During yeast dough fermentation, such as the high-sucrose bread-making process, the yeast cells are subjected to considerable osmotic stress, resulting in poor outcomes. Invertase is important for catalyzing the irreversible hydrolysis of sucrose to free glucose and fructose, and decreasing the catalytic activity of the invertase may reduce the glucose osmotic stress on the yeast. In this study, we performed structural design and site-directed mutagenesis (SDM) on the Saccharomyces cerevisiae invertase (ScInV) in an Escherichia coli expression system to study the catalytic activity of ScInV mutants in vitro. In addition, we generated the same mutation sites in the yeast endogenous genome and tested their invertase activity in yeast and dough fermentation ability. Our results indicated that appropriately reduced invertase activity of yeast ScInV can enhance dough fermentation activity under high-sucrose conditions by 52%. Our systems have greatly accelerated the engineering of yeast endogenous enzymes both in vitro and in yeast, and shed light on future metabolic engineering of yeast.

• Klíčová slova
• Fermentation ability, Invertase activity, Protein engineering, Site-directed mutagenesis,
• MeSH
• fermentace MeSH
• glukosa metabolismus   MeSH
• invertasa * genetika   metabolismus   MeSH
• proteinové inženýrství MeSH
• Saccharomyces cerevisiae * metabolismus   MeSH
• sacharosa metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glukosa MeSH
• invertasa * MeSH
• sacharosa MeSH

Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.

• Klíčová slova
• Cell wall invertase, cytokinins, drought stress, ethylene, source–sink relationships, tomato.,
• MeSH
• buněčná stěna enzymologie   MeSH
• Chenopodium genetika   metabolismus   MeSH
• ektopická exprese * MeSH
• fotosyntéza MeSH
• geneticky modifikované rostliny genetika   metabolismus   MeSH
• invertasa genetika   metabolismus   MeSH
• listy rostlin metabolismus   MeSH
• období sucha * MeSH
• regulace genové exprese u rostlin * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• Solanum lycopersicum enzymologie   genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• invertasa MeSH
• rostlinné proteiny MeSH

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.

• MeSH
• biotechnologie metody   MeSH
• časové faktory MeSH
• Cryptococcus enzymologie   metabolismus   MeSH
• imobilizované buňky enzymologie   metabolismus   MeSH
• invertasa izolace a purifikace   MeSH
• koncentrace vodíkových iontů MeSH
• kultivační média chemie   MeSH
• pufry MeSH
• sacharosa MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• invertasa MeSH
• kultivační média MeSH
• pufry MeSH
• sacharosa MeSH

After periodate oxidation of its glycosidic component, invertase was covalently bound onto three types of modified solid supports: glycidyl methacrylate, styrene-divinylbenzene copolymers, and bead cellulose. Direct reaction of the invertase aldehyde groups that were formed with amino groups of the support and use of the modified Ugi reaction have been employed as immobilization procedures. Apart from binding methods, the important effects of the buffer, support, conditions of periodate oxidation, and the length of the spacer on the activity of the enzyme conjugate have been investigated. Superior conjugate activity was obtained, via modified Ugi reaction, by the immobilization of a suitably oxidized invertase to a styrene-divinylbenzene copolymer having free amino groups.

• Publikační typ
• časopisecké články MeSH

Salinization of water and soil has a negative impact on tomato (Solanum lycopersicum L.) productivity by reducing growth of sink organs and by inducing senescence in source leaves. It has been hypothesized that yield stability implies the maintenance or increase of sink activity in the reproductive structures, thus contributing to the transport of assimilates from the source leaves through changes in sucrolytic enzymes and their regulation by phytohormones. In this study, classical and functional physiological approaches have been integrated to study the influence of metabolic and hormonal factors on tomato fruit sink activity, growth, and yield: (i) exogenous hormones were applied to plants, and (ii) transgenic plants overexpressing the cell wall invertase (cwInv) gene CIN1 in the fruits and de novo cytokinin (CK) biosynthesis gene IPT in the roots were constructed. Although salinity reduces fruit growth, sink activity, and trans-zeatin (tZ) concentrations, it increases the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) during the actively growing period (25 days after anthesis). Indeed, exogenous application of the CK analogue kinetin to salinized actively growing fruits recovered sucrolytic activities (mainly cwInv and sucrose synthase), sink strength, and fruit weight, whereas the ethylene-releasing compound ethephon had a negative effect in equivalent non-stressed fruits. Fruit yield was increased by both the constitutive expression of CIN1 in the fruits (up to 4-fold) or IPT in the root (up to 30%), owing to an increase in the fruit number (lower flower abortion) and in fruit weight. This is possibly related to a recovery of sink activity in reproductive tissues due to both (i) increase in sucrolytic activities (cwInv, sucrose synthase, and vacuolar and cytoplasmic invertases) and tZ concentration, and (ii) a decrease in the ACC levels and the activity of the invertase inhibitor. This study provides new functional evidences about the role of metabolic and hormonal inter-regulation of local sink processes in controlling tomato fruit sink activity, growth, and yield under salinity.

