 2.31
The rate of plant fibre hydrolysis was measured by assaying reducing sugar production by the method of Lever (1972).
The hydrolysis reaction was performed at 40 C for 2 days using Setaria stem with in vivo digestibility of 64.7%.
LEGENDS
Figure 1 - Restriction maps of CelD and CelE.
Abbreviations for restriction enzymes:
E,EcoRI; B,Bg1II; K,KpnI; P,PvuII, X,XhoI; D,DraI.
Figure 2 - Restriction maps of celA, celB and celC.
Figure 3 - Cross-hybridization of three cellulase cDNA inserts by Southern blot analysis. Plasmids containing celA (A), celB (B) and celC (C) were cut with .EcoRI and Xhol (the cDNA cloning sites) and fractionated on 1% (w/v) agarose gel. Digoxigenin-labelled RNA probes generated from 3'-region-deleted cDNA clones were used for hybridization : celA ' probe, left blot; celC probe, right blot. Large arrows indicate the cDNA inserts being hybridized. The bands indicated by small arrows are the cloning vector being hybridized, as the RNA probes contain part of the sequence from the vector. Numbers on the margins indicate the sizes, in kb, of molecular markers (BstEII fragments of λDNA) .
Figure 4 - Congo-red staining assay of the celD enzyme activity on lichenan and laminarin. Two microlitres of crude enzyme extract were placed onto wells cut in the agarose plates containing lichenan or laminarin as described in Methods. After incubation at 39°C for 2 hr and staining with Congo red, hydrolysis of substrates is indicated by presence of a yellow halo in the red background around the well.
Figure 5 - Analysis of products of cellulosic compounds hydrolysed by the celD enzyme. (a) Crude cell lysate was prepared from E. coli harbouring plasmid pCNP4.1 and low-molecular-mass compounds were removed by spin- dialysis using a Centricon-10 tube (Amicon). The enzyme preparation was incubated with cellodextrins: [cellotriose (G3), cellotetraose (G4) and cellopentaose
SUBSTITUT ΈE SHE (G5), each 2 mg ml"1] or with 1% (w/v) Avicel (C) as described in Methods. Partial hydrolysis of cellodextrins is shown to illustrate the intermediate products. Hydrolysis products were identified by their Rf values on a TLC plate. Authentic cellodextrins (S) are shown in the rightmost lane and the positions of glucose (G,), cellobiose (G2), G3 and G4 are indicated, (b) Illustration of the catalytic mode of the celD enzyme on cellotetraose.
Figure 6 - Analysis of hydrolysis products of the celA, celB and CelC enzymes on cellulosic compounds. (a) Crude cell lysates were prepared from E. coli harbouring recombinant plasmids ( celA, celB and celC) and the small molecules were removed by spin-dialysis using Centricon-10 tubes (Amicon). The enzyme preparations were incubated with cellodextrins (2 mg ml-1); cellotriose (G3), cellotetraose (G4) and cellopentaose (G5) or with 1% (w/v) Avicel (C) as described in Methods. Products were identified by TLC. Complete hydrolysis of cellodextrins by the celA enzyme and partial hydrolysis by the celB and celC enzymes are shown. Authentic cellodextrins (S) are shown in the rightmost lane. (b) Illustration of the catalytic mode of the three cloned enzymes on cellotetraose.
Figure 7 - Effect of pH on the activity of the recombinant cellulases. Cellulase assays were performed at 40°C in 50 mM Na-citrate (pH4-7) or 25mM Tris-Cl/50mM NaCl (pH7.5-9.5) containing 1% Avicel for 22 hours.
Figure 8 - Effect of incubation temperature on the activity of the recombinant cellulases. Cellulase assays were performed in 50 mM Na-citrate (pH 5 or 6) containing 1% Avicel for 22 hours. pH5. -*-.;pH6 -•- Figure 9 - Time course of cellulase hydrolysis by the cloned celA enzyme. Cellulase hydrolysis was performed at 39°C in 50mM Na-citrate (pH6.0) containing 1% Avicel. The celA enzyme (1-12μL) was added to the reaction of a final volume of 500μl.
Figure 10 - Time course of cellulose hydrolysis by the cloned celD enzyme. Cellulose hydrolysis was performed at 39°C in 50mM Na-citrate (pH6.0) containing 1% Avicel. The celD enzyme (1-12μL) was added to the reaction of a final volume of 500μL.
Figure 11 - Effect of celA and celD enzymes in combination on the rate of crystalline cellulose hydrolysis. Cellulose hydrolysis was performed at 39°C in 50mM Na-citrate (pH6) containing 1% Avicel for 4 hr.
Figure 12 - Restriction map of celD cDNA and its deletion mutants. The positions of the cleavage sites of EcoRI (E ) , Bg/II (B ) , Kpnl (K ) , PauII(P) and -YΛoI(X) are shown. The positions of deletion mutants of celD are indicated by solid bars and numbers in kbp corresponding to the positions in pCNP4.1. The enzyme activity of the clones was determined on substrate- containing agarose gel plates and cellulose-binding capacity was determined with Avicel: +, active, -, inactive; ND, not determined, CMC, CM-cellulose; Xyn, xylan; Av, Avicel: CB, cellulose-binding.
SUBSTITUTE SHEET Figure 13 - Characterisation of Neocallimastix patriciarum celD nucleotide sequence.
"A" - Single base induces frame shift and TAA stop codon "B" - Apparent original TAG stop codon.
N-terminal of 3-galactosidase α- peptide.
359 amino acid catalytic cellulase domain.
Catalytic domains present in triplicate.
Predicted amino acid identity of each catalytic domain is >95%.
Catalytic domains show most identity with sub-family A4 cellulases (A4 comprises only endoglucanases from anaerobic bacteria, including
Butyrivibrio, Prevotella and
Ruminococcus) .
Serine-, threonine- and proline-rich linker sequence separating each of the catalytic domains.
Cysteine-rich repeats of unknown function. Repeats show limited identity with analogous carboxy- terminal domains encoded by Neocallimastix patriciarum xynA cDNA.
AT-rich 3' untranslated sequence and polyA transcription terminator.
