Purification And Characterization Of Peroxidase From Ligustrum Lucidum Ait

Peroxidase (POD, EC1.11.1.7) is an oxidase that contains a hemachrome group and which exits extensively in every living creature like animal, plant, and microorganism. It also contains metal ions such as iron or copper and have not identical structure and catalytic mechanism. It serves a number of different functions.In this article, Prification, thermostability, and some characteristics of POD from leaves and fruits of Ligustrum lucidum Ait. were investigated. The result showed that:1) After sulfate-acetone co-precipitation, Sephadex G-100 gel filtration chromatography and DEAE- cellulose ion-exchange column chromatography, one peroxidase from fruits of Ligustrum lucidum Ait. was obtained. The yield and purification fold was 13.3% and 48-fold respectively.And the molecular weight of the POD was 22 kDa. Another isoperoxidase from apoplast proteins of leaves of Ligustrum lucidum Ait. was also purified, which the molecular weight was 62 kDa.2)The crude peroxidase from leaves of Ligustrum lucidum Ait. had high thermostability. Its activity remained 61% at the condition of 60℃in 60 min. Under 35℃, 40℃, 45℃, or 50℃, the fruit POD activity remained more than 90% in 1h, but lost completely after heated for 5min at 80℃, while it remained 71% at 70℃. The appropriate concentration of trehalose andβ- cyclodextrin could protect the fruit POD thermostability. With trehalose andβ- cyclodextrin, the thermostability of the fruit POD at 60℃rose up 11% and 9.8% respectively. The optimum pH of the POD in Ligustrum lucidum Ait was 6.0.3) The optimum pH of the purified POD from fruits of Ligustrum lucidum Ait. was 6.5; the optimum temperature is 40℃; the thermostability decreased. The activity remained 26% after heated 5 min at 70℃; it remained 44% at the condition of 60℃disposing 60 min. Its apparent Km of guaiacol and hydrogen peroxide was 6.2 mmol/L and 3.85 mmol/L respectively.4) Two kinds of dipeptides, e.g. Ala-Asp and Lys-Val, could improve the thermostability of the purified POD at 60℃significantly. 75μmol/L trehalose could also improve the thermostability. However,β- cyclodextrin had no effect on the purified POD and PEG-6000 restrained the POD activity.