Molecular cloning and functional characterization of a sucrose isomerase (isomaltulose synthase) gene from Enterobacter sp. FMB-1

Cha, Jaeho; Jung, Jong‐Hyun; Park, S.E.; Cho, M.H.; Seo, Dong-Ho; Ha, Suk‐Jin; Yoon, Jong-Won; Lee, Ok‐Hwan; Kim, Y.C.; Park, C.S.

doi:10.1111/j.1365-2672.2009.04295.x

Cited by 26 publications

(13 citation statements)

References 44 publications

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“…The recombinant SIase was located in the periplasmic space, unlike SIases of Enterobacter sp. FMB-1, P. rubrum, and S. plymuthica [9,13,26]. The facility of substrate transfer and product export could result in less product inhibition.…”

Section: Conversion Of Sucrose Into Isomaltulose By Recombinant E Comentioning

confidence: 97%

“…LX3, P. Mesoacidophila MX-45, and Enterobacter sp. FMB-1, showed significant decreases in enzyme activity at pH below 5.0 [4,6,9]. Thus, the stability of the present SIase would be less likely to be affected when cells experienced lower pH and pO 2 conditions [10], and enzymes that could tolerate acidic environment would help to reduce the chemical reaction between sugars and proteins [25].…”

Section: Enzymatic Characterization Of the Recombinant Sucrose Isomerasementioning

confidence: 99%

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

Cai

Qing

et al. 2010

Appl Biochem Biotechnol

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The sucrose isomerase (SIase) gene from an efficient strain of Erwinia rhapontici NX-5 for isomaltulose hyperproduction was cloned and overexpressed in Escherichia coli. Protein sequence alignment revealed that SIase was a member of the glycoside hydrolase 13 family. The molecular mass of the purified recombinant protein was estimated at 66 kDa by SDS-PAGE. The SIase had an optimal pH and temperature of 5.0 and 30 °C, respectively, with a K (m) of 257 mmol/l and V (max) of 48.09 μmol/l/s for sucrose. To the best of our knowledge, the recombinant SIase has the most acidic optimum pH for isomaltulose synthesis. When the recombinant E. coli (pET22b- palI) cells were used for isomaltulose synthesis, almost complete conversion of sucrose (550 g/l solution) to isomaltulose was achieved in 1.5 h with high isomaltulose yields (87%). The immobilized E. coli cells remained stable for more than 30 days in a "batch"-type enzyme reactor. This indicated that the recombinant SIase could continuously and efficiently produce isomaltulose.

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Section: Conversion Of Sucrose Into Isomaltulose By Recombinant E Comentioning

confidence: 97%

Section: Enzymatic Characterization Of the Recombinant Sucrose Isomerasementioning

confidence: 99%

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

Cai

Qing

et al. 2010

Appl Biochem Biotechnol

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“…Many bacterial species sucrose isomerase producing has been reported, including Serratia plymuthica, Erwinia rhapontici, Enterobacter sp., Klebsiella singaporensis, Raultella planticola, Enterobacter sp., Pantoea dispersa, and Pseudomonas mesoacidophila (Goulter et al, 2012;Mu et al, 2014a). Several of sucrose isomerase coding genes have been cloned, expressed and characterized (Cha et al, 2009;Park et al, 2010;Mu et al, 2014b). In general, sucrose isomerases are functioning in near-neutral pH and are thermolabile.…”

Section: Introductionmentioning

confidence: 99%

Cloning of sucrose isomerase encoding gene from Klebsiella singaporensis ISB-36 and its expression in Pichia pastoris

Bach¹,

Anh²,

Thuy³

et al. 2020

Vietnam J. Biotechnol.

