Production Of High Fructose Corn Syrup Research Articles

Construction and investigation of multi-enzyme immobilized matrix for the production of HFCS.

Enzymes are biological molecules that act as catalysts and speed up the biochemical reactions. The world's biotechnological ventures are development of enzyme productiveness, and advancement of novel techniques for thriving their shelf existence. Nowadays, the most burning questions in enzyme technology are how to improve the enzyme productivity and reuse them. The immobilization of enzymes provides an excellent scope to reuse the enzymes several times to increase productivity. The main aim of the present study is the establishment of an immobilized multi-enzyme bio-system engineering process for the production of High-fructose corn syrup (HFCS) with an industrial focus. In this study, multi-enzyme such as α-amylase, glucoamylase and glucose isomerase were immobilized in various support matrices like sodium alginate, sawdust, sugarcane bagasse, rice bran and combination of alginate with cellulosic materials. The activities of the immobilized multi-enzyme system for the production of HFCS from the starch solution were determined. The multi-enzyme immobilized in sodium alginate shows better fructose conversion than free enzyme. Among the support matrices, multi-enzyme immobilized in sawdust produced total 80.74 mg/mL of fructose from starch solution and it was able to be used in several production cycles. On the other hand, multi-enzyme immobilized in combination of sodium alginate and sawdust produced the maximum amount of fructose (total 84.82 mg/mL). The free enzyme produced 17.25 mg/mL of fructose from the starch solution in only a single cycle. In this study a new fixed bed immobilized multi-enzyme bioreactor system was developed for the production of HFCS directly from starch. This finding will create a new opportunity for the application of immobilized multi-enzyme systems in many sectors of industrial biotechnology.

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.

Termofilik Anoxybacillus gonensis glukoz izomerazının DEAE-Sefaroz üzerine iyonik ve kovalent immobilizasyonu

High fructose corn syrup (HFCS), which is produced by the conversion of one sugar into another (glucose to fructose), has a marketing value. Hence, different glucose isomerases [(GI) (D-xylose ketol isomerase, EC 5.3.1.5)] isolated from different sources (macro-and microorganisms) were researched until today. In addition, the cost reduction of GI production for industrial applications has been investigated and applied with different techniques. Enzyme immobilization approaches have prominent features because they allow enzymes to be used repeatedly. In the current study, Anoxybacillus gonensis G2T glucose isomerase (AgoGI) (wild type) were immobilized with ionic and covalent binding on DEAE-sepharose matrix. Afterward, kinetic and biochemical parameters of the immobilized enzymes were evaluated. The pH and temperature parameters, in which the ionic and covalent immobilized enzymes showed the best activity, were determined as 6.50 and 85 °C, respectively. The kinetic data (Vmax and Km) of ionic bound AgoGI on DEAE-sepharose were 4.85±2.09 μmol/min/mg protein and 130,57±5,42 mM, as covalent immobilized AgoGI on the same matrix were 40.51± 0.81 μmol/min/mg protein µmol/min and 127,28±2,96 mM, respectively. Consequently, the usage of DEAE-sepharose for both covalent and ionic immobilization as immobilization matrix did not exhibit any negative effects on biochemical and kinetic parameters of glucose isomerase. Therefore, immobilized AgoGI on DEAE-sepharose was an excellent and promising tool for HFCS production.

Glucose Isomerase: Functions, Structures, and Applications

Glucose isomerase (GI, also known as xylose isomerase) reversibly isomerizes D-glucose and D-xylose to D-fructose and D-xylulose, respectively. GI plays an important role in sugar metabolism, fulfilling nutritional requirements in bacteria. In addition, GI is an important industrial enzyme for the production of high-fructose corn syrup and bioethanol. This review introduces the functions, structure, and applications of GI, in addition to presenting updated information on the characteristics of newly discovered GIs and structural information regarding the metal-binding active site of GI and its interaction with the inhibitor xylitol. This review provides an overview of recent advancements in the characterization and engineering of GI, as well as its industrial applications, and will help to guide future research in this field.

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.

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 showedthat 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 603h at a constant flow rate. It showedthat the immobilized cells exhibited good operational stability and the yield of D-fructose retained above 42% within 603h. The space-time yield of high fructose corn syrup reached 3.84kgL-1day-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.

