Cloning and Characterization of a Sucrose Isomerase from Erwinia rhapontici NX-5 for Isomaltulose Hyperproduction

Li, Sha; Cai, Heng; Qing, Yujia; Ren, Ben; Xu, Hong; Zhu, Hongyang; Yao, Jun

doi:10.1007/s12010-010-9015-z

Cited by 39 publications

(32 citation statements)

References 30 publications

(17 reference statements)

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“…The calculated molecular weight for the monomeric protein is 70.84 kDa, slightly larger than the observed mass (66.5 kDa) by SDS-PAGE analysis. Compared to our previous report [29], the new recombinant protein does not harbor the N-terminal peptide sequence derived from the expression vector.…”

Section: Resultsmentioning

confidence: 92%

“…To facilitate protein crystallization, we modified the recently reported expression plasmid pET-22b-palI by truncating the gene encoding the N-terminal peptide sequence [29]. Briefly, the gene of Erwinia rhapontici NX-5 was amplified using the primers F: 5’-GGCGTTC CATATG GATTCTCAAGGATTG-3’; R: 5’-CCG CTCGAG CGGATTAAGTTTATAAAT-3’ ( Nde I and Xho I restriction sites are underlined) and pET-22b-palI as the template.…”

Section: Methodsmentioning

confidence: 99%

“…coli BL21 (DE3) strain as described previously [29] at 297 K by the addition of 0.5 mM lactose in refined culture medium (pH 7.0) consisting of sucrose 10 g/L, yeast extract 20 g/L, NaCl 9 g/L, KH 2 PO 4 3 g/L, and MgSO 4 0.5 g/L. The cells were harvested by centrifugation after growth for 11 h. Cell pellets were lysed by sonication in buffer A (10 mM HEPES, pH 8.0, 500 mM NaCl, 5 mM β-mercaptoethanol, and 10% glycerol).…”

Section: Methodsmentioning

confidence: 99%

“…The sucrose isomerase activities of the NX-5 proteins were measured using a high performance liquid chromatography method [29]. In brief, 0.5 mg of purified enzyme solution was mixed with 20 g/L of sucrose in a 1 mL reaction volume containing 50 mM PBS, pH 6.0.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we have characterized an isomaltulose synthase from Erwinia rhapontici NX-5 (named NX-5 in this work) that predominantly produces isomaltulose (~87%) [28,29]. Compared to other isomaltulose synthases, NX-5 shows more acidic tolerance and higher product selectivity on sucrose isomerization.…”

Section: Introductionmentioning

confidence: 99%

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The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

et al. 2013

PLoS ONE

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Sucrose isomerase NX-5 from Erwinia rhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonas mesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations.

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Section: Resultsmentioning

confidence: 92%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

et al. 2013

PLoS ONE

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Bioinspired Production of Antibacterial Sucrose Isomerase‐Sponge for the Synthesis of Isomaltulose

Qiu

et al. 2016

Adv Synth Catal

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Isomaltulose is a new functional sweetener that possesses biological characteristics and can be obtained by enzymatic synthesis using sucrose as substrate. In this study, the sucrose isomerase (SIase) was successfully immobilized on the sponge synthesized with ϵ‐poly‐l‐lysine and gelatin. The inhibition zones proved that the sponge possessed a significant bacteriostatic function, which can effectively prevent the collapse of the scaffolds of the immobilized enzymes. The immobilized SIase activity reached up to 71.58 U g−1 (the SIase loading was about 153.43 mg g−1), and the activity recovery was approximately 84.50%. After immobilization, the optimum pH of SIase decreased from 6.0 to 5.5 and the optimum temperature increased from 30 °C to 40 °C. The affinity of SIase to substrate was basically unchanged. Immobilized SIase still exhibited more than 90% sucrose conversion after 13 consecutive cycles, which indicated that it had a good operational stability. Furthermore, the immobilized SIase has the potential for isomaltulose production, with 200 g L−1 sucrose solution as its substrate in the food industry. Isomaltulose was isolated in 83.58% yield and high purity (97.3%).magnified image

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Efficient production of isomaltulose using engineered Yarrowia lipolytica strain facilitated by non‐yeast signal peptide‐mediated cell surface display

Liu,

Li,

Chen

et al. 2024

J Sci Food Agric

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BackgroundIsomaltulose is a ‘generally recognized as safe’ ingredient and is widely used in food, pharmaceutical and chemical industries. Exploration and development of efficient technologies are essential for enhancing isomaltulose yield.ResultsIn this study, a simple and efficient surface display platform mediated by a non‐yeast signal peptide was developed in Yarrowia lipolytica and was utilized to efficiently produce isomaltulose from sucrose. We discovered that the signal peptide SP1 of sucrose isomerase from Pantoea dispersa UQ68J (PdSI) could guide SIs anchoring to the cell surface of Y. lipolytica, demonstrating a novel and simple cell surface display strategy. Furthermore, the PdSI expression level was significantly increased through optimizing the promoters and multi‐site integrating genes into chromosome. The final strain gained 451.7 g L−1 isomaltulose with conversion rate of 90.3% and space–time yield of 50.2 g L−1 h−1.ConclusionThis study provided an efficient way for manufacturing isomaltulose with high space–time yield. This heterogenous signal peptide‐mediated cell surface display strategy featured with small fusion tag (~2.2 kDa of SP1), absence of enzyme leakage in fermentation broth, and ample room for optimization, providing a convenient way to construct whole‐cell biocatalysts to synthesize other products and broadening the array of molecular toolboxes accessible for engineering Y. lipolytica.This article is protected by copyright. All rights reserved.

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Cloning and Characterization of a Sucrose Isomerase from Erwinia rhapontici NX-5 for Isomaltulose Hyperproduction

Cited by 39 publications

References 30 publications

The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

Bioinspired Production of Antibacterial Sucrose Isomerase‐Sponge for the Synthesis of Isomaltulose

Efficient production of isomaltulose using engineered Yarrowia lipolytica strain facilitated by non‐yeast signal peptide‐mediated cell surface display

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