(1 -> 3, 1 -> 4)-beta-glucanase of enhanced stability

Abstract

A modified cereal (1→3,1→4)-&bgr;-glucanase is produced by the method of single point substitution in a native cereal (1→3,1→4)-&bgr;-glucanase enzyme, whereby the substitution:

Description

BACKGROUND OF THE INVENTION

[0001] Barley quality encompasses a range of physical and chemical attributes, depending on whether the grain is to be used in the preparation of malt for brewing purposes, in the formulation of stockfeed, or as a component of human foods. Currently, specifications of barley quality are tailored primarily for the malting and brewing industries, in which germinated barley (malt) is the principal raw material. The quality specifications include such parameters as grain size, dormancy, malt extract, grain protein content, development of enzymes for starch degradation in malt and (1→3,1→4)-&bgr;-glucan content. Malt extract is a widely-used quality indicator. It is an estimate of the percentage of malted grain that can be extracted with hot water. Barley breeders and growers strive to produce grain with high malt extract values, because greater extract percentages provide higher levels of materials for subsequent fermentative growth by yeast during brewing. Malt extract values are influenced both by the composition of the ungerminated barley and by the speed and extent of endosperm modification during malting. Given the central role of cell walls as a potential barrier against the free diffusion of starch- and protein-degrading enzymes from the scutellum or from the aleurone to their substrates in cells of the starchy endosperm, it is not surprising that wall composition and the ability of the grain to rapidly produce enzymes that hydrolyse wall constituents are important determinants of malt extract values.

[0002] The major constituents of endosperm cell walls of barley are the (1→3,1→4)-&bgr;-glucans, which account for approximately 70% by weight of the walls (Fincher, 1975). In the germinating grain (1→3,1→4)-&bgr;-glucanases function to depolymerise (1→3,1→4)-&bgr;-glucans of cell walls during endosperm mobilisation.

[0003] Total (1→3,1→4)-&bgr;-glucan in ungerminated barley grain is not highly correlated with malt extract (Henry 1986; Stuart et al, 1988). However, the residual (1→3,1→4)-&bgr;-glucan in malted barley is highly correlated, in a negative sense, with malt extract (Bourne et al, 1982; Henry 1986; Stuart et al, 1988), and this residual polysaccharide reflects a combination of the initial (1→3,1→4)-&bgr;-glucan levels in the barley and, more importantly, the capacity of the grain to rapidly produce high levels of (1→3,1→4)-&bgr;-glucanase during malting (Stuart et al, 1988). The (1→3,1→4)-&bgr;-glucanase potential of barley cultivars is also dependent on both genotype and environment, although environmental conditions during grain maturation appear to be particularly important in the development of the enzymes (Stuart et al, 1988). Monoclonal antibodies specific for barley (1→3,1→4)-&bgr;-glucanases have been used in enzyme-linked immunosorbent assays (ELISA) that may be useful for the quantitation of (1→3,1→4)-&bgr;-glucanase levels in large numbers of barley lines generated in breeding programs (HØj et al, 1990). Furthermore, mutant barleys with altered (1→3,1→4)-&bgr;-glucan content (Aastrup 1983; Molina-Cano et al, 1989) or (1→3,1→4)-&bgr;-glucanase potential will be useful in future studies on the effects of these components on malting quality and may be valuable in breeding programmes.

[0004] The ability of the (1→3;1→4)-&bgr;-glucanases [E.C. 3.2.1.73] to retain enzymic activity at elevated temperatures (thermostability) is of extreme importance during the utilization of barley in the malting and brewing industries. Malt quality, as measured by the ‘malt extract’ index, is highly dependent on the ability of the grain to rapidly synthesize high levels of the enzyme during germination (Stuart et al, 1988). High levels of (1→3;1→4)-&bgr;-glucanases are also desirable in the brewing process, where residual (1→3;1→4)-&bgr;-glucans in malt extracts can adversely effect wort and beer filtration due to their propensity to form aqueous solutions of high viscosity. These residuals can also contribute to the formation of certain hazes or precipitates at elevated ethanol concentrations or low temperatures in the final beer (Woodward and Fincher, 1983). The elevated temperatures used during commercial malting and brewing lead to rapid and extensive inactivation of these enzymes. The high temperatures (up to 85°) of commercial kilning processes destroy greater than 60% of the enzyme activity and much of the remaining enzyme is inactivated by the hot water used for malt extraction (Brunswick et al, 1987), Loi et al, 1987). It is therefore highly desirable to develop commercial strains of barley that express a thermostable (1→3;1→4)-&bgr;-glucanase enzyme, or to produce the (1→3;1→4)-&bgr;-glucanase enzymes exogenously as an additive to be used in the brewing process.

[0005] Barley (1→3;1→4)-&bgr;-glucans also pose problems in the stockfeed industry. In poultry formulations prepared from cereal grains, (1→3;1→4)-&bgr;-glucans significantly raise the viscosity of the gut contents of chickens. This impairs digestion and slows growth rates, and results in sticky faecal droppings that make hygienic handling of eggs and carcases difficult (Fincher and Stone, 1986). This application would require the enzyme to be stable at a range of pHs, particularly in the pH region of the foregut. It would also be an advantage for the enzyme to be sufficiently thermostable to withstand the steam pelleting processes widely used in stockfeed manufacture.

[0006] Thus it is envisaged that (1→3,1→4)-&bgr;-glucanase of amino acid sequence modified so as to provide enhanced thermostability and/or pH stability will have a variety of industrial uses, either by means of barley expressing the modified enzyme, or by addition of the modified enzyme to barley being processed.

[0007] There has been considerable interest in inserting (1→3,1→4)-&bgr;-glucanase genes into brewing yeasts, in the expectation that low level, constitutive expression would lead to the secretion of active enzyme and the depolymerisation of residual (1→3,1→4)-&bgr;-glucan during fermentation (Hinchliffe, 1988). A barley (1→3,1→4)-&bgr;-glucanase cDNA (Fincher et al, 1986) fused with a mouse &agr;-amylase signal peptide is expressed and secreted from yeast under the direction of the yeast alcohol dehydrogenase I gene promoter (Jackson et al, 1986). Although the gene for isoenzyme EII has not yet been isolated, the availability of almost full length CDNA for use as a probe means that such isolation can readily be carried out using conventional methods.

