Sucrose Isomerase Research Articles

High-Level Expression of Sucrose Isomerase in Bacillus subtilis Through Expression Element Optimization and Fermentation Optimization.

Sucrose isomerase is an important food enzyme that catalyzes the isomerization of sucrose into isomaltulose, a functional sugar widely used in food industry, while the production level of sucrose isomerase in food safe host strains was much lower than industrial requirement. Bacillus subtilis is an excellent host strain for recombinant protein expression, which owns the characteristics of powerful secretory capability and generally recognized as safe state. In this study, the expression of sucrose isomerase in B. subtilis was improved through expression element optimization and fermentation optimization. Firstly, the extracellular chaperone PrsA was overexpressed to enhance extracellular folding of sucrose isomerase, which improved the recombinant expression level by 80.02%. Then, the protein synthesis level was optimized through promoter screening, improving the recombinant expression level by 60.40%. On the basis of strain modification, the fermentation conditions including nitrogen source, carbon source, metal ion, pH and temperature were optimized successively in shake-flask. Finally, the 3 L bioreactor cultivation condition was optimized and yielding a sucrose isomerase activity of 862.86 U/mL, the highest level among the food safety strains. This study provides an effective strategy to improve the expression level of food enzymes in B. subtilis.

Development of an engineered Bacillus subtilis strain for antibiotic-free sucrose isomerase production.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered Bacillus subtilis strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the B. subtilis strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 UmL-1 in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 UmL-1 during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered B. subtilis strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.

A Critical Review on Immobilized Sucrose Isomerase and Cells for Producing Isomaltulose.

Isomaltulose is a novel sweetener and is considered healthier than the common sugars, such as sucrose or glucose. It has been internationally recognized as a safe food product and holds vast potential in pharmaceutical and food industries. Sucrose isomerase is commonly used to produce isomaltulose from the substrate sucrose in vitro and in vivo. However, free cells/enzymes were often mixed with the product, making recycling difficult and leading to a significant increase in production costs. Immobilized cells/enzymes have the following advantages including easy separation from products, high stability, and reusability, which can significantly reduce production costs. They are more suitable than free ones for industrial production. Recently, immobilized cells/enzymes have been encapsulated using composite materials to enhance their mechanical strength and reusability and reduce leakage. This review summarizes the advancements made in immobilized cells/enzymes for isomaltulose production in terms of refining traditional approaches and innovating in materials and methods. Moreover, innovations in immobilized enzyme methods include cross-linked enzyme aggregates, nanoflowers, inclusion bodies, and directed affinity immobilization. Material innovations involve nanomaterials, graphene oxide, and so on. These innovations circumvent challenges like the utilization of toxic cross-linking agents and enzyme leakage encountered in traditional methods, thus contributing to enhanced enzyme stability.

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

Isomaltulose is a 'generally recognized as safe' ingredient and is widely used in the food, pharmaceutical and chemical industries. The exploration and development of efficient technologies is essential for enhancing isomaltulose yield. In the present study, a simple and efficient surface display platform mediated by a non-yeast signal peptide was developed in Yarrowia lipolytica and 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 a conversion rate of 90.3% and a space-time yield of 50.2 g L-1 h-1. The present study provides an efficient way for manufacturing isomaltulose with a high space-time yield. This heterogenous signal peptide-mediated cell surface display strategy featured with small fusion tag (approximately 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. © 2024 Society of Chemical Industry.

Analyzing Current Trends and Possible Strategies to Improve Sucrose Isomerases' Thermostability.

Due to their ability to produce isomaltulose, sucrose isomerases are enzymes that have caught the attention of researchers and entrepreneurs since the 1950s. However, their low activity and stability at temperatures above 40 °C have been a bottleneck for their industrial application. Specifically, the instability of these enzymes has been a challenge when it comes to their use for the synthesis and manufacturing of chemicals on a practical scale. This is because industrial processes often require biocatalysts that can withstand harsh reaction conditions, like high temperatures. Since the 1980s, there have been significant advancements in the thermal stabilization engineering of enzymes. Based on the literature from the past few decades and the latest achievements in protein engineering, this article systematically describes the strategies used to enhance the thermal stability of sucrose isomerases. Additionally, from a theoretical perspective, we discuss other potential mechanisms that could be used for this purpose.

Construction and Catalytic Study of Affinity Peptide Orientation and Light Crosslinking Immobilized Sucrose Isomerase.

