Directed homoenzymatic fragmentation of the plant protopectin complex: Assessment criteria

Introduction. The functional basis of protopectin complex may be represented as a network of regions that consist of homogalacturonan sequences and a base of rhamnogalacturonans-I, i.e. rhamnosyl-containing branching sites. Enzymatic isolation of these regions is possible only at a certain minimal native degree of polymerization. The research objective was to develop a system of criteria for assessing the potential applicability of the enzymatic transformation of plant protopectin complex. Study objects and methods. The research featured the polymerization degree of the homogalacturonan regions within the protopectin complex and produced a system of assessment criteria for the enzymatic fragmentation potential of the protopectin complex. The theoretical calculations were based on the values of the mass fractions of rhamnosyl and galacturonide residues in plant cell walls. The result was a new polymerization degree analytical function. Results and discussion. The ratio of the mass fractions of rhamnosyl and galacturonide residues in the water-insoluble plant tissue served as a dimensionless criterion of applicability. The rational condition for the dimensionless criterion of applicability was based on the fundamental constraint for homogalacturonan regions in the protopectin complex. It was expressed by a fundamental inequation. The rational area for determining the numerical values of the applicability criterion was presented as


INTRODUCTION
The biopolymer complex of plant tissue cell walls is a complex conglomerate of intertwined branched supramolecular networks of the protopectin complex and the hemicellulose. The complex is permeated with cellulose microfibrils and protein extensin ( Fig. 1) [1,2]. All its components are linked to each other by ester, salt, combined, and hydrogen bonds. Each component possesses valuable physicochemical properties with a good potential for food industry [3][4][5][6][7].
Pectins have the most attractive and numerous functional properties among all the carbohydrates of plant cell walls [5,8]. They owe these useful properties due to their molecular structure. In their native form, pectins have a water-insoluble supramolecular structure called the protopectin complex. The structure is an extended and highly branched linear and lateral network of polymer fragments (Fig. 2). Lateral branches also have a complex structure and can be interconnected with salt and borate bonds [9][10][11][12][13][14][15][16][17][18].
Homogalacturonans are linear polymeric fragments of α-D(+)-galacturonic acid residues, linked by (1 → 4)-glycosidic bonds (Fig. 3) [19,20]. Each residue contains a carboxyl group, which naturally may exist in a free, esterified, or amidated state. Free carboxyl groups are capable of dissociation, while acquiring a partial negative charge. Carboxyl groups esterified with methanol demonstrate inactivated charge formation. Amidated carboxyl groups, due to the donor-acceptor bond of the lone-pair electrons, accept cation H+ and acquire a partial positive charge.
In positions C1 and C2, hydroxyl groups can form glycosidic bonds with the residues of xylose, ribose, arabinose, and galactose, as well as ester bonds with carboxylic acids and aromatic compounds. The state and total amount of carboxyl groups in the pectin molecule fragment define the physicochemical properties of pectins, while the degree and the nature of the substitution of hydroxyl groups define the inhibition degree.
The practical use of pectins depends on the chemical structure of homogalacturonans.
Ramnogalacturonan-I is the second most common fragment of pectins. Its content can reach 45% in sugar beet pectin [5,19,20]. These fragments include sequences from the residue of α-L-rhamnose and α-D(+)-galacturonic acid, linked by a (1 → 4)-glycosidic bond. In the rhamnosyl residue, the pair can be linked with other pairs or with the end of the homogalacturonan by a rhamnosyl-uronic (1 → 2)-glycosidic bond. In the uronic residue, the pair can be linked with other pair by a rhamnosyl-uronic (1 → 2)-glycosidic bond or with the end of homogalacturonan by a uronic-uronic (1 → 4)-glycosidic bond. As a result, rhamnosyl residues of rhamnogalacturonan-I are the branching zones of the pectin molecule, where free functional groups can form glycosidic bonds with either residues of neutral sugars, or their polymer sequences, i.e. arabinans, galactans, arabinogalactans, and galactoarabinans-I and II (Fig. 4).
The basis of the protopectin complex of plant tissue cell walls is a network of regions formed by linear sequences of homogalacturonans and rhamnogalacturonans-I. Of course, this assumption excludes two types of lateral branches: the rhamnosilfree lateral branches (rhamnogalacturonan-II), which may contain residues of L-rhamnose and/or α-D(+)-galacturonic acid with proportion of ≤ 2-3%, and branches formed by neutral sugars and their oligo-and polymers [16,19]. Molecular properties of homogalacturonan fragments define the physicochemical properties of plant pectin. Therefore, enzymatic fragmentation is the most effective method for the protopectin complex. It is a selective hydrolytic cleavage of rhamnosyl-uronide (1 → 2) and (1 → 4) glycosidic bonds. However, the physicochemical properties of pectin also depend on the polymerization degree of the fragmentation products [21]. The maximal possible degree of polymerization depends on the polymerization degree of the native homogalacturonan fragments in the protopectin complex. In each specific case, the experimental determination of this indicator is a difficult resource-and time-consuming task.
Therefore, a criteria assessment would be the optimal approach to evaluate the potential efficiency of the directed enzymatic fragmentation of a particular plant protopectin complex. Such assessment can also define the boundary conditions that determine the degree of the targeted physicochemical properties of the fermentolysis products. This approach could also determine the conditions for processing any plant tissue or its derivatives. The approach consists of some stepby-step stages. The first stage was a system of criteria for assessing the transformation potential of a plant biopolymer complex [22].
As a next stage, the present research objective was to develop a system of criteria for assessing the enzymatic transformation potential of a plant biopolymer complex as in the case of pectin substances. The research included the following tasks: -developing the abovementioned assessment criteria system, based on the use of zoned criteria space; -developing a system of boundary conditions for the classification of plant raw materials according to the applicability of the enzymatic transformation of its protopectin complex.