[0008] We have now determined the three dimensional structure of (1→3,1→4)-&bgr;-glucanase isoenzyme EII and (1→3)-&bgr;-glucanase isgenzyme GII (E.C.3.2.1.39), and have identified regions of the structures of these enzymes which are candidates for modification in order to provide enhanced thermal and pH stability, as well as suitable point mutations for achieving such stabilisation. We have found that the 3-dimensional structures of these two enzymes, which share only 50% sequence homology, are remarkably similar in their structural framework, and that their active sites are also surprisingly similar, despite the difference in substrate specificity.

SUMMARY OF THE INVENTION

[0009] According to a first aspect, the invention provides a (1→3,1→4)-&bgr;-glucanase of enhanced thermostability and/or pH stability.

[0010] In a second aspect, the invention provides an isolated DNA sequence encoding a (1→3,1→4)-&bgr;-glucanase of enhanced thermostability and/or pH stability, and plasmids, expression vectors, and transgenic plants comprising said sequence. Preferably the expression host is E. coli or Saccharomyces cereviseae; preferably the transgenic plant is barley. It will be clearly understood that barley grain from plants encoding the improved enzyme is within the scope of this invention.

[0011] In a third aspect, the invention provides a method selected from the group consisting of malting, brewing and stockfeed processing, comprising the step of

[0012] a) using barley expressing the (1→3,1→4)-&bgr;-glucanase of this invention as a starting material, or

[0013] b) adding (1→3,1→4)-&bgr;-glucanase of this invention to a grain to be processed.

[0014] In a fourth aspect, the invention provides a composition for use in malting, brewing, or stockfeed processing, comprising the improved (1→3,1→4)-&bgr;-glucanase of the invention, together with carriers acceptable for use in processing of beverages or of stockfeeds.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The invention will now be described in detail by way of reference only to the following non-limiting examples, and to the figures, in which

[0016] FIG. 1 shows a stereo view of the alpha carbon trace of the polypeptide backbone of the EII and GII glucanase enzymes. The heavy lines represent the EII enzyme and the lighter lines represent the GII enzymes. The active site groove runs north to south, and the C- and N-termini are indicated, as are the two putative active site residues glutamic acids at residues 232 and 288 (using EII sequence numbers).

[0017] FIG. 2 shows the sequence comparison of the EII (lower line) and GII (upper line) glucanase enzymes based on the 3-dimensional structure, with the sequence given using the three letter code for amino acids. Residue numbers at the start of each line are the sequence numbers of the two enzymes. The secondary structure elements of both enzymes are given above the GII sequence and below the EII sequence (see text for notation used in the description of the tertiary structure).

[0018] &agr; represents alpha helices; &bgr; represents beta sheets; A and B represent additional alpha helices and beta sheets to those of a typical &agr;/&bgr; barrel.

[0019] FIG. 3 is a schematic drawing of the (1→3,1→4)-&bgr;-glucanase EII enzyme. The elements with arrow heads represent beta sheet structure and the elements with a curled tape coil represent alpha helices. Some of the smaller beta sheets are not drawn. Elsewhere the chain is represented as a rope. The black dots represent amino acid locations where thermostable mutants have been proposed (see text).

[0020] FIG. 4 is a schematic drawing of the (1→3)-&bgr;-glucanase GII enzyme. The elements with arrow head represent beta sheet structure and the elements with a curled tape coil represent alpha helices. Some of the smaller beta sheets are not drawn. Elsewhere the chain is represented as a rope. The black dots represent amino acids locations around the active site groove which confer the specific activity of the enzymes. It is proposed to modify these amino acids to change the specificity of the GII enzyme into that of the EII enzyme.

[0021] FIG. 5 shows a comparison between stability of (1→3,1→4)-&bgr;-glucanase isoenzyme EII with that of (1→3)-&bgr;-glucanase isoenzyme GII at pH 3.5.

[0022] FIG. 6 compares the stabilities of (1→3,1→4)-&bgr;-glucanase isoenzymes EII with that of (1→3)-&bgr;-glucanase isoenzyme GII at 50°.

[0023] FIG. 7 compares the stabilities of (1→3,1→4)-&bgr;-glucanase isoenzyme EII with that of (1→3)-&bgr;-glucanase isoenzyme GII at increasing temperatures.

[0024] FIG. 8 compares the stabilities of wildtype (1→3,1→4)-&bgr;-glucanase isoenzyme EII and mutant H300P on heating for 15 minutes.

[0025] FIG. 9 compares the stabilities of wildtype (1→3,1→4)-&bgr;-glucanase isoenzyme EII and mutant H300P at 48° C.

[0026] FIG. 10 compares the stabilities of wildtype (1→3,1→4)-&bgr;-glucanase isoenzyme EII and mutant H300P during mashing at 55° C.

[0027] The (1→3;1→4)-&bgr;-glucanases catalyse the hydrolysis of (1→4)-&bgr;-glucosyl linkages in (1→3;1→4)-&bgr;-glucans, only where the alucosyl residue is substituted at the C(O)3 position, as follows: 1

[0028] The glucosyl residues are represented by G, (1→3)- and (1→4)-&bgr;-linkages by 3 and 4, respectively, and the reducing terminus (red) of the polysaccharide chain is indicated. Thus the enzymes have an absolute requirement for adjacent (1→3)- and (1→4)-&bgr;-linked glucosyl residues in their substrates. The (1→3)-&bgr;-glucanases [EC 3.2.1.39] are able to hydrolyse the single (1→3)-&bgr;-linkages found in (1→3;1→4)-&bgr;-glucans, but can catalyse the hydrolysis of (1→3)-&bgr;-glucosyl linkages in (1→3)-&bgr;-glucans, as follows: 2

[0029] Arrows indicate the hydrolysis of (1→3)-&bgr;-linkages between glucosyl residues (G).

[0030] Furthermore it is known that the (1→3)-&bgr;-glucanase isoenzyme GII is more thermostable, pH stable and protease resistant than the (1→3;1→4)-&bgr;-glucanase EII enzyme. Thus using the three dimensional structures of these enzymes, we can create more stable forms of the (1→3;1→4)-&bgr;-glucanase by the following methods:

[0031] (a) transferring the structural elements that generate the heat stability of the (1→3)-&bgr;-glucanase, on to the (1→3;1→4)-&bgr;-glucanase.