• Klíčová slova
• Cell wall invertase, cytokinins, fruit, salinity, sink activity, tomato.,
• MeSH
• biomasa MeSH
• buněčná stěna enzymologie   MeSH
• chlorid sodný farmakologie   MeSH
• cytokininy metabolismus   MeSH
• exprese genu MeSH
• geneticky modifikované rostliny MeSH
• invertasa genetika   metabolismus   MeSH
• kořeny rostlin účinky léků   genetika   metabolismus   MeSH
• květy účinky léků   genetika   metabolismus   MeSH
• kyselina abscisová metabolismus   MeSH
• ovoce účinky léků   genetika   metabolismus   MeSH
• regulátory růstu rostlin farmakologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sacharosa metabolismus   MeSH
• salinita MeSH
• sekvestrace uhlíku MeSH
• Solanum lycopersicum účinky léků   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorid sodný MeSH
• cytokininy MeSH
• invertasa MeSH
• kyselina abscisová MeSH
• regulátory růstu rostlin MeSH
• rostlinné proteiny MeSH
• sacharosa MeSH

Pollination in flowering plants is initiated by germination of pollen grains on stigmas followed by fast growth of pollen tubes representing highly energy-consuming processes. The symplastic isolation of pollen grains and tubes requires import of Suc available in the apoplast. We show that the functional coupling of Suc cleavage by invertases and uptake of the released hexoses by monosaccharide transporters are critical for pollination in tobacco (Nicotiana tabacum). Transcript profiling, in situ hybridization, and immunolocalization of extracellular invertases and two monosaccharide transporters in vitro and in vivo support the functional coupling in supplying carbohydrates for pollen germination and tube growth evidenced by spatiotemporally coordinated expression. Detection of vacuolar invertases in maternal tissues by these approaches revealed metabolic cross talk between male and female tissues and supported the requirement for carbohydrate supply in transmitting tissue during pollination. Tissue-specific expression of an invertase inhibitor and addition of the chemical invertase inhibitor miglitol strongly reduced extracellular invertase activity and impaired pollen germination. Measurements of (competitive) uptake of labeled sugars identified two import pathways for exogenously available Suc into the germinating pollen operating in parallel: direct Suc uptake and via the hexoses after cleavage by extracellular invertase. Reduction of extracellular invertase activity in pollen decreases Suc uptake and severely compromises pollen germination. We further demonstrate that Glc as sole carbon source is sufficient for pollen germination, whereas Suc is supporting tube growth, revealing an important regulatory role of both the invertase substrate and products contributing to a potential metabolic and signaling-based multilayer regulation of pollination by carbohydrates.

• MeSH
• 1-deoxynojirimycin analogy a deriváty   farmakologie   MeSH
• biologické modely MeSH
• hexosy metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• invertasa antagonisté a inhibitory   metabolismus   MeSH
• klíčení účinky léků   MeSH
• opylení účinky léků   MeSH
• proteiny přenášející monosacharidy genetika   metabolismus   MeSH
• pylová láčka účinky léků   enzymologie   růst a vývoj   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• reprodukovatelnost výsledků MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sacharidy farmakologie   MeSH
• tabák enzymologie   genetika   metabolismus   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-deoxynojirimycin MeSH
• hexosy MeSH
• inhibitory enzymů MeSH
• invertasa MeSH
• miglitol MeSH Prohlížeč
• proteiny přenášející monosacharidy MeSH
• rostlinné proteiny MeSH
• sacharidy MeSH

