AU4299593A - Recombinant cellulases - Google Patents

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 β-1,4-glucosidic bonds. Conversion of cellulose to simple sugars (cellobiose and glucose) involves at least two types of hydrolases: endoglucanases which hydrolyse internal β-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 β-1,4-linkedxylose units. The enzymatic cleavage of β-1,4-xylosidic linkages is performed by endo-β-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-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 (MUC) and have no or little depolymerising activity against CM-cellulose. Similarly, many xylanases exclusively attack β-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 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 polysaccharide 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 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. 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 mRNAs in order to splice out introns. Therefore, enzymatically functional protein cannot normally be synthesised in clones obtained from a fungal genomic library.

The cDNA cloning approach can be used to overcome the post-transcriptional modification problem in E. coli . 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 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, λgt11 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 exocellobiohydrolase (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 , Neocallimastix 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-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.

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-sodium citrate, pH7.0, 1mM-EDTA and 0.1 M-β-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/HCl/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 β-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 × 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 (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-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 × 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 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 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 β-glucosidase. Other cellulosic substrates tested were lichenan (a mixed glucan containing β-1 , 4 and β-1 , 3 linkages) and laminarin (predominantly β-1,3-glucan). The celD enzyme had very high activity towards lichenan (Table 1) and produced a large hydrolysis zone on lichenancontaining 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-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 3-5 glucose units) to glucose and cellobiose. Its catalytic mode on these cellulosic substrates is of a typical endoglucanase (ie. it cleaved β-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%) 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 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 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^ˉ1 per mg protein (the crude cell lysate preparation), compared to 24.3 μg min^ˉ1 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 × 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 β-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 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).

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Table 1 Activity of the cloned cellulases on various substrates.

Specific activity

Substrate (nmol product min^-1 (mg protein)^-1 )

celA celB celC CelD celE

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 0 0 0 0 pNPC 0 1.3 0 169 80 MUG ND ND ND 0 ND

MUC ND ND ND 944 150

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 p-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 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 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 spindialysis using a Centricon-10 tube (Amicon). The enzyme preparation was incubated with cellodextrins: [cellotriose (G₃), cellotetraose (G₄) and cellopentaose (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 R_f values on a TLC plate. Authentic cellodextrins (S) are shown in the rightmost lane and the positions of glucose (G₁), 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 ) , KpnI (K ), PauII(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 substratecontaining 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.

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 β-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 Reproduced hereinbelow are sequence listings wherein -

SEQ ID NO: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 β-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

1 ATGACCATGA TTACGCCAAG CTCGAAATTA ACCCTCACTA AAGGGAACAA AAGCTGGAGC

61 TCCACCGCGG TGGCGGCCGC TCTAGAACTA GTGGATCCCC CGGGCTGCAG GAATTCGGCA

121 CGAGCTCCAA TCCGTGATAT TTCATCCAAA GAATTAATTA AAGAAATGAA TTTCGGTTGG

181 AATTTAGGTA ATACTTTAGA TGCTCAATGT ATTGAATACT TAAATTATGA TAAGGATCAA

241 ACΓGCTTCTG AAACTTGCTG GGGTAATCCA AAGACTACTG AAGATATGTT CAAGGTTTTA

301 ATGGATAACC AATTTAATGT TTTCCGTATT CCAACTACTT GGTCTGGTCA CTTCGGTGAA

361 GCTCCAGATT ACAAGATTAA TGAAAAATGG TTAAAGAGAG TTCATGAAAT TGTTGATTAT

421 CCATACAAGA ATGGAGCTTT CGTTATCTTA AATCTTCACC ATGAAACTTG GAACCATGCC

481 TTCTCTGAAA CTCTTGACAC TGCCAAGGAA ATCTTAGAAA AGATTTGGTC TCAAATTGCT

541 AAAGAATTTA AGGATTATGA TGAACACTTA ATTTTTGAAG GATTAAACGA ACCAAGAAAG

601 AATGATACTC CAGTTGAATG GACTGGTGGT GATCAAGAAG GATGGGATGC TGTTAATGCT

661 ATGAATGCCG TTTTCTTAAA GACTATTCGT AGTTCTGGTG GTAATAATCC AAAGCGTCAT

721 CTTATGATCC CTCCATATGC TGCTGCTTGT AATGAAAATT CATTCAAGAA CTTTATTTTC

781 CCAGAAGATG ATGACAAGGT TATTGCTTCT GTTCATGCTT ATGCTCCATA CAACTTTGCC

841 TTAAATAATG GTGAAGGAGC TGTTGATAAG TTTGATGCTG CTGGTAAGAA AGATCTTGAA

901 TGGAACATTA ACTTAATGAA GAAGAGATTT GTTGATCAAG GTATTCCAAT GATTCTTGGT

961 GAATATGGTG CCATGAATCG TGATAATGAA GAAGATCGTG CAGCTTGGGC TGAATTCTAC

1021 ATGGAAAAGG TCACTGCTAT GGGAGTTCCA CAAGTCTGGT GGGATAATGG TATCTTTGAA

1081 GGTACCGGTG AACGTTTTGG TCTTCTTGAT CGTAAAAACT TAAAGATTGT TTATCCAACT

1141 ATCGTTGCTG CTTTACAAAA GGGAAGAGGT TTAGAAGTCA ATGTTGTTCA TGCTATTGAA

1201 AAAAAAOCAG AAGAAOCAAC TAAAACTACT GAACCAGTTG AACCAACTGA AACTACTAGT

1261 CCAGAAGAAC CAGCTGAAAC TACTAATCCA GAAGAAOCAA CCGGTAATAT TCGTGATATT

1321 TCATCTAAQG AATTAATTAA AGAAATGAAT TTCGGTTGGA 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 GAAGAACCAA 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 AACTAAAAAA

SEQ ID NO:1 celD

4081 AAAAAAAAAA AACTCGAG

SEQ ID NO:1

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

TCTGGTTAGATAACACTGGTGGTAACGGTTCTATGACTCTCGGTAGTGGTGCAACTTTCA I W L D N T G G N G S M T L G S G A T F

AGGCTGAATGGAATGCAGGΓGTTAACCGTGGTAACTTCCTTGCCCGTCGTGGTCTTGACT

K A E W N A A V N R G N F L A R R G L D

TCGGTTCTCAAAAGAAGGCAACCGATTACGACTACATTGGATTAGATTATGCTGCTACTT

F G S Q 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 L V E Y Y I I E D W V D

GGGTTCCAGATGCACAAGGAAAAATG GTAACCATTGATGGAGCTCAATATAAGATTTTCC W V P D A Q G K M V T I D G A Q Y K I F

AAATGGATCACACTGGTCCAACTATCAATGGTGGTAGTGAAACCTTTAAGCAATACTTCA Q M D H T G P T I N G G S E T F K Q 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 Q S S G V A D V T L 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

Claims (36)

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.