Molecular characterization of non-heme bromoperoxidases from gracilaria species
1
Issued Date
2023
Copyright Date
1993
Language
eng
File Type
application/pdf
No. of Pages/File Size
xviii, 200 leaves : ill. (some col.)
Access Rights
restricted access
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (Ph.D. (Biochemistry))--Mahidol University, 1993
Suggested Citation
Tuangporn Suthiphongchai Molecular characterization of non-heme bromoperoxidases from gracilaria species. Thesis (Ph.D. (Biochemistry))--Mahidol University, 1993. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/89634
Title
Molecular characterization of non-heme bromoperoxidases from gracilaria species
Alternative Title(s)
การศึกษาคุณสมบัติระดับโมเลกุลของเอ็นไซม์โบรโมเปอร์ออกซิ เดสที่ไม่มีฮีมในสาหร่าย Gracilaria species
Author(s)
Abstract
Haloperoxidase, the peroxidase enzyme that can catalyze peroxidative halogenation in the presence of hydrogen peroxide, should offer a wide range of possibility for commercial applications, especially in chemical synthesis and analytical diagnosis. In general peroxidases from many sources so far are hemoprotein. In last decade, many non-heme haloperoxidases are found mainly bromoperoxidases from marine algae. These enzymes are more resistant to azide, cyanide and hydrogen peroxide than the heme containing ones, pointing to some advantages with respect to commercial applications. Red algae from the Gulf of Thailand were examined for haloperoxidatic activity. Seven species, viz., Gracilaria changii, G. edulis, G. firma, G. fisheri, G. irregularis,G. salicornia, and G. tenuistipitata showed bromoperoxidatic activity. The enzyme extracts showed both bromoperoxidatic activity and peroxidatic activity (in the presence of bromide ion). The isoenzyme patterns on NDPAGE were different from species to species, and there were peroxidatic and bromoperoxidatic activity bands which did not correspond. Purification and characterization of bromoperoxidases from G. fisheri are described. Three BPO isoenzymes were purified namely B1, B3 and B3L with the specific activity of 15.7, 311.4 and 198 U/mg protein, respectively. These three purified enzymes were different in several properties. B3 and B3L were monomer with molecular weight of 68 kd whereas B1 was oligomer of 560 kd with the same subunit size as B3 and B3L. The isoelectric point of B1 B3 and B3L were 7, 6.6 and 3.2, respectively. The amino acid compositions of B1 and B3 were similar whereas that of B3L were significantly different. However, all three showed to contain higher acid amino acids (Glx, Asx) than basic amino acids (Lys, Arg, His). The kinetic studies performed under optimum pH at 5.8 showed that the Km for MCD, H202 and KBr of B1 were 0.7 uM, 0.03 mM and 0.1 mM, of B3 were 0.7 uM, 0.1 mM and 0.2 mM and of B3L were 2 uM, 0.3 mM and 0.14 mM. These enzyme are remarkably thermostable especially B3 and B3L. In the studies of prosthetic group, these three enzymes were shown to be non-heme proteins by the absence of Soret band. The enzymes were deactivated by dialysis against EDTA at low pH and these deactivated enzymes were reactivated specifically only by vanadium not other metal ions nor heme. Furthermore, the purified enzymes showed to contain vanadium by atomic absorption spectrometer and the vanadium content in the enzyme samples were related to enzyme activity. Thus, these 3 BPOs were concluded to be non-heme bromoperoxidase containing vanadium as prosthetic group. The purified B1 contained 0.18 mole of vanadium per mole of enzyme subunit, B3 and B3L contained 0.6 mole of V per mole of enzyme. The fully vanadium activated purified enzymes, B1, B3 and B3L, contained about one mole of vanadium per mole of enzyme (subunit) Although these enzymes were less resistant to azide and cyanide compared to other vanadium BPOs, the enzymes were highly resistant to H(,2)0(,2), the advantage in application purposes.
Degree Name
Doctor of Philosophy
Degree Level
Doctoral Degree
Degree Department
Faculty of Science
Degree Discipline
Biochemistry
Degree Grantor(s)
Mahidol University