Figure 14 - pNX-Tac construct
SUBSTITUTE SHEET Reproduced hereinbelow are sequence listings wherein -
SEQ ID N0:1 refers to nucleotide sequence of
Neocallimastix patriciarum celD cDNA. The sequence underlined is derived from pBluescript SK-vector and the EcoRI adaptor used for cDNA cloning.
SEQ ID NO:2 refers to translated sequence of domains I and II of Neocallimastix patriciarum celD cDNA. Translated polypeptide includes the N-terminus of the 3-galactosidase α-peptide (derived from nucleotides 1-111) and amino acids derived from the 5' oligonucleotide linker (nucleotides 112-124) used in cDNA library construction.
SEQ ID NO:3 refers to translated sequence of domain III of Neocallimastix patriciarum celD cDNA.
SEQ ID NO:4 refers to the sequence of the modified xylanase cDNA in pNX-Tac.
celD 39
1 TGACCATGA TTACGCCAAG CTCGAAATTA ACCCTCACTA AAGGGAACAA AAGCTGGAGC
61 TCCACCGCGG TGGCGGCCGC TCTAGAACTA GTGGATCCCC CGGGCTGCAG GAATTCGGCA
121 CGAGCTCCAA TCCGTGATAT TTCATCCAAA GAATTAATTA AAGAAATGAA TTTCGGTTGG
181 AATTTAGGTA ATACTTTAGA TGC CAATGT ATTGAATACT TAAATTATGA TAAGGATCAA
241 ACΓGCTTCTG AAACTTGCTG GGGTAATCCA AAGACTACTG AAGATATGTT CAAGGTTTTA
301 ATGGATAACC AATTTAATGT TTTCCGTATT CCAACTACTT GGTCTGGTCA CTTCGGTGAA
361 GCTCCAGATT ACAAGATTAA GAAAAATGG TTAAAGAGAG TTCATGAAAT TGTTGATTAT
421 CCATACAAGA A GGAGCTTT CCΪTTA CTTA AATCT CACC ATGAAACTTG GAACCATGCC
481 TTCTCTGAAA CTCTTGACAC TGCCAAGGAA ATCTTAGAAA AGATTTGGTC TCAAATTGCT
541 AAAGAATTTA AGGATTATGA GAACACTTA ATTTTTGAAG GATTAAACGA ACCAAGAAAG
601 AATGATACTC CAGTTGAATG GACTGGTGGT GA CAAGAAG GATGGGATGC TGTTAATGCT
661 ATGAATGCCG TTTTCTTAAA GACTATTCGT AGTTCTGG G GTAATAATCC AAAGCGTCAT
721 CTTATGATCC CTCCATA GC TGCTGCTTGT AATGAAAATT CATTCAAGAA CTTTATTTTC
781 CCAGAAGATG ATGACAAGGT TA TGCTTCT GTTCATGCTT ATGCTCCATA CAACTTTGCC
841 TTAAATAATG G GAAGGAGC TGTTGATAAG TTTGA GCTG CTGGTAAGAA AGATCTTGAA
901 GGAACATTA ACTTAATGAA GAAGAGATTT GTTGATCAAG GTATTCCAAT GATTCTTGGT
961 GAATATGGTG CCATGAATCG TGATAATGAA GAAGATCGTG CAGCT GGGC TGAATTC AC
1021 ATQGAAAAGG TCACTGCTAT GGGAGTTCCA CAAGTCTGGT GGGATAATGG TATCTTTGAA
1081 GGTACCGGTG AACGTTTTGG TCTTCTTGAT CGTAAAAACT TAAAGATTGT TTATCCAACT
1141 ATCGTTGCTG CTTTACAAAA GGGAAGAGGT TTAGAAGTCA ATGTTGTTCA TGCTAT GAA
1201 AAAAAAOCAG AAGAAOCAAC TAAAACTACT GAACCAGTTG AACCAAC GA AACTACTAGT
1261 CCAGAAGAAC CAGCTGAAAC TACTAA CCA GAAGAAOCAA CCGGTAATAT CGTGATATT
1321 CATCTAAQG AATTAATTAA ACY-AATGAAT TTCGGT GGA ATTTAGGTAA TACTTTAGAT
1381 GCTCAATGTA TTGAATACTT AAATTATGAT AAGGATCAAA CTGCTTCTGA AACTTGCTGG
1441 GGTAATCCAA AGACTACTGA AGATATGTTC AAGGTTTTAA TGGATAACCA ATTTAATGTT
1501 TTCCGTATTC CAACTACTTG GTCTGGTCAC TTCGGTGAAG CTOCAGATTA CAAGATTAAT
1561 GAAAAATGGT TAAAGAGAGT TCATGAAATT GTTGATTATC CATACAAGAA TGGAGCTTTC
1621 GTTATCTTAA ATCTTCACCA TGAAACTTGG AACCATGCTT TCTCTGAAAC TCTTGACACT 1681 GCCAAGGAAA TTTTAGAAAA GATTTGGTCT CAAATTGCTG AAGAATTTAA GGATTATGAT 1741 GAACACTTAA TTTTTGAAGG ATTAAACGAA CCAAGAAAGA ATGATACTCC AGTTGAATCC 1801 ACTGGTGGTG ATCAAGAAGG ATGGGATGCT GTTAATGCTA TGAATGCCGT TTTCTTAAAG 1861 ACTATTCGTA GTTCTGGTGG TAATAATCCA AAGCGTCATC TTATGATCCC TCCATATGCT 1921 GCTGCTTGTA ATGAAAATTC ATTCAAGAAC TTTATTTTCC CAGAAGATGA TGACAAGGTT 1981 ATTGCTTCTG TTCATGCTTA TGCTCCATAC AACTTTGCCT TAAATAATGG TGAAGGAGCT 2041 GTTGATAAGT TTGATGCCGC TGGTAAGAAT GACCTTGAAT GGAATATTAA CTTAATGAAG 2101 AAGAGATTTG TTGATCAAGG TATTCCAATG ATTCTTGGTG AATATGGTGC CATGAATCGT 2161 GATAATGAAG AAGATCGTGC AGCTTGGGCT GAATTCTACA TGGAAAAGGT CACTGCTATG 2221 GGAGTTCCAC AAGTCTGGTG GGATAATGGT ATCTTTGAAG GTACCGGTGA ACGTTTTGGT 2281 CTTCTTGATC GTAGAAACTT AAAGATTGTT TATCCAACTA TCGTTGCTGC TTTACAAAAG 2341 GGAAGAGGTT TAGAAGTCAA TGTTGTTCAT GCTGTTGAAA AAAAAACCAG AAGAACCAAC 2401 TAAGACTACT GAACCAGTTG AACCAACTGA AACTACTAGT CCAGAAGAAC CAACTGAAAC 2461 TACTAATCCA GAAGAAOCAA CCGGTAATAT TCGTGATATT TCATCTAAGG AATTAATTAA 2521 AGAAATGAAT TTCGGTTGGA ATTTAGGTAA TACTTTAGAT GCTCAATGTA TTGAATACTT 2581 AAATTATGAT AAGGATCAAA CTGCTTCTGA AACTTGCTGG GGTAATCCAA AGACTACTGA 2641 AGATATGTTC AAGGTTTTAA TGGATAACCA ATTTAATGTT TTCCGTATTC CAACTACTTG 2701 GTCTGGTCAC TTCGGTGAAG CTCCAGATTA CAAGATTAAT GAAAAATGGT TAAAGAGAGT 2761 TCATGAAATT GTTGATTATC CATACAAGAA TGGAGCTTTC GTTATCTTAA ATCTTCACCA 2821 TGAAACTTGG AACCATGCTT TCTCTGAAAC TCTTGACACT GCCAAGGAAA TTTTAGAAAA 2881 GATTTGGTCT CAAATTGCTG AAGAATTTAA GGATTATGAT GAACACTTAA TTTTTGAAGG 2941 ATTAAACGAA CCAAGAAAGA ATGATACTCC AGTTGAATGG ACTGGTGGTG ATCAAGAAGG 3001 ATGGGATGCT GTTAATGCTA TGAATGCCGT TTTCTTAAAG ACTATTCGTA GTTCTGGTGG 3061 T.AATAATCCA AAGCGTCATC TTATGATCCC TCCATATGCT GCTGCTTGTA ATGAAAATTC 3121 ATTCAAGAAC TTTATTTTCC CAGAAGATGA TGACAAGGTT ATTGCTTCTG TTCATGCTTA 3181 TGCTCCATAC AACTTTGCCT TAAATAATGG TGCAGGAGCT GTTGATAAGT TTGATGCCGC 3241 TGGTAAGAAA GATCTTGAAT GGAACATTAA CTTAATGAAG AAGAGATTTG TTGATCAAGG 3301 TATTCCAATG ATTCTTGGTG AATATGGTGC CATGAACCGT GATAATGAAG AAGAACGTGC 3361 TACATGGGCT GAATTCTACA TGGAAAAGGT CACTGCTATG GGAGTTCCAC AAGTCTGGTG 3421 GGATAATGGT GTCTTTGAAG GTACCGGTGA ACGTTTTGGT CTTCTTGATC GTAAAAACTT 3481 AAAGATTGTT TATCCAACTA TCGTTGCTGC TTTACAAAAG GGAAGAGGTT TAGAAGTTAA 3541 GGTTGTTCAT GCAAATGAAG AAGAAACAGA AGAATGTTGG TCTGAAAAGT ATGGTTATGA 3601 ATGTTGTTCT CCTAACAATA CTAAGGTTGT AGTCAGTGAT GAAAGTGGAA ATTGGGGTGT 3661 TGAAAATGGT AATTGGTGTG GTGTTCTTAA ATACACTGAA AAATGTTGGT CACTTCCATT 3721 TGGATACCCA TGTTGTCCAC ATTGTAAGGC TCTTACTAAG GATGAAAATG GTAAATGGGG 3781 AGAAGTAAAT GGTGAATGGT GTGGTATTGT TGCTGATAAA TGTTAGATTA TAAAATAAAA 3841 ATAAATAGAT TTTGTTATGA AAATTATTAA TGAATAATAA ATAAAATAGA AAATTTTATA 3901 TAAACATATT TCTAATAAAA GATGTAATTA TGTATTTTTT GTTTCTTATT CTTTCAAATA 3961 AAAAAAGTAA GGAAAGAAAA TATATAAAAT AAAAAAAAAA AATAAATAAA TAAATATTTT 4021 AATTATTTTT TTTTTACTAA AAAAAAGGAA TTTAATTAAA ATATAATTAA AA TAAAAAA
SEQ ID NO:l
SUBSTITUTE SHEET celD
4081 AAAAAAAAAA AACTCGAG
SEQ ID NO:l
SUBSTITUTE SHEET celD .oml/2 Translated. Secjuence
Sequence Range: 1 Co 2400
10 20 30 40 50
* * * * *
ATG ACC ATG ATT ACG CCA AGC TCG AAA TTA ACC CTC ACT AAA GGG AAC AAA AGC TAC TGG TAC TAA TGC GGT TOG AGC TTT AAT TGG GAG TGA TTT CCC TTG TTT TCG M T M I T P S S K L T L T K G N K S> a a a a a TRANSLATION OF CELD DOM1/2 a a a a a >
60 70 80 90 100
* * * * *
TGG AGC TCC ACC GCG GTG GOG GCC GCT CTA GAA CTA GTG GAT CCC COG GGC TGC ACC TCG AGG TGG CGC CAC CGC CGG CGA GAT CTT GAT CAC CTA GGG GGC CCG ACG W S S T A V A A A L E L V D P P G C> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
110 120 130 140 150 160
•» * * « • *
AGG AAT TOG GCA CGA GCT CCA ATC CGT GAT ATT TCA TCC AAA GAA TTA ATT AAA TCC TTA AGC CGT GCT CGA GGT TAG GCA CTA TAA AGT AGG TTT CTT AAT TAA TTT R N S A R A P I R D I S S K E L I K> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
170 180 190 200 210
GAA ATG AAT TTC GGT TGG AAT TTA GGT AAT ACT TTA GAT GCT CAA TGT ATT GAA CTT TAC TTA AAG CCA ACC TTA AAT OCA TTA TGA AAT CTA CGA GTT ACA TAA CTT E M N F G W N L G N T L D A Q C I E> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
220 230 240 250 260 270
<* •* * * * •*
TAC TTA AAT TAT GAT AAG GAT CAA ACT GCT TCT GAA ACT TGC TGG GGT AAT CCA ATG AAT TTA ATA CTA TTC CTA GTT TGA CGA AGA CTT TGA ACG ACC CCA TTA GGT Y L N Y D K D Q T A S E T C W G N P> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
280 290 300 310 320
* * • » «
AAG ACT ACT GAA GAT ATG TTC AAG GTT TTA ATG GAT AAC CAA TTT AAT GTT TTC TTC TGA TGA CTT CTA TAC AAG TTC CAA AAT TAC CTA TTG GTT AAA TTA CAA AAG K T T E D M F K V L M D N Q F N V F> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
330 340 350 360 370
•» * * * *
CGT ATT CCA ACT ACT TGG TCT GGT CAC TTC GGT GAA GCT CCA GAT TAC AAG ATT GCA TAA GGT TGA TGA ACC AGA CCA GTG AAG CCA CTT CGA GGT CTA ATG TTC TAA R I P T T W S G H F G E A P D Y K I> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
380 390 400 410 420 430
« * * * * *
