Properties of Invertases in Sugar Storage Tissues of Citrus Fruit and Changes in Their Activities during Maturation

Both acid and alkaline invertases were present in immature juice sacs of satsuma mandarin (Citrus‘Unshu Marcovitch”) fruit, in which sugar content was low. Maturing and mature juice sacs, in which sugar content increased steadily with time, were characterized by the presence of alkaline invertase and the absence of acid invertase. When the immature juice sacs were homogenized with 0.2 M sodium phosphate-citrate buffer (pH 8.0), almost all of the acid invertase activity was found in the solubilized fraction, whereas almost all of the alkaline invertase activity was present in the insoluble fraction. The distribution of alkaline invertase between the solubilized and insoluble fractions changed with the development of fruit. The acid invertase had a molecular weight of 69,000, optimum pH of 4.8–5.3, and K_m value for sucrose of 7.3 mM. The alkaline invertase had a molecular weight of 200,000, pH optimum of 7.2–7.7, and K_m value of 35.7 mM. The hydrolysing activities of both enzymes for raffinose were considerably less than those for sucrose. The alkaline invertase had lower activity for raffinose than the acid invertase.     

Acid and Neutral Invertases in the Mesocarp of Developing Muskmelon (Cucumis melo L. cv Prince) Fruit

Acid and neutral invertases were found in the mesocarp of developing muskmelon (Cucumis melo L. cv Prince) fruit and the activities of these enzymes declined with maturation of the fruit, concomitantly with the accumulation of sucrose. Neutral invertase was only present in the soluble fraction and acid invertase was present in both the soluble and cell-wall fractions. The cell-wall fraction contained three types of acid invertase: a NaCl-released invertase; an EDTA-released invertase, and a tightly bound invertase that still remained on the cell wall after treatment with NaCl and EDTA. The soluble acid and neutral invertases could be separated from one another by chromatography on DEAE-cellulose and they exhibited clear differences in their properties, namely, in their pH optima, substrate specificity, K_m values for sucrose, and inhibition by metal ions. The EDTA-released invertase and the soluble acid invertase were similar with regard to their chromatographic behavior on DEAE-cellulose, but the NaCl-released invertase was different because it was adsorbed to a column of CM-cellulose. The soluble acid invertase and two cell-wall bound invertases had very similar characteristics with regard to optimal pH and temperature, K_m value for sucrose, and substrate specificity.     

Acid Invertase of Melon Fruits: Immunochemical Detection of Acid Invertases

Soluble (S) and cell wall-bound (CW) forms of acid invertases(AIs) were partially purified from mesocarp tissue of melon(Cucumis melo L. cv. AMS) fruits and some properties of theenzymes were examined. The optimum pH values for activity were5.5 and 4.5 for S- and CW-AI, respectively. K_m values of S-and CW-AIs for sucrose were 4.2 and 1.7 mM, and those for raffinosewere 20.0 and 10.5 mM, respectively. The band of a polypeptideof about 70 kDa was detected with antibodies against AI of tomatofruits (CW form) and of mung bean seedlings (S form) by immunoblotanalysis. The 70-kDa polypeptide was isolated from each fractionand further analyzed by limited proteolytic digestion with Staphylococcusaureus V8 protease. The digested polypeptides generated almostidentical profiles. The localization of CW-AI was also studiedand it was found to be an extracellular enzyme. The activitiesof S- and CW-AIs per gram fresh weight increased and reacheda maximum on day 10 after anthesis, and then they decreasedrapidly during maturation of fruits. The relative levels ofS- and CW-AI polypeptides were correlated with the levels ofactivities of S- and CW-AIs. These results show that the decreasein activity of AI during maturation of melon fruits is due toa decline in levels of AI polypeptides in the fruits. (Received April 1, 1992; Accepted September 7, 1992)     

Aroma Components of Fresh Sugar Cane Juice

To study the behavior of Bifidobacterium toward oxygen, oxygen uptake was investigated in detail. The cells of Bifidobacterial strains absorbed considerable amounts of oxygen. The exogenous oxygen uptake activity changed depending upon the period of incubation. Bifidobacterial cells also had high endogenous oxygen uptake, which was, in B. longum strains, as high as about 80% of the exogenous oxygen uptake activity. Bifidobacterial cells accumulated considerable amounts of polysaccharide, which was associated with cellular growth. By incubating the cultivated cells in a glucose-free medium, the endogenous oxygen uptake activity was decreased with a decrease of intracellular polysaccharide. Therefore it was postulated that the high endogenous oxygen uptake activity of Bifidobacterium was owing to the metabolism of intracellular polysaccharide. The enzymatic activity, which was involved in the mechanism of oxygen uptake, was also investigated.     

