ImmobilizationBinding capacity and leakage of different carriers

Generally, the best immobilization rates were achieved on the IB-HIS carriers with affinity binding of the lipase via a His6-tag. However, no conclusion can be drawn concerning the functionalization of the carriers or polarity that promoted binding of PCI_Lip. IB-HIS-1 is classified as very hydrophobic due to the high amount of butyl groups and low amount of Ni-IDA. It displays a similar binding capacity as IB-HIS-20 functionalized with ammonia groups and Ni-NTA, which is classified as very hydrophilic. The particle size reached from 150 to 710 µm, while the supplier offers no information on the pore size. Beyond all tested IB-HIS carriers, the particle size is mostly similar, only IB-HIS-15 with 250–400 µm, IB-HIS-16 with 20–40 µm, and IB-HIS-17 with 100–250 µm deviate from the average of 150–710 µm. Studies on immobilized lipases from Mucor miehei and Rhizopus oryzae revealed that the particle size of the support has no influence on the binding capacity. The main factor concerning binding capacity was found to be the pore size of the support material, which may also influence the activity of the immobilized enzyme (Gustafsson et al. 2012). The advantage of these supports is immobilization by affinity using the His6-tag fused to PCI_Lip to immobilize the enzyme. Compared to other immobilization techniques such as adsorptive or covalent binding methods, affinity immobilization uses highly selective interactions between the support and enzyme. This offers a controlled orientation of the enzyme avoiding inactivation by blocking the active site. Further, the mostly peripheral position of affinity tags reduces conformational changes of the enzyme due to immobilization (Anboo et al. 2022). The relative specific activity and hydrolysis profiles of free and immobilized PCI_Lip WT are very similar, which rules out major conformational changes and supports the minimal impact of this immobilization technique. The polar cellulose matrix of IB-HIS-16 might be one reason for its overall low binding capacity. Mostly hydrophobic support surfaces are described as more suitable for effective immobilization of lipases due to their interfacial activation (Deon et al. 2020). Even though, this affinity binding is described as rather weak (Homaei et al. 2013), for PCI_Lip immobilized on the IB-HIS carriers also other factors seemed to have an impact on the binding strength. Putatively, there might be further non-covalent interactions of the lipase with the surface of the support besides the binding of the His6-tag to the Ni2+-ion. The hydrophobic, mesoporous polystyrene support ECR 1090 M was marginally better than the hydrophilic amino C2-derivatized methacrylate support ECR 8806 M, even though the latter had a much lower surface area and smaller pore size. Adsorption is the most favorable immobilization method for industrial applications because of its low cost and simplicity (Ishak et al. 2025). Though the two supports investigated in this study do not seem to be the optimal supports for PCI_Lip WT. Some studies report an increased specific activity of immobilized lipases, which is attributed to the interfacial activation of the lipase being immobilized in its open conformation (Mokhtar et al. 2020). The low amount of lipase immobilized to the adsorptive carriers may influence specific activity of PCI_Lip positively, as the enzyme carries a putative lid domain responsible for interfacial activation (Broel et al. 2022). Both covalent carriers were used in the recycling experiment with glutaraldehyde as the linker and spacer between enzyme and support. This was reported to have positive effects on the activity of immobilized enzymes. By using a spacer in covalent immobilization, the enzyme remains more flexible and steric hindrance is avoided (Zhang et al. 2013). Further, the activity of PCI_Lip after a harsher immobilization treatment should be investigated. As covalent immobilization is described to have a reduced loss of enzyme (Homaei et al. 2013), it is possible that besides the covalent immobilization of PCI_Lip, some of the enzyme was absorptively attached to the support and was washed off during the cycle.

