Data of fixed-target pink-beam serial synchrotron crystallography at the Pohang Light Source II.

Pink-beam serial synchrotron crystallography (SSX) is beneficial in terms of X-ray flux and overcoming partial reflection compared with SSX using a monochromatic beam. The fixed-target (FT) scanning method can minimize the physical damage on the crystal sample when delivering the crystals to the X-ray interaction point. Additionally, general researchers can easily access the experiment since no specialized sample transfer technology is needed. The fixed-target pink-beam SSX at the 1C beamline at the Pohang Light Source II (PLS-II) was previously demonstrated using a newly developed magnetic-based sample holder. The room-temperature structure of glucose isomerase and lysozyme were determined using FT pink-beam SSX. Meanwhile, the SSX dataset for glucose isomerase and lysozyme images containing the high X-ray background and multi-crystal hits. These data can be tentatively used to develop an indexing algorithm and practice processing the SX data. This study used detailed information on the diffraction data of fixed-target pink-beam SSX at PLS-II to access the raw data and process the information.

Promotion of lactose isomerization to fructose and lactulose in one batch by immobilized enzymes on bacterial cellulose membranes.

The production of the sugars fructose and lactulose from lactose using the enzymes ß-galactosidase and glucose isomerase immobilized on bacterial cellulose (BC) membranes has been investigated. Lactose is hydrolyzed by ß-galactosidase at 30 °C to glucose and galactose at a high conversion rate, while at the same temperature, glucose isomerase is not effective in converting the produced glucose to fructose. The rate of the isomerization reaction of glucose to fructose at 70 °C has been studied. Two types of enzyme immobilization were investigated: immobilization in one stage and immobilization in two stages. The results showed that BC membrane increased three-fold the yield and the reaction rate of fructose and lactulose production from lactose. The noteworthy enhancement of BC membranes' impact on the isomerization reaction by immobilized enzymes grants permission for a novel research avenue within the context of white biotechnology development. Additionally, this effect amplifies the role of BC in sustainability and the circular economy.

Enhanced High-Fructose Corn Syrup Production: Immobilizing Serratia marcescens Glucose Isomerase on MOF (Co)-525 Reduces Co2+ Dependency in Glucose Isomerization to Fructose.

The escalating demand for processed foods has led to the widespread industrial use of glucose isomerase (GI) for high-fructose corn syrup (HFCS) production. This reliance on GIs necessitates continual Co2+ supplementation to sustain high catalytic activity across multiple reaction cycles. In this study, Serratia marcescens GI (SmGI) was immobilized onto surfaces of the metal-organic framework (MOF) material MOF (Co)-525 to generate MOF (Co)-525-GI for use in catalyzing glucose isomerization to generate fructose. Examination of MOF (Co)-525-GI structural features using scanning electron microscopy-energy dispersive spectroscopy, Fourier-transform infrared spectroscopy, and ultraviolet spectroscopy revealed no structural changes after SmGI immobilization and the addition of Co2+. Notably, MOF (Co)-525-GI exhibited optimal catalytic activity at pH 7.5 and 70 °C, with a maximum reaction rate (Vmax) of 37.24 ± 1.91 µM/min and Km value of 46.25 ± 3.03 mM observed. Remarkably, immobilized SmGI exhibited sustained high catalytic activity over multiple cycles without continuous Co2+ infusion, retaining its molecular structure and 96.38% of its initial activity after six reaction cycles. These results underscore the potential of MOF (Co)-525-GI to serve as a safer and more efficient immobilized enzyme technology compared to traditional GI-based food-processing technologies.

Data of the subatomic resolution structure of glucose isomerase complexed with xylitol inhibitor.