STUDY OBJECTS AND METHODS
The protopectin complex of the plant tissue consists of three main types of fragments: homogalacturonan, rhamnogalacturonan-I, and rhamnogalacturonan-II. The latter type was disregarded as its mass fraction in the protopectin complex is ≤ 2%.
Rhamnogaracturonan-I has linear polynalacturonan sites. As a result, the homogalanic component of the protopectin complex can be considered as part of rhamnogalacturonan-I fragments.
A pectin molecule can be classified as rhamnogalacturonan-I only if, in addition to the homogalacturonan component, it contains at least one branch formed by at least one rhamnosyl residue. Consequently, a polymer molecule has at least two homogalacturonan regions with at least one terminal link (rhamnosyl residue) each.
Linear and homogalacturonan regions of the molecular network alternate in the protopectin complex in a particular order. This order presumably depends on the taxonomy of the raw material and the function   [20] of the plant parts. The structural features of the fragments of rhamnogalacturonan-I are such that the natural boundaries of the homogalacturonan regions are L-rhamnose residues connected to the terminal uronid links (1 → 2) and (1 → 4) by glycosidic bonds. The fragment can be roughly described by the following sequence: "terminal link of homogalacturonanrhamnose residue (the branching starts) -branching site -rhamnose residue (the branching ends)homogalacturonan region -… -section of homogalacturonan -rhamnose residue (the branching starts) -branching site -rhamnose residue (the branching ends) -terminal link of homogalacturonan".
In the simplest case, the rhamnogalacturonan-I fragment has only one branching site ( 1 r b = ). Depending on its structure, the rhamnogalacturonan-I can include only one rhamnosyl residue ( 1 Rh z = ). In a more complex case, the rhamnogalacturonan-I may contain several rhamnosyl residues ( Rh z q = , where 1, 2, 3, ... q = ), which alternate with galacturonid residues (Fig. 5).
The number of branching sites may also depend, to some extent, on the plant species and the functional type of the plant tissue. Figure 5 features no fragments of rhamnogalacturonan-I as their lateral branches are represented mainly by the nonuronic component.
The conditional assumption is that the uronidecontaining part of rhamnogalacturonan-I is completely determined by the following variables: HG n is total homogalacturonan sites, Rh n is total rhamnosyl units in the branching sites, is total uronid residues in the branching sites, Rh z is number of rhamnosyl residues per branching site, is number of uronid residues per branching site, and br n is total branch sites. Table 1 demonstrates the numerical values of the variables in particular cases of the distribution of homogalacturonan and branching sites in Fig. 5.
The ratios in Table 1 can be expressed by the following formulae: The structure of the rhamnogalacturonan-I fragments suggests that the main structural unit is the amount of rhamnosyl residues in the branching sites. As a result, formulae (1) and (2) take the following form: Rh Rh HG Rh Rh n n z n z z Based on the data in Table I, Thus, the final formula (4) is: These dependences give an approximate quantitative idea of the structure of rhamnogalacturonan-I. For their practical use, they have to be linked to the real chemical composition of a particular raw material.
The line of reasoning follows the next path.
Considering that the molecular weight of the rhamnosyl residue is Rh M (Da) and the mass fraction of rhamnose in the composition of the natively insoluble part of the raw material is Rh ω (%), the amount of rhamnosyl residues in the mass of the natively insoluble part of the raw material m (g) can be calculated according to the formula below: where a is the atomic mass unit (1.66053892×10 -24 g/Da). A combination of formulae (6) and (7) gives the number of moles of α-D(+)-galacturonic acid residues in the branch sites: Consequently, the mass fraction of α-D(+)galacturonic acid residues in the insoluble part of the raw material in the branching sites is: where GalA M is the molar mass of α-D(+)-galacturonic acid residue, Da.
The conditional assumption is that all the residues of α-D(+)-galacturonic acid in the insoluble part belong exclusively to the protopectin complex and are present only in the composition of homogalacturonan fragments and branch points of rhamnogalacturonan-I. Then, the mass fraction of α-D(+)-galacturonic acid residues in homogalacturonan fragments can be calculated as follows: As the plant tissue grows, the protopectin complex of cell walls and intercellular spaces changes continuously. As a result, the structure of the complex becomes heterogeneous. Assuming that all homogalacturonan regions of the protopectin complex are a native component of rhamnogalacturonan fragments, the whole protopectin complex can be represented as consisting almost entirely of rhamnogalacturonan-I fragments. The length of the homogalacturonan regions differs in different parts of the protopectin complex. Consequently, a particular homogalacturonan molecular mass is in fact a certain mean value. The molecular weight of any arbitrarily taken (i-th) homogalacturonan region of the protopectin complex is related to its polymerization degree by the following ratio: where i k is the polymerization degree of the i-th homogalacturonan region.
Consequently, the formula for the average molecular weight of homogalacturonan sites is as follows: A combination of formulae (3) and (13) gives the following result: As a result, the rational condition for criterion ν is: