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Monolithic Bioreactors for Macromolecules 273
260 nm. Hydrolyzed DNA/RNA was collected at outlet of CIM disk monolithic
column, and RT-PCT or PCR was performed as described in the literature (14,15).
Products of RT-PCR and PCR were analyzed with gel electrophoresis (16).
The results of RT-PCR and PCR on DNA or RNA hydrolyzed by enzyme
reactors are presented in Fig. 6.
4. Notes
1. Choice of immobilization method highly depends on (i) activated groups of
monolith, (ii) immobilization conditions and (iii) enzyme to be immobilized.
2. Changing the substrate concentration enables calculation of kinetics parameters
v max and K m using Michaelis–Menten equation (see Fig. 2).
v = v max ·
S
K m + S (2)
where v = initial velocity (the instantaneous velocity, d[P]/dt at given substrate
concentration); v max = maximum biological activity; [S] = fixed substrate concentration;
K m = Michaelis–Menten constant.
v max = k 3 × E (3)
where [E] = amount of immobilized enzyme; k 3 = rate constant for the breakdown
of enzyme substrate complex, turnover constant, catalytic rate constant.
3. Apparent values are used as absolute values cannot be determined due to the nature
of the polymeric substrate, the unknown number and type of different substrate
binding sites, and the unclear relationship between the absorbance signal and the
actual catalytic events.
4. K m and k 3 values of free DNase were 0.07 g/l and 76 dA 260 nm /min/mg, respectively
(9).
5. Addition of a benzamidine hydrochloride in immobilization buffer prevents
undesired autodigestion of trypsin.
Acknowledgments
Ministry of higher education, science and technology supported this work.
We thank N. Berginc, J. Kuplenk and J. Jančar for technical assistance.
References
1. Švec, F., Tennikova, T.B. and Deyl, Z. (2003) Monolithic Materials: Preparation,
Properties, and Applications. Elsevier, Amsterdam.
2. Podgornik, A. and Štrancar, A. (2005) Biotechnology Annual Review, vol. 11, 1st
ed. Ed: El-Gewely, M.R. Elsevier, Amsterdam, pp. 281–333.