The production of lignopolyols by enzymatic glycerolysis is still little explored in the literature due to the implications related to the presence of lignin in the reaction medium, such as inhibition of enzymatic activity due to changes in pH and viscosity of the medium, among other shortcomings (Cajnko et al. 2021; Araujo et al. 2023b).
Before the first cycle the enzyme presented a value of 174.0 U/g, after the first cycle the enzyme presented an average loss of 43.22% of activity. This loss of activity is common after cycles of use, as the biocatalyst is often subjected to atypical environments for its best action, such as changes in pH during the reaction process, time, temperature, substrate concentration and products. The products formed during the process can inhibit and inactivate the biocatalyst, among other physical and chemical factors of the process to which it is subjected (Balen et al. 2015; Mulalee et al. 2015; Meneses et al. 2019; Ortiz et al. 2019; Girão Neto et al. 2023). The recycling of the immobilized enzyme was realized in the exact first reaction condition. After the reuse this loss of activity was even greater for the reactions that had lignin incorporated in the reaction medium, about 70, 94.05, 93.08% for the 5, 10 and 15 wt% lignin content, respectively. This did not occur for the enzymatic glycerolysis reaction in the absence of lignin, which showed a total loss of 48%. The effect of lignin proportion on the biocatalyst (N435) can be seen in Fig. 1 and detailed in Table 1.
The enzymatic activity of N435 was also investigated in a fed process for propionic acid production through the esterification reaction of benzyl propionate. After the first cycle was analyzed, values above 90% substrate conversion into product were found in the different acid: alcohol ratios tested in the study. In the second cycle, the biocatalyst's activity and the substrate's conversion into a product were drastically reduced due to the reaction medium's alteration and the protein content leaching in the support (Meneses et al. 2019). Mulalee et al. (2015) evaluated the potential of the Novozym 435 enzyme in producing biodiesel through the esterification process of oleic acid, ethanol, oleic acid, and methanol. Both tests led to conversions of over 90%. However, the biocatalyst suffered changes in morphology when ethanol was used as the absolute alcohol in the reaction due to the presence of water (5%) and molecular structure, obtaining better results when methanol was used with absolute alcohol, obtaining reuse of 13 cycles and 10 cycles when renewable resources were used to obtain biodiesel.
Sousa et al. (2022) compared commercial lipases, including N435, with non-commercial lipases in synthesizing octyl esters at different concentrations of biocatalyst, reaction time, reuse, and economic evaluation for this process. They conducted this study to economically evaluate the feasibility of using these biocatalysts in synthesis. They concluded that despite the high cost of commercial enzymes, their productivity is high, above 90%, when low loads are used to carry out the synthesis, making their use economically feasible. However, alternative biocatalysts have also shown promising results for synthesizing octyl esters, such as dry fermented solid (DFS), but at very high concentrations compared to commercial lipases.
Table 1
N435 activity before and after transesterification cycles in the absence and presence of lignin in different percentages.
| Condition | Novozym esterification activity (U/g) |
| - | 0 wt.% | 5 wt.% | 10 wt.% | 15 wt.% |
| Before reaction | 174.0 ± 12.7 | - | - | - | - |
| After the 1st transesterification cycle | - | 100.9 ± 4.6 | 102.9 ± 12.2 | 112.9 ± 7.1 | 81.3 ± 13.6 |
| After the 2nd transesterification cycle | - | 92.0 ± 9.0 | 52.9 ± 13.4 | 10.3 ± 7.5 | 12.1 ± 4.7 |
This behavior can also be seen in the qualitative analysis of the protein content in the support by optical microscopy (Fig. 2) and FTIR of the lignopolyols (Fig. 3).
Before the first transesterification cycle, the filling of the support by lipase, highlighted in a dark brown color (Fig. 2A), was observed. After use and reuse, the protein content was leached from the support, and at higher lignin concentrations, this process could be observed in the first cycle. The structural complexity of lignin may have affected the desorption of the protein and the reaction medium to which it was subjected, in addition to the contact with the substrate for a long reaction time. Although the N435 enzyme is widely and successfully used in esterification and transesterification reactions, the immobilized content was strongly reduced after use and reuse, corroborating the previously eluted results (Surendran et al. 2018; Meneses et al. 2019; Cajnko et al. 2021).
The complex molecular structure of lignin, composed of irregular phenylpropanoids, influences the inactivation of enzymes through steric hindrance, blocking the biocatalyst's access to the structure and thus hindering the efficiency of the catalysis (Zhang et al. 2022). Furthermore, lignins from different origins, chemical compositions, and molecular structures can cause variations in the adsorption of protein content on enzyme supports, reducing their catalytic activity, and phenolic compounds can be the leading causes of non-specific inhibition and inactivation of enzymes (Kim et al. 2011). As this content has yet to be explored in the literature, there is still no solid information on the mechanism and effect of lignin on enzymes (Li and Zheng 2017). The presence of lignin in the transesterification process results in the leaching of high protein content, reducing the conversion of the substrate into the product.
FTIR analysis provides valuable insights into the chemical composition of lignopolyols and indicates the enzyme's catalytic activity. In Fig. 3, lignopolyols exhibit a prominent band related to the hydroxyl group (3500 to 3200 cm− 1) after the first cycle (Gurgel et al. 2021; Araujo et al. 2023b).
The spectra after the second cycle reveal a substantial reduction in the band intensity related to the hydroxyl group for lignopolyols containing 10% and 15% lignin. This reduction is associated with the substrate with which the biocatalyst came into contact, again inhibiting its catalytic potential during this reaction period, making the desired conversion into new esters difficult, resulting in a loss of enzymatic activity more significant than 90% at the end of the cycle. The decrease in biocatalyst activity over its reuse cycle is an expected phenomenon. The presence of lignin significantly influences the enzymatic activity of N435 (Girão Neto et al. 2023). This effect becomes apparent as the lignin concentration in the reaction medium increases. For polyols with 0% and 5% lignin contents, there was no significant loss of enzymatic activity affecting the conversion of substrate into product, as evidenced by FT-IR spectra (Fig. 3) in the hydroxyl region and the data presented in (Table 1), can be used a third cycle for activity tests. It is crucial to highlight that the differential behavior in lignopolyols with higher lignin contents suggests a possible detrimental interaction between lignin and the biocatalyst over time. The comprehensive analysis conducted in this study suggests a direct correlation between the presence of lignin and the activity of the enzyme N435 (Li and Zheng 2017). The increase in lignin concentration demonstrates a consistent trend of reduced catalytic efficiency, highlighting the crucial importance of the interaction between lignin and the enzyme in transesterification reactions.
Novozym 435 is commonly used in esterification reactions, as it shows better results in converting the substrate, such as the production of biopolymers. Although it is widely used for this purpose, the use of enzymes in producing lignopolyols has yet to be explored (Baek et al. 2020; Morya et al. 2021). Araujo et al. (2023b) carried out an innovative study using Novozym 435 and Candida antarctica free Cal B lipase as catalysts in transesterification reactions, producing promising results in obtaining biopolymers to produce rigid polyurethane foams. However, the use of microorganisms or enzymes in the lignin depolymerization process is widely found in the literature (Nguyen et al. 2021). This study is a pioneer in exploring the reuse of N435 in the transesterification reaction in the presence of lignin.
Despite its remarkable use in this context, this enzyme should be used more in producing lignopolyols. The use of the enzyme in the lignin depolymerization process should be further documented in the literature.