Glucose isomerase (GI) is a crucial enzyme in industrial processes, including the production of high-fructose corn syrup, biofuels, and other renewable chemicals. Understanding the mechanisms of GI inhibition by GI inhibitors can offer valuable insights into enhancing production efficiency. We previously reported the subatomic resolution structure of Streptomyces rubiginosus GI (SruGI) complexed with a xylitol inhibitor, determined at 0.99 Å resolution, was reported. Structural analysis showed that the xylitol inhibitor is partially bound to the M1 binding site at the SruGI active site, enabling it to distinguish the xylitol-bound and -free state of SruGI. This structural information demonstrates that xylitol binding to the M1 site causes a conformational change in the metal binding site and the substrate binding channel of SruGI. Herein, detailed information on data collection and processing procedures of the subatomic resolution structure of the SruGI complexed with xylitol was reported.

Xylitol binding to the M1 site of glucose isomerase induces a conformational change in the substrate binding channel.

Glucose isomerase (GI) is extensively used in the food industry for production of high-fructose corn syrup and for the production of biofuels and other renewable chemicals. Structure-based studies on GI inhibitors are important for improving its efficiency in industrial applications. Here, we report the subatomic crystal structure of Streptomyces rubiginosus GI (SruGI) complexed with its inhibitor, xylitol, at 0.99 Å resolution. Electron density map and temperature factor analysis showed partial binding of xylitol to the M1 metal binding site of SruGI, providing two different conformations of the metal binding site and the substrate binding channel. The xylitol molecule induced a conformational change in the M2 metal ion-interacting Asp255 residue, which subsequently led to a conformational change in the side chain of Asp181 residue. This led to the positional shift of Pro25 by 1.71 Å and side chain rotation of Phe26 by 21°, where located on the neighboring protomer in tetrameric SruGI. The conformation change of these two residues affect the size of the substrate-binding channel of GI. Therefore, xylitol binding to M1 site of SruGI induces not only a conformational changes of the metal-binding site, but also conformational change of substrate-binding channel of the tetrameric SruGI. These results expand our knowledge about the mechanism underlying the inhibitory effect of xylitol on GI.

Enzymatic and selective production of alkyl α-d-glucopyranosides by the α-glucosyl transfer enzyme derived from Xanthomonas campestris WU-9701.

Several alkyl glucosides exhibit various bioactivities. 1-Octyl ß-d-glucopyranoside produced by organic synthesis is used as a nonionic surfactant. However, no convenient method has been developed for the selective production of alkyl α-glucosides (α-AGs), such as 1-octyl α-d-glucopyranoside (α-OG). Therefore, we developed a simple method for selective production of α-AGs using the glucosyl transfer enzyme XgtA, (E.C. 3.2.1.20), derived from Xanthomonas campestris WU-9701. When 0.80 M alkyl alcohol and 2.5 units XgtA were incubated in 2.0 mL of 30 mM HEPES-NaOH buffer (pH 8.0) containing 1.2 M maltose at 45 °C, a specific α-AG corresponding to each alkyl alcohol (C2-C10) was detected. Under the standard conditions, we examined the selective production of α-OG from 1-octanol and maltose using XgtA. The reaction product was isolated and identified as α-OG via 1H nuclear magnetic resonance and nuclear overhauser effect spectroscopy analyses. No other glucosylated products, such as maltotriose, were detected in the reaction mixture. Under the standard conditions at 45 °C for 96 h, 243 mM α-OG (71 g/L) was produced in one batch production. Moreover, the addition of glucose isomerase to the reaction mixture decreased the concentration of glucose released via the reaction and increased the amount of α-OG produced; 359 mM α-OG (105 g/L) was maximally produced at 96 h. In conclusion, this study demonstrates the selective production of α-AGs using a simple enzymatic reaction, and XgtA has the potential to selectively produce various α-AGs.

Mining and Characterization of Thermophilic Glucose Isomerase Based on Virtual Probe Technology.

