Glutaraldehyde is a well known covalent crosslinker that is often used to improve the enzyme immobilization process by creating covalent attachment points (Rodrigues et al. 2021). It reacts with amine groups on the surface of proteins / particles and in so doing promotes structural integrity of the enzyme by connecting it to the solid support, to neighboring proteins, or to itself. If you are interested in the many different applications of glutaraldehyde I recommend this review (Barbosa et al. 2013) also available here for those without a subscription. In the case of immobilized lactase, glutaraldehyde is known to be beneficial during immobilization on a wide variety of materials (Jafri et al. 2017). In this project we used lactase as a model enzyme to find which carriers in Solidzymes’ collection benefit from pre-activation with glutaraldehyde.
Experimental Design
Thirty one carriers that contained amine groups on their surfaces were selected from Solidzymes’ collection. Two 25 mg samples of each carrier were soaked with phosphate buffer pH 7.0 overnight finally equilibrating all carriers into the pH 6.8 – 7.5 range. One sample of each pair was then activated with aldehyde groups by the addition of 0.5 mL water containing 1% glutaraldehyde. Glutaraldehyde was allowed to react with mixing for 1 hr. During this reaction many samples that were not already brown changed to a brown color signaling the formation of aromatic bonds on the surface of these carriers (Figure 1).
Figure 1. Examples of browning during glutaraldehyde activation of carriers. The appearance of browning scored in Table 1 was as follows (a) “ ? “ unknown browning (b) “ – “ little or no browning (c) “ + “ some browning (d) “ ++ “ dark browning
After one hour the glutaraldehyde was thoroughly washed off with phosphate buffer. One milligram of lactase (4% w/w relative to carrier) in phosphate buffer pH 7.0 was given to all samples (control and glutaraldehyde modified). These samples were allowed to bind overnight then the percentage of unbound lactase remaining was determined by measuring the absorbance of the supernatants at 280 nm. Some samples enjoyed an increase in binding due to glutaraldehyde modification and this corresponded somewhat to the degree of browning observed (Table 1). Other samples did not bind lactase either with or without glutaraldehyde. Seven of these poor performers were conveniently excluded from further experimentation bringing the total number of samples for activity measurements down to forty eight.
Excess lactase was washed off of these forty eight samples and the activity of immobilized lactase was determined as described in the Lactase Case Study post except that the reaction temperature was increased to 50oC. After initial activity measurements the samples were stored at 50oC in reaction buffer and activity was measured again after seven days and after fourteen days. This allowed comparison of the thermal stability of lactase immobilized on controls by ionic interactions versus lactase immobilized covalently on glutaraldehyde modified samples (Table 1).
Table 1. Summary of the results for twenty four carriers modified with glutaraldehyde and compared to unmodified controls. Twenty were improved in stability or activity.
Results
Lactase performance was improved on a majority of the selected carriers. In most cases this was due to improved lactase stability as samples with covalent stabilization lost less activity during the first week at 50oC (Figure 2a). Interestingly, some carriers such as DIAION HPA512L and Duolite A568 did not have improved stability with covalent attachment. However, they did have improved overall activity with the glutaraldehyde treatment (Figure 2b). This increase in activity did not correspond to an increase in lactase loading suggesting that glutaraldehyde activation did not increase the number of binding sites. Therefore, the most likely explanation is that glutaraldehyde modification of the surface reduced lactase activity loss during the process of immobilization on these carriers.
Although most carriers benefitted from glutaraldehyde crosslinking there were a few exceptions such as Applexion 5251 and Accuspheres #2 and #3 that did not have improved performance. The performance of Accuspheres #2 and #3 controls, in particular, suggests that these carriers already increased the thermal stability of lactase without the need for glutaraldehyde. This could be attributable to the large, hydrophilic, polymer coating on these carriers. This coating has been previously reported to stabilize glucose isomerase without the need for glutaraldehyde (Scheels 2007).
Figure 2. Representative graphs for activity over time of samples from Table 1. (a) DIAION CR20 – lactase stability was improved by glutaraldehyde. (b) DIAION HPA512L – lactase activity was improved by glutaraldehyde. (c) Accuspheres #2 activity and stability were good and not improved by glutaraldehyde.
Conclusion
This project successfully demonstrated the benefits of covalent crosslinking during lactase immobilization. Twenty carriers were ultimately identified as improved by glutaraldehyde surface modification. Thus, this project paves the way for a more robust enzyme immobilization screening service at Solidzymes that includes an option for glutaraldehyde modification of these carriers. In addition, some carriers were identified that stabilized lactase at relatively high temperatures even in the absence of covalent crosslinking. These anion exchange based materials might be of interest to customers who want to move away from covalent immobilization.