Production Of High-fructose Syrup Research Articles

Bioproduction of high-allulose-fructose syrup from d-glucose catalyzed by recombinant E. coli co-expressing glucose isomerase and d-allulose 3-epimerase

High-fructose syrup (HFS) is a sugar substitute widely used in food and beverages and can also be used as an inexpensive substrate for production of rare sugars, such as d-allulose (or d-psicose) etc. Biotransformation of HFS to produce d-allulose contained HFS, called High-allulose-fructose syrup (HAFS), can be used as a value added sweetener with healthy functions. The recombinant Escherichia coli expressing glucose isomerase (GI) from Caldicellulosiruptor bescii DSM 6725 was constructed and used for production of HFS from d-glucose. A dual enzyme expression system for expressing above GI and d-allulose 3-epimerase (DAE or d-psicose 3-epimerase, DPE) from Ruminococcus sp. 5_1_39BFAA was constructed for production of HAFS from d-glucose. The recombinant strains developed had strong substrate tolerance and high capacity in production of HFS and HAFS. The cultivation conditions of above strains were made, and the biotransformation conditions of HAFS catalyzed by the cells of two recombinant strains expressing GI and DAE, respectively, or by the cells of the single strain co-expressing GI and DAE were optimized. Under the optimum conditions, about 18.6, 110.4 and 163.8 g/L d-allulose can be obtained from 100, 600 and 900 g/L d-glucose respectively. The HAFS containing about 18.2% (163.8 g/L) d-allulose, 36.3% (326.7 g/L) d-fructose and 45.5% (409.5 g/L) d-glucose was obtained at the initial d-glucose concentration of 900 g/L catalyzed by the cells of the single strain co-expressing GI and DAE. The production process developed in this study was simple, highly efficient, and can provide reference for the production of HAFS.

Modeling and evaluation of the sucrose-degrading activity of recombinantly produced oligo-1,6-glucosidase from A. gonensis

Fructose is the most preferred sugar to provide benefits for sweetening and health. As many industrial enzymes are used to produce High Fructose Syrup (HFS), it is vital to explore alternative enzymes for fructose production. Oligo-α-1,6-glucosidase (O-1-6-glucosidase) hydrolyzes non-reducing ends of isomaltooligosaccharides, panose, palatinose, and an a-limit dextrin by breaking α-1,6-glucoside bonds, although it generally has no activity on α-1,4-glucoside bonds of maltooligosaccharides. In this study, sucrose-hydrolyzing activity of O-1-6-glucosidase of thermophilic A. gonensis was evaluated. For this purpose, O-1-6-glucosidase gene region of A. gonensis was cloned in the pET28(a)+ expression vector, the expression product was purified, modeled, and biochemically characterized. The optimal activity of the enzyme was to be at pH 7.0 and 60 °C. The enzyme activity was halved at the end of the 276th h at 60 °C. The enzyme maintained its activity even after 300 h at pH 6.0-10.0. The values of Km , Vmax , kcat, and kcat/Km were determined as 44.69 ± 1.27 mM, 6.28 ± 0.05 µmoL/min/mg protein, 6.70 1/s and 0.15 1/mMs−1, respectively. While Zn2+, Cu2+, Pb2+, Ag2+, Fe3+, Hg2+, and Al2+ metal ions inhibited O-1-6-glucosidase, Mn2+, Fe2+, and Mg2+ ions activated the enzyme. Consequently, A. gonensis O-1-6-glucosidase (rAgoSuc2) has interesting properties, especially for HFS production.

Optimization of production and partial purification of inulinase from Bacillussubtilis