[0032] (b) modifying the (1→3;1→4)-&bgr;-glucanase using general principles of protein structure and stability (Matthews, 1987).

[0033] (c) engineering a thermostable or pH stable (1→3;1→4)-&bgr;-glucanase enzyme by transforming the (1→3)-&bgr;-glucanase into the (1→3;1→4)-&bgr;-glucanase. This is done by transferring elements of the catalytic site of the (1→3;1→4)-&bgr;-glucanase enzyme on to the (1→3)-&bgr;-glucanase enzyme.

[0034] (d) engineering a thermostable (1→3,1→4)-&bgr;-glucanase and (1→3)-&bgr;-glucanase by creating cysteine pairs which can form disulphide bonds across the C and N terminals.

[0035] A combination of two or more of these methods may be used.

[0036] For each of these methods knowledge of the protein structures is an important prerequisite. This knowledge enables us to separate differences between the two enzymes which govern substrate specificity from those for thermal and pH stability. It also enables us to predict which kind of changes to the sequence which will enhance the stability of the secondary structure elements. Random mutagenesis of glucanase genes will invariably reduce the stability of the protein by disrupting its structure, or may cause inactivation of the enzyme. This is due to the inability of current methods to predict protein folding and catalytic activity from amino acid sequence information alone.

EXAMPLE 1

Determination of the 3-Dimensional Structure of the Glucanase Enzymes

[0037] We have determined the 3-dimensional structure of (1→3;1→4)-&bgr;-glucanase isoenzyme EII (hereafter called EII) and (1→3)-&bgr;-glucanase isoenzyme GII (hereafter called GII) to Sigh resolution (2.2 Å) by X-ray crystallographic techniques described by Blundell and Johnson (1979).

[0038] In Appendix 3 we have set out the 3-dimensional coordinates and mean thermal vibration parameters (isotropic B values) of the two enzymes, as determined from the crystallographic refinement of the X-ray diffraction data obtained from single crystals of each enzyme.

[0039] The EII and GII glucanase structures have essentially identical &agr;/&bgr; barrel folds (FIG. 1). Minor perturbations are found in the loops mainly at positions where there are sequence insertions and deletions. A sequence comparison is set out in FIG. 2. The active site groove, which runs along the full length of the upper surface of the molecule perpendicular to the barrel axis, is almost identical in the central region of the groove, and different in detail towards the ends of the groove. The carboxylate groups of the two putative active site glutamates (Chen et al, 1993) are positioned in an identical way some 7 Å apart. Also around these residues are a ring of residues which are totally conserved in all plant (1→3)-&bgr;-glucanases known (Xu et al, 1992 and sequences from the Genbank database). Details of the structure, which is a novel type of &agr;/&bgr; barrel are given below.

[0040] In FIG. 2 elements of the secondary structure have been identified alongside the sequence alignment of the two enzymes. We shall refer to the beta barrel strands as &bgr;i and the major (longest) helices connecting the beta strands as &agr;1, where i goes from 1 to 8. Minor &bgr; sheet and &agr; helices are referred to as Bi and Ai, respectively if they appear after the strand &bgr;i and before &bgr;i+1, and a further subscript a or b, if more than one occur.

[0041] Looking at the glucanase tertiary structure from above, down the barrel axis (the long axis of the elliptical barrel running east west), the active site groove runs north to south on the upper face of the molecule, as shown in FIGS. 3 and 4.

[0042] The N-terminal starts under the molecule entering the east side of the barrel as &bgr;1 and emerges on the upper surface and the heads back towards the bottom surface as &agr;1 (traversing the outside of the molecule) to meet &bgr;2, where this motif is repeated for strands &bgr;2 to &bgr;4, building the upper half of a conventional &agr;/&bgr; barrel (note that for the third &agr;/&bgr; loop there are two helices).

[0043] The lower half of the barrel has more elaborate secondary structural elements, not previously observed in other &agr;/&bgr; barrel structures. There is what could be called a subdomain built around the helix &agr;6. This helix runs perpendicular to the groove axis and at the southern end of the groove and is supported by three two stranded antiparallel &bgr; sheet ‘fingers’ (B5 on the upper surface, B7 on the underneath surface and B6 at the southern end of the groove) and three small helices (A5 at the western side and A6a and A6a at the eastern side of the groove). This subdomain, which forms a platform for the residues making up the lower half of the groove, is different in detail (possibly arising from the difference in specificity) between the EII and GII enzymes; for example the helix A5 is missing in GII.

[0044] The C-terminal strand, consisting of some 30 residues, starts after the strand &bgr;8, and has an unusual turn which involves a cis peptide bond between residues Phe 275 and Ala 276 (a cis proline could not accommodate this type of turn). This turn allows the loop of residues from 276 to 286 to position the glutamate at 288, which is in a small helical turn &agr;8, at the appropriate orientation to act as a catalytic acid group. The C-terminal strand then finds its way down to the underside of the molecule between the helices &agr;1 and &agr;7 to within 4.2 Å from the N-terminus.

EXAMPLE 2

Identification of Sites of Contact with Substrate

[0045] In order to observe which amino acids in the substrate-binding groove contacted the substrate, the structure of glucanase GII was determined after soaking crystals with 1→3 linked oligosaccharides. Three sites were found where glucose units of monomer or disaccharides bind to the protein The coordinates of these sites are listed in Appendix 2. This establishes the orientation of the substrate within the groove, and that some of the proposed changes to GII are important for substrate binding.

