Premium
Stabilization of restriction endonuclease Bam HI by cross‐linking reagents
Author(s) -
Dubey A. K.,
Bisaria V. S.,
Mukhopadhyay S. N.,
Ghose T. K.
Publication year - 1989
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260331013
Subject(s) - glutaraldehyde , chemistry , denaturation (fissile materials) , endonuclease , reagent , kinetics , thermal stability , enzyme , chromatography , nuclear chemistry , biochemistry , organic chemistry , physics , quantum mechanics
Bacillus amyloliquefaciens H produces a restriction endonuclease enzyme Bam Hl which is heat labile even at low temperatures. Studies were conducted to enhance thermal stability of Bam Hl using cross‐linking reagents, namely, glutaraldehyde, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS), and dimethyl 3,3′‐dithiobispropionimidate (DTBP). Reaction with glutaraldehyde did not result in a preparation with enhanced thermal stability. However, the DMA‐, DMS‐, and DTBP‐cross‐linked preparations of Bam HI exhibited significant improvement in thermal stability. Studies on thermal denaturation of the cross‐linked enzyme preparations revealed that these do not follow a true first‐order kinetics A possible deactivation scheme has been proposed in which the enzyme has been envisaged to go through a fully active but more susceptible transient state which, on prolonged heat exposure, exhibits a first‐order decay kinetics. At 35°C, which is close to the optimum reaction temperature of 37°C for Bam Hl activity, the half‐line of DMA‐, DMS‐, and DTBP‐cross‐linked preparations were 4.0, 5.25, and 5.5 h, respectively, whereas the native enzyme exhibited a half‐line of 1.2 h only. The apparent values of deactivation rate constants for native, DMA‐, DMS‐, and DTBP‐cross‐linked Bam Hl were 1.13, 0.39, 0.29, and 0.26 h −1 , respectively, at the same temperature, and the apparent values of activation energies for denaturation of native, DMA‐, DMS‐, and DTBP‐cross‐linked Bam Hl were 2.63, 5.24, 6.55, and 9.2 kcal/mol, respectively. The DTBP‐cross‐linked Bam HI was, therefore, the best heat‐stable preparation among those tested. The unusually low values of activation energies for denaturation of Bam Hl represent their highly thermolabile nature compared to other commonly encountered enzymes such as trypsin, having activation energies of more than 40 kcal/mol for their denaturation.