The use of inulinase enzyme for production of high fructose syrups (HFS) found more attention during that last few decades due to the less efforts and cost required compared with the traditional two-step method. In the current study, a bacterial isolate have been selected for its potential inulinase productivity. It was identified using 16S rRNA as Bacillus subtilis Inu and given the accession number “OK444102″ in the database of NCBI. Statistical methods; Taguchi Orthogonal Array and Box-Behnken, have been applied for the improvement inulinase production and the data revealed maximum enzyme activity titer (70.8 U/ml) at 15.61 g/l, sucrose; 15.94 g/l, molasses; 5.2 g/l, peptone; 5.2, pH; 32.5 °C, incubation temperature and 6 days, incubation period). The produced enzyme was purified using aqueous two phase system (ATPS) composed of Poly ethylene glycol (PEG) conjugated with salt solution (PEG/salt). The two phases in the system were studied and the data revealed that PEG 6000 (15%)/Sodium sulfate (15%) was the most effective system in the partitioning inulinase enzyme by 13.5 purification fold and 285% yield recovery in the top PEG rich phase. The isolated Bacillus subtilis is an efficient strain for production of inulinase compared with the other conducted studies.

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.

Inulinase Producing Yeast Isolated from Kepok Banana Peels (Musa acuminata x balbisiana)

Inulinase enzyme (EC 3.2.1.80) is an enzyme capable of hydrolyzing inulin and is used as a catalyst in the production of High Fructose Syrup (HFS) and fructooligosaccharides (FOS). Inulinase enzymes can be produced by yeasts found in waste such as kepok banana peels (Musa acuminata x balbisiana). The aims of this study were to obtain the inulinase enzyme-producing yeast from kepok banana peels and to determine the effect of commercial inulin concentration and incubation time on enzyme activity. This research was conducted experimentally using a factorial completely randomized design with factors being inulin concentration of 1% (K1), 3% (K2) and 5% (K3) and incubation time of 6th (W1), 12th (W2), 18th (W3), and 24th hours (W4). The results showed that there were 4 isolates of inulinolytic yeasts obtained from kepok banana peels, namely YP1, YP2, YP4, and YP6. However, the most potential isolate to be used in inulinase production was YP2 isolate because it had the highest inulinolytic ratio value of 1.96. The results of inulinase activity showed that only inulin substrate with concentration of 5% (K3) had a significant or significant effect on inulinase production.

Membrane-bound D-mannose isomerase of acetic acid bacteria: finding, characterization, and application.

D-Mannose isomerase (EC 5.3.1.7) catalyzing reversible conversion between D-mannose and D-fructose was found in acetic acid bacteria. Cell fractionation confirmed the enzyme to be a typical membrane-bound enzyme, while all sugar isomerases so far reported are cytoplasmic. The optimal enzyme activity was found at pH 5.5, which was clear contrast to the cytoplasmic enzymes having alkaline optimal pH. The enzyme was heat stable and the optimal reaction temperature was observed at around 40 to 60˚C. Purified enzyme after solubilization from membrane fraction showed the total molecular mass of 196 kDa composing of identical four subunits of 48 kDa. Washed cells or immobilized cells were well functional at nearly 80% of conversion ratio from D-mannose to D-fructose and reversely 20-25% of D-fructose to D-mannose. Catalytic properties of the enzyme were discussed with respect to the biotechnological applications to high fructose syrup production from konjac taro.

An innovative strategy of recycling miscellaneous waste carbohydrates from high-fructose syrup production for Pichia pastoris fermentation

Recycling waste carbon source for fermentation is of great interests and high practical value. However, the process suffered some problems like unclear carbohydrate composition, low nutrition content, presence of various contaminants and high cost for pretreatment. Currently, the high-fructose syrup (HFS) production industry in China generates a large amount of waste liquid containing miscellaneous waste carbohydrates (MWC), which has a notable environmental impact. To recycle MWC for Pichia pastoris fermentation, the composition of MWC was analyzed and the fermentation processes were optimized. The composition of MWC was determined as 802.3 g L−1 total carbohydrate, 480 g L−1 glucose, 92 g L−1 fructose, 103.6 g L−1 maltose, 36.8 g L−1 maltotriose and 89.9 g L−1 other oligosaccharides with different degrees of polymerization (DPs 4–16). After the fermentation medium and conditions for pGAP/pAOX1-P. pastoris were optimized, MWC were employed as the alternative carbon source to culture the two strains to produce endo-β-1,3-glucanase in 7-L bioreactors. Using MWC for pGAP-P. pastoris culture, the dry cell weight (DCW) and endo-β-1,3-glucanase activity reached 69.1 g L−1 and 171.8 U mL−1, respectively, which were the same levels as in the batch using glucose as the carbon source. When using MWC and the self-designed DO-Stat-Time feeding strategy for pAOX1-P. pastoris culture, the maximum DCW and enzyme activity reached 83.6 g L−1 and 212.3 U mL−1, which improved 41.4% and 34.9%, respectively compared with the control group. This study established cleaner production procedures for both HFS and P. pastoris fermentation industries by considerably reducing environmental pollution and decreasing the production cost.