Recycling of immobilized PCI_Lip

During the recycling, seven immobilisates could be divided into two groups. The activity of the first group exhibits a similar development as the activity observed for Candida antarctica lipase B (Cal B) immobilized on bagasse for the production of biodiesel (Cui and Di Cai 2018). A minor increase during the first cycles may be due to a rearrangement of the immobilized lipase into a superior conformation or position. This could be observed especially when there was a longer storage period up to the following measurement (Gustafsson et al. 2012). The microenvironment around the immobilized PCI_Lip keeps it stable to retain a relative activity above 60% in the beginning. Accessibility of the active site does not seem to be heavily impacted by immobilization to these support materials. However, the stabilization of the enzyme by immobilization can be caused by increased rigidity of the enzyme, hindering conformational changes during the catalysis (Stepankova et al. 2013). This may cause lower initial activity than the free PCI_Lip. But, immobilization to these support materials seemed to result in smaller conformational changes with little impact on the activity than the other four immobilisates. The second group of immobilized enzymes may face restricted active site accessibility or mass transfer limitations, causing the relatively low activity compared to the free enzyme (Robescu and Bavaro 2025). The increase within the first cycles was probably caused by diffusional limitations of the substrate and product. For the recycling, the activity of immobilized PCI_Lip was measured photometrically, using pNPO as substrate. Even though the supernatant has been totally removed after each cycle and the carriers have been washed, a slight yellow coloring of the carriers remained, indicating that at least pNP could not be removed completely. The increased activity could be attributed to the potential retention of substrate in the immobilisate matrix from previous cycles and elution in the further cycles. Mass transfer limitations are common constraints in enzyme immobilization. For efficient use of immobilized enzymes, free diffusion of substrate and product is necessary. The mobility of both can be restricted by the functionalization of the carrier surface and the pore size of the support. Diffusional limitations can severely impact enzyme activity by reducing the reaction rate, fortifying substrate inhibition and the formation of pH gradients (Hanefeld et al. 2009; Zdarta et al. 2018). In the case of these four IB-HIS carriers, the pore size does not seem to be suitable for the immobilization of PCI_Lip, as the product was held back. Generally, a smaller pore size leads to a larger surface area, which can increase enzyme loading, but it can inhibit mass transfer. Therefore, the ideal support material has to be evaluated for every enzyme immobilization individually (Chen et al. 2019). Selective individual increases in relative activity during the recycling can be described through longer storage periods overnight between the cycles. Storage was conducted in the standard buffer, which seemed to regenerate inactivated, immobilized lipase. These increases in activity are probably assigned to a combination of restricted mass transfer and enzyme regeneration. Chen and Wu describe the regeneration of an immobilized lipase in biodiesel production. The washing removed substrate molecules that stuck to the immobilized enzyme, causing its inactivation (Chen and Wu 2003). In our case, longer storage may have removed perturbing molecules from PCI_Lip. After the recycling experiment, IB-HIS-19 was chosen as the best carrier for further investigations of the immobilized PCI_Lip. The affinity binding of PCI_Lip offered a simple way of immobilization where no hazardous chemicals were needed. This carrier had already displayed a high binding capacity in combination with low leakage. In the recycling experiments, it stood out with high initial activity, which was kept up for five cycles with a slow decline afterward.

