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Aim of present study was to develop biological catalysts of L-arabinose isomerase (L-AI) by immobilizing on four different supports such as multiwalled carbon nanotube (MWCNT), graphene oxide (GOx), Santa Barbara Amorphous (SBA-15) and mobile composite matter (MCM-41). Also, comparative analysis of the developed catalysts was performed to evolve the best in terms of transformation efficiency for D-tagatose production. The developed nano-enzyme conjugates (NECs) were characterized using the high resolution transmission electron microscopy (HR-TEM) and elemental analysis was performed by energy dispersive X-ray spectroscopy (EDS). The functional groups were investigated by Fourier transform infra red spectroscopy. Also, the thermo gravimetric analysis (TGA) was employed to plot a thermal degradation weight loss profile of NECs. The conjugated L-AI with MWCNT and GOx were found to be more promising immobilized catalysts due to their ability to provide more surface area. Conversion of D-Galactose to D-Tagatose at moderate temperature and pH was observed to attain the equilibrium level of transformation (~50%). On the contrary, NECs prepared using SBA-15 and MCM-41 as support matrix were unable to reach the equilibrium level of conversion. Additionally, the developed NECs were suitable for reuse in multiple batch cycles. Thus, promising nanotechnology coupled with biocatalysis made the transformation of D-Galactose into D-tagatose more economically sustainable., Competing Interests: Declaration of competing interest Authors declare that there is no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

D-Galactose is an abundant carbohydrate monomer in nature and widely exists in macroalgae, plants, and dairy wastes. D-Galactose is useful as a raw material for biomass fuel production or low-calorie sweetener production, attracting increased attention. This article summarizes the studies on biotechnological processes for galactose utilization. Two main research directions of microbial fermentation and enzyme-catalyzed conversion from galactose-rich biomass are extensively reviewed. The review provides the recent discoveries for biofuel production from macroalgae, including the innovative methods in the pretreatment process and technological development in the fermentation process. As modern people pay more attention to health, enzyme technologies for low-calorie sweetener production are more urgently needed. D-Tagatose is a promising low-calorie alternative to sugar. We discuss the recent studies on characterization and genetic modification of L-arabinose isomerase to improve the bioconversion of D-galactose to D-tagatose. In addition, the trends and critical challenges in both research directions are outlined at the end. KEY POINTS: • The value and significance of galactose utilization are highlighted. • Biofuel production from galactose-rich biomass is accomplished by fermentation. • L-arabinose isomerase is a tool for bioconversion of D-galactose to D-tagatose., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

d-Tagatose is a rare monosaccharide that is used in products in the food industry as a low-calorie sweetener. To facilitate biological conversion of d-tagatose, the agarolytic enzyme complexes based on the principle of the cellulosome structure were constructed through dockerin-cohesin interaction with the scaffoldin. The construction of agarolytic complexes composed of l-arabinose isomerase caused efficient isomerization activity on the agar-derived sugars. In a trienzymatic complex, the chimeric β-agarase (cAgaB) and anhydro-galactosidase (cAhgA) from Zobellia galactanivorans could synergistically hydrolyze natural agar substrates and l-arabinose isomerase (LsAraA Doc) from Lactobacillus sakei 23K could convert d-galactose into d-tagatose. The trienzymatic complex increased the concentration of d-tagatose from the agar substrate to 4.2 g/L. Compared with the monomeric enzyme, the multimeric enzyme showed a 1.4-fold increase in tagatose production, good thermostability, and reusability. A residual activity of 75% remained, and 52% of conversion was noted after five recycles. These results indicated that the dockerin-fused chimeric enzymes on the scaffoldin successfully isomerized d-galactose into d-tagatose with synergistic activity. Thus, the results demonstrated the possibility of advancing efficient strategies for utilizing red algae as a biomass source to produce d-tagatose in the industrial food field that uses marine biomass as the feedstock.