Development of a sustainable route for the production of high-fructose syrup from the polyfructan inulin.

The authors used mesoporous silica microspheres as a support for the immobilization of inulinase from Aspergillus brasiliensis MTCC 1344 by the process of cross‐linking. Under optimized operating conditions of pH 6.0, particle/enzyme ratio of 2.0:1.0 and glutaraldehyde concentration of 7 mM, a maximum immobilization yield of 90.7% was obtained after a cross‐linking time of 12.25 h. Subsequently, the cross‐linked inulinase was utilized for the hydrolysis of 5% inulin, and a maximum fructose concentration of 31.7 g/L was achieved under the optimum conditions of pH 6.0 and temperature 60°C in 3 h. Furthermore, on performing reusability studies during inulin hydrolysis, it was observed that the immobilized inulinase could be reused up to 10 subsequent cycles of hydrolysis, thus providing a facile and commercially attractive process of high‐fructose syrup production.

The Important Roles Played in Substrate Binding of Aromatic Amino Acids in Exo-Inulinase From Kluyveromyces cicerisporus CBS 4857.

Inulinase is a member of the glycoside hydrolase family 32 (GH32). It catalyzes the randomly hydrolyzation of 2,1-β-D-fructosidic linkages in inulin and plays a role in the production of high-fructose syrup. In this study, detailed roles of the conserved residues W79, F113, M117, R181, C239, and W334 of the exo-inulinase from Kluyveromyces cicerisporus CBS4857 (KcINU1) in substrate binding and stabilization were evaluated by in silico analysis and site-directed mutagenesis. These residues belong to the conserved WG, FSGSMV, RDP, ECP, and WQY regions of the GH32 and are located around the catalytic pocket of KcINU1. Zymogram assay showed relatively weaker band for F113W and similar band for M117A compared to the wild-type enzyme toward inulin and sucrose, whereas all other variants showed no observable stain on the native polyacrylamide gel electrophoresis. These results were further confirmed with the dinitrosalicylic acid colorimetric method. It showed that the residual activities of F113W toward inulin and sucrose were 33.8 ± 3.3% and 96.2 ± 5.5%, respectively, and that of M117A were 103.8 ± 1.3% and 166.5 ± 12%, respectively. Results from fluorescence spectra indicated that there is a significant conformational change that happened in F113W compared to the wild-type enzyme, while M117A exhibited limited impact although the quenching effect was increased.

Thermostability of Aspergillus niger inulinase from sugar beet molasses in the submerged fermentation and determination of its kinetic and thermodynamic parameters

Inulinases are commonly used for the production of high-fructose syrup, fructooligosaccharides, and inulooligosaccharides and also in the food and pharmaceutical industries. Besides, inulinases are also utilized for the fabrication of various high-added-value products such as bioethanol, lactic acid, citric acid, sugar alcohols, single-cell oils and proteins, 2,3-butanediol, gluconic acid, and butanol. Besides, before using an enzyme in a bioprocess, the determination of its properties is very essential. Therefore, this study evaluated the thermostability (30–80 °C for 0.5–6 h) of Aspergillus niger inulinase produced from sugar beet molasses in the shake flask fermentation and determined its kinetic and thermodynamic features. Based on the thermostability test of inulinase, it was stable at 30 °C and 40 °C and activity retained approximately 78.5% and 21.8% at 50 °C and 60 °C at the end of 6-h incubation time. Besides, half-life and D-value of enzyme reduced with increasing temperature, but increased with increasing incubation time at a fixed temperature. Q10-values were generally below one and reduced with increasing temperature. Nevertheless, inactivation energy and Z-value of inulinase were between 203.8 and 293.2 kJ/mol and between 17.05 and 20.99 °C to varying incubation times. Enthalpy and entropy of inulinase decreased with increasing temperature. The free energy of inulinase was unstable with increasing temperature depending on its inactivation energy. Consequently, inulinase can be used for biocatalytic strategies in various bioprocesses at relatively high temperatures.

