CA2139099A1 - Recombinant cellulases - Google Patents

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 bac-teriophage expression vector selected from .lambda.ZAP, .lambda.ZAPII 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 bi-ologically functional cellulases in E. coli cells. There is also provided various cDNA molecules which may be utilised in the abovementioned method.

Description

W094/00578 ~l 39 ~ 9 9 ~ PCT/AU93/00307 .

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 ~-1,4-glucosidic bonds. Conversion of cellulose to simple sugars (cellobiose and glucose) involves at least two types of hydrolases:
endoglucanases which hydrolyse internal ~-1,4-glucosidic linkages in less ordered regions of cellulose and exoglucanases tmainly cellobiohydrolases) which cleave cellobiosyl units from non-reducing ends of cellulose chains. Xylan, similar to the structure of cellulose, consists of a backbone of ~-1,4-linked xylose units. The enzymatic cleavage of ~-1,4-xylosidic linkages is performed by endo-~-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 ~-1, 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 tMUC) and have no or little depolymerising activity against CM-cellulose. Similarly, many xylanases exclusively attack ~-1,4-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 ~139~99 `
W094/00578 PCT/AU93/003~-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 Neocallimastix 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 tQ be one of the key steps in achieving this goal.

W094/00578 21 ~ o g ~ PCT/AU93/00307 -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 Neocal l imastix 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., 1g88; 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;

WO 94/00578 ~ 1 3 q ~ pcr/Au93/oo3o~

(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 65 (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. col i . However this approach for isolation of cellulase genes from fungal genomic libraries with functional expression of 15 cellulase is usually not possible. This is because fungi are eucaryotic microorganisms. Most eucaryotic genes contain introns and E. col i is unable to perform post-transcriptional modification of mRNAs in order to splice out introns. Therefore, enzymatically 20 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. col i . However, cellulases in fungi are usually 25 glycosylated and glycosylation is often required for biological activity of many glycosylated enzymes. E.
col i lacks a glycosylation mechanism. This problem can be solved if the cloned gene is transferred to an eucaryotic organism, such as yeast. Other problems 30 which are often encountered in obtaining a biologically functional protein from a cDNA clone in E. col i 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 35 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 W094/00578 ~ 1 3 9 a 9 9 PCT/AU93/00307 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 51 217-224 (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, ~gt11 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 W094/00578 2134 ~ ~ 9 PCT/AU93/0030-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);

W O 94/00578 PC~r/A U93/00307 213'qOg9 (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 (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 hurleyensis, Neocallimastix stanthorpensis, 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

W094/00578 % 1 3q Q 9 9 PCT/AU93/003~-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) t 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) t mRNA to cDNA by the enzyme reverse transcriptase. The first strand of cDNA may be synthesised using reverse transcriptase and the second strand of the cDNA may be synthesised using E. coli DNA
polymerase I. The cDNA may subsequently be fractionated to a suitable size and may be ligated to the bacteriophage expression vector, preferably ~ZAP or ~ZAPII. The cDNA library may then be amplified after packaging in vitro, using any suitable host bacterial W094/00578 ~1 3 q o ~ 9 PCT/AU93/00307 ._ cell such as a suitable strain of E. col i .
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 AZAPII.
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 col i using an expression vector such as that described above. ~ZAP and ~ZAP II are examples of such expression vectors.
Therefore the term "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 W094/00578 2 ~ ~q 9 g ` PCT/AU93/0030~

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 col i 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. col i 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 40C, 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 40C, respectively.

W094/00578 %13~ ogg PCT/AU93/00307 .. _ (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 Neocal l imastix 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 col i PLK-F and XL1-Blue obtained from Stratagene. E col i 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 N2.
Powdered mycelia were homogenised in guanidinium thiocyanate solution (4M guanidinium thiocyanate, 0.5%
(w/v) sodium lauryl sarcosine, 25 mM-sodium citrate, pH7.0, 1mM-EDTA and 0.1 M-~-mercaptoethanol) using a mortar and pestle for 5 min and ther. 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 2 13 q 09 g 12 (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 4C 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 8Ø 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 techniaues.
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 screeninq 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 ~ZAPII 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 ~-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 ~- 213~9 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 sequencinq.
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 2 rapid mini-preparation method as follows.
One millilitre of phage lysate from liquid culture was incubated with RNAase A (10~g ml~') and DNasel (1~g ml~l) at 37C for 1 h and with proteinase K (1 mg ml~l) at 37C for 3 h and then extracted with phenol/chloroform.
The DNA was precipitated by ethanol, digested with EcoR1 and Xhol (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 50C 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~l , 0.2 mg herring sperm DNA ml~l (1 x Denhardt~s solution is 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone). High-stringency washing 21~q ~99 W094/00578 ~ PCT/AU93/0030' was performed in 0.1 x SSC/0.1% (w/v) SDS at 68C (1 X
SSC is 0.15 M-NaCl, 15 mM-sodium citrate).
EnzYme assaYs, cellulose-bindinq 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 39C 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 (H3PO4-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-bindin5 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 200~1 of pre-washed 5% (w/v) Avicel in 50 mM-sodium citrate (pH 5.7) at 0C with continuous shaking for 1h.
The unbound protein was removed after centrifugation and the Avicel pellet was washed three times with 50mM-W094/00578 2~ ~q O PCT/AU93/00307 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 39C
in 50 mM-sodium citrate (pH 5.7) with 1% (w/v) Avicel or cellodextrins (2 mg ml~l) 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 AZAPII 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 W094/00578 , PCT/AU93/0030' 213~ O~g ~
` 16 of cellulase in the supernatant of the recombinant bacteriophage lysates to Avicel followed by incubation at 39C 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 EcoR1-EcoR1 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 W094/00578 ~ 1 3 q O 9 9 PCT/AU93/00307 ._ .

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 ~-glucosidase. Other cellulosic substrates tested were lichenan (a mixed glucan containing ~-1,4 and ~-1,3 linkages) and laminarin (predominantly ~-1,3-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 ~-1,4-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-S
glucose units) to glucose and cellobiose. Its catalytic mode on these cellulosic substrates is of a typical endoglucanase (ie. it cleaved ~-1,4-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%) W094/00578 ~ l ~q 0 9 ~ PCT/AU93/003 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 30C-60C. The celA and celD enzymes are active preferably at 30C-50C. The recombinant enzymes remain active in hydrolysis of Avicel at 39C 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 celD
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 W094/00578 ~1 3 4 0 9 g ~ i PCT/AU93/00307 .~

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-bindinq 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 I per mg protein (the crude cell lysate preparation), compared to 24.3 ~g min I per mg protein for the enzyme added without prior absGrption. 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 WO 94/00578 ~ l 3 q ~ 9 9 ~ ,~ , PCI/AU93/00307 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 3I region of celD cDNA and domain III by exonuclease III digestion from both the 5' region and 3I 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 W094/00578 ~1 ~q ~ PCT/AU93/00307 . _ 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 funqus, 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%) W094/00578 2 ~ 3 q O 9 g ~ PCT/AU93/0030' 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 col i 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 10l 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 fortransfer into rumen bacteria.
Other potential applications of these W094/00578 ~139 ~9 PCT/AUg3/00307 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 cel D
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 ~-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.
35 The use of celA and celD as qenetic material for modification of some economicallY imPortant micro-orqanisms for imProvement of cellulose utilisation ~13 q O 9 g I PCT/AU93/0030~

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.