EXAMPLE 3

Proposed Modification of the (1→3,1→4)-&bgr;-Glucanase of Barley to Increase the Thermostability of the Enzyme

[0046] The following amino acid changes are proposed for enhancing the thermostability of (1→3,1→4)-&bgr;-glucanase EII, based on the 3-dimensional structure of the EII and GII enzymes. Some of the changes proposed involve substituting the GII amino acids that could be responsible for stabilizing that protein. These substitutions are based on the principle that the proposed changes will not alter the specificity of the enzyme (leave the active site groove unaltered), and where changes would not lead to deleterious changes in the 3-dimensional structure of the protein. Where possible glycines have been replaced by prolines or alanines in helices (Matthews et al, 1987) in order to stiffen the amino acid chain and reduce the entropy of the unfolded protein. Negatively charged residues have been attached to the N-termini of helices to stabilise them (Nicholson et al, 1988, Eijsink et al, 1992). Ion pairs have been introduced to increase the binding energy of the folded protein, and lysines changed. to arginines to prevent glycation and improve stability (Mrabet et al, 1992) by increasing the hydrogen-bonding with other parts of the protein. EI and EII refer to the isozymes of (1→3,1→4)-&bgr;-glucanase and GI to GVI refer to the isozymes of the (1→3)-&bgr;-glucanase (Xu et al, 1992). The location of these substitutions are shown on FIG. 3. The mutation is described using the following notation: eg. the mutation Ala 14 Ser represents the mutation of the Alanine residue to a Serine at position 14 in the amino acid sequence (FIG. 3). The conventional 3 letter code for amino acids is used. 1 Mutation comments Ala 14 Ser as in GII, GV, GVI to stabilise helix &agr;1 Ala 15 Arg as in GII, GIV, GV ion pair with Asp 36 at end of groove Thr 17 Asp as in GII to form ion pair with Met 298 Lys in GII Lys 23 Arg as in GI to GIV, H-bond to O46 Lys 28 Arg Asn 36 Asp as in GI, GII, GIV, GVI, EI, to stabilise helix &agr;2, ibid Gly 44 Arg as in GI, GII, GV, GVI Gly 45 Asn as in GII, solvent exposed Gly 53 Asp as in GI, GII, GIII, GV, forms a stable ion pair with Arg 31 Gly 53 Glu Lys 74 Arg as in GI, GV Gln 78 Arg as in GI, GII Ala 79 Pro as in GI, GII, GVI, surface residue Lys 82 Arg Ala 95 Asp as in GIII, ion pair with Arg 128 at end of groove, Asn in GII Gly 97 Pro Phe 85 Tyr OH of Tyr H-bonds to O 76 Lys 107 Arg as in GI, GII, GIII, GIV Gly 111 Ala as in GII, helix residue Gly 119 Pro Lys 122 Arg conserved in all except GVI, H-bond to O 161 and O 120 Ser 128 Arg as in GI to GV Gly 133 Ala as in GII, on the lip of the groove, could have packing problems here with Thr 144 Gly 145 Asn different conformation in GII Gly 152 Thr as in GII, His 221 will clash with Thr so need to change His to Ala Pro 153 Asp as in GII, see below for ion pair Gln 156 Arg as in GII, need Pro 153 Asp for ion pair Asn 162 Gly Gly 185 Asn as in GII, stabilised by Asp 183 Ala 191 Pro as in GII, buried (near surface) Gly 193 Ala wrong dihedrals for a Pro Gly 199 Pro as in GI, GII has a different loop conformation solvated, so could be modified. Ala 200 Gly Gly 202 Thr as in GII, H-bond to Thr 194 and Arg 197 space for Pro here. Gly 219 Glu as in GI to GVI, ion pair with Arg 265 might need Glu 266 Lys Lys 220 Arg as in GI H-bonds to O139 His 221 Ala as in GII, ibid Gly 223 Ala as in GII (buried) Ser 224 Pro as in GI to GV Lye 227 Arg as in GI, GIV, GV, ion pair with Glu 268 Gly 238 Ala as in GI, GII, GIV, GV, could clash with Asn 290 Gly 239 Gln as in GIII wrong dihedrals form a Pro Ala 242 Gly Gly 260 Glu ion pair with Arg 261 or Pro Pro 267 Arg as in GII Gly 268 Glu as in GII, could for ion pair with Arg 227 (peptide flipped wrt GII) Gly 286 Ala as in GII or Asp to stabilise helix &agr;7 Gln 289 Arg as in GII, GIV, GV Met 298 Lys as in GI, GII, GIV, GV, ibid His 300 Pro as in GI to GV

[0047] Of the above proposed modification the following ion pairs have to be substituted at the same time. 2 Ala 15 Arg and Asn 36 Asp Thr 17 Asp and Met 298 Lys Ala 95 Asp and Ser 128 Arg Pro 153 Asp and Gln 156 Arg Lys 227 Arg and Gly 268 Arg Gly 152 Thr and His 221 Ala

[0048] It will be clearly understood that, subject to this requirement for concurrent substitution of ion pairs, combinations of two or more of the proposed modifications may be used.

[0049] An additional class of mutations is proposed in which the main chain torsion angle about the N and C&agr; atoms is greater than 0°. In this case a replacement by a Gly residue is energetically more favourable, particularly at the C terminal of an &agr;-helix (Aurora et al., 1994). These mutations are: 3 Asn 162 Gly as in GI, GII, GV, GVI, EI, terminus of helix &agr;5 Ala 200 Gly as in GIII, GIV, GV, GIV, main chain torsion angles Ala 242 Gly Main chain torsion angles Met 298 Gly Main chain torsion angles

EXAMPLE 4

Proposed Modification of the (1→3)-&bgr;-Glucanase of Barley to Alter its Catalytic Activity to that of (1→3,1→4)-&bgr;-Glucanase and Increase the Thermostability and pH Stability of the Enzyme

[0050] As mentioned before the most noticeable feature of both the GII and EII enzymes is a deep groove across one face of the molecule. This appears to be the substrate binding site. Using structural information from both the GII and EII enzymes it is possible to determine which amino acid residues are likely to control substrate specificity. Furthermore, as these two enzymes are very similar in structure it is possible to graft the loops from one enzyme on to the more heat and pH stable framework of the other to change the specificity.

[0051] We propose replacing the GII loops which form the sides and bottom of the cleft by the corresponding amino acids from the EII enzyme. These changes are as follows: 4 residue 8       Ile→Ser, residue 34      Phe→Ala, residue 208     Ala→Thr, residue 209     Met→Thr, residue 213     Val→Phe residue 128-137 Ile-Arg-Phe-Asp- (SEQ. ID NO:1) Glu-Val-Ala-Asn-Ser-Phe→ Val-Ser-Gln-Ala-Ile-Leu- Gly-Val-Phe-Ser, residue 171-179 Phe-Ala-Tyr-Arg- (SEQ. ID NO:2) Asp-Asn-Pro-Gly-Ser→ Leu-Ala-Trp-Ala-Tyr-Asn- Pro-Ser-Ala and residue 283-291 Thr-Gly-Asp-Ala- (SEQ. ID NO:3) Thr-Glu-Arg-Ser-Phe→ Asp-Ser-Gly-Val-Glu-Gln- Asn-Trp

[0052] Some or all of these changes are necessary. The skilled person will readily be able to test the effectiveness of the substitutions.

