SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO

1. A method is given whereby the course of hydrolysis of sucrose by live yeast cells may be followed with precision equal to that found when invertase solutions prepared from autolyzed yeast are used to cause inversion. 2. The practical value of the equation of Nelson and Hitchcock as a means of following the course of enzymic hydrolysis of sucrose is hereby extended. 3. The inversion of sucrose by live yeast cells and by extracted invertase has been quantitatively compared. 4. The course of hydrolysis of sucrose by the invertase of Fleischmann''s yeast has been found to be identical in vivo and in vitro.     

SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H⁺) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen''s α-glucosidase (maltase), was found.     

FORMATION OF AUXIN IN YEAST CULTURES

We have found far more auxin in the culture media of bakers'' yeast than was obtained by Kögl and Kostermans from the cells themselves. The production of auxin by yeast cells resembles the formation observed in other organisms such as Rhizopus and Rhizobium which also form auxins in their culture media. The auxin yield was found to increase with the concentration of sucrose and to decrease with the concentration of peptone. An inverse relation with the rate of cell multiplication was observed. Enlarged and elongated cells appeared only in those media which contained considerable amounts of auxin. The total auxin yield in the various cultures was found to be directly proportional, below pH 5, to the hydrogen ion concentration. Thus, it was proposed that certain growth conditions favor the breakage of the link between auxin and its protein carrier (Skoog and Thimann) 1940) and consequently accelerate the rate of excretion of auxin into the growth medium.     

The development of invertase activity in slices of the root of Beta vulgaris L. washed under aseptic conditions

1. When disks of root tissue from sugar or red beet (Beta vulgaris L.) are washed in running aerated tap water the sucrose contained in them disappears and glucose and fructose are formed. 2. Invertase activity in the disks has been measured by a polarimetric method. Freshly cut tissue has a very low activity, but a considerable increase occurs during the first 3–4 days of washing, the final activity being sufficient to hydrolyse the sucrose contained in the disk within a few hours. 3. Disks of red beet have been cut and shaken in water under aseptic conditions. Sucrose breakdown and invertase development still took place. Microbial contamination is therefore not responsible. 4. Trisaccharides that appear in sugar-beet disks during the washing process have been isolated and identified; their formation also suggests that a higher-plant invertase is acting. 5. The significance of these results is discussed in relation to protein synthesis in washed storage-tissue slices, and the occurrence of high invertase activity in growing plant cells.     

Non-specific β-fructofuranosidase (inulase) from Kluyveromyces fragilis: Batch and continous fermentation,simple recovery method and some industrial properties

The yeast Kluyveromyces fragilis was grown in continuous culture on a complex medium containing sucrose as the carbon source and limiting nutrient. The inulase yield was 7000 μg hexose/min.mg biomass, determined with 4% sucrose at 50°C and pH 5.0. Maximum amount of enzyme was formed at the lowest dilution rate (0.09 h^?1) tested. Any increase in dilution rate caused a severe reduction in activity per unit biomass due to carbon catabolite repression. Inulase yields were constant in the pH range 3.5 to 6.0 and in the dissolved oxygen tension range 2.5 to 40% of saturation. Continuous cultivation on sucrose produced twice as much inulase as the previously used method of batch cultivation on the expensive substrate inulin. A high quality industrial grade inulase was prepared by autolysis of the yeast cells, ultrafiltration of the supernatant and acetone precipitation. Under the assay conditions used the preparation showed an activity ratio toward sucrose and inulin of 10.5 as compared to 1600 for baker's yeast invertase. Further comparison with invertase showed that it is at least as resistant to substrate inhibition, as thermostable and has slightly less transferase activity. These results suggest that K. fragilis inulase may represent an alternative to Saccharomyces cerevisiae invertase presently used in industry.     

Tris–sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61

The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris–sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.     

A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells

Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acer pseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific. At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The K_m for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDPglucose pyrophosphorylase, which is shown to be an effective `PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly 1/10 the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible regulation and energetic differences between the sucrose synthase and invertase pathways are discussed.     