W094/00578 213 4~g I PCT/AU93/00307 Plasmid pCNP4.1 in E col i strain XL1-Blue has been deposited at the International Depository Australian Government Analytical Laboratories on June ~ 22, 1992 under accession number N92/27543.
Plasmid pCNP1 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).

W094/00578 21~9 U9~ ~j PCT/AU93/00307 REFERENCES:
BAUCHOP, T. (1981). The anaerobic fungi in rumen fiber digestion. Agriculture and Environment 6, 339-348.
BÉGUIN, P. (1990). Molecular biology of cellulose degradation. Annual Review of Microbiology 44, 219-248.
BERGER, E., JONES, W.A., JONES, D.T. & WOODS, D.R.
(1989). Cloning and sequencing of an endoglucanase (end I) gene from Butyrivibrio fibrisolvens H17c. Molecular and G e n e r a 1 Genetics 219, 193-198.
CAVICCHIOLI, R. & WATSON, K. (1991). Molecular cloning, expression and characterisation of endoglucanase genes from Fibrobacter succinogenes ARL Applied and Environmental Microbiology 57, 359-365.
DEHORITY, B.A. (1991). Cellulose degradation in ruminants in Biosynthesis and biodegradation of cellulose, edited by Haigler, C.H. and Weimer, P.S., pp 327-351. New York, Marcel Dekker.
DESHPANDE ET AL. (1988) In Methods in Enzomology, 160, pp 126-130.
EL-GOGARY, S. LEITE, A., CRIVELLARO, O., EVELEIGH, D.E.
& EL-DORRY, H. (1989). Mechanism by which cellulose triggers cellobiohydrolase I gene expression in Trichoderma reesei. Proceedings of the National Academy of Sciences of the United States of America 86, 6138-6141.
FLINT, H.J., McPHERSON, C.A. & BISSET, J. t1989).
Molecular cloning of genes from Ruminococcus flavefaciens encoding xylanase and ~(1, 3-, 1, 4-) glucanase activities. A p p 1 i e d a n d Environmental Microbiology 55, 1230-1233.

.

W094/00578 21 3 q ~ g ~ PcT/Aug3/on307 FLINT, H.J., McPHERSON, C.A. & MARTIN, J. (1991).
Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus ~ flavefaciens 17 cloned in pUC13. Journal of General Microbiology 137, 123-129.
FOONG, E., HAMAMOTO, T., SHOSEYOV, O. & DOI, R.H.
(1991). Nucleotide sequence and characteristics of endoglucanase gene engB
from Clostridium cellulovorans. Journal of General Microbiology 137, 1729-1736.
GILBERT, H.J., HAZLEWOOD, G.P., LAURIE, J.L., ORPIN, C.G. & XUE, G.P. (1992). Xylanase A from the rumen anaerobic fungus Neocallimasrix patriciarum contains two homologous catalytic domains. Molecular Microbiology 6(15), 2065-2072.
GILKES, N.R., HENRISSAT, B., KILBURN, D.G., MILLER, R.C.JR. & WARREN, R.A.J. (1991). Domains in microbial ~-1, 4-glycanases, sequence conservation, function, and enzyme families, Microbiological Review 55, 303-315.
GILKES, N.R., WARREN, R.A.J., MILLER, R.C.JR. &
KILBURN, D.G. (1988). Precise excision of the cellulose binding domains from two Cellulomonas fimicellulases by a homologous protease and the effect on carlaysis. The Journal of Biological Chemistry. 263, 10401-10407.
GOMEZ DE SEGURA, B.G. & FEVRE, M. (1991). Cell wall hydrolases from Neocallimastix frontaris:
production and regulation. Fourth International Mycological Congress, 185.
GOYAL, A., GHOSH, B. & EVELEIGH, D. (1991).
Characteristics of fungal cellulases.
Bioresource Technology 36, 37-50.

W094/00578 ~1 3q 0 g ~ PCT/AU93/0030' HAZLEWOOD, G.P., DAVIDSON, K., LAURIE, J.I., ROMANIEC, M.P.M. & GILBERT, H.J. (1990). Cloning and sequencing of the celA gene encoding endoglucanase A of Butyrivibrio fibrisolvens strain A46. Journal of General Microbiology 136, 2089-2097.
HOWARD, G.T. & WHITE, B.A. (1988). Molecular cloning and expression of cellulase genes from Ruminococcus albus 8 in Escherichia coli bacteriophage Applied and E n vi r o n m e n t a l Microbiology 54, 1752-1755.
LAKE, B.D. & GOODWIN, H.J. (1976). Lipids, In Chromatographic and Electropherotic Techniques, vol. 1, 4th Edition, pp345-366.
Edited by I. Smith and J.W.T. Seakins, Bath:
The Pitman Press.
LEVER, M. (1972). Anal. Biochem. 47, 273-279.
LI, X. & CLAZA, R.E. (1991). Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188. Applied and Environmental Microbiology 57, 3331-3336.
MEINKE, A., GILKES, N.R., KILBURN, D.G., MILLER, R.C.JR. & WARREN, R.A.J. (1991). Multiple domains in endoglucanase B Journal of Bacteriology 173, 7126-7135.
MESSNER, R. & KUBICEK, C.P. (1991). Carbon source control of cellobiohydrolase I and II
formation by Applied and Environmental Microbiology 57, 630-635.
ORPIN, C.G. & MUNN, E.A. (1986). Neocallimastix patriciarum sp. nov., a new member of the Neocallimasticaceae inhabiting the rumen of sheep. Transactions of the British Mycological Society 86, 178-180.
ROBSON, L.M. & CHAMBLISS, G.H. (1989). Cellulases of bacterial origin. Enzyme and Microbial Technology 11, 626-644.

~1~4099 W094/00578 ~ PCT/AU93/00307 -ROMANTEC, M.P.M., DAVIDSON, K., WHITE, B.A., HAZLEWOOD, G.P. (1989). Cloning of Ruminococcus albus endo-~-1, 4-glucanase and xylanase genes.
Letters in Applied Bacteriology 9, 101-104.
SAARILAHTI, H.T., HENRISSAT, B. & PALVA, E.T. (1990).
CelS: a novel endoglucanase identified from Erwinia carotovora subsp. carotovora. Gene 90, 9-14.
SAMBROOK, J., FRITSCH, E.F. & MANIATIS, T. (1989).
Molecular Cloning: A Laboratory Manual, 2nd Edition, New York: Cold Spring Harbour Laboratory Press.
SAUL, D.J., WILLIAMS, L.C., GRAYLING, R.A., CHAMLEY, L.W., LOVE, D.R. & BERGQUIST, P.L. (1990).
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. Applied Microbiology and Biotechnology 27, 50-56.
TABOR, S. & RICHARDSON, C. (1987). DNA sequence analysis with a modified bacteriphage T7 DNA
polymerase. Proceedings of the National Academy of Sciences of the United States of America 84, 4767-4771.
TAYLOR, K.A., CROSBY, B., McGAVIN, M., FORSBERG, C.W. &
THOMAS, D.Y. (1987). Characteristics of the endoglucanase encoded by a cel gene from Bacteroides succinogenes expressed in Escherichia coli. Applied and Environmental Microbiology 53, 41-46.