A novel affinity peptide orientation and light-controlled covalent immobilized method was developed. Sucrose isomerase (SI) was selected as the model enzyme. Molecular simulation was first performed to select the targeted immobilization region. Subsequently, a short peptide (H2N-VNIGGX-COOH, VG) with high affinity to this region was rationally designed. Thereafter, 4-benzoyl-l-phenylalanine with the photosensitive group of benzophenone was introduced. Then, the affinity between the ligand and the SI was validated using molecular dynamics simulation. Thereafter, the SI was directionally immobilized onto the surface of the epoxy resin (EP) guided by VG via photo-crosslinking, and thus the oriented photo-crosslinking enzymes were obtained. The enzymatic activity, thermostability, and reusability of the affinity directional photo-crosslinked immobilized sucrose isomerase (hv-EP-VG-SI) were systematically studied. The oriented immobilization enzymes were significantly improved in recycling and heat resistance. Moreover, hv-EP-VG-SI retained more than 90% of the original activity and 50% of the activity after 11 cycles.

Thermostability improvement of sucrose isomerase PalI NX-5: a comprehensive strategy.

To increase the thermal stability of sucrose isomerase from Erwinia rhapontici NX-5, we designed a comprehensive strategy that combines different thermostabilizing elements. We identified 19 high B value amino acid residues for site-directed mutagenesis. An in silico evaluation of the influence of post-translational modifications on the thermostability was also carried out. The sucrose isomerase variants were expressed in Pichia pastoris X33. Thus, for the first time, we report the expression and characterization of glycosylated sucrose isomerases. The designed mutants K174Q, L202E and K174Q/L202E, showed an increase in their optimal temperature of 5°C, while their half-lives increased 2.21, 1.73 and 2.89 times, respectively. The mutants showed an increase in activity of 20.3% up to 25.3%. The Km values for the K174Q, L202E, and K174Q/L202E mutants decreased by 5.1%, 7.9%, and 9.4%, respectively; furthermore, the catalytic efficiency increased by up to 16%. With the comprehensive strategy followed, we successfully obtain engineered mutants more suitable for industrial applications than their counterparts: native (this research) and wild-type from E. rhapontici NX-5, without compromising the catalytic activity of the molecule.

Engineering cyanobacteria for converting carbon dioxide into isomaltulose

Isomaltulose is a promising functional sweetener with broad application prospects in the food industry. Currently, isomaltulose is mainly produced through bioconversion processes based on the isomerization of sucrose, the economic feasibility of which is influenced by the cost of sucrose feedstocks, the biocatalyst preparation, and product purification. Cyanobacterial photosynthetic production utilizing solar energy and carbon dioxide represents a promising route for the supply of sugar products, which can promote both carbon reduction and green production. Previously, some cyanobacteria strains have been successfully engineered for synthesis of sucrose, the main feedstock for isomaltulose production. In this work, we introduced different sucrose isomerases into Synechococcus elongatus PCC 7942 and successfully achieved the isomaltulose synthesis and accumulation in the recombinant strains. Combinatory expression of an Escherichia coli sourced sucrose permease CscB with the sucrose isomerases led to efficient secretion of isomaltulose and significantly elevated the final titer. During a 6-day cultivation, 777 mg/L of isomaltulose was produced by the engineered Synechococcus cell factory. This work demonstrated a new route for isomaltulose biosynthesis utilizing carbon dioxide as the substrate, and provided novel understandings for the plasticity of cyanobacterial photosynthetic metabolism network.

Enhancement of healthful novel sugar contents in genetically engineered sugarcane juice integrated with molecularly characterized ThSyGII (CEMB-SIG2)

Enhancement of sugar contents and yielding healthful sugar products from sugarcane demand high profile scientific strategies. Previous efforts to foster manipulation in metabolic pathways or triggering sugar production through combating abiotic stresses fail to yield high sugar recovery in Saccharum officinarum L. Novel sucrose isomers trehalulose (TH) and isomaltulose (IM) are naturally manufactured in microbial sources. In pursuance of novel scientific methodology, codon optimized sucrose isomerase gene, Trehalulose synthase gene II(CEMB-SIG2) cloned under dual combined stem specific constitutive promoters in pCAMBIA1301 expression vector integrated with Vacuole targeted signal peptide (VTS) to concentrate gene product into the vacuole. The resultant mRNA expression obtained by Real Time PCR validated extremely increased transgene expression in sugarcane culms than leaf tissues. Overall sugar estimation from transgenic sugarcane lines was executed through refractometer. HPLC based quantifications of Trehalulose (TH) alongside different internodes of transgenic sugarcane confirmed the enhancement of boosted sugar concentrations in mature sugarcane culms. Trehalulose synthase gene II receptive sugarcane lines indicated the unprecedented impressions of duly combined constitutive stem regulated promoters. Transgenic sugarcane lines produce highest sugar recovery percentages, 14.9% as compared to control lines (8.5%). The increased sugar recovery percentage in transgenic sugarcane validated the utmost performance and expression of ThSyGII gene .High Profile Liquid chromatography based sugar contents estimation of Trehalulose (TH) and Isomaltulose (IM) yielded unprecedented improvement in the whole sugar recovery percentage as compared to control lines.⁠

