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.

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.

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.

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.

Analysis of oscillatory rocking curve by dynamical diffraction in protein crystals.

High-quality protein crystals meant for structural analysis by X-ray diffraction have been grown by various methods. The observation of dynamical diffraction in protein crystals is an interesting topic because dynamical diffraction generally occurs in perfect crystals such as Si crystals. However, to our knowledge, there is no report yet on protein crystals showing clear dynamical diffraction. We wonder whether the perfection of protein crystals might still be low compared with that of high-quality Si crystals. Here, we present observations of the oscillatory profile of rocking curves for protein crystals such as glucose isomerase crystals. The oscillatory profiles are in good agreement with those predicted by the dynamical theory of diffraction. We demonstrate that dynamical diffraction occurs even in protein crystals. This suggests the possibility of the use of dynamical diffraction for the determination of the structure and charge density of proteins.

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.

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.

Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn2+, but not in the presence of Mg2+. Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn2+ at the M2 site. Glucose and Mn2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition.

Production of d-mannose from d-glucose by co-expression of d-glucose isomerase and d-lyxose isomerase in Escherichia coli.

BACKGROUND: d-Mannose is not only the epimer of d-glucose at the C-2 position, but also the aldose isomer of d-fructose. Because of its physiological properties and health benefits, d-mannose has attracted public interest. It has been confirmed that d-mannose has broad applications in food, cosmetics, and pharmaceutical industries. According to the Izumoring strategy, d-glucose isomerase (d-GI) and d-lyxose isomerase (d-LI) play important roles in the conversions of d-fructose from d-glucose and of d-mannose from d-fructose respectively. In this study, a one-step enzyme process of d-mannose production from d-glucose has been constructed by co-expression of the d-GI from Acidothermus cellulolyticus and d-LI from Thermosediminibacter oceani in Escherichia coli BL21(DE3) cells. RESULTS: The co-expression system exhibits maximum activity at pH 6.5 and 65 °C with Co2+ supplement. It is relatively thermostable at less than 65 °C. When the reaction reaches equilibrium, the ratio of d-glucose, d-fructose, and d-mannose is approximately 34 : 49.6 : 16.4. By using this co-expression system, about 60.0 g L-1 d-mannose is obtained from 400 g L-1 d-glucose in 8 h. CONCLUSION: This co-expression of d-GI and d-LI system provides a novel and efficient approach for d-mannose production. © 2018 Society of Chemical Industry.

Crystal structure of glucose isomerase in complex with xylitol inhibitor in one metal binding mode.

Glucose isomerase (GI) is an intramolecular oxidoreductase that interconverts aldoses and ketoses. These characteristics are widely used in the food, detergent, and pharmaceutical industries. In order to obtain an efficient GI, identification of novel GI genes and substrate binding/inhibition have been studied. Xylitol is a well-known inhibitor of GI. In Streptomyces rubiginosus, two crystal structures have been reported for GI in complex with xylitol inhibitor. However, a structural comparison showed that xylitol can have variable conformation at the substrate binding site, e.g., a nonspecific binding mode. In this study, we report the crystal structure of S. rubiginosus GI in a complex with xylitol and glycerol. Our crystal structure showed one metal binding mode in GI, which we presumed to represent the inactive form of the GI. The metal ion was found only at the M1 site, which was involved in substrate binding, and was not present at the M2 site, which was involved in catalytic function. The O2 and O4 atoms of xylitol molecules contributed to the stable octahedral coordination of the metal in M1. Although there was no metal at the M2 site, no large conformational change was observed for the conserved residues coordinating M2. Our structural analysis showed that the metal at the M2 site was not important when a xylitol inhibitor was bound to the M1 site in GI. Thus, these findings provided important information for elucidation or engineering of GI functions.

Beet molasses-based feeding strategy enhances recombinant thermostable glucose isomerase production by Escherichia coli BL21 (DE3).

