Lignin is the second most abundant biopolymer on earth, after cellulose. The use of this raw material as a template for phenolic polymer synthesis has potential applications in the production of polymeric dispersants, soil conditioning agents, phenolic resins or adhesives, and laminates, among others. Several methods have been developed for the production of lignin containing phenolic resins (Tock et al, 1987; Matt and Doucet, 1988), many of which employ formaldehyde to hydroxymethylate lignin and /or phenol in order to crosslink the polymer. However, these lignin phenol resins are as toxic as formaldehyde itself. The enzymatic polymerisation of phenols has proven to be an attractive alternative for the synthesis of phenolic resins. In the presence of H2O2, peroxidases catalyse the oxidation of phenols that eventually give rise to higher molecular weight polymers (Xu et al, 1995; Nicel and Wright, 1997). The copolymerization of phenols with Kraft lignin has been performed in aqueous-organic solvent mixtures catalysed by horseradish peroxidase (Blinkovsky and Dorkick, 1993; Popp et al, 1993). The enzyme horseradish peroxidase, when encapsulated in reversed micelles, was able to catalyse the polymerisation of phenolic compounds and aromatic amines (Rao et al, 1993).

This article deals with the enzymatic copolymerization of lignin with cresol in revered micelles. The objective here is not so much to examine the potential application of the polymer, but rather to assess the feasibility of the reaction in terms of kinetics and monomer conversions and to measure the gross characteristics of the polymer, especially its molecular weight and distribution.

Materials. Horseradish peroxidase (EC 1.11.1.7) type II (HRP) was purchased from Shanghai Lizu Orient Technology Corp. Ltd., with activity of 250 units/mg, assuming an enzyme molecular weight of 40,000. Other chemicals used in this work were of the highest purity commercially available. Polystyrene molecular weight standards were obtained from Polysciences, Inc. (Warington, PA). The lignin used in this work was the straw-pulp-lignin obtained from black liquor or alkali liquor.

Analytical Methods. For soluble dimethylformamide fractions, the molecular weights of the resulting polymer and lignin were determined by gel permeation chromatography (GPC, C-R7A, Warters, Milford, MA). The concentration of unreacted phenols was measured by HPLC. Fourier Transfer Infra-Red spectra were performed using a Nicolet Magna-IR 750 instrument with the sample as KBr pellet. Thermal analyses were conducted using a differential scanning calorimeter (DSC) SR-1 (Beijing Analysis instrument Factory).

Copolymerization Procedure. Enzymatic polymerisation was carried out in reversed micelles and a typical reaction was carried out as follows. First, phenol and the solution of enzyme-in-buffer were added to a solution of lignin-reversed micelles, and the solution so obtained was incubated in a constant temperature bath for 10-20 min at a defined temperature, e.g., 25°C. Then, certain amount of hydrogen peroxide, e.g., 200m m, was microinjected to initiate the copolymerization of lignin with phenol. The range of enzyme concentration was from 1.0025x10^-7 mol/L to 8.7525x10^-7 mol/L; the range of water to surfactant molar ratio (W/O) was from 29.06 to 90.94; the range of phenol concentration was from 62.04 to 141.05 mg/mL; the range of lignin concentration was from 46.4 to 135.6 mg/mL; the range of surfactant concentration (CTAB) was from 0.863 to 1.637 mol/L; the ratio of n-butanol to isooctane was from 2.0 to 4.0.

The oxidation of phenols by peroxidase in the presence of H₂O₂ produces phenoxy radicals that are subjected to a number of different non-enzymatic reactions. In the absence of other phenols or other hydrogen donors, phenoxy radicals can undergo radical transfer or termination (coupling) reactions to give phenolic homopolymers. In the presence of other phenols, however, radical transfer and coupling reactions can occur between different phenolic groups. One can expect three possible reactions to be catalysed by peroxidase in the presence of a monomeric phenol (Blinkovsky and Dorkick, 1993): (1) phenol-phenol interactions; (2) lignin-phenol interactions; (3) lignin-lignin interactions. The first case is undesirable in view of modifying lignin properties. The second is the most desirable. The third is less likely to occur, as peroxidase prefers low molecular weight substrates (Saunders et al, 1964), although in the absence of phenols, the enzyme will catalyse lignin oxidation (Kaplan, 1979). The goal of our research was to gain an insight in the way of controlling molecular weight and distribution of copolymer resulting from the enzymatic reaction with reversed micelles, in other words, to optimise the enzymatic copolymerization of lignin with monomeric phenols.