W094/00578 ~ ~ 3 ~ o g 9 PCT/AUg3/0030-TEATHER, R.M. & WOOD, P.J. (1982). Use of Congo Red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Applied and Environmental Microbiology 43, 777-780.
TOMME, P., VAN TILBEURGH, H., PETTERSSON, G., VAN
DAMME, J., VANDEKERCKHOVE, J., KNOWLES, J., TEERI, T. & CLAEYSSENS, M. (1988). Studies of the cellulolytic system of Trichoderma reesei QM 9414. European Journal Biochemistry 170, 575-581.
VAN TILBEURGH, H., TOMME, P.M CLAEYSSENS, M., BHIKHABHAI, R. & PETTERSSON, G. (1986).
Limited proteolysis of the cellobiohydrolase I
from Trichoderma reesei. FEBS Letters 204, 223-227.
WILLIAMS, A.G. & ORPIN, C.G. (1987). Polysaccharide-degrading enzymes formed by three species of anaerobic rumen fungi grown on a range of carbohydrate substrates. Canadian Journal of Microbiology 33, 418-426.
WOOD, T.M., McCRAE, S.I., WILSON, C.A., BHAT, K.M. &
GOW, L.A. (1988). Biochemistry and genetics of cellulose degradation, edited by Aubert, J.-P., Beguin, P. & Millett, J. pp.31-52, Academic Press, London.
XUE, G.P. & MORRIS, R. (1992). Expression of the neuronal surface glycoprotein Thy-l does not follow appearance of its mRNA in developing mouse Purkinje cells. Journal of Neurochemistry 58, 430-440.
XUE, G.P., ORPIN, C.G., GOBIUS, K . S ., AYLWARD, J.H. &
SIMPSON, G.D. (1992). Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli. Journal of General Microbiology ( in press).

W094/00578 ~134 9 9 1"~ PCT/AU93/00307 YAGUE, E., BÉGUIN, P. & AUBERT, J.-P. (1990).
Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum. Gene 89, 61-67.

W0 94/00578 Q ~ 3a~ 0 ~ 9 PCI /AU93/0030~

Tab1e1 Activity of the cioned cellulases on various substrates.

Specific activity Slubstrate (nmol product min ' (mg protein)~' ) celA celB celC ceID ce~E

CM-cellulose 466 54 21 4929 1256 Avicel 196 1.4 0.9 179 50 Amorphous 1874 6.2 20 812 ND
cellulose Xylan 0 0 0 466 124 Lichenan 13600 ND ND 14312 ND
pNPG 0 pNPC 0 1.3 0 169 80 ND- Not Determined Crude cell Iysate preparations were used for the measurement of enzyme activities as described in Methods. The values given are csentative of at least three assays and are e..p,cised as nmol product (reducing sugar or ~nitrophenol released) min~' (mg protein)~ ' .

WO 94/00578 . PCI`/AU93/00307 - 21~9099 Table 2 Effect of celA and celD enzymes and pNX-Tac xylanase in combination onhydrolysis 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 within vivo digestibility of 64.7%.

W094/00578 2 1~ ~ 9 9 PCT/AU93/0030' LEGENDS
Figure 1 - Restriction maps of CelD and CelE.
Abbreviations for restriction enzymes:
E,EcoRI; B,BglII; 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 cel C ( C ) were cut with EcoRI and XhoI ( 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 39C 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 SUBST~UTE SHEET ¦

W094/00578 213 q O 9 9 i PCT/AU93/00307 -(Gs)~ each 2 mg ml~'] 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 ~f values on a TLC plate. Authentic cellodextrins (S) - are shown in the rightmost lane and the positions of glucose (Gl), 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. col i 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~'); 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 40C in 50 mM Na-citrate (pH4-7) or 25mM
Tris-C1/50mM NaCl (pH7.5-9.5) containing 1% Avicel for 2 2 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 2? hours.
pH5.-*-.;pH6 -.-¦ SU~5S~ JTE S5~EET

W094/00578 ~ 3 9 ~ 9 9 PCT/AU93/00307 Figure 9 - Time course of cellulase hydrolysls by the cloned celA enzyme. Cellulase hydrolysis was performed at 39C 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 500~1.

Figure 10 - Time course of cellulose hydrolysis by the cloned celD enzyme. Cellulose hydrolysis was performed at 39C 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.

Figure 11 - Effect of celA and celD enzymes in combination on the rate of crystalline cellulose hydrolysis. Cellulose hydrolysis was performed at 39C
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), KpnI(K), ~auII(P) and XhoI(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.

¦ SU~5TiTUT~ S!5~T ¦

W094/00578 ~1 3 q ~ 9 9 ~ ~ , i PCT/AU93/00307 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 ~-galactosidase ~-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 ~ SU~STI, UT~ ET

W094/00578 21 3q 0 ~ 9 PCT/AU93/0030 Reproduced hereinbelow are sequence listings wherein -SEQ ID NO:1 refers to nucleotide sequence ofNeocallimastix patriciarum celD cDNA. The sequence underlined is derived from pBluescript SK-vector and the EcoR1 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 ~-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.

¦ SUBSTITUTE SI;EE

.213q~gg . , ~I~ PC~r/A U93/00307 Ce1D
1 .~TCACCATGA TTACGCCAAG CTCGAAATTA ACCCTCACTA AAGGGAACAA AAGCTGGAGC
61 ~C~CCGCGG ~G~ ~GC TCTAGAACTA GTGGATCCCC CG~OCl~CAG GAATTCGGCA
121 C_ CTCCAA TCCGTGATAT TTCATCCAAA GAA~TAATTA AAGAAATGAA l11CGC11~G

241 A.-~ -~G AAACTTGCTG GGGTAATCCA A`AGACTACTG AAGATATGTT CAAGGTTTTA
301 ATGGATAACC AATTTAATGT ~1~CCG1ATT CCAACTAC~T ~-OG-~A C1 .C~-1~AA