[0053] Again combinations of two or more of these proposed modifications may be used.

[0054] Doan and Fincher (1992) showed that relative to the EI enzyme, EII is more thermostable because of the carbohydrate at residue 190. We propose to introduce a carbohydrate attachment site into the modified GII enzyme to enhance the thermostability. The mutations required are 189-191 Gln-Pro-Gly→Asn-Ala-Ser

[0055] FIG. 4 is a schematic drawing of the GII enzyme structure showing locations of the proposed mutations.

EXAMPLE 5

Construction of Mutant Glucanases

[0056] Construction of the proposed mutant glucanases may be effected using the polymerase chain reaction (PCR)-based megaprimer method (Sarkar & Sommers, 1990), and single site mutants of the isozymes EI and EII have already been produced in this way by one of us (Doan and Fincher, 1992). Briefly, for each site mutant or short series of adjacent mutations one oligonucleotide is synthesised which contains the complementary sequence required for the mutation(s) and sufficient flanking regions to anneal to the wild type cDNA. This oligonucleotide is extended against the cDNA template with a DNA polymerase. PCR is used to amplify the mutant section of cDNA, and then this is inserted back into the plasmid containing the original cDNA. For multiple mutations this process is repeated to produce the final construct. Alternatively, commercially-available site directed mutagenesis kits based on the unique site elimination method (Deng and Nickoloff, 1992) can be used.

[0057] We currently have the cDNAs for the EII and GII enzymes which form the starting points for the mutagenesis (Doan and Fincher, 1992; HØj et al, 1989). For the purposes of demonstrating improved stability or altered specificity of these enzymes and for production of the enzymes in quantity, the proteins can then be expressed in E. coli (Wynn et al, 1992) using the plasmid ET or other vectors or in insect cells (e.g. Sf9 cells) using a Baculovirus system (Doan & Fincher, 1992). A person skilled in the art will be aware of a variety of other suitable expression systems. For example, yeast would be a suitable host, and such an engineered yeast could be used directly in the brewing process. The availability of the gene encoding (1→3,1→4)-&bgr;-glucanase isoenzyme EI and near full-length cDNAs for isoenzymes EI and EII (Slakeski et al, 1990) presents an opportunity to accelerate or enhance (1→3,1→4)-&bgr;-glucanase development in germinated grain through gene technology. Increased enzyme activity might be achieved by several means, for example, by splicing more efficient promoters onto the gene, by altering the existing promoter to enhance expression levels, by the use of translational enhancers, or by increasing the copy number of the genes.

[0058] Two more steps are required for the mutant enzymes to be incorporated into barley and expressed in a spatially and temporally appropriate manner. These are construction of a barley glucanase gene with the appropriate control of expression, and the insertion of the gene into a viable barley plant. The sequence the EII gene, including the promoter regions and the coding region and the signal peptide has been determined (Wolf, 1991). Thus for correct expression of the mutant glucanases we will replace a portion of this gene by the corresponding portion of a mutant cDNA using the above methods. It is expected that transformation of barley, that is to regenerate a fertile transgenic barley plant, will be possible in the near future. Foreign or manipulated DNA can be integrated into the barley genome in a stable form (Lazzeri et al, 1991) and fertile plants can be regenerated from single protoplasts (Jahne et al, 1991a, b). Among the cereals related to barley, rice can now be routinely transformed, and transformation of both wheat and maize has been reported. Methods for effecting transformation of monocotyledonous plants such as barley using biolistic techniques are widely used, and whole plants of transgenic barley have been grown. Barley has recently been transformed using the biolistic microprojectile gun procedure (Wan and Lemaux, 1994).

EXAMPLE 6

[0059] i) Stability of GII and EII at pH 3.5

[0060] (1→3)-&bgr;-glucanase isoenzyme GII (9.2 &mgr;g/ml) and (1→3,1→4)-&bgr;-glucanase isoenzyme EII (0.23 mg/ml) were incubated in 100 mM sodium acetate buffer at pH 3.5 in the presence of bovine serum albumin at 37° C. (0.5 mg/ml) Residual enzyme activities (At) were determined and compared to the initial activity at t=0 (Ao). The results are illustrated in FIG. 5. GII shows markedly greater stability with time at pH 3.5 than does EII. (Note: at pH 4.3 the enzymes differ only slightly in their stability and exhibit only minimal loss of activity; data not shown).

[0061] ii) Stabilities of GII and EII at 50° C.

[0062] (1→3)-&bgr;-glucanase isoenzyme GII (16 &mgr;g/ml) and (1→3;1→4)-&bgr;-glucanase isoenzyme EII (19 &mgr;g/ml) were incubated in 50 mm sodium acetate buffer at pH 5.0 in the presence of bovine serum albumin (1 mg/ml) at 50° C. Residual enzyme activities (At) were determined and compared to the initial activity at t=0 (Ao). The results are illustrated in FIG. 6. GII is very much more stable at 50° C. than is EII.

[0063] iii) Stabilities of GII and EII at Increasing Temperatures

[0064] (1→3)-&bgr;-glucanase isoenzyme GII (16 &mgr;g/ml) and (1→3;1→4)-&bgr;-glucanase isoenzyme EII (19 &mgr;g/ml) were incubated in 50 mM sodium acetate buffer at pH 5.0 at the indicated temperature for 15 min. Residual enzyme activities (At) were determined and compared to the initial activity at t=0) (Ao). The results are illustrated in FIG. 7. EII is stable only up to 40° C., while GII is stable up to 50° C.

EXAMPLE 7

Size-directed Mutagenesis

[0065] Of the possible mutations listed in Example 3, the following alterations were considered to be the most likely to improve stability. The alterations are based on: 5 1. creation of ion pairs: Gly 53 Asp Gly 53 Glu Thr 17 Asp; Met 298 Lys Ala 95 Asp; Ser 128 Arg 2. removal of potential glycation sites: Lys 122 Arg Lys 23 Arg Lys 74 Arg 3. reduction in entropy of unfolded state: Gly 44 Arg Gly 223 Ala Ala 79 Pro 4. hydrophobic effects: Phe 85 Tyr

[0066] Site-directed mutagenesis was carried out by the unique restriction enzyme site elimination procedure using a U.S.E. Mutagenesis Kit (Pharmacia) with double-stranded plasmid DNA as a template. Appropriate mutagenic primers were designed to generate the mutations and were synthesized on a standard DNA synthesizer. All oligonucleotide primers were phosphorylated at their 5′-end before use, and the mutagenesis procedure was performed essentially as prescribed by the manufacturer. Mutants were confirmed by dideoxynucleotide sequencing using a Sequence version 2.0 sequencing Kit (U.S. Biochemical Co.). 6 The following EII mutants were produced and confirmed by sequence analysis: Lys 74 Arg Gly 44 Arg Phe 85 Arg Gly 53 Glu Lys 122 Arg Lys 23 Arg Ala 79 Pro In addition, we have also made the following mutants: Gly 223 Ala Gly 53 Asp

EXAMPLE 8

Expression of Mutant Enzymes in E. coli

[0067] The mutant cDNA inserts in the expression plasmid pMAL-c2 were transformed in E. coli DH5&agr; cells, and grown overnight at 37° C. in LB containing 0.2% glucose and 100 &mgr;g/ml ampicillin. Aliquots of the cell suspension were sub-cultured into the same medium and grown at 37° C. with vigorous shaking to an optical density at 600 nm of 0.5, induced for 3 h with 1 mM isophenyl-&bgr;-thiogalactoside and lysed with lysozyme treatment and freeze/thawing. After removal of cell debris by centrifugation, enzyme activity was measured either in the unpurified extract or following purification.

[0068] The following EII mutants have been expressed in E. coli and the expressed proteins have been confirmed to be of the correct size:

[0069] Lys 122 Arg

[0070] Phe 85 Tyr

[0071] Gly 44 Arg

EXAMPLE 9

Purification of Recombinant Fusion Proteins

[0072] For the purification of the wild-type enzyme, crude extract from 1 litre culture was diluted 10-fold with 15 mM Tris-Hcl buffer, pH 8.0 and applied at a flow rate of 2.5 ml/min to a DEAE-Sepharose Fast Flow (Pharmacia) column (3×11.5 cm) equilibrated with 25 mM Tris-HCl buffer, pH 8.0. After washing the column exhaustively, bound proteins were eluted with a linear 0-250 mM NaCl gradient in 1.2 litre equilibration buffer. Fractions containing significant enzyme activity were pooled, desalted and adjusted to 25 mM NaAc, pH 5.0. After exhaustive washing, bound proteins were eluted with a linear 0-200 mM NaCl gradient in 1 litre equilibration buffer. The fractions containing pure protein were pooled to give 5.0 mg active fusion protein.

[0073] Mutant enzymes were all purified by a single ion-exchange chromatography step employing a shallow salt gradient elution. The crude extract from 4 to 5 litre culture was diluted 10 fold with 15 mM Tris-HCl (pH 8.0) and applied at a flow rate of 2.5-3.0 ml/min to a DEAE-Sepharose column (5×21 cm) equilibrated with 12.5 mM Tris-HCl (pH 8.5). After exhaustive washing, bound proteins were eluted with a 1.9 litre linear 0-80 mM NaCl gradient at a flow rate of 2.0 ml/min. Fractions containing pure fusion protein were located by SDS-PAGE, pooled, concentrated and adjusted to 2.5 mM sodium acetate (pH 5.0) by ultrafiltration before clarification by centrifugation.

EXAMPLE 10

Activity of Expressed Enzymes

[0074] (1→3,1→4)-&bgr;-Glucanase activity was measured viscometrically at 40° C., using 5 mg/ml barley (1→3,1→4)-&bgr;-glucan in 50 mM sodium acetate pH 5.0 as substrate. A unit of activity is defined as the amount of enzyme causing an increase of 1.0 in the reciprocal specific viscosity (&Dgr;1/&eegr;sp) per minute. Specific activity is expressed as the activity per mg protein.

[0075] The activities of the following mutant enzymes have been measured and compared with the activity of the expressed wild type enzyme: 7 Lys 122 Arg activity same as wild type Phe 85 Pyr activity approx. 70% of wild type Gly 44 Arg activity very low

EXAMPLE 11

Thermostability Assays

[0076] Aliquots of wild type or mutant fusion proteins were diluted with 50 mM sodium acetate buffer, pH 5.5 and incubated at temperatures ranging from 40° C. to 60° C. for 15 min. Samples incubated at 0° C. were used as controls. Residual enzyme activity was determined viscometrically with 550 &mgr;l (1→3,1→4)-&bgr;-glucan substrate, as described for Example 10.

[0077] References listed herein are identified on the following pages.

[0078] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

EXAMPLE 12

Increased Thermostability of Isoenzyme EII by Site-Directed Mutagenesis

[0079] Stability of (1→3,1→4)-&bgr;-glucanase isoenzyme EII (mutant H300P)

[0080] The cDNA encoding (1→3,1→4)-&bgr;-glucanase isoenzyme EII was subjected to site-directed mutagenesis using the unique site elimination method (Deng and Nickoloff, 1992), to generate mutant H300P. The mutagenesis procedure was performed using a modified pET-3a vector containing the wild type (1→3,1→4)-&bgr;-glucanase isoenzyme EII cDNA as a template, which enables the rapid purification of expressed foreign proteins using a nickel-based affinity resin (Hochuli et al, 1987). The expressed mutant H300P showed an increase in the T50 value (the temperature at which only 50% of the initial activity remains) of approximately 3.8° C., after heating for 15 minutes at various temperatures. This is illustrated in FIG. 8.

[0081] An additional test for increased thermostability was provided by following the residual activity (At) of wild type isoenzyme EII and the corresponding mutant H300P over time at 48° C. The results are shown in FIG. 9. Finally, as a further indication of increased thermostability in a commercial context, activity of the wild type and mutant (1→3,1→4)-&bgr;-glucanase isoenzyme EII was measured over time in a simulated mashing experiment at 55° C. Briefly, mashing conditions were simulated by stirring malted, dried barley grain in water at 55° C. for 40 minutes to inactivate any endogenous (1→3,1→4)-&bgr;-glucanase activity, and then wild type or mutant H300P enzyme was added to the mash and residual activity (At) was monitored over time. The results are shown in FIG. 10.

EXAMPLE 13

Further Mutants Expected to Enhance Thermostability

[0082] 8 Met 7 Val as GI GII GIII, allow loop 7-12 to pack tighter against C-terminus Ala 9 Gly as GII GIII GV GVI, allow loop 7-12 to pack tighter against C-terminus Ala 15 Pro as GIII GVI Met 21 Leu as GI-GVI, prevent close contact with Met 298 (or Lys) Phe 22 Tyr as GI-GVI, buried H-bond with Val 30 Asn 25 Lys as GI-GIV, cover hydrophobic patch Gly 26 Asn as GV, GVI, rigidify helix capping residue Gly 240 Ala rigidify loop Asn 279 Asp stronger H-bonds Ser 285 Pro rigidify loop Val 287 Pro rigidify loop Asn 290 His as GI GIV, His would pack tighter Phe 294 Tyr could H-bond to Asn 25 OD1 Asn 297 Asp as GI GII GVI, tighter H-bond in loop Met 298 Gly Main chain torsion angles suit Gly Val 301 Ala as GI-GIII, change water structure    307 Asn extend C terminus to make a salt bridge with Lys 28 Ala 176 Arg and Gly 286 Asp ion pair Ser 237 Phe and Asn 279 Ser close packed bridge across or Trp C-terminal tail

[0083] As the N and C termini are close to each other it would be possible to tie down the C terminus by linking the ends together. The shortest linker with a structurally reasonable conformation is Ala-Ala-Gly (or Gly-Pro-Gly or combinations). As helix a6 and strand b7 are buried in the protein, new N and C termini at Val 226 and Gly 223 will not reduce the thermostability of the protein. Furthermore the new termini could form an ion pair.

[0084] References

[0085] Aastrup S. Carlsberg Res. Comnmun., 1983 48 307-316

[0086] Aurora, R., Srinivasan, R., and Rose, G. D. Science, 1994 264 1126-1130

[0087] Bamforth, C. W. Brewers Digest, 1983 57 22-27

[0088] Blundell, T. L. and Johnson, L. N. Protein Crystallography, 1976, Academic Press, London

[0089] Bourne, D. T., Powlessland, T. and Wheeler, R. E. j. Inst. Brew., 1982 88 371-375

[0090] Brunswick,P., Manners, D. J. and Stark, J. R. J. Inst. Brew., 1987 93 181-186

[0091] Chen, L., Fincher, G. B. and HØj, P. B. J. Biol. Chem., 1993 268, in press

[0092] Deng, W. P. and Nickoloff, J. A. Analytical Biochemistry, 1992 200 81

[0093] Doan, D. N. P. and Fincer, G. B. FEBS Lett, 1992 309 265-271

[0094] Eijsink, V. G. H, Vriend, G., van den Burg, B., van der Zee, J. R. and Venema, G. Prot. Eng., 1992 5 165-170

[0095] Fincher, G. B. J. Inst. Brewing, 1975 81 116-122

[0096] Fincher, G. B., Lock, P. A., Morgan, M. M., Lingelbach, E., Wettenhall, R. E. H., Mercer, J. f. B., Brandt, A. and Thomsen, K. K. Proc. Natl. Acad. Sci. USA, 1986 83 2081-2085

[0097] Fincher, G. B. and Stone, B. A. Adv. Cer. Sc. Techn., 1986 8 207-295

[0098] Henry, R. J. J. Cereal Sci., 1986 4 269-277

[0099] HØj, P. B., Hartman, D. J., Morrice, N. A., Doan, D. N. P. and Fincher, G. B. Plant Mol. Biol., 1989 13 31-42

[0100] HØj, P. B., Hoogenraad, N. J., Hartman, D. J., Yanakena, H. and Fincher, G. B. J. Cereal Science, 1990 11 261-268

[0101] Jahne, A., Lazzeri, P. A., Jager-Gussen, M. and Lorz, H. Theor. Appl. Genet, 1991 82 74-80

[0102] Lazzeri, P. A., Brettschneider, R., Luhrs, R., and Lorz, H. Theor. Appl. Genet., 1991 81 437-444

[0103] Loi, L., Barton, P. A. and Fincher, G. B. J. Cer. Sci., 1987 5 45-50

[0104] Matthews, B. W. Biochemistry, 1987 26 6885-6888

[0105] Matthews, B. W., Nicholson, H., Becktel, W. J. Proc. Natl. Acad. Sci. U.S.A., 1987 84 6663-6667

[0106] Mrabet, N. T. et al Biochem., 1992 31 2239-2253

[0107] Nicholson, H., Becktel, W. J., Matthews, B. W. Nature (London), 1988 336 651-656

[0108] Powell, W., Caligari, P. D. S., Swanston, J. S. and Jinks, J. L. Theoretical and Applied Genetics, 1989 71 461-466

[0109] Sarkar, G and Sommers, S. S. BioTequniques, 1990 4 404-407

[0110] Slakeski, N., Baulcombe, D. C., Devos, R. M. Ahluwalia, B., Doan, D. N. P. and Fincher G. B. Mol. Gen. Genet., 1990 224 437-449

[0111] Stuart, I. M., Loi, L. and Fincher, G. B. J. Cer. Sci. 1988 7 61-71

[0112] Wan, Y and Lemaux, P. G. Plant Physiology, 1994 104 37-48

[0113] Wolf, N. Plant Physiol., 1991 96 1382-1384

[0114] Woodward, J. R. and Fincher, G. B. Brewers Digest, 1983 58 28-32

[0115] Wynn, R. M, Davie, J. R., Cox, R. P. and Chuang, D. T. J. Bio. Chem. 1992 267 12400-12403

[0116] Xu, P., Wang, J. and Fincher, G. B. Gene (Amst.), 992 120 157-165

Claims

1. A plant (1→3,1→4)-&bgr;-glucanase enzyme of enhanced thermostability and/or pH stability, said enzyme being modified by transfer of a non-homologous protein sequence of different substrate specificity.