AAT GAA AAA TGG TTA AAG AGA GTT CAT GAA ATT GTT GAT TAT CCA TAC AAG AAT TTA CTT TTT ACC AAT TTC TCT CAA GTA CTT TAA CAA CTA ATA GGT ATG TTC TTA E K W L K R V H E I V D Y P Y K N> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
440 450 460 470 480
GGA GCT TTC GTT ATC TTA AAT CTT CAC CAT GAA ACT TGG AAC CAT GCC TTC TCT CCT CGA AAG CAA TAG AAT TTA GAA GTG GTA CTT TGA ACC TTG GTA CGG AAG AGA G A F V I L N L H H E T W N H A F S> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
490 500 510 520 530 540
SEQ ID NO: 2
SUBSTITUTE SHEET celD doml/2 Translated Sequence 42
GAA ACT CTT GAC ACT GCC AAG GAA ATC TTA GAA AAG ATT TGG TCT CAA ATT GCT CTT TGA GAA CTG TGA CGG TTC CTT TAG AAT CTT TTC TAA ACC AGA GTT TAA CGA E T L D T A K E I L E K I W S Q l A> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a -y
550 560 570 580 590
AAA GAA TTT AAG GAT TAT GAT GAA CAC TTA ATT TTT GAA GGA TTA AAC GAA CCA TTT CTT AAA TTC CTA ATA CTA CTT GTG AAT TAA AAA CTT CCT AAT TTG CTT GGT K E F K D Y D E H L I F E G L N E P> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
600 610 620 630 640
* * * * *
AGA AAG AAT GAT ACT CCA GTT GAA TGG ACT GGT GGT GAT CAA GAA GGA TGG GAT TCT TTC TTA CTA TGA GGT CAA CTT ACC TGA CCA CCA CTA GTT CTT CCT ACC CTA R K D T P V E W T G G D Q E G W D> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
650 660 670 680 690 700
* * * * * *
GCT GTT AAT GCT ATG AAT GCC GTT TTC TTA AAG ACT ATT CGT AGT TCT GGT GGT CGA CAA TTA CGA TAC TTA CGG CAA AAG AAT TTC TGA TAA GCA TCA AGA CCA CCA A V N A M N A V F L K T I R S S G G> a a a a a "TRANSLATION OF CELD DQMl/2 a a a a a >
710 720. 730 740 750
* * * * -*
AAT AAT CCA AAG CGT CAT CTT ATG ATC CCT CCA TAT GCT GCT GCT TGT AAT GAA TTA TTA GGT TTC GCA GTA GAA TAC TAG GGA GGT ATA CGA CGA CGA ACA TTA CTT N N P K R H L M I P P Y A A A C N E> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
760 770 780 790 800 810
• * * « * *
AAT TCA TTC AAG AAC TTT ATT TTC CCA GAA GAT GAT GAC AAG GTT ATT GCT TCT TTA AGT AAG TTC TTG AAA TAA AAG GGT CTT CTA CTA CTG TTC CAA TAA CGA AGA - N S F K N F I F P E D D D K V I A S> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
820 830 840 850 860
* * * * *
GTT CAT GCT TAT GCT CCA TAC AAC TTT GCC TTA AAT AAT GGT GAA . GCT GTT CAA GTA CGA ATA CGA GGT ATG TTG AAA CGG AAT TTA TTA CCA CTT CCT CGA CAA V H A Y A P Y N F A L N N G E G A V> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
870 880 890 900 910
* * * * «
GAT AAG TTT GAT GCT GCT GGT AAG AAA GAT CTT GAA TGG .AAC ATT AAC TTA ATG CTA TTC AAA CTA CGA CGA CCA TTC TTT CTA GAA CTT ACC TTG TAA TTG AAT TAC D K F D A A G K K D L E W N I N L M> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
920 930 940 950 960 970
* * * * * *
AAG AAG AGA TTT GTT GAT CAA GGT ATT CCA ATG ATT CTT GGT GAA TAT GGT GCC TTC TTC TCT AAA CAA CTA GTT CCA TAA GGT TAC TAA GAA CCA CTT ATA CCA CGG K K R F V D Q G I P M I L G E Y G A> a a TRANSLATION OF CELD DQMl/2 a a a a a >
980 990 1000 1010 1020
* * * * *
ATG AAT CGT GAT AAT GAA GAA GAT CGT GCA GCT TGG GCT GAA TTC TAC ATG GAA TAC TTA GCA CTA TTA CTT CTT CTA GCA CGT CGA ACC CGA CTT AAG ATG TAC CTT M N R D N E E D R A A W A E F Y M E> a a a a a TRANSLATION OF CELD DQMl/2.. a a a a a >
SEQ ID NO: 2
j SUBSTITUTE SHEET celD doml/2 Translated Sequence 3
1030 1040 1050 1060 1070 1080
* * * * * *
AAG GTC ACT GCT ATG GGA GTT CCA CAA GTC TGG TGG GAT AAT GGT ATC TTT GAA TTC CAG TGA CGA TAC CCT CAA GGT GTT CAG ACC ACC CTA TTA CCA TAG AAA CTT K V T A M G V P Q V W W D N G I F E> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
1090 1100 1110 1120 1130
* * * * *
GGT ACC GGT GAA CGT TTT GGT CTT CTT GAT CGT AAA AAC TTA AAG ATT GTT TAT CCA TGG CCA CTT GCA AAA CCA GAA GAA CTA GCA TTT TTG AAT TTC TAA CAA ATA G T G E R F G L L D R K N L K I V > a a a a a TRANSLATION OF CELD Dθt.1/2 a a a a a >
1140 1150 1160 1170 1180
* * * * *
CCA ACT ATC GTT GCT GCT TTA CAA AAG GGA AGA GGT TTA GAA GTC AAT GTT GTT GGT TGA TAG CAA OGA CGA AAT GTT TTC CCT TCT CCA AAT CTT CAG TTA CAA CAA P T I V A A L Q K G R G L E V N V V> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