Fructose, which is produced by the isomerization of glucose isomerase, is a crucial precursor for the biosynthesis of rare sugars. In this study, thermophilic glucose isomerases (GI) from Caldicellulosiruptor acetigenus (CAGI), Thermoanaerobacter thermocopriae (TTGI), and Thermotoga petrophila (TPGI) were screened from GenBank database by a virtual probe and were successfully expressed in Escherichia coli BL21(DE3). The results of characterization demonstrated that the optimal pH for CAGI and TTGI were 8.0 and were maintained at 80% in a slightly acidic environment. The relative residual activities of CAGI and TTGI were found to be 40.6% and 52.6%, respectively, following an incubation period of 24 h at 90 ℃. Furthermore, CAGI and TTGI exhibited superior catalytic performance that their reaction equilibrium both reached only after an hour at 85 ℃ with 200 g/L glucose, and the highest conversion rates were 54.2% and 54.1%, respectively. This study identifies competitive enzyme candidates for fructose production in the industry with appreciable cost reduction.

Carbohydrate microcapsules tailored and grafted for covalent immobilization of glucose isomerase for pharmaceutical and food industries.

Carrageenan is one of the most common carbohydrates utilised in the entrapment industry to immobilise cells and enzymes. However, it lacks functionality. Carrageenan has been grafted to produce fructose by covalently immobilising glucose isomerase (GI). Fructose is one of the most widely used sweeteners in beverages, food production, and the pharmaceutical business. Up to 91.1 U g-1 gel beads are immobilised by the grafted beads. Immobilized GI has a Vmax of 13.8 times that of the free enzyme. pH of immobilized GI was improved from 6.5-7 to 6-7.5 that means more stability in wide pH range. Also, optimum temperature was improved and become 65-75 °C while it was at 70 °C for free enzyme. The immovability and tolerance of the gel beads immobilised with GI over 15 consecutive cycles were demonstrated in a reusability test, with 88 percent of the enzyme's original activity retained, compared to 60 percent by other authors. These findings are encouraging for high-fructose corn syrup producers.

Novel Recyclable UCST-Type Immobilized Glucose Isomerase Biocatalyst with Excellent Performance for Isomerization of Glucose to Fructose.

The development of a suitable immobilization strategy to improve the performance of immobilized glucose isomerase for the isomerization of glucose to fructose is crucial to promoting the industrial production of high-fructose syrup. In this work, a novel recyclable upper critical solution temperature (UCST)-type mVBA-b-P(AAm-co-AN)@glucose isomerase biocatalyst (PVAA@GI) was prepared, and the immobilized glucose isomerase could capture the glucose substrate through the affinity of 4-vinylbenzeneboronic acid (4-VBA) and the glucose substrate, which led to the enhanced substrate affinity and catalytic efficiency of the PVAA@GI. The biocatalyst exhibited excellent stability in pH, thermal, storage, and recycling compared to the free enzyme. The mVBA-b-P(AAm-co-AN)@glucose isomerase biocatalyst displayed reversibly soluble-insoluble characteristics with temperature change, which was in the soluble state during the enzyme reaction process but could be recovered in an insoluble form by lowering the temperature after the reaction. The highest fructose production rate reached 62.79%, which would have potential application in the industrial production of high-fructose syrup.

Selective and high-yield production of ethyl α-d-glucopyranoside by the α-glucosyl transfer enzyme of Xanthomonas campestris WU-9701 and glucose isomerase.