421 CCATAC~AAGA ATCGAGC m C~ALC~A AATCTTCACC ATGAAACTTG GAACCATCCC
481 1 ~ AAA CICTTGAC~C TY CCAAGGAA ATCTTAGAAA A~A1~GG~C TCAAATTGCT
541 AAAGAATTTA AG~A--A.GA TGAACACTTA ATTTTTGAAG GATTAAACGA ACCAACAAAG
601 AATGATACTC CAC~TGAATG GAC-CC-~- GATCAACAAG CATGGGATGC TGTTAATGCT
661 ATGAATGCCG ~ AAA GAC~A~CS- AG11~1GG~ GTAATAATCC AAAGCC~AT
721 C~TATGATCC C~C~A~AIaC ~ ~l AATGAAAATT CATTCAACAA CTTTATTTTC
781 CCAGAAGATG AToAC;AGCT A-1OC11~- GTTCATGCIT A-G~-CCATA CAA~1--GCC
8q1 TTAAATAATG G~AAGGAGC TGTTGATAAG 1--GA~GC~G C~GGTAAGAA AGATCTTGAA
901 TGGAACATTA A~ 1AATGAA GAAGAGA m Gl1GA~AAG C~A1~ AT GA1~11GC1 961 GAA~h~G-~ CCATGAATCG TGATAATGAA GAAGATCGTG C~AGC11G~GC TGAATTCTAC
1021 ATGCAAAhGG TCAL1~C1AT GGGAGTTCCA CAAGrCTGGT GGGATA~TGG TATCTTTG~AA
1081 GGTACCGGTG AAC~l-ll~ AT CGTAAAAACT TAAACATTGT TTATCCAACT
1141 A-C~11~1~ CTTTACAAAA GGCAAGAGGT TTAGAAGTCA AlGl-lCllCA TGCTATTGAA
1201 Aa~AAA~cAG AAGaACCAAC TAAAACTACT GAACCAGTTG AACCAACTGA AACTACTAGT
1261 ccAoaAoAAC CAGCIGAAAC TACTAATCCA GAAGAACCAA C~GGTAATAT TCGTGATATT
1321 1~-~AGG AATTAATTAA AGAAATGAAT 11C~C11~A ATITAGGTAA TAC~TAGAT
1381 GC~CAATGTA TTGAATACTT AAA11A1GAI AAGGATCAAA C1GC11~1GA AA 11GC-GG
1441 GGTAATCCAA ACACTACTGA AGA-h1C11C AAGGTTTTAA TGGATAACCA ATTTAATGTT
1501 11~C~1A11~ CAACTACTTG ~1C1G~AC llC~GlGAAG CTCCACATTA CAAGATThAT
1561 GA`AAAATG'GT TAAAGAGACT TCATGAAATT ~11~A11ATC CATACAACAA TGGAGCTITC
1621 ~11AIC11AA ATCrTCACCA TGAAAC`TTGG AAC~Al~11 TCrCrGaAAC TCTTGACACT
1681 GCCAAGGAAA TTITAGAAAA GA~lG~l~l CM ATTGCTG AAGAATTTAA GGATTATGAT
1741 GAACACTTAA TlSTTGaAGG ATTAAAQGAA CCAAGAAAGA ATGATAC~CC AGTTGAATCC
1801 AL1~1~G1G ATCAAGAAGG A1G~GA~GC1 GTTAATGCTA TGAATGC~GT TTTCTTAAAG
1861 AC1~11~1A ~11~1G~1G~ TAATAATCCA AAG~ A1C 11Al~AnCCC 1~_ArA1~L1 1921 GC~G~ll~lA ATGAAAATTC ATTCAAGAAC lllAllll~C CAGAAGATGA TGACAAGGTT
1981 A11~C11~1G 11~A1G~11A GC-CCA1..C A~L-11G~C- TAAATAATGG TGAAGS;OCT
2041 GTTGATAAGT 11~A1G W ~` TGGTAAGAAT GACCTTGAAT GGAATATTAA CTThATGAAG
2101 AAGAGATTTG TTGATCAAGG TATTCCAATG All~l1G~G AATATGGTGC CATG MTCGT
2161 GATAAT~AAG AACA1C~1GC AGC11G~GC1 GAATTCTACA CGAUAAGGT CA~1GC1AIG
2221 GG`AGTTCCAC AAG1~C-G GGATAATGGT ATCTTTGAAG GTAC~ rGA AC~ Gl 2281 ~11~11~Al~ GTAGAAACTT AAA~A11~11 TATCCAACTA Y~11GClGC TTTACAAAAG
2341 GGAAGAGGTT TAGAAGTCAA 1~11~11~AT G-1~11~AAA AAAAAACCAG AAGAACCAAC
2401 TAAGACTACT GAACCAGTTG AACC;~C1GA AACTACTAGT CCAGAAGAAC C`AACTGAAAC
2461 TACTAATC~A GAAGAACCAA CCGGTAATAT TCGrGATATT TCATCTAAGG AATTAATTAA
2521 AGAAATGAAT 11C~11GGA ATTTAGGT M TACTTTAGAT GCTC M TGTA TTGAATACTT
2581 AAA11A1Ghl AAGGATCAAA C1~11~1GA hhL-11V~1~G GGTAATCCAA AGACTACTGA
2641 AGA1A1~11C M GGTTTTAA TGGATAACCA ATITAATC~T 11~1A11~ CAACTAC~TG
2701 C1~ ~AC -C~C1~AG CTCCAGATTA CAAGATTAAT GAAA M TGGT TAAAGAGACT
2761 TCATGA M TT ~11GA~1~ CATACAAG M TGGAGCTTTC C11A~11AA ATCTqCACCA
2821 TGAAAC$TGG AAC~A~G~11 lC~C~C~AC TCrTCDCACT GCCAAGGAAA ITTTAGAAAA
2881 GA1--~1C1 CAAA`1~ ~ AAGAATTTAA ~GA11AIGAT GAACAC~T M TTTTTGAAGG
2941 ATTAAACGAA CCMGA~AAGA ATGATACTCC AGTTGMTGG AC1~1G~1C ATC MGAAGG
3001 A1G~3~A-1GC1 GTTAATGCTA TGAATGCCGT TrTCITAAAG A~A1~CG1A ~11~1G~1GG
3061 TAATAATCCA AAG~G1~Ar~ TTATGATCCC ~AlA1~1 GL1~C11~1A ATGAAAATTC
3121 ATT~AAGAAC 11~A1111CG CACAAGATGA TGACAAGGTT AllGL1l~lG l~A~GC~lA
3181 1~L1C~ATAC AAC111GL~81 TAAATAATGG TGCAGGAGCT GTTGAT MGT ~1CAr~CGG

3301 lAl 1CC~ATG Al 1~11~G1G AATATGGTGC CATGAACC~GT CATAAT~AAG AAGAACGTGC
3361 TACATGGGCT GAATTCTACA TæAAAAGGT C~ACTGCTATG GCAGTTCCAC AAG1~1~GLG
3421 C~ATAATGGT ~1~111~AAG GTACCGGIGA AL~1111~1 ~11~11 ~ATC G~AAAAAC~T
3481 AAAGATTGTT TATC~ACTA 1~11~1GC TTTACAA MG GGMGAGGTT TACAAGITAA
3541 G~11C11CAT G~AAATGAAG AACAAACAGA AGAATGTTGG TCTGAA MGT ATGGTTATGA
3601 Al~-ll~ll~l C~TAACAATA CTAAGGT~GT AGTCAGTGAT GAAAGTGG M A11~GGG1~1 3661 TG~AAATGGT AA11G~1~1G Cl~11~11~A ATACACTGAA AAA~11G~1 CACTTCCATT
- 3721 TGGATACCCA '1~11C1 AC ATTGTAAGGC TCTTACTAAG 6ATGAAAATG GTAAATGCGG
3781 AGAAGTAAAT GGTGAATGGT ~l~lAl~Cl TG~TGATAAA TGITAGATTA TAAAATAAAA
3841 ATAAATAGAT ll~l~AlGA AAATTATTAA TGAATAATAA ATAAAATAGA AAATTTTATA
3901 TAAACATATT TCTAATA~AA 6ATCTAATTA TGTATTTTTT 6-1~11ATT CITTCAAATA
- 3961 AAAAAAGTAA GÆAAAGAAAA TATATAAAAT AAAAAAAAAA AATAAATAAA TAAATATTTT
4021 AATTATTTTT TTTTTA(`.TAA AAAAAA~AA TTTAATTAAA ATATAATTAA AA('TAAAAAA

SEQ ID NO:1 `~~-SUB~i T ~ JTE SffEET ~

WO 94/00578 ~ - PCI/AU93/00307 213qO99 40 .