Robust and recyclable cross-linked enzyme aggregates of sucrose isomerase for isomaltulose production

A novel cross-linked enzyme aggregates (CLEAs) catalyst was produced by precipitation and cross-linking sucrose isomerase (SIase) for isomaltulose production. The effects of precipitants and cross-linkers on the catalytic performance of the CLEAs were first evaluated. Then, bovine serum albumin (BSA) was used as additive and two immobilized enzymes, cross-linked SIase aggregates (CLSIAs) and CLSIAs-BSA were obtained. All the immobilized preparations exhibited superior thermal stability, pH tolerance, and storage stability compared to the soluble SIase, and showed excellent reusability. These samples still retained more than 61% of their initial activity after ten reuse cycles, with CLSIAs-BSA retaining up to 91.7%. The conversion ratios of sucrose into isomaltulose using CLSIAs-BSA reached 88.4 and 81.2% with sucrose and sugar cane juice as substrate, respectively. Therefore, CLSIAs are a highly effective biocatalyst for the preparation of isomaltulose with great potential for industrial applications.

Enhanced Extracellular Production and Characterization of Sucrose Isomerase in Bacillus subtilis with Optimized Signal Peptides

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, which is an important functional sugar widely used in the food industry. However, the lack of safe and efficient expression systems for recombinant SIase has impeded its production and application. In this study, enhanced expression of a SIase from Klebsiella sp. LX3 (referred to as KsLX3-SIase) was achieved in Bacillus subtilis WB800N, by optimizing the signal peptides. First, 13 candidate signal peptides were selected using a semi-rational approach, and their effects on KsLX3-SIase secretion were compared. The signal peptide WapA was most efficient in directing the secretion of KsLX3-SIase into the culture medium, producing a specific activity of 23.0 U/mL, as demonstrated by shake flask culture. Using a fed-batch strategy, the activity of KsLX3-SIase in the culture medium was increased to 125.0 U/mL in a 5-L fermentor. Finally, the expressed KsLX3-SIase was purified and was found to have maximum activity at 45 °C and pH 5.5. Its Km for sucrose was 267.6 ± 18.6 mmol/L, and its kcat/Km was 10.1 ± 0.2 s−1mM−1. These findings demonstrated an efficient expression of SIase in B. subtilis, and this is thought to be the highest level of SIase produced in a food-grade bacteria to date.

Sustainable isomaltulose production in Corynebacterium glutamicum by engineering the thermostability of sucrose isomerase coupled with one-step simplified cell immobilization.

Sucrose isomerase (SI), catalyzing sucrose to isomaltulose, has been widely used in isomaltulose production, but its poor thermostability is still resisted in sustainable batches production. Here, protein engineering and one-step immobilized cell strategy were simultaneously coupled to maintain steady state for long-term operational stabilities. First, rational design of Pantoea dispersa SI (PdSI) for improving its thermostability by predicting and substituting the unstable amino acid residues was investigated using computational analysis. After screening mutagenesis library, two single mutants (PdSIV280L and PdSIS499F) displayed favorable characteristics on thermostability, and further study found that the double mutant PdSIV280L/S499F could stabilize PdSIWT better. Compared with PdSIWT, PdSIV280L/S499F displayed a 3.2°C-higher Tm, and showed a ninefold prolonged half-life at 45°C. Subsequently, a one-step simplified immobilization method was developed for encapsulation of PdSIV280L/S499F in food-grade Corynebacterium glutamicum cells to further enhance the recyclability of isomaltulose production. Recombinant cells expressing combinatorial mutant (RCSI2) were successfully immobilized in 2.5% sodium alginate without prior permeabilization. The immobilized RCSI2 showed that the maximum yield of isomaltulose by batch conversion reached to 453.0 g/L isomaltulose with a productivity of 41.2 g/l/h from 500.0 g/L sucrose solution, and the conversion rate remained 83.2% after 26 repeated batches.