Activities of twelve hydrolytic enzymes in the digestive tract of young rabbits before weaning (4 weeks old) and adult rabbits (3 months old) were measured. The principal digestive enzymes in both groups of rabbits appeared to be amylase (EC 3.2.1.1), maltase (EC 3.2.1.20), pectinase (EC 3.2.1.15) and proteinases. The stomach of young rabbits contained most of the lipolytic activity and 45.7% of the total proteolytic activity of the digestive tract. The highest specific activities (per g digesta) of amylase, maltase and proteinase in young rabbits were found in the small intestine. Total activities (per segment) of amylase and maltase in the small intestine and the caecum were similar. Activities of cellulase (EC 3.2.1.4), inulinase (EC 3.2.1.7) and beta-glucosidase (EC 3.2.1.21) were low and activity of pectinase was fairly high in all segments of the digestive tract. The highest activity of urease (EC 3.5.1.5) was found in the caecum. Enzymic profiles of the colonic chymus resembled those of the caecum. Total hydrolytic activity was lower in the colon than in the caecum. Specific activities of amylase and invertase (EC 3.2.1.26) were lower and those of inulinase and lactase (EC 3.2.1.23) higher in 4-week-old rabbits than in 3-month-old rabbits. Gastric proteinase represented almost half of the total proteolytic activity of the digestive tract, whereas lipolytic activity of gastric contents was not found in measurable quantities in adult rabbits. The caecal contents of adult rabbits contained most of the total activity of lipase (EC 3.1.1.3), cellulase, xylanase (EC 3.2.1.32), pectinase, lactase, invertase, beta-glucosidase and urease present in the digestive tract.(ABSTRACT TRUNCATED AT 250 WORDS)

• MeSH
• alfa-glukosidasy metabolismus   MeSH
• amylasy metabolismus   MeSH
• beta-galaktosidasa metabolismus   MeSH
• cékum enzymologie   MeSH
• endopeptidasy metabolismus   MeSH
• glykosidhydrolasy metabolismus   MeSH
• insulinasa metabolismus   MeSH
• invertasa MeSH
• kolon enzymologie   MeSH
• králíci metabolismus   MeSH
• polygalakturonasa metabolismus   MeSH
• tenké střevo enzymologie   MeSH
• věkové faktory MeSH
• žaludek enzymologie   MeSH
• zvířata MeSH
• Check Tag
• králíci metabolismus   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• alfa-glukosidasy MeSH
• amylasy MeSH
• beta-galaktosidasa MeSH
• endopeptidasy MeSH
• glykosidhydrolasy MeSH
• insulinasa MeSH
• invertasa MeSH
• polygalakturonasa MeSH

The activity of enzymes in the digestive tract is an important parameter for appropriate digestive tract function. Feed mixtures can be adjusted to support enzymatic activity in different parts of the digestive tract. Flaxseed and hemp seed are commodities and significant sources of nutrition, and their addition to feed could change enzymatic activity in the digestive tract and improve nutritional intake. The aim of this study was to determine the effects of flaxseed, hemp seed and a combination of both on basic enzymes in the polysaccharidase group, such as amylase, cellulase, pectinase, xylanase and inulinase; basic enzymes in the disaccharidase group, including maltase, invertase and lactase; proteinases and lipases in the digestive tract of broiler chickens. During the experiment, the control group was fed a diet without flaxseed or hemp seed. The diet of the second group contained 80 g/kg flaxseed, the diet of the third group contained 40 g/kg hemp seed, and the diets of the fourth to sixth groups contained 80 and 30 g/kg, 80 and 40 g/kg and 80 and 50 g/kg flaxseed and hemp seed, respectively. Enzyme activity was found to depend on the location in the digestive tract and the composition of the feed mixture (P < 0.05). Most enzymatic conversion occurs in the ileum, where the addition of flaxseed and hemp seed to the diet increased most enzyme activities, namely, amylase, cellulase, pectinase, xylanase, maltase, invertase, proteinase and lipase activities. The highest values of enzyme activity were found in groups IV-VI fed a combination of flaxseed and hempseed, especially in chickens fed diet VI (flaxseed and hemp seed at 80 and 50 g/kg). Growth performance results confirmed the enzyme activity results, as the weights of the chickens increased after the addition of flaxseed and/or hemp seed. The findings have economic implications, suggesting that feeding a diet with a combination of flaxseed and hemp seed is beneficial.