celD

4081 AAAAA~ AACI~G

SEQ ID N0: 1 ¦ SlU~ JTE SHEET ~

WO 94/00578 21 3 q ~ ~ ~ PCI`/AU93/00307 ` F ~

celD doml/2 Tran~lated sequence S~-~n~e Range: 1 to 2400 ATG ACC ATG ATT ACG C'CA AGC TCG AAA TTA ACC CTC ACT AAA GGG AAC AAA AGC
TAC TGG TAC TAA TGC GGT TCG AGC TIT AAT TGG GAG TGA TTT CCC TTG TIT TCG
M T M I T P S S K L T L T K G N K S>
a_a a_a_a T~NCr~TIC~ OF CELD DOMl/2 a a a a a_~

.
TGG AGC TCC ACC GCG GTG GCG GCC GCT CTA GAA CTA GTG GAT CCC CCG GGC TGC
ACC TCG AGG TGG CGC CAC CGC CGG CGA GAT CIT 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 _TRANSLATIaN OF CELD D0Ml/2 a a_a a a >

~ ~ ~ .
AGG AAT TCG GCA C'GA GCT CCA ATC CCT GAT ATT TCA TCC AAA GAA TTA ATT AAA
TCC TTA AGC C'GT GCT CGA GGT TAG GCA CTA TAA AGT AGG TIT CTT AAT TAA TTT
R N S A R A P I R D I S S K E L I R>
a_a_a_a~TEtANSL~TI~ OF CELD DOt~lt2 a_a a a_a_>

GAA ATG AAT TTC GGl IGG AAT TTA GGT AAT ACT TTA GAT GCT CAA TGT ATT GAA
CTT TAC TTA AAG C'CA ACC TTA AAT CCA TTA TGA AAT CTA CGA GIT ACA TAA C'TT
E M N F G W N L G N T L D A Q C I E~
a a_a a_a _TRANS ~TICN OF CELD D0Ml/2_a a a~_a_a_>

TAC TTA AAT TAT GAT AAG GAT CAA ACT GCT TCT GAA ACT TGC TGG GGT AAT C'CA
ATG AAT TTA ATA CTA TTC CTA GIT TGA CGA AGA CTT TGA ACG ACC C'CA TTA
Y L N Y D R D Q T A S E T C W G N P>
a a a a a _TRANSLATICN OF CELD D0Ml/2 a a_a a a_>

AAG ACT ACT GAA GAT ATG TTC AAG GIT TTA ATG GAT AAC CAA TTT AAT GIT TTC
TTC TGA TGA CTT CTA TAC AAG TTC CAA AAT TAC CTA TTG GIT 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 _TRANSI~ATICN OF CELD D0Ml/2 a_a a a a >

--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 Cl~ 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~rR~NCr~TIat7 OF OE LD D0t51/2 a a a a_a_>

~
AAT GAA AAA TGG TTA AAG AGA GIT CAT GAA ATT GTT GAT TAT CCA TAC AAG AAT
TTA CTT TTT ACC AAT TTC TCT CAA GTA CIT TAA CAA CTA ATA GGT ATG TTC TTA
N E K W L K R V H E I V D Y P Y K .N>
_a_a_a a a _TR~NCr~TION OF CELD D0tSl/2_a a a_a_a_>

~ . ~ ~ .
GGA GCT TTC GIT ATC TTA AAT CTT CAC CAT GAA ACT TGG AAC CAT GCC TTC TCT
CCT CGA AAG CAA TAG AAT TTA GAA GTG GTA C,TT 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_TRANSLPTION OF CELD D0Ml/2_a_a_a_a a_>
490 soo 510 520 530 S40 SEQ ID NO:~

~ SUe~g~ JTE SHEET ¦

WO 94/00578 2 1 3 q ~ 9 9 Pc~r/Aug3/oo307 ceLD doml/2 Translate~ Sequence 4 2 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 I A, _ a _ a _ a _ a _ a _TRANSLATION OF CELD DOMlt2 a a a _ a a 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 R D Y D E H L I F E G L N E P~
_a a _ a _ a a _ TRANSLATION OF CELD DOMl/2 a a a a a _ 600 610 620 630 6g0 AGA AAG AAT GAT AiCT CCA GTT GAA TGG ACT GGT GGT GAT CAA GAA GGA TGG GAT
TCT TTC TTA CTA TGA GGT C M CTT ACC TGA CCA CCA CTA GTT CTT CCT ACC CTA
R K N D T P V E W T G G D Q E G W D>
a _ a_a_ a a_ TRANSLATION OF CELD D0Mlt2 aaaa a _ ~ . . ~ . ~
GCT GTT M T GC,T ATG AAT GCC GTT TTC TTA AAG ACT ATT CIGT AGT TCT GGT G
C~GA CAA TTA CGA TAC TTA CGG CAA M G M T TrC T~A TAA GCA TCA AGA CCA CCA
A V N A MN A V F L K T I R S S G G>
_ a a a _ a a _TRANSLATION OF CELD DOMl/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 D0ffl/2 aa_ a _ a _a_>

~ ~ .
AAT TCA TIC AAG AAC TTTATTTTC CCA GAA GAT GAT GAC A~G GTT ATT GCT TCT
TTA AGT AAG TIC TrG AAA T M AAG GGT CTT CTA CTA CTG TIC C M T M 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 DOMl/2 a a aa a _ >

~ ~ ~
GTT C'AT GCT TAT GCT CC~ TAC AAC TTT GCC TTA M T M T GGT GAA ~ GCT GTT
GAA GTA CGA ATA CGA GGT ATG TTG AM CGG M T 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_ TRANS~ATION OF CELD DOMl/2 a a _ a a a _ GAT AAG TrT GAT GCT GCT GGT AAG M A GAT CTT GAA TCG AAC ATT M C TTA ATG
CTA TTC AAA CTA CGA CGA CCA TTC TTT CTA GAA CTT ACC TTG $AA TTG AAT TAC
D X F D A A G K K D L - E W N I N L M~
a a a _ a a_ TRANSLATION OF ~~Frn D0Ml/2 a a a a a _ AAG AAG AGA TTT GTT GAT CAA GGT ATT CcA ATG ATT cTT GGT G~A T~T GGT GCC
TTC TTCTCT AAA CAA CTA GTT C'CA TAA GGT TAC TAA GAA CCA crT ATA CCA CGG
K R R F V D Q G I P M I L G E Y G A>
_ a _ a_aa_ TRANSLATION OF ~rn D0Ml/2 _ aa_ a _a a _ ATG AAT CG$ GAT AAT GAA GAA GAT CGT GCA GCT TGG GCT GAA TTC TAC ATG GAA
TAC 'TTA GCA CTA TTA CTTCTT 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>
aaa_aa_ TRANSLATION OF CELD D0M1/2 a a _ aa_ a _ SEQ ID NO:2 ~ SUB8TITUTE 8HEET ~

WO 94/00578 ~, ~ . PCI`/AU93/00307 -celD doml/2 Tr;~ncl ~Itffl Se~auence 4 3 1030 10gO 1050 1060 1070 1080 tr ~ ~
AAG GTC ACT GCT ATG GGA GIT CCA CAA GTC TGG TGG G~T AAT GGT ATC TTT GAA
TTC CAG TGA CGA TAC CCT CAA GGT GTT CAG ACC ACC CTA TTA CCA TAG AAA cTr K V T A M G V P Q V W W D N G I F E>
a_a_a a_a TRANSLATICN OF CELD DOM1/2 _a a a a~a_>
1090 ~00 1110 1120 1130 ~ . ^ . ~

