Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1995-08-16
2003-05-13
Zeman, Mary K. (Department: 1631)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C424S094100, C424S094200, C426S615000
Reexamination Certificate
active
06562340
ABSTRACT:
The present invention relates to an enzyme feed additive and in particular to such an additive which can decrease the feed conversion ratio of a cereal-based feed and/or increase its digestibility.
Improvements in animal feeds to enable animals to digest them more efficiently are constantly being sought. One of the main concerns is to improve the feed conversion ratio (FCR) of a feed without increasing its cost per unit weight. The FCR is the ratio of the amount of feed consumed relative to the weight gain of an animal. A low FCR indicates that a given amount of feed results in a growing animal gaining proportionately more weight. This means that the animal is able to utilise the feed more efficiently. One way in which the FCR of a feed can be improved is to increase its digestibility.
There are various constraints on the digestibility of the nutritional components of a feed such as its starch, fat, protein and amino acid content. These constraints include:
(i) the viscosity of materials present in the animal's gut. Such viscosity is due, at least in part, to soluble non-starch polysaccharides such as mixed-linked &bgr;-glucans and arabinoxylans;
(ii) entrapment of nutrients within the cell walls of the feed, particularly those of the aleurone layer in cereals. Such entrapment is caused by the high levels of non-starch polysaccharides in the cell walls of cereals which are relatively resistant to break-down by the animal's digestive system. This prevents the nutrients entrapped within the cells from being nutritionally available to the animal; and
(iii) a deficiency in endogenous enzyme activity, both of the animal and of the gut microbial population particularly in a young animal.
The above problems which interfere with digestibility are particularly noticeable in the case of cereal-based diets, and in particular those having a high barley content.
Due to the problem of poor digestibility of nutrients from the feed, it is normally necessary to formulate feeds to contain higher levels of energy providing materials in order to meet the nutritional demands of animals. Such energy providing materials conventionally include starch, fat, sugars, fibre etc. The requirement of including these energy providing materials, or sources of such materials, in a feed adds a considerable extra cost which is disadvantageous from an economic view point.
In an attempt to solve the problem of poor digestibility of cereal-based feeds, it is known to include enzyme supplements such as &bgr;-glucanases or xylanases in animal feeds. For example, WO 91/04673 discloses a feed additive for alleviating malabsorption syndrome in poultry which causes reduced digestion. The additive includes a cellulase and a xylanase. JP-A-60-75238 discloses a feed for domestic animals which contains an enzyme cocktail including protease-, cellulase-, amylase- and lipase-activities. This reference speculates that these various enzyme activities enable fermentation microbes to grow and that these become useful nutritional components of the feed.
Whole cellulase is a mixture of different enzymes which cooperate to hydrolyze cellulose (&bgr;-1,4-D-glucan linkages) and/or derivatives thereof (e.g. phosphoric acid swollen cellulose) and give as primary products compounds such as glucose, cellobiose, and cellooligo-saccharides. Whole cellulase is made up of several different enzyme classifications including enzymes having exo-cellobiohydrolase activity, endoglucanase activity and &bgr;-glucosidase activity.
For example, the whole cellulase produced by the fungus
Trichoderma longibrachiatum
comprises two exo-cellobiohydrolases, CBHI and CBHII, at least three endoglucanases, EGI, EGII and EGIII, and at least one &bgr;-glucosidase. A representative fermentation from
T. longibrachiatum
may produce a whole cellulase including by protein weight 45-55% CBHI, 13-15% CBHII, 11-13% EGI, 8-10% EGII, 1-4% EGIII and 0.5-1% BG. However, it should be noted that actual concentrations of a specific cellulase component will vary according to numerous factors, including fermentation conditions, substrate concentrations and strain type. Thus, in a representative fermentation,
Trichoderma longibrachiatum
produces a whole cellulase having from 58-70% of cellobiohydrolases.
Each endoglucanase of
T. longibrachiatum
has its own distinct characteristics. Thus, EGI in addition to cellulase activity is known to hydrolyze xylan. EGII and EGIII by comparison do not show significant xylanase activity, at least according to azo-xylan native PAGE overlay. Further, it is known that EGI, EGII and EGV contain structurally distinct cellulose binding domains (CBD's). On the other hand, EGIII does not appear to contain a structurally distinct binding domain and has been shown to have a lower affinity for crystalline cellulose compared to EGI or EGII.
WO 92/06209 discloses processes for transforming the filamentous fungus
Trichoderma reesei
(now called “
T. longibrachiatum
”) which involves the steps of treating a
T. reesei
strain with substantially homologous linear recombinant DNA to permit homologous transformation and then selecting the resulting
T. reesei
transformants. For instance, transformants are described in which certain targeted genes are deleted or disrupted within the genome and extra copies of certain native genes such as those encoding EGI and EGII are homologously recombined into the strain. It is noted in this reference that cellulase compositions obtained from strains deficient in CBHI and CEHII components are useful as components of a detergent cleaning composition. Such cellulase compositions are of course relatively enriched
When used in vivo, endoglucanases and cellobiohydrolases are considered to act synergistically in the hydrolysis of cellulose to small cello-oligosaccharides (mainly cellobiose), which are subsequently hydrolysed to glucose by the action of &bgr;-glucosidase. In addition to hydrolyzing the &bgr;-1,4 linkages in cellulose, endo-1,4-&bgr;-glucanase (EC 3.2.1.4) will also hydrolyze 1,4 linkages in &bgr;-glucans also containing 1,3-linkages. The endoglucanases act on internal linkages to produce cellobiose, glucose and cello-oligosaccharides. The cellobiohydrolases act on the chain ends of cellulose polymers to produce cellobiose as the principal product.
Whole cellulase obtained from
T. longibrachiatum
has been used in combination with barley in fields such as brewing and in animal nutrition for several years. One of the benefits of adding cellulases to barley-based diets for livestock is to increase the digestibility of various components present in the diet including protein and amino acids. As a result, dietary input costs can be reduced without loss of performance, and excretion of nitrogen in the manure can be significantly reduced. This reduces the environmental impact of intensive livestock farming.
Endosperm cell walls of barley contain a high proportion of high molecular weight, water-soluble mixed-linked &bgr;-(1,3)(1,4)-glucans. When solubilised, these poly-saccharides cause an increase in the solution's viscosity. For example, if barley is fed to broiler chickens, this leads to a relatively high level of viscosity in the region of their gastrointestinal tract, which results in reduced efficiency of digestion and growth depression.
Organisms which produce or express cellulose enzyme complexes often also express xylanase activity. For example, two different xylanase enzymes produced by
T. longibrachiatum
have been identified. The purification of these two different xylanases, one referred to as high pI xylanase (having a pI of about 9.0) and the other referred to as low pI xylanase (having a pI of about 5.2), as well as the cloning and sequencing of the gene for each xylanase is described in detail in WO 92/06209 and WO 93/24621. FIG. 16 of this document sets out the deduced amino acid sequences for both the low pI and high pI gene products. Example 22 also teaches how to create
T. longibrachiatum
strains which over-express the low pI and high pI xylanase genes.
As mentioned ab
Bedford Michael R.
Clarkson Kathleen A.
Collier Katherine D.
Fowler Timothy
Larenas Edmund A
Clow Lori A.
Genencor International Inc.
Genencor International, Inc
Zeman Mary K.
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