Optimization of various encapsulation systems for efficient immobilization of actinobacterial glucose isomerase

Abstract Glucose isomerase (GI) converts glucose into fructose by a reversible reaction. This reaction is industrially valuable for the production of high fructose corn syrup (HFCS) which is used as a sweetener by food and beverage industries. GI being an expensive enzyme necessitates its usage in immobilized form for increasing its reusability and economic viability. Global HFCS production accounts to 10 million tons which is highest among products yielded by immobilized process. Current study evaluates different encapsulation systems for immobilization of GI produced by Streptomyces sp. T.S.A.KP which was found to be Streptomyces enissocaesilis (Accession number MN911386 ). This is the first report of extracellular GI production by a strain of Streptomyces enissocaesilis. GI was immobilized using chitosan, alginate and agar to enhance its stability and conversion efficiency. The concentration of polymeric gel with cross linking agent was optimized to get ideal pore size for maximum GI activity. Immobilization on chitosan significantly increased (P

A Novel Glucose Isomerase from Caldicellulosiruptor bescii with Great Potentials in the Production of High-Fructose Corn Syrup.

Glucose isomerase (GI) that catalyzes the conversion of D-glucose to D-fructose is one of the most important industrial enzymes for the production of high-fructose corn syrup (HFCS). In this study, a novel GI (CbGI) was cloned from Caldicellulosiruptor bescii and expressed in Escherichia coli. The purified recombinant CbGI (rCbGI) showed neutral and thermophilic properties. It had optimal activities at pH 7.0 and 80°C and retained stability at 85°C. In comparison with other reported GIs, rCbGI exhibited higher substrate affinity (Km = 42.61 mM) and greater conversion efficiency (up to 57.3% with 3M D-glucose as the substrate). The high catalytic efficiency and affinity of this CbGI is much valuable for the cost-effective production of HFCS.

Development of Some Properties of a Thermophilic Recombinant Glucose Isomerase by Mutation

Site-directed mutagenesis was made to obtain a glucose isomerase (GI) working at high temperatures and low pH values and having high affinity to the substrate and more pH and termal stability in comparison with the native enzyme. For this purpose, GI gene from Geobacillus caldoxylosilyticus TK4 was cloned to pET-28a(+) vector and H99Q, V184T and D102N mutations were performed. Biochemical features of the recombinant and mutant enzymes were identified. These mutations enabled the mutant enzymes to increase the optimum temperature and KM and decrease the optimum pH and Vmax values of the reaction. Furthermore, the mutant proteins had more thermal and pH stability than that of the recombinant GI. The mutant enzymes showed the highest activities in the presence of Co2+, Cu2+ and Mn2+ and were more resistant to some metals than the recombinant GI. In conclusion, the site-directed mutations led to improve the performance of the recombinant enzyme, which means that the mutant enzyme (TK4GI mutation) may be practicable in high fructose corn syrup production process.

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.

High-level expression of Thermobifida fusca glucose isomerase for high fructose corn syrup biosynthesis

Glucose isomerase (GIase), an efficient enzyme in the isomerization of d-glucose to d-fructose, has been widely used in food processing. In this study, an efficient expression system for a Thermobifida fusca GIase (GIaseTfus) in Escherichia coli was firstly designed via a two-stage feeding strategy for improving expression level. The cultivation strategy was performed at an exponential feeding rate during the pre-induction phase, followed by a gradient-decreasing feeding rate at the induction phase in a 3-L fermenter. During this process, the effect of induction conditions and the complex nitrogen supplementation in feeding solutions on GIaseTfus production were investigated and optimized. Under the optimal conditions, the yield of GIaseTfus reached 124.1 U/mL, which is the highest expression level of GIase by recombinant E. coli reported to date. Additionally, the obtained GIaseTfus was performed to produce high fructose corn syrup (HFCS) with conversion approacing 55 % from glucose (45 %, w/v) to fructose. According to the molecular dynamic simulation, a number of hydrogen bonds existed in the enzyme-substrate complex could stablilize the transient states, and a appreciate reaction distance of M1 catalytic site and oxygen atom of glucose make the reaction proceed easily, thus resulting in the efficient biosynthesis of HFCS. The function of GIaseTfus renders it a valuable catalyst for HFCS-55 (containing 55 % d-fructose) manufacturing, the most favorable industrial product of HFCS. The efficient expression of GIaseTfus and its efficient HFCS production lays the foundation for its proming industrial application.

