(1→3, 1→4)—&bgr;-glucanase of enhanced...

Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase

Reexamination Certificate

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C435S069100, C530S372000

Reexamination Certificate

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06277615

ABSTRACT:

BACKGROUND OF THE INVENTION
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 or 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.
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.
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 immunoadsorbent 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-Cona 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.
The ability of the (1→3;1→4)-&bgr;-glucanases [E. C. 3.2.1.73] to retain enzymatic 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 enzyme exogenously as an additive to be used in the brewing process.
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.
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.
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.
We have now determined the three dimensional structure of (1→3,1→4)-&bgr;-glucanase isoenzyme EII and (1→3,1→4)-&bgr;-glucanase isoenzyme 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
According to a first aspect, the invention provides a (1→3,1→4)-&bgr;-glucanase of enhanced thermostability and/or pH stability.
In a second aspect, the invention provides an isolated DNA sequence encoding (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 cerevisae;
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.
In a third aspect, the invention provides a method se

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