we immobilized enzymes on epoxy or amino resins, and just find decreased thermal stbaility (compared with their free forms). We are wondering whether the covalent bonds are formed. However, we did not really detect protein release~ anyone has such experience as well? Covalently bonded enzyme showed worse thermal stability? I did not find such cases reported~
Hi Yuhong Mao,
Because 7α-Hydroxydehydroepiandrosterone is a metabolite of dehydroepiandrosterone at cytochrome CYP7B1 in brain (which we've studied in our lab, - see at http://www.xenobiovir.com/), we investigated enzyme-kinetics of the reactions occurred, as as the termostability of properties of an enzyme immobilized on epoxy resin, which was modified by different quality percentage concentrations of chitosan to obtain different crowding and charge conditions of the microenvironment.
Obviously, in our studies (as well in those of Qiong Yang et al. in RSC Adv., 2017, 7:38028-38036 DOI: 10.1039/C7RA06544B), the thermal stability of immobilized 7α-HSDH was BETTER than the thermal stability of the free enzymes. Free 7α-HSDH is highly sensitive to temperature and a significant decline in activity was seen when incubation was at 27 °C. The relative activity of 7α-HSDH is 44.00%. The activity of free 7α-HSDH is hardly detectable when it was treated at 45 °C for 2 h. As an explanation, after the enzyme was immobilized on the carrier, the rigidity of the enzyme domain structures is strengthened and the flexibility of the enzyme is weakened. External forces are smaller in influencing the enzyme configuration.
To explain an obvious discrepancy between the above and yours results as regarding the thermal stability of the immobilized 7α-HSDH, please be cautioned that termostability results show that it depends on chitosan, and that the distribution profiles of chitosan within the microbeads is highly dependent on pore size!
Good luck,
Ilya B. Tsyrlov
https://en.wikipedia.org/wiki/Ilya_B._Tsyrlov
Hi,
It is a relatively common situation. You are using the wrong immobilization technique for your enzyme. In this case, the immobilization may be occurring by a disfavorable zone or is involving a key amino residue(s) for catalysis/stability. Also, immobilization protocol may be conducted at the wrong pH, which negatively alters the enzyme structure during the immobilization. Thus, the immobilized enzyme structure is prone to denaturation. Some suggestions:
add a competitive inhibitor during the immobilization
slightly vary the pH
try other immobilization strategies
Try heterofuntional supports, in order to promote a specific enzyme orientation during the immobilization
Carlos Vera hey, thanks for sharing your idea~ in our case, the initial activity of immobilized enzyme is quite high, although a bit lower than the free one, while I would say it is acceptable~ However, its thermal stability decreased more secirously than its free form, so it confused me a lot. As read from many publications, the stability against pH varioation, thermal treatment, etc., of covalently bonded enzyme is generally improved, which is contrary to our case~
Yuhong, taking into account your previous comment, I think you are overloading the catalyst. So, most of the enzyme is not immobilized. When you use the techniques mentioned in your first post, the maximum support loading is around 30 mg protein/1g of support(3%). Therefore, it is expected a low specific activity, because most of the catalyst is an inert mass.
On the other hand, of course, there is a science bias: most of the researchers report positive results. We have somewhat broad experience in immobilization, so I can properly tell you not all immobilization techniques provide always an increase in enzyme stability. At the end of the day, enzyme immobilization is just a trial and error process.