1190 1200 1210 1220 1230 1240
* •* * * * *
CAT GCT ATT GAA AAA AAA CCA GAA GAA CCA ACT AAA ACT ACT GAA CCA GTT GAA GTA CGA TAA CTT TTT TTT GGT CTT CTT GGT TGA TTT TGA TGA CTT GGT CAA CTT H A I E K K P E E P T K T T E P V E> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
1250 1260 1270 1280 1290
• * * * «
CCA ACT GAA ACT ACT AGT CCA GAA GAA CCA GCT GAA ACT ACT AAT CCA GAA GAA GGT TGA CTT TGA TGA TCA GGT CTT CTT GGT CGA CTT TGA TGA TTA GGT CTT CTT P T E T T S P E E P A E T T N P E E> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
1300 1310 1320 1330 1340 1350
CCA ACC GGT AAT ATT OGT GAT ATT TCA TCT AAG GAA TTA ATT AAA GAA ATG AAT GGT TGG CCA TTA TAA GCA CTA TAA AGT AGA TTC CTT AAT TAA TTT CTT TAC TTA P T G N I R D I S S K E L I K E M > a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
1360 1370 1380 1390 1400
* * * * *
TTC GGT TGG AAT TTA GGT AAT ACT TTA GAT GCT CAA TGT ATT GAA TAC TTA AAT AAG CCA ACC TTA AAT CCA TTA TGA AAT CTA CGA GTT ACA TAA CTT ATG AAT TTA F G W N L G N T I- D A Q C I E Y N> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
1410 1420 1430 1440 1450
* * * * *
TAT GAT AAG GAT CAA ACT GCT TCT GAA ACT TGC TGG GGT AAT CCA AAG ACT ACT ATA CTA TTC CTA GTT TGA OGA AGA CTT TGA ACG ACC CCA TTA GGT TTC TGA TGA Y D K D Q T A S E T C W G N P K T T> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
1460 1470 1480 1490 1500 1510
* * * * * *
GAA GAT ATG TTC .AAG GTT TTA ATG GAT AAC CAA TTT AAT GTT TTC CGT ATT CCA CTT CTA TAC AAG TTC CAA AAT TAC CTA TTG GTT AAA TTA CAA AAG GCA TAA GGT E D M F K V L M D N Q F N V F R I P> a a a a a TRANSLATION OF CELD DQMl/2 a a a a a >
1520 1530 1540 1550 1560
* * * * * .
ACT ACT TGG TCT GGT CAC TTC GGT GAA GCT CCA GAT TAC AAG ATT AAT GAA AAA
SEQ ID NO:2
SUBSTITUTE SH celD doml/2 Translated Sequence
'TGA TGA ACC AGA CCA GTG AAG CCA CTT CGA GGT CTA ATG TTC TAA TTA CTT TTT
T T W S G H F G E A P D Y K I N E K> ___a__a___a__a__a__TRANSLATICN OF CELD DQMl/2 a a a a a --
1570 1580 1590 1600 1610 1620
TGG TTA AAG AGA GTT CAT GAA ATT GTT GAT TAT CCA TAC AAG AAT GGA GCT TTC ACC AAT TTC TCT CAA GTA CTT TAA CAA CTA ATA GGT ATG TTC TTA CCT CGA AAG W L K R V H E I V D Y P Y K N G A F> a a a a a TRANSLATION OF CELD DOMl/2___a__a__a_a__a__>
1630 1640 1650 1660 1670
* * * * *
GTT ATC TTA AAT CTT CAC CAT GAA ACT TGG AAC CAT GCT TTC TCT GAA ACT CTT CAA TAG AAT TTA GAA GTG GTA CTT TGA ACC TTG GTA CGA AAG AGA CTT TGA GAA I L N L H H E T W N H A F S E T L> a a a a a TRANSLATION OF CELD DQMl/2 a a a a *
1680 1690 1700 1710 1720
GAC ACT GCC AAG GAA ATT TTA GAA AAG ATT TGG TCT CAA ATT GCT GAA GAA TTT CTG TGA CGG TTC CTT TAA AAT CTT TTC TAA ACC AGA GTT TAA CGA CTT CTT AAA D T A K E I L E K I W S Q I A E E F> a a a a a TRANSLATION OF CELD DOMl/2 a a a a a >
1730 1740 1750 1760 1770 1780
* * * * * *
AAG GAT TAT GAT GAA CAC TTA ATT TTT GAA GGA TTA AAC GAA CCA AGA AAG AAT TTC CTA ATA CTA CTT GTG AAT TAA AAA CTT CCT AAT TTG CTT GGT TCT TTC TTA K D Y D E H L I F E G L N E P R K N> a a a a a TRANSLATION OF CELD DOMl/2 a a a a a >
1790 1800 1810 1820 1830
* * * * *
GAT ACT CCA GTT GAA TCC ACT GGT GGT GAT CAA GAA GGA TGG GAT GCT GTT AAT CTA TGA GGT CAA CTT AGG TGA CCA CCA CTA GTT CTT CCT ACC CTA CGA CAA TTA D T P V E S T G G D Q E G W D A V N> a a a a a TRANSLATION OF CELD DOMl/2 a a a a a >
1840 1850 1860 1870 1880 1890
* * -* * * *
GCT ATG AAT GCC GTT TTC TTA AAG ACT ATT CGT AGT TCT GGT GGT AAT AAT CCA CGA TAC TTA CO--? CAA AAG AAT TTC TGA TAA GCA TCA AGA CCA CCA TTA TTA GGT A M N A V F L K T I R S S G G N N P a a a a a TRANSLATION OF CELD DOMl/2 a a a a a >
1900 1910 1920 1930 1940
* * * * *
AAG CGT CAT CTT ATG ATC CCT CCA TAT GCT GCT GCT TGT AAT GAA AAT TCA TTC TTC GCA GTA GAA TAC TAG GGA GGT ATA CGA CGA CGA ACA TTA CTT TTA AGT AAG K R H L M I P P Y A A A C N E N S F> a a a a a TRANSLATION OF CELD DOMl/2 a a a a a >