Tuning the catalytic performances of a sucrose isomerase for production of isomaltulose with high concentration.

Obtaining a sucrose isomerase (SIase) with high catalytic performance is of great importance in industrial production of isomaltulose (a reducing sugar). In order to obtain such SIase mutant, a high-throughput screening system in microtiter plate format was developed based on a widely used 2,4-dinitrosalicylic acid (DNS) method for determination of reducing sugar. An SIase from Erwiniasp. Ejp617 (ErSIase) was selected to improve its catalytic efficiency. After screening of ~ 8000 mutants from a random mutagenesis library, Q209 and R456 were identified as beneficial positions. Saturation mutagenesis of the two positions resulted in a double-site mutant ErSIase_Q209S-R456H that showed the highest catalytic efficiency, and its specific activity reached 684 U/mg that is 17.5-fold higher than that of the wild-type ErSIase. By employing the lyophilized Escherichia coli (E. coli) cells harboring ErSIase_Q209S-R456H, a high space-time yield (STY = 3.9kg/(L·d)) was achieved toward 600g/L sucrose. Furthermore, the in silico analysis suggested that the hydrogen bond network was improved and steric hindrance was reduced due to the beneficial substitutions.Key points• A sucrose isomerase mutant with high catalytic efficiency was obtained.• The highest space-time yield was achieved toward high-concentration sucrose.• The optimized H-bond network contributed to the enhanced catalytic efficiency.

Immobilization of Sucrose Isomerase from Erwinia sp. with Graphene Oxide and Its Application in Synthesizing Isomaltulose.

Sucrose isomerase (SIase) is a key enzyme used for the production of isomaltulose from sucrose. In this study, an SIase gene from Erwinia sp. Ejp617 (ErSIase) was heterologously expressed in Escherichia coli BL21(DE3), and the recombinant ErSIase was served as biocatalyst combined with the graphene oxide (GO) as carrier for ErSIase immobilization. The Fourier transform infrared spectroscopy, transmission electron microscope, and confocal laser microscopy analyses showed that ErSIase was successfully immobilized on the surface of GO to form ErSIase-GO. The loading capacity of ErSIase on GO reached up to 460mg/g with a specific activity of 727.04 U/mg protein when the optimal immobilization time of 12h and the ErSIase/GO ratio of 7.4:4 (w/w) were applied. A high conversion rate of 95.3% was reached from sucrose to isomaltulose using ErSIase-GO as biocatalyst with 600g/L sucrose as substrate, after 180min at 40°C and pH 6.0. Moreover, stabilities of the immobilized ErSIase-GO in the aspects of thermal, pH, and storage were improved, and its activity after 10 batches still remained around 80% under the optimal conditions. The Km value of ErSIase-GO was 29.32mM, and the kcat/Km was increased to 27.34s-1mM-1 when 0.1% (w/v) detergent NP40 was added. These results indicated that the ErSIase was well immobilized onto GO, and the ErSIase-GO is a promising biocatalyst with high operational stability and catalytic activity for industrial production of isomaltulose.

Direct Isomaltulose Synthesis From Beet Molasses by Immobilized Sucrose Isomerase

Isomaltulose is becoming a focus as a functional sweetener for sucrose substitutes; however, isomaltulose production using sucrose as the substrate is not economical. Low-cost feedstocks are needed for their production. In this study, beet molasses (BM) was introduced as the substrate to produce isomaltulose for the first time. Immobilized sucrose isomerase (SIase) was proved as the most efficient biocatalyst for isomaltulose synthesis from sulfuric acid (H2SO4) pretreated BM followed by centrifugation for the removal of insoluble matters and reducing viscosity. The effect of different factors on isomaltulose production is investigated. The isomaltulose still achieved a high concentration of 446.4 ± 5.5 g/L (purity of 85.8%) with a yield of 0.94 ± 0.02 g/g under the best conditions (800 g/L pretreated BM, 15 U immobilized SIase/g dosage, 40°C, pH of 5.5, and 10 h) in the eighth batch. Immobilized SIase used in repeated batch reaction showed good reusability to convert pretreated BM into isomaltulose since the sucrose conversion rate remained 97.5% in the same batch and even above 94% after 11 batches. Significant cost reduction of feedstock costs was also confirmed by economic analysis. The findings indicated that this two-step process to produce isomaltulose using low-cost BM and immobilized SIase is feasible. This process has the potential to be effective and promising for industrial production and application of isomaltulose as a functional sweetener for sucrose substitute.

