„Schiffe Versenken“: Optimierung einer hochaktiven Saccharose-Isomerase

The sucrose isomerase SmuA from Serratia plymuthica catalyses the production of isomaltulose, an artificial sweetener used in the food industry. To suppress the formation of the hygroscopic by-product trehalulose we applied a semirational protein engineering strategy inspired by the “battleship” board game. After seven cycles of introducing amino acid exchanges around the active site and investigating their influence on the enzymatic product profile we obtained a triple mutant that showed 2.3 times less trehalulose formation but had retained high catalytic efficiency.

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

Background Sugar 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). Results The 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. Conclusions The 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.

Stem Vacuole-targeted Sucrose Isomerase Enhances Sugar Accumulation in Sorghum

Background: Sugar accumulation 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, has shown great potential for biofuel. In this study, sorghum was investigated as a C4 diploid model for crops with more complicated genomes such as maize and sugarcane. To enhance sugar accumulation, the sucrose isomerase (SI) gene, driven by stem-specific promoters (A2 or LSG) with a vacuole-targeted signal peptide, was transformed into the sorghum inbred line (Tx430).Results: The study demonstrated that transgenic lines of grain sorghum, containing 50-60% isomaltulose, accumulated sevenfold (804 mM) more total sugar than the control Tx430 did (118 mM) in stalks. Subsequently, the elite engineered lines (A5, and LSG9) were crossed with sweet sorghum (R9188, and Rio). Total sugar contents (over 750 mM), were significantly 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 higher than that of the field-grown sugarcane (normal range 600-700 mmol/L). Additionally, physiological characterization demonstrated that the superior progenies had notably higher rates of photosynthesis, sucrose transport, and sink strength than the controls.Conclusions: The genetic engineering approach has significantly enhanced total sugar content in grain sorghum (T0, and T1) and hybrid sorghum (F1, and F2), demonstrating that sorghum can accumulate sugar contents as high or higher than sugarcane. This research puts sorghum in the spotlight and frontier as a biofuel crop, particularly as it is a shorter duration crop. The substantial increase in sugar content would lead to enormous 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 accumulation is inheritable and shed light on improvement for other sugar crops.

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.

Stem vacuole-targetted sucrose isomerase enhances sugar accumulation in sorghum

Background: Sugar accumulation is critically important in determining sugar crop productivity. Sorghum, especially high biomass sweet sorghum has shown great potential for biofuel. However, improvement in sugar content has been stagnant among sugar crops for decades. In this study, sorghum was investigated as a C4 diploid model for more complicated genomes such as maize and sugarcane. To promote sugar accumulation in sorghum, the sucrose isomerase (SI) gene, driven by stem-specific promoters A1 (A) or LSG2 (L) with a signal peptide, was designed to target the stem vacuole in grain sorghum inbred line (Tx430).Results: The study demonstrated that transgenic lines of grain sorghum can accumulate isomaltulose which accounted for 50-60% of total sugar (up to 1012 mM) in stalks. While the average sugar content is 118 mM in the control Tx430. Subsequently, the best-engineered line (L9) was crossed with an elite sweet sorghum variety (Rio). The total sugar contents were significantly higher in both F1 (up to 763 mM) and F2 (up to 821 mM) progenies than the sweet sorghum Rio (485 mM), representing 57% and 69% increase respectively. Those total sugar contents in those engineered sorghum lines are higher than in the field-grown sugarcane (600-700 mM). Physiological characterization demonstrated that the superior progenies of F2 hybrids had notably higher rates of photosynthesis, sucrose transport, and sink strength than controls.Conclusion: The genetic engineering approach has significantly enhanced total sugar content in grain sorghum and hybrids of (sweet X grain) sorghum. This research has put sorghum in the spotlight and frontier as a biofuel crop. More importantly, our results prove that the phenotype of high sugar content is heritable in the grain sorghum as well as hybrids. The massive increase in sugar accumulation would lead to enormous financial benefits for industrial and biofuel use. This study would have a substantial impact on renewable energy due to the supreme capacity of total sugar accumulation in transgenic sorghum.