Effects of Inhibitors on Kinetic Properties of Invertase from Saccharomyces cerevisiae

Sirisatesuwon, Chollada; Ninchan, Boontiwa; Sriroth, Klanarong

doi:10.1007/s12355-019-00757-2

Cited by 8 publications

(7 citation statements)

References 19 publications

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“…) and K m (0.11 M) indicated that DPEase possesses a high substrate a nity and demonstrates e cient conversion of D-fructose to D-psicose. These results are in line with previous studies emphasizing the importance of low K m values in indicating high enzyme-substrate a nity(Ninchan et al 2017;Sirisatesuwon et al 2020). Interestingly, a unique three-dimensional structure of A. tumefaciens DPEase contains the Mn 2+ ion binding site as same the structure of DPEase that was constructed from A. tumefaciens C58 in this research as shown in Fig.9, resulting to the high activity of DPEase under the presenting of Mn 2+ ion in the reaction.…”

supporting

confidence: 93%

Production, Purification, Characterization, and Safety Evaluation of Constructed Recombinant D-Psicose 3-Epimerase

Watthanasakphuban,

Sr,

Pinmanee

et al. 2024

Preprint

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Background D-psicose 3-epimerase (DPEase) is a potential catalytic enzyme for D-psicose production. D-psicose, also known as D-allulose is a low-calorie sweetener that has garnered considerable attention for healthy alternative sweetener due to its remarkable physicochemical properties. This research focused on an in-depth investigation of the expression of the constructed DPEase gene from Agrobacterium tumefaciens in Escherichia coli for D-psicose synthesis, with a strong emphasis on scalability. Experimentally, this research created the recombinant enzyme, explored the optimization of gene expression systems and protein purification strategies, and then investigated the enzymatic characterization to establish a robust and scalable process for D-psicose production. Additionally, the optimization of D-psicose production using the recombinant DPEase was also studied. Finally, the produced D-psicose syrup was underwent acute toxicity evaluation to provide scientific evidence supporting its safety.Results The optimization of DPEase expression involved the utilization of Mn2+ as a cofactor, fine-tuning isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, and controlling induction temperature. The purification process was strategically designed to facilitate industrial-scale operations. A nickel column and an elution buffer containing 200 mM imidazole were employed, resulting in purified DPEase with a remarkable 21.03-fold increase in specific activity compared to the crude extract. The optimum D-psicose conversion conditions of purified DPEase were performed at pH 7.5 and 55°C under Mn2+ addition as the cofactor. Kinetic parameters of purified DPEase, Vmax and Km, were determined, demonstrating the high substrate affinity and efficiency of DPEase, which are crucial factors for achieving cost-effective for D-psicose production that was confirmed by binding site of fructose-DPEase-Mn2+ structure. Stability studies were conducted to ensure the enzyme's robustness during storage, which the glycerol supplementation and storage at -20°C were identified as effective strategies for maintaining its stability. D-psicose production using purified DPEase was investigated across a range of fructose concentrations, with a focus on optimizing yields for industrial applications. Moreover, the results from acute toxicity study showed no toxicity to rats, supporting the safety of mixed D-fructose-D-psicose syrup produced using recombinant DPEase.Conclusions These findings have direct and practical implications for the industrial-scale production of D-psicose, a valuable rare sugar with a broad range of applications in the food and pharmaceutical industries. Our research advances the understanding of DPEase biocatalysis and offers a roadmap for the successful scale-up production of rare sugars, opening new avenues for their utilization in various industrial processes.

