Food or edible material: processes – compositions – and products – Products per se – or processes of preparing or treating... – Basic ingredient is starch based batter – dough product – etc.
Reexamination Certificate
1998-11-10
2003-05-06
Prouty, Rebecca E. (Department: 1652)
Food or edible material: processes, compositions, and products
Products per se, or processes of preparing or treating...
Basic ingredient is starch based batter, dough product, etc.
C426S635000, C435S183000, C435S200000, C435S203000, C435S208000, C435S209000, C435S252300, C435S254300, C435S320100
Reexamination Certificate
active
06558728
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the provision of &agr;-glucuronidase in pure, isolated form or as a recombinantly produced enzyme and the use;hereof as a xylan side-group hydrolysing enzyme, in particular in food manufacturing, as a feed additive or in processing of cellulosic pulps.
TECHNICAL BACKGROUND AND PRIOR ART
Biodegradation of hemicellulose requires accessory enzyme activities that remove non-xylose substituents, in particular glucuronic acid substituents, from the xylan backbone in addition to endoxylanases and &bgr;-xylosidases. The content of glucuronic acid substituents present in xylans varies with the sources of xylan such as hardwood xylans, softwood xylans and cereal xylans.
The configuration of hardwood xylan is presented in FIG.
1
. The backbone of hardwood xylan consists of (1,4)-&bgr;-D-linked xylopyranose residues. About every tenth xylose unit carries a single, terminal side chain consisting of 4-O-methylglucuronic acid attached directly to the 2-position of xylose. Seven out of 10 xylose residues contain an O-acetyl group at C-2, at C-3, or at both positions (Timell, 1967). Additionally, hardwood xylans contain minor amounts of rhamnose and galacturonic acid residues (Puls and Schuseil, 1993). O-acetyl-4-O-methyl-glucurono-xylan comprise between 10-35% of hardwoods (Timell, 1967).
Softwood xylans consist of a backbone of &bgr;-1,4-linked D-xylopyranose residues. There is one 4-O-methylglucuronosyl residue attached to C-2 per 5-6 xylose units. Instead of acetyl-substituents as found in hardwoods, softwood xylans contain alfa-L-arabinofuranose residues directly linked to C-3 of the xylose. One, arabi-nose occurs per eight or nine xylose units, see
FIG. 2
(Timell, 1967). Arabino-4-O-methyl-glucuronoxylan amounts to 10-15% of the softwood hemicelluloses (Puls and Schuseil, 1993; Timell, 1967).
Cereal xylans, which are commonly referred to as arabinoxylans consist of a linear backbone of (1,4)-&bgr;-D-linked xylopyranose units to which alfa-L-arabinofuranosyl substituents are attached at O-2 or O-3 or at both of these positions,
FIG. 3
(Voragen et al., 1992). There are two main types of arabinoxylans in annual plants. In the endosperms there is a highly branched form which basically is devoid of glucuronic acid. In the more lignified tissues, much less branched forms substituted with additional glucuronic acid residues and/or 4-O-methylether thereof as well as galactose units are found. Glucuronic acid residues are linked to O-2 on the xylan backbone (Viikari et al., 1993)
The amount of glucuronic acid in arabinoxylan varies considerably between cereals of different origins and between fractions of individual cereals. Glucuronoarabinoxylans are found in rice and sorghum and in the bran fraction of wheat, rye and barley and in a number of lignocellulosic by-products like e.g. sunflower meal. There are alsoacetyl groups present in cereal arabinoxylans, particularly in sorghum. They are ester-linked to O-2, O-3 or O-2,3 of xylose. In addition, phenolic acids like ferulic and coumaric acid have been found ester bound to the arabinofuranosyl residues of arabinoxylans (Voragen et al., 1992).
There is currently a considerable industrial interest in the enzymatic hydrolysis of xylans either with the aims of having complete hydrolysis to obtain xylose or with the objective of obtaining enzymatic pulping and bleaching in paper manufacturing.