Microbial industrial enzymes: An annotated selection of World Wide Web sites relevant to the topics in microbial biotechnology.

https://link.springer.com/chapter/10.1007/978-3-319-52287-6_28 This review article in an e-book provides an excellent overview to the field of industrial enzymes. In addition to the current commonly used enzymes, it contains information of emerging enzymes of interest. http://www.novozymes.com/-/media/Project/Novozymes/.../Enzymes_at_work.pdf This web brochure produced by Novozymes provides many examples of industrial enzymes, their origins and applications. http://www1.lsbu.ac.uk/water/enztech/detergent.html This web article is a little dated but it nevertheless provides a good overview of how enzymes are used on washing formulations. https://www.aasv.org/shap/issues/v18n2/v18n2p90.html Phytase is an enzyme that releases phosphate from phytate, a hexaphosphate ester of inositol. Because much phosphate in grain and seeds is stored in phytate, phytase enzyme is added to animal feed to help release phosphate from the feed. https://www.ocl-journal.org/articles/ocl/pdf/2017/04/ocl170015.pdf Lipases, mostly obtained from fungi, is used extensively in industry. Major uses are in detergents and in food oil processing. https://www.heraproject.com/files/22-F-07_PROTEASE_HERA_Final%20Edition%20(unsecured%20-%20PDFA-1b).pdf Subtilisin is a protease, originally obtained from strains of Bacillus. It is used in laundry, dishwashing and contact lens cleaning applications. The current document contains much information relevant to the industrial use of subtilisin. https://www.chemistryworld.com/podcasts/chymosin/3007682.article Chymosin is an important enzyme used in the conversion of milk to cheese. It was originally obtained from the digestive tracts of young ruminant animals. More recently, it is produced in recombinant form by microbial fermentation processes. https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0507-6 Cellulases are important industrial enzymes in biomass conversion. A major organism long studied for it enzymes for degrading cellulose is the fungus Trichoderma reesei. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769773/ This review article discusses the uses of amylases, mostly in converting various food products such as starches in corn, wheat, rice, and tapioca. A very large process currently is the conversion of corn starch to glucose by amylase coupled to the isomerization of glucose to fructose. https://www.researchgate.net/publication/315803185_Xylanases_and_their_industrial_applications_A_review This Research Gate website contains an abstract on xylanases and a link to the full review article on the topic. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347563/ This review article covers enzymes used in the production of semi-synthetic beta-lactam antibiotics. The major focus is on protein expression and engineering. https://www.creative-enzymes.com/similar/glucose-isomerase_319.html Glucose isomerase, also known as xylose isomerase, is widely used in the production of high-fructose corn syrup. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/pectinase Pectinases are widely used in producing clarified fruit juices. The pectinases hydrolyze linkages in the pectic substance found in the cell walls of plants. https://www.novozymes.com/en Novozymes is a major enzyme producing company, headquartered in Denmark. http://biosciences.dupont.com DuPont is a very large producer of enzymes with currently 900 commercial products on the market. https://www.dupont.com/products-and-services/industrial-biotechnology/industrial-enzymes-bioactives/primagreen.html Many enzymes are used in textile processing and clothing finishing. This website addresses are group of enzymes used in those applications. http://animalnutrition.dupont.com/productsservices/feed-enzymes/ Animal nutrition enzymes are largely derived from bacteria and aid in the animal digestion of their foods. The enzymes are principally phytase, proteases, and carbohydrate hydrolyzing enzymes. https://www.basf.com/global/en/products/cross-industry-solutions/enzymes.html The German chemical company BASF made a large entry into industrial enzymes with the the purchase of the Verenium Corporation. https://www.amano-enzyme.com/uk/about-company/ Amano produces microbial enzymes for use in food, supplement, and medical diagnostic industries.

Design of bypass-simulated moving bed chromatography for reduced purity requirements

Bypass-Simulated moving bed (BP-SMB) chromatography, a process characterized by the possibility of over-purifying the raffinate and/or the extract product followed by blending with the feed, is studied. The BP-SMB provides additional degrees of freedom; the opportunity to increase productivity; increased robustness and operational flexibility compared to obtaining reduced purities directly from the SMB. Based on the local-equilibrium theory, explicit equations to calculate productivity are derived for systems following a linear isotherm based on the minimum switch time. It is shown that the BP-SMB offers productivity advantage compared to a reduced-purity SMB specifically at low purity requirements, high selectivity and high efficiency conditions. The effect of column efficiency and feed concentration for systems following non-linear isotherms are studied using numerical simulations. The production of high-fructose corn syrup, an industrial application of BP-SMB, is analysed. For the HFCS separation, it is shown that a BP-SMB operation while not providing any advantages in terms of productivity, provides operational flexibility and robustness.