A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene pal-2 from Raoultella terrigena was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the E. coli system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (Km) and maximum reaction rate (Vmax) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the Raoultella genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.

Stem vacuole-targetted sucrose isomerase enhances sugar content in sorghum

BackgroundSugar content is critically important in determining sugar crop productivity. However, improvement in sugar content has been stagnant among sugar crops for decades. Sorghum, especially sweet sorghum with high biomass, shown great potential for biofuel, has lower sugar content than sugarcane. To enhance sugar content, the sucrose isomerase (SI) gene, driven by stem-specific promoters (A2 or LSG) with a vacuole-targetted signal peptide, was transformed into the sorghum inbred line (T×430).ResultsThe study demonstrated that transgenic lines of grain sorghum, containing 50–60% isomaltulose, accumulated up to eightfold (1000 mM) more total sugar than the control T×430 did (118 mM) in stalks of T0 generation. Subsequently, the elite engineered lines (A5, and LSG9) were crossed with sweet sorghum (Rio, and R9188). Total sugar contents (over 750 mM), were notably higher in F1, and F2 progenies than the control Rio (480 mM). The sugar contents of the engineered lines (over 750 mM), including T0, T1, F1, and F2, are surprisingly higher than that of the field-grown sugarcane (normal range 600–700 mmol/L). Additionally, analysis of physiological characterization demonstrated that the superior progenies had notably higher rates of photosynthesis, sucrose transportation, and sink strength than the controls.ConclusionsThe genetic engineering approach has dramatically enhanced total sugar content in grain sorghum (T0, and T1) and hybrid sorghum (F1, and F2), demonstrating that sorghum can accumulate as high or higher sugar content than sugarcane. This research illustrates that the SI gene has enormous potential on improvement of sugar content in sorghum, particularly in hybirds and sweet sorghum. The substantial increase on sugar content would lead to significant financial benefits for industrial utilization. This study could have a substantial impact on renewable bioenergy. More importantly, our results demonstrated that the phenotype of high sugar content is inheritable and shed light on improvement for other sugar crops.

Studies on Biological Production of Isomaltulose Using Sucrose Isomerase: Current Status and Future Perspectives

Isomaltulose, as a safe sucrose substitute, is widely used as a functional sweetener due to its promising properties, such as slower digestion, prolonged energy release, and less cariogenicity. The transformation of sucrose to isomaltulose by free sucrose isomerase (SIase) or microbial cells harboring the SIase gene has received considerable attention in the industry. Heterologous expression of SIase in food-safe grade strains has become a hot topic due to its broad applicability in the food industry. Thanks to rapid developments in genetic engineering technology, SIases from different sources have been heterogeneously expressed in Escherichia coli, which significantly increased the enzyme’s titer. This review presents a systematic and detailed summary of the contemporary biotechnological approaches employed for isomaltulose production, including the source, structural determination, catalysis mechanism, heterologous expression, catalytic reaction condition optimization of SIase, and immobilization of cells. In addition, protein engineering, heterologous expression in food-grade safety strains, fermentation optimization strategies, and immobilization techniques of SIase are introduced in detail. Towards the end, the review is wrapped up with the concluding remarks, and future strategies are outlined for improving the biological production of isomaltulose. Summary of biological isomaltulose production from sucrose catalyzed by sucrose isomerase.

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

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.

Characterization of a recombinant sucrose isomerase and its application to enzymatic production of isomaltulose.

To characterize a recombinant isomerase that can catalyze the isomerization of sucrose into isomaltulose and investigate its application for the enzymatic production of isomaltulose. A sucrose isomerase gene from Erwinia sp. Ejp617 was synthesized and expressed in Escherichia coli BL21(DE3). The enzymatic characterization revealed that the optimal pH and temperature of the purified sucrose isomerase were 6.0 and 40°C, respectively. The enzyme activity was slightly activated by Mn2+and Mg2+, but partially inhibited by Ca2+, Ba2+, Cu2+, Zn2+ and EDTA. The kinetic parameters of Km and Vmax for sucrose were 69.28mM and 118.87 U/mg, respectively. The time course showed that 240.9g/L of isomaltulose was produced from 300g/L of sucrose, and the yield reached 80.3% after bioreaction for 180min. This recombinant enzyme showed excellent capability for biotransforming sucrose to isomaltulose at the substrate concentration of 300g/L. Further investigations should be carried out focusing on selection of suitable heterologous expression system with the aim to improve its expression level.