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Sugar beet molasses is a low-value byproduct from the sugar industry. It contains significant amounts of sucrose (approx. 50% (w/w)), which can be used for many different applications, for example, as feedstock for the production of fuel (as ethanol) and biobased chemicals such as 5-hydoxymethyl furfural (HMF). To produce platform chemicals, sucrose is hydrolyzed into its monomeric C6 sugars: glucose and fructose. When comparing the hydrolysis rates of molasses with a pure sucrose solution, the specific reaction rate is much slower (Qp/x,60min = 93 and 70 gprod L−1 h−1 gcell−1 for pure sucrose and crude molasses, respectively) at the same sucrose concentration (300 g/L) and process conditions. To clarify why molasses inhibits the enzymatic hydrolysis rate, the influence of its viscosity and inorganic and organic composition was investigated. Also, the effects of molasses and treated molasses on pure enzymes, invertase (from Saccharomyces cerevisiae, 0.05 mg/mL), compared with hydrolysis using whole cells of Baker's yeast (3 mg/mL), were tested. The results indicate an inhibitory effect of potassium (Qp/x,60min = 76 gprod L−1 h−1 gcell−1), generally at high salt concentrations (Qp/x,60min = 67 gprod L−1 h−1 gcell−1), which could be correlated to the solution's high salt concentrations and possibly the synergistic effects of different ions when applying concentrations that were four times that in the molasses. Also, the viscosity and sucrose purity seem to have an effect, where pure sucrose solutions and thick juice from the sugar mill yielded higher hydrolysis rates (Qp/x,60min = 97 gprod L−1 h−1 gcell−1) than molasses-type solutions with a higher viscosity (Qp/x,60min = 70–74 gprod L−1 h−1 gcell−1). Attempting to further understand the effects of different components on the invertase activity, an in silico investigation was performed, indicating that high salt concentrations affected the binding of sucrose to the active site of the enzyme, which can result in a lower reaction rate. This knowledge is important for future scale-up of the hydrolysis process, since reduced hydrolysis rates require larger volumes to provide a certain productivity, requiring larger process equipment and thereby higher investment costs. [ABSTRACT FROM AUTHOR]

Production of fructose is important especially in the food industries where fructose is used as a sweetener in preference to sucrose. This work studied some cultural conditions for the production of invertase by Aspergillus niger. In a preliminary experiment, all isolated fungal spores were incubated in 0.2 M sucrose solution and the spores of Aspergillus niger produced the highest invertase activity of 72.8U/mg protein and was therefore selected for further work. Inversion of sucrose by spores of Aspergillus niger was then studied after various incubation periods. Incubation for 6 hours with shaking was best for enzyme production at which 94.6 U/mg protein of the enzyme was produced. Enzyme yields at 1 and 2 h of incubation were very low while incubation for elevated periods of 7 and 8 h resulted in moderate enzyme yields of 68.3 and 60.4 U/mg protein respectively. Lower sucrose concentrations of 0.3 and 0.4 M were almost completely inverted while higher concentrations showed decreases in enzyme activities. Invertase from Aspergillus niger was incubated at various temperatures and it was found that at 10oC, only 26.5 U/mg protein of the enzyme activity was produced. This level increased gradually with increases in temperature until 30oC at which the highest enzyme activity of 72.8 U/mg protein was produced. This level reduced progressively with increases in temperature until it reached a temperature of 50oC at which very low activity was obtained. It was shown that pH 5.0 was the best for invertase production by the test fungus. [ABSTRACT FROM AUTHOR]

Invertases catalyze the hydrolysis of sucrose into fructose and glucose and can be employed as an alternative in producing high fructose syrup. In this study, we reported the heterologous expression of an invertase gene (GspInv) of Gongronella sp. w5 in Komagataella pastoris. GspInv activity reached 147.6 ± 0.4 U/mL after 5 days of methanol induction. GspInv is invertase with a high specific activity of 2,776.1 ± 124.2 U/mg toward sucrose. GspInv showed high tolerance to sucrose (IC 50 = 1.2 M), glucose (IC 50 > 2 M), fructose (IC 50 = 1.5 M), and a variety of metal ions that make it an ideal candidate for high fructose syrup production. A carbohydrate-binding module was sequence-optimized and fused to the N-terminus of GspInv. The fusion protein had the highest immobilization efficiency at room temperature within 1 h adsorption, with 1 g of cellulose absorption up to 8,000 U protein. The cellulose-immobilized fusion protein retained the unique properties of GspInv. When applied in high fructose syrup preparation by using 1 M sucrose as the substrate, the sucrose conversion efficiency of the fused protein remained at approximately 95% after 50 h of continuous hydrolysis on a packed bed reactor. The fused protein can also hydrolyze completely the sucrose in sugarcane molasses. Our results suggest that GspInv is an unusual invertase and a promising candidate for high fructose syrup preparation. [ABSTRACT FROM AUTHOR]

Background An 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. Results In 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. Conclusions The 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. [ABSTRACT FROM AUTHOR]

A gene encoding Uninv, an invertase active in the neutral pH range, was isolated from a metagenomic library and the enzyme was biochemically characterized. The enzyme had an optimum pH of 6.5 and an optimum temperature of 50°C. It showed maximal ~37% sequence identity to all invertases identified so far. Uninv displayed typical Michaelis-Menten kinetics with substrate inhibition of sucrose, but it retained 50% activity at sucrose concentrations up to 1,000 mM. Uninv had no transfructosylating activity even at high sucrose concentrations. These properties will make this enzyme useful in the food industry. [ABSTRACT FROM AUTHOR]

The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 °C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 °C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 °C. The values for kinetic parameters were determined as: 984 and 98 mM for $$ K_{m}^{\text{app}} $$ of immobilized and free re-INVB, respectively. $$ K_{\text{cat}}^{\text{app}} $$ values for immobilized and free enzymes were 6.1 × 102 and 1.2 × 104 s−1, respectively, and immobilized re-INVB showed $$ V_{\max }^{\text{app}} $$ of 158.73 μmol h min−1 mg−1. Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 °C and 70% at 40 °C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme. [ABSTRACT FROM AUTHOR]

This study focused on the purification and characterization of an extracellular β-d-fructofuranosidase or invertase from Aspergillus sojae JU12. The protein was purified by size exclusion chromatography with 5.41 fold and 10.87% recovery. The apparent molecular mass of the enzyme was estimated to be ~ 35 kDa using SDS-PAGE and confirmed by deconvoluted mass spectrometry. The fungal β-d-fructofuranosidase was suggested to be a monomer by native PAGE and zymography, and was found to be a glycoprotein possessing 68.92% carbohydrate content. The products of enzyme hydrolysis were detected by thin layer chromatography and revealed the monosaccharide units, d-glucose and d-fructose. β-d-fructofuranosidase showed enhanced activity at broad pH 4.0–9.0 and activity at a temperature range from 30 to 70 °C, while the enzyme was stable at pH 8.0 and 40 °C, respectively. The β-d-fructofuranosidase activity was lowered by metal ion inhibitors Ag2+ and Hg2+ whereas elevated by SDS and β-ME. The fungal β-d-fructofuranosidase was capable of hydrolyzing d-sucrose and the kinetics were determined by Lineweaver–Burk plot with Km of 10.17 mM and Vmax of 0.7801 µmol min−1. Additionally, the extracellular β-d-fructofuranosidase demonstrated tolerance to high ethanol concentrations indicating its applicability in the production of alcoholic fermentation processes. [ABSTRACT FROM AUTHOR]