Aminopeptidase (AP) activity in ripe but firm fruit of
Kiwifruit (
Aminopeptidases (APs), particularly those from microbial sources, are important food processing enzymes and are widely used to modify proteins in food [
Generally, there are many studies on seed aminopeptidases [
Ripe but firm kiwifruit (
The protein concentration in extracts was determined based on the Coomassie brilliant blue dye-protein binding principle [
Aminopeptidase activity was determined as described below unless indicated otherwise using L-leucine-
The effect of temperature on AP activity was determined in three different experiments. To find the optimum temperature for the enzyme activity, AP activity in crude extracts of the whole fruit was determined at different incubation temperatures ranging from
The effect of pH on AP activity in the crude extracts of fruit was determined by replacing the potassium phosphate buffer at pH 8.0 in the assay mixture, with the three buffer mixtures (25.0 mM acetic acid, 25.0 mM MES, and 50.0 mM Tris) at different pH values ranging from 6 to 10 as described in [
Crude enzyme extracts were preincubated with 0.45 ml of 0.1 M potassium phosphate buffer (pH 8) in the presence of different inhibitors or activators for 30 min at
The crude enzyme extracts were pre-incubated at
The whole kiwifruit (550 g) was cut into small pieces and homogenized in 225 ml of 0.1 M of potassium phosphate buffer (pH 8.0) supplemented with 1% (w/v) insoluble PVPP, 5% (v/v) glycerol, and 3 mM DTT (extraction buffer). The homogenate was filtered through 2 layers of synthetic cloth. The filtrate was centrifuged at 10,000
Statistical analysis of the data was performed using STATISTIX 8.0 software. The comparison between treatments was analysed using one-way analysis of variance (ANOVA). Where a statistical significance was observed, a Tukey’s Honest Significance Difference (HSD) test was performed to determine how significant from the appropriate zero the values were. Standard errors were calculated and graphically represented as symmetrical error bars.
In preliminary experiments, when crude extracts from the whole fruit had been prepared with sodium phosphate or potassium phosphate buffer (pH 7.0), AP activity was not detectable. Kiwifruit contains more than 80 volatile aroma, and flavour compounds including terpenses, esters, aldehydes, alcohols with varying levels of monoterpenes, and phenolic compounds [
AP activity was found in all parts of the fruit of
Aminopeptidase activity in different parts of kiwifrui
Type of tissue | Total activity (units/g fresh weight) | Specific activity (units/mg soluble protein) |
---|---|---|
Outer pericarp | ||
Inner pericarp | ||
Core | ||
Seed |
AP activity in crude extracts of the whole kiwifruit was most active at alkaline pH (Figure
Effect of pH on aminopeptidase activity in extracts of the whole fruit of
Kiwifruit AP was most active at
Effect of temperature on the aminopeptidase activity in the crude extracts prepared from the whole fruit of
Effect of temperature on the stability of aminopeptidase activity in crude extracts prepared from the whole fruit of
The presence of 1 mM of 1,10-phenanthroline, EDTA-Na2 and iodoacetamide inhibited kiwifruit aminopeptidase activity (Figure
Effect of proteolytic enzyme inhibitors and activators on aminopeptidase activity in crude extract of the whole fruit of
The observed inhibition of kiwifruit AP activity by metal chelators such as 1,10-phenanthroline and EDTA suggested the involvement of a metal ion in the active site of the enzyme. Similar effects were also reported in the studies on leucine aminopeptidases of potato [
The effects on kiwifruit AP activity of
Effect of divalent cations on aminopeptidase activity in crude extracts of the whole fruit of
Two major peaks of AP activity were separated using DEAE cellulose column chromatography: the unadsorbed and adsorbed fractions (Figure
Elution profile from a DEAE cellulose column of aminopeptidase (AP) activity and protein content in a concentrated fraction of ammonium sulfate precipitation of crude extracts of