The data of copolymerization rates are given in Table 1. Before the data was collected, some experiments had to be repeated to determine the range of experimental error (± 11%). The concentration of p-cresol is not an absolute value; however, it can be used to estimate conversion.

Where R1 is the size of internal standard, microlitre; R2 is the height of p-cresol which is a height of a peak in HPLC, centimetre; R3 is the height of phenyl standard which is also a height of a peak in HPLC, centimetre; R4 is the size of sample, millilitre; R5 is the relative concentration of unreacted p-cresol; R6 is the conversion of p-cresol, %; time is sampling time, i.e., the reaction time, second; RT represents reaction temperature, and CE, concentration of enzyme HRP, mol/L.

Conversion of p-cresol was 94.5% at 60 second in run No. 35 with an enzyme concentration of 1.33x10^-7 mol/L. Comparison of run 35 with run 1, one can find that effect of temperature on reaction rate is significant, at 60 seconds run 35 has a conversion of 94.5% with a higher temperature, 35°C, it is much higher than 88.2% of run 1 at 150 second with a lower temperature, 20°C, although the enzyme of Run 35 is much lower than that of Run 1.

Copolymerization did not occur when the enzyme concentration was reduced to 2.5x10^-8 mol/L; a possible explanation is inhibition by hydrogen peroxide. Very high reaction rates as obtained has also been claimed by Rao et al (1993) in the enzymatic polymerisation of phenols in reversed micellar system of dioctyl sodium sulfosuccinate (AOT)-water-isooctane, a significant difference being observed between polymerisation in reversed micelles and in other monophasic organic systems. The fact that enzyme molecule occupancy per micelle is seldom greater than unity indicates that the catalyst dispersion is extremely high, leading to efficient utilisation. The reversed micellar system may sustain the growing chain in solution very well, and it has a significant effect on keeping the growing chain in solution and in promoting continued polymerisation. In addition to provide a high degree of dispersion to the enzyme, the surfactant does appear to interact with the monomer and polymeric species, promoting the reaction. Perhaps this is a consequence of the surfactant (CTAB) induced positioning of the substrate (cresol) at the interface and the easier accessibility to the enzyme active site.

Figure 1 shows the FTIR spectra of the free (or native) lignin and lignin-p-cresol resin. As shown, strong differences were observed in the 470 to 3300 cm^-1 range. For example, there is a strong absorbance peak at 2850 cm^-1 in (B), but the same one does not appear in (A). The peak at 2850 cm^-1 is benzene CH3 asymmetric stretch; the peak at 2925 cm^-1 in (B) becomes bigger than that in (A), this peak being a benzene ring CH3 symmetric stretch. Finally, in (B), sample exhibits significant differences in peaks at 1593, 1506, 1463, 1218, 1126, 1101,1035 and 470 cm^-1. The peak at 3400 cm^-1 shifts to lower wave number; it might be a distribution of the hydrogen bonds between polycresol. Hence, significant differences between the lignin-cresol copolymer and free lignin are most likely due to the distributions of incorporation of cresol into lignin or/and polycresol segments in the copolymer. Blinkovsky et al (1993) observed that the FTIR spectrum of lignin-cresol is strongly different from that of polycresol obtained in aqueous-organic solvent mixtures, but not in reversed micelles.

Figure 2 shows differential calorimetric scans for lignin-cresol resin and free lignin, respectively. An exotherm is also observed for lignin-cresol copolymer in (A) of figure 2 at about 275°C, which indicates that some crosslinking and /or branching is taking place at a higher temperature. This heat flow is irreversible as indicated by the absence of heat flow in a second heating (data not shown). However, no exotherms appear up to about 400 °C for straw-pulp-lignin in (b), and this is different from Kraft lignin. In (a) an endotherm at 96°C is shown that may indicate a glass transition. Generally, straw-pulp-lignins from different sources have different glass transition temperatures, in the range from 127 to 193°C; however, in our sample of straw-pulp-lignin no glass transition temperature was observed and the reason for this is unclear. Thus, copolymerization of lignin with cresol lowers the endothermic glass transition temperature. This is consistent with published reports performed in non-aqueous media (Blinkovsky and Dordick, 1993). Therefore, the incorporation of phenol into lignin does not appear to destroy the native structure of the lignin; albeit bound phenols can radically alter its thermal properties.