2. A plant (1→3,1→4)-&bgr;-glucanase in which the amino acid sequence of said enzyme:

(a) is modified to comprise structural elements of plant (1→3)-&bgr;-glucanase, said structural elements conferring improved heat stability;

(b) is modified at sites other than the active site to stabilise helices, to increase binding energy of the folded protein, to increase hydrogen bonding, and/or to prevent glycation; or

(c) is modified by creating cysteine pairs which can form disulphide bonds across the C and N terminals.

3. A (1→3,1→4)-&bgr;-glucanase according to claim 2, in which two or more of the modifications (a) to (c) are present.

4. A (1→3,1→4)-&bgr;-glucanase according to claim 1 comprising the structural framework of the enzyme (1→3)-&bgr;-glucanase and elements of the catalytic site of (1→3,1→4)-&bgr;-glucanase.

5. An enzyme according to claim 4 additionally comprising modification (b) and/or modification (c) of claim 2.

6. A (1→3,1→4)-&bgr;-glucanase according to claim 1, in which the amino acid sequence of (1→3,1→4)-&bgr;-glucanase isoenzyme EII is modified to comprise one or more substitutions selected from the group consisting of:

Ala 14 Ser

Ala 15 Arg

Thr 17 Asp

Lys 23 Arg

Lys 28 Arg

Asn 36 Asp

Gly 44 Arg

Gly 45 Asn

Gly 53 Asp

Gly 53 Glu

Lys 74 Arg

Gln 78 Arg

Ala 79 Pro

Lys 82 Arg

Ala 95 Asp

Gly 97 Pro

Phe 85 Tyr

Lys 107 Arg

Gly 111 Ala

Gly 119 Pro

Lys 122 Arg

Ser 128 Arg

Gly 133 Ala

Gly 145 Asn

Gly 152 Thr

Pro 153 Asp

Gln 156 Arg

Asn 162 Gly

Gly 185 Asn

Ala 191 Pro

Gly 193 Ala

Gly 199 Pro

Ala 200 Gly

Gly 202 Thr

Gly 219 Glu

Lys 220 Arg

His 221 Ala

Gly 223 Ala

Ser 224 Pro

Lys 227 Arg

Gly 238 Ala

Gly 239 Gln

Ala 242 Gly

Gly 260 Glu

Pro 267 Arg

Gly 268 Glu

Gly 286 Ala

Gly 286 Asp

Gln 289 Arg

Met 298 Lys

His 300 Pro

subject to the proviso that the following ion pairs must both be substituted:

9 Ala 15 Arg and Asn 36 Asp Thr 17 Asp and Met 298 Lys Ala 95 Asp and Ser 128 Arg Pro 153 Asp and Gln 156 Arg Lys 227 Arg and Gly 268 Arg Gly 152 Thr and His 221 Ala.

7. A (1→3,1→4)-&bgr;-glucanase according to claim 4 in which amino acids in the loops forming the sides and bottom of the active site cleft of (1→3)-&bgr;-glucanase GII are replaced by corresponding amino acids from (1→3,1→4)-&bgr;-glucanase EII, as follows:

10 residue 8 Ile → Ser, residue 34 Phe → Ala, residue 208 Ala → Thr, residue 209 Met → Thr, residue 189-191 Gln-Pro-Gly → Asn-Ala-Ser residue 128-137 Ile-Arg-Phe-Asp-Glu-Val-Ala-Asn-Ser-Phe → Val-Ser- Gln-Ala-Ile-Leu-Gly-Val-Phe-Ser (SEQ. ID NO: 1),

residue 171-179 Phe-Ala-Tyr-Arg-Asp-Asn-Pro-Gly-Ser→Leu-Ala-Trp-Ala-Tyr-Asn-Pro-Ser-Ala (SEQ. ID NO: 2) and

residue 283-291 Thr-Gly-Asp-Ala-Thr-Glu-Arg-Ser-Phe→Asp-Ser-Gly-Val-Glu-Gln-Asn-Trp (SEQ. ID NO: 3)

8. A (1→3,1→4)-&bgr;-glucanase according to claim 6 comprising one or more of the following substitutions:

Gly 53 Asp

Gly 53 Glu

Thr 17 Asp; Met 298 Lys

Ala 95 Asp; Ser 128 Arg

Lys 122 Arg

Lys 23 Arg

Lys 74 Arg

Gly 44 Arg

Gly 223 Ala

Ala 89 Pro

Phe 85 Tyr

9. A (1→3,1→4)-&bgr;-glucanase according to any one of claims 6 to 8, additionally comprising the mutation 189-191 Gln-Pro-Gly-→Asn-Ala-Ser.

10. A (1→3,1→4)-&bgr;-glucanase according to claim 6 comprising the substitution Lys 122→Arg and/or the substitution Phe 85→Tyr.

11. A DNA molecule whose sequence encodes a (1→3,1→4)-&bgr;-glucanase according to any one of claims 1 to 10.

12. A plasmid comprising a DNA sequence according to claim 11.

13. An expression vector comprising a DNA sequence according to claim 11.

14. A transgenic plant comprising a DNA sequence according to claim 11.

15. A transgenic plant according to claim 14, selected from the group consisting of barley, wheat, rice, and maize.

16. A transgenic plant according to claim 15, which is barley.

17. A process selected from the group consisting of malting, brewing and stockfeed processing, comprising the step of:

(a) using barley expressing the (1→3,1→4)-&bgr;-glucanase of any one of claims 1 to 10 as a starting material; or

(b) adding the (1→3,1→4)-&bgr;-glucanase of any one of claims 1 to 10 to a grain to be processed.

18. A composition for use in malting, brewing, or stockfeed processing, comprising the (1→3,1→4)-&bgr;-glucanase of any one of claims 1 to 10, together with a carrier acceptable for use in processing of beverages or of stockfeeds.

19. A beverage produced using a composition according to claim 18.

20. A stockfeed produced using a composition according to claim 18.

21. Grain produced by a transgenic plant according to claim 15.

22. Barley grain produced by a transgenic barley according to claim 16.