CCA TGG CCA CTT GCA AAA CCA GAA GAA CTA GCA TTT TTG AAT .1~ TAA CAA ATA
G T G E R F G L L D R K N L K I V Y>
a_a_a a_ a_ TR ~ LATI~ OF OELD D0t~11/2 a a a a_a_>
1140 llS0 . 1160 1170 1180 .. .. . .
CCA ACT ATC GTT GCT GCT TTA CAA AAG GGA AGA G~T TTA GAA GTC AAT GTT GTT
GST TGA TAG CAA CGA CGA AAT GTT TTC CCT TCT CCA AAT C~- 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_~ TRANSLATI~ OF OELD D0MV2-a a a a_~ >

.. .
CAT GCT ATT GAA AAA AAA CCA GAA GAA CCA ACT AAA ACT ACT GAA CCA GTT GAA
GTA CGA~rAA CTT TTT TTT .T CTT CTT GGT TGA TTT TGA TGA CTT GGT CAA CTT
H A I E K K P E E P T ~ T T E P V E>
a a_a a_aT~tA~TION OF CELD D0M1/2_a a a_a_a_~

~ .
CCA ACT~GAA ACT ACT AGT CCA GAA GAA CCA GCT GAA ACT ACT AAT CCA GAA GAA
GGT TGA CTT TGA TGA TCA GCT CTT CIT oeT 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~TRANSl~TIC~ OF OELD DOM1/2 a a a a a_>

CCA A GGT AAT ATT CGT GAT ATT TCA TCT AAG GAA TTA A~T AAA GAA ATG AAT
GGT TGG CCA TTA TAA GCA CTA TAA AGT AGA l-C CTT AAT TAA TTT CTT TAC TTA
P T G N I R D I S S ~ E L I K E M N>
a_a_a_a_a ~`'~TI~LD ~1/2 a_a a a_a_>

TTC GGT TGG AAT TTA GGT AAT ACT TTA GAT GCT CAA TGT ATT GAA TAC TTA AAT
AAG CCA ACC TTA AAT CCA TrA TGA AAT CT~ CGA G~T ACA TAA CTT ATG AAT TTA
F G W N L G N T L D A Q C I E Y L N>
a a_a a_a n2uLn~TIcN OF CELD D4M1/2 a a a_a_a_>

~ ~ ~
TAT GAT AAG GAT CAA ACT GCT TCT GAA ACT TGC TGG GGT AAT CCA AAG ACT ACT
ATA CTA TTC CTA GTT ~GA CGA AGA CTT TGA ACG ACC CCA TTA GGT ITC TGA TGA
Y D K D Q T A S E T C W G N P K T T>
a ~ ~ ~_~.T~TI~OFOE~/2 a_~ a_>
1460 lq70 1480 1490 1500 1510 GAA GAT ATG TTC AAG GTT TTA ATG GAT AAC CAA T~T 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 X V L M D N Q F N V F R I P>
a a ~ a_a_ TR~TIC~N OF CELD DOtS1/2_ a a_a_a_a_>
1520 1530 lSg0 1550. lS60 ~
. ACT ACT TGG TCT GGT CAC TrC GGT GAA GCT CCA GAT TAC AAG ATT AAT GAA AAA

SEQ ID NO:2 i ;3UBSTITUTE 8HEET ~

W O 94/00578 - PC~r/A U93/0030-213~09g `
: ` 44 celD doml/2 TranslaLed Sequence TGA TGA ACC AGA CCA GTG AAG CCA CTT CGA GGT CTA ATG 1~ TAA TTA crr TTT
T T W S G H F G E A p D Y K I N E K>
a a a a_a_TRA~SLATION OF CELD DOM1/2_a_a a a a_~
1570 lS80 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 C(~A MG
W L K R V H E I V D y p y K N G A F>
a_a a aa_ TRANSLATION OF CELD D0Ml/2 a a a_aa>

~ ~ ~
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
.VIL N L H H E T W N H A F S E T L>
_a_aa_a_a _TRANSLATION OF~ErD DOMl/2 _ aa_aaa>

~ ~ . ^ .
GAC ACT GCC AAG GAA ATT TTA GM AAG ATT ~;G TCT CAA ATT GCT GAA GM TTT
CTG TGA CGG TTC CTT TAA MT CTT TTC TM ACC AGA GTT TAA CGA CTT CTT AAA
D T A X E I L E K I W S Q I A E E F>
aaaaa _TRANSLATION OF CELD DOM1~2 a_aaaa_>

~ ^ --AAG GAT TAT GAT GAA CAC ITA ATT TTT GM GGA TTA AAC GAA CCA AGA AAG AAT
TTC CTA ATA CTA CTT GTG AAT TAA MA CTT CCT AAT TTG CTT GGT TCT TTC TTA
R D Y D E H L I F E G L N E P R R N>
aaaa_a_ TRANSLATION OF CELD DOMl/2 aaaaa_>

GAT ACT CCA GTT GAA TCC ACT GGT GGT GAT CM GAA GGA TGG GAT GCT GTT AAT
CTA TGA GGT CAA CTT AGG TGA CCA CCA CTA GTT CTT CCT ACC CTA CGA CM TTA
DTPVEST G G D Q E G W D A V N>
aaaaa _TRANSLATION OF CELD D0Ml/2 aaaaa>
18q0 1850 1860 1870 1880 1890 GCT ATG AAT GCC GTT TTC TTA AAG ACT ATT CGT AGT TCT GGT GGT AAT MT CCA
CGA TAC TTA ~ CM AAG AAT TTC TGA TAA GCA TCA AGA CCA CCA ~TA TTA GGT
A M N A V F L K T I R S S G G N N P~
a_a_aaa_ TRANSLATION OF CELD D0Ml/2. a aa_a_a>

~ ~ .
AAG CGT CAT CTT ATG ATC CCT CCA TAT GCT GCT GCT TGT AAT GAA AAT TCA TTC
TTC GCA GTA GM TAC TAG GGA GGT ATA CGA CGA CGA ACA TTA CTT TTA AGT MG
K R H L M I P P Y A A A C N E N S F>
aaa_aa_ TRANSLATION OF CELD D0Ml/2 a_ aaaa>

~ ~ --AAG AAC rrr ATI' ~C CCA GAA GAT GAT G~C AAG Grr ATI GCT TCT GTT CAT GCT
TTC ~ AAA TM MG 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>
aaa a a _TRANSLATION OF CELD DOMlt2 _ a_ a_a_a a_, ~~ ~ --TAT GCT CCA TAC MC TIT GCC TTA MT AAT GGT GAA GGA GCT GTT GAT AAG TTT
ATA CGA GGT ATG Tl~, MM C~;G AAT TTA TTA CCA CTT CCT CGA CAA CTA TTC A~A
Y . A P Y N F A L N N G E G A V D ~C F>
aa a aa_ TRANSLATION OF CELD DOM1/2 a_aaaa_, SEQ I D NO:2 ¦ SUBSTITUTE SHEET ~

W O 94/00578 21 3 ~ O 9 9 ~ PC~r/A U93/00307 ,_ ~

celD doml/2 Tr~nc]are~ SY~-~n~e ~ ~ ~
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 X R~
_ a a _ a a _ a _TRANSLATION OF CELD DOMl/2 a a _ a _ a _ a _ TTT GTT GAT CAA GGT ATT CCA ATG ATT CTT GGT GAA TAT GGT GCC ATG AAT CGT
AAA CAA CTA GIT CCA TAA GGT TAC TAA G~A CCA CTT ATA CCA 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 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 _ ~ ~ . . .
GCT ATG GGA GTT CCA CAA GTC ~CG TGG GAT AAT GGT ATC TTT GAA GGT ACC GGT
CGA TAC CCT CAA GGT GTT CAG ACC ACC CTA TTA CCA TAG AAA CTT CCA TGG CCA
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 OOMl/2 a a a _ a _ a _ >