Ethyl α-d-glucopyranoside (α-EG) is detected in sake (Japanese rice wine), that has moisturizing and skin conditioning effects. The production of α-EG by fermentation or enzymatic synthesis to date generates unwanted by-products such as maltooligosaccharides and/or organic acids. In this study, we employed a reaction involving selective α-glucosylation of ethanol by the α-glucosyl transfer enzyme (XgtA) of Xanthomonas campestris WU-9701. Under standard conditions, when 0.80 M ethanol and 1.2 M maltose were used as substrates with XgtA (2.5 units) and incubated in 30 mM HEPES-NaOH buffer (pH 8.0) at 45°C, only one form of ethyl glucopyranoside was selectively obtained as a product. The isolated product was identified as ethyl α-d-glucopyranoside by 1H NMR, 1H-1H COSY, and NOESY analyses. In the reaction mixture, other glucosylated products such as maltotriose and ethylmaltoside were not detected. Under optimum conditions, 180 mM (37.5 g/L) α-EG was produced in one batch production for 80 h. Further, the reaction rate of α-EG production decreased with an increase in glucose, especially more than 500 mM. In contrast, the addition of glucose isomerase decreased the concentration of glucose and was useful for maintaining a glucose concentration of less than 500 mM in the reaction mixture. Thus, owing to the enzymatic reaction with XgtA and glucose isomerase, as much as 260 mM (54.1 g/L) α-EG was produced in one batch production for 100 h. Altogether, this study reports the highest concentration of α-EG produced by enzymatic reaction.

Bioconversion of Lactose into Glucose-Galactose Syrup by Two-Stage Enzymatic Hydrolysis.

Fermentation technology enables the better use of resources and the conversion of dairy waste into valuable food products. The aim of this study is to evaluate the conversion rate of glucose into fructose by immobilised glucose isomerase (GI) in sweet and acid whey permeates for glucose-galactose syrup production. The experiments demonstrated that the highest concentration of glucose and galacto-oligosaccharides (GOSs) in sweet and acid whey permeates was reached by GODO-YNL2 ß-galactosidase, 32 ± 2% and 28 ± 1%, respectively. After glucose isomerisation, the highest fructose yield was 23 ± 0.3% and 13 ± 0.4% in sweet and acid whey permeates, where Ha-Lactase 5200 ß-galactosidase was used for lactose hydrolysis in sweet and acid whey permeates. Finally, the results of this study highlight the potential for two-stage enzymatic hydrolysis to increase the sweetness of glucose-galactose syrup made from sweet and acid whey permeates.

Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4.

Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 Å resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.

Enhanced catalytic efficiency and thermostability of glucose isomerase from Thermoanaerobacter ethanolicus via site-directed mutagenesis.

Glucose isomerase (GI) is a key enzyme in the preparation of high fructose corn syrup (HFCS). In this study, a mutant TEGI-M-L38 M/V137 L (TEGI-M2) of glucose isomerase (TEGI-M) originated from Thermoanaerobacter ethanalicus CCSD1 was obtained by site-directed mutagenesis. The TEGI-M2 showed an optimal activity at 85 ℃ and pH 6.5 with the divalent cations Co2+ and Mg2+. The structural differences between TEGI-M and TEGI-M2 were investigated based on the homology modeling and molecular docking, to elucidate the mechanism of improvement in the enzymatic properties. Compared with the original enzyme, the TEGI-M2 showed a 2.0-fold increased enzyme activity and a decreased Km from 234.2 mM to 85.9 mM. Finally, the application of mutant TEGI-M2 in HFCS one-step biosynthesis was attempted, resulting in a d-fructose yield of 67.3 %, which was 14.3 % higher than that of TEGI-M. This improved catalytic performance of TEGI-M2 was of great importance for the industrial preparation of d-fructose in one-step process.

Production of D-Allose From D-Allulose Using Commercial Immobilized Glucose Isomerase.

Rare sugars are regarded as functional biological materials due to their potential applications as low-calorie sweeteners, antioxidants, nucleoside analogs, and immunosuppressants. D-Allose is a rare sugar that has attracted substantial attention in recent years, owing to its pharmaceutical activities, but it is still not widely available. To address this limitation, we continuously produced D-allose from D-allulose using a packed bed reactor with commercial glucose isomerase (Sweetzyme IT). The optimal conditions for D-allose production were determined to be pH 8.0 and 60°C, with 500 g/L D-allulose as a substrate at a dilution rate of 0.24/h. Using these optimum conditions, the commercial glucose isomerase produced an average of 150 g/L D-allose over 20 days, with a productivity of 36 g/L/h and a conversion yield of 30%. This is the first report of the successful continuous production of D-allose from D-allulose by commercial glucose isomerase using a packed bed reactor, which can potentially provide a continuous production system for industrial applications of D-allose.