Characterization of immobilized PCI_Lip

For PCI_Lip WT the advantage of affinity immobilization becomes clear. The immobilization on the peripheral affinity tag of PCI_Lip did not cause severe conformational changes or hinder the flexibility of the enzyme. The affinity tag of PCI_Lip ensures controlled orientation of the enzyme without the risk of activity loss caused by blocking the active site. The attachment of the enzyme on the His6-tag causes only minor conformational changes as opposed to other immobilization techniques. Either adsorption or covalent binding always affects the surface of the enzyme, sometimes even at multiple points. This enhances the stability of the enzyme by making it more rigid. However, the increased rigidity may cause reduction or even loss of activity (Anboo et al. 2022). Besides acting as the chelating agent of the divalent nickel ion the nitrilotriacetic acid is a spacer between the carrier surface and the enzyme. This enlarges the orientation space of the enzyme further reducing conformational restrictions. Also the higher flexibility of the enzyme leads to a stronger binding (Alács et al. 2024). The slight decrease of activity of immobilized PCI_Lip WT is caused by the interaction of the lipase with the carrier surface. Besides the attachment via the His6-tag parts of the enzyme surface can interact with functional groups of the carrier surface by means of hydrophobic and van-der-Waals interactions as well as hydrogen bonds (Alács et al. 2024; Tzialla et al. 2009). The additional adsorptive interactions further stabilized the immobilized PCI_Lip. This goes along with the increased T5060 values of the immobilized enzyme compared to the free one. However, the increased stability engendered a slight reduction of the activity. Further hydrolysis profiles of the two immobilized double mutants S163M+L302G and I245F+L302G have been compared to their free versions. These mutants have been created to shift the selectivity of PCI_Lip to an increased hydrolysis of short- to medium-chain fatty acids and also reduce the hydrolysis of long-chain fatty acids. Therefore, amino acids mainly in the binding pocket of PCI_Lip have been substituted (Henrich et al. 2026). For S163M+L302G the activity for its preferential substrates pNPV and pNPH is strongly increased. Several studies describe an increased activity of lipases when immobilized (Cui and Di Cai 2018; Deon et al. 2020; Dong et al. 2021; Tzialla et al. 2009; Zhao et al. 2008). This is often explained by the immobilization of the lipases in their most active state. Most lipases including PCI_Lip share the common feature of a lid domain that covers the active site and opens up, when in contact to a hydrophobic interphase. Especially when using adsorptive immobilization on hydrophobic supports the lipase can be immobilized in its open conformation showing the highest activity. The permanent open conformation of a lipase enables free access of the substrate to the active site and causes considerably higher activity often called hyperactivation (Blanco et al. 2004; Deon et al. 2020). Even though the amino acid exchanges of S163M+L302G are located near and inside the substrate channel, they seem to affect the overall conformation of PCI_Lip. The implementation of methionine and glycine may have changed the conformation of the lipase. This might be in a way that adsorptive interactions are enhanced that favor the open conformation and cause hyperactivation. As mutation L302G is present in both investigated mutants, the hyperactivation seems to be associated with mutation S163M. This one is positioned a little aside the substrate channel and has previously been shown to alter the chain length selectivity towards short- to medium-chain fatty acids (Henrich et al. 2026). The mutation is located further away from the active site, near the substrate channel and the lid domain. It may therefore influence the conformation of the lid domain without noticing beforehand but refining the mutants’ properties when immobilized. The free form of PCI_Lip mutant I245F+L302G is most active against pNPV and pNPH, but already exhibits reduced activity compared to the WT (Henrich et al. 2026). Immobilization of this mutant reduced its activity by almost half and simultaneously shifted its selectivity. Both substitutions are located in the substrate channel but replacing isoleucine with phenylalanine and leucine with glycine seems to impact on the overall enzyme conformation, leading to a diminished enzyme activity when immobilized. The combination of amino acid substitutions and immobilization lead to a reduced activity in this case. These minor changes can also impact enzyme selectivity. Several factors have been described to putatively influence selectivity when an enzyme is immobilized. The outcome is hard to predict and has to be tested for each enzyme/carrier combination individually. Potential reasons for an alters selectivity in a specific enzyme/support combination may be diffusional limitations, the micro-environment, structural modifications, or a changed rigidity (Rodrigues et al. 2013). In this case, the substitution of isoleucine to the bulkier phenylalanine in combination with the immobilization may downgrade the free diffusion of the substrate molecules into the active site leading to the reduction of activity. A similar change of selectivity has been observed for a lipase from R. miehei when immobilized to a resin. This immobilized lipase became more active on short-chain fatty acids as well (Lee and Parkin 2001). The examination of the hydrolysis profiles of PCI_Lip WT and two double mutants underlined that every enzyme/support combination accounts for different results. Immobilization of WT to HB-HIS-19 displayed only minor changes in activity, while immobilization of S163M+L302G showed a hyperactivation and I245F+L302G exhibited reduced activity combined with altered selectivity.

Among other aspects, the aim of this study was to improve the stability of PCI_Lip. Therefore, thermodynamic stability and kinetic stability of the lipase have been investigated. Thermodynamic stability refers to the enzyme conformation and its tendency to denature, while kinetic stability deals with irreversible inactivation and the loss of activity (Iyer and Ananthanarayan 2008). The introduction of two other amino acids in the free double mutants led to an increase in the T5060 value compared to the WT with a T5060 value of 29.6 ± 2.8 °C. Exchanging leucine for glycine at position 302 raised the semi-inactivation temperature for 1.5 °C (Broel et al. 2022). Both double mutants carry this mutation, which has been shown to have a positive effect on the T5060 value. The increase of kinetic stability in the free double mutants compared to the WT is likely a side effect of mutating selected amino acids in or near the substrate channel. Xie et al. have chosen a similar approach to stabilize Cal B. They chose against established approaches that target increased structural rigidity, restricted conformational flexibility or boost the interactions of unstable domains. Since the active site of an enzyme is a rather fragile part, nevertheless vital to catalytic activity, they aimed to stabilize this part of the enzyme by exchanging amino acids near the active site. This strategy led to a mutant with a semi-inactivation temperature 12 °C above that of the WT in the Cal B (Xie et al. 2014). The mutations in the substrate channel stabilized the active site of PCI_Lip, which led to an increased kinetic stability of the free double mutants. The combination of amino acid exchanges seemed to have increased the semi-inactivation temperature even more. Similar effects have been observed in a creatinase, where the combination of mutations led to an increase in T50 (Bian et al. 2024). Immobilization of PCI_Lip further enhanced the kinetic stability of the enzyme. The semi-inactivation temperature of the WT and both mutants was increased by about 6–7 °C through immobilization. As the increase is similar in all three variants it can be attributed to immobilization, and the individual differences have no major impact here. The enhanced thermal stability was a result of the rigidification of PCI_Lip through immobilization. A more rigid enzyme can resist the denaturing effects of higher temperatures better than the free enzyme with high flexibility (Khan 2021). Our findings go along with literature data showing that immobilization of lipases leads to an enhanced thermostability of the respective enzymes (Wilson et al. 2006; Xie et al. 2014; Costantini and Califano 2021). The enhanced stability of PCI_Lip through immobilization that simultaneously keeps its activity offers the chance for industrial applications. Immobilized PCI_Lip could be implemented in the biotechnological production of biodiesel. Therefore, the current findings could be combined with previous findings, showing that chain length selectivity of PCI_Lip can be altered by protein engineering (Henrich et al. 2026). This can offer a chance to find a variant suitable to hydrolysis of long-chain fatty acids with improved stability when immobilized to a support material such as IB-HIS-19.