~ . . . ~ ~
GAA CGT TTT GGT CTT CIT GAT CGT AGA AAC TTA AAG ATT GTT TAT CCA ACT ATCCTT GCA AAA CCA G~A 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 ~ NS1~TION OF CELD DOMl/2 a a a a a 2330 23q0 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 _SRANSLATION OF CELD DOMl/2 a a a a a _ >

~ ~
GAA AAA AAA ACC AGA AGA ACC AAC
CTT TTT TTT TGG TCT TCT TGG TTG
E X K T R R T N>
_ TRANSLATION OF CELD DOMn _ >

SEQ ID NO:2 - ¦1 8U~ 111 iJTE SHEET

WO 94/00578 21 3q 0 99 ~ ` PCI'/AU93/0030' celD dom3 46 SF~nre Range: 1 to 1437 CCA GAA GAA CCA ACT AAG ACT A GAA CCA GTT GAA CCA ACT GAA ACT ACT AGT
CTT CTT GGT TGA ~1l~ TGA TGA CTT GGT CAA CTT GGT TGA CTT TGA TGA TCA
P E E P T K T T E P V E P T E T T S>
a _ a _ a a _ TRANSLATION OF CELD DOM3 TRUNC_a _ a a _ a a >

CCA GAA GAA CCA ACT GAA ACT ACT AAT CCA GAA GAA CCA ACC GGT AAT ATT CGT
GGT CTT CTT GGT TGA CTT TGA TGA TTA GGT CTT CTT GGT TGG CCA TTA TAA GCA
P E E P T E T T N P E E P T G N I R~
_ a _ a a___a TRANSLATION OF CELD DOM3 TRUNC_a a _ a _ a _ a _ >

GAT ATT TCA TCT AAG GAA TTA ATT AAA GAA ATG AAT TTC GGT TGG AAT TTA GGT
CTA TAA AGT AGA TTC CTT AAT TAA TTT CTT TAC TTA AAG CCA ACC TTA AAT CCA
D I S S K E L I K E M N F , G W N L- G>
_ a a a a _ TRU~L~TION OF CELD DOM3 TRUNC_a a a a a _ >

AAT ACT TTA GAT GCT CAA TGT ATT GAA TAC TTA AAT TAT GAT AAG GAT CAA ACT
TTA TGA AAT CTA CGA G,TT ACA TAA CTT ATG AAT T,TA ATA CTA TTC CTA GTT TGA
N T L D A Q C I E Y L N Y D K D Q T>
a a, a a _ TR~N5LATION OF CELD DOM3 TRUNC_a _ a _ a _ a a _ >

GCT TCT GAA ACT TGC TGG GGT AAT CCA AAG ACT ACT GAA GAT ATG TTC AAG GTT
CGA AGA CTT TGA ACG ACC CCA TTA GGT TTC TGA TGA CTT CTA TAC AAG TTC CAA
A S E T C W G N P K T T E D M F K V>
a a a a, TRANSLATION OF CELD DCM3 TRUNC_a a _ a _ a a _ >

TTA ATG GAT AAC CAA TTT AAT GTT TTC CGT ATT CCA ACT ACT TGG TCT G~T CAC
AAT TAC CTA TTG GTT AAA TTA CAA AAG GCA TAA GGT TGA TGA ACC AGA CCA GTG
L M D N Q F N V F R I P T T W S G H>
a a a a TRANSLATION OF CELD DOM3 TRUNC_a a a a a _ >
330 3g0 350 360 370 TTC GGT GAA GCT CCA GAT TAC AAG ATT AAT GAA AAA TGG TTA AAG AGA GTT CAT
AAG CCA CTT CGA GGT CTA ATG TTC TAA TTA CTT TTT ACC AAT TTC TCT CAA GTA
F G E A P D Y K I N E K W L K R V H>
a _ a _ a a TRANSLATION OF CELD DOM3 TFUNC_a a _ a a a _ >

GAA ATT GTT GAT TAT CCA TAC AAG AAT GGA GCT TTC GTT ATC TTA AAT CTT CAC
TAA CAA CTA ATA GGT ATG TTC TTA CCT CGA AAG CAA TAG AAT TTA GAA GTG
E I V D Y P Y K N G A F V I L N L H>
a~a~a,_a_~ANSL~TICi~J OF OEID ~M3 TRDNC_a a_a a a CAT GAA ACT TGG AAC CAT GCT TTC TCT GAA ACT CTT GAC ACT GCC AAG GAA ATT
GTA CTT TGA ACC TTG GTA CGA AAG AGA CTT TGA GAA CTG TGA CGG TTC CTT TAA
H E T W N H A F S E T L D T A K E I>
a _ a a _ a _ TFU~E~LATION OF CELD DOM3 TRUNC_a a a a a _ >
490 500 510 520 530 5q0 SEQ ID NO: 3 SUB~TITUTE SHEET

WO 94/00~78 213 4 0 ~ 9 PC~r/AU93/00307 celD dom3 TTA GAA AAG ATT TGG TCT CAA ATT GCT GAA GAA TTT AAG GAT TAT GAT GAA CAC
AAT CTT ~1-l~ TAA ACC AGA GTT TAA CGA CTT CTT AAA TTC CTA ATA CTA CTT GTG
L E R I W S Q I A E E F R D Y D E H>
a _ a _ a _ a TRANSLATION OF CELD DOM3 TRoNC_a a a a a >
550 s60 570 s80 sgo TTA ATT TTT GAA GGA TTA AAC GAA CCA AGA AAG AAT GAT ACT CCA GTT GAA TGG
AAT TAA AAA CTT CCT AAT TTG CTT GGT TCT TTC TTA CTA TGA GGT CAA CTT ACC
L I F E G L N E P R R N D T P V E W~
_ a _ a _ a _ a, TRANSLATION OF CELD DOM3 TRU,NC_a a a a a >

ACT GGT GGT GAT CAA GAA GGA TGG GAT GCT GTT AAT GCT ATG AAT GCC GTT TTC
TGA CCA CCA CTA GTT CTT CCT ACC CTA CGA CAA TTA CGA TAC TTA CGG CAA AAG
T G G D Q E G W D A V N A M N A V F>
a a a a TRANSLATION OF ~Fr-n DOM3 TRU~C_a, a a _ a a >
6s0 660 670 680 690 700 TTA AAG ACT ATT C~-l~ AGT TCT GGT AAT AAT CCA AAG CGT CAT CTT ATG ATC
AAT TTC TGA TAA GCA TCA AGA CCA CCA TTA TTA GGT TTC GCA GTA GAA TAC TAG
L R T I R S S G G N N P R R H L M I>
a a _ a_ a _ TRANSLATION OF CELD DOM3 TRUNC_a a a a a 710 720 730 740 7so CCT CCA TAT GCT GCT GCT TGT AAT GAA AAT TCA TTC AAG AAC TTT ATT TTC CCA
GGA GGT ATA CGA CGA CGA ACA TTA CTT TTA AGT AAG TTC TTG AAA TAA AAG GGT
P P Y A A A C N E N S F R N F I F P~
a a _ a a TRANSLATION OF CELD DOM3 TRUNC_a _ a a _ a a _ >

