Thermostability of the vanadium bromoperoxidase from Corallina officinalis

Abstract

The vanadium bromoperoxidases c a t a l y s e a h a l i d e -a s s i s t e d d i s p r o p o r t i o n a t i o n o f H, O, [1,2] t o y i e l d an i n t e r m e d i a t e t h a t can undergo f u r t h e r o x i d a t i o n t o produce s i n g l e t oxygen o r promote b r o m i n a t i o n o f a s u i t a b l e o r g a n i c s u b s t r a t e . Bromoperoxidases o f t h i s t y p e a r e widespread i n m a r i n e a l g a e [3], p a r t i c u l a r l y t h e r e d a l g a e and brown a l g a e . We have r e p o r t e d r e c e n t l y [4] t h e i s o l a t i o n and c h a r a c t e r i z a t i o n o f t h e vanadium bromoperoxidase f r o m t h e r e d macroalga CoraZZina officinazis. T h i s i s an e x t r e m e l y l a r g e o l i g o m e r i c p r o t e i n o f M r about 740000 c o n s i s t i n g o f p r o b a b l y 12 i d e n t i c a l s u b u n i t s . Nevertheless, t h e enzyme showed a h i g h degree o f t h e r m o s t a b i l i t y r e t a i n i n g a p p r e c i a b l e a c t i v i t y (approx. 50%) a t 60-70°C and w i t h t h e a c t i v i t y r e s t o r e d on re-adjustment t o 25°C. Repeated c y c l e s o f t h i s t r e a t m e n t gave s i m i l a r changes i n a c t i v i t y [4]. Even a t 90°C f o r 20 min some a c t i v i t y was r e t a i n e d b y c e l l e x t r a c t s and a l t h o u g h t h e p u r i f i e d enzyme was i n a c t i v e a f t e r t h i s t r e a t m e n t i n b o t h i n s t a n c e s t h e a c t i v i t y was f u l l y r e s t o r e d o v e r 30 min a t 25°C. The vanadium bromoperoxidase f r o m t h e brown a l g a AscophyZZum nodosum [5] and t h e l i c h e n Xanthoria par+tina [6] were a l s o t o l e r a n t t o 50°C .and, p a r t i c u l a r l y t h e former, r e t a i ned a p p r e c i a b l e a c t i v i t y a t 70°C; however, r e s t o r a t i o n o f a c t i v i t y on r e a d j u s t m e n t t o l o w e r temperature d i d n o t o c c u r w i t h t h e AscophyZZum enzyme and f o r t h e Xanthoria enzyme was n o t i n v e s t i g a t e d . The vanadium bromoperoxidase f r o m a r e d a l g a Cerarniurn ruhrurn was n o t t h e r m o s t a b l e [7]. G e n e r a l l y speaking o n l y a few enzymes o f l o w M r a r e h i g h l y t o l e r a n t o f i n c r e a s e d temperature and e x h i b i t r e v e r s i b l e d e n a t u r a t i o n so t h e o l i g o m e r i c bromoperoxidase f r o m CoraZLina i s n o t a b l e i n t h i s r e s p e c t . The o l i g o m e r i c s t r u c t u r e o f t h e CoraZZina officinalis enzyme was n o t a p p a r e n t l y d i s r u p t e d b y exposure t o 60°C s i n c e enzyme e q u i 1 i b r a t e d t o t h i s temperature and s u b j e c t e d t o non-denaturing PAGE a t 60°C showed t h e same e l e c t r o p h o r e t i c p r o p e r t i e s as r e p o r t e d p r e v i o u s l y f o r t h e enzyme f r o m PAGE s t u d i e s c a r r i e d o u t a t l a b o r a t o r y temperature d u r i n g p r o t e i n c h a r a c t e r i z a t i o n [4]. The a c t i v a t i o n energy f o r response t o temperature o v e r t h e range 2O"C-5O0C ...

A vanadium-dependent bromoperoxidase in the marine red alga Kappaphycus alvarezii (Doty) Doty displays clear substrate specificity

“…Vanadium-containing bromoperoxidases (BPOs) specific for Br À and I À are widespread in marine macroalgae such as Corallina (Itoh et al, 1987a;Sheffield et al, 1993) and Ascophyllum (Vilter, 1984;Krenn et al, 1989;Butler, 1998). Some of these enzymes display low activity towards Cl À (Soedjak and Butler, 1990), and modification of enzyme can change the halide ion specificity (Ohshiro et al, 2004).…”

Immobilisation of bromoperoxidase from Corallina officinalis

“…This protein showed remarkable thermostability, retaining appreciable activity at 60-70°C and recovering the original activity on re-equilibration to 25°C either in repeated cycles of this temperature regime or after loss of activity at 90°C (Sheffield et al, 1993b). The enzyme also showed good tolerance to acetone, ethanol, methanol and propan-l -01 retaining 60% of activity after exposure for 24h to 30% by volume of the organic solvent.…”

Halogenation and Vanadium Haloperoxidases

“…This form of multimerization was also suggested in biochemical studies of other brown algal haloperoxidases (Almeida et al, 2001). Much like the brown algal VBPO, the 64 kDa VBPO isolated from the red alga C. pillulifera and C. officinalis form important homododecameric complexes of 12 subunits of 784 kDa and 768 kDa, respectively (Isupov et al, 2000;Itoh, Izumi, and Yamada, 1986;Sheffield, Smith, and Harry, 1993;Shimonishi et al, 1998).…”