Thermodynamic stability of PCI_Lip and its mutants

Thermofluor assays provide the possibility to examine the structural stability of a protein and the influence of its environment. While for the kinetic stability the hydrolytic activity of PCI_Lip was monitored, the thermodynamic stability monitored the structural denaturation. The melting temperature observed in the Thermofluor assays refers to the temperature at which half of the enzyme is denatured (Iyer and Ananthanarayan 2008). The comparison of kinetic and thermodynamic stability of PCI_Lip displays that the enzyme structures that are essential for the hydrolysis unfolded before the whole enzyme scaffold denatured. Compared to Cal B with TM of 54.5 °C, which is often used for industrial purposes, PCI_Lip offers a higher thermostability (Kim et al. 2010). The elevated TM brings advantages for putative industrial applications. In industrial processes, higher temperatures are used as reaction rates are increased and substrate solubility is better. In this case, lipases from thermophile organisms offer much higher thermostability; for example, a lipase from Thermomyces lanuginosus has a TM of 93 °C (Xiang et al. 2023). However, microbial lipases mainly catalyze the hydrolysis of long-chain fatty acids (Chandra et al. 2020). The advantage of PCI_Lip and especially the two double mutants is their selectivity for the hydrolysis of medium-chain fatty acids and its mutants offer a selectivity for short-chain fatty acids (Wang et al. 2025; Henrich et al. 2026). The Thermofluor method applied in this study offered the chance to improve the thermostability of this lipase with special selectivity for short- to medium-chain fatty acids to meet the requirements for an industrial application. An increase in TM with higher buffer concentration has also been observed for other proteins. Higher buffer strength leads to a higher concentration of ions in the hydration shell of the enzyme and increases water activity on the protein surface. Both give a better structural order and reduce conformational flexibility, resulting in a more thermostable enzyme (Boivin et al. 2013; Kalisz et al. 1997). The chelating agent EDTA can be added to an enzyme solution in order to avoid oxidation of SH-moieties (Boivin et al. 2013). For PCI_Lip, the addition of EDTA did not help to stabilize the enzyme. The addition of salts is a common tool to elevate enzyme stability. Generally, the stabilizing effect of neutral salts is ascribed to ions binding to charged groups on the protein surface and a competition for solubilization in water. Both enhance the hydrophobic interactions of the protein, boosting its stability. While some ions with chaotropic effects rather destabilize enzymes, ions with kosmotropic effects support stabilization. The effects of each ion on enzyme stability are described by the Hofmeister series (Silva et al. 2018). In this study, chloride, bromide, and iodide as anions were tested on the stability of PCI_Lip. Bromide has a less stabilizing effect on the lipase than chloride, but iodide has a more stabilizing effect. Within the Hofmeister series, chloride is claimed to have no effect on the water structure; bromide is described as an anion with slightly chaotropic effect (Lavelle and Fresco 2003). This chaotropic influence of bromide in comparison to chloride is displayed by PCI_Lip with a lower TM. According to the Hofmeister series, iodide should be more destabilizing. At this point, the series is not in accordance with our results. By screening the three most common sodium halogenides, iodide displayed the highest increase in TM. In the case of Hofmeister series for cations, larger ones with lower charge are considered to salt out proteins, while smaller ones with a higher charge, salt proteins (Kherb et al. 2012). According to the Hofmeister series, in this study, Mg2+ should stabilize the protein best and K+ least good. However, the melting temperatures of PCI_Lip WT were highest with the highest concentration of KCl. With increasing concentration of Mg2+, TM decreased. Calcium, as another divalent ion, barely affected the stability of PCI_Lip even though it is in similar position as magnesium in the Hofmeister series. This discordance also goes along with the results of magnesium. Ammonium sulfate combines two ions that are supposed to have a positive impact on the enzyme stability (Silva et al. 2018). At this point, it is to mention that the Hofmeister series acts as a general guideline with some conformity to stabilizing ions for PCI_Lip. Effects of ions on enzyme stability are highly individual. This is reflected by the deviance from this theoretical series in our data. For PCI_Lip, an increase in TM appears at higher concentrations, but other salts at the respective concentrations enhance stability even more. Polyols in general are known to stabilize proteins. Even though the mechanism behind this effect has not yet been fully elucidated, it is traced back to the promotion of hydrophobic interactions within the enzymes, making them more