Commercial Invertase Research Articles

Production and characterization of intracellular invertase from Saccharomyces cerevisiae (OL629078.1), using cassava-soybean as a cost-effective substrate

The growing global market for industrial enzymes has led to a constant search for efficient, cost-effective methods for their production. This study reports the production of invertase using inexpensive and readily available agro-materials. Starch-digesting enzymes extracted from malted unkilned sorghum were used to hydrolyze cassava starch supplemented with 2% whole soybean. The production of intracellular invertase by Saccharomyces cerevisiae OL629078.1 in cassava-soybean and yeast sucrose broth was compared. The purification and characterization of invertase produced using the low-cost medium were also reported. The results showed that there was a 4.1-fold increase in the units of invertase produced in cassava-soybean medium (318.605 U/mg) compared to yeast sucrose broth medium (77.6 U/mg). The invertase produced was purified by chromatographic methods up to 5.53-fold with a recovery of 62.6%. Estimation of the molecular weight with gel filtration indicated a molecular weight of 118 kDa. The enzyme demonstrated its maximum activity at 50 °C and there was no decrease in its activity following a 1-h incubation at this temperature. At a pH of 5.0, the enzyme demonstrated optimal activity and it maintained over 60% of its activity in the acid range (pH 3–6). The Michalis-Menten constants Km and Vmax of intracellular invertase were 5.85 ± 1.715 mM and 6.472 ± 2.099 U/mg, respectively. These results suggest that Saccharomyces cerevisiae grown on cassava-soybean is a viable, cost-effective alternative for commercial invertase production, which can be explored for biotechnological processes.

How fructophilic lactic acid bacteria may reduce the FODMAPs content in wheat-derived baked goods: a proof of concept

BackgroundFODMAPs (Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) intake is associated with the onset of irritable bowel syndrome symptoms. FODMAPs in wheat-derived baked goods may be reduced via bioprocessing by endogenous enzymes and/or microbial fermentation. Because of the inherent enzyme activities, bread made by baker’s yeast and sourdough may result in decreased levels of FODMAPs, whose values are, however, not enough low for people sensitive to FODMAPs.ResultsOur study investigated the complementary capability of targeted commercial enzymes and metabolically strictly fructophilic lactic acid bacteria (FLAB) to hydrolyze fructans and deplete fructose during wheat dough fermentation. FLAB strains displayed higher fructose consumption rate compared to conventional sourdough lactic acid bacteria. Fructose metabolism by FLAB was faster than glucose. The catabolism of mannitol with the goal of its reuse by FLAB was also investigated. Under sourdough conditions, higher fructans breakdown occurred in FLAB inoculated doughs compared to conventional sourdough bacteria. Preliminary trials allowed selecting Apilactobacillus kunkeei B23I and Fructobacillus fructosus MBIII5 as starter candidates, which were successfully applied in synergy with commercial invertase for low FODMAPs baking.ConclusionsResults of this study clearly demonstrated the potential of selected strictly FLAB to strongly reduce FODMAPs in wheat dough, especially under liquid-dough and high oxygenation conditions.

Effects of Inhibitors on Kinetic Properties of Invertase from Saccharomyces cerevisiae

The understanding of the kinetic properties of invertase activity is that it is always produced during yeast fermentation and helps in the pretreatment and raw material preparation of ethanol production which usually uses molasses to achieve high efficiency of ethanol yield and productivity. Normally, the activity of enzymes is limited by many parameters including invertase. Thus, this research focused on some inhibitors that affected invertase activity produced from Saccharomyces cerevisiae, using cultivation on different carbon sources (pure sucrose solution and sugarcane molasses) compared with commercial invertase. Metal contamination from fertilizer during sugarcane cultivation has resulted in significant levels of calcium ions (Ca2+), potassium ions (K+), and magnesium ions (Mg2+) in harvested sugarcane at approximately 0.09, 0.18, and 0.05% (w/w), respectively. Consequently, harvested sugarcane and contamination from sugar processing were investigated in the current study, particularly the levels of the metal ions; Ca2+, K+, and Mg2+ that contaminated molasses. Moreover, the substrate and enzyme concentration were studied. The results showed that both crude invertases (sucrose and molasses invertase) had lower activity (lower Vmax and higher Km) than commercial invertase, probably due to enzyme impurities. Furthermore, the substrate and enzyme concentrations also affected the invertase activity, with increased values of V0 and Qp indicating increased invertase activity at a sucrose concentration less than 100 g/L. Furthermore, increasing the crude invertase concentration had a negative effect on enzyme activity because of the greater interference by the impurities in sugar invertase compared to commercial invertase. Moreover, all metal ions were identified as competitive inhibitors and severely inhibited the activity of invertase at 0.6% (w/v) of metal ion concentration, especially with Ca2+ contamination.

Complete sucrose hydrolysis by heat-killed recombinant Pichia pastoris cells entrapped in calcium alginate.