1950 1960 1970 1980 1990
* * * * *
AAG AAC TTT ATT TTC CCA GAA GAT GAT GAC AAG GTT ATT GCT TCT GTT CAT GCT TTC TTG .AAA TAA AAG GGT CTT CTA CTA CTG TTC CAA TAA CGA AGA CAA GTA CGA K N F I F P E D D D K V I A S V H A> a a a a a TRANSLATION OF CELD DOMl/2. _. a a a a a >
2000 2010 2020 2030 2040 2050
* * * * * •
TAT GCT CCA TAC AAC TTT GCC TTA AAT AAT GGT GAA GGA GCT GTT GAT AAG TTT ATA CGA GGT ATG TTG AAA CGG AAT TTA TTA CCA CTT CCT CGA CAA CTA TTC AAA Y A P Y N F A L N N G E G A V D K F> a a a a a TRANSLATION OF CELD DOMl/2__a___a_a a a >
SEQ ID NO: 2
SUBSTITUTE SHEET celD doml/2 Translated Sequence
2060 2070 2080 2090 2100
* * * * *
GAT GCC GCT GGT AAG AAT GAC CTT GAA TGG AAT ATT AAC TTA ATG AAG AAG AGA CTA CGG CGA CCA TTC TTA CTG GAA CTT ACC TTA TAA TTG AAT TAC TTC TTC TCT D A A G K N D L E W N I N L M K K R> a a a a a TRANSLATION OF CELD DOMl/2 a a a a a ->
2110 2120 2130 2140 2150 2160
* * * * * *
TTT GTT GAT CAA GGT ATT CCA ATG ATT CTT GGT GAA TAT GGT GCC ATG AAT CGT AAA CAA CTA GTT CCA TAA GGT TAC TAA GAA CCA CTT ATA OCA CGG TAC TTA GCA F V D Q G I P M I L G E Y G A M N R a a a a a TRANSLATION OF CELD DOMl/2 a a a a a >
2170 2180 2190 2200 2210
* * * * *
GAT AAT GAA GAA GAT CGT GCA GCT TGG GCT GAA TTC TAC ATG GAA AAG GTC ACT CTA TTA CTT CTT CTA GCA CGT CGA ACC CGA CTT AAG ATG TAC CTT TTC CAG TGA D N E E D R A A W A E F Y M E K V T> a a a a a TRANSLATION OF CELD DOMl/2 a a a a a >
2220 2230 2240 2250 2260
* * * * *
GCT ATG GGA GTT CCA CAA GTC TGG TGG GAT AAT GGT ATC TTT GAA GCT ACC GGT CGA TAC CCT CAA GGT GTT CAG ACC ACC CTA TTA CCA TAG AAA CTT CCA TGG OCA A M G V P Q V W W D N G I F E G T G> a a a a a TRANSLATION OF CELD DOMl/2 a a a a a >
2270 2280 2290 2300 2310 2320
GAA CGT TTT GGT CTT CTT GAT CGT AGA AAC TTA AAG ATT GTT TAT CCA ACT ATC CTT GCA AAA CCA GAA GAA CTA GCA TCT TTG AAT TTC TAA CAA ATA GGT TGA TAG .E R F G L L D R R N L K I V Y P T I> a a a a a TRANSLATION OF CELD DOMl/2 a a a a a >
2330 2340 2350 2360 2370
* * * * *
GTT GCT GCT TTA CAA AAG GGA AGA GGT TTA GAA GTC AAT GTT GTT CAT GCT GTT CAA CGA CGA AAT GTT TTC CCT TCT CCA AAT CTT CAG TTA CAA CAA GTA CGA CAA V A A L Q K G R G L E V N V V H A V> a a a a a TRANSLATION OF CELD DOMl/2 a a a a a >
2380 2390 2400
* * «
GAA AAA AAA ACC .AGA AGA ACC AAC CTT TTT TTT TGG TCT TCT TGG TTG- E K K T R R T N> TRANSLATION OF CELD D0K1__>
SEQ ID NO: 2
SUBSTITUTE SHEET celD dom3 46
Sequence
ATT CGT
TTA GGT C ^TT TA MC T NTA AA pG CC GA A wCC TT N A AA L T . CC G A >
GAT CAA ACT
DOM3 TRUNC_a a a a a >
TCT GGT CAC
>
SEQ I D NO : 3
SUBSTITUTE SHEET 47 celD doπβ
SEQ ID NO: 3 48 celD doπβ P
1030 1040 1050 1060 1070 1080
* * * * * *
GTC TGG TGG GAT AAT GCT GTC TTT GAA GCT ACC GGT GAA CGT TTT GGT CTT CTT CAG ACC ACC CTA TTA CCA CAG AAA CTT CCA TGG CCA CTT GCA AAA CCA GAA GAA V W W D N G V F E G T G E R F G L L> a a a a TRANSLATION OF CELD DOM3 TRUNC_a a a a_ a >
1090 1100 1110 1120 1130
* * * * *
GAT CGT AAA AAC TTA AAG ATT GTT TAT CCA ACT ATC GTT GCT GCT TTA CAA AAG CTA GCA TTT TTG AAT TTC TAA CAA ATA GCT TGA TAG CAA CGA CGA AAT GTT TTC D R K N L K I V Y P T I V A A L Q K> a a a a TRANSLATION OF CELD DOM3 TRUNC a a a a a _>
1140 1150 1160 1170 1180
* * * * *
GGA AGA GGT TTA GAA GTT AAG GTT GTT CAT GCA AAT GAA GAA GAA ACA GAA GAA CCT TCT CCA AAT CTT CAA TTC CAA CAA GTA CCT TTA CTT CTT CTT TCT CTT CTT G R G L E V K V V H A N E E E T E E> a a a a TRANSLATION OF CELD DOM3 TRUNC a a a a a __
1190 1200 1210 1220 1230 1240
• * * * * *
TGT TGG TCT GAA AAG TAT GGT TAT GAA TGT TGT TCT CCT AAC AAT ACT AAG GTT ACA ACC AGA CTT TTC ATA CCA ATA CTT ACA ACA AGA GGA TTG TTA TGA TTC CAA C W S E K Y G Y E C C S P N N T K V> a a a a TRANSLATION OF CELD DOM3 TRUNC a a a a a >
1250 1260 1270 1280 1290
* * * * *
GTA GTC AGT GAT GAA ACT GGA AAT TGG GGT GTT GAA AAT GGT AAT TGG TGT GGT CAT CAG TCA CTA CTT TCA CCT TTA ACC CCA CAA CTT TTA CCA TTA ACC ACA CCA V V S D E S G N W G V E N G N C G> a a a a TRANSLATION OF CELD DOM3 TRUNC a a a a a >