GAA GAT GAT GAC AAG GTT ATT GCT TCT GTT CAT GCT TAT GCT CCA TAC AAC TTT
. CTT CTA CTA CTG TTC CAA TAA CGA AGA CAA GTA CGA ATA CGA GGT ATG TTG AAA
E D D D R V I A S V H A Y A P Y N F>
_ a a a a TRANSLATION OF ~F~n DOM3 TRUNC_a a _ a _ a _ a >

GCC TTA AAT AAT GGT GCA GGA GCT GTT GAT AAG TTT GAT GCC GCT GGT AAG AA~
CGG AAT TTA TTA CCA CGT CCT CGA CAA CTA TTC AAA CTA CGG CGA CCA TTC TTT
A L N N G A G A V D R F D A A G K R>
a a a a _ TRANSLATION OF CELD DoM3 TRUNC_a a a a a _ >
870 880 890 900 91o ~ ~ ~
GAT CTT GAA TGG AAC ATT AAC TTA ATG AAG AAG AGA TTT GTT GAT CAA GGT ATT
CTA GAA CTT ACC TTG TAA TTG AAT TAC TTC TTC TCT AAA CAA CTA GTT CCA TAA
D L E W N I N L M R R R F V D Q G I>
_ a a a a _ TRANSLATION OF ~FTn ~OM3 TRUNC_a _ a, a a a _ >
920 930 940' 950 960 970 ~ ~ ~

CCA ATG ATT CTT GGT GAA TAT GGT GCC ATG AAC CGT GAT AAT GAA GAA GAA CGT
GGT TAC TAA GAA CCA CTT ATA CCA CGG TAC TTG GCA CTA TTA CTT CTT CTT GCA
P M I L G E Y G A M N R D N E E E '~
a, a a _ a _ TRANSLATION OF CELD DOM3 TRUNC_a a a a a _ 980 990 lOoO lolO 1020 ~ ~ --GCT ACA TGG GCT GAA TTC TAC ATG GAA AAG GTC ACT GCT ATG GGA GTT CCA CAA
- CGA TGT ACC CGA CTT AAG ATG TAC CTT TTC CAG TGA CGA TAC CCT CAA GGT GTT
A T W A E F Y M E R V T A M G V P Q>
_ a _ a a a _ TRANSLATION OF CELD DOM3 TRUNC_a a _ a a a SEQ ID NO:3 ~ 5Ut~ JTE~ 8HEET ~

PCI`/AU93/0031`"
21~9~

celD dom3 P

GTC TGG TGG GAT AAT GGT GTC TTT GAA GGT ACC GGT GAA CGT TTT GGT CTT CTT
CAG ACC ACC CTA TTA CCA CAG A~A 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 D0M3 TRUNC_a a _ a _ a a _ GAT CGT AAA AAC TTA AAG ATT GTT TAT CCA ACT ATC GTT GCT GCT TTA CAA AAG
CTA GCA TTT TTG AAT ITC TAA CAA ATA GGr TGA TAG CAA CGA CGA AAT GTT TTC
D R X N L X 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 GGA AGA GGT TTA GAA GTT AAG ~1-l GTT CAT GCA AAT GAA GAA GAA ACA GAA GAA
CCT TCT CCA AAT CTT CAA TTC CAA CAA GTA CGT TTA CTT CTT CTT TGT 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 O~ CELD DOM3 TRUNC_a a a a a 1190 1200 1210 1220 1230 12~0 TGT TGG ~CT GAA A~G 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 X Y G Y E C C S P N N T K V>
a a a a TRANSLATION OF CELD D0M3 TRUNC_a a a a a _ GTA GTC AGT GAT GAA AGT 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 W C G>
a a a a _ TRANSLATION OF CELD DOMB TRUNC_a a a a a _ 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 AGT GAA GGT 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 DOM3 TRUNC_a _ a a _ a a _ 1360 1370 1380 1390 lg00 CCA CAT TGT AAG GCT CTT ACT AAG GAT GAA AAT GGT AAA TGG GGA GAA GTA AAT
GGT GTA ACA TTC CGA GAA TGA TTC CTA CTT TTA CCA TTT ACC CCT CTT CAT TTA
P H C X A L T K D E N G K W G E V N>
a a a _ a _ TRANSLATION OF CELD DoM3 TRUNC_a a _ a a a _ 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 X C>
_ a _ TRANSLATION OF CELD DOM3 TRUNC _ a _ >

SF.Q ID NO: 3 3 ~3UE~8~ 1TE SHEET

W094/00578 ~1 3q ~q~ pcr/Aug3/oo3o7 -ATGGCTAGC AATGG~AAAAAG~
M A S ~ G X ~

TTACTGTCGGTAATGGACAAAACCAACATAAGGGTGTCA~CGATGGT~TCAGTTATGAAA
F ~ v G ~ G Q ~ Q ~ K G V N D G F S Y E

TCTGGTTAGATAACACTGGTG~TAACGGTTCTATGACTCTCGGTAGTGGTGCAACTTTCA
I W L D N T G G ~ G S M T L G S G A T F

AGGCTGAATGGAATGCAGCTGTTAACCGTGGTAACTTCCrTGCCCGTCGTGGTCTTGACT
K A E U N A A V N ~ G N F L A R ~ G ~ D

TCGGTTCTCAAAA~AAGGCAACCGATTACGAC~ACATTGGATTAGATTATGCTGCTACTT
F G S ~ K K A T D Y D ~ I G L D Y A

ACAAACAAACTGCCAGTGCAAGTGGTAACTCCCGTCTCTGTGTATACGGATGGTTCCAA~
Y K Q T A S A S G ~ S ~ L C V Y G W F Q

ACCGTGGACTTAATGGGGTTCCTTTAGTAGAATACTACAT~ATTGAAGATTGGGTTGACT
N R G L N G V P ~ V E Y Y I I E D W V D

GGGTTCCAGATGCACAAGGAAAAATG~TAACCATTGATGGAGCTCAATATAAGATTTTCC
W V ~ D A Q G R M v T I D G A Q Y ~ I F

AAATGGATcAcAcTGGTccAAcTATcAATGGTGGTAGTGAAAccTTTAAG~AATAcTTcA
~ M D ~ T G P T I N G G S E T F K Q Y

GTGTCCÇTCAACAAAAGAGAACTTCTGGTCATATTACTGTCTCAGATCACTTTAAGGAAT
S v R Q Q ~ ~ T S G ~ I T V ~ D ~ F ~ E

GGGCCAAACAAGGTTGGGGTATTGGTAACCTT~A~GAAGTTGCTTTGAACGCCGAAG~TT
~ A ~ Q G W G I G N L Y E V A ~ ~ A E G

G~CAAAGTAGTGGTGTTGCTGATGTCACCTTATTAGATGTTTACACAACTCCAAAGGGTT
W Q S S G V A D Y T L L D V Y T T P K G

CTAGTCCAGCC~CCTCTGCCGC~CCTCGT TM
S S P A T S A A P R

SEQ ID NO:4 SUBSTITUTE SHEET

Claims (27)

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 .lambda.ZAP, .lambda.ZAPII 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 .lambda.ZAPII.

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 produced from a recombinant cellulase cDNA construct contained in an E
coli host cell and 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 patriciarum 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.