Acid and alkaline invertases in suspension cultures of sugar beet cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K_m values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K_m values for sucrose.     

An invertase inactivator in maize endosperm and factors affecting inactivation

A protein present in the developing endosperm of maize (Zea mays L.) causes a loss of invertase activity under certain conditions of incubation. This protein, designated an inactivator, inactivates invertase I of maize even in the presence of other proteins. No inactivation of invertase II of maize or yeast invertase has been observed. The inactivator and invertase I are found only in the endosperm. The quantity of inactivator increases in the normal endosperm during development while invertase I activity decreases. However, the altered levels of invertase I activity in several endosperm mutant lines do not result from different quantities of inactivator. The inactivator can decrease invertase I activity during a preincubation period before addition of sucrose; inactivation is noncompetitive. Invertase I activity decreases curvilinearly with an increase in inactivator concentration. At high buffer concentrations or low inactivator concentrations in the reaction mixture, a latent period is observed when invertase I is not inactivated. Inactivation increases with an increase in temperature and a decrease in pH.     

The genetics of a putative social trait in natural populations of yeast

The sharing of secreted invertase by yeast cells is a well‐established laboratory model for cooperation, but the only evidence that such cooperation occurs in nature is that the SUC loci, which encode invertase, vary in number and functionality. Genotypes that do not produce invertase can act as ‘cheats’ in laboratory experiments, growing on the glucose that is released when invertase producers, or ‘cooperators’, digest sucrose. However, genetic variation for invertase production might instead be explained by adaptation of different populations to different local availabilities of sucrose, the substrate for invertase. Here we find that 110 wild yeast strains isolated from natural habitats, and all contained a single SUC locus and produced invertase; none were ‘cheats’. The only genetic variants we found were three strains isolated instead from sucrose‐rich nectar, which produced higher levels of invertase from three additional SUC loci at their subtelomeres. We argue that the pattern of SUC gene variation is better explained by local adaptation than by social conflict.     

Effect of cultivation conditions on invertase production by hyperproducing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> isolates

Invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) finds major uses in confectionery and in the production of invert syrup. In the present study, we report on invertase production by wild cultures of Saccharomyces cerevisiae. The yeast strains were isolated from dates available in a local market. Five hyperproducing yeast strains (>100- fold higher invertase activity) were kinetically analysed for invertase production. Saccharomyces cerevisiae strain GCA-II was found to be a better invertase-yielding strain than all the other isolates. The values of Q_p and Y_p/s for GCA-II were economical as compared to other Saccharomyces cultures. The effect of sucrose concentration, rate of invertase synthesis, initial pH of fermentation medium and different organic nitrogen sources on the production of invertase under submerged culture conditions was investigated. Optimum concentrations of sucrose, urea and pH were 3, 0.2 (w/v), and 6 respectively. The increase in the enzyme yield obtained after optimization of the cultural conditions was 47.7%.     

THE ACTIVITY OF YEAST INVERTASE AS A FUNCTION OF OXIDATION-REDUCTION POTENTIAL

The activity of yeast invertase as a function of oxidation-reduction potential has been investigated using a large number of oxidants and reductants. The activity is constant over the range of E_h from –270 to +600 mv., but above E_h = +600 mv. there is a sharp decrease in activity reaching 0 at E_h = +1,000 mv. The inhibiting action of strong oxidants is upon the enzyme rather than on the substrate and appears to be essentially irreversible Experiments indicate that the inhibiting action of strong oxidants on invertase is primarily related to their high oxidation-reduction potential rather than to a specific toxic action unrelated to E_h. The effects of oxidation-reduction potential upon invertase activity are independent of the purity of the enzyme, since they are the same for commercial invertases, fresh bakers'' yeast, powdered bakers'' yeast, brewers'' yeast, and highly purified invertase. Possible mechanisms involved in the inactivation of invertase by oxidants are discussed.     

A sucrose binding protein homologue from soybean affects sucrose uptake in suspension-cultured transgenic tobacco cells

We have obtained Nicotiana tabacum transgenic cell lines expressing a sucrose binding protein (sbp) homologue gene from soybean (Glycine max L.), designated s-64, either in the sense or antisense orientation. Sense cell lines over-accumulated the S-64 protein, whereas the antisense cell lines had reduced levels of the endogenous homologue protein. Sucrose uptake experiments were conducted by incubating suspension-cultured tobacco cells with radiolabeled sucrose at pH 4.5 or 7.0. Raising the extracellular pH to 7.0 caused an inhibition of radiolabeled carbon uptake efficiency, which was attributed to the pH-sensitivity of cell-wall invertase (EC 3.2.1.26), H⁺/hexose transporter and/or H⁺/sucrose symporter activities. Because SBP-mediated sucrose uptake has been shown to be insensitive to extracellular pH in yeast, we performed the sucrose uptake experiments in sense and antisense cultured cells at pH 7.0. Under this condition, the level of SBP homologue correlated with the efficiency of radiolabeled uptake by the transgenic tobacco cells. Furthermore, manipulation of S-64 levels altered sucrose-cleaving activities in a metabolic compensatory manner. Enhanced accumulation of S-64 caused an increase in intracellular sucrose synthase (cleavage, EC 2.4.1.13) activity with a concomitant decline in cell-wall invertase activity. This result may reflect a metabolic adjustment of the sense cell lines caused by its high efficiency of direct sucrose uptake as disaccharide. In contrast, the level of cell-wall invertase activity was remarkably increased in antisense cells, favoring the invertase-dependent sugar uptake system. Collectively, these results may establish a functional link between radiolabeled influx and S-64 accumulation, suggesting that SBP affects sucrose uptake in suspension-cultured cells.     

Invertases in Oat Seedlings: SEPARATION, PROPERTIES, AND CHANGES IN ACTIVITIES IN SEEDLING SEGMENTS

The soluble invertase activity in etiolated Avena seedlings was highest at the apex of the coleoptile and much lower in the primary leaf, mesocotyl, and root. The activity in all parts of the seedling consisted of two invertases (I and II) which were separated by chromatography on diethylaminoethylcellulose. Both enzymes appeared to be acid invertases, but they differed in molecular size, pH optimum, and the kinetic parameters K_m and V_max of their action on sucrose, raffinose, and stachyose. Invertase II had low stability at pH 3.5 and below, and exhibited high sensitivity to Hg²⁺, with complete inhibition by 2 micromolar HgCl₂. Segments of coleoptiles incubated in water lost about two-thirds of the total invertase activity after 16 hours. The loss of activity was due primarily to a decrease in the level of invertase II. The loss of invertase was decreased by indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and α-naphthaleneacetic acid but not by β-naphthaleneacetic acid and p-chlorophenoxyisobutyric acid. Conditions that inhibited auxin-induced growth of the segments (20 millimolar CaCl₂ and 200 millimolar mannitol) also blocked the auxin effect on invertase loss.     

Production of a novel transfructosylating enzyme from Bacillus macerans EG-6

A new bacterium producing a novel transfructosylating enzyme was isolated from soil and designated as Bacillus macerans EG-6. Various culture conditions for enzyme production were optimized in a flask culture. 1% (w/v) sucrose as a carbon source and a mixed nitrogen source (1% yeast extract, 1% polypeptone, and 0.5% ammonium chloride) gave the best enzyme production. Addition of phosphate and magnesium ion into the medium enhanced the enzyme yield. Optimum culture pH and temperature were 7.0 and 37?°C, respectively. Under optimal culture conditions, transfructosylating enzyme was rapidly produced in the early growth period, thereafter invertase activity was predominant as the culture proceeded. Using the culture filtrate, production of fructooligosaccharides from sucrose was preliminarily carried out. In a low sucrose concentration (200?g/l), transfructosylating activity competes with invertase activity in sucrose utilization. Subsequently, low fructooligosaccharide yield (20%) was achieved due to liberation of high amounts of glucose and fructose. The best oligosaccharide yield (43%) was achieved when 500?g/l sucrose was utilized.     

Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus

The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0–6.0 and 55–65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0–10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²⁺ (161 %), Co²⁺ (68 %) and Mg²⁺ (61 %) and was inhibited by Al³⁺, Ag⁺, Fe²⁺ and Fe³⁺. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K_M = 22 mM). However, in the presence of Mn²⁺, the apparent affinity and V_max for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V_max was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.     

The naturally occurring silent invertase structural gene suc2 zero contains an amber stop codon that is occasionally read through

Summary The yeast invertase structural gene SUC2 has two naturally occurring alleles, the active one and a silent allele called suc2°. Strains carrying suc2° are unable to ferment sucrose and do not show detectable invertase activity. We have isolated suc2° and found an amber codon at position 232 of 532 amino acids. However, transformants carrying suc2° on a multicopy plasmid were able to ferment sucrose and showed detectable invertase activity. Full-length invertase was found in gels stained for active invertase and in immunoblots. Therefore we concluded that the amber codon is occasionally read as an amino acid. The calculated frequency of read-through is about 4% of all translation events.     

Continuous inversion of sucrose by gel-entrapped yeast cells

The feasibility of immobilizing invertase (β-d-fructofuranosidase; EC 3.2.1.26) from Saccharomyces cerevisiae cells by various methods was examined. The yeast cells were adapted for maximal invertase activity by growth in a medium containing 0.2% glucose and 1% lactate. There was no permeability barrier for the enzyme in the whole cells. Entrapment in acrylamide polymerized by gamma-rays (200 kR) was observed to be most effective, with retention of 85% of the activity. The evaluation of the properties of the immobilized invertase indicated that the kinetic values were not appreciably altered despite a broad pH optimum. The enzyme was more stable to both heat and gamma-radiation. The immobilized cells could be used repeatedly in a packed bed reactor system for inversion of sucrose without observable loss in activity for over one month.     

An-Overview on invertase in sugarcane

Saccharum officinarum is one of the most cultivated hybrid varieties among the sugarcane varieties. In sugarcane plant sucrose is the major carbohydrate which can be stored and transported. Different physiological and biochemical studies on this crop report that invertase activity and sucrose concentration some how are key limiting step in the process of sucrose accumulation. Significant efforts have been made in relation to the sucrose cycle by altering the sucrose phosphate synthetase, sucrose synthetase and invertase. In sugarcane two types of invertase enzymes have been reported on the basis of pH and cellular localization. Invertase breaks the sucrose into hexoses as a source of energy and carbon. It has also been reported that this enzyme is involved in the process of cell differentiation and plant development. Progress has been made for the understanding of invertase activity and its role in sugarcane plant. With the help of biotechnology it is possible to target the desired gene with genetic engineering approach to increase sucrose content by careful manipulation of invertase (enzyme) gene to increase the sucrose yield in sugarcane. Purpose of this mini review is to high-light the role of invertase in sugarcane and how to overcome sucrose recovery in sugarcane.     

A membrane-associated isozyme of invertase in yeast precursor of the external glycoprotein

An enzymatic test is described which allows the localization of yeast invertase activity directly on sodium dodecyl sulfate gels. When crude membrane fractions are prepared from Saccharomyces cerevisiae cells which are actively synthesizing external invertase, these membranes show an activity band on sodium dodecyl sulfate gels additional to the external and the internal invertase. In the soluble fraction this form was completely absent. It has a molecular weight of approximately 190 000 and was 50 000 smaller than the external invertase. It showed kinetic characteristics of a precursor of the external enzyme. Thus it appeared transiently, when yeast cells were shifted from a condition of non-synthesizing external invertase to one where the enzyme was synthesized. When the increase in the external enzyme slowed down after some time, the membrane-associated form almost completely disappeared.The addition of tunicamycin to yeast cells synthesizing external invertase inhibited further synthesis of the enzyme by 97%; also the formation of the membrane-associated form was strongly inhibited. The amount of it present before the addition of tunicamycin completely disappeared in the presence of the antibiotic. The precursor form, therefore, seems to possess already those carbohydrate parts which contain N-acetylglucosamine and are transferred via dolichyl phosphate-bound intermediates. The membrane-associated precursor amounts to less than 5% of the total invertase activity of a yeast cell.