rigid, which increases thermostability (Balcão and Vila 2015). The addition of glucose, trehalose, and glycerol as polyols increased the thermostability of PCI_Lip. The highest increase in TM was observed with the addition of 10% glycerol by 2 °C. Research is ambivalent about the effects of DMSO on protein structures and stability. On the one hand, it is described to have positive effects on protein stability, being a popular and widely used cosolvent in bioassays. On the other hand, in some studies, its destabilizing effect on proteins even at low level concentrations is shown (Cubrilovic and Zenobi 2013; Landreh et al. 2014). PCI_Lip seems to be another example for DMSO having destabilizing effects on the enzyme. Still, this does not mean that DMSO generally affects protein stability in a negative way, as Chan et al. found that the impact of DMSO on enzyme stability is highly protein-dependent. In this case, the individual interactions with charged states and electrostatic repulsions caused destabilization of avidin at 4% DMSO but boosted the stability of a bacterial cytochrome at a concentration of 40% (Chan et al. 2017). PEG had a destabilizing effect on PCI_Lip while high molecular weight, PEG 20k, was used. Most literature describes PEG as crowding agents with positive effects on stability. However, recently, some studies showcased some negative effects. It has become clear that, similar to DMSO, effects are protein-dependent and also the size of PEG matters. Conformational changes caused by high molecular PEG have been proven in a tRNA-synthetase from E. coli (Liebau et al. 2024). As high molecular PEG has an amphiphilic character, this might affect hydrophobic parts of the lipase causing destabilization and eventually a lower TM. Some amino acids have been reported to inhibit protein aggregation and support renaturation (Boivin et al. 2013). Therefore, the impact on TM of PCI_Lip by adding betaine, glycine, and proline respectively has been investigated. In this case, none of the amino acids had a positive impact on thermal stability. Even though the improvement of thermostability has not been the initial aim of the protein engineering approach, we decided to investigate the two mutants with the best changes in the hydrolysis profiles in this study. These investigations gave deeper insights in the stability of these mutants for putative industrial applications of a microbial lipase highly selective for short- to medium-chain fatty acids. We suggest that the mutation I245F is causing enhanced stability as the mutation L302G appears in both of the tested mutants. Even though the enhancement of stability was not the initial intention of the protein engineering, the results are in line with other studies aiming to improve thermostability by the exchange of amino acids. The mechanisms underlying the stabilization of an enzyme by the introduction of other amino acids are versatile. In this case, it seems likely that the increased stability could be attributed to the formation of new π-π-interactions with F165 stabilizing this rather flexible region (Bai et al. 2020; Bian et al. 2024). This mutation is located in the substrate channel. Xie and co-workers followed a similar strategy to improve the kinetic stability of Cal B by mutating amino acids in flexible regions near the active site. This increased the rigidity of the enzyme in an area vital for its activity, increasing its half-life time 13-fold (Xie et al. 2014). Finally, the thermostability of PCI_Lip WT and both double mutants was investigated using the best buffer systems with the best additives. The results show that the improvement of the composition of the enzyme solution is a good tool to improve the properties of an enzyme for a potential industrial application. Glycerol, glycine, and trehalose were most effective in increasing TM in comparison to the pure buffer. All in all, citrate buffer with glucose, glycerol, glycine, NaCl, and trehalose could increase TM to 71 ± 0 °C and PPB with glycerol, glycine, and trehalose reached the same TM. The improvement of thermodynamic stability of PCI_Lip by selection of appropriate buffer systems gives rise to a broad use of this lipase in biotechnological processes. The high stability makes the lipase a good candidate for a biocatalyst in the production of chemical or pharmaceutical compound, as these reactions are often conducted under elevated temperatures.

Storage stability of free and immobilized PCI_Lip

An increase of activity of free PCI_Lip WT within the first days after purification has been observed beforehand. We assume that this is attributed to an ongoing refolding of the enzyme during the first days of storage as heterologous expression also leads to the formation of inclusion bodies besides some amount of soluble lipase. Spontaneous refolding is also known for other proteins like different fibronectin domains (Shah et al. 2017). A decrease of activity over a long storage period is commonly observed for various enzymes. Another factor is the storage temperature that can affec