BackgroundAn ideal immobilized biocatalyst for the industrial-scale production of invert sugar should stably operate at elevated temperatures (60-70°C) and high sucrose concentrations (above 60%, w/v). Commercial invertase from the yeast Saccharomyces cerevisiae is thermolabile and suffers from substrate inhibition. Thermotoga maritima β-fructosidase (BfrA) is the most thermoactive and thermostable sucrose-hydrolysing enzyme so far identified and allows complete inversion of the substrate in highly concentrated solutions.ResultsIn this study, heat-killed Pichia pastoris cells bearing N-glycosylated BfrA in the periplasmic space were entrapped in calcium alginate beads. The immobilized recombinant yeast showed maximal sucrose hydrolysis at pH 5–7 and 90°C. BfrA was 65% active at 60°C and had no activity loss after incubation without the substrate at this temperature for 15 h. Complete inversion of cane sugar (2.04 M) at 60°C was achieved in batchwise and continuous operation with respective productivities of 4.37 and 0.88 gram of substrate hydrolysed per gram of dry beads per hour. The half-life values of the biocatalyst were 14 and 20 days when operated at 60°C in the stirred tank and the fixed-bed column, respectively. The reaction with non-viable cells prevented the occurrence of sucrose fermentation and the formation of by-products. Six-month storage of the biocatalyst in 1.46 M sucrose (pH 5.5) at 4°C caused no reduction of the invertase activity.ConclusionsThe features of the novel thermostable biocatalyst developed in this study are more attractive than those of immobilized S. cerevisiae cells for application in the enzymatic manufacture of inverted sugar syrup in batch and fixed-bed reactors.

Investigation of Some Kinetic Properties of Commercial Invertase from Yeast

The effect of substrate concentration, pH, temperature and inhibitor (silver nitrate) on the activity of the enzyme invertase was studied using sucrose as the substrate. The invertase acted on the sucrose and converted it to product, glucose and fructose. From Lineweaver-Burk Plot, Vmax was found to be 1 mM/min and the Km approximately 24 mM. At temperature range of 25EšC to 650EšC, the optimum activity was at 450EšC. For a pH range of 3.5-6.0, the pH optimum activity was at 5.5. Inhibition studies showed that silver nitrate inhibited invertase competitively with apparent Km of 150 mM and Ki value of 160 mM.

Biotransformation of pineapple juice sugars into dietetic derivatives by using a cell free oxidoreductase from Zymomonas mobilis together with commercial invertase

An easy procedure for cell free biotransformation of pineapple juice sugars into dietetic derivatives was accomplished using a commercial invertase and an oxidoreductase from Zymomonas mobilis. First, pineapple juice sucrose was quantitatively converted into glucose and fructose by invertase, thus increasing the concentration of each monosaccharide in the original juice to almost twice. In a second step, glucose–fructose oxidoreductase (GFOR) transformed glucose into gluconolactone, and fructose into the low calorie sweetener sorbitol. The advantage of using GFOR is simultaneous reduction of fructose and oxidation of glucose, allowing the continuous regeneration of the essential coenzyme NADP(H), that is tightly bound to the enzyme. The yield of GFOR catalyzed sugar conversion depends on initial pH and control of pH during the reaction. At optimal conditions (pH control at 6.2) a maximum of 80% (w/v) sugar conversion was obtained. Without pH control, GFOR is inactivated rapidly due to gluconic acid formation. Therefore, conversion yields are relatively low at the natural pH of pineapple juice. The application of this process might be more advantageous on juices of other tropical fruits (papaya, jackfruit, mango) due to their naturally given higher pH.

Production of high-fructose syrup using immobilized invertase in a membrane reactor

The aim of this work is to evaluate the performance of immobilized invertase in a continuous process using a membrane bioreactor (MB) at a feeding rate of 1.6 h −1 and at an agitation of 100 rpm. Commercial invertase (BIOINVERT ®) was adsorbed on polystyrene beads of DOWEX ®-anionic resin and used for sucrose hydrolysis in the MB coupled with an ultrafiltration (UF) or a microfiltration (MF) membrane. Considering the reactor in a 20 h continuous steady-state operation, the yields attained were 100% (mean activity of 0.88 mmol/h mg Enzyme) and 99% (mean activity of 1.02 mmol/h mg Enzyme) when a UF and a MF-membrane were used, respectively. No leakage of enzyme from the support was detected. The inverted sugar attained was submitted to a chromatographic separation through a column packed with DOWEX-50W 8x100 resulting in a 70% high-fructose syrup (HFS).

Cereal fructans: Hydrolysis by yeast invertase, in vitro and during fermentation

Cereal fructans were isolated from wheat flour and fractionated into tri-, tetra-, penta- and the higher molecular weight oligo- and polysaccharides. These five fructan fractions were used as substrates for both commercial invertase and bakers' yeast and were compared with sucrose and inulin. Optimal pH for hydrolysis with invertase was in the range 4·5 to 5·0 for all substrates except inulin, which showed an optimum pH at 6·0. The hydrolytic activity of both invertase and bakers' yeast towards the various fructan oligomers decreased with an increase in their molecular size. In comparison with sucrose, the five fructan fractions were hydrolysed at the following rates: tri- (14–17%); tetra- (10–12%); penta- (9%) and the two higher-molecular-weight fractions between 2 and 7%. Enzyme kinetics were investigated using di-, tri- and tetrasaccharides as substrates. The hydrolysis of cereal fructans by bakers' yeast during fermentation of doughs prepared from wheat flour was also investigated. During 1 hour of fermentation, most of the tri-, tetra and pentasaccharides disappeared, amounting to half of the fructan concentration initially present.

Stabilisation of invertases by treatment with cross‐linking reagents

AbstractCommercial baker's yeast invertase concentrate, used in the manufacture of a variety of confectionary products, was treated with various chemical cross‐linking agents in an attempt to improve its thermal stability to that of the more useful invertase from Candida utilis, which has been shown previously to have high thermal stability. Only glutaraldehyde was successful but at the expense of a loss of enzyme activity and a three‐fold increase in Km. Dimethylsuberimidate stabilised commercial invertase only slightly and the other reagents tried, either had no effect upon stability or caused a destabilisation of the enzyme. C. utilis invertase can be stabilised even further against thermal denaturation by dimethylsuberimidate. A similar stabilisation was found when this enzyme was heated in the presence of ≥ 10 mM‐CaCl2.

Note on measurement of activity of commercial invertase

Note on measurement of activity of commercial invertase