1300 1310 1320 1330 1340 1350
* * * * * *
GTT CTT AAA TAC ACT GAA AAA TGT TGG TCA CTT CCA TTT GGA TAC CCA TGT TGT CAA GAA TTT ATG TGA CTT TTT ACA ACC ACT GAA GCT AAA CCT ATG GGT ACA ACA V L K Y T E K C W S L P F G Y P C C> a a a a TRANSLATION OF CELD D0M3 TRUNC a a a a a >
1360 1370 1380 1390 1400
* * * * *
CCA CAT TGT AAG GCT CTT ACT AAG GAT GAA AAT GCT AAA TGG GGA GAA CTA AAT GGT GTA ACA TTC CGA GAA TGA TTC CTA CTT TTA CCA TTT ACC CCT CTT CAT TTA P H C K A L T K D E N G K G E V > a a a a TRANSLATION OF CELD DOM3 TRUNC a a a a a >
1410 1420 1430
* * *
GGT GAA TGG TGT GGT ATT GTT GCT GAT AAA TGT
CCA CTT ACC ACA CCA TAA CAA CGA CTA TTT ACA
G E W C G I V A D K C> a TRANSLATION OF CELD D0M3 TRUNC a >
SEQ ID NO: 3
SUBSTITU ATGGCTAGC AATGGTAAAAAGT M A S N G K K
TTACTGTCGGTAATGGACAAAACCAACATAAGGGTGTCAACGATGGTTTCAGTTATGAAA F T V G N G Q N Q H K G V N D G F S Y E
TCTGGTTAGATAACACTGGTGGTAACGGTTCTATGACTCTCG^TAGTGGTGCAACTTTCA I W L D N T G G N G S M T L G S G A T F
AGGCTGAATGGAATGCAGGΓGTTAACCGTG;TAACTTCCTTGCCCGTCGTGGTCTTGACT
K A E N A A V N R G N F L A R R G L D
TCGGTTCTCAAAAGAAGGCAACCGATTACGACTACATTGGATTAGATTATGCTGCTACTT
F G S K K A T D Y D Y I G L D Y A A T
ACAAACAAACTGCCAGTGCAAGTGGTAACTCCCGTCTCTGTGTATACGGATGGTTCCAAA Y K Q T A S A S G N S R L C V Y G W F Q
ACCGTGGACTTAATGGCGTTCCTTTAGTAGAATACTACATCATTGAAGATTGGGTTGACT N R G L N G V P V E Y Y I I E D V7 V D
GGGTTCCAGATGCACAAGGAAAAATG5TAACCATTGATGGAGCTCAATATAAGATTTTCC W V P D A G K M V T I D G A Y K I F
AAATGGATCACACTGGTCCAACTATCAATGGTGGTAGTGAAACCTTTAAGCAATACTTCA Q M D H T G P T I N G G S E T F K Y F
GTGTCCGTCAACAAAAGAGAACTTCTGGTCATATTACTGTCTCAGATCACTTTAAGGAAT S V R Q Q K R T S G H I T V S D H F K E
GGGCCAAACAAGGTTGGGGTATTGGTAACCTTTATGAAGTTGCTTTGAACGCCGAAGGTT W A K Q G W G I G N L Y E V A L N A E G
GGCAAAGTAGTGGTGTTGCTGATGTCACCTTATTAGATGTTTACACAACTCCAAAGGGTT W S S G V A D V T L D V Y T T P K G
CTAGTCCAGGCACCTCTGCCGCTCCTCGT TAA S S P A T S A A P R
SEQ ID NO: 4
SUBSTITUTE SHEET

Claims

CLAIMS :
1. A method of cloning of cellulase clones from an anaerobic rumen fungus including the steps of:
(i) cultivation of an anaerobic rumen fungus;
(ii) isolating total RNA from the culture in step (i);
(iii) isolating poly A+ mRNA from the total
RNA referred to in step (ii);
(iv) constructing a cDNA expression library;
(v) ligating cDNA to a bacteriophage expression vector selected from λZAP, λZAPII or vectors of similar properties;
(vi) screening of cellulase positive recombinant clones in a culture medium incorporating cellulase by detection of cellulase hydrolysis; and
(vii) purifying cellulase positive recombinant clones.
2. A method as claimed in claim 1 wherein the expression vector is λZAPII.
3. A method as claimed in claim 1 wherein the detection of enzyme hydrolysis is carried out using a colour indicator Congo red.
4. A method as claimed in claim 1 wherein after production of cellulase positive clones the cDNA insert in such clones were excised into p Bluescript SK(-) using helper phage.
5. A method as claimed in claim 4 wherein the helper phage is R408 helper phage.
6. Cellulase positive recombinant clones produced by the method of claim 1.
7. Recombinant cellulase clones containing cellulase cDNAs derived from N. patriciarum, having the property of production of biologically functional cellulases in E coli .
8. Recombinant cellulase clone pCNP4.1 in E coli strain XL1-Blue deposited at the Australian Government Analytical Laboratories on June 22, 1992 under accession number N92/27543.
9. An isolated DNA molecule including a DNA sequence essentially corresponding to pCNP4.1 cellulase cDNA as shown in SEQ ID NO:1 including DNA sequences capable of hybridizing thereto.
10. A polypeptide including amino acid sequence of pCNP4.1 cellulase essentially as shown in SEQ ID NO:2 and SEQ ID NO:3.