D-Tagatose is a ketohexose, which presents unique properties as a low-calorie functional sweetener possessing a sweet flavor profile similar to D-sucrose and having no aftertaste. Considered a generally recognized as safe (GRAS) substance by FAO/WHO, D-tagatose can be used as an intermediate for the synthesis of other optically active compounds as well as an additive in detergent, cosmetic, and pharmaceutical formulations. This study reports important features for L-arabinose isomerase (EC 5.3.1.4) (L-AI) use in industry. We describe arabinose (araA) gene virulence analysis, gene isolation, sequencing, cloning, and heterologous overexpression of L-AI from the food-grade GRAS bacterium Enterococcus faecium DBFIQ E36 in Escherichia coli and assess biochemical properties of this recombinant enzyme. Recombinant L-AI (rL-AI) was one-step purified to homogeneity by Ni 2+ -agarose resin affinity chromatography and biochemical characterization revealed low identity with both thermophilic and mesophilic L-AIs but high degree of conservation in residues involved in substrate recognition. Optimal conditions for rL-AI activity were 50 °C, pH 5.5, and 0.3 mM Mn 2+ , exhibiting a low cofactor concentration requirement and an acidic optimum pH. Half-life at 45 °C and 50 °C were 1427 h and 11 h, respectively, and 21.5 h and 39.5 h at pH 4.5 and 5.6, respectively, showing the high stability of the enzyme in the presence of a metallic cofactor. Bioconversion yield for D-tagatose biosynthesis was 45% at 50 °C after 48 h. These properties highlight the technological potential of E. faecium rL-AI as biocatalyst for D-tagatose production.

L-arabinose isomerase (L-AI) (EC 5. 3. 1. 4. L-AI) that mediates the isomerization of D-galactose to D-tagatose was isolated from Lactobacillus brevis (MF 465792), and was further purified and characterized. Pure enzyme with molecular weight of 60.1 kDa was successfully obtained after the purification using Native-PAGE gel extraction method, which was a monomer in solution. The L-AI was found to be stable at 45-75 °C, and at pH 7.0-9.0. Its optimum temperature and pH was determined as 65 °C and 7.0, respectively. Besides, we found that Ca 2+ , Cu 2+ , and Ba 2+ ions inhibited the enzyme activity, whereas the enzyme activity was significantly enhanced in the presence of Mg 2+ , Mn 2+ , or Co 2+ ions. The optimum concentration of Mn 2+ and Co 2+ was determined to be 1 mM. Furthermore, we characterized the kinetic parameters for L-AI and determined the K m (129 mM) and the V max (0.045 mM min - 1 ) values. Notably, L. brevisL-AI exhibited a high bioconversion yield of 43% from D-galactose to D-tagatose under the optimal condition, and appeared to be a more efficient catalyst compared with other L-AIs from various organisms.

L-Arabinose isomerase isolated from Geobacillus stearothermophilus (GSAI) was modified to improve its substrate specificity for D-galactose for the production of D-tagatose, a potential reduced-energy sweetener. Among the selected residues, mutation at residue 18 produced a mutant strain, H18T, which exhibited increased activity for D-galactose compared with the wild-type (WT) enzyme. Analysis of the substrate specificity of H18T showed a 45.4% improvement for D-galactose. Replacing histidine with threonine at residue 18 resulted in approximately 2.7-fold and 1.8-fold higher substrate binding and catalytic efficiency, respectively, for D-galactose. Further enhancement of the specific activity and catalytic efficiency of H18T for D-galactose by up to 2.7-fold and 4.3-fold, respectively, was achieved by adding borate during L-arabinose isomerase catalysis. Moreover, H18T showed thermostability and no destabilization was detected, which is promising for the industrial production of D-tagatose., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed., Results: The coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn(2+) at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (k cat/K m) for L-arabinose and D-galactose were 8.7 mM(-1) min(-1) and 1.0 mM(-1) min(-1) respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L(-1) and 250 g L(-1) D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h)., Conclusions: In this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn(2+). Its substrate specificity was understood deeply by carrying out molecular modelling and docking studies. When the recombinant E. coli expressing the AI was used as a biocatalyst, D-tagatose could be produced efficiently in a simple one-pot biotransformation system.

The gene encoding L-arabinose isomerase from food-grade strain Pediococcus pentosaceus PC-5 was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and characterized. It was optimally active at 50 °C and pH 6.0. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its maximal activity evaluated at 0.6 mM Mn(2+) or 0.8 mM Co(2+). Interestingly, this enzyme was distinguished from other L-AIs, it could not use L-arabinose as its substrate. In addition, a three-dimensional structure of L-AI was built by homology modeling and L-arabinose and D-galactose were docked into the active site pocket of PPAI model to explain the interaction between L-AI and its substrate. The purified P. pentosaceus PC-5 L-AI converted D-galactose into D-tagatose with a high conversion rate of 52% after 24 h at 50 °C, suggesting its excellent potential in D-tagatose production., (Crown Copyright © 2013. Published by Elsevier GmbH. All rights reserved.)