From molasses to syrup: Engineering ultrafiltration membrane surface to improve invertase reusability

A by-product from membrane-mediated molasses refining process mainly contains sucrose, glucose, fructose and KCl, which is an ideal raw material to produce high fructose syrup (HFS) by invertase. Ultrafiltration (UF) membrane can achieve invertase reuse while protein adsorption on the membrane not only decreases the membrane permeability, but also induces enzyme activity loss. In this work, the effect of glucose/fructose and KCl on the catalytic behavior of invertase was investigated. Then the reusability of invertase for sucrose hydrolysis was evaluated by different UF membranes, and mussel-inspired surface modification was employed to further improve the invertase reusability. The results showed that the presence of KCl in the raw materials greatly decreased the reaction rate, and thus desalination and concentration of the raw materials should be conducted before enzymatic hydrolysis to improve the enzymatic hydrolysis efficiency, save energy and reduce reactor volume. Moreover, it was found that the sucrose conversion decline with enzyme reuse by UF was caused by enzyme adsorption in/on the membrane and/or its penetration through the membrane leading to overall enzyme loss in the retentate. A polyether sulfone (PES) membrane with molecular weight cut-off (MWCO) of 10 kDa (UP010) was selected to recycle invertase because it enabled complete invertase retention and relatively high permeate flux. When l-cysteine (Cys) was grafted on the polydopamine-coated UP010 membrane, it could reduce invertase adsorption on the membrane, and thus keep high sucrose conversion (93–95%) in four enzyme reuse cycles because the grafted Cys greatly increased the negative charge and hydrophilicity of the membrane surface. This work not only offered an alternative for HFS production by membrane technology but also developed a novel strategy to construct antifouling surface to retard enzyme adsorption.

Identification and Immobilization of an Invertase With High Specific Activity and Sucrose Tolerance Ability of Gongronella sp. w5 for High Fructose Syrup Preparation.

Invertases catalyze the hydrolysis of sucrose into fructose and glucose and can be employed as an alternative in producing high fructose syrup. In this study, we reported the heterologous expression of an invertase gene (GspInv) of Gongronella sp. w5 in Komagataella pastoris. GspInv activity reached 147.6 ± 0.4 U/mL after 5 days of methanol induction. GspInv is invertase with a high specific activity of 2,776.1 ± 124.2 U/mg toward sucrose. GspInv showed high tolerance to sucrose (IC50 = 1.2 M), glucose (IC50 > 2 M), fructose (IC50 = 1.5 M), and a variety of metal ions that make it an ideal candidate for high fructose syrup production. A carbohydrate-binding module was sequence-optimized and fused to the N-terminus of GspInv. The fusion protein had the highest immobilization efficiency at room temperature within 1 h adsorption, with 1 g of cellulose absorption up to 8,000 U protein. The cellulose-immobilized fusion protein retained the unique properties of GspInv. When applied in high fructose syrup preparation by using 1 M sucrose as the substrate, the sucrose conversion efficiency of the fused protein remained at approximately 95% after 50 h of continuous hydrolysis on a packed bed reactor. The fused protein can also hydrolyze completely the sucrose in sugarcane molasses. Our results suggest that GspInv is an unusual invertase and a promising candidate for high fructose syrup preparation.

Production, purification and physicochemical characterization of D-xylose/glucose isomerase from Escherichia coli strain BL21.