Immobilization of recombinant Escherichia coli cells expressing glucose isomerase using modified diatomite as a carrier for effective production of high fructose corn syrup in packed bed reactor.

To improve the operational stability of glucose isomerase in E. coli TEGI-W139F/V186T, the immobilized cells were prepared with modified diatomite as a carrier and 74.1% activity of free cells was recovered after immobilization. Results showed that the immobilized cells still retained 86.2% of the initial transformational activity after intermittent reused 40 cycles and the yield of D-fructose reached above 42% yield at 60 °C. Moreover, the immobilized cells were employed in the continuous production of High Fructose Corn Syrup (HFCS) in a recirculating packed bed reactor for 603 h at a constant flow rate. It showed that the immobilized cells exhibited good operational stability and the yield of D-fructose retained above 42% within 603 h. The space-time yield of high fructose corn syrup reached 3.84 kg L-1 day-1. The investigation provided an efficient immobilization method for recombinant cells expressing glucose isomerase with higher stability, and the immobilized cells are a promising biocatalyst for HFCS production.

Room-Temperature Structure of Xylitol-Bound Glucose Isomerase by Serial Crystallography: Xylitol Binding in the M1 Site Induces Release of Metal Bound in the M2 Site.

Glucose isomerase (GI) is an important enzyme that is widely used in industrial applications, such as in the production of high-fructose corn syrup or bioethanol. Studying inhibitor effects on GI is important to deciphering GI-specific molecular functions, as well as potential industrial applications. Analysis of the existing xylitol-bound GI structure revealed low metal occupancy at the M2 site; however, it remains unknown why this phenomenon occurs. This study reports the room-temperature structures of native and xylitol-bound GI from Streptomyces rubiginosus (SruGI) determined by serial millisecond crystallography. The M1 site of native SruGI exhibits distorted octahedral coordination; however, xylitol binding results in the M1 site exhibit geometrically stable octahedral coordination. This change results in the rearrangement of metal-binding residues for the M1 and M2 sites, the latter of which previously displayed distorted metal coordination, resulting in unstable coordination of Mg2+ at the M2 site and possibly explaining the inducement of low metal-binding affinity. These results enhance the understanding of the configuration of the xylitol-bound state of SruGI and provide insights into its future industrial application.

Crystal structure of the metal-free state of glucose isomerase reveals its minimal open configuration for metal binding.

Glucose/xylose isomerase catalyzes the reversible isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This enzyme is not only involved in sugar metabolism but also has industrial applications, such as in the production of high fructose corn syrup and bioethanol. Various crystal structures of glucose isomerase have shown the binding configuration of the substrate and its molecular mechanism; however, the metal binding mechanism required for the isomerization reaction has not been fully elucidated. To better understand the functional metal binding, the crystal structures of the metal-bound and metal-free states of Streptomyces rubiginosus glucose isomerase (SruGI) were determined at 1.4 Å and 1.5 Å resolution, respectively. In the meal-bound state of SruGI, Mg2+ is bound at the M1 and M2 sites, while in the metal-free state, these sites are occupied by water molecules. Structural comparison between the metal binding sites of the metal-bound and metal-free states of SruGI revealed that residues Glu217 and Asp257 exhibit a rigid configuration at the bottom of the metal binding site, suggesting that they serve as a metal-binding platform that defined the location of the metal. In contrast, the side chains of Glu218, His220, Asp255, Asp257, and Asp287 showed configuration changes such as shifts and rotations. Notably, in the metal-free state, the side chains of these amino acids are shifted away from the metal binding site, indicating that the metal-binding residues exhibit a minimal open configuration, which allows metal binding without large conformational changes.

Safety evaluation of the food enzyme xylose isomerase from the genetically modified Streptomyces rubiginosus strain DP-Pzn37.

