Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-05-09
2003-09-16
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S101000
Reexamination Certificate
active
06620604
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to sulfohydrolases, such as galactan sulfohydrolases, such as nu- and mu-carrageenan sulfohydrolases. The present invention is directed to the amino acid and nucleotide sequences of sulfohydrolases. The present invention is further directed to enzymatic modification of sulfated compounds, such as galactans. For example, the enzymatic modification may involve tailoring of the properties of sulfated galactans, such as gelling properties, such as by removal of sulfate groups and creation of a bridge between ring positions in a saccharide structure of the galactan. The present invention is further directed to processes of extracting nu-carrageenan from seaweed. The present invention is also directed to enzymatically modified compounds.
2. Discussion of Background
Hydrocolloids, which may be broadly defined as substances that yield a gel in the presence of water, are used in part for their rheological properties, and may also provide benefits in stability.
There are several classes of hydrocolloids. One categorization approach breaks these classes into exudates, such as gum arabic, ghatti, karaya, talha, and tragacanth; extracts, such as alginate from brown seaweeds, agar, carrageenan, and furcelleran from red seaweeds, and konjak (glucomannan), guar, pectin and arabinogalactan from land plants; biopolymers, such as xanthan; chemically modified hydrocolloids, such as the cellulosics, including carboxymethyl cellulose, hydroxypropyl cellulose, and carboxymethylhydroxymethyl cellulose; and intermediate forms, such as microcrystalline cellulose.
Red seaweeds are known sources of industrial gelling and thickening cell-wall sulfated galactans referred to as agar and carrageenans. They consist of a linear backbone of galactopyranose residues linked by alternating alpha(1→3) and beta(1→4) linkages. While all &bgr;-linked residues are in the D-configuration, the alpha(1→4)-linked galactose units are in the L-configuration in agars and in the D-configuration in carrageenans.
Agar is extracted from dried algae by more or less hot alkaline solutions (100-120° C.). After filtration, agar solutions are allowed to gel de-watered by pressing, dried, and ground, as disclosed in ARMISEN et al., “Production, Properties and Uses of Agar”,
Production and Utilization of Products from Commercial Seaweeds
, FAO Fisheries Technical Paper, 288, pp. 1-57 (1987), the disclosure of which is herein incorporated by reference in its entirety. Seaweed sources for agar extraction include the genera Gelidium, Pterocladia, Gelidiella, and Gracilaria, as disclosed in STANLEY, “Production, Properties and Uses of Carrageenan”,
Production and Utilization of Products from Commercial Seaweeds
, FAO Fisheries Technical Paper, 288, pp. 116-146 (1987), the disclosure of which is herein incorporated by reference in its entirety.
Carrageenan is itself a generic name for a family of natural water-soluble sulfated galactans isolated from red seaweeds. The thickening and gelation properties exhibited by carrageenans are useful in food and cosmetic formulations, as disclosed in THERKELSEN, “Carrageenan”,
Industrial Gums: Polysaccharides and their Derivatives
, 3rd ed., pp. 145-180, (1993), and DeRUITER et al., “Carrageenan Biotechnology”,
Trends in Food Science
&
Technology
, Vol. 8, pp. 389-395 (1997), both of which are herein incorporated by reference in their entireties.
Seaweed sources for carrageenan include the genera Eucheuma (such as
E. spinosum, E. cottonii
(=
Kappaphycus alvarezii
), and
E. denticulatum
), Chondrus (such as
C. crispus
), Calliblepharis (such as
C. jubata
), and Gigartina (such as
G. radula
and
G. stellata
).