Cell lysate of Escherichia coli strain BL21 showed significant D-glucose isomerase activity. The rate of glucose conversion was increased up to 40% when cells were induced with 1% D-xylose. E. coli BL21 xylose isomerase (ECXI-BL21) was purified to homogeneity, up to 1.9-fold with overall 10.88% enzyme yield by heat shock, salting out and electro-elution. The molecular mass of ECXI-BL21 was estimated as 43.9kDa on SDS-PAGE. pHopt. and Topt. of the enzyme were calculated as 7.0 and 50°C, respectively. Activation energy (E a) of ECXI-BL21 was 45kJ/mol. Enzyme was stable from 30 to 55°C and at pH range 6.0-8.0. ECXI-BL21(holo) was activated by 10mM magnesium (35%), 0.5mM cobalt (20%) and manganese (25%), and 0.5/10mM Mn2+/Mg2+ (50%) and Co2+/Mg2+ (30%) as compared to ECXI-BL21(apo). Catalytic affinity (K m) of ECXI-BL21 for D-glucose was calculated as 0.82mM, while maximum velocity (V max) of the reaction D-glucose(aldo) ⇌ D-fructose(keto) was 108μmol/mg/min. D-fructose formed was identified on silica gel plate. This thermophilic enzyme, T m = 75°C, has great potential for high fructose syrup production used in food and soft drink industries.

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.

Production of Invertase from Actinomycetes Isolated from Soils in Eliogbolo and Oyigbo, Rivers State using Submerged Fermentation Technique

The use of commercial sources of invertase for the production of High fructose syrup and other pharmaceutical formulations has been a problem due to its high ash content and cost implication, therefore there is a need to explore bacterial species that can produce invertase. In this study, actinomycetes strains were isolated from soils from a botanical garden, sugar cane sites and garden egg at Elogbolo and Oyigbo respectively. Yeast extract agar; Czapeks Dox agar and Arginine glycerol agar were used for the isolation of actinomycetes. The study showed that Czapeks Dox agar gave the highest population count followed by Yeast extract and Arginine glycerol agar. The dominated genus of actinomycetes observed were species of Streptomyces designated as OZMU 1; OZMU 2; OZMU 3 and OZMU 4. The results showed that the reducing sugar content of the Streptomyces sp. had similar reducing sugar liberation and consumption behavior without any lag phase, with OZMU 1 concentration peak at 12 h (0.12 g/l), and OZMU 3 (0.15 g/l). But OZMU 2 indicated a lag phase from 6-12h, increased from 12-24 h (0.18 g/l), but activity declined dramatically at 36 h and 48 h. The reducing sugar content for Streptomyces sp. OZMU 4 revealed longer lag phase, which was almost constant throughout the investigation period from 6-36 h. But at 48 h (0.06 g/l) a decline in the reducing sugar content was noticed compared to the other isolates, indicating that all the available reducing sugar were completely consumed by the other strains. The research work revealed the ability of indigenous actinomycetes strains to produce invertase enzyme which has high conversion efficiency and can be used in various pharmaceutical and nutraceutical industries.

Immobilization of the glucose isomerase from Caldicoprobacter algeriensis on Sepabeads EC-HA and its efficient application in continuous High Fructose Syrup production using packed bed reactor

The glucose isomerase GICA from Caldicoprobacter algeriensis was immobilized by ionic adsorption on polymethacrylate carriers (Sepabeads EC-EA and EC-HA) or covalent attachment to glyoxal agarose. The Sepabeads EC-HA yielded the highest recovery of activity (89%). The optimum temperature and pH of immobilized GICA were 90 °C and 7.0, respectively, similar to the corresponding values of free enzyme. Nevertheless, the adsorbed enzyme displayed higher relative activity at acidic pH, greater thermostability, and better storage stability, compared to the free form. Moreover, the immobilized enzyme showed an excellent operational stability, in 15 successive 3 h reaction cycles at 85 °C under a batch reactor, preserving 83% of its initial activity. Interestingly, a continuous process for High Fructose Syrup (HFS) production was established with the adsorbed GICA using a packed bed reactor during eleven days at 70 °C. HPAEC-PAD analysis showed a maximum bioconversion rate of 49% after 48 h of operation.