The food enzyme is a d-xylose aldose-ketose-isomerase (EC 5.3.1.5) produced with the genetically modified Streptomyces rubiginosus strain DP-Pzn37 by Danisco US Inc. Although the production strain contains antibiotic resistance genes, the food enzyme was shown to be free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in an immobilised form for the isomerisation of glucose for the production of high fructose syrups. Residual amounts of total organic solids (TOS) are eliminated by the use of an immobilised food enzyme and further removed by the purification steps applied during the production of high fructose syrups using the immobilised enzyme; consequently, dietary exposure was not calculated. Genotoxicity tests did not raise safety concerns. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 85.2 mg TOS/kg body weight (bw) per day, the highest dose tested. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the immobilisation process and the removal of total organic solids during the production of high fructose syrups, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Purification and kinetic behavior of glucose isomerase from Streptomyces lividans RSU26.

Glucose isomerase (GI), an enzyme with deserved high potential in the world market. GI plays a major role in high Fructose Corn Syrup Production (HFCS). HFCS is used as a sweetener in food and pharmaceutical industries. Streptomyces are well-known producers of various industrially valuable enzymes, including Glucose isomerase. Currently, recombinant strains have been available for the production of various enzymes, but it has limitation in the large scale production. Therefore, identifying effective streptomyces strains have emerged. The current study, the novel S. lividans RSU26 was isolated from a marine source and optimized its potential to produce glucose isomerase at different physical and chemical conditions. The optimum pH and temperature for GI and biomass production were 7.5 and 35 °C, respectively at 96 h. Characterization study revealed that the approximate molar mass of GI was 43 kDa for monomeric and 170 kDa for tetrameric forms. Kinetic behavior exhibits Km, and Vmax values for the conversion of fructose to glucose conversion were 48.8 mM and 2.54 U mg-1 at 50 °C and glucose to fructose were 29.4 mM and 2.38 U mg-1 at 65 °C protein, respectively. Therefore, the present study suggested that the wild-type S. lividans RSU26 has strong potential to produce glucose isomerase for various industrial applications.

Application of mannitol producing Leuconostoc citreum TR116 to reduce sugar content of barley, oat and wheat malt-based worts.

Achieving a high monosaccharide composition in malt wort is instrumental to achieve successful lactic acid bacteria fermentation of malt based beverages. The conversion of monosaccharides to alternative metabolites such as the sweet polyol, mannitol with heterofermentative strains presents a novel approach for sugar reduction and to compensate for the loss of sweetness. This work outlines the application of an adopted mashing regimen with the addition of exogenous enzymes to produce wort with high fructose content which can be applied to different malted grain types with consistently efficacious monosaccharide production for bacterial fermentation. The so produced worts are then fermented with Leuconostoc citreum TR116 a mannitol hyper-producer. Malted barley, oat and wheat were mashed to stimulate protein degradation and release of free amino acids along with the enzymatic conversion of starch to fermentable sugars. Amyloglucosidase and glucose isomerase treatment converted di- and oligo-saccharides to glucose and provided a moderate fructose concentration in malt worts which was consistent across the three cereals. Fructose was completely depleted during fermentation with Lc. Citreum TR116 and converted to mannitol with high efficiency (>90%) while overall sugar reduction was >25% in all malt worts. Differences in amino acid composition of malt worts did not significantly affect growth of Lc. Citreum TR116 but did affect the formation of the aroma compounds diacetyl and isoamyl alcohol. Organic acid production and acidification of wort was similar across cereal substrates and acetic acid formation was linked to yield of mannitol. The results suggest that differences in amino acid and fructose content of malt worts considerably change metabolite formation during fermentation with Lc. Citreum TR116, a mannitol hyper-producer. This work gives new insight into the development of consumer acceptable malt based beverages which will provide further options for the health conscious and diabetic consumer, an important step in the age of sugar overconsumption.