Design of operational temperature for immobilized glucose isomerise using an accelerated inactivation method

Abstract Thermal inactivation of immobilized glucose isomerase in a concentrated glucose solution was investigated in the batch mode and temperature range of 83–95 °C, which is substantially higher than the temperature used in the industrial production of high-fructose corn syrup. Simultaneous evaluation of all inactivation data showed that first-order kinetics with the Arrhenius temperature dependence of the rate constant provided a good approximation of the biocatalyst stability under the investigated conditions. The model parameters were then used to predict the operational temperature for this biocatalyst in the production of high-fructose corn syrup based on the set operational life-time of the biocatalyst. The simulation predicted a window of operational temperature of 60–65 °C, which corresponds very well with the industrial applications of this biocatalyst. This observation demonstrates that the multi-temperature method of enzyme inactivation can provide a good estimate of biocatalyst process stability and is thus a useful tool in the development of biocatalytic processes.

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.

Immobilization of Recombinant Glucose Isomerase for Efficient Production of High Fructose Corn Syrup.

Glucose isomerase is the important enzyme for the production of high fructose corn syrup (HFCS). One-step production of HFCS containing more than 55% fructose (HFCS-55) is receiving much attention for its industrial applications. In this work, the Escherichia coli harboring glucose isomerase mutant TEGI-W139F/V186T was immobilized for efficient production of HFCS-55. The immobilization conditions were optimized, and the maximum enzyme activity recovery of 92% was obtained. The immobilized glucose isomerase showed higher pH, temperature, and operational stabilities with a K m value of 272mM and maximum reaction rate of 23.8mMmin-1. The fructose concentration still retained above 55% after the immobilized glucose isomerase was reused for 10cycles, and more than 85% of its initial activity was reserved even after 15 recycles of usage at temperature of 90°C. The results highlighted the immobilized glucose isomerase as a potential biocatalyst for HFCS-55 production.

Properties of a novel thermostable glucose isomerase mined from Thermus oshimai and its application to preparation of high fructose corn syrup.

Glucose isomerase (GI) is used in vitro to convert d-glucose to d-fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d-glucose among the screened GIs. Its optimum temperature reached 95°C and could retain more than 80% of initial activity in the presence of 20mM Mn2+ at 85°C for 48h. The Km and kcat/Km values for ToGI were 81.46mM and 21.77min-1mM-1, respectively. Furthermore, the maximum conversion yield of 400g/L d-glucose to d-fructose at 85°C was 52.16%. Considering its excellent high thermostability and ameliorable application performance, ToGI might be promising for realization of future industrial production of HFCS at elevated temperature.

Biochemical characterization of a novel glucose isomerase from Anoxybacillus gonensis G2T that displays a high level of activity and thermal stability

In the continuing search for novel enzymes suitable for the production of high fructose corn syrup (HFCS), a new glucose isomerase (GI) from the thermophile Anoxybacillus gonensis G2T is described. The gene encoding this GI (AgoG2GI) was cloned and then engineered for heterologous expression in Escherichia coli. The recombinant enzyme was purified from the heat treated cell-free extract by anion exchange chromatography followed by hydrophobic interaction chromatography. The purified enzyme showed optimal activity at 85 °C and pH 6.5. The steady state parameters of Km and kcat with d-glucose were found to be 146.08 ± 9.50 mM and 36.47 ± 2.01 (1/s), respectively. l-arabinose, d-ribose and d-mannose also served as substrates for the enzyme with comparable kinetic parameters. AgoG2GI requires the divalent cations of Co2+, Mn2+ and Mg2+ for its maximal activity and thermostability. The results reported here are indicative of a new GI with desirable kinetics and stability parameters for the efficient production of HFCS at industrial scale.

ChemInform Abstract: Industrial Use of Immobilized Enzymes

AbstractReview: 351 refs.

Immobilised enzymes in biorenewables production

Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, such as the production of High-Fructose Corn Syrup, but these are still rather rare. Fortunately, there is a rapid expansion in the research efforts that try to improve this, driven by a combination of economic and ecological reasons. This review focusses on those efforts, by looking at attempts to use fatty acids, carbohydrates, proteins and lignin (and their building blocks), as substrates in the synthesis of biorenewables using immobilised enzymes. Therefore, many examples (390 references) from the recent literature are discussed, in which we look both at the specific reactions as well as to the methods of immobilisation of the enzymes, as the latter are shown to be a crucial factor with respect to stability and reuse. The applications of the renewables produced in this way range from building blocks for the pharmaceutical and polymer industry, transport fuels, to additives for the food industry. A critical evaluation of the relevant factors that need to be improved for large-scale use of these examples is presented in the outlook of this review.