Directionally modulating the product chain length of an inulosucrase by semi-rational engineering for efficient production of 1-kestose

Ni, Dawei; Zhang, Shuqi; Huang, Zhaolin; Xu, Wei; Zhang, Wenli; Mu, Wanmeng

doi:10.1016/j.enzmictec.2022.110085

Cited by 7 publications

(5 citation statements)

References 33 publications

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“…Similar to the wild type, mutant M5 predominantly accumulated oligosaccharides with a polymerization degree lower than GF5, without accumulating larger oligosaccharides (Figure B). Some studies involving the molecular modification of inulosucrase to control product chain length have resulted in decreased molecular weights. − It is challenging to improve the molecular weight of produced inulin. Remarkably, the molecular weight of the produced inulin reached 3.5 × 10 6 g/mol, surpassing that produced by the wild-type enzyme (Figure C).…”

Section: Resultsmentioning

confidence: 99%

“…Despite extensive efforts to modify the product spectrum of inulosucrase through molecular engineering, most mutants have shown a reduction in the molecular weight of the products. , Notably, the R544W mutant of Lactobacillus reuteri 121 inulosucrase primarily produced kestose, nystose, and 1F-fructofuranosyl nystose, as documented by Ni et al Conversely, only a limited number of site-directed mutagenesis studies have aimed to enhance catalytic properties. For instance, Charoenwongpaiboon et al developed a G237P mutant of L.…”

Section: Introductionmentioning

confidence: 99%

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Multistrategy Engineering of an Inulosucrase to Enhance the Activity and Thermostability for Efficient Production of Microbial Inulin

Ni,

Zhang,

Huang

et al. 2024

J. Agric. Food Chem.

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Inulin has found commercial applications in the pharmaceutical, nutraceutical, and food industries due to its beneficial health effects. The enzymatic biosynthesis of microbial inulin has garnered increasing attention. In this study, molecular modification was applied to Lactobacillus mulieris UMB7800 inulosucrase, an enzyme that specifically produces high-molecular weight inulin, to enhance its catalytic activity and thermostability. Among the 18 variable regions, R5 was identified as a crucial region significantly impacting enzymatic activity by replacing it with more conserved sequences. Site-directed mutagenesis combined with saturated mutagenesis revealed that the mutant A250 V increased activity by 68%. Additionally, after screening candidate mutants by rational design, four single-point mutants, S344D, H434P, E526D, and G531P, were shown to enhance thermostability. The final combinational mutant, M5, exhibited a 66% increase in activity and a 5-fold enhancement in half-life at 55 °C. These findings are significant for understanding the catalytic activity and thermostability of inulosucrase and are promising for the development of microbial inulin biosynthesis platforms.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Multistrategy Engineering of an Inulosucrase to Enhance the Activity and Thermostability for Efficient Production of Microbial Inulin

Ni,

Zhang,

Huang

et al. 2024

J. Agric. Food Chem.

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“…When compared to the literature, the obtained I-FOS concentration of 401 g L −1 and yield of 0.50 g I-FOS per g applied sucrose exceed the concentrations and yields of previous I-FOS productions performed with bacterial inulosucrases (Table 3 ). To the best of our knowledge, no I-FOS concentrations higher than 307 g L −1 (Ni et al 2022 ) and I-FOS yields higher than 0.45 g per g substrate (Díez-Municio et al 2013 ) have been achieved using bacterial inulosucrases. Final I-FOS concentrations comparable to our results were achieved with scFOS-producing enzymes of fungal origin (Table 3 ).…”

Section: Discussionmentioning

confidence: 99%

High-yield production and purification of prebiotic inulin-type fructooligosaccharides

2022

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Due to the health-promoting effects and functional properties of inulin-type fructooligosaccharides (I-FOS), the global market for I-FOS is constantly growing. Hence, there is a continuing demand for new, efficient biotechnological approaches for I-FOS production. In this work, crude inulosucrase InuGB-V3 from Lactobacillus gasseri DSM 20604 was used to synthesize I-FOS from sucrose. Supplementation with 1 mM CaCl2, a pH of 3.5–5.5, and an incubation temperature of 40 °C were found to be optimal production parameters at which crude inulosucrase showed high conversion rates, low sucrose hydrolysis, and excellent stability over 4 days. The optimal process conditions were employed in cell-free bioconversion reactions. By elevating the substrate concentration from 570 to 800 g L−1, the I-FOS concentration and the synthesis of products with a low degree of polymerization (DP) could be increased, while sucrose hydrolysis was decreased. Bioconversion of 800 g L−1 sucrose for 20 h resulted in an I-FOS-rich syrup with an I-FOS concentration of 401 ± 7 g L−1 and an I-FOS purity of 53 ± 1% [w/w]. I-FOS with a DP of 3–11 were synthesized, with 1,1-kestotetraose (DP4) being the predominant transfructosylation product. The high-calorie sugars glucose, sucrose, and fructose were removed from the generated I-FOS-rich syrup using activated charcoal. Thus, 81 ± 5% of the initially applied I-FOS were recovered with a purity of 89 ± 1%.

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“…The authors should, therefore, include details about some food interventions that have been excluded in the study. 3 Another issue is that it has been reported that the stimulating effect of 1-kestose in humans can differ according to age, so a subgroup comparison for age should be performed in this study. 4,5 However, these interesting details were not addressed.…”

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confidence: 99%

Letter: 1‐kestose, the smallest fructooligosaccharide component, protection for mild to moderate ulcerative colitis patients

peng

Chen

2023

Aliment Pharmacol Ther

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Editors,We read with great interest the publication by Ikegami et al. 1 Their study found that oral 1-kestose was well tolerated and provided clinical improvement for patients with mild to moderate ulcerative colitis (UC) through modulation of the gut microbiome. This study is important and interesting. We nonetheless have some concerns regarding AUTH O R CO NTR I B UTI O N Schen peng: Conceptualization (lead); data curation (lead); resources (lead); software (lead); supervision (lead); writing -review and editing (lead).chen fuchao: Project administration (supporting); resources (supporting).

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Directionally modulating the product chain length of an inulosucrase by semi-rational engineering for efficient production of 1-kestose

Cited by 7 publications

References 33 publications

Multistrategy Engineering of an Inulosucrase to Enhance the Activity and Thermostability for Efficient Production of Microbial Inulin

Multistrategy Engineering of an Inulosucrase to Enhance the Activity and Thermostability for Efficient Production of Microbial Inulin

High-yield production and purification of prebiotic inulin-type fructooligosaccharides

Letter: 1‐kestose, the smallest fructooligosaccharide component, protection for mild to moderate ulcerative colitis patients

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