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supporting

confidence: 93%

Production, Purification, Characterization, and Safety Evaluation of Constructed Recombinant D-Psicose 3-Epimerase

Watthanasakphuban,

Sr,

Pinmanee

et al. 2024

Preprint

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“…Research in the literature has shown that certain metal ions, specifically calcium (Ca 2+ ), potassium (K + ), and magnesium (Mg 2+ ), have an effect on the activity of the invertase enzyme. , M1 is a YEPD medium. M2 was prepared as a comparison medium to distinguish the effect of the presence of metal ions from the effect of using fruit pulp.…”

Section: Resultsmentioning

confidence: 99%

“…The activity of many enzymes, including invertase, can be affected by the presence of specific metal ions. In particular, the activity of invertase may be influenced by calcium ions (Ca 2+ ) and magnesium ions (Mg 2+ ) . These ions have the potential to interact with the enzyme, possibly altering its structure or its interaction with the substrate, leading to changes in the enzyme’s activity.…”

Section: Resultsmentioning

confidence: 99%

“…However, the precise effect can vary and would necessitate experimental determination. It is also crucial to note that while certain metal ions can enhance enzyme activity, others may inhibit it …”

Section: Resultsmentioning

confidence: 99%

“…It is also crucial to note that while certain metal ions can enhance enzyme activity, others may inhibit it. 31 The impact of different metal ions on the immobilized invertase was examined in this study. Results indicated that Co 2+ ions exhibited the inhibition of immobilized enzyme activity, while other metal ions showed slightly varying degrees of inhibitory effects (shown in Figure 8B).…”

Section: Determination Of the Kinetic Constantmentioning

confidence: 99%

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Industrial Approach to Invertase Production from Fruit Waste for Enhanced Efficiency and Conservation

Dokuzparmak

2024

ACS Omega

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This study investigates the commercial viability of repurposing fruit waste for enzyme production, specifically focusing on the invertase enzyme derived from Saccharomyces cerevisiae. By utilizing fruit pulp that incorporates mulberry, carob, Figure, and grape pulp as a nutrient source, it is observed that the culture medium containing carob pulp exhibits the highest invertase activity. Specifically, the invertase activity in this medium is approximately 2.5 times greater (12.90 U/mg protein) than that observed in the peptone medium (5.98 U/mg protein). The extract undergoes several purification steps, including ultrafiltration, ammonium sulfate precipitation, dialysis, and ion-exchange chromatography (purification ratio: 12.11 times, yield: 26.93%). The purified enzyme is immobilized using alginate beads, improving pH and thermal stability. The immobilized enzyme exhibits optimal activity between pH 3.50 and pH 7.00, thereby broadening the enzyme's high-activity pH range. The thermal stability of the immobilized invertase enzyme is significantly improved, especially at 65 °C. Activity studies in the presence of metal ions and certain chemicals have been conducted. The immobilized enzyme's activity increases by approximately 40% in the presence of Ca 2+ and Mg 2+ , and the immobilized enzyme maintains its activity in the presence of detergents such as SDS, Tween-20, and organic solvents like ethanol and methanol. The potential for the reuse of immobilized invertase was investigated under standard assay conditions. After 20 cycles, the immobilized enzyme was found to retain 80% of its initial activity. Overall, the study establishes the commercial potential of fruit pulp, typically discarded in fruit juice production, as a valuable source for obtaining an invertase enzyme. Furthermore, this study also aims to develop a suitable purification process for invertase in the fruit juice industry. By harnessing fruit waste and implementing innovative enzyme production strategies, industries can enhance their efficiency, reduce their environmental footprint, and optimize resource utilization.

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Biosynthesis of polyhydroxyalkanoates from sugarcane molasses by recombinant Ralstonia eutropha strains

Sohn

Park

et al. 2021

Korean J. Chem. Eng.

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Effects of Inhibitors on Kinetic Properties of Invertase from Saccharomyces cerevisiae

Cited by 8 publications

References 19 publications

Production, Purification, Characterization, and Safety Evaluation of Constructed Recombinant D-Psicose 3-Epimerase

Production, Purification, Characterization, and Safety Evaluation of Constructed Recombinant D-Psicose 3-Epimerase

Industrial Approach to Invertase Production from Fruit Waste for Enhanced Efficiency and Conservation

Biosynthesis of polyhydroxyalkanoates from sugarcane molasses by recombinant Ralstonia eutropha strains

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