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Given potential health benefits including low glycemic index, tooth friendly, suitable to infants, elderly and diabetic patients, isomaltulose was considered as a promising alternative sweetener to sucrose. Due to the presence of liposaccharide endotoxin in Serratia plymuthica CBS 574.44, a Gram-negative bacterium, and minute amount of formaldehyde carried over, purification of isomaltulose requires rigorous controls in industry. To reduce the cost associated with product purification, here we propose the use of recombinant enzyme in isomaltulose production. The mature gene coding for sucrose isomerase synthase (K.SI36.PalI) from Klebsiella singarporensis ISB 36, which isolated from woodborer in Vietnam, was expressed in Pichia pastoris X33. The nucleotide sequence of K.SI36.PalI gene was similar to AY040843.1 of Klebsiella sp. LX3 except one nucleotide C1025 in AY040843.1 replaced by T1025 in K.SI36.PalI. This leads to single amino acid difference in deduced protein sequence (from 342Ser to 342Phe). Furthermore, the addition of two amino acids (Glu and Phe) was observed at N-terminus. The calculated molecular weight of sucrose isomerase from K.SI36.PalI was 67.46 kDa and the pI was 6.55. There was one potential glycosylation site at 466Asn. The maximum sucrose isomerase activity in the culture broth reached 36,6 U.mL-1in 1 L shake-flask. The purified recombinant enzyme was most active at 40°C and pH 7.0. At the optimum condition, within 6 hours, the enzyme converted 94% of sucrose in a 40% sucrose solution into isomaltulose. This was the first study on the expression of sucrose isomerase synthase gene in P. pastoris, and the results showed the efficient conversion of sucrose isomerase recombinant.

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“…LX3 [ 7 ], Klebsiella pneumoniae NK33-98-9[ 8 ], Pantoea dispersa UQ68J [ 9 ], Enterobacter sp. FMB-1 [ 10 ], Erwinia rhapontici NX-5 [ 4 , 11 , 12 ], Pseudomonas mesoacidophila MX-45 [ 13 ]. A series of the corresponding genes have been cloned and designated pal I [ 5 , 7 , 8 , 10 , 11 ], smu A [ 6 ], sim [ 9 ] and mut B [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

“…FMB-1 [ 10 ], Erwinia rhapontici NX-5 [ 4 , 11 , 12 ], Pseudomonas mesoacidophila MX-45 [ 13 ]. A series of the corresponding genes have been cloned and designated pal I [ 5 , 7 , 8 , 10 , 11 ], smu A [ 6 ], sim [ 9 ] and mut B [ 13 ]. During the isomerization of sucrose to isomaltulose and trehalulose, small amounts of glucose and fructose are produced as by-products [ 5 , 7 , 9 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis

Duan

Cheng

et al. 2016

PLoS ONE

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The sucrose isomerase of Serratia plymuthica AS9 (AS9 PalI) was expressed in Escherichia coli BL21(DE3) and characterized. The half-life of AS9 PalI was 20 min at 45°C, indicating that it was unstable. In order to improve its thermostability, six amino acid residues with higher B-factors were selected as targets for site-directed mutagenesis, and six mutants (E175N, K576D, K174D, G176D, S575D and N577K) were designed using the RosettaDesign server. The E175N and K576D mutants exhibited improved thermostability in preliminary experiments, so the double mutant E175N/K576D was constructed. These three mutants (E175N, K576D, E175N/K576D) were characterized in detail. The results indicate that the three mutants exhibit a slightly increased optimal temperature (35°C), compared with that of the wild-type enzyme (30°C). The mutants also share an identical pH optimum of 6.0, which is similar to that of the wild-type enzyme. The half-lives of the E175N, K576D and E175N/K576D mutants were 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. Kinetic studies showed that the Km values for the E175N, K576D and E175N/K576D mutants decreased by 6.6%, 2.0% and 11.0%, respectively, and their kcat/Km values increased by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, we found that the E175N, K576D and E175N/K576D mutants displayed slightly improved isomaltulose yields, compared with the wild-type enzyme. Therefore, the mutants produced in this study would be more suitable for industrial biosynthesis of isomaltulose.

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Molecular cloning and functional characterization of a sucrose isomerase (isomaltulose synthase) gene from Enterobacter sp. FMB-1

Cited by 26 publications

References 44 publications

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

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

Cloning of sucrose isomerase encoding gene from Klebsiella singaporensis ISB-36 and its expression in Pichia pastoris

Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis

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