&agr;-Glucuronidases are produced naturally by several micro-organisms, and up till now isolation of the enzyme from 7 different sources has been described. These source organisms are:
Trichoderma reesei
Rut C-30 (Siika-aho et al., 1994),
Trichoderma viride
(Ishihara et al., 1990),
Aspergillus niger
(Uchida et al., 1992b),
Thermoascus aurantiacus
(Khandke et al., 1989),
Agaricus bisporus
(Korte, 1990), Thermoanaerobacterium sp. strain JW/SL-YS485 (Shao et al., 1995) and
Helix pomatia
(snail) (Kawabata et al., 1995).
The &agr;-glucuronidases isolated from
A.niger
and
Helix pomatia
are intracellular enzymes whereas the others are extracellular enzymes. A molecular weight above 90 kDa are common for these isolated &agr;-glucuronidases. They all have an acidic isoelectric point and pH optimum. The fungal &agr;-glucuronidases, except the Agaricus enzyme are single polypeptide chains, while the bacterial &agr;-glucuronidase, the Agaricus and the snail enzyme are dimers. Isoenzymes have only been found in
Aspergillus niger
(Uchida et al., 1992b).
In general, the specific activity of &agr;-glucuronidase decreases with increasing chain length of the substrate. Only the
Thermoascus aurantiacus
enzyme has any activity on polymeric xylan. The &agr;-glucuronidase from
Helix pomatia
is the only isolated enzyme which had activity towards p-nitro-phenyl-&agr;-D-glucuronide.
Several reports have shown a synergistic effect between xylanase, &bgr;-xylosidase and &agr;-glucuronidase in the breakdown of glucuronoarabinoxylans (Siika-aho et al., 1994). &agr;-Glucuronidase therefore has potential application in the total hydrolysis of hemicellulose to produce xylose (Uchida et al., 1992a).
In WO 93/11296 is disclosed a method for the enzymatic treatment of cellulosic pulps where an &agr;-glucuronidase preparation is used to remove metal ions (bound through the carboxylic groups) from the pulp. This improves the bleaching properties of the pulp. Through removal of the glucuronic acid groups the pulp is also-rendered more susceptible to xylanase treatment.
WO 94/21785 discloses xylanases which are useful in food production and which may enhance the utilization of animal feed. However, there is no specific teaching of the use of an &agr;-glucuronidase for such purposes.
Thus, the prior art is silent with respect to improvements which can be obtained specifically by use of &agr;-glucuronidases in the manufacturing of food products including bakery products, and as an additive to enhance the utilization and digestibility of animal feeds. However, as it is described herein, a highly advantageous effect of &agr;-glucuronidase was demonstrated in animal feed ingredients with respect to improving the nutritional value of animal feedstuffs e.g containing annual plants which will result in an enhanced utilization of feed and accordingly of conversion rate for such feedstuffs. Furthermore, nutritionally and quality improving effects of &agr;-glucuronidase alone or in combination with other enzyme activities in dietary fiber-containing food products, such as doughs and finished bakery products including in particular bakery products based on wholemeal, were demonstrated.
Whereas enzyme preparations having &agr;-glucuronidase activity presently can be obtained from microorganisms, in particular fungal species, which produce the enzyme naturally, such preparations are crude in the sense that they contain several other enzyme activities such as cellulase or protease activity. Such impure enzyme preparations are industrially less feasible, since the associated non-&agr;-glucuronidase enzyme activities may be undesirable in several application areas and since it is difficult or impossible to provide enzyme products having a well-defined and standardized &agr;-glucuronidase activity.
Evidently, enzyme preparations only having, or substantially only having &agr;-glucuronidase activity may be provided by extensive purification from media in which &agr;-glucuronidase producing organisms have been cultivated (when the enzyme is secreted out of the cell) or by isolating the enzyme from cells producing the enzyme naturally (when the enzyme is accumulated intracellularly). For specific application areas such as in the food industry such purified, naturally produced &agr;-glucuronidase preparations may be useful.
However, the provision of &agr;-glucuronidase in this manner in industrially needed amounts and at a feasible cost level is not possible due to the low amount of the enzyme being produced naturally in the above source organisms.
It is therefore an important aspect of the present invention to provide the means for producing
de Vries Ronald Peter
Poulsen Charlotte Horsmans
Visser Jacob
Zargahi Masoud R.
Danisco A/S
Hunton & Williams
Prouty Rebecca E.
Rao Manjunath
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