Carrageenans are linear, partially sulfated galactans mainly composed of repeating dimers of an alpha(1-4)-linked D-galactopyranose or 3,6-anhydro-D-galactopyranose residue and a beta(1-3)-linked D-galactopyranose residue. As noted above, agars are likewise linear, partially sulfated galactans of similar structure, except that the alpha(1-4)-linked galactopyranose residue is in the L-form. Carrageenan occurs in several structures that differ primarily in the number and placement of sulfate groups on the dimer backbone, and in whether the individual residues of the dimer are present in the left hand (
4
C
1
) or right-hand (
1
C
4
) ‘chair’ configuration. These structures include kappa (&kgr;), iota (&igr;), lambda (&lgr;), theta (&thgr;), mu (&mgr;), and nu (&ngr;), as shown below. The iota-, kappa-, and theta-carrageenans contain 3,6-anhydro bridges, whereas the nu-, mu-, and lambda-carrageenans do not have this bridge. Furthermore, the conformation of the alpha-linked unit of nu-, mu-, and lambda-carrageenans is different from the anhydrobridge-containing carrageenans, preventing sufficient helix aggregation. Helix aggregation is important because helices facilitate formation of gels. In this regard, kappa-carrageenan forms firm, brittle gels, whereas iota-carrageenan forms elastic, soft gels, and whereas lambda-carrageenan is a non-gelling thickening agent.
The amount of SO
3
−
in carrageenans can be considerable and vary between 0 and 41% (w/w), resulting in highly negatively charged polymers. Ideal kappa-, iota-, and lambda-carrageenan dimers respectively have 1, 2, and 3 sulfate esters groups, resulting in typical sulfate contents of respectively 22%, 32%, and 38% (w/w). However, large variations in sulfate can occur in commercial extracts due to differences in seaweed species or batches. The sulfate ester linkages are chemically very stable, and there are no apparent or practical chemical methods to modify the sulfate level or distribution without also lowering the molecular weight of the polymer, except for the removal of 6-O-sulfate from precursor carrageenans as is done during alkaline treatment.
Carrageenan is typically extracted commercially from red seaweeds by boiling in aqueous solution, sometimes under alkaline conditions, followed by filtration, concentration, precipitation, and drying. Precipitation may either be by alcohol addition, or by gelling with salts followed by pressing of the gel, as discussed in STANLEY, “Production, Properties and Uses of Carrageenan”,
Production and Utilization of Products from Commercial Seaweeds
, FAO Fisheries Technical Paper, 288, pp. 116-146 (1987), the disclosure of which is herein incorporated by reference in its entirety. Semi-refined carrageenans are also produced by treating seaweeds with alkali followed by thorough rinsing with water. These treatments improve the gelling characteristics of the carrageenan preparation and remove most of the proteins, pigments and small metabolites; such preparations also contain other polymers such as cellulosic materials, as discussed in HOFFMANN et al., “Effect of Isolation Procedures on the Molecular Composition and Physical Properties of
Eucheuma Cottonii
Carrageenan”,
Food Hydrocolloids
, 9, pp. 281-289 (1995), the disclosure of which is herein incorporated by reference in its entirety.
The non-gelling mu- and nu-carrageenans are the natural precursors present in seaweed of, respectively, kappa- and iota-carrageenans, and have a sulfate ester group at the C-6 position of the alpha(1-4)-linked D-galactopyranose residue of the dimeric unit. It has been generally assumed that elimination of the sulfate from the C-6 sulfate ester of the precursors, and formation of the 3,6-anhydro bridge, occur concomitantly during the strong alkaline treatment.
The functional properties (including helix formation, rheological properties, and applications) of the different carrageenans are determined by (1) molecular weight of the polymer (2) the number of sulfate ester groups and their place of substitution of the carbon backbone, and (3) the number of 3,6-anhydro-galactose residues, as discussed in THERKELSEN, “Carrageenan”,
Industrial Gums: Polysaccharides and their Derivatives
, 3rd edition, pp. 145-180 (1993); VIEBKE et al., “Characterization of Kappa- and Iota-Carrageenan Coils and Helice
De Ruiter Gerhard
Genicot Sabine
Kloareg Bernard
Penninkhof Bea
Potin Phillipe
Centre National de la Recherche Scientifique "CNRS"
Patterson Jr. Charles L.
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