Evaluation of carbon sources for the production of inulinase by Aspergillus niger A42 and its characterization

Inulinases are used for the production of high-fructose syrup and fructooligosaccharides, and are widely utilized in food and pharmaceutical industries. In this study, different carbon sources were screened for inulinase production by Aspergillus niger in shake flask fermentation. Optimum working conditions of the enzyme were determined. Additionally, some properties of produced enzyme were determined [activation (Ea)/inactivation (Eia) energies, Q10 value, inactivation rate constant (kd), half-life (t1/2), D value, Z value, enthalpy (ΔH), free energy (ΔG), and entropy (ΔS)]. Results showed that sugar beet molasses (SBM) was the best in the production of inulinase, which gave 383.73U/mL activity at 30°C, 200rpm and initial pH 5.0 for 10days with 2% (v/v) of the prepared spore solution. Optimum working conditions were 4.8 pH, 60°C, and 10min, which yielded 604.23U/mL, 1.09inulinase/sucrase ratio, and 2924.39 U/mg. Additionally, Ea and Eia of inulinase reaction were 37.30 and 112.86kJ/mol, respectively. Beyond 60°C, Q10 values of inulinase dropped below one. At 70 and 80°C, t1/2 of inulinase was 33.6 and 7.2min; therefore, inulinase is unstable at high temperatures, respectively. Additionally, t1/2, D, ΔH, ΔG values of inulinase decreased with the increase in temperature. Z values of inulinase were 7.21°C. Negative values of ΔS showed that enzymes underwent a significant process of aggregation during denaturation. Consequently, SBM is a promising carbon source for inulinase production by A. niger. Also, this is the first report on the determination of some properties of A. niger A42 (ATCC 204,447) inulinase.

A novel thermostable and efficient Class II glucose isomerase from the thermophilic Caldicoprobacter algeriensis: Biochemical characterization, molecular investigation, and application in High Fructose Syrup production.

A novel glucose isomerase gene from the thermophilic Caldicoprobacter algeriensis, encoding a polypeptide of 438 residues, was identified, cloned and successfully expressed in E. coli. The purified enzyme (GICA) was a homotetramer of about 200 kDa displaying the highest activity at pH 7.0 and 90 °C and retaining 97% of its maximum activity at pH 6.5. The enzyme showed an excellent thermostability with a half-life of 6 min at 100 °C. Interestingly, GICA had a very high affinity of 40 mM and catalytic efficiency of 194 min-1 mM-1 toward d-glucose at 90 °C. A maximum of 54.7% d-glucose to d-fructose conversion was achieved by GICA at 85 °C making it an attractive candidate for HFCS-55 production. The primary sequence inspection and molecular modeling studies revealed that the thermal stability of GICA could be attributed to the presence of extra charged residues at the surface like E108 and Q408 increasing surface charge interactions. Moreover, a serine at position 56 near to P58 could establish hydrogen bond strengthening the dimer attachment. The high catalytic efficiency and affinity of GICA could be ascribed to the presence of amino acid like E108 and K62 that created more charges around the catalytic site entry.

Safety evaluation of the food enzyme glucose isomerase from Streptomycesmurinus (strain NZYM-GA).

The food enzyme is a glucose isomerase (d‐xylose aldose‐ketose‐isomerase; EC 5.3.1.5) produced with a non‐genetically modified Streptomyces murinus strain NZYM‐GA by Novozymes A/S. The glucose isomerase is intended only to be used in an immobilised form in glucose isomerisation for the production of high fructose syrups. Residual amounts of total organic solids are 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 a safety concern. 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 to occur 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.

High fructose syrup production from mixed food and beverage waste hydrolysate at laboratory and pilot scales

Conversion of food and beverage (F&B) waste into high fructose syrup (HFS) was proposed in this study. Hydrolysate obtained from saccharification of mixed F&B waste is rich in glucose (260.3±7.8gL−1) and fructose (54.1±1.6gL−1) but also contains trace preservatives, caffeine, colourants, ions and soluble proteins. Over 99% of these impurities were removed by adsorption and ion exchange chromatography, followed by enzymatic isomerisation where glucose was converted to reach 50% of fructose content. Pilot scale downstream processing was successfully conducted with more than 89% of sugars recovered from the hydrolysate. Mass balance analysis indicated an overall conversion yield of 0.08kg HFS per kg of mixed F&B waste and meanwhile the HFS also conforms to industrial standards. This proposed process is believed to promote the development of a circular economy by recycling F&B waste as a renewable resource for HFS production.