关键词: 枇杷, 过氧化物酶, 分离纯化, 酶学性质, 酶促褐变

Abstract: Peroxidase in the acetone powder of loquat (Eriobotrya japonica Lindl. cv. Zaozhong 6) fruit was extracted and purified through ammonium sulfate fractionation, dialysis and chromatography on DEAE-cellulose (DE-32). Two enzyme activity peaks were obtained, and the purified enzyme from the second activity peak determined to be homogeneous by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE. The specific activity of the purified enzyme was 710.5 U/mg. Molecular weight of enzyme subunit was 22.6 kDa and the pI value was calculated to be 3.6 by isoelectric focusing. The optimum pH and optimum temperature of the enzyme were pH 5.0 and 35℃, respectively. The stability of the enzyme was investigated, and the results show ed that the enzyme was stable relatively in a pH range from 2.6 to 10.0 and at temperatures <45℃. Kinetic studies showed that the reaction catalyzed by the enzyme followed the typical behavior of ordered mechanism of two-substrate reaction. The K_mapp values of 4-methylcatechol and H₂O₂ were 20.58 and 12.04 mmol/L, respectively. Several compounds including phenolic compounds were used as the substrates of the enzyme for specificity study. Meanwhile, the effects of metal ions on the enzyme were studied. The result indicated Mg ²⁺, Ca²⁺, Cu²⁺, Fe²⁺, Ba²⁺ and Pb²⁺ activated the enzyme, while Al³⁺, Mn²⁺, Zn²⁺ and Hg²⁺ show ed various degrees of inhibitory effects on the enzyme. The effects of twelve inhibitors on this peroxidsae were also assayed, and this study may provide some enzymatic inhibitors for post-harvest treatment to delay such browning.

Key words: loquat fruit, peroxidase, isolation and purification, enzyme property, enzymic browning