• Klíčová slova
• Enzymes, Flax, Hemp, Nutrition, Poultry,
• MeSH
• alfa-glukosidasy MeSH
• amylasy MeSH
• Cannabis * MeSH
• celulasy * MeSH
• dieta veterinární   MeSH
• fyziologie výživy zvířat MeSH
• gastrointestinální trakt MeSH
• invertasa MeSH
• krmivo pro zvířata analýza   MeSH
• kur domácí MeSH
• len * MeSH
• polygalakturonasa MeSH
• potravní doplňky MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alfa-glukosidasy MeSH
• amylasy MeSH
• celulasy * MeSH
• invertasa MeSH
• polygalakturonasa MeSH

In a multiple deletion mutant hxt1deltahxt2deltahxt3delta hxt4deltasnf3delta of Saccharomyces cerevisiae growing on a 2% glucose, high-affinity glucose-uptake (low Km) was exhibited throughout growth on glucose in contrast to the wild-type, which exhibited the usual low-affinity to high-affinity transition as the glucose in the medium was consumed. Elevated levels of invertase activity throughout growth on glucose, in this mutant as compared to the wild-type, indicate that glucose repression may be impaired. However, in a mutant containing only the HXT2 gene (hxt1deltahxt3deltahxt4deltasnf3delta), invertase levels were similar to those in the wild-type. It is likely, therefore, that some of these putative glucose transporters, such as HXT2, also have regulatory roles in cellular metabolism. In triple hexose-kinase mutants, rapid (200-ms) measurements of initial glucose-uptake revealed high-affinity glucose uptake (Km approx. 2 mmol/L) while measurements on the slower 5-s scale clearly demonstrate that uptake is not linear over this longer period. These results suggest that this high-affinity component does not require a functional hexose-kinase.

• MeSH
• biologický transport MeSH
• glukosa metabolismus   MeSH
• glykosidhydrolasy metabolismus   MeSH
• invertasa MeSH
• kinetika MeSH
• proteiny přenášející monosacharidy genetika   fyziologie   MeSH
• Saccharomyces cerevisiae enzymologie   genetika   metabolismus   MeSH
• sekvenční delece fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glukosa MeSH
• glykosidhydrolasy MeSH
• invertasa MeSH
• proteiny přenášející monosacharidy MeSH

UNLABELLED: Baker's yeast (Saccharomyces cerevisiae) cells were magnetically modified with magnetic iron oxide particles prepared by microwave irradiation of iron(II) sulfate at high pH. The modification procedure was very simple and fast. Both non-cross-linked and glutaraldehyde cross-linked magnetic cells enabled efficient sucrose conversion into glucose and fructose, due to the presence of active intracellular invertase. The prepared magnetic whole-cell biocatalyst was stable; almost the same catalytic activity was observed after 1-month storage at 4°C. Simple magnetic separation and stability of the developed biocatalyst enabled its reusability without significant loss of enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Magnetic whole yeast cell biocatalyst containing intracellular invertase in its natural environment has been prepared. Magnetic properties enable its easy separation from reaction mixture. Magnetically modified Saccharomyces cerevisiae cells have been used for invert sugar production, hydrolysing sucrose into glucose and fructose. The described magnetization procedure employing microwave-synthesized iron oxide microparticles is a low-cost and easy-to-perform alternative to already existing magnetization techniques.

• MeSH
• biokatalýza MeSH
• fruktosa metabolismus   MeSH
• glukosa metabolismus   MeSH
• hydrolýza MeSH
• imobilizované buňky MeSH
• invertasa metabolismus   MeSH
• magnetické jevy * MeSH
• mikrovlny MeSH
• Saccharomyces cerevisiae enzymologie   metabolismus   MeSH
• sacharosa metabolismus   MeSH
• železité sloučeniny * MeSH
• železnaté sloučeniny MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferric oxide MeSH Prohlížeč
• fruktosa MeSH
• glukosa MeSH
• invert sugar MeSH Prohlížeč
• invertasa MeSH
• sacharosa MeSH
• železité sloučeniny * MeSH
• železnaté sloučeniny MeSH