Plastid Development and Ultrastructure in Yellowish Mutants of Sugar Cane

The comparisons of the ultrastructures of plastids in yellowish mutant and yellowish-green striped mutant and in normal green plant from tissue culture of sugar cane were made. There was no difference found in the structure and development of chloroplasts between the normal green leaves and the green tissue of striped leaves, but the plastid in the yellowish tissue of two kinds of mutants were anomalous. They had not a fully developed system of grana and stroma lameilae as in the normal green leaves. This aberrant plastid contained only some vesicles, a few lamellae and more or less clearly defined ribosome particles and DNA-like mierofibrils, while some stacking and swelling of thylakoids were often observed. In some sections of this aberrant plastid a bunchy lamellae and cross connective fibrils between parallel lamellae were often found too. However, the mixed cell which contains both of normal chloroplast and defective plastid together was never found in the leaves of the mutant plants. It was suggested that yellowish and yellowish-green striped leaves from tissue culture of sugar cane might be caused by nuclear gene mutation.     

Acetic Acid Bacterial Biota of the Pink Sugar Cane Mealybug, Saccharococcus sacchari, and Its Environs

Saccharococcus sacchari is the primary colonizer of the developing “sterile” tissue between the leaf sheath and stem of sugar cane. The honeydew secreted by the mealybugs is acidic (about pH 3) and supports an atypical epiphytic microbiota dominated by acetobacter-like bacteria and acidophilic yeast species. However, Erwinia and Leuconostoc species predominate within the leaf sheath pocket region when the mealybugs die out. The unidentified acetobacters were readily isolated from S. sacchari throughout its life cycle and from other genera of mealybugs on sugar cane and various other plants, both above and below ground. No other insect present on sugar cane was a significant vector of acetic acid bacteria. The major factors restricting microbial diversity within the environs of mealybugs were considered to be yeast activity along with bacterial production of acetic acid, ketogluconic acids, and gamma-pyrones, in association with their lowering of pH. The microbial products may aid in suppressing the attack by the parasitic mold Aspergillus parasiticus on mealybugs but could act as attractants for the predatory fruit fly Cacoxenus perspicax.     

应用果胶酶澄清甘蔗汁的研究

以甘蔗为原料,采用果胶酶和自然澄清方法进行甘蔗汁澄清比较,结果表明,酶促效果好于自然澄清,100ml甘蔗汁添加200U果胶酶在25℃、12h或45℃、1h条件下,均可使甘蔗汁的澄清度由5%提高到95%,粘度由4.0mPa·s下降到1.1mPa·s。     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Effects of Cadmium on Antioxidant Enzyme Activities in Sugar Cane

Sugar cane (Saccharum officinarum L. cv. Copersucar SP80-3280) seedlings were grown in nutrient solution with varying concentrations (0, 2 and 5 mM) of cadmium chloride for 96 h. Leaves were analysed for catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) activities. Although a clear effect of CdCl₂ on plant growth was observed, the activity of SOD was not altered significantly. However, the CAT activity decreased as the concentration of CdCl₂ increased. GR exhibits a significant increase in activity at 2 and 5 mM CdCl₂. CAT and SOD isoenzymes were further characterised by analysis in non-denaturing PAGE. Activity staining for SOD revealed up to seven isoenzymes in untreated control and 2 mM CdCl₂ treated plants, corresponding to Cu/Zn-SOD isoenzymes. At 5 mM CdCl₂, only six Cu/Zn-SOD isoenzymes were observed. No Fe-SOD and Mn-SOD isoenzymes were detected. For CAT, one band of activity was observed.     

糖和脱落酸及乙烯互作及其与植物生长发育的关系

从分子角度介绍和讨论了植物(主要是拟南芥)生长发育过程中,植物糖信号的复杂信号网络和作用,特别是它与脱落酸和乙烯合成的关系.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves

A procedure is described for the purification of phosphoenolpyruvatecarboxylase (EC 4.1.1.31 [EC] ) and NADP-dependent malic enzyme (EC1.1.1.40 [EC] ) from sugar cane leaves. Each enzyme was purified tohomogeneity as judged by sodium dodecyl sulfate-polyacrylamidegel electro-phoresis, with about 30% yield. Phosphoenolpyruvatecarboxylase was purified 54-fold. A molecular weight of 400,000and a homotetrameric structure were determined for the nativeenzyme. The purified carboxylase had a specific activity of20.0 {diaeresis}mol (mg protein)_–1 min_–1, and wasactivated by glucose-6-phosphate and inhibited by L-malate.K_m values at pH 8.0 for phosphoenolpyruvate and bicarbonatewere 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-foldpurified, exhibited a specific activity of 71.2 {diaeresis}mol(mg protein)_–1 min_–1 and was characterized as ahomotetramer with native molecular weight of 250,000. Purifiedmalic enzyme showed an absolute specificity for NADP⁺ and requireda divalent metal ion for activity. K_m values of 0.33 and 0.008mM for L-malate and NADP⁺, respectively, were determined. Thisenzyme was inhibited by several organic acids, including ketoand amino acids; while succinate and citrate increased the enzymeactivity when assayed with 10{diaeresis}M L-malate. The effectsshown by amino acids and by citrate were dependent on pH, beinghigher at pH 8.0 than at pH 7.0. (Received October 26, 1988; Accepted February 3, 1989)