11. Cellulases produced from the recombinant cellulase clones of claim 6.
12. Cellulases produced from the recombinant cellulase clones of claim 7.
13. celA enzyme produced from a recombinant cellulase cDNA construct contained in an E coli host cell and having activity against crystalline and amorphous cellulose and other cellulosic substrates.
14. celD enzyme being a multifunctional cellulase having activity as an endoglucanase, cellobiohydrolase and also as a xylanase.
15. A DNA construct containing a DNA sequence as claimed in claim 9 operably linked to regulatory regions capable of directing the expression of a polypeptide having cellulase activity in a suitable expression host.
16. A transformed microbial host capable of the expression of fungal cellulase harbouring the cellulase construct of claim 15.
17. A polypeptide having cellulase activity produced by expression using a microbial host of claim 16.
18, A polypeptide including amino acid sequences derived from the polypeptide of claim 17.
19. Plasmid pCNP1 contained in E coli XLl-Blue lodged at the Australian Government Analytical
Laboratories on June 22, 1993 under accession number N93/28000.
20. An isolated cDNA molecule which encodes a functional Neocallimastix cellulase.
21. An isolated cDNA molecule which encodes a functional Neocallimastix pa tri ciarum cellulase.
22 A DNA construct containing a celA cDNA operably linked to regulatory regions capable of directing the expression of a polypeptide having cellulase activity in a suitable host.
23. celD cDNA capable of being truncated to code for three catalytically active domains having endoglucanase, cellobiohydrolase and xylanase activity respectively.
24. celA cDNA having a restriction map as shown in
FIG 2 including cellulase cDNAs which hybridise thereto.
25, celB cDNA having a restriction map as shown in
FIG 2 including cellulase cDNAs which hybridise thereto.
26. celC cDNA having a restriction map as shown in
FIG 2 including cellulase cDNAs which hybridise thereto.
27 celD cDNA having a restriction map as shown in
FIG 1 including cellulase cDNAs which hybridise thereto.
28. celE cDNA having a restriction map as shown in
FIG 1 including cellulase cDNAs which hybridise thereto.
29 Deletion mutants of celD cDNA having restriction maps as shown in FIG 12.
30. An enzyme composition including:
(i) celA enzyme produced from a recombinant cellulase clone contained in an E coli host cell and having activity against crystalline and amorphous cellulose and other cellulose substrates; and
(ii) celD enzyme capable of being truncated to code for three catalytically active domains having endoglucanase, cellobiohydrolase and xylanase activity respectively.
31. A combination of a recombinant cellulase derived from N. patriciarum in E coli and a recombinant xylanase derived from N. patriciarum in E coli .
32. An enzyme composition including -
(i) celA enzyme produced from a recombinant cellulase clone contained in an E coli host cell and having activity against crystalline and amorphous cellulose and other cellulose substrates; and
(ii) celD enzyme capable of being truncated to code for three catalytically active domains having endoglucanase, cellobiohydrolase and xylanase activity respectively.
(iii) A xylanase enzyme encoded by pNX Tac essentially as shown in Fig. 13 and SEQ ID NO: 4.
33. An enzyme composition including - (i) celD enzyme capable of being truncated to code for three catalytically active domains having endoglucanase, cellobiohydrolase and xylanase activity respectively,
(ii) A xylanase enzyme encoded by pNX Tac essentially as shown in Fig. 13 and SEQ ID NO:4.
34. An enzyme composition including - (i) celA enzyme produced from a recombinant cellulase clone contained in an E coli host cell and having activity against crystalline and amorphous cellulose and other cellulose substrates; and
(ii) A xylanase enzyme encoded by pNX Tac essentially as shown in Fig. 13 and SEQ ID NO: 4.
35. A polypeptide derived from the celD cDNA of claim 23.
36. A polypeptide derived from the celA cDNA of claim 24.
37 A polypeptide derived from the celD cDNA of claim 27.
EP93912457A 1992-06-24 1993-06-24 Recombinant cellulases. Withdrawn EP0649471A4 (en)

Applications Claiming Priority (3)

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AUPL3096/92 1992-06-24
AUPL309692 1992-06-24
PCT/AU1993/000307 WO1994000578A1 (en) 1992-06-24 1993-06-24 Recombinant cellulases

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US5824533A (en) * 1995-05-19 1998-10-20 University Of Georgia Research Foundation, Inc. Orpinomyces xylanase proteins and coding sequences
WO2000061625A1 (en) * 1999-04-09 2000-10-19 Human Genome Sciences, Inc. 48 human secreted proteins
GB9908077D0 (en) * 1999-04-09 1999-06-02 Babraham Inst Novel carbohydrate binding domain
DE10038969A1 (en) * 2000-08-10 2002-03-14 Infineon Technologies Ag Semiconductor component with diagnostic device, diagnostic element and circuit arrangement for diagnosis
US7226772B2 (en) * 2002-09-17 2007-06-05 Geneway Biotechnology Corporation Recombinant xylanases derived from anaerobic fungi, and the relevant sequences, expression vectors and hosts
EP1612267A1 (en) * 2004-07-02 2006-01-04 GBF Gesellschaft für Biotechnologische Forschung mbH Cellulases from rumen
FR3000500A1 (en) * 2013-01-02 2014-07-04 Julien Sylvestre Obtaining microorganisms e.g. yeast, by feeding folivore monkey with diet comprising cellulose compound, recovering sample taken from microbial flora of digestive tract of monkey, and culturing sample in medium with cellulose compound
CN109750015B (en) * 2019-03-27 2023-05-23 云南师范大学 Xylanase mutant with improved thermostability and application thereof

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WO1989009259A1 (en) * 1988-03-24 1989-10-05 Novo-Nordisk A/S A cellulase preparation
WO1991010732A1 (en) * 1990-01-19 1991-07-25 Novo Nordisk A/S An enzyme exhibiting cellulase activity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989009259A1 (en) * 1988-03-24 1989-10-05 Novo-Nordisk A/S A cellulase preparation
WO1991010732A1 (en) * 1990-01-19 1991-07-25 Novo Nordisk A/S An enzyme exhibiting cellulase activity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9400578A1 *

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CA2139099A1 (en) 1994-01-06
FI945994A0 (en) 1994-12-21

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