The aim of this work was to develop an effective fed-batch feeding strategy to enhance recombinant glucose isomerase (r-GI) production by recombinant Escherichia coli BL21 (DE3) pLysS on an industrially relevant feedstock without the application of an exogenous inducer. Following the batch operation (0 < t < 7 H), the effects of pulse and/or continuous feeding of hydrolyzed beet molasses were investigated under five different feeding strategies. The two most promising strategies with respect to r-GI activity were (i) PM-0.05, designed with one pulse feed (t = 7 H) followed by a continuous feed and (ii) 2PMF -0.05, designed with two consecutive pulse feeds (t = 7 and 10 H) followed by a continuous feed. The continuous feeding of molasses for both fermentation strategies employed the same precalculated feeding rate, µo = 0.05 H-1 . The maximum r-GI activities exhibited by PM-0.05 and 2PMF -0.05 were 29,050 and 30,642 U dm-3 , respectively. On the one hand, compared to PM-0.05 r-GI activity reached its maximum within a shorter cultivation time (∆tmax = 2 H) at 2PMF -0.05, which could be preferable in terms of manufacturing costs and possible risks; on the other hand, PM-0.05 is a simpler fermentation regime compared to 2PMF -0.05 with respect to manipulations that should be considered in large-scale production.

Immobilized Trienzymatic System with Enhanced Stabilization for the Biotransformation of Lactose.

The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, ß-galactosidase from Bacillus circulans, l-arabinose (d-galactose) isomerase from Enterococcus faecium, and d-xylose (d-glucose) isomerase from Streptomyces rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L. Immobilized activity yields were over 90% in all cases. With the purpose of increasing thermostability of derivatives, two post-immobilization treatments were performed: alkaline incubation to favor the formation of additional covalent linkages, and blocking of excess oxirane groups by reacting with glycine. The greatest thermostability was achieved when alkaline incubation was carried out for 24 h, producing l-arabinose isomerase-Eupergit C derivatives with a half-life of 379 h and d-xylose isomerase-Eupergit C derivatives with a half-life of 554 h at 50 °C. Preliminary assays using immobilized and stabilized biocatalysts sequentially to biotransform lactose at pH 7.0 and 50 °C demonstrated improved performances as compared with soluble enzymes. Further improvements in ketohexose productivities were achieved when the three single-immobilizates were incubated simultaneously with lactose in a mono-reactor system.

Production of d-allulose from d-glucose by Escherichia coli transformant cells co-expressing d-glucose isomerase and d-psicose 3-epimerase genes.

BACKGROUND: d-Allulose is a novel and low-calorie rare monosaccharide that is a C-3 epimer of d-fructose. Because of its excellent physiological properties and commercial potential, d-allulose has attracted researchers' interests. Based on the Izumoring strategy, d-allulose is converted from d-fructose by d-psicose 3-epimerase (DPEase), while d-fructose is converted from d-glucose by d-glucose isomerase (GIase). In this study, we created a cellular system capable of converting d-glucose to d-allulose in a one-step process that co-expressed the GIase from Acidothermus cellulolyticus and the DPEase from Dorea sp. CAG. RESULTS: The co-expression plasmid pETDuet-Dosp-DPE/Acce-GI was generated and transformed into Escherichia coli BL21(DE3) cells. The recombinant co-expression cells exhibited maximum catalytic activity at pH 6.5 and 75 °C. These cells were thermostable at less than 60 °C. The addition of Co2+ significantly increased the catalytic activity by 10.8-fold. When the reaction equilibrium was reached, the ratio of d-glucose, d-fructose and d-allulose was approximately 6.5:7:3, respectively. CONCLUSION: A recombinant co-expression strain that catalysed the bioconversion of d-allulose from d-glucose in a one-step process was created and characterised. When adding 500 g L-1 d-glucose as a substrate, 204.3 g L-1 d-fructose and 89.1 g L-1 d-allulose were produced. © 2016 Society of Chemical Industry.

Wavelet-Based Analytical Algorithm for Solving Steady-State Concentration in Immobilized Glucose Isomerase of Packed-Bed Reactor Model.

Wavelet method is a recently developed tool in applied mathematics. The mathematical model of the steady-state immobilized enzyme electrodes is discussed. This theoretical model is based on one-dimensional heat conduction equations containing a non-linear term related to Michaelis-Menten kinetics. An efficient Chebyshev wavelet-based technique is applied to solve the non-linear diffusion equation for the steady-state condition. A simple expression of the substrate concentration is obtained as a function of the Thiele modulus [Formula: see text] and [Formula: see text](kinetic parameter). The wavelet results are compared with the numerical and HPM solutions and found to be in good agreement.

Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase.

A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na2SiO3, 600